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A Chemical Approach to Assess the Impact of Post-translational Modification on MHC Peptide Binding and Effector Cell Engagement
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A Chemical Approach to Assess the Impact of Post-translational Modification on MHC Peptide Binding and Effector Cell Engagement
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  • Joey J. Kelly
    Joey J. Kelly
    Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
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  • Nathaniel Bloodworth
    Nathaniel Bloodworth
    Division of Clinical Pharmacology, Department of MedicineVanderbilt University Medical Center, Nashville, Tennessee 37240, United States
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  • Qianqian Shao
    Qianqian Shao
    Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
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  • Jeffrey Shabanowitz
    Jeffrey Shabanowitz
    Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
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    Orcidhttps://orcid.org/0000-0001-5750-3539
  • Donald Hunt
    Donald Hunt
    Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
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  • Jens Meiler
    Jens Meiler
    Division of Clinical Pharmacology, Department of MedicineVanderbilt University Medical Center, Nashville, Tennessee 37240, United States
    Institute of Drug Discovery, Faculty of MedicineUniversity of Leipzig, Leipzig, SAC 04103, Germany
    Center for Structural Biology Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Chemistry Vanderbilt University, Nashville, Tennessee 37232, United States
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  • Marcos M. Pires*
    Marcos M. Pires
    Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-5676-0725
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ACS Chemical Biology

Cite this: ACS Chem. Biol. 2024, 19, 9, 1991–2001
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https://doi.org/10.1021/acschembio.4c00312
Published August 16, 2024

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Abstract

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The human major histocompatibility complex (MHC) plays a pivotal role in the presentation of peptidic fragments from proteins, which can originate from self-proteins or from nonhuman antigens, such as those produced by viruses or bacteria. To prevent cytotoxicity against healthy cells, thymocytes expressing T cell receptors (TCRs) that recognize self-peptides are removed from circulation (negative selection), thus leaving T cells that recognize nonself-peptides. Current understanding suggests that post-translationally modified (PTM) proteins and the resulting peptide fragments they generate following proteolysis are largely excluded from negative selection; this feature means that PTMs can generate nonself-peptides that potentially contribute to the development of autoreactive T cells and subsequent autoimmune diseases. Although it is well-established that PTMs are prevalent in peptides present on MHCs, the precise mechanisms by which PTMs influence the antigen presentation machinery remain poorly understood. In the present work, we introduce chemical modifications mimicking PTMs on synthetic peptides. This is the first systematic study isolating the impact of PTMs on MHC binding and also their impact on TCR recognition. Our findings reveal various ways PTMs alter antigen presentation, which could have implications for tumor neoantigen presentation.

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Introduction

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To maintain homeostasis, the human immune system must efficiently recognize and destroy cells that have accumulated genetic mutations or have been invaded by pathogenic microorganisms. (1,2) Self-identification to the immune system is a primary mechanism deployed by human cells to flag the presence of nonself-proteins or proteins produced by genetic lesions. (3) To this end, presentation of antigenic peptidic fragments via the major histocompatibility complex (MHC) to surveying immune cells serves as a key system to recognize diseased cells (Figure 1A). (2) MHC is present in the membrane of every nucleated cell and is responsible for presenting both antigenic and endogenous protein fragments to the extracellular space. The recognition of peptide-MHC complexes (pMHCs) by T cell receptors (TCRs) (4) initiates an immune cell response, the nature of which depends on the type of the T cell (primarily CD4+ and CD8+ cells). (5)

Figure 1

Figure 1. (A) Schematic representation of the process involving the proteolytic processing of cytosolic protein into peptides. Subsequent steps lead to the loading of the peptides onto MHC molecules that are then transported to cell surface for presentation. (B). Chemical structures of sarsWT and the PTM modified variants. (C) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. Data are represented as mean ± SD (n = 3). P-values were determined by a two-tailed t-test (**** p < 0.0001, ns = not significant). (D) A general schematic representation of how the orientation of the PTM within the presented peptide could negatively impact binding to MHC molecules (right) compared to the unmodified peptide (left). Note that modifications can also occur on the termini of the peptides.

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Critically, precise recognition and binding of pMHCs by TCRs must operate with a high level of fidelity since the response to self-peptides in healthy cells can result in cellular injury. (6) Through negative selection, thymocytes expressing TCRs that bind tightly to self-peptides are removed from circulation. (4,7) In theory, changes to the primary sequence of a protein could yield autoreactive pMHC as long as the primary sequence was not part of the negative selection process. Some primary sequence changes can be permanent such as amino acid changes due to gene mutations, while others can be transient, like post-translational modifications (PTMs). In healthy states, enzymatic PTMs are added by specific sets of enzymes to modulate protein activity, localization, and interactions of the protein with other cellular components. (8) Because PTMs are covalent modifications, most are stable enough to persist through protease digestion and loading onto MHC molecules, potentially leading to the presentation of modified peptide during immune surveillance. (9)
Remarkably, human proteins containing PTMs have been shown to be excluded from negative selection, despite the potential that pMHCs with PTM-modified peptides to yield autoreactive cells. (10) It has been hypothesized that this exclusion is due to the relatively low abundance of PTMs in healthy and young adults in the prime phase of negative selection, compared to the unmodified parent proteins. However, thymus involution after adolescence may contribute to age-related autoimmune diseases. (11,12) Additionally, various factors, such as imbalances of PTM writers and erasers in diseased states, cellular stresses, or aging, can dramatically increase the prevalence of PTMs across the proteome. (13) Collectively, the increased level of pMHCs bearing PTMs mean that a higher proportion of potentially autoreactive cells is present. (14−20) In other words, when peptides with PTM modifications are presented, the immune system may perceive them as nonself, triggering a pathogenic autoimmune response. (21−24) A recent example of this phenomenon involved cysteine carboxyethylation by a cellular metabolite, leading to a pathogenic neoantigen presented on pMHC and resulting in autoreactive T cell responses. (25)
Various PTMs have been previously found in pMHCs. (26−34) Notably, increased conversion of arginine to citrulline in the myelin sheath has been shown to lead to the development of self-reactive T cells that exacerbate the progression of autoimmune diseases such as Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS). (35−40) Moreover, there is mounting evidence that pMHCs presenting peptides with PTMs are prevalent in tumors. (41−44) A recent report revealed that PTMs can shape the antigenic landscape. (44) Despite the importance of PTMs in potentially generating autoreactive peptides, several aspects of PTM biology in this field remain poorly defined. One primary barrier to better understanding the impact of PTMs on pMHCs has been the complexity of assigning the role of PTMs in the context of the many steps leading to MHC presentation. These processes may include changes in a protein half-life, altered proteolytic processing to generate presentable peptides, or binding affinity of individual peptides to MHC molecules. Among these factors, peptide binding affinity to MHC molecules is a primary determinant of the peptide repertoire within pMHCs. (45,46) Here, we systematically isolate the impact of PTMs on the ability of the peptides to bind to MHC molecules and be recognized by cognate T cells.

Results and Discussion

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In general, PTMs can be challenging to detect in the context of complex biological systems, and the levels of PTMs can change in response to cellular cues. (47) Traditional cellular assays do not typically establish the ratio of PTM proteins relative to their unmodified counterparts. (48) These challenges are amplified when analyzing PTMs on peptides from pMHCs. (49) Extracting peptides from isolated MHCs involves challenging separation steps and downstream mass spectrometry analysis. (50) On the other hand, a top-down approach of treating cells with PTM-modified proteins also poses a challenge because exogenous proteins are not readily processed to yield peptides that are presented by MHC I molecules. (51) Even if the peptides get processed, the ultimate pMHC presentation involves multiple factors including peptide half-life, (52) uptake efficiency, or peptide binding affinity to MHC. Consequently, assessment of how various naturally occurring PTMs impact peptide binding to MHCs and recognition by TCRs has not been systematically conducted.
To address these challenges, we based our approach on the RMA-S cell line, which lacks the TAP importer. (53,54) TAP is essential for delivering peptides from the cytosol to the endoplasmic reticulum. By incubating peptides with RMA-S cells at lower temperatures, we allowed peptides to associate with surface-bound MHCs (Figure S1). Upon increasing the temperature, we observed the stabilization of pMHCs on the cell surface. The quantity of MHC on the cell surface correlated with the binding affinity (55−60) of the peptide to the MHC molecule, quantified via a fluorescently labeled anti-MHC antibody. (61) Given the features of this well-established cell line, we anticipated that RMA-S cells would effectively reveal the impact of PTMs on peptides that bind to MHC with high affinity. Moreover, RMA-S cells have been used extensively for isolating the affinity of a peptide for MHC.
To start, we sought to identify a peptide from SARS-CoV-2 spike protein that could be displayed on MHC molecules. We used NetMHCPan4.0, (62) an algorithm that estimates the propensity of peptides to bind MHC, to select a peptide sequence (ESIVRFPNI, referred to as sarsWT) for stabilization of the specific MHC molecule (H-2Kb) on the surface of RMA-S cells. The peptide was synthesized using standard solid-phase peptide synthesis techniques. The culture medium of the RMA-S cells was supplemented with sarsWT at 26 °C to enable association of the peptide with surface bound MHCs. Following sarsWT incubation, the temperature was raised to 37 °C. Subsequently, the cells were incubated with APC conjugated antimouse H-2Kb antibody and analyzed by flow cytometry where fluorescence is expected to correspond with the amount of sarsWT bound to the MHC. The peptide SNFVSAGI (cntPEP) was used as a negative control since it has been reported to not interact appreciably with H-2Kb. (63) Various concentrations of sarsWT were used to optimize the RMA-S stabilization assay with the goal of increasing the number of pMHC molecules formed on the surface of the cells (Figure S2). We found an increased fluorescence signal corresponding to increasing concentrations of sarsWT, indicating that the peptide bound and stabilized the MHC on the surface of the cell. The amount of stable pMHCs that formed at the surface also saturated at 50 μM of peptide and showed an EC50 of 1.7 μM. These results confirmed the suitability of RMA-S to report on the stabilization of the pMHC complex by sarsWT.
Another parameter to consider is the time in which the MHC binding peptide has to associate with the MHC to reach MHC saturation. To evaluate this time frame, the workflow described above was conducted; however, the peptide incubation time at 37 °C was varied from 2 to 6 h (Figure S3). The results showed that the fluorescent signal increased with increased incubation periods. This is likely due to accumulation of stabilized pMHCs in the presence of excess high-affinity peptide. Nevertheless, a 6 h incubation at 37 °C provided the highest signal-to-noise ratio and was used for downstream assays.
With an optimized protocol in hand, we then synthesized a panel of peptides containing PTMs to investigate their potential impact on MHC binding (Figure 1B). We envisioned that modifying residues on sarsWT would be representative of how the selected modifications may impact binding of different MHC peptides. The specific PTMs chosen were a proline to hydroxyproline, (64,65) arginine to citrulline, (66) and N-terminus acetylation (67) modifications, as they are naturally occurring PTMs. By performing the RMA-S stabilization assay, we found that the citrullination and hydroxy-proline modifications had no significant effect on MHC binding, but the N-terminal acetylation modification completely disrupted MHC binding (Figure 1C). Gratifyingly, there is precedence for N-acetylated peptides to be displaced in MHC class I. (68,69) These results demonstrate that naturally occurring PTMs can alter pMHC complex formation and influence the antigen presentation pathway depending on the interface of the peptide and the MHC molecule (Figure 1D).
Next, we shifted our focus to the peptide epitope SIINFEKL (ovaWT) from the model antigen ovalbumin (OVA) to interrogate how other PTMs impact MHC binding as well as TCR recognition. (70) To this end, we synthesized a new library of OVA peptides containing PTMs on its serine and lysine residues and performed a concentration scan to assess MHC binding using the RMA-S stabilization assay (Figure 2A). Interestingly, there was a significant decrease in MHC binding when serine was phosphorylated despite predictions that this site does not contribute significantly to binding (Figure 2B). (71) Our results highlight the potential deficiency of current models to predict how PTMs could impact peptide binding to MHC molecules given how PTM products can be structurally distinct relative to the side chains of the canonical amino acids.

Figure 2

Figure 2. (A) Chemical structures of ovaWT and the PTM modified variants. (B) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. (C) RMA-S cells were incubated with peptide and B3Z T cells overnight at an effector to target ratio of 1:1. β-galactosidase expression was then measured via the colorimetric reagent CPRG on a plate reader at 570 nm. (D) Schematic representation of how PMTs can impact engagement with TCRs. Data are represented as mean ± SD (n = 4). P-values were determined by a two-tailed t test (**** p < 0.0001)

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The side chain of lysine (K7) on ovaWT was explored next. Lysine was decorated with different types of PTMs that included three different charge states. Previously, the same lysine on SIINFEKL had been altered with various groups including fluorophores and caging groups. (72−74) Our data showed considerable accommodation of structural alterations to the lysine side chain (Figure 2B). With larger modifications such as biotin, binding levels are altered but the change is not significant. These findings can be explained by the solvent exposed nature of lysine on the pMHC complex and the length of the lysine side chain, which may reduce structurally unfavorable interactions with the MHC molecule (Figure 1D). (75) Critically, for ovaWT the anchor residues that are more important for the overall binding affinity to MHC molecules (Phe5 and Leu8) are not amendable to conventional PTMs. (76)
We then sought to detect changes in peptide binding via a competition assay using the parent cell line (RMA) that has a competent TAP system. To this end, we performed a competition assay using the panel of OVA peptides barring PTM mark in live RMA cells. The assay was performed by coincubating RMA cells individually with PTM-modified peptides and also ovaFl (SIINFEK(FITC)L), in which the γ position of lysine is modified with fluoresceine. ovaFl has long been utilized by the field to interrogate binding of untagged peptides for MHC molecules. A decrease in cellular fluorescence should indicate higher association levels with MHC molecules, thereby informing on the relative affinity of the PTM-modified molecules. Critically, this assay retains MHC molecules within a physiologically relevant context and this alternative readout should complement the RMA-S MHC stabilization assay. As expected, the trend in binding affinity in the competition experiment closely mirrored our results with the RMA-S stabilization assay indicating that the RMA-S assay can accurately report on relative MHC binding affinities in a cellular context (Figure S4). As further evidence and to specifically detect PTM-modified peptides bound on MHC, ovaWT and ovaK7m3 treated RMA-S cells had their MHC-bound peptides eluted and identified using mass spectrometry. The canonical binding peptide, ovaWT, was clearly detected after elution from live RMA-S cells (Figures S5 and S6). Likewise, the ovaK7m3 was detected by mass spectrometry and this provides direct evidence that trimethylation of K7 does not disrupt binding of this peptide to MHC (Figures S7 and S8). While PTMs on solvent exposed residues could potentially retain binding to MHCs, their overall impact may be more strongly reflected in the pMHC engagement with TCRs. TCRs recognize T cell exposed motifs of the bound peptide within pMHCs. (77,78) Therefore, PTMs at these positions may have a greater impact on T cell activation.
To assess the impact of ovaWT PTMs on TCR recognition, we utilized the B3Z T cell hybridoma cell line that contains an OVA specific TCR along with a NFAT-LacZ reporter gene that encodes for β-galactosidase on an IL-2 inducible promoter. (79) Upon B3Z recognition of the OVA pMHC complex on RMA-S cells, IL-2 production promotes β-galactosidase expression, which can hydrolyze chlorophenol red-β-D-galactopyranoside (CPRG), and the resulting color change is representative of activation levels. Our results showed that the PTMs have a much more significant effect on TCR recognition than on peptide binding to MHC (Figure 2C). While relatively modest disruption of TCR recognition was seen for increasing the degree of methylation on the lysine residue, near complete disruption of TCR recognition was seen for all PTMs that imparted a change in charge of either the serine or lysine residue. Additionally, the reduction in TCR recognition for both the phosphoserine and biotinylated OVA cannot be fully explained solely by their decrease in MHC binding. Instead, the PTMs that are well accommodated for MHC binding could be displayed away from the binding cleft and alter engagement with TCRs on cognate T cells (Figure 2D).
To confirm the relevance of these findings in a broader context, we also performed the T cell activation assay on DC2.4 dendritic cells, which also express H-2Kb. Unlike the RMA-S cells, peptides were incubated with DC2.4 cells at 37 °C and were expected to load onto MHC molecules if they display sufficient affinity toward H-2Kb. Satisfyingly, the pattern of T cells activation with PTM modified ovaWT peptides showed a similar profile with DC2.4 cells as RMA-S cells (Figure S9). Our results confirmed that the impact of PTMs on T cell activation may be consistent across multiple cell types. Finally, we sought to investigate whether we could assess PTMs on a peptide that has potential pathological implications. To this end, a number of autoimmune diseases, including RA and MS, have been described to involve the citrullination of arginine. (80) Citrullination is carried out by peptidylarginine deiminases (PADs) and higher levels of citrullinated proteins have been found in older mice relative to young mice. (81) A shift in higher levels of citrullination past the full scope of negative selection could provide a pathway for autoreactivity. Using a peptide originating from a known PAD substrate myelin basic protein (RTAHYGSL, mbpWT) as the baseline (Figure 3A), we found that there was a statistically significant increase in peptide binding to MHC molecules upon citrullination (Figure 3B). This result is consistent with the possibility that PTMs can impact presentation of peptides in the context of disease-linked peptides.

Figure 3

Figure 3. (A) Chemical structures of mbpWT and the PTM modified variant. (B) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. (C) NetMHCpan 4.1 predicted binding scores of tumor associated peptides. %Rank is the prediction score for comparing MHC binding across random peptides where a %Rank of 1 indicates that a peptide scored in the top 1% of random peptides. A %Rank of <2 or <0.5 are the cutoffs for weak binding or strong binding to MHC respectively. A %Rank >2 indicates nonbinders. (D) A heat map of wild-type and PTM modified peptides relative MHC binding analyzed by using the RMA-S stabilization assay. RMA-S cells were incubated with peptide at indicated concentration of peptides and detected via flow cytometry with APC conjugated antimouse H-2Kb antibodies. Abbreviations: Cit (citrullination), Yp (phosphorylated tyrosine), Kac (lysine acetylated at the γ position), NAc (acetylation at the α position), and Sp (phosphorylated serine). Data are represented as mean ± SD (n = 4). P-values were determined by a two-tailed t test (**** p < 0.0001).

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We then sought to uncover if PTMs are playing a role in influencing the presentation of cancer antigens. However, only a limited number of studies have identified a wide range of PTM modified MHC peptides presented on cancer cells. (43,44) Additionally, the specific impact of these PTMs on peptide-MHC binding remains unclear. To this end, we synthesized 10 PTM modified MHC peptides previously identified on cancer cells and compared their binding affinity to MHC to their respective unmodified versions. (43,44) Out of this library, only 3 PTM modified peptides were found to bind to MHC at the biologically relevant concentrations, highlighting the need for secondary validation assays for peptide libraries identified through mass spectrometry screens (Figure 3C,D). Interestingly, out of the peptides that bound, the PTM versions all showed significant improvement in MHC stabilization on RMA-S cells suggesting that PTMs may enhance the presentation of cancer associated peptides (Figure S10).
To gain additional insight into how PTM s might alter peptide binding affinity to H-2Kb, we simulated peptide binding to H-2Kb using ROSETTA and FlexPepDock. FlexPepDock has been previously benchmarked against MHC-I bound peptides and is capable of generating models with subangstrom accuracy. (82,83) One advantage of FlexPepDock over contemporary machine learning methods is its ability to incorporate noncanonical amino acids and PTM residues by generating custom ROSETTA parameters files; this enables FlexPepDock to generate accurate models of MHC-I bound epitopes containing such residues. (84,85) Furthermore, the binding energy metrics calculated by this application (the reweighted_sc score term) can serve a surrogate for peptide binding affinity to MHC-I. (86) We simulated the binding of the SARS-CoV-2-derived peptide to H-2Kb and generated results recapitulating experimentally derived binding affinity changes (Figure 4). ROSETTA correctly predicts decreased binding affinity for the top-scoring decoys after N-terminal acetylation (corresponding to an increase in the average reweighted_sc term; ROSETTA energy metrics are inversely proportional to binding affinity). ROSETTA predicts either no change or a modest increase in binding affinity for the other two PTMs assessed (Figure 4A). When the top-scoring model for each peptide is visually inspected, we noted significant alterations in the peptide backbone structure for the acetylated N-terminus peptide variant largely confined to the H-2Kb N-terminus binding pocket (Figure 4B), a region that is largely responsible for dictating epitope binding specificity and that cannot easily accommodate large conformational changes. (9) Conversely, R5 citrullinated and P7 hydroxylated variants are located outside the H-2Kb binding pockets and regions tolerant of larger conformational changes (Figure 4C).

Figure 4

Figure 4. Modeling the sars peptide with and without PTMs using FlexPepDock. (A) N-terminal acetylation results in higher reweighted_sc values (and thus lower likelihood of binding) compared to the wild-type peptide, consistent with experimental results (one-way ANOVA and Holm-Sidak posthoc; individual values correspond to the top 0.5% of models generated, and bars represent mean score term values). (B) Top peptide models generated for each epitope positioned in the H-2Kb binding cleft. (C) Side chain modifications compared to the unmodified epitope for the SARS-CoV-2 peptide. Modeling the ovalbumin-derived peptide with and without PTMs using FlexPepDock. (D) Phosphorylation of the N-terminal serine residue generates models with higher average reweighted_sc terms, similar to N-terminal acetylation of the SARS-CoV-2 peptide (one-way ANOVA and Holm-Sidak posthoc; individual values correspond to the top 0.5% of models generated, and bars represent mean score term values). (E) Superimposed peptide backbones for the top peptide models generated for each PTM. (F) Detailed side-chain configurations for the top-scoring models generated by FlexPepDock.

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We repeated this analysis on the ovalbumin-derived peptide and each PTM (Figure 4D). Phosphorylation of the N-terminus serine generates structures with higher (and therefore less favorable) score terms, again likely due to disruption of the N-terminus peptide-binding pocket (Figure 4E). Peptide models with the remaining PTMs scored either equivalently or slightly better than their wild-type counterparts. While largely consistent with experimental data, ROSETTA predicts modest increases in peptide binding favorability that are not observed experimentally. This may be a result of biases inherent in ROSETTA’s scoring weights that have not been fully optimized for unusual or noncanonical amino acids such as those modeled here. Alternatively, the assay used for assessing binding affinity may be insufficiently sensitive to detect small changes above certain affinity thresholds. Modifications at the lysine in position 7 alter T cell activation in vitro as illustrated in Figure 2C. In silico, these altered residues do not interact appreciably with peptide binding pockets but instead occur in the region displayed by MHC-I to patrolling T cells (Figure 4F). Methylated and acetylated lysine alters residue charge and hydrophobicity; succinylation adds a negative charge (as opposed to lysine’s normally positively charged side chain); and biotinylation results in the addition of a large aliphatic heteropolycyclic side chain very different in size and character from the native lysine residue.
Finally, we performed a similar analysis with the mbp peptide with and without R1 citrullination and generated in silico results that recapitulated experimental findings (Figure 3). Citrullination at R1 significantly increases the reweighted_sc metrics for the models generated by ROSETTA (Figure S11A). While this modification occurs at the N-terminus binding pocket, it does not appreciably alter peptide backbone configuration (Figure S11A, in contrast to the effects of N-terminal acetylation of the sars peptide). It does, however, alter side chain hydrophobicity and charge. Given that the citrullinated variant is charge-neutral (compared to arginine’s 1+ charge) and given H-2Kb’s preference for hydrophobic or charge-neutral residues at binding pockets, it is reasonable to expect this modification may enhance peptide binding affinity.
Increasingly, there are efforts dedicated to developing therapeutic agents that harness a patient’s immune system against cancerous lesions. (87−89) A prominent example involves the modulation of the programmed death-1 (PD-1) system and its cognate programmed death-ligand 1 (PD-L1). Cancers can often hijack this set of proteins to maintain growth while suppressing the patient immune response against transformed cells. Immune checkpoint inhibitors that disrupt the association of PD-1 and PD-L1 have shown potent anticancer activity by a number of mechanisms but primarily via increased T cell engagement. (90,91) Neoantigens arising from genetic lesions likely provide a pool of antigens that can be presented by cancer MHCs on the cell surface during checkpoint therapy. Importantly, our results suggest that therapeutic agents that increase the presentation of peptides through an altered balance of PTM marks could potentially complement the targeting antigenic pool.
Considering the observed roles of PTMs in MHC binding and recognition by TCRs, it is conceivable that the balance of PTM addition by “writers” and PTM removal by “erasers” can be central in immunological health. Any imbalance that could be driven by age or manufactured by therapeutic interventions could result in pathological state. For example, proteins could naturally exist in high abundance of nonacetylated states on lysine side chains past the peak period of negative selection of T cells. After cancer development, there is an increased level of acetylation, but those marks need to be removed by “erasers” called Lysine Deacetylases (KDAC) to reduce an anticancer immunological response against the cancerous cells. Upon the administration of KDAC inhibitors, a larger pool of acetylated peptide would be presented. As our data showed, the acetylation of lysine that interacts with TCRs would escape negative selection and provide an anticancer immunological response. Such “cryptic” antigens could potentially be driving the pharmacological effect of some PTM modulators in cancer patients. To this end, there are a high number of ongoing studies evaluating the combination of KDAC inhibitors and PD-1/PDL-1 inhibitors across many types of difficult-to-treat tumors. (92−98) We are currently working toward using this strategic platform to demonstrate how KDAC inhibitors directly lead to an immunopeptidome that reveal cryptic antigens able to enhance antitumor immunological activity.

Conclusions

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In conclusion, the data presented here demonstrate that naturally occurring PTMs can drastically impact the antigen presentation pathway by altering either their affinity toward MHC molecules or TCR recognition by cognate T cells. In our assays, we showed that some PTMs can disrupt peptide binding to MHC, but primarily on residues that directly engage with MHCs. Conversely, PTMs on residues whose side chains are positioned away from the MHC binding face are more sensitive to TCR recognition due to the altered binding modality with TCRs. To the best of our knowledge, our study is the first systematic analysis of the impact that PTMs have on MHC binding in a whole cell context and how PTMs can also alter T cell activation using live target and effector cells. Provided that negative selection is age-dependent and becomes increasingly diminished past adolescence, it is evident that PTMs could be causal events that result in autoimmune diseases.

Methods

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Mammalian Cell Culture

RMA-S cells were a kind gift from Dr. John Sampson. RMA-S cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 50 IU/mL penicillin, 50 μg/mL streptomycin,1X MEM nonessential amino acid solution (ThermoFisher) and cultured in a humidified atmosphere of 5% CO2 at 37 °C. B3Z cells were kindly provided by Dr. Aaron Esser-Kahn and maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 50 IU/mL penicillin, 50 μg/mL streptomycin and cultured in a humidified atmosphere of 5% CO2 at 37 °C.

RMA-S Stabilization Assay

105 RMA-S cells were seeded in a treated 96 well plate at 37 °C overnight. The next day, RMA-S cells were moved to a 26 °C incubator for 24–48 h. Following the incubation period, cells were incubated with peptides in culture media at indicated concentrations for 1 h at 26 °C before being moved to the 37 °C incubator for 6 h. The media was then replaced with a 1:100 dilution of APC-labeled antimouse H-2Kd/H-2Dd antibody in culture media for 1 h at 4 °C. Cells were removed from the well plate by vigorous pipetting, fixed with 2% formaldehyde solution, and analyzed using the Attune NxT Flow Cytometer (Thermo Fischer) equipped with a 637 nm laser with 670/14 nm bandpass filter.

Mild Acid Elution

109 RMA-S cells were treated with 20 μM peptide at 26 °C for 1 h. Cells were then warmed to 37 °C for 6 h. RMA-S cells were collected by spinning down at 500 x g for 5 min and thoroughly washed with 1X PBS Buffer pH 7.4 (Invitrogen). Next, 15 mL of mild acid elution (MAE) buffer (0.131 M citric acid, 0.066 M Na2HPO4, 150 mM NaCl, 0.3 μM Aprotinin, and 5 mM iodoacetamide at pH 3.3) was added to RMA-S cells for 2 min. RMA-S cells were then spun down at 4000 x g for 5 min and the supernatant was collected, lyophilized, and stored at −80 °C until further use.

Mass Spectrometry Analysis

The emitter tip of the analytical column was laser-pulled to produce an opening of 2–5 μm, and a 2 mm kasil frit was used in place of the irregular reverse phase (RP) resin. The precolumns (100 μm i.d. x 360 μm O.D. fused silica) were packed to 7 cm with 10 μm C18 beads, and the analytical columns (75 μm i.d. x 360 μm o.d. fused silica) were packed to 10 cm with 3 μm C18 beads. Ova samples and 100 fmol each of the internal standards (Angio and Vaso) were loaded onto the precolumn using a pressure vessel for a 15 min desalting rinse at a flow rate of 100 nL/min with 0.1% acetic acid (AcOH) in water before connecting to the analytical column with a 2 cm Teflon tubing. Reverse phase separation was conducted at a flow rate of 100 nL/min by HPLC using 0.1% AcOH for solvent A and 0.1% AcOH in 60% acetonitrile for solvent B, and a gradient as follows: 0% to 60% solvent B in 60 min, 60% to 100% solvent B in 2 min, 100% solvent B for 4 min, 0% in 2 min, followed by a 22 min equilibration with 100% solvent A. The peptides eluted from the analytical column were electrosprayed into an LTQ-Orbitrap mass spectrometer. MS1 spectra were acquired with a resolution of 60,000, an AGC target of 5e5 and scan range of 300 to 2000 m/z in the Orbitrap analyzer, followed by low resolution data dependent MS2 acquisition in the ion trap with normal scan rate. Only precursor ions with charge +2 and +3 were selected for fragmentation. The top 2 most abundant precursor ions, as well as the targeted +1 and +2 precursor masses for the ovaWT and ovaK7m3 peptides, were selected for collision-activated dissociation (CAD) in the ion trap analyzer with an AGC target of 1e4, a normalized collision energy of 35%, an activation time of 30 ms, and a 2.0 m/z isolation window. If the same precursor ion was selected three times or was detected twice within a 20-s repeat duration, the ion was dynamically excluded for 15 s. The presence of ovaWT and ovaK7m3 peptides was verified by manually inspecting the targeted MS2 spectra for the expected fragmentations and fragment ion masses.

B3Z T Cell Activation

105 RMA-S cells were seeded in a treated 96 well plate at 37 °C overnight. The following day the culture media was replaced with media containing indicated concentration of peptide along with 105 B3Z cells in culture media and coincubated overnight. Cells were then spun down at 500xg for 5 min and washed with 1X PBS a total of two times. Lysis buffer containing 0.2% saponin, 500 μM CPRG reagent, 100 mM MgCl2, and 100 mM β-mercaptoethanol in 1X PBS was added to each well. After 2–4 h absorbance 570 was recorded using a BioTek Epoch 2 microplate reader.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.4c00312.

  • Additional figures including RMA-S (workflow scheme, concentration scan with sarsWT, and time scan), DC2.4 T Cell activation, modeling of citrullination, and mass spectrometry results from MHC peptides; materials/methods; and synthetic peptide characterization (mass spectrometry and analytical HPLC traces) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Joey J. Kelly - Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
    • Nathaniel Bloodworth - Division of Clinical Pharmacology, Department of MedicineVanderbilt University Medical Center, Nashville, Tennessee 37240, United States
    • Qianqian Shao - Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
    • Jeffrey Shabanowitz - Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States;  Orcidhttps://orcid.org/0000-0001-5750-3539
    • Donald Hunt - Department of Chemistry University of Virginia Charlottesville, Virginia 22904, United States
    • Jens Meiler - Division of Clinical Pharmacology, Department of MedicineVanderbilt University Medical Center, Nashville, Tennessee 37240, United States;  Institute of Drug Discovery, Faculty of MedicineUniversity of Leipzig, Leipzig, SAC 04103, Germany;  Center for Structural Biology Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Chemistry Vanderbilt University, Nashville, Tennessee 37232, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This study was supported by the NIH grant 2R35GM124893 (M.M.P.), GM037537 (D.H.), NIH 5T32HL144446-05 (N.B.), 5R01AI141661-05 (J.M.), and 5R01DA046138-05 (J.M.).

References

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    A review. The functionality of proteins is greatly extended by a diverse array of post-translational modifications (PTMs), many of which are recognized by the immune system. Notably, a significant proportion of peptides presented to T cells by the major histocompatibility complex in vivo are post-translationally modified. Since the cellular mechanisms that introduce and control protein modifications can differ between health and disease, the assocd. changes in antigen presentation have the potential to alter immune responses. A no. of such situations have been implicated with infection, inflammation, autoimmune disease, and cancer, and the investigation of PTMs that affect antigen recognition has provided insight in disease progression as well as raising prospects for novel approaches in immunotherapy.
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    Marcilla, M.; Alpizar, A.; Lombardia, M.; Ramos-Fernandez, A.; Ramos, M.; Albar, J. P. Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue. Mol. Cell Proteomics 2014, 13, 462– 474,  DOI: 10.1074/mcp.M113.034314
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    Bassani-Sternberg, M.; Braunlein, E.; Klar, R.; Engleitner, T.; Sinitcyn, P.; Audehm, S.; Straub, M.; Weber, J.; Slotta-Huspenina, J.; Specht, K.; Martignoni, M. E.; Werner, A.; Hein, R.; D, H. B.; Peschel, C.; Rad, R.; Cox, J.; Mann, M.; Krackhardt, A. M. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat. Commun. 2016, 7, 13404,  DOI: 10.1038/ncomms13404
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    Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry
    Bassani-Sternberg, Michal; Braeunlein, Eva; Klar, Richard; Engleitner, Thomas; Sinitcyn, Pavel; Audehm, Stefan; Straub, Melanie; Weber, Julia; Slotta-Huspenina, Julia; Specht, Katja; Martignoni, Marc E.; Werner, Angelika; Hein, Ruediger; H. Busch, Dirk; Peschel, Christian; Rad, Roland; Cox, Juergen; Mann, Matthias; Krackhardt, Angela M.
    Nature Communications (2016), 7 (), 13404CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) anal., we survey the melanoma-assocd. immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.
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    Alpizar, A.; Marino, F.; Ramos-Fernandez, A.; Lombardia, M.; Jeko, A.; Pazos, F.; Paradela, A.; Santiago, C.; Heck, A. J.; Marcilla, M. A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens. Mol. Cell Proteomics 2017, 16, 181– 193,  DOI: 10.1074/mcp.M116.063800
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    A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens
    Alpizar, Adan; Marino, Fabio; Ramos-Fernandez, Antonio; Lombardia, Manuel; Jeko, Anita; Pazos, Florencio; Paradela, Alberto; Santiago, Cesar; Heck, Albert J. R.; Marcilla, Miguel
    Molecular & Cellular Proteomics (2017), 16 (2), 181-193CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
    As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I mols. can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the mol. determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands assocd. with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar mol. features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To det. the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes assocd. to different HLA-B mols. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This mol. understanding of the presentation of phosphopeptides by HLA-B mols. provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.
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    Malaker, S. A.; Penny, S. A.; Steadman, L. G.; Myers, P. T.; Loke, J. C.; Raghavan, M.; Bai, D. L.; Shabanowitz, J.; Hunt, D. F.; Cobbold, M. Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia. Cancer Immunol Res. 2017, 5, 376– 384,  DOI: 10.1158/2326-6066.CIR-16-0280
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    Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia
    Malaker, Stacy A.; Penny, Sarah A.; Steadman, Lora G.; Myers, Paisley T.; Loke, Justin C.; Raghavan, Manoj; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Cobbold, Mark
    Cancer Immunology Research (2017), 5 (5), 376-384CODEN: CIRACV; ISSN:2326-6066. (American Association for Cancer Research)
    Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-assocd. peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three exptl. approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be assocd. with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equiv. unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR.
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    Marino, F.; Mommen, G. P.; Jeko, A.; Meiring, H. D.; van Gaans-van den Brink, J. A.; Scheltema, R. A.; van Els, C. A.; Heck, A. J. Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07. J. Proteome Res. 2017, 16, 34– 44,  DOI: 10.1021/acs.jproteome.6b00528
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    Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07
    Marino, Fabio; Mommen, Geert P. M.; Jeko, Anita; Meiring, Hugo D.; van Gaans-van den Brink, Jacqueline A. M.; Scheltema, Richard A.; van Els, Cecile A. C. M.; Heck, Albert J. R.
    Journal of Proteome Research (2017), 16 (1), 34-44CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Alterations in protein post-translational modification (PTMs) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related Human Leukocyte Antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line the authors detected 149 HLA class I peptides harboring mono- and/or di-methylated arginine residues by mass spectrometry. A striking preference was obsd. in presentation of arginine (di)methylated peptides for HLA-B*07 mols., likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored di-methylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been assocd. with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra the authors found most of the peptides to be asym. di-methylated, most likely by CARM1. These data expand the authors' knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides, and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and di-methylated arginine residues may therefore offer a novel target for immunotherapy.
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    Mohammed, F.; Stones, D. H.; Zarling, A. L.; Willcox, C. R.; Shabanowitz, J.; Cummings, K. L.; Hunt, D. F.; Cobbold, M.; Engelhard, V. H.; Willcox, B. E. The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status. Oncotarget 2017, 8, 54160– 54172,  DOI: 10.18632/oncotarget.16952
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    Mangalaparthi, K. K.; Madugundu, A. K.; Ryan, Z. C.; Garapati, K.; Peterson, J. A.; Dey, G.; Prakash, A.; Pandey, A. Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I. J. Proteins Proteom 2021, 12, 151– 160,  DOI: 10.1007/s42485-021-00066-x
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    The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting
    Malmstrom Vivianne; Catrina Anca I; Klareskog Lars
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    Patients with rheumatoid arthritis can be divided into two major subsets characterized by the presence versus absence of antibodies to citrullinated protein antigens (ACPAs) and of rheumatoid factor (RF). The antibody-positive subset of disease, also known as seropositive rheumatoid arthritis, constitutes approximately two-thirds of all cases of rheumatoid arthritis and generally has a more severe disease course. ACPAs and RF are often present in the blood long before any signs of joint inflammation, which suggests that the triggering of autoimmunity may occur at sites other than the joints (for example, in the lung). This Review summarizes recent progress in our understanding of this gradual disease development in seropositive patients. We also emphasize the implications of this new understanding for the development of preventive and therapeutic strategies. Similar temporal and spatial separation of immune triggering and clinical manifestations, with novel opportunities for early intervention, may also occur in other immune-mediated diseases.
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    Curran, A. M.; Naik, P.; Giles, J. T.; Darrah, E. PAD enzymes in rheumatoid arthritis: pathogenic effectors and autoimmune targets. Nat. Rev. Rheumatol 2020, 16, 301– 315,  DOI: 10.1038/s41584-020-0409-1
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    Yang, L.; Tan, D.; Piao, H. Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology. Neurochem. Res. 2016, 41, 1845– 1856,  DOI: 10.1007/s11064-016-1920-2
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    Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology
    Yang, Lei; Tan, Dewei; Piao, Hua
    Neurochemical Research (2016), 41 (8), 1845-1856CODEN: NEREDZ; ISSN:0364-3190. (Springer)
    Multiple sclerosis (MS) is a multifactorial demyelinating disease characterized by neurodegenerative events and autoimmune response against myelin component. Citrullination or deimination, a post-translational modification of protein-bound arginine into citrulline, catalyzed by Ca2+ dependent peptidylarginine deiminase enzyme (PAD), plays an essential role in physiol. processes include gene expression regulation, apoptosis and the plasticity of the central nervous system, while aberrant citrullination can generate new epitopes, thus involving in the initiation and/or progression of autoimmune disorder like MS. Myelin basic protein (MBP) is the major myelin protein and is generally considered to maintain the stability of the myelin sheath. This review describes the MBP citrullination and its consequence, as well as offering further support for the "inside-out" hypothesis that MS is primarily a neurodegenerative disease with secondary inflammatory demyelination. In addn., it discusses the role of MBP citrullination in the immune inflammation and explores the potential of inhibition of PAD enzymes as a therapeutic strategy for the disease.
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    Hill, J. A.; Southwood, S.; Sette, A.; Jevnikar, A. M.; Bell, D. A.; Cairns, E. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J. Immunol 2003, 171, 538– 541,  DOI: 10.4049/jimmunol.171.2.538
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    Jaeger, A. M.; Stopfer, L. E.; Ahn, R.; Sanders, E. A.; Sandel, D. A.; Freed-Pastor, W. A.; Rideout, W. M., 3rd; Naranjo, S.; Fessenden, T.; Nguyen, K. B.; Winter, P. S.; Kohn, R. E.; Westcott, P. M. K.; Schenkel, J. M.; Shanahan, S. L.; Shalek, A. K.; Spranger, S.; White, F. M.; Jacks, T. Deciphering the immunopeptidome in vivo reveals new tumour antigens. Nature 2022, 607, 149– 155,  DOI: 10.1038/s41586-022-04839-2
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    Yi, X.; Liao, Y.; Wen, B.; Li, K.; Dou, Y.; Savage, S. R.; Zhang, B. caAtlas: An immunopeptidome atlas of human cancer. iScience 2021, 24, 103107  DOI: 10.1016/j.isci.2021.103107
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    Kacen, A.; Javitt, A.; Kramer, M. P.; Morgenstern, D.; Tsaban, T.; Shmueli, M. D.; Teo, G. C.; da Veiga Leprevost, F.; Barnea, E.; Yu, F.; Admon, A.; Eisenbach, L.; Samuels, Y.; Schueler-Furman, O.; Levin, Y.; Nesvizhskii, A. I.; Merbl, Y. Post-translational modifications reshape the antigenic landscape of the MHC I immunopeptidome in tumors. Nat. Biotechnol. 2023, 41, 239– 251,  DOI: 10.1038/s41587-022-01464-2
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    Sette, A.; Vitiello, A.; Reherman, B.; Fowler, P.; Nayersina, R.; Kast, W. M.; Melief, C. J.; Oseroff, C.; Yuan, L.; Ruppert, J.; Sidney, J.; del Guercio, M. F.; Southwood, S.; Kubo, R. T.; Chesnut, R. W.; Grey, H. M.; Chisari, F. V. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol 1994, 153, 5586– 5592,  DOI: 10.4049/jimmunol.153.12.5586
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    The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes
    Sette, Alessandro; Vitiello, Antonella; Reherman, Barbara; Fowler, Patricia; Nayersina, Ramin; Kast, W. Martin; Melief, Cornelis J. M.; Oseroff, Carla; Yuan, Lunli; et al.
    Journal of Immunology (1994), 153 (12), 5586-92CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
    The relation between binding affinity for HLA class I mols. and immunogenicity of discrete peptide epitopes was analyzed in 2 different exptl. approaches. In the 1st approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approx. 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approx. 500 nM (preferably 50 nM or less) apparently dets. the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, the data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.
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    Post-translational modifications in signal integration
    Deribe, Yonathan Lissanu; Pawson, Tony; Dikic, Ivan
    Nature Structural & Molecular Biology (2010), 17 (6), 666-672CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    A review. Post-translational modifications of proteins and the domains that recognize these modifications play central roles in creating a highly dynamic relay system that reads and responds to alterations in the cellular microenvironment. Here, the authors review the common principles of post-translational modifications and their importance in signal integration underlying epidermal growth factor receptor signaling and endocytosis, DNA-damage responses, and immunity.
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    Modification-specific proteomics: Strategies for characterization of post-translational modifications using enrichment techniques
    Zhao, Yingming; Jensen, Ole N.
    Proteomics (2009), 9 (20), 4632-4641CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. More than 300 different types of protein post-translational modifications (PTMs) have been described, many of which are known to have pivotal roles in cellular physiol. and disease. Nevertheless, only a handful of PTMs have been extensively investigated at the proteome level. Knowledge of protein substrates and their PTM sites is key to dissection of PTM-mediated cellular processes. The past several years have seen a tremendous progress in developing mass spectrometry (MS)-based proteomics technologies for global PTM anal., including numerous studies of yeast and other microbes. Modification-specific enrichment techniques combined with advanced MS/MS methods and computational data anal. have revealed a surprisingly large extent of PTMs in proteins, including multi-site, cooperative modifications in individual proteins. The authors review some of the current strategies employed for enrichment and detection of PTMs in modification-specific proteomics.
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    Solleder, M.; Guillaume, P.; Racle, J.; Michaux, J.; Pak, H. S.; Muller, M.; Coukos, G.; Bassani-Sternberg, M.; Gfeller, D. Mass Spectrometry Based Immunopeptidomics Leads to Robust Predictions of Phosphorylated HLA Class I Ligands. Mol. Cell Proteomics 2020, 19, 390– 404,  DOI: 10.1074/mcp.TIR119.001641
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    Hassan, C.; Kester, M. G.; Oudgenoeg, G.; de Ru, A. H.; Janssen, G. M.; Drijfhout, J. W.; Spaapen, R. M.; Jimenez, C. R.; Heemskerk, M. H.; Falkenburg, J. H.; van Veelen, P. A. Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes. J. Proteomics 2014, 109, 240– 244,  DOI: 10.1016/j.jprot.2014.07.009
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    Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes
    Hassan, Chopie; Kester, Michel G. D.; Oudgenoeg, Gideon; de Ru, Arnoud H.; Janssen, George M. C.; Drijfhout, Jan W.; Spaapen, Robbert M.; Jimenez, Connie R.; Heemskerk, Mirjam H. M.; Falkenburg, J. H. Frederik; van Veelen, Peter A.
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    Knowledge of the accurate copy no. of HLA class I presented ligands is important in fundamental and clin. immunol. Currently, the best copy no. detns. are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amt. of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurifn. and filtration steps, are not well defined and must, therefore, be estd. In addn., such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepd. and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate detn. of specific MHC class I-presented ligands. Our study pinpoints the immunopurifn. step as the origin of the rather extreme losses during sample pretreatment and offers a soln. to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy no. and thus allows improvement of vaccine design and strategies for immunotherapy.
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    Ljunggren, H. G.; Stam, N.; Ohlen, C.; Neefjes, J. J.; Hoglund, P.; Heemels, M. T.; Bastin, J.; Schumacher, T.; Townsend, A.; Karre, K.; Ploegh, H. L. Empty Mhc Class-I Molecules Come out in the Cold. Scand J. Immunol 1990, 32, 403– 403,  DOI: 10.1038/346476a0
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    Jurtz, V.; Paul, S.; Andreatta, M.; Marcatili, P.; Peters, B.; Nielsen, M. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J. Immunol 2017, 199, 3360– 3368,  DOI: 10.4049/jimmunol.1700893
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    NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data
    Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten
    Journal of Immunology (2017), 199 (9), 3360-3368CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
    Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I mols. Peptide binding to MHC mols. is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC mols. has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing no. of MHC-presented peptides identified by mass spectrometry have been reported contg. information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification of naturally processed ligands, cancer neoantigens, and T cell epitopes.
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    Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins
    Huizinga, Tom W. J.; Amos, Christopher I.; van der Helm-van Mil, Annette H. M.; Chen, Wei; van Gaalen, Floris A.; Jawaheer, Damini; Schreuder, Geziena M. T.; Wener, Mark; Breedveld, Ferdinand C.; Ahmad, Naila; Lum, Raymond F.; de Vries, Rene R. P.; Gregersen, Peter K.; Toes, Rene E. M.; Criswell, Lindsey A.
    Arthritis & Rheumatism (2005), 52 (11), 3433-3438CODEN: ARHEAW; ISSN:0004-3591. (John Wiley & Sons, Inc.)
    Objective. The main genetic risk factor for rheumatoid arthritis (RA), the HLA region, has been known for 25 years. Previous research has demonstrated, within the RA population, an assocn. between HLA-DRB1 alleles carrying the shared epitope (SE) and antibodies directed against cyclic citrullinated peptides (anti-CCP antibodies). We undertook this study to make the first comparison of SE allele frequencies in the healthy population with those in RA patients who do or do not harbor anti-CCP antibodies. Methods. HLA-DRB1 typing was performed in 408 RA patients from the Leiden Early Arthritis Clinic (the Leiden EAC; a Dutch population-based inception cohort in which disease course was followed up over time), in 423 healthy Dutch controls, and in 720 affected members of 341 US multiplex (sibpair) families of Caucasian origin from the North American RA Consortium (NARAC) with well-established disease and fulfilling the American College of Rheumatol. classification criteria for RA. The presence of anti-CCP antibodies was detd. by ELISA. Results. For the Leiden EAC, the odds ratio (OR) describing the assocn. of 2 copies of the SE allele with anti-CCP positivity (using no copies of the SE allele in the healthy control group as the referent) was 11.79 (P < 0.0001), while the OR for 1 SE allele was 4.37 (P < 0.0001). No assocn. with the SE was obsd. in the Dutch anti-CCP-neg. RA patients. For the NARAC families, linkage and assocn. anal. revealed the SE to be assocd. only with anti-CCP-pos. disease and not with anti-CCP-neg. disease. Stratified analyses indicated that anti-CCP antibodies primarily mediated assocn. of the SE with joint damage or disease persistence. Conclusion. HLA-DRB1 alleles encoding the SE are specific for disease characterized by antibodies to citrullinated peptides, indicating that these alleles do not assoc. with RA as such, but rather with a particular phenotype.
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    van der Gracht, A. M. F.; de Geus, M. A. R.; Camps, M. G. M.; Ruckwardt, T. J.; Sarris, A. J. C.; Bremmers, J.; Maurits, E.; Pawlak, J. B.; Posthoorn, M. M.; Bonger, K. M.; Filippov, D. V.; Overkleeft, H. S.; Robillard, M. S.; Ossendorp, F.; van Kasteren, S. I. Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes. ACS Chem. Biol. 2018, 13, 1569– 1576,  DOI: 10.1021/acschembio.8b00155
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    Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes
    van der Gracht Anouk M F; de Geus Mark A R; Sarris Alexi J C; Bremmers Jessica; Maurits Elmer; Pawlak Joanna B; Posthoorn Michelle M; Filippov Dmitri V; Overkleeft Herman S; van Kasteren Sander I; Camps Marcel G M; Ossendorp Ferry; Ruckwardt Tracy J; Bonger Kimberly M; Robillard Marc S
    ACS chemical biology (2018), 13 (6), 1569-1576 ISSN:.
    Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.
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    Fremont, D. H.; Stura, E. A.; Matsumura, M.; Peterson, P. A.; Wilson, I. A. Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 2479– 2483,  DOI: 10.1073/pnas.92.7.2479
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    Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove
    Fremont, Daved H.; Stura, Enrico A.; Matsumura, Masazumi; Peterson, Per A.; Wilson, Ian A.
    Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (7), 2479-83CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Sequence anal. of peptides naturally presented by major histocompatibility complex (MHC) class I mols. has revealed allele-specific motifs in which the peptide length and the residues obsd. at certain positions are restricted. Nevertheless, peptides contg. the std. motif often fail to bind with high affinity or form physiol. stable complexes. Here the authors present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb mol. at 2.5-Å resoln. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water mol. that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residues at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallog. and immunol. studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.
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    Swee, L. K.; Guimaraes, C. P.; Sehrawat, S.; Spooner, E.; Barrasa, M. I.; Ploegh, H. L. Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 1428– 1433,  DOI: 10.1073/pnas.1214994110
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    Fremont, D. H.; Stura, E. A.; Matsumura, M.; Peterson, P. A.; Wilson, I. A. Crystal-Structure of an H-2k(B)-Ovalbumin Peptide Complex Reveals the Interplay of Primary and Secondary Anchor Positions in the Major Histocompatibility Complex Binding Groove. P Natl. Acad. Sci. USA 1995, 92, 2479– 2483,  DOI: 10.1073/pnas.92.7.2479
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    MHC/peptide binding studies indicate hierarchy of anchor residues
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    Cellular Immunology (1993), 151 (1), 158-67CODEN: CLIMB8; ISSN:0008-8749.
    MHC class I mols. present octa- or nonapeptides derived from cellular proteins. Such peptides adhere to strict rules, which are individual to each MHC allele. Synthetic peptides conforming to these rules or peptides being at variance at crit. residues were assayed for binding to MHC class I mols. The binding assay employed the peptide-induced stabilization of MHC mols. of RMA-S cells. Most proline-free peptides conforming to the allele-specific motifs of Kb or Db bind to the resp. mols., whereas peptides missing only 1 of the 2 allele-specific anchor residues lost their capacity to stabilize class I mols. on RMA-S cells. The residues allowed at anchor positions of the Kb motif are not equal in their binding efficiency and can be ordered in a hierarchic row. Residues at nonanchor positions may also influence efficiency of peptide binding or may require deviations from the std. peptide length.
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    Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene
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    Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (9), 3972-6CODEN: PNASA6; ISSN:0027-8424.
    The bacterial β-galactosidase gene (lacZ) was used as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/Ab complex, was transfected with the NF-AT-lacZ construct. LacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacol. agents, to the physiol. peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concns. of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. Thus, TCR-mediated activation can be readily measured in single T cells and, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concn. of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.
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    Curran, A. M.; Girgis, A. A.; Jang, Y.; Crawford, J. D.; Thomas, M. A.; Kawalerski, R.; Coller, J.; Bingham, C. O., 3rd; Na, C. H.; Darrah, E. Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis. Nat. Commun. 2023, 14, 1061,  DOI: 10.1038/s41467-023-36620-y
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    Martinod, K.; Witsch, T.; Erpenbeck, L.; Savchenko, A.; Hayashi, H.; Cherpokova, D.; Gallant, M.; Mauler, M.; Cifuni, S. M.; Wagner, D. D. Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J. Exp Med. 2017, 214, 439– 458,  DOI: 10.1084/jem.20160530
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    Liu, T.; Pan, X.; Chao, L.; Tan, W.; Qu, S.; Yang, L.; Wang, B.; Mei, H. Subangstrom accuracy in pHLA-I modeling by Rosetta FlexPepDock refinement protocol. J. Chem. Inf Model 2014, 54, 2233– 2242,  DOI: 10.1021/ci500393h
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    Subangstrom Accuracy in pHLA-I Modeling by Rosetta FlexPepDock Refinement Protocol
    Liu, Tengfei; Pan, Xianchao; Chao, Li; Tan, Wen; Qu, Sujun; Yang, Li; Wang, Bochu; Mei, Hu
    Journal of Chemical Information and Modeling (2014), 54 (8), 2233-2242CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    Flexible peptides binding to human leukocyte antigen (HLA) play a key role in mediating human immune responses and are also involved in idiosyncratic adverse drug reactions according to recent research. However, the structural detns. of pHLA complexes remain challenging under the present conditions. In this paper, the performance of a new peptide docking method, namely FlexPepDock, was systematically investigated by a benchmark of 30 crystd. structures of peptide-HLA class I complexes. The docking results showed that the near-native pHLA-I models with peptide bb-RMSD less than 2 Å were ranked in the top 1 model for 100% (70/70) docking cases, and the subangstrom models with peptide bb-RMSD less than 1 Å were ranked in the top 5 lowest-energy models for 65.7% (46/70) docking cases. Furthermore, 10 out of 70 docking cases ranked the subangstrom all-atom models in the top 5 lowest-energy models. The results showed that the FlexPepDock can generate high-quality models of pHLA-I complexes and can be widely applied to pHLA-I modeling and mechanism research of peptide-mediated immune responses.
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    Raveh, B.; London, N.; Schueler-Furman, O. Sub-angstrom modeling of complexes between flexible peptides and globular proteins. Proteins 2010, 78, 2029– 2040,  DOI: 10.1002/prot.22716
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    Sub-angstrom modeling of complexes between flexible peptides and globular proteins
    Raveh, Barak; London, Nir; Schueler-Furman, Ora
    Proteins: Structure, Function, and Bioinformatics (2010), 78 (9), 2029-2040CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large no. of rotatable bonds in flexible peptides relative to typical ligand mols., and the fact that different peptides assume different backbone conformations within the same binding site. In this study, the authors present Rosetta FlexPepDock, a novel tool for refining coarse peptide-protein models that allows significant changes in both peptide backbone and side chains. The authors obtain high resoln. models, often of sub-angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide-protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with at. accuracy. In addn., the authors' protocol has improved modeling quality for the important application of cross docking to PDZ domains. The authors anticipate that the ability to create high resoln. models for a wide range of peptide-protein complexes will have significant impact on structure-based functional characterization, controlled manipulation of peptide interactions, and on peptide-based drug design.
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    Bloodworth, N.; Barbaro, N. R.; Moretti, R.; Harrison, D. G.; Meiler, J. Rosetta FlexPepDock to predict peptide-MHC binding: An approach for non-canonical amino acids. PLoS One 2022, 17, e0275759  DOI: 10.1371/journal.pone.0275759
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    Rosetta FlexPepDock to predict peptide-MHC binding: An approach for non-canonical amino acids
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    PLoS One (2022), 17 (12), e0275759CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
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    A review Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body's immunol. activity against tumors. Cytotoxic T lymphocyte-assocd. mol.-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clin. activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or mols. targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clin. trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.
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    Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.; Leming, P. D.; Spigel, D. R.; Antonia, S. J.; Horn, L.; Drake, C. G.; Pardoll, D. M.; Chen, L.; Sharfman, W. H.; Anders, R. A.; Taube, J. M.; McMiller, T. L.; Xu, H.; Korman, A. J.; Jure-Kunkel, M.; Agrawal, S.; McDonald, D.; Kollia, G. D.; Gupta, A.; Wigginton, J. M.; Sznol, M. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J. Med. 2012, 366, 2443– 2454,  DOI: 10.1056/NEJMoa1200690
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  • Abstract

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    Figure 1

    Figure 1. (A) Schematic representation of the process involving the proteolytic processing of cytosolic protein into peptides. Subsequent steps lead to the loading of the peptides onto MHC molecules that are then transported to cell surface for presentation. (B). Chemical structures of sarsWT and the PTM modified variants. (C) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. Data are represented as mean ± SD (n = 3). P-values were determined by a two-tailed t-test (**** p < 0.0001, ns = not significant). (D) A general schematic representation of how the orientation of the PTM within the presented peptide could negatively impact binding to MHC molecules (right) compared to the unmodified peptide (left). Note that modifications can also occur on the termini of the peptides.

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    Figure 2

    Figure 2. (A) Chemical structures of ovaWT and the PTM modified variants. (B) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. (C) RMA-S cells were incubated with peptide and B3Z T cells overnight at an effector to target ratio of 1:1. β-galactosidase expression was then measured via the colorimetric reagent CPRG on a plate reader at 570 nm. (D) Schematic representation of how PMTs can impact engagement with TCRs. Data are represented as mean ± SD (n = 4). P-values were determined by a two-tailed t test (**** p < 0.0001)

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    Figure 3

    Figure 3. (A) Chemical structures of mbpWT and the PTM modified variant. (B) Flow cytometry analysis of RMA-S cells treated with specific peptide (20 μM) detected by APC conjugated antimouse H-2Kb antibody. (C) NetMHCpan 4.1 predicted binding scores of tumor associated peptides. %Rank is the prediction score for comparing MHC binding across random peptides where a %Rank of 1 indicates that a peptide scored in the top 1% of random peptides. A %Rank of <2 or <0.5 are the cutoffs for weak binding or strong binding to MHC respectively. A %Rank >2 indicates nonbinders. (D) A heat map of wild-type and PTM modified peptides relative MHC binding analyzed by using the RMA-S stabilization assay. RMA-S cells were incubated with peptide at indicated concentration of peptides and detected via flow cytometry with APC conjugated antimouse H-2Kb antibodies. Abbreviations: Cit (citrullination), Yp (phosphorylated tyrosine), Kac (lysine acetylated at the γ position), NAc (acetylation at the α position), and Sp (phosphorylated serine). Data are represented as mean ± SD (n = 4). P-values were determined by a two-tailed t test (**** p < 0.0001).

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    Figure 4

    Figure 4. Modeling the sars peptide with and without PTMs using FlexPepDock. (A) N-terminal acetylation results in higher reweighted_sc values (and thus lower likelihood of binding) compared to the wild-type peptide, consistent with experimental results (one-way ANOVA and Holm-Sidak posthoc; individual values correspond to the top 0.5% of models generated, and bars represent mean score term values). (B) Top peptide models generated for each epitope positioned in the H-2Kb binding cleft. (C) Side chain modifications compared to the unmodified epitope for the SARS-CoV-2 peptide. Modeling the ovalbumin-derived peptide with and without PTMs using FlexPepDock. (D) Phosphorylation of the N-terminal serine residue generates models with higher average reweighted_sc terms, similar to N-terminal acetylation of the SARS-CoV-2 peptide (one-way ANOVA and Holm-Sidak posthoc; individual values correspond to the top 0.5% of models generated, and bars represent mean score term values). (E) Superimposed peptide backbones for the top peptide models generated for each PTM. (F) Detailed side-chain configurations for the top-scoring models generated by FlexPepDock.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVCntLnF&md5=6a0c2487ab6e20446ced5faf9e0b4474
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      Pishesha, N.; Harmand, T. J.; Ploegh, H. L. A guide to antigen processing and presentation. Nat. Rev. Immunol 2022, 22, 751– 764,  DOI: 10.1038/s41577-022-00707-2
      3
      A guide to antigen processing and presentation
      Pishesha, Novalia; Harmand, Thibault J.; Ploegh, Hidde L.
      Nature Reviews Immunology (2022), 22 (12), 751-764CODEN: NRIABX; ISSN:1474-1733. (Nature Portfolio)
      A review. Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4+ T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) mols. in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8+ T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II mols., which present antigenic peptides to CD8+ T cells and CD4+ T cells, resp. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degrdn., destroyed and ultimately displayed as peptides in complex with MHC mols. for T cell recognition.
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    4. 4
      Huppa, J. B.; Davis, M. M. T-cell-antigen recognition and the immunological synapse. Nat. Rev. Immunol 2003, 3, 973– 983,  DOI: 10.1038/nri1245
      4
      T-cell-antigen recognition and the immunological synapse
      Huppa, Johannes B.; Davis, Mark M.
      Nature Reviews Immunology (2003), 3 (12), 973-983CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)
      Much excitement of the past five years in the area of T-cell-antigen recognition has centered around the immunol. synapse-a complex cellular structure that forms at the interface of a T cell and a cell that expresses the appropriate peptide-MHC complexes. Thanks to new imaging technologies, we are now beginning to understand the role of cell-surface mols. and some of their attendant signalling modules in the context of cell-to-cell communication. Progress has been so rapid that T-cell-antigen recognition might be the first system in which the mol. basis of cell-cell recognition is understood.
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    5. 5
      Gorentla, B. K.; Zhong, X. P. T cell Receptor Signal Transduction in T lymphocytes. J. Clin. Cell Immunol. 2012, 2012, 5,  DOI: 10.4172/2155-9899.s12-005
      There is no corresponding record for this reference.
    6. 6
      Jameson, S. C. Maintaining the norm: T-cell homeostasis. Nat. Rev. Immunol 2002, 2, 547– 556,  DOI: 10.1038/nri853
      There is no corresponding record for this reference.
    7. 7
      Klein, L.; Kyewski, B.; Allen, P. M.; Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol 2014, 14, 377– 391,  DOI: 10.1038/nri3667
      There is no corresponding record for this reference.
    8. 8
      Conibear, A. C. Deciphering protein post-translational modifications using chemical biology tools. Nat. Rev. Chem. 2020, 4, 674– 695,  DOI: 10.1038/s41570-020-00223-8
      8
      Deciphering protein post-translational modifications using chemical biology tools
      Conibear, Anne C.
      Nature Reviews Chemistry (2020), 4 (12), 674-695CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)
      A review. Proteins carry out a wide variety of catalytic, regulatory, signalling and structural functions in living systems. These modifications modulate protein activity, structure, location and interactions, and, thereby, control many core biol. processes. Aberrant post-translational modifications are markers of cellular stress or malfunction and are implicated in several diseases. Therefore, gaining an understanding of which proteins are modified, at which sites and the resulting biol. consequences is an important but complex challenge requiring interdisciplinary approaches. One of the key challenges is accessing precisely modified proteins to assign functional consequences to specific modifications. Chem. biologists have developed a versatile set of tools for accessing specifically modified proteins by applying robust chemistries to biol. mols. and developing strategies for synthesizing and ligating proteins. This Review provides an overview of these tools, with selected recent examples of how they have been applied to decipher the roles of a variety of protein post-translational modifications. Relative advantages and disadvantages of each of the techniques are discussed, highlighting examples where they are used in combination and have the potential to address new frontiers in understanding complex biol. processes.
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    9. 9
      Sandalova, T.; Sala, B. M.; Achour, A. Structural aspects of chemical modifications in the MHC-restricted immunopeptidome; Implications for immune recognition. Front Chem. 2022, 10, 861609  DOI: 10.3389/fchem.2022.861609
      There is no corresponding record for this reference.
    10. 10
      Raposo, B.; Merky, P.; Lundqvist, C.; Yamada, H.; Urbonaviciute, V.; Niaudet, C.; Viljanen, J.; Kihlberg, J.; Kyewski, B.; Ekwall, O.; Holmdahl, R.; Backlund, J. T cells specific for post-translational modifications escape intrathymic tolerance induction. Nat. Commun. 2018, 9, 353,  DOI: 10.1038/s41467-017-02763-y
      10
      T cells specific for post-translational modifications escape intrathymic tolerance induction
      Raposo Bruno; Merky Patrick; Urbonaviciute Vilma; Holmdahl Rikard; Backlund Johan; Raposo Bruno; Lundqvist Christina; Ekwall Olov; Yamada Hisakata; Niaudet Colin; Viljanen Johan; Kihlberg Jan; Kyewski Bruno; Ekwall Olov
      Nature communications (2018), 9 (1), 353 ISSN:.
      Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.
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    11. 11
      Srinivasan, J.; Lancaster, J. N.; Singarapu, N.; Hale, L. P.; Ehrlich, L. I. R.; Richie, E. R. Age-Related Changes in Thymic Central Tolerance. Front Immunol 2021, 12, 676236  DOI: 10.3389/fimmu.2021.676236
      There is no corresponding record for this reference.
    12. 12
      Lancaster, J. N.; Keatinge-Clay, D. E.; Srinivasan, J.; Li, Y.; Selden, H. J.; Nam, S.; Richie, E. R.; Ehrlich, L. I. R. Central tolerance is impaired in the middle-aged thymic environment. Aging Cell 2022, 21, e13624  DOI: 10.1111/acel.13624
      There is no corresponding record for this reference.
    13. 13
      Li, W.; Li, F. F.; Zhang, X.; Lin, H. K.; Xu, C. Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment (vol 7, 31, 2022). Signal Transduct Tar 2022, 7, 422,  DOI: 10.1038/s41392-021-00825-8
      There is no corresponding record for this reference.
    14. 14
      Michaelsson, E.; Malmstrom, V.; Reis, S.; Engstrom, A.; Burkhardt, H.; Holmdahl, R. T-Cell Recognition of Carbohydrates on Type-Ii Collagen. J. Exp Med. 1994, 180, 745– 749,  DOI: 10.1084/jem.180.2.745
      There is no corresponding record for this reference.
    15. 15
      Sidney, J.; Vela, J. L.; Friedrich, D.; Kolla, R.; von Herrath, M.; Wesley, J. D.; Sette, A. Low HLA binding of diabetes-associated CD8+T-cell epitopes is increased by post translational modifications. Bmc Immunol 2018, 19, 12,  DOI: 10.1186/s12865-018-0250-3
      There is no corresponding record for this reference.
    16. 16
      McAdam, S. N.; Fleckenstein, B.; Rasmussen, I. B.; Schmid, D. G.; Sandlie, I.; Bogen, B.; Viner, N. J.; Sollid, L. M. T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: Critical role for asparagine deamidation. J. Exp Med. 2001, 193, 1239– 1246,  DOI: 10.1084/jem.193.11.1239
      There is no corresponding record for this reference.
    17. 17
      Mamula, M. J.; Gee, R. J.; Elliott, J. I.; Sette, A.; Southwood, S.; Jones, P. J.; Blier, P. R. Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins. J. Biol. Chem. 1999, 274, 22321– 22327,  DOI: 10.1074/jbc.274.32.22321
      17
      Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins
      Mamula, Mark J.; Gee, Renelle J.; Elliott, James I.; Sette, Alessandro; Southwood, Scott; Jones, Paul-James; Blier, Peter R.
      Journal of Biological Chemistry (1999), 274 (32), 22321-22327CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp93 to Leu94 occurs through the β-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiol. conditions. These post-translationally modified peptides may serve as an early immunol. stimulus in autoimmune disease.
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    18. 18
      Kim, J. K.; Mastronardi, F. G.; Wood, D. D.; Lubman, D. M.; Zand, R.; Moscarello, M. A. Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis. Mol. Cell Proteomics 2003, 2, 453– 462,  DOI: 10.1074/mcp.M200050-MCP200
      18
      Multiple sclerosis: An important role for post-translational modifications of myelin basic protein in pathogenesis
      Kim, J. K.; Mastronardi, F. G.; Wood, D. D.; Lubman, D. M.; Zand, R.; Moscarello, M. A.
      Molecular and Cellular Proteomics (2003), 2 (7), 453-462CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)
      Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a no. of sites and was elevated in MS; phosphorylation was obsd. in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes obsd. in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amts. of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.
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    19. 19
      Raposo, B.; Merky, P.; Lundqvist, C.; Yamada, H.; Urbonaviciute, V.; Niaudet, C.; Viljanen, J.; Kihlberg, J.; Kyewski, B.; Ekwall, O.; Holmdahl, R.; Backlund, J. T cells specific for post-translational modifications escape intrathymic tolerance induction. Nat. Commun. 2018, 9, 353,  DOI: 10.1038/s41467-017-02763-y
      19
      T cells specific for post-translational modifications escape intrathymic tolerance induction
      Raposo Bruno; Merky Patrick; Urbonaviciute Vilma; Holmdahl Rikard; Backlund Johan; Raposo Bruno; Lundqvist Christina; Ekwall Olov; Yamada Hisakata; Niaudet Colin; Viljanen Johan; Kihlberg Jan; Kyewski Bruno; Ekwall Olov
      Nature communications (2018), 9 (1), 353 ISSN:.
      Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.
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    20. 20
      Doyle, H. A.; Mamula, M. J. Autoantigenesis: the evolution of protein modifications in autoimmune disease. Curr. Opin Immunol 2012, 24, 112– 118,  DOI: 10.1016/j.coi.2011.12.003
      There is no corresponding record for this reference.
    21. 21
      Clement, C. C.; Moncrieffe, H.; Lele, A.; Janow, G.; Becerra, A.; Bauli, F.; Saad, F. A.; Perino, G.; Montagna, C.; Cobelli, N.; Hardin, J.; Stern, L. J.; Ilowite, N.; Porcelli, S. A.; Santambrogio, L. Autoimmune response to transthyretin in juvenile idiopathic arthritis. JCI Insight 2016, 1, e85633  DOI: 10.1172/jci.insight.85633
      There is no corresponding record for this reference.
    22. 22
      Clement, C. C.; Nanaware, P. P.; Yamazaki, T.; Negroni, M. P.; Ramesh, K.; Morozova, K.; Thangaswamy, S.; Graves, A.; Kim, H. J.; Li, T. W.; Vigano, M.; Soni, R. K.; Gadina, M.; Tse, H. Y.; Galluzzi, L.; Roche, P. A.; Denzin, L. K.; Stern, L. J.; Santambrogio, L. Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery. Immunity 2021, 54, 721– 736,  DOI: 10.1016/j.immuni.2021.02.019
      There is no corresponding record for this reference.
    23. 23
      Clement, C. C.; Osan, J.; Buque, A.; Nanaware, P. P.; Chang, Y. C.; Perino, G.; Shetty, M.; Yamazaki, T.; Tsai, W. L.; Urbanska, A. M.; Calvo-Calle, J. M.; Ramsamooj, S.; Ramsamooj, S.; Vergani, D.; Mieli-Vergani, G.; Terziroli Beretta-Piccoli, B.; Gadina, M.; Montagna, C.; Goncalves, M. D.; Sallusto, F.; Galluzzi, L.; Soni, R. K.; Stern, L. J.; Santambrogio, L. PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes. Sci. Immunol. 2022, 7, eabl3795  DOI: 10.1126/sciimmunol.abl3795
      There is no corresponding record for this reference.
    24. 24
      Sidney, J.; Vela, J. L.; Friedrich, D.; Kolla, R.; von Herrath, M.; Wesley, J. D.; Sette, A. Low HLA binding of diabetes-associated CD8+ T-cell epitopes is increased by post translational modifications. BMC Immunol. 2018, 19, 12,  DOI: 10.1186/s12865-018-0250-3
      There is no corresponding record for this reference.
    25. 25
      Zhai, Y.; Chen, L.; Zhao, Q.; Zheng, Z. H.; Chen, Z. N.; Bian, H.; Yang, X.; Lu, H. Y.; Lin, P.; Chen, X.; Chen, R.; Sun, H. Y.; Fan, L. N.; Zhang, K.; Wang, B.; Sun, X. X.; Feng, Z.; Zhu, Y. M.; Zhou, J. S.; Chen, S. R.; Zhang, T.; Chen, S. Y.; Chen, J. J.; Zhang, K.; Wang, Y.; Chang, Y.; Zhang, R.; Zhang, B.; Wang, L. J.; Li, X. M.; He, Q.; Yang, X. M.; Nan, G.; Xie, R. H.; Yang, L.; Yang, J. H.; Zhu, P. Cysteine carboxyethylation generates neoantigens to induce HLA-restricted autoimmunity. Science 2023, 379, eabg2482  DOI: 10.1126/science.abg2482
      There is no corresponding record for this reference.
    26. 26
      Petersen, J.; Purcell, A. W.; Rossjohn, J. Post-translationally modified T cell epitopes: immune recognition and immunotherapy. J. Mol. Med. 2009, 87, 1045– 1051,  DOI: 10.1007/s00109-009-0526-4
      26
      Post-translationally modified T cell epitopes: immune recognition and immunotherapy
      Petersen, Jan; Purcell, Anthony W.; Rossjohn, Jamie
      Journal of Molecular Medicine (Heidelberg, Germany) (2009), 87 (11), 1045-1051CODEN: JMLME8; ISSN:0946-2716. (Springer)
      A review. The functionality of proteins is greatly extended by a diverse array of post-translational modifications (PTMs), many of which are recognized by the immune system. Notably, a significant proportion of peptides presented to T cells by the major histocompatibility complex in vivo are post-translationally modified. Since the cellular mechanisms that introduce and control protein modifications can differ between health and disease, the assocd. changes in antigen presentation have the potential to alter immune responses. A no. of such situations have been implicated with infection, inflammation, autoimmune disease, and cancer, and the investigation of PTMs that affect antigen recognition has provided insight in disease progression as well as raising prospects for novel approaches in immunotherapy.
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    27. 27
      Marcilla, M.; Alpizar, A.; Lombardia, M.; Ramos-Fernandez, A.; Ramos, M.; Albar, J. P. Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue. Mol. Cell Proteomics 2014, 13, 462– 474,  DOI: 10.1074/mcp.M113.034314
      There is no corresponding record for this reference.
    28. 28
      Bassani-Sternberg, M.; Braunlein, E.; Klar, R.; Engleitner, T.; Sinitcyn, P.; Audehm, S.; Straub, M.; Weber, J.; Slotta-Huspenina, J.; Specht, K.; Martignoni, M. E.; Werner, A.; Hein, R.; D, H. B.; Peschel, C.; Rad, R.; Cox, J.; Mann, M.; Krackhardt, A. M. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat. Commun. 2016, 7, 13404,  DOI: 10.1038/ncomms13404
      28
      Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry
      Bassani-Sternberg, Michal; Braeunlein, Eva; Klar, Richard; Engleitner, Thomas; Sinitcyn, Pavel; Audehm, Stefan; Straub, Melanie; Weber, Julia; Slotta-Huspenina, Julia; Specht, Katja; Martignoni, Marc E.; Werner, Angelika; Hein, Ruediger; H. Busch, Dirk; Peschel, Christian; Rad, Roland; Cox, Juergen; Mann, Matthias; Krackhardt, Angela M.
      Nature Communications (2016), 7 (), 13404CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) anal., we survey the melanoma-assocd. immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.
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    29. 29
      Alpizar, A.; Marino, F.; Ramos-Fernandez, A.; Lombardia, M.; Jeko, A.; Pazos, F.; Paradela, A.; Santiago, C.; Heck, A. J.; Marcilla, M. A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens. Mol. Cell Proteomics 2017, 16, 181– 193,  DOI: 10.1074/mcp.M116.063800
      29
      A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens
      Alpizar, Adan; Marino, Fabio; Ramos-Fernandez, Antonio; Lombardia, Manuel; Jeko, Anita; Pazos, Florencio; Paradela, Alberto; Santiago, Cesar; Heck, Albert J. R.; Marcilla, Miguel
      Molecular & Cellular Proteomics (2017), 16 (2), 181-193CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
      As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I mols. can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the mol. determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands assocd. with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar mol. features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To det. the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes assocd. to different HLA-B mols. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This mol. understanding of the presentation of phosphopeptides by HLA-B mols. provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.
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    30. 30
      Malaker, S. A.; Penny, S. A.; Steadman, L. G.; Myers, P. T.; Loke, J. C.; Raghavan, M.; Bai, D. L.; Shabanowitz, J.; Hunt, D. F.; Cobbold, M. Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia. Cancer Immunol Res. 2017, 5, 376– 384,  DOI: 10.1158/2326-6066.CIR-16-0280
      30
      Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia
      Malaker, Stacy A.; Penny, Sarah A.; Steadman, Lora G.; Myers, Paisley T.; Loke, Justin C.; Raghavan, Manoj; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Cobbold, Mark
      Cancer Immunology Research (2017), 5 (5), 376-384CODEN: CIRACV; ISSN:2326-6066. (American Association for Cancer Research)
      Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-assocd. peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three exptl. approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be assocd. with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equiv. unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR.
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    31. 31
      Marino, F.; Mommen, G. P.; Jeko, A.; Meiring, H. D.; van Gaans-van den Brink, J. A.; Scheltema, R. A.; van Els, C. A.; Heck, A. J. Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07. J. Proteome Res. 2017, 16, 34– 44,  DOI: 10.1021/acs.jproteome.6b00528
      31
      Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07
      Marino, Fabio; Mommen, Geert P. M.; Jeko, Anita; Meiring, Hugo D.; van Gaans-van den Brink, Jacqueline A. M.; Scheltema, Richard A.; van Els, Cecile A. C. M.; Heck, Albert J. R.
      Journal of Proteome Research (2017), 16 (1), 34-44CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Alterations in protein post-translational modification (PTMs) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related Human Leukocyte Antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line the authors detected 149 HLA class I peptides harboring mono- and/or di-methylated arginine residues by mass spectrometry. A striking preference was obsd. in presentation of arginine (di)methylated peptides for HLA-B*07 mols., likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored di-methylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been assocd. with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra the authors found most of the peptides to be asym. di-methylated, most likely by CARM1. These data expand the authors' knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides, and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and di-methylated arginine residues may therefore offer a novel target for immunotherapy.
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    32. 32
      Mohammed, F.; Stones, D. H.; Zarling, A. L.; Willcox, C. R.; Shabanowitz, J.; Cummings, K. L.; Hunt, D. F.; Cobbold, M.; Engelhard, V. H.; Willcox, B. E. The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status. Oncotarget 2017, 8, 54160– 54172,  DOI: 10.18632/oncotarget.16952
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      Sandalova, T.; Sala, B. M.; Achour, A. Structural aspects of chemical modifications in the MHC-restricted immunopeptidome. Implications for immune recognition, Front Chem. 2022, 10, 861609  DOI: 10.3389/fchem.2022.861609
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      Mangalaparthi, K. K.; Madugundu, A. K.; Ryan, Z. C.; Garapati, K.; Peterson, J. A.; Dey, G.; Prakash, A.; Pandey, A. Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I. J. Proteins Proteom 2021, 12, 151– 160,  DOI: 10.1007/s42485-021-00066-x
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      Ireland, J.; Herzog, J.; Unanue, E. R. Cutting edge: Unique T cells that recognize citrullinated peptides are a feature of protein immunization. J. Immunol 2006, 177, 1421– 1425,  DOI: 10.4049/jimmunol.177.3.1421
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      Huseby, E. S.; Sather, B.; Huseby, P. G.; Goverman, J. Age-dependent T cell tolerance and autoimmunity to myelin basic protein. Immunity 2001, 14, 471– 481,  DOI: 10.1016/S1074-7613(01)00127-3
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      Malmstrom, V.; Catrina, A. I.; Klareskog, L. The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting. Nat. Rev. Immunol 2017, 17, 60– 75,  DOI: 10.1038/nri.2016.124
      37
      The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting
      Malmstrom Vivianne; Catrina Anca I; Klareskog Lars
      Nature reviews. Immunology (2017), 17 (1), 60-75 ISSN:.
      Patients with rheumatoid arthritis can be divided into two major subsets characterized by the presence versus absence of antibodies to citrullinated protein antigens (ACPAs) and of rheumatoid factor (RF). The antibody-positive subset of disease, also known as seropositive rheumatoid arthritis, constitutes approximately two-thirds of all cases of rheumatoid arthritis and generally has a more severe disease course. ACPAs and RF are often present in the blood long before any signs of joint inflammation, which suggests that the triggering of autoimmunity may occur at sites other than the joints (for example, in the lung). This Review summarizes recent progress in our understanding of this gradual disease development in seropositive patients. We also emphasize the implications of this new understanding for the development of preventive and therapeutic strategies. Similar temporal and spatial separation of immune triggering and clinical manifestations, with novel opportunities for early intervention, may also occur in other immune-mediated diseases.
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      Curran, A. M.; Naik, P.; Giles, J. T.; Darrah, E. PAD enzymes in rheumatoid arthritis: pathogenic effectors and autoimmune targets. Nat. Rev. Rheumatol 2020, 16, 301– 315,  DOI: 10.1038/s41584-020-0409-1
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      Yang, L.; Tan, D.; Piao, H. Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology. Neurochem. Res. 2016, 41, 1845– 1856,  DOI: 10.1007/s11064-016-1920-2
      39
      Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology
      Yang, Lei; Tan, Dewei; Piao, Hua
      Neurochemical Research (2016), 41 (8), 1845-1856CODEN: NEREDZ; ISSN:0364-3190. (Springer)
      Multiple sclerosis (MS) is a multifactorial demyelinating disease characterized by neurodegenerative events and autoimmune response against myelin component. Citrullination or deimination, a post-translational modification of protein-bound arginine into citrulline, catalyzed by Ca2+ dependent peptidylarginine deiminase enzyme (PAD), plays an essential role in physiol. processes include gene expression regulation, apoptosis and the plasticity of the central nervous system, while aberrant citrullination can generate new epitopes, thus involving in the initiation and/or progression of autoimmune disorder like MS. Myelin basic protein (MBP) is the major myelin protein and is generally considered to maintain the stability of the myelin sheath. This review describes the MBP citrullination and its consequence, as well as offering further support for the "inside-out" hypothesis that MS is primarily a neurodegenerative disease with secondary inflammatory demyelination. In addn., it discusses the role of MBP citrullination in the immune inflammation and explores the potential of inhibition of PAD enzymes as a therapeutic strategy for the disease.
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      Hill, J. A.; Southwood, S.; Sette, A.; Jevnikar, A. M.; Bell, D. A.; Cairns, E. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J. Immunol 2003, 171, 538– 541,  DOI: 10.4049/jimmunol.171.2.538
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      Ramarathinam, S. H.; Croft, N. P.; Illing, P. T.; Faridi, P.; Purcell, A. W. Employing proteomics in the study of antigen presentation: an update. Expert Rev. Proteomics 2018, 15, 637– 645,  DOI: 10.1080/14789450.2018.1509000
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      Jaeger, A. M.; Stopfer, L. E.; Ahn, R.; Sanders, E. A.; Sandel, D. A.; Freed-Pastor, W. A.; Rideout, W. M., 3rd; Naranjo, S.; Fessenden, T.; Nguyen, K. B.; Winter, P. S.; Kohn, R. E.; Westcott, P. M. K.; Schenkel, J. M.; Shanahan, S. L.; Shalek, A. K.; Spranger, S.; White, F. M.; Jacks, T. Deciphering the immunopeptidome in vivo reveals new tumour antigens. Nature 2022, 607, 149– 155,  DOI: 10.1038/s41586-022-04839-2
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      Yi, X.; Liao, Y.; Wen, B.; Li, K.; Dou, Y.; Savage, S. R.; Zhang, B. caAtlas: An immunopeptidome atlas of human cancer. iScience 2021, 24, 103107  DOI: 10.1016/j.isci.2021.103107
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      Kacen, A.; Javitt, A.; Kramer, M. P.; Morgenstern, D.; Tsaban, T.; Shmueli, M. D.; Teo, G. C.; da Veiga Leprevost, F.; Barnea, E.; Yu, F.; Admon, A.; Eisenbach, L.; Samuels, Y.; Schueler-Furman, O.; Levin, Y.; Nesvizhskii, A. I.; Merbl, Y. Post-translational modifications reshape the antigenic landscape of the MHC I immunopeptidome in tumors. Nat. Biotechnol. 2023, 41, 239– 251,  DOI: 10.1038/s41587-022-01464-2
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      Garstka, M. A.; Fish, A.; Celie, P. H.; Joosten, R. P.; Janssen, G. M.; Berlin, I.; Hoppes, R.; Stadnik, M.; Janssen, L.; Ovaa, H.; van Veelen, P. A.; Perrakis, A.; Neefjes, J. The first step of peptide selection in antigen presentation by MHC class I molecules. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 1505– 1510,  DOI: 10.1073/pnas.1416543112
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      Sette, A.; Vitiello, A.; Reherman, B.; Fowler, P.; Nayersina, R.; Kast, W. M.; Melief, C. J.; Oseroff, C.; Yuan, L.; Ruppert, J.; Sidney, J.; del Guercio, M. F.; Southwood, S.; Kubo, R. T.; Chesnut, R. W.; Grey, H. M.; Chisari, F. V. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol 1994, 153, 5586– 5592,  DOI: 10.4049/jimmunol.153.12.5586
      46
      The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes
      Sette, Alessandro; Vitiello, Antonella; Reherman, Barbara; Fowler, Patricia; Nayersina, Ramin; Kast, W. Martin; Melief, Cornelis J. M.; Oseroff, Carla; Yuan, Lunli; et al.
      Journal of Immunology (1994), 153 (12), 5586-92CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
      The relation between binding affinity for HLA class I mols. and immunogenicity of discrete peptide epitopes was analyzed in 2 different exptl. approaches. In the 1st approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approx. 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approx. 500 nM (preferably 50 nM or less) apparently dets. the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, the data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.
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      Deribe, Y. L.; Pawson, T.; Dikic, I. Post-translational modifications in signal integration. Nat. Struct Mol. Biol. 2010, 17, 666– 672,  DOI: 10.1038/nsmb.1842
      47
      Post-translational modifications in signal integration
      Deribe, Yonathan Lissanu; Pawson, Tony; Dikic, Ivan
      Nature Structural & Molecular Biology (2010), 17 (6), 666-672CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      A review. Post-translational modifications of proteins and the domains that recognize these modifications play central roles in creating a highly dynamic relay system that reads and responds to alterations in the cellular microenvironment. Here, the authors review the common principles of post-translational modifications and their importance in signal integration underlying epidermal growth factor receptor signaling and endocytosis, DNA-damage responses, and immunity.
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      Zhao, Y.; Jensen, O. N. Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques. Proteomics 2009, 9, 4632– 4641,  DOI: 10.1002/pmic.200900398
      48
      Modification-specific proteomics: Strategies for characterization of post-translational modifications using enrichment techniques
      Zhao, Yingming; Jensen, Ole N.
      Proteomics (2009), 9 (20), 4632-4641CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. More than 300 different types of protein post-translational modifications (PTMs) have been described, many of which are known to have pivotal roles in cellular physiol. and disease. Nevertheless, only a handful of PTMs have been extensively investigated at the proteome level. Knowledge of protein substrates and their PTM sites is key to dissection of PTM-mediated cellular processes. The past several years have seen a tremendous progress in developing mass spectrometry (MS)-based proteomics technologies for global PTM anal., including numerous studies of yeast and other microbes. Modification-specific enrichment techniques combined with advanced MS/MS methods and computational data anal. have revealed a surprisingly large extent of PTMs in proteins, including multi-site, cooperative modifications in individual proteins. The authors review some of the current strategies employed for enrichment and detection of PTMs in modification-specific proteomics.
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      Solleder, M.; Guillaume, P.; Racle, J.; Michaux, J.; Pak, H. S.; Muller, M.; Coukos, G.; Bassani-Sternberg, M.; Gfeller, D. Mass Spectrometry Based Immunopeptidomics Leads to Robust Predictions of Phosphorylated HLA Class I Ligands. Mol. Cell Proteomics 2020, 19, 390– 404,  DOI: 10.1074/mcp.TIR119.001641
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      Hassan, C.; Kester, M. G.; Oudgenoeg, G.; de Ru, A. H.; Janssen, G. M.; Drijfhout, J. W.; Spaapen, R. M.; Jimenez, C. R.; Heemskerk, M. H.; Falkenburg, J. H.; van Veelen, P. A. Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes. J. Proteomics 2014, 109, 240– 244,  DOI: 10.1016/j.jprot.2014.07.009
      50
      Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes
      Hassan, Chopie; Kester, Michel G. D.; Oudgenoeg, Gideon; de Ru, Arnoud H.; Janssen, George M. C.; Drijfhout, Jan W.; Spaapen, Robbert M.; Jimenez, Connie R.; Heemskerk, Mirjam H. M.; Falkenburg, J. H. Frederik; van Veelen, Peter A.
      Journal of Proteomics (2014), 109 (), 240-244CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
      Knowledge of the accurate copy no. of HLA class I presented ligands is important in fundamental and clin. immunol. Currently, the best copy no. detns. are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amt. of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurifn. and filtration steps, are not well defined and must, therefore, be estd. In addn., such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepd. and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate detn. of specific MHC class I-presented ligands. Our study pinpoints the immunopurifn. step as the origin of the rather extreme losses during sample pretreatment and offers a soln. to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy no. and thus allows improvement of vaccine design and strategies for immunotherapy.
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      Colbert, J. D.; Cruz, F. M.; Rock, K. L. Cross-presentation of exogenous antigens on MHC I molecules. Curr. Opin Immunol 2020, 64, 1– 8,  DOI: 10.1016/j.coi.2019.12.005
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      Lee, J. M.; Hammaren, H. M.; Savitski, M. M.; Baek, S. H. Control of protein stability by post-translational modifications. Nat. Commun. 2023, 14, 201,  DOI: 10.1038/s41467-023-35795-8
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      Ljunggren, H. G.; Stam, N.; Ohlen, C.; Neefjes, J. J.; Hoglund, P.; Heemels, M. T.; Bastin, J.; Schumacher, T.; Townsend, A.; Karre, K.; Ploegh, H. L. Empty Mhc Class-I Molecules Come out in the Cold. Scand J. Immunol 1990, 32, 403– 403,  DOI: 10.1038/346476a0
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      Schumacher, T. N.; Heemels, M. T.; Neefjes, J. J.; Kast, W. M.; Melief, C. J.; Ploegh, H. L. Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro. Cell 1990, 62, 563– 567,  DOI: 10.1016/0092-8674(90)90020-F
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      Saito, Y.; Peterson, P. A.; Matsumura, M. Quantitation of peptide anchor residue contributions to class I major histocompatibility complex molecule binding. J. Biol. Chem. 1993, 268, 21309– 21317,  DOI: 10.1016/S0021-9258(19)36925-X
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      Ross, P.; Holmes, J. C.; Gojanovich, G. S.; Hess, P. R. A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88. Vet. Immunol. Immunopathol. 2012, 150, 206– 212,  DOI: 10.1016/j.vetimm.2012.08.012
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      Apostolopoulos, V.; Haurum, J. S.; McKenzie, I. F. MUC1 peptide epitopes associated with five different H-2 class I molecules. Eur. J. Immunol. 1997, 27, 2579– 2587,  DOI: 10.1002/eji.1830271017
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      Cerundolo, V.; Elliott, T.; Elvin, J.; Bastin, J.; Rammensee, H. G.; Townsend, A. The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules. Eur. J. Immunol. 1991, 21, 2069– 2075,  DOI: 10.1002/eji.1830210915
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      De Silva, A. D.; Boesteanu, A.; Song, R.; Nagy, N.; Harhaj, E.; Harding, C. V.; Joyce, S. Thermolabile H-2Kb molecules expressed by transporter associated with antigen processing-deficient RMA-S cells are occupied by low-affinity peptides. J. Immunol 1999, 163, 4413– 4420,  DOI: 10.4049/jimmunol.163.8.4413
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      Feltkamp, M. C.; Vierboom, M. P.; Kast, W. M.; Melief, C. J. Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity. Mol. Immunol 1994, 31, 1391– 1401,  DOI: 10.1016/0161-5890(94)90155-4
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      Elvin, J.; Potter, C.; Elliott, T.; Cerundolo, V.; Townsend, A. A method to quantify binding of unlabeled peptides to class I MHC molecules and detect their allele specificity. J. Immunol Methods 1993, 158, 161– 171,  DOI: 10.1016/0022-1759(93)90210-X
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      Jurtz, V.; Paul, S.; Andreatta, M.; Marcatili, P.; Peters, B.; Nielsen, M. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J. Immunol 2017, 199, 3360– 3368,  DOI: 10.4049/jimmunol.1700893
      62
      NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data
      Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten
      Journal of Immunology (2017), 199 (9), 3360-3368CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
      Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I mols. Peptide binding to MHC mols. is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC mols. has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing no. of MHC-presented peptides identified by mass spectrometry have been reported contg. information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification of naturally processed ligands, cancer neoantigens, and T cell epitopes.
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      Dubey, P.; Hendrickson, R. C.; Meredith, S. C.; Siegel, C. T.; Shabanowitz, J.; Skipper, J. C. A.; Engelhard, V. H.; Hunt, D. F.; Schreiber, H. The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68. J. Exp Med. 1997, 185, 695– 705,  DOI: 10.1084/jem.185.4.695
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      Kjellen, P.; Brunsberg, U.; Broddefalk, J.; Hansen, B.; Vestberg, M.; Ivarsson, I.; Engstrom, A.; Svejgaard, A.; Kihlberg, J.; Fugger, L.; Holmdahl, R. The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256–270 glycopeptide to H-2Aq and H-2Ap molecules. Eur. J. Immunol. 1998, 28, 755– 767,  DOI: 10.1002/(SICI)1521-4141(199802)28:02<755::AID-IMMU755>3.0.CO;2-2
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      Rosloniec, E. F.; Whittington, K. B.; Zaller, D. M.; Kang, A. H. HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen. J. Immunol 2002, 168, 253– 259,  DOI: 10.4049/jimmunol.168.1.253
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      Huizinga, T. W.; Amos, C. I.; van der Helm-van Mil, A. H.; Chen, W.; van Gaalen, F. A.; Jawaheer, D.; Schreuder, G. M.; Wener, M.; Breedveld, F. C.; Ahmad, N.; Lum, R. F.; de Vries, R. R.; Gregersen, P. K.; Toes, R. E.; Criswell, L. A. Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins. Arthritis Rheum 2005, 52, 3433– 3438,  DOI: 10.1002/art.21385
      66
      Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins
      Huizinga, Tom W. J.; Amos, Christopher I.; van der Helm-van Mil, Annette H. M.; Chen, Wei; van Gaalen, Floris A.; Jawaheer, Damini; Schreuder, Geziena M. T.; Wener, Mark; Breedveld, Ferdinand C.; Ahmad, Naila; Lum, Raymond F.; de Vries, Rene R. P.; Gregersen, Peter K.; Toes, Rene E. M.; Criswell, Lindsey A.
      Arthritis & Rheumatism (2005), 52 (11), 3433-3438CODEN: ARHEAW; ISSN:0004-3591. (John Wiley & Sons, Inc.)
      Objective. The main genetic risk factor for rheumatoid arthritis (RA), the HLA region, has been known for 25 years. Previous research has demonstrated, within the RA population, an assocn. between HLA-DRB1 alleles carrying the shared epitope (SE) and antibodies directed against cyclic citrullinated peptides (anti-CCP antibodies). We undertook this study to make the first comparison of SE allele frequencies in the healthy population with those in RA patients who do or do not harbor anti-CCP antibodies. Methods. HLA-DRB1 typing was performed in 408 RA patients from the Leiden Early Arthritis Clinic (the Leiden EAC; a Dutch population-based inception cohort in which disease course was followed up over time), in 423 healthy Dutch controls, and in 720 affected members of 341 US multiplex (sibpair) families of Caucasian origin from the North American RA Consortium (NARAC) with well-established disease and fulfilling the American College of Rheumatol. classification criteria for RA. The presence of anti-CCP antibodies was detd. by ELISA. Results. For the Leiden EAC, the odds ratio (OR) describing the assocn. of 2 copies of the SE allele with anti-CCP positivity (using no copies of the SE allele in the healthy control group as the referent) was 11.79 (P < 0.0001), while the OR for 1 SE allele was 4.37 (P < 0.0001). No assocn. with the SE was obsd. in the Dutch anti-CCP-neg. RA patients. For the NARAC families, linkage and assocn. anal. revealed the SE to be assocd. only with anti-CCP-pos. disease and not with anti-CCP-neg. disease. Stratified analyses indicated that anti-CCP antibodies primarily mediated assocn. of the SE with joint damage or disease persistence. Conclusion. HLA-DRB1 alleles encoding the SE are specific for disease characterized by antibodies to citrullinated peptides, indicating that these alleles do not assoc. with RA as such, but rather with a particular phenotype.
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    67. 67
      Yague, J.; Alvarez, I.; Rognan, D.; Ramos, M.; Vazquez, J.; de Castro, J. A. An N-acetylated natural ligand of human histocompatibility leukocyte antigen (HLA)-B39. Classical major histocompatibility complex class I proteins bind peptides with a blocked NH(2) terminus in vivo. J. Exp Med. 2000, 191, 2083– 2092,  DOI: 10.1084/jem.191.12.2083
      There is no corresponding record for this reference.
    68. 68
      Sun, M.; Liu, J.; Qi, J.; Tefsen, B.; Shi, Y.; Yan, J.; Gao, G. F. Nalpha-terminal acetylation for T cell recognition: molecular basis of MHC class I-restricted nalpha-acetylpeptide presentation. J. Immunol 2014, 192, 5509– 5519,  DOI: 10.4049/jimmunol.1400199
      There is no corresponding record for this reference.
    69. 69
      de Haan, E. C.; Wauben, M. H.; Wagenaar-Hilbers, J. P.; Grosfeld-Stulemeyer, M. C.; Rijkers, D. T.; Moret, E. E.; Liskamp, R. M. Stabilization of peptide guinea pig myelin basic protein 72–85 by N-terminal acetylation-implications for immunological studies. Mol. Immunol 2004, 40, 943– 948,  DOI: 10.1016/j.molimm.2003.10.015
      There is no corresponding record for this reference.
    70. 70
      Shastri, N.; Gonzalez, F. Endogenous generation and presentation of the ovalbumin peptide/Kb complex to T cells. J. Immunol 1993, 150, 2724– 2736,  DOI: 10.4049/jimmunol.150.7.2724
      There is no corresponding record for this reference.
    71. 71
      Koch, C. P.; Perna, A. M.; Pillong, M.; Todoroff, N. K.; Wrede, P.; Folkers, G.; Hiss, J. A.; Schneider, G. Scrutinizing MHC-I binding peptides and their limits of variation. PLoS Comput. Biol. 2013, 9, e1003088  DOI: 10.1371/journal.pcbi.1003088
      There is no corresponding record for this reference.
    72. 72
      van der Gracht, A. M. F.; de Geus, M. A. R.; Camps, M. G. M.; Ruckwardt, T. J.; Sarris, A. J. C.; Bremmers, J.; Maurits, E.; Pawlak, J. B.; Posthoorn, M. M.; Bonger, K. M.; Filippov, D. V.; Overkleeft, H. S.; Robillard, M. S.; Ossendorp, F.; van Kasteren, S. I. Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes. ACS Chem. Biol. 2018, 13, 1569– 1576,  DOI: 10.1021/acschembio.8b00155
      72
      Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes
      van der Gracht Anouk M F; de Geus Mark A R; Sarris Alexi J C; Bremmers Jessica; Maurits Elmer; Pawlak Joanna B; Posthoorn Michelle M; Filippov Dmitri V; Overkleeft Herman S; van Kasteren Sander I; Camps Marcel G M; Ossendorp Ferry; Ruckwardt Tracy J; Bonger Kimberly M; Robillard Marc S
      ACS chemical biology (2018), 13 (6), 1569-1576 ISSN:.
      Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.
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    73. 73
      Fremont, D. H.; Stura, E. A.; Matsumura, M.; Peterson, P. A.; Wilson, I. A. Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 2479– 2483,  DOI: 10.1073/pnas.92.7.2479
      73
      Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove
      Fremont, Daved H.; Stura, Enrico A.; Matsumura, Masazumi; Peterson, Per A.; Wilson, Ian A.
      Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (7), 2479-83CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Sequence anal. of peptides naturally presented by major histocompatibility complex (MHC) class I mols. has revealed allele-specific motifs in which the peptide length and the residues obsd. at certain positions are restricted. Nevertheless, peptides contg. the std. motif often fail to bind with high affinity or form physiol. stable complexes. Here the authors present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb mol. at 2.5-Å resoln. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water mol. that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residues at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallog. and immunol. studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.
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    74. 74
      Swee, L. K.; Guimaraes, C. P.; Sehrawat, S.; Spooner, E.; Barrasa, M. I.; Ploegh, H. L. Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 1428– 1433,  DOI: 10.1073/pnas.1214994110
      There is no corresponding record for this reference.
    75. 75
      Fremont, D. H.; Stura, E. A.; Matsumura, M.; Peterson, P. A.; Wilson, I. A. Crystal-Structure of an H-2k(B)-Ovalbumin Peptide Complex Reveals the Interplay of Primary and Secondary Anchor Positions in the Major Histocompatibility Complex Binding Groove. P Natl. Acad. Sci. USA 1995, 92, 2479– 2483,  DOI: 10.1073/pnas.92.7.2479
      There is no corresponding record for this reference.
    76. 76
      Deres, K.; Beck, W.; Faath, S.; Jung, G.; Rammensee, H. G. MHC/peptide binding studies indicate hierarchy of anchor residues. Cell Immunol 1993, 151, 158– 167,  DOI: 10.1006/cimm.1993.1228
      76
      MHC/peptide binding studies indicate hierarchy of anchor residues
      Deres, Karl; Beck, Werner; Faath, Stefan; Jung, Guenther; Rammensee, Hans Georg
      Cellular Immunology (1993), 151 (1), 158-67CODEN: CLIMB8; ISSN:0008-8749.
      MHC class I mols. present octa- or nonapeptides derived from cellular proteins. Such peptides adhere to strict rules, which are individual to each MHC allele. Synthetic peptides conforming to these rules or peptides being at variance at crit. residues were assayed for binding to MHC class I mols. The binding assay employed the peptide-induced stabilization of MHC mols. of RMA-S cells. Most proline-free peptides conforming to the allele-specific motifs of Kb or Db bind to the resp. mols., whereas peptides missing only 1 of the 2 allele-specific anchor residues lost their capacity to stabilize class I mols. on RMA-S cells. The residues allowed at anchor positions of the Kb motif are not equal in their binding efficiency and can be ordered in a hierarchic row. Residues at nonanchor positions may also influence efficiency of peptide binding or may require deviations from the std. peptide length.
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    77. 77
      Bremel, R. D.; Homan, E. J. Frequency Patterns of T-Cell Exposed Amino Acid Motifs in Immunoglobulin Heavy Chain Peptides Presented by MHCs. Front. Immunol. 2014, 5, 541,  DOI: 10.3389/fimmu.2014.00541
      There is no corresponding record for this reference.
    78. 78
      Reddehase, M. J.; Rothbard, J. B.; Koszinowski, U. H. A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature 1989, 337, 651– 653,  DOI: 10.1038/337651a0
      There is no corresponding record for this reference.
    79. 79
      Karttunen, J.; Shastri, N. Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 3972– 3976,  DOI: 10.1073/pnas.88.9.3972
      79
      Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene
      Karttunen, Jaana; Shastri, Nilabh
      Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (9), 3972-6CODEN: PNASA6; ISSN:0027-8424.
      The bacterial β-galactosidase gene (lacZ) was used as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/Ab complex, was transfected with the NF-AT-lacZ construct. LacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacol. agents, to the physiol. peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concns. of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. Thus, TCR-mediated activation can be readily measured in single T cells and, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concn. of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.
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    80. 80
      Curran, A. M.; Girgis, A. A.; Jang, Y.; Crawford, J. D.; Thomas, M. A.; Kawalerski, R.; Coller, J.; Bingham, C. O., 3rd; Na, C. H.; Darrah, E. Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis. Nat. Commun. 2023, 14, 1061,  DOI: 10.1038/s41467-023-36620-y
      There is no corresponding record for this reference.
    81. 81
      Martinod, K.; Witsch, T.; Erpenbeck, L.; Savchenko, A.; Hayashi, H.; Cherpokova, D.; Gallant, M.; Mauler, M.; Cifuni, S. M.; Wagner, D. D. Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J. Exp Med. 2017, 214, 439– 458,  DOI: 10.1084/jem.20160530
      There is no corresponding record for this reference.
    82. 82
      Liu, T.; Pan, X.; Chao, L.; Tan, W.; Qu, S.; Yang, L.; Wang, B.; Mei, H. Subangstrom accuracy in pHLA-I modeling by Rosetta FlexPepDock refinement protocol. J. Chem. Inf Model 2014, 54, 2233– 2242,  DOI: 10.1021/ci500393h
      82
      Subangstrom Accuracy in pHLA-I Modeling by Rosetta FlexPepDock Refinement Protocol
      Liu, Tengfei; Pan, Xianchao; Chao, Li; Tan, Wen; Qu, Sujun; Yang, Li; Wang, Bochu; Mei, Hu
      Journal of Chemical Information and Modeling (2014), 54 (8), 2233-2242CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      Flexible peptides binding to human leukocyte antigen (HLA) play a key role in mediating human immune responses and are also involved in idiosyncratic adverse drug reactions according to recent research. However, the structural detns. of pHLA complexes remain challenging under the present conditions. In this paper, the performance of a new peptide docking method, namely FlexPepDock, was systematically investigated by a benchmark of 30 crystd. structures of peptide-HLA class I complexes. The docking results showed that the near-native pHLA-I models with peptide bb-RMSD less than 2 Å were ranked in the top 1 model for 100% (70/70) docking cases, and the subangstrom models with peptide bb-RMSD less than 1 Å were ranked in the top 5 lowest-energy models for 65.7% (46/70) docking cases. Furthermore, 10 out of 70 docking cases ranked the subangstrom all-atom models in the top 5 lowest-energy models. The results showed that the FlexPepDock can generate high-quality models of pHLA-I complexes and can be widely applied to pHLA-I modeling and mechanism research of peptide-mediated immune responses.
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    83. 83
      Raveh, B.; London, N.; Schueler-Furman, O. Sub-angstrom modeling of complexes between flexible peptides and globular proteins. Proteins 2010, 78, 2029– 2040,  DOI: 10.1002/prot.22716
      83
      Sub-angstrom modeling of complexes between flexible peptides and globular proteins
      Raveh, Barak; London, Nir; Schueler-Furman, Ora
      Proteins: Structure, Function, and Bioinformatics (2010), 78 (9), 2029-2040CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large no. of rotatable bonds in flexible peptides relative to typical ligand mols., and the fact that different peptides assume different backbone conformations within the same binding site. In this study, the authors present Rosetta FlexPepDock, a novel tool for refining coarse peptide-protein models that allows significant changes in both peptide backbone and side chains. The authors obtain high resoln. models, often of sub-angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide-protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with at. accuracy. In addn., the authors' protocol has improved modeling quality for the important application of cross docking to PDZ domains. The authors anticipate that the ability to create high resoln. models for a wide range of peptide-protein complexes will have significant impact on structure-based functional characterization, controlled manipulation of peptide interactions, and on peptide-based drug design.
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    84. 84
      Bloodworth, N.; Barbaro, N. R.; Moretti, R.; Harrison, D. G.; Meiler, J. Rosetta FlexPepDock to predict peptide-MHC binding: An approach for non-canonical amino acids. PLoS One 2022, 17, e0275759  DOI: 10.1371/journal.pone.0275759
      84
      Rosetta FlexPepDock to predict peptide-MHC binding: An approach for non-canonical amino acids
      Bloodworth, Nathaniel; Barbaro, Natalia Ruggeri; Moretti, Rocco; Harrison, David G.; Meiler, Jens
      PLoS One (2022), 17 (12), e0275759CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Computation methods that predict the binding of peptides to MHC-I are important tools for screening and identifying immunogenic antigens and have the potential to accelerate vaccine and drug development. However, most available tools are sequence-based and optimized only for peptides contg. the twenty canonical amino acids. This omits a large no. of peptides contg. non-canonical amino acids (NCAA), or residues that undergo varied post-translational modifications such as glycosylation or phosphorylation. These modifications fundamentally alter peptide immunogenicity. Similarly, existing structure-based methods are biased towards canonical peptide backbone structures, which may or may not be preserved when NCAAs are present. Rosetta FlexPepDock ab-initio is a structure-based computational protocol able to evaluate peptide-receptor interaction where no prior information of the peptide backbone is known. We benchmarked FlexPepDock ab-initio for docking canonical peptides to MHC-I, and illustrate for the first time the method's ability to accurately model MHC-I bound epitopes contg. NCAAs. FlexPepDock ab-initio protocol was able to recapitulate near-native structures (≤1.5Å) in the top lowest-energy models for 20 out of 25 cases in our initial benchmark. Using known exptl. binding affinities of twenty peptides derived from an influenza-derived peptide, we showed that FlexPepDock protocol is able to predict relative binding affinity as Rosetta energies correlate well with exptl. values (r = 0.59, p = 0.006). ROC anal. revealed 80% true pos. and a 40% false pos. rate, with a prediction power of 93%. Finally, we demonstrate the protocol's ability to accurately recapitulate HLA-A*02:01 bound phosphopeptide backbone structures and relative binding affinity changes, the theor. structure of the lymphocytic choriomeningitis derived glycosylated peptide GP392 bound to MHC-I H-2Db, and isolevuglandin-adducted peptides. The ability to use non-canonical amino acids in the Rosetta FlexPepDock protocol may provide useful insight into crit. amino acid positions where the post-translational modification modulates immunol. responses.
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    85. 85
      Tivon, B.; Gabizon, R.; Somsen, B. A.; Cossar, P. J.; Ottmann, C.; London, N. Covalent flexible peptide docking in Rosetta. Chem. Sci. 2021, 12, 10836– 10847,  DOI: 10.1039/D1SC02322E
      There is no corresponding record for this reference.
    86. 86
      Alam, N.; Schueler-Furman, O. Modeling Peptide-Protein Structure and Binding Using Monte Carlo Sampling Approaches: Rosetta FlexPepDock and FlexPepBind. Methods Mol. Biol. 2017, 1561, 139– 169,  DOI: 10.1007/978-1-4939-6798-8_9
      There is no corresponding record for this reference.
    87. 87
      Marin-Acevedo, J. A.; Dholaria, B.; Soyano, A. E.; Knutson, K. L.; Chumsri, S.; Lou, Y. Next generation of immune checkpoint therapy in cancer: new developments and challenges. J. Hematol. Oncol. 2018, 11, 39,  DOI: 10.1186/s13045-018-0582-8
      87
      Next generation of immune checkpoint therapy in cancer: new developments and challenges
      Marin-Acevedo, Julian A.; Dholaria, Bhagirathbhai; Soyano, Aixa E.; Knutson, Keith L.; Chumsri, Saranya; Lou, Yanyan
      Journal of Hematology & Oncology (2018), 11 (), 39/1-39/20CODEN: JHOOAO; ISSN:1756-8722. (BioMed Central Ltd.)
      A review Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body's immunol. activity against tumors. Cytotoxic T lymphocyte-assocd. mol.-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clin. activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or mols. targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clin. trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.
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    88. 88
      Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.; Leming, P. D.; Spigel, D. R.; Antonia, S. J.; Horn, L.; Drake, C. G.; Pardoll, D. M.; Chen, L.; Sharfman, W. H.; Anders, R. A.; Taube, J. M.; McMiller, T. L.; Xu, H.; Korman, A. J.; Jure-Kunkel, M.; Agrawal, S.; McDonald, D.; Kollia, G. D.; Gupta, A.; Wigginton, J. M.; Sznol, M. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J. Med. 2012, 366, 2443– 2454,  DOI: 10.1056/NEJMoa1200690
      88
      Safety, activity, and immune correlates of anti-PD-1 antibody in cancer
      Topalian, Suzanne L.; Hodi, F. Stephen; Brahmer, Julie R.; Gettinger, Scott N.; Smith, David C.; McDermott, David F.; Powderly, John D.; Carvajal, Richard D.; Sosman, Jeffrey A.; Atkins, Michael B.; Leming, Philip D.; Spigel, David R.; Antonia, Scott J.; Horn, Leora; Drake, Charles G.; Pardoll, Drew M.; Chen, Lieping; Sharfman, William H.; Anders, Robert A.; Taube, Janis M.; McMiller, Tracee L.; Xu, Haiying; Korman, Alan J.; Jure-Kunkel, Maria; Agrawal, Shruti; McDonald, Daniel; Kollia, Georgia D.; Gupta, Ashok; Wigginton, Jon M.; Sznol, Mario
      New England Journal of Medicine (2012), 366 (26), 2443-2454CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      Background: Blockade of programmed death 1 (PD-1), an inhibitory receptor expressed by T cells, can overcome immune resistance. We assessed the antitumor activity and safety of BMS-936558, an antibody that specifically blocks PD-1. Methods: We enrolled patients with advanced melanoma, non-small-cell lung cancer, castration-resistant prostate cancer, or renal-cell or colorectal cancer to receive anti-PD-1 antibody at a dose of 0.1 to 10.0 mg per kg of body wt. every 2 wk. Response was assessed after each 8-wk treatment cycle. Patients received up to 12 cycles until disease progression or a complete response occurred. Results: A total of 296 patients received treatment through Feb. 24, 2012. Grade 3 or 4 drug-related adverse events occurred in 14% of patients; there were three deaths from pulmonary toxicity. No max. tolerated dose was defined. Adverse events consistent with immune-related causes were obsd. Among 236 patients in whom response could be evaluated, objective responses (complete or partial responses) were obsd. in those with non-small-cell lung cancer, melanoma, or renal-cell cancer. Cumulative response rates (all doses) were 18% among patients with non-small-cell lung cancer (14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses lasted 1 yr or more in patients with 1 yr or more of follow-up. To assess the role of intratumoral PD-1 ligand (PD-L1) expression in the modulation of the PD-1-PD-L1 pathway, immunohistochem. anal. was performed on pretreatment tumor specimens obtained from 42 patients. Of 17 patients with PD-L1-neg. tumors, none had an objective response; 9 of 25 patients (36%) with PD-L1-pos. tumors had an objective response (P = 0.006). Conclusions: Anti-PD-1 antibody produced objective responses in approx. one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; the adverse-event profile does not appear to preclude its use. Preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective response.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtV2rs7fN&md5=7c13f228ef92b6089982fc878d3a394b
    89. 89
      Tumeh, P. C.; Harview, C. L.; Yearley, J. H.; Shintaku, I. P.; Taylor, E. J.; Robert, L.; Chmielowski, B.; Spasic, M.; Henry, G.; Ciobanu, V.; West, A. N.; Carmona, M.; Kivork, C.; Seja, E.; Cherry, G.; Gutierrez, A. J.; Grogan, T. R.; Mateus, C.; Tomasic, G.; Glaspy, J. A.; Emerson, R. O.; Robins, H.; Pierce, R. H.; Elashoff, D. A.; Robert, C.; Ribas, A. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014, 515, 568– 571,  DOI: 10.1038/nature13954
      89
      PD-1 blockade induces responses by inhibiting adaptive immune resistance
      Tumeh, Paul C.; Harview, Christina L.; Yearley, Jennifer H.; Shintaku, I. Peter; Taylor, Emma J. M.; Robert, Lidia; Chmielowski, Bartosz; Spasic, Marko; Henry, Gina; Ciobanu, Voicu; West, Alisha N.; Carmona, Manuel; Kivork, Christine; Seja, Elizabeth; Cherry, Grace; Gutierrez, Antonio J.; Grogan, Tristan R.; Mateus, Christine; Tomasic, Gorana; Glaspy, John A.; Emerson, Ryan O.; Robins, Harlan; Pierce, Robert H.; Elashoff, David A.; Robert, Caroline; Ribas, Antoni
      Nature (London, United Kingdom) (2014), 515 (7528), 568-571CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clin. responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8+ T cells (termed adaptive immune resistance). Here we show that pre-existing CD8+ T cells distinctly located at the invasive tumor margin are assocd. with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quant. immunohistochem., quant. multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumors, patients responding to treatment showed proliferation of intratumoral CD8+ T cells that directly correlated with radiog. redn. in tumor size. Pre-treatment samples obtained from responding patients showed higher nos. of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumor margin and inside tumors, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate anal., we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumor regression after therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are neg. regulated by PD-1/PD-L1-mediated adaptive immune resistance.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFanu7jL&md5=df43fa2237e9e66d99ad230159e0fe9e
    90. 90
      Riaz, N.; Morris, L.; Havel, J. J.; Makarov, V.; Desrichard, A.; Chan, T. A. The role of neoantigens in response to immune checkpoint blockade. Int. Immunol. 2016, 28, 411– 419,  DOI: 10.1093/intimm/dxw019
      There is no corresponding record for this reference.
    91. 91
      Gubin, M. M.; Zhang, X.; Schuster, H.; Caron, E.; Ward, J. P.; Noguchi, T.; Ivanova, Y.; Hundal, J.; Arthur, C. D.; Krebber, W. J.; Mulder, G. E.; Toebes, M.; Vesely, M. D.; Lam, S. S.; Korman, A. J.; Allison, J. P.; Freeman, G. J.; Sharpe, A. H.; Pearce, E. L.; Schumacher, T. N.; Aebersold, R.; Rammensee, H. G.; Melief, C. J.; Mardis, E. R.; Gillanders, W. E.; Artyomov, M. N.; Schreiber, R. D. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014, 515, 577– 581,  DOI: 10.1038/nature13988
      91
      Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens
      Gubin, Matthew M.; Zhang, Xiuli; Schuster, Heiko; Caron, Etienne; Ward, Jeffrey P.; Noguchi, Takuro; Ivanova, Yulia; Hundal, Jasreet; Arthur, Cora D.; Krebber, Willem-Jan; Mulder, Gwenn E.; Toebes, Mireille; Vesely, Matthew D.; Lam, Samuel S. K.; Korman, Alan J.; Allison, James P.; Freeman, Gordon J.; Sharpe, Arlene H.; Pearce, Erika L.; Schumacher, Ton N.; Aebersold, Ruedi; Rammensee, Hans-Georg; Melief, Cornelis J. M.; Mardis, Elaine R.; Gillanders, William E.; Artyomov, Maxim N.; Schreiber, Robert D.
      Nature (London, United Kingdom) (2014), 515 (7528), 577-581CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The immune system influences the fate of developing cancers by not only functioning as a tumor promoter that facilitates cellular transformation, promotes tumor growth and sculpts tumor cell immunogenicity, but also as an extrinsic tumor suppressor that either destroys developing tumors or restrains their expansion. Yet, clin. apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte assocd. antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clin. benefits-including durable responses-to patients with different malignancies. However, little is known about the identity of the tumor antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumor-specific. Here we use genomics and bioinformatics approaches to identify tumor-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumor rejection comparably to checkpoint blockade immunotherapy. Although mutant tumor-antigen-specific T cells are present in progressively growing tumors, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumor rejection. These results reveal that tumor-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.
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    92. 92
      Woods, D. M.; Sodre, A. L.; Villagra, A.; Sarnaik, A.; Sotomayor, E. M.; Weber, J. HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade. Cancer Immunol Res. 2015, 3, 1375– 1385,  DOI: 10.1158/2326-6066.CIR-15-0077-T
      92
      HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade
      Woods, David M.; Sodre, Andressa L.; Villagra, Alejandro; Sarnaik, Amod; Sotomayor, Eduardo M.; Weber, Jeffrey
      Cancer Immunology Research (2015), 3 (12), 1375-1385CODEN: CIRACV; ISSN:2326-6066. (American Association for Cancer Research)
      Expression of PD-1 ligands by tumors and interaction with PD-1-expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this coinhibitory axis have proven clin. successful in the treatment of metastatic melanoma, non-small cell lung cancer, and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDAC) have shown clin. success in the treatment of some hematol. malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance antitumor immune responses. Here, we show that class I HDAC inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 h. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared with control and single-agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade.
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    93. 93
      Gameiro, S. R.; Malamas, A. S.; Tsang, K. Y.; Ferrone, S.; Hodge, J. W. Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells. Oncotarget 2016, 7, 7390– 7402,  DOI: 10.18632/oncotarget.7180
      There is no corresponding record for this reference.
    94. 94
      Llopiz, D.; Ruiz, M.; Villanueva, L.; Iglesias, T.; Silva, L.; Egea, J.; Lasarte, J. J.; Pivette, P.; Trochon-Joseph, V.; Vasseur, B.; Dixon, G.; Sangro, B.; Sarobe, P. Enhanced anti-tumor efficacy of checkpoint inhibitors in combination with the histone deacetylase inhibitor Belinostat in a murine hepatocellular carcinoma model. Cancer Immunol Immunother 2019, 68, 379– 393,  DOI: 10.1007/s00262-018-2283-0
      There is no corresponding record for this reference.
    95. 95
      Burke, B.; Eden, C.; Perez, C.; Belshoff, A.; Hart, S.; Plaza-Rojas, L.; Delos Reyes, M.; Prajapati, K.; Voelkel-Johnson, C.; Henry, E.; Gupta, G.; Guevara-Patino, J. Inhibition of Histone Deacetylase (HDAC) Enhances Checkpoint Blockade Efficacy by Rendering Bladder Cancer Cells Visible for T Cell-Mediated Destruction. Front. Oncol. 2020, 10, 699,  DOI: 10.3389/fonc.2020.00699
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    96. 96
      Baretti, M.; Yarchoan, M. Epigenetic modifiers synergize with immune-checkpoint blockade to enhance long-lasting antitumor efficacy. J. Clin Invest 2021, 131, e151002  DOI: 10.1172/JCI151002
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    97. 97
      Truong, A. S.; Zhou, M.; Krishnan, B.; Utsumi, T.; Manocha, U.; Stewart, K. G.; Beck, W.; Rose, T. L.; Milowsky, M. I.; He, X.; Smith, C. C.; Bixby, L. M.; Perou, C. M.; Wobker, S. E.; Bailey, S. T.; Vincent, B. G.; Kim, W. Y. Entinostat induces antitumor immune responses through immune editing of tumor neoantigens. J. Clin Invest 2021, 131, e138560  DOI: 10.1172/JCI138560
      There is no corresponding record for this reference.
    98. 98
      Zhang, P.; Du, Y.; Bai, H.; Wang, Z.; Duan, J.; Wang, X.; Zhong, J.; Wan, R.; Xu, J.; He, X.; Wang, D.; Fei, K.; Yu, R.; Tian, J.; Wang, J. Optimized dose selective HDAC inhibitor tucidinostat overcomes anti-PD-L1 antibody resistance in experimental solid tumors. BMC Med. 2022, 20, 435,  DOI: 10.1186/s12916-022-02598-5
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

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    • Additional figures including RMA-S (workflow scheme, concentration scan with sarsWT, and time scan), DC2.4 T Cell activation, modeling of citrullination, and mass spectrometry results from MHC peptides; materials/methods; and synthetic peptide characterization (mass spectrometry and analytical HPLC traces) (PDF)


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