Dissecting the Ability of Siglecs To Antagonize Fcγ Receptors

Fcγ receptors (FcγRs) play key roles in the effector function of IgG, but their inappropriate activation plays a role in several disease etiologies. Therefore, it is critical to better understand how FcγRs are regulated. Numerous studies suggest that sialic acid-binding immunoglobulin-type lectins (Siglecs), a family of immunomodulatory receptors, modulate FcγR activity; however, it is unclear of the circumstances in which Siglecs can antagonize FcγRs and which Siglecs have this ability. Using liposomes displaying selective ligands to coengage FcγRs with a specific Siglec, we explore the ability of Siglec-3, Siglec-5, Siglec-7, and Siglec-9 to antagonize signaling downstream of FcγRs. We demonstrate that Siglec-3 and Siglec-9 can fully inhibit FcγR activation in U937 cells when coengaged with FcγRs. Cells expressing Siglec mutants reveal differential roles for the immunomodulatory tyrosine-based inhibitory motif (ITIM) and immunomodulatory tyrosine-based switch motif (ITSM) in this inhibition. Imaging flow cytometry enabled visualization of SHP-1 recruitment to Siglec-3 in an ITIM-dependent manner, while SHP-2 recruitment is more ITSM-dependent. Conversely, both cytosolic motifs of Siglec-9 contribute to SHP-1/2 recruitment. Siglec-7 poorly antagonizes FcγR activation for two reasons: masking by cis ligands and differences in its ITIM and ITSM. A chimera of the Siglec-3 extracellular domains and Siglec-5 cytosolic tail strongly inhibits FcγR when coengaged, providing evidence that Siglec-5 is more like Siglec-3 and Siglec-9 in its ability to antagonize FcγRs. Additionally, Siglec-3 and Siglec-9 inhibited FcγRs when coengaged by cells displaying ligands for both the Siglec and FcγRs. These results suggest a role for Siglecs in mediating FcγR inhibition in the context of an immunological synapse, which has important relevance to the effectiveness of immunotherapies.

Table S2.Viral vector primers to install SphI and PacI.S4 Table S3.Antibodies used in flow cytometry staining of cells.S5              Table S1.Forward (FWD) and reverse (RVS) site-directed mutagenesis primers.

Figure S1 .
Figure S1.Siglec expression on primary peripheral blood human neutrophils S6 and monocytes and U937 cells.

Figure
Figure S2.Creation of cell lines.S7

Figure S7 .
Figure S7.Siglecs do not intrinsically inhibit FcR activation whenS12 stimulated with liposomes.

Figure S9 .
Figure S9.Creation of cells expressing Siglecs with the critical arginineS14 residue required for binding mutated.

Figure S10 .
Figure S10.Calcium flux of Siglec-3 and FcR engagement on separate S15 liposomes or WT cells.

Figure S12 .
Figure S12.Phosphorylation of downstream protein Erk is decreased when S17 Siglecs-3 or -9 are co-engaged with FcRs.

Figure S2 .
Figure S2.Creation of cell lines.(a) Schematic illustrating the deletion of a Siglec of interest from U937 cells using CRISPR/Cas9 followed by the viral transduction of the cells with empty vector (Siglec -/-) or WT Siglec.(b) Flow cytometry histograms illustrating the staining for the presence of a Siglec after CRISPR/Cas9 genetic knockout.(c) Expression of Siglec in CRISPR/Cas9 cells after viral transduction of empty lentiviral vector (Siglec -/-) or WT Siglec as determined by flow cytometry staining.

Figure S3 .
Figure S3.Development of mouse monoclonal anti-trinitrophenyl IgG antibodies.C57BL/6J mice were repeatedly immunized with TNP:KLH intraperitoneally before total splenocytes were isolated and fused with Sp2/0 cells using PEG.HAT resistant hybridomas were screened for secretion of TNP specific antibody by ELISA with TNP:BSA coated plates.TNP specific hybridomas of different isotopes were isolated and cloned by limiting dilution.

Figure S5 .
Figure S5.Optimization of FcR dependent TNP-liposome binding to cells.(a,b) Raw flow cytometry histograms (a) and plotted MFI quantification (b) of liposomal nanoparticles loaded with 0, 0.03, 0.1, or 0.3 mol % of TNP-PEG-DSPE with 0.1 mol % AF647-PEG-DSPE binding to cells pre-incubated anti-TNP-IgG2c.Binding (MFI) values from a control liposome bearing only 0.1 mol % AF647-PEG-DSPE were subtracted from the plotted MFI values.(c,d) TNP-liposome binding histogram (c) and MFI quantification (d) to cells pre-treated with anti-TNP-IgG2c with or without Fc block.Data plots in b and c are presented as median with 95% CI.Statistical analysis was performed on three technical replicates using an unpaired Student's t-test.

Figure S6 .
Figure S6.FcR expression on human monocytes derived from primary peripheral blood.(a) Flow cytometry representative histograms of FcRI, FcRII, FcRIII expression on monocytes isolated from human blood.(b) Quantification of three technical replicates MFI values plotted.Error bars are presented as median with 95% CI.

Figure S7 .
Figure S7.Siglecs do not intrinsically inhibit FcR activation when stimulated with liposomes.(a) Schematic of liposomes used to measure overall activation of cells.(b,c,d,e) Representative calcium flux of Siglec-3 -/-and WT Siglec-3 (b), Siglec-5 -/-and WT Siglec-5 (c), Siglec-7 -/-and WT Siglec-7 (d), and Siglec-9 -/-and WT Siglec-9 (e) after administration of the indicated liposomes.Cells were loaded with INDO-1-AM followed by an anti-TNP-IgG2c antibody.The pink arrow represents addition of the liposome ten seconds after acquisition on the flow cytometer.(f) Area under the calcium flux curves (AUC) from TNP liposome administration plotted with naked liposome AUC subtracted in Siglec -/-cells (Black) or WT Siglec cells (colour).Values are normalized to the amount of TNP liposome activation in Siglec -/-cells.Error bars are presented as median with 95% CI and P values were determined using unpaired Student's t-tests for six technical replicates per condition.

Figure S8 .
Figure S8.Three independent experiments showing Siglec-3, -9, and -7 inhibition of FcRs.(a) Liposomes used for stimulation of cells.(b,c,d) Area under the calcium flux curve (AUC) after administration of the indicated liposomes to Siglec-3 cell lines (b), Siglec-9 cell lines (c), and Siglec-7 cell lines (d).Each data point represents an average of three technical replicates and each pair of data points was taken on three different days with the background, Naked liposomes, subtracted.The P values were measured using paired Student's t-tests.

Figure S10 .
Figure S10.Calcium flux of Siglec-3 and FcR engagement on separate liposomes or WT U937 cells.(a) Representative calcium flux curves of U937 WT Siglec-3 cells loaded with INDO-1-Am followed by anti-IgG2c antibody then stimulated with the indicated liposomes.The pink arrow represents the addition of liposomes 10 seconds after acquisition on the flow cytometer.(b) The amount of cellular activation (AUC) from the calcium curves quantified and plotted.(c,d) Representative flow cytometry calcium flux curves (c) and quantified area under the curves (d) of WT U937 cells administered the indicated liposomes.P values were quantified using unpaired Student's t-test and error bars are represented by median with 95% CI for three technical replicates.

Figure S11 .
Figure S11.Liposomes pre-complexed with anti-TNP antibody bind and stimulate U937 cells.(a,b) Flow cytometry histogram (a) and plotted MFI values (b) of AF647 and TNP-containing liposomes pre-complexed with 0, 0.16, 0.33, 0.66, 1.33, or 2.66 µM of anti-TNP-IgG2c, (c,d) Representative calcium flux curves (c) and quantified area under the curve (d) of U937 WT cells stimulated with the indicated liposomes pre-complexed with 0.66 µM of antibody.The pink arrow represents the addition of liposomes 10 seconds after acquisition on the flow cytometer.P values were quantified using unpaired Student's t-test and error bars are represented by median with 95% CI for three technical replicates.

Figure S20 .
Figure S20.Lipid insertion of TNP-PEG-DSPE and/or Sig-3L-PEG-DSPE into K562 cells.(a) Schematic of lipid insertion into K562 Cells.(b) AUC from calcium flux of Siglec-3 -/-or WT Siglec-3 cells given the indicated stimulating cells.The AUC values are with Naked cells area subtracted and normalized to the amount of activation from TNP-PEG-DSPE loaded cells.P values were obtained using unpaired Student's t-tests on four technical replicates and error bars are represented as median with 95% CI.