Fig 2: Sialylation influences FasL mediated cell death by altering intracellular phosphorylation events.A, phosphoproteomics workflow for analysis of WT or ST6GAL1−/− Jurkat cells treated with FasL. B, Volcano plot of differentially phosphorylated peptides detected in ST6GAL1−/− relative to WT Jurkat cells treated with 100 ng/mL FasL. Phosphopeptide IDs that were compatible with RoKAI analysis are annotated in red. Companion plot for cells that were not treated with FasL is presented in Figure S4. C, differential activity of the kinases and phosphatases in ST6GAL1−/− relative to WT Jurkat cells identified via RoKAI analysis of the dataset from panel B. D, GO biological process terms associated with the kinases and phosphatases identified in C. Figure generated using ShinyGO V. 0.85 (55). All data is the result of four technical replicates submitted for phosphoproteomic analysis.

Fig 3: α2-6-sialoglycan deficiency enhances localization dynamics of the Fas Receptor.A, workflow for evaluating surface presentation and spatial organization of FasR on WT or ST6GAL1−/− Jurkat cells via imaging flow cytometry. B, surface expression levels of FasR as a function of ST6GAL1 expression, exposure to FasL (100 ng/mL), and live vs. apoptotic status as measured by the BB515 channel on the imaging flow cytometer (same dataset as imaging data in subsequent panels). Gating scheme for live vs. apoptotic as in Figure 2B. FMO = fluorescent minus one staining control. C, Quantification of results from B. D, diffusivity of signal from the fluorescent anti-FasR antibody. E, representative individual cell images from the same groups of cells as reported in B. Data recorded via imaging flow cytometry. Grayscale layer corresponds to lightloss, which is analogous to a brightfield image of the cell. Green layer corresponds to the fluorescence signal from the anti-FasR antibody. F, Quantification of data from D. G, complementary fluoresence microscopy images for the experiment described in A. Scale bar, 10 μm. For plots in (C) and (F), results are reported as mean SD from three experiments. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, p > 0.05 = ns = not significant. Two-way ANOVA with Tukey post hoc test.

Fig 4: FasL increases apoptosis and cell death in the α2-6-sialoglycan deficient T cell model compared to other TNF receptor superfamily ligands.A, workflow for evaluating WT or ST6GAL1−/− Jurkat cell death induced by TRAIL, TNF-α, and FasL. Annexin V (recognizes exposed phosphatidylserine) and DAPI (intercalates DNA) report on apoptosis and cell death, respectively. B, flow cytometry plots for Annexin V and DAPI staining of WT or ST6GAL1−/− Jurkat cells treated with 50 ng/mL TRAIL, 100 ng/mL TNF-α, or 100 ng/mL FasL. Bottom left gates (blue) = live cells, bottom right gates (orange) = apoptotic cells, and top right gate (red) = dead cells. Percent values indicate the portion of total singlet cell events within each quadrant. C, D, and E, quantification of data from (B). F, immunoblot against cleaved (cl.) caspase 3 (18 kDa) with β-actin shown as loading control (45 kDa). For plots in C–E, results are reported as mean SD from three experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001, p > 0.05 = ns = not significant. Two-way ANOVA with Tukey post hoc test.

Fig 5: Loss of cell surface sialic acids on stimulated primary human T cells increases cell death in response to FasL.A, workflow for evaluating programmed cell death in primary human T cells following stimulation, desialylation, and exposure to FasL. B, FasR expression on unstimulated (day 0) vs. stimulated (day 6) human T cells. C, Staining of primary human T cells with SNA for detection of α2-6-sialoglycans following exposure to recombinant VC sialidase (550 mU), heat inactivated (HI) VC sialidase, or no enzyme. D, flow cytometry plots for Annexin V and DAPI staining of stimulated primary human CD4+ and CD8+ T cells treated with 0, 10, or 100 ng/mL FasL and VC sialidase (550 mU) or HI VC sialidase. Bottom left elliptical gates (blue) = live cells, square gates (orange and red) = apoptotic and dying cells respectively. Percent values indicate the proportion of total single cell events within each gate. E. Quantification of data from D. For plots in B, C, and E, results are reported as mean SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Student’s t test, two-tailed (B) or one-way ANOVA with Tukey post hoc test (C and E). For plots in D (and Fig. S7) minor adjustments to the position of gates for the three populations were made to fully capture cell events in the relevant populations due to variation in positioning across different human donors. All gates used for data quantification across all donors are presented in Figures S8 and S9.