Figure 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.