Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity

Abstract

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8⁺ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8⁺ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8⁺ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

Keywords: CD8(+) T cells; Mgat5; antigen sensitivity; chronic infection; galectin 3; glycosylation; immune regulation; interleukin 10.

Figure 1. LCMV cl13 infection reduces the antigen sensitivity of CD8⁺ T cells in an IL-10 dependent manner by impairing proximal TCR signaling

A) Percentage of IFN-γ⁺ P14 cells at day 8 post-infection with LCMV Arm or LCMV cl13 in the indicated host was determined following ex vivo stimulation with titrated concentrations of GP_33-41 peptide. Data (Mean ± SEM) are normalized to the proportion of IFN-γ⁺ cells at saturating peptide concentration (10 nM). B) Summary (mean ± SEM) of EC₅₀ for IFN-γ⁺ P14 cells. C) Antigen sensitivity as in Figure 1A at day 8 post-infection with LCMV Arm in the indicated host. D) Summary (mean ± SEM) of EC₅₀ for IFN-γ⁺ P14 cells. E) Representative histograms of expression of PD-1 on P14 cells at day 8 post-infection with indicated strain of LCMV. Shaded histogram represents isotype control. F) Summary (mean ± SEM) of geometric mean fluorescence intensity (gMFI) of PD-1 expression on P14 cells. G) Antigen sensitivity as in Figure 1A for P14 cells mock transduced or expressing shRNAs targeting FF_luc or Il10ra. H) Summary (mean ± SEM) of EC₅₀ for IFN-γ⁺ P14 cells. I) Immunoblot analysis of P14 cell lysates at day 8 post-infection with indicated strains of LCMV from the indicated host. Cells were stimulated by CD3-crosslinking for the indicated time and equivalent amounts of total protein loaded into each lane. Total and phospho(p)-proteins were probed, as indicated. J) Same as I), but using PMA stimulation. Data in A-H represent 3 mice per group and are representative of at least 2 independent experiments. Data in B, D, F and H were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. *p<0.05, **p<0.01, *** p<0.001, ****p<0.0001. Data in I and J are representative of at least 2 independent experiments from 3 pooled mice per group. See also Figure S1 and S2.

Figure 2. IL-10 restricts the co-localization of TCR with CD8 co-receptor and enhances N-glycan branching of CD8⁺ T cells

A) Schematic of FRET approach. B) Summary (mean ± SEM) of FRET Units for P14 cells at day 8 post-infection with indicated strain of LCMV in the indicated host. C) Schematic of Mgat5-modified N-glycan and respective galectin and PHA-L binding sites. D) Relative Mgat5 expression in day 8 effector P14 cells from indicated conditions compared to naїve P14 cells (mean ± SEM). E) Representative histogram of PHA-L binding on effector P14 cells at day 8 post-infection. Shaded histogram represents fluorescence minus one (FMO) control F) Summary (mean ± SEM) of gMFI of PHA-L binding. G) Immunoblot of α/β TCR following pull-down with PHA-L conjugated beads. H) Relative Mgat5 expression in day 7 P14 cells following infection with P. yoellii – GP₃₃ compared to naïve P14 cells (mean ± SEM). I) Relative MGAT5 expression in total CD8⁺ T cells isolated from human patients with chronic HCV infection compared to a naïve cohort (mean ± SEM). J) Representative histogram of Gal3 binding on P14 cells at day 8 post-infection with indicated strain of LCMV. Shaded histogram represents FMO control. Refer to panel E for color legend. K) Summary (mean ± SEM) of gMFI of Gal3 expression on P14 cells. Data in B, D, E, F, H, J, K represent 3 mice per group and are representative of at least 2 experiments. Data G are from at least 2 pooled mice per group and representative of 3 experiments. Data in I are from 5 patients per group. Data in B, D, F and K were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. Data in H and I were analyzed by two-tailed unpaired T-test. *p<0.05, ****p<0.0001. See also Figure S3.

Figure 3. Inhibition of galectin binding rescues T cell function during LCMV cl13 infection

A) Antigen sensitivity as in Figure 1A of P14 cells from day 8 post-infection with the indicated strain of LCMV in WT hosts. Cells were stimulated with or without 50 mM D-Lactose. B) Summary (mean ± SEM) of EC₅₀ for IFN-γ⁺ P14 cells. C) Summary FRET units (mean ± SEM) with or without 50 mM D-Lactose treatment. D) Immunoblot analysis as in Figure 1I following treatment with or without 50 mM D-Lactose. E) Antigen sensitivity in P14 cells isolated from LCMV Arm infection with or without treatment with D-Lactose as in Figure 1A. F) Summary (mean ± SEM) EC₅₀ for IFN-γ⁺ P14 cells. Data in A, B and C, E, F represent 3 mice per group and are representative of at least 3 experiments. Data in D represent total protein of 3 pooled mice per group and is representative of at least 2 experiments. Data in B and C were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. Data in F were analyzed by two-tailed unpaired T-test. ****p<0.0001. See also Figure S4.

Figure 4. Gal3 regulates antigen sensitivity and limits T cell function during LCMV cl13 infection

A) Antigen sensitivity as in Figure 1A for P14 cells at day 8 post-infection with the indicated LCMV strain in the indicated host. B) Summary (mean ± SEM) EC₅₀ for IFN-γ⁺ P14 cells. C) Summary (mean ± SEM) FRET Units. D) Representative histograms of PHA-L binding on P14 cells. Shaded histogram represents FMO control. E) Summary (mean ± SEM) gMFI of PHA-L binding. F) and G) Plaque Forming Units (PFU) per gram of indicated organ at day 8 post-infection with indicated strains of LCMV in the indicated host. Data in A-E represent 3 mice per group and are representative of 2 experiments. Data in F and G represent 6 mice per group pooled from 2 experiments. Data in B, C, and E were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. **p<0.01, ****p<0.0001. See also Figure S5.

Figure 5. Mgat5 knockdown rescues CD8⁺ T cell antigen sensitivity during LCMV cl13 infection

A) Antigen sensitivity as in Figure 1G for P14 cells expressing an shRNA targeting Mgat5, FF_luc or mock transduced. B) Summary (mean ± SEM) of EC₅₀ for IFN-γ⁺ P14 cells. C) Representative histograms of PD-1 expression, shaded histogram represents isotype control. D) Summary (mean ± SEM) gMFI of PD-1 expression on P14 cells. E) Representative histograms of Gal3 binding on P14 cells, shaded histogram represents FMO control. Refer to panel C for color legend. F) Summary (mean ± SEM) gMFI of Gal3 binding on P14 cells. G) Representative histograms of PHA-L binding on P14 cells, shaded histogram represents FMO control. Refer to panel C for color legend. H) Summary (mean ± SEM) gMFI of PHA-L expression on P14 cells. Data represent 3 mice per group and are representative of 2 experiments. Data in B, D and F, H were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. IL-10 directly regulates Mgat5 expression and reduces antigen sensitivity in a T cell intrinsic manner

A) Relative Mgat5 expression in CD8⁺ T cells activated ex vivo in the presence of increasing doses of IL-10 for 8 days compared to untreated (0) group (mean ± SEM). B) Representative histograms of PHA-L binding to CD8⁺ T cells, shaded histogram represents FMO control. C) Summary (mean ± SEM) gMFI of PHA-L on CD8⁺ T cells following IL-10 treatment. D) Relative Mgat5 expression in CD8⁺ T cells activated ex vivo and cultured for 8 days with exposure to 200 ng/mL of IL-10 on the first day (d1), first and second day (d1+d2) or all days of culture (All) compared to untreated (none) group (mean ± SEM). E) Representative histograms of PHA-L binding to CD8⁺ T cells for cells treated as in (D), shaded histogram represents FMO control. F) Summary (mean ± SEM) gMFI of PHA-L on CD8⁺ T cells following IL-10 treatment as in (D). G) Relative Mgat5 expression in CD8⁺ T cells activated ex vivo in the presence or absence of IL-10 and/or STAT3 inhibitor (WP1066) compared to untreated group (mean ± SEM). H) Summary (mean ± SEM) gMFI of PHA-L on CD8⁺ T cells. I) Summary (mean ± SEM) gMFI of Gal3 binding on CD8⁺ T cells following incubation with exogenous Gal3. J) Summary (mean ± SEM) gMFI of Gal3 binding on P14 cells transduced or mock-transduced as indicated K) Summary (mean ± SEM) gMFI of PHA-L binding on P14 cells transduced or mock-transduced as indicated. Data represent 3 mice per group and are representative of 2 experiments. Data in A, C, D, F, G, H, I, J and K were analysed by one-way ANOVA with Tukey’s post-hoc analysis of multiple comparisons. Data in A and C are indicated as significant relative to untreated (0 ng/mL) group only. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S6.