Sel1L, an Endoplasmic Reticulum Associated Degradation Adaptor, Regulates CD8+ Tcell Fate

CD8⁺ T cells are critical players in protective immunity against intracellular pathogens and cancer. Upon activation with cognate antigen, CD8⁺ T cells undergo marked increase in protein synthesis and significant proteome remodeling as they clonally expand and acquire effector function. However, little is known about the mechanisms by which CD8⁺ T cells maintain protein homeostasis (proteostasis) during T cell differentiation and which proteostatic pathways regulate T cell fate. Proteostasis is maintained through two main pathways: ER-associated degradation (ERAD) and the unfolded protein response (UPR). While UPR mediators have been implicated in CD8⁺ T cell immune responses, nothing is known about the role of ERAD in CD8⁺ T cell immunity.

To interrogate proteostasis following CD8⁺ T cell activation in vivo, we utilized the murine model of acute viral infection LCMV-Armstrong (LCMV) to evaluate endoplasmic reticulum (ER) stress and proteostatic pathway activation over the course of initial effector differentiation. We found that endogenous LCMV-specific CD8⁺ T cells experience dynamic ER stress as measured by an increase in ER size [fold change (F.C.) ER Tracker median fluorescence intensity (MFI) relative to D0: D5 7.48± 4.91 *p<0.05; D8: 0.86±1.78, n.s., one-way ANOVA Dunnett's multiple comparisons test] and protein aggregation (Proteostat F.C. relative to D0: D5 1.95± 0.24 *p<0.05; D8 1.6 ±0.11, n.s.) over the course of infection with a peak at day 5 post-infection (p.i.), largely resolving by day 8 p.i. Furthermore, we assessed activation of the UPR pathways and found induction of UPR mediators XBP1s (F.C. relative to D0 D5: 3.96 ± 1.88 **p<0.01 D8 1.60±0.36 n.s.) and CHOP (F.C. relative to D0: D5 12.47±0.32 ***p<0.001; D8 2.58±1.5, n.s.) in LCMV-specific CD8⁺ T cells at the highest levels on day 5 p.i., which largely resolved by day 8 p.i.. Together, these data suggest that activated virus-specific CD8⁺ T cells experience dynamic ER stress and upregulation of proteostatic pathways during the course of CD8⁺ T cell differentiation. To test the cell-intrinsic role of ERAD in CD8⁺ T cell immune responses, we utilized TCR-transgenic CD8⁺ T cells that were specific for LCMV (termed P14 cells) and were deficient in Sel1L, a critical adaptor of the ERAD complex that recognizes misfolded proteins in ER and recruits them to be retro-translocated to cytosol for proteasomal degradation. We adoptively transferred Sel1L-deficient P14 cells mixed in a 1:1 ratio with wildtype (WT) P14 cells into cogenically disparate mice that were subsequently infected with LCMV. By day 15 p.i., Sel1L-deficient P14 represented only 29.50% of donor cells in peripheral blood, a fraction which was reduced to 7.5% by day 45 p.i.. These data suggest that Sel1L/ERAD is required for CD8⁺ T cell survival and memory formation following acute viral infection. To investigate the mechanism by which Sel1L/ERAD regulated CD8⁺T cell fate, we performed RNA-sequencing on WT and Sel1L-deficient P14 cells at day 8 p.i.. Pathway analysis revealed a reduction in pathways corresponding to T-cell metabolism, specifically 'mitochondrial respiratory chain' (FDR 1.59E^-03) and 'oxidative phosphorylation'(FDR 2.45E^-08), as well as an increase in apoptotic terms. Furthermore, immune signature analysis suggested Sel1L-deficient P14 cells lack memory precursors CD8⁺ T cells due to enrichment in immune signature of KLRG1hi effector CD8⁺ T cells (FDR 1.34E^-18). Functional validation by flow cytometry revealed that Sel1L-deficient P14 cells are significantly reduced in their ability to generate memory precursor CD8⁺ T cells (9.05%±3.84 vs. 6.12%±4.28 ***p<0.001, paired t-test) compared to WT on day 8 p.i. and that D6 in-vitro activated Sel1L-deficient CD8⁺ T cells exhibit reduced mitochondrial membrane potential relative to WT (TMRM MFI FC relative to WT 0.39±0.29 *p<0.05, unpaired t-test)

Our studies demonstrate that virus-specific CD8⁺ T cells experience dynamic ER stress in vivo during an acute viral infection and that Sel1L/ERAD is required for antigen-specific CD8⁺ T cell persistence following an infection. Ongoing mechanistic studies will help determine how Sel1L/ERAD promotes CD8⁺ T cell survival and persistence.

No relevant conflicts of interest to declare.