TCR stimulation strength is inversely associated with establishment of functional brain-resident memory CD8 T cells during persistent viral infection

<div><p>Establishing functional tissue-resident memory (T_RM) cells at sites of infection is a newfound objective of T cell vaccine design. To directly assess the impact of antigen stimulation strength on memory CD8 T cell formation and function during a persistent viral infection, we created a library of mouse polyomavirus (MuPyV) variants with substitutions in a subdominant CD8 T cell epitope that exhibit a broad range of efficiency in stimulating TCR transgenic CD8 T cells. By altering a subdominant epitope in a nonstructural viral protein and monitoring memory differentiation of donor monoclonal CD8 T cells in immunocompetent mice, we circumvented potentially confounding changes in viral infection levels, virus-associated inflammation, size of the immunodominant virus-specific CD8 T cell response, and shifts in TCR affinity that may accompany temporal recruitment of endogenous polyclonal cells. Using this strategy, we found that antigen stimulation strength was inversely associated with the function of memory CD8 T cells during a persistent viral infection. We further show that CD8 T_RM cells recruited to the brain following systemic infection with viruses expressing epitopes with suboptimal stimulation strength respond more efficiently to challenge CNS infection with virus expressing cognate antigen. These data demonstrate that the strength of antigenic stimulation during recruitment of CD8 T cells influences the functional integrity of T_RM cells in a persistent viral infection.</p></div

Characterizing the T cell response to analogues of a subdominant CD8 T cell epitope.

<p>(A) RMA/S cells were cultured overnight at 26°C. Cells were incubated with the indicated dose of peptide for 1 h at RT followed by incubation at 37C for 2 h. Table shows the EC₅₀ of each analogue peptide for MHC-I binding and stabilization. (B) RMA/S cells were cultured overnight at 25°C in 100 μM of peptide. Cells were then resuspended in fresh media, incubated at 37C, and sampled hourly for 4 h. Table shows the half-life of the analogue peptides for binding to MHC-I. (C-D) Splenocytes were isolated from a naïve TCR-V mouse and stimulated with TagV analogue peptides at the indicated concentrations for 5 h at 37°C. Nuclear Nur77 (C) and intracellular IFNγ expression (D) shown. Table shows the EC₅₀ of each analogue peptide for expression of these activation markers. All data plotted as percent of maximal expression ± SD.</p

Brain-T_RM cells primed with lower stimulation exhibit increased recall potential.

<p>(A) 1 x 10³ donor TCR-V CD8 T cells were adoptively transferred into recipient C57BL/6 mice. Mice were infected with 2 x 10⁶ PFU of MuPyV via hind footpads the following day and given depleting anti-CD8α on day 10 p.i. and once/week for 3 weeks. Mice were challenged i.c. with MuPyV.TagV virus on day 30 p.i. and sacrificed five days post-challenge. (B) Number of TCR-V cells in the brain with or without secondary challenge (left panel) and fold change of TCR-V cells in mice receiving secondary challenge compared to day 30 averages (right panel). (C) IFNγ and TNFα co-expression from TCR-V cells stimulated for 5 h ex vivo with 1 μM TagV peptide. Mean ± SD plotted. N = 6–15 mice from 2–5 independent experiments. *, p < 0.05; ANOVA with Tukey’s test for significance.</p

TCR stimulation strength does not affect CD8 T cell memory differentiation in the spleen during acute infection.

<p>TCR-V cells were transferred i.v. into B6 mice one day prior to infection with TagV analogue viruses and sacrificed at day 8 p.i. (A) Number of TCR-V cells in the spleen. (B) Percent of TCR-V cells that are KLRG1^hiCD127^lo (left panel) or KLRG1^loCD127^hi (right panel). Representative dot plots shown. (C-D) Expression of T-bet (C) and eomes (D) by splenic TCR-V cells. Representative histograms shown. Gray shaded histograms represent fluorescence-minus-one controls. (E) IFNγ and TNFα co-expression (left panel) and Nur77 expression (right panel) in splenocytes stimulated for 5 h ex vivo with 1 μM TagV peptide. Mean ± SD plotted. N = 9–18 mice from 3–6 independent experiments.</p

Memory splenic CD8 T cells primed with lower TCR stimulation exhibit superior recall responses.

<p>(A-B) TCR-V cells were adoptively transferred into B6 mice one day prior to infection with cognate Tag V or TagV analogue viruses, then sacrificed at day 30 p.i. (A) Number of TCR-V cells in the spleen. (B) IFNγ and TNFα co-expression (left panel) and Nur77 expression (right panel) in splenocytes stimulated for 5 h ex vivo with 1 μM TagV peptide. (C-D) Intracellular IFNγ (left panel) and Nur77 (right panel) expression by TCR-V cells isolated from the spleen at day 8 p.i. (C) or day 30 p.i. (D) stimulated for 5 h ex vivo with varying concentrations of the cognate TagV peptide. (E) 1 x 10³ donor TCR-V CD8 T cells were transferred into B6 mice. Mice were infected with 2 x 10⁶ PFU of MuPyV via hind footpads the following day, challenged i.p. with 5 x 10⁷ 116A1 cells at day 30 p.i., and sacrificed five days post-challenge. (F) Number of TCR-V cells in the spleen with or without secondary challenge and fold change of TCR-V cells in mice receiving secondary challenge compared to day 30 averages. (G) IFNγ and TNFα co-expression in splenocytes stimulated for 5 h ex vivo with 1 μM TagV peptide. Mean ± SD plotted. N = 6–18 mice from multiple independent experiments. *, p < 0.05; ****, p < 0.0001; ANOVA with Tukey’s test for significance.</p

TagV analogues.

<p>TagV analogues.</p

Infection by analogues of the subdominant TagV epitope does not affect the endogenous anti-MuPyV response.

<p>(A) 1 x 10³ TCR-V CD8 T cells were adoptively transferred i.v. into B6 mice. Mice were infected with 2 x 10⁶ PFU of MuPyV via hind footpads the following day and sacrificed at day 8 or day 30 p.i. (B) Viral load in the spleen at day 8 (top panel) or day 30 (bottom panel) p.i. (C) Number of LT359-specific CD8 T cells in the spleen at day 8 p.i. (top panel) or day 30 p.i. (bottom panel). (D) Percent of LT359-specific CD8 T cells that are KLRG1^hiCD127^lo (black) and KLRG1^loCD127^hi (gray) at day 8 (top panel) or day 30 (bottom panel) p.i. Mean ± SD plotted: no significant differences. N = 12–18 mice from 4–6 independent experiments.</p