Figure 4. SVY antigen in B.breve bacteria stimulates an immunogenic T cell response.

(A) CD8⁺ T cells were isolated and expanded from Jackson and Taconic splenocytes with K^bSVY aAPCS. On day 7, cells were harvested and stained for antigen specificity. Data represent 1 of 5 independent experiments. (B) CD8⁺ T cells were harvested and expanded as in A. Data show percentage of K^bSVY⁺ CD8⁺ T cells. P value = 0.0025, measured by a 1-tailed, paired t test. N = 5. (C) Total cell numbers for CD8⁺ T cell enrichment and expansion with K^bSVY aAPCs. P value = 0.0214, measured by 1-tailed, paired t test. N = 5. (D) Jackson and Taconic mouse CD8⁺ T cells stimulated as in A. On day 7, cells were harvested and stained with a titration of K^bSVY. Results are reported as percentage of peptide-MHC positivity relative to that observed at 10 μg pMHC. Jackson (blue) and Taconic (red) peptide stains were compared at a given concentration. P value = 0.0071, measured by 2-way ANOVA and Bonferroni’s post hoc test for multiple comparisons. N = 3. (E and F) Taconic mice were immunized by intraperitoneal injection of αCD40 on day –8 and subcutaneous injection of K^bSVY on day –7. (E) On day 0, control or immunized mice were injected intravenously with target and nontarget autologous splenocytes mixed at a 1:1 ratio. Spleens were harvested from mice on day 1, and the ratio of target to nontarget cells was analyzed. P < 0.0001 by 2-tailed, unpaired t test. N = 3 mice/group. (F) Taconic mice were sacrificed on day 1. Spleens and draining lymph nodes were harvested and stained for K^bSVY specificity. P = 0.019 by 2-tailed, unpaired t test. N = 6 immunized, 3 not immunized. (G) Jackson and Taconic mice were cohoused for 7 days or separately housed with littermates. CD8⁺ T cells from splenocytes stimulated as in A and harvested and stained for antigen specificity on day 7. Antigen specificity measured by subtracting noncognate antigen reactivity. P value = 0.0014 by 1-tailed t test. N = 5 Taconic only; N = 8 cohoused animals. (H) Jackson and Taconic mice were either cohoused for 7 days or housed with their respective littermates. CD8⁺ T cells were isolated and enriched and expanded with K^bOVA aAPCS and harvested and stained for antigen specificity on day 7. Antigen specificity was measured by subtracting the noncognate antigen specificity. No statistical difference seen by 1-tailed, unpaired t test. N = 4. All cells gated on live CD8⁺ cells. All data represent mean ± SEM.