Human tonsil CD8 T_FR inhibit T_FH and GC B cell function.

<p>Tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, CD3+CD8-CXCR5+ T_FH, and CD19+CD38+ GC B cells and X4- or R5-spinoculated. Cells were then co-cultured at indicated ratios for 2 days and analyzed. (A) IL-21 production by T_FH with increasing (left to right) number of CD8 T_FR (n = 4). (B) Representative examples from X4- and R5-spinoculations showing IL-21 production by T_FH alone, 1:1 with CD8 T_FR, 1:1 with CD8 T_FR and anti-Tim3 antibody (500 ng/μl; right panels), and 1:1 with CD8 T_FR and an isotype control antibody (500 ng/μl). (C) Results from a total of 6 tonsils (isotype n = 3) as described in B. (D) IgG production in X4-spinoculated cultures with 2.5 μg/mL CpG-B stimulation in CD8 T_FR, T_FH, and B cell co-cultures as measured by ELISA. All co-cultures are 1:1 (n = 7). Statistical significance was determined by non-parametric Wilcoxon matched-pairs tests (B) or one-way ANOVA (Friedman test, C) and is displayed as * = p<0.05, ** = p<0.01 and *** = p<0.001.</p

Most human tonsil follicular homing CD8 T cells are CD8 T_FR.

<p>Disaggregated tonsil cell cultures were mock-spinoculated or spinoculated with X4- or R5-tropic HIV and cultured for 2 days (n = 6). (A) Of the viable CD3+CD8+ population expressing the follicular phenotype CXCR5+CCR7-, the percent CD44^hi (CD8 T_FR) and all other CD3+CD8+ (CD8 conv) in mock- and HIV-spinoculated cultures is shown. (B) The percent CCR7 expression on CD8 T_FR (red) and CD8 conv (blue) compared to an FMO control (black). Graphs depict median and range. Statistical significance was determined by non-parametric one-way ANOVA (Friedman test) and is displayed as * = p<0.05.</p

CD8 T_FR induces T_FH apoptosis via HLA-E.

<p>Human tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, and T_FH, spinoculated with R5-tropic HIV, and cultured for 2 days. (A) Representative flow plots showing the percent of Annexin-V+ T_FH in co-culture at 1:1 ratio with CD8 T_FR or CD8 conv 2 days after R5-spinoculatoin. (B) Results from a total of 6 tonsil for mock-, X4-, and R5-spinoculation (isotype n = 3) in A. Co-cultures were also performed with HLA-E blocking antibody or isotype controls (500 ng/μl). (C) Number of T_FH per microliter on day 0 and day 2 when cultured alone (circle, triangle), 1:1 with CD8 T_FR (square), or 1:1 with conventional CD8 T cells (upside down triangle) (n = 6). Statistical significance was determined by Wilcoxon matched-pairs tests and is displayed as * = p<0.05 and ** = p<0.01.</p

CD8 T_FR suppress IL-21 production in T_FH via Tim-3 and induce apoptosis via HLA-E in SIV-infected rhesus macaques.

<p>Disaggregated cells from lymphoid tissues of SIV-infected rhesus macaques (n = 2) were sorted for T_FH and CD8 T_FR and co-cultured at a 1:1 ratio for 2 days with or without HLA-E blocking antibody and analyzed by flow cytometry. (A) Flow gating showing the percent T_FH producing IL-21, and (B) percent T_FH expressing Annexin-V (n = 2).</p

Most rhesus macaque CXCR5+CCR7- CD8 T cells are CD8 T_FR.

<p>Disaggregated cells from lymph node and spleen of SIV-uninfected (n = 6) and SIV-infected (n = 6) rhesus macaques were analyzed by flow cytometry. (A) Percentages of CD8 T_FR and CD8 conv from all CD3+CD8+CXCR5+CCR7- are shown for SIV-uninfected and SIV-infected animals. (B) The percent CCR7+ cells in CD8 T_FR and CD8 conv cell populations(C) Representative images of rhesus macaque spleen tissue staining. CD20 staining was performed to determine follicular (F) and extrafollicular (EF) regions. (D) The percentage of CXCR5+ CD8 T cells in follicular and extrafollicular regions as determined from images similar to C. Left plot (white bars) show uninfected rhesus macaques, right plot (black bars) show chronically infected rhesus macaques. LN = lymph node, Mes = mesenteric, Ing = inguinal, Ax = axial, and Sp = spleen. Graphs depict median and range. Statistical significance was determined by non-parametric one-way ANOVA (Friedman test) and is displayed as *** = p<0.001.</p