TFH Cells Induced by Vaccination and Following SIV Challenge Support Env-Specific Humoral Immunity in the Rectal-Genital Tract and Circulation of Female Rhesus Macaques

Abstract

T follicular helper (T_FH) cells are pivotal in lymph node (LN) germinal center (GC) B cell affinity maturation. Circulating CXCR5⁺ CD4⁺ T (cT_FH) cells have supported memory B cell activation and broadly neutralizing antibodies in HIV controllers. We investigated the contribution of LN SIV-specific T_FH and cT_FH cells to Env-specific humoral immunity in female rhesus macaques following a mucosal Ad5hr-SIV recombinant priming and SIV gp120 intramuscular boosting vaccine regimen and following SIV vaginal challenge. T_FH and B cells were characterized by flow cytometry. B cell help was evaluated in T_FH-B cell co-cultures and by real-time PCR. Vaccination induced Env-specific T_FH and Env-specific memory (ESM) B cells in LNs. LN Env-specific T_FH cells post-priming and GC ESM B cells post-boosting correlated with rectal Env-specific IgA titers, and GC B cells at the same timepoints correlated with vaginal Env-specific IgG titers. Vaccination expanded cT_FH cell responses, including CD25⁺ Env-specific cT_FH cells that correlated negatively with vaginal Env-specific IgG titers but positively with rectal Env-specific IgA titers. Although cT_FH cells post-2^nd boost positively correlated with viral-loads following SIV challenge, cT_FH cells of SIV-infected and protected macaques supported maturation of circulating B cells into plasma cells and IgA release in co-culture. Additionally, cT_FH cells of naïve macaques promoted upregulation of genes associated with B cell proliferation, BCR engagement, plasma cell maturation, and antibody production, highlighting the role of cT_FH cells in blood B cell maturation. Vaccine-induced LN T_FH and GC B cells supported anti-viral mucosal immunity while cT_FH cells provided B cell help in the periphery during immunization and after SIV challenge. Induction of T_FH responses in blood and secondary lymphoid organs is likely desirable for protective efficacy of HIV vaccines.

Keywords: B cell help; SIV vaccine; TFH cells; humoral immunity; rhesus macaque.

Figure 1

Vaccine-induced T_FH cells with GC phenotype and Env-specific T_FH cells in LNs. (A) Gating strategy used to identify LN and GC-T_FH cells in LN of rhesus macaques. (B) BCL6⁺ was used to identify T_FH cells with GC phenotype. (C) The AIM assay was used to identify LN and GC Env-specific T_FH cells as described in Methods. Dynamics of (D) total LN T_FH cells and (E) GC-T_FH cells assessed at pre, 3 days and 2 weeks post-2^nd prime (week 13 + 3 days and week 15), and 3 days and 2 weeks post-2^nd boost (week 38 + 3 days and week 40) and represented as magnitude of response. (F) Comparison of the frequencies of BCL6 expressing total LN and GC-T_FH cells at pre and 3 days following 2^nd prime and 2^nd boost immunizations. Dynamics of (G) BCL6⁺ GC-T_FH cells, (H) Dynamics of Env-specific LN T_FH cells, and (I) Env-specific GC-T_FH cells over the course of immunization. Statistics were generated using the Wilcoxon signed rank test for animals within the same group or the Mann-Whitney U test for animals from different groups (D–I). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Figure 2

Vaccination induced GC B cell populations indicate efficient GC maturation. (A) Total B cells in LN were identified as CD20⁺ (green gate); GC B cells as BCL6⁺ B cells (blue gate) and GC memory B cells as IgD^- CD27⁺ GC B cells (red gate). (B) Centrocytes (CC) and Centroblasts (CB) were gated on GC B cells and defined respectively as Ki67^{neg or dim} and Ki67^hi. Env-specific B cells were identified as gp120⁺ and gated either on total CD20⁺ B cells or GC memory B cells. Dynamics of (C) GC B cells, (D) centroblasts, and (E) centrocytes assessed at pre, 3 days and 2 weeks post-2^nd prime (week 13 + 3 days and week 15), and 3 days and 2 weeks post-2^nd boost (week 38 + 3 days and week 40) and represented as magnitude of response. Negative correlations were seen between (F) Env-specific LN T_FH and (G) Env-specific GC T_FH cells and GC B cells at week 38 + 3 days. (H) Magnitude of GC Env-specific memory B cells over the course of vaccination. Paired analysis of non-normalized GC Env-specific memory B cell frequencies were performed on samples collected at (I) pre- and day 3 following immunizations and (J) pre- and 2 weeks following immunizations. (K) Magnitude of total Env-specific B cell response over the course of vaccination. (L) Paired analysis of non-normalized total Env-specific B cell frequencies within animals that had LNs collected 2 weeks post-immunizations. Statistics were generated using the Wilcoxon signed rank test for animals from the same group (I, J, L) or the Mann-Whitney U test for animals from different groups (C–E, H, K). Correlation analyses were performed using Spearman correlation (F–G, M). * indicates P < 0.05, ** indicates p < 0.01.

Figure 3

LN cellular subsets rapidly induced by immunization correlate with SIV-specific antibody responses at mucosal sites. Correlation between total GC B cells elicited at (A) day 3 post-2^nd adeno and vaginal Env-specific IgG responses at week 16 and (B) day 3 post-2^nd boost and vaginal Env-specific IgG responses at week 41. No correlation was seen between GC B cells elicited at (C) 2 weeks post-adeno and vaginal Env-specific IgG titers at week 16 or (D) GC B cells elicited at 2 weeks post-boost and vaginal Env-specific IgG titers at week 41. (E) LN Env-specific T_FH cells induced at day 3 post-2^nd adeno strongly correlated with Env-specific IgA titers in rectal secretions at week 16. Rectal Env-specific IgA antibody titers at week 41 positively correlated with B cell populations associated with GC maturation including (F) centrocytes and (G) GC Env-specific memory B cells. Correlation analyses were performed using Spearman correlations.

Figure 4

Vaccine induced cT_FH cells and Env-specific cT_FH cells in peripheral blood of immunized rhesus macaques. (A) gating strategy for identification of cT_FH (CXCR5⁺ CD4⁺ T cells) and PD-1⁺ cT_FH cells. Dynamics of (B) cT_FH cells and (C) PD-1⁺ cT_FH cells over the course of immunization represented as magnitude of response. (D) Identification of Env-specific cT_FH cells by flow cytometry using the activation-induced marker CD25. Circulating T_FH cells were either stimulated with Env-pooled peptides or remained unstimulated. (E) Dynamics of CD25⁺ Env-specific cT_FH cells over the course of immunization represented as magnitude of response and (F) paired analysis within animals that had blood collected at pre-vaccination, week 15 and week 41. (G) Dynamics of CD25⁺ PD-1⁺ Env-specific cT_FH cells over the course of immunization represented as magnitude of response and (H) paired analysis within animals that had blood collected at pre-vaccination, week 15 and week 41. Statistics were generated using the Wilcoxon signed rank test for animals within the same group (B, C, E–H) or the Mann-Whitney U test for animals from different groups (B, C, E, G). * indicates P < 0.05, ** indicates p < 0.01.

Figure 5

Circulating T_FH cell subpopulations positively correlate with GC T_FH cell subpopulations. Correlations between frequencies of (A) PD-1⁺ cT_FH cells elicited at day 3 post-2^nd adeno and total LN T_FH cells induced at same time point, (B) BCl6⁺ GC-T_FH cells induced at week 40, 2 weeks after the last boost, and CCR7⁺ PD-1⁺ cT_FH cells at week 41, 3 weeks after the last boost. Correlation analyses were performed using Spearman correlations.

Figure 6

Circulating T_FH cells support plasma cell phenotype in co-cultured autologous B cells. CXCR5⁺ CD4⁺ cT_FH cells were isolated from blood samples of rhesus macaques obtained at necropsy and co-cultured with autologous enriched B cells. B cells were also co-cultured alone or in the presence of CXCR5^- CD4⁺ enriched T cells (non-cT_FH cells). Samples were included from both SIV-infected and protected animals for general assessment of T_FH/B-cell help capacity with robust statistical power. B cells were analyzed by flow cytometry after 7 days of co-culture. CD38 expression could not be assessed in all T_FH co-cultured B cells while intracellular staining with anti-Ki67 could only be performed in samples with relatively high cell counts. Frequencies of (A) CD138⁺ B cells (n=17), (B) CD138⁺ CD38⁺ B cells (n=13), (C) Ki67⁺ B cells (n=6) and (D) CD27⁺ IgD^- memory B cells (n=17) were compared between cT_FH+B cells, non-cT_FH+B cells and B cells alone conditions. cT_FH-dependent (E) IgA and (F) IgG responses were assessed in supernatants of the three different co-cultures by ELISA. Squares: protected animals; circles: chronically infected animals; solid symbols: vaccinated animals; open symbols: non-vaccinated animals; X: naïve animals. Statistical analysis was performed using the Wilcoxon Sign-rank test in all panels. * indicates P < 0.05, ** indicates p < 0.01, *** indicates < 0.001.

Figure 7

Relationship between circulating T_FH cell subpopulations prior to SIV challenge and plasma viral loads. Total cT_FH cells at week 41 were positively correlated with geometric means of (A) acute (weeks 1–6 post-infection) and (B) chronic (weeks 8–32 post-infection) viral loads. PD-1⁺ cT_FH cells at week 41 positively correlated with geometric means of (C) acute and (D) chronic viral loads. Correlation analyses were performed using Spearman correlations.

Figure 8

B-cell help capacity of cT_FH cells from chronically infected and uninfected rhesus macaques. Phenotypic comparison of B cells co-cultured with cT_FH cells (CXCR5⁺ CD4⁺ T cells), non-cT_FH cells (CXCR5^- CD4⁺ T cells) or cultured alone between chronically infected animals and protected animals. CD38 expression could not be assessed in all T_FH co-cultured B cells. B cell frequencies were assessed for (A) CD138⁺ B cells (n=17, 11 infected and 6 protected), (B) CD138⁺ CD38⁺ B cells (n=13, seven infected and six protected) and (C) CD27⁺ IgD^- memory B cells (n=17, 11 infected and six protected). (D) IgA release in supernatants of different co-cultures between infected and uninfected macaques. Squares: protected animals; circles: chronically infected animals; solid symbols: vaccinated animals; open symbols: non-vaccinated animals; X: naïve animals. Analyses comparing different groups of macaques used the Mann Whitney U test (A–D). Analyses within the same group of macaques used the Wilcoxon Sign-rank test (A–D). * indicates P < 0.05, ** indicates p < 0.01, *** indicates < 0.001.