Early T Follicular Helper Cell Responses and Germinal Center Reactions Are Associated with Viremia Control in Immunized Rhesus Macaques

Abstract

T follicular helper (T_FH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident T_FH cells have been fully characterized in human immunodeficiency virus (HIV) infection. However, the role of GC T_FH cells in GC B cell responses following various simian immunodeficiency virus (SIV) vaccine regimens in rhesus macaques (RMs) has not been fully investigated. We characterized GC T_FH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIV_M766Gag/Pro/gp120-TM and SIV_M766&CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/SIVGag/rhesus interleukin 12 (IL-12) plus SIV_M766&CG7V gD-gp120 proteins formulated in alum phosphate (DNA&Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC T_FH and GC B cell dynamics including correlation analyses supported a significant role for early GC T_FH cells in providing B cell help during initial phases of GC formation. GC T_FH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia postinfection. Our results highlight the importance of early GC T_FH cell responses for robust GC maturation and generation of long-lasting SIV-specific humoral responses at mucosal and systemic sites. Further investigation of GC T_FH cell dynamics should facilitate development of an efficacious HIV vaccine.IMPORTANCE The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (T_FH) cells are CD4⁺ T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of T_FH cells in eliciting prolonged virus-specific humoral responses, we vaccinated rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV, mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced T_FH responses elicited at day 3 postimmunization and robust GC maturation were strongly associated. Further, early T_FH-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC T_FH responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.

Keywords: T follicular helper cell; germinal center; rhesus macaque; simian immunodeficiency virus; vaccine.

FIG 1

Schematic representation of immunization protocol. Fifty rhesus macaques were primed at weeks 0 and 12 with (Ad5hr)-SIV_M766gp120-TM and Ad5hr-SIV₂₃₉Gag. Immunized macaques were divided into two groups and boosted at weeks 24 and 36 with ALVAC/Env (ALVAC-SIV_M766Gag/Pro and SIV_M766&CG7V gD-gp120 proteins formulated in alum hydroxide) or DNA&Env (DNA with SIVenv/SIVGag/rhesus IL-12 plus SIV_M766&CG7V gD-gp120 proteins formulated in alum phosphate). The control group (n = 10) received empty Ad5hr vector at priming and adjuvant only at boosting. At week 42, weekly repeated low-dose SIV_mac251 challenges of all animals were initiated. Inguinal LNs were sampled 4 weeks prior to the first immunization. Three groups of animals had LN biopsy specimens collected, respectively, at days 3, 7, and 14 after the second prime and after the second boost. IN, intranasal; O, oral; IT, intratracheal; IM, intramuscular; IR, intrarectal.

FIG 2

Phenotypic and functional characterization of GC-resident T follicular helper (T_FH) cells in immunized rhesus macaques. (A) GC T_FH cells were defined as CCR7⁻ CXCR5⁺ PD-1^hi (red gate), gated on the CD4⁺ CD3⁺ T cell population. CCR7⁻ CXCR5⁺ PD-1^low/int cells (blue gate) were classified as non-GC T_FH cells. (B) IL-21⁺ Env-specific GC T_FH cells were identified after stimulation with Env pooled peptides. Unstimulated cells were used for gate definition of stimulated cells. PMA-ionomycin stimulation was performed as a positive control for cytokine release.

FIG 3

Gating strategy for identification of GC B cells, centroblasts (CBs), centrocytes (CCs), and GC Env-specific memory (ESM) B cells. (A) The follicular B cell population was defined as CD20⁺. GC B cells were defined as CD20⁺ IgD⁻ BCL6⁺. CBs were gated on GC B cells and defined as CD20⁺ BCL-6⁺ Ki67^hi; CCs were defined as CD20⁺ BCL-6⁺ Ki67^low/int/neg. (B) GC ESM B cells were gated on the GC B cell population and identified as CD27⁺ gp120⁺. The percentage of the GC ESM B cells depicted is based on frequency of the parent population (CD27⁺ B cells). Positive staining thresholds were set with fluorescence minus one (FMO) controls.

FIG 4

Evaluation of GC-resident T_FH and GC-resident B cells in LNs of vaccinated rhesus macaques over the course of immunization. (A) Analysis of GC resident T_FH (CXCR5⁺ PD-1^hi CD4⁺ T cells) cells over the course of immunization. Because LN biopsy specimens were collected from three different groups of macaques, respectively, at day 3, 7, and 14 after each immunization, percentages of GC T_FH cells were normalized to preimmunization frequencies and levels are reported as magnitude of response. Comparisons were performed by the Mann-Whitney test. (B) Comparison of GC T_FH cell nonnormalized percentages of paired samples from macaques with LNs collected at day 3 following immunization using the Wilcoxon signed-rank test. (C) Analysis of the magnitude of response of GC B cells. GC B cell responses were normalized to preimmunization frequencies and comparisons were assessed by the Mann-Whitney test. (D) Comparison of GC B cell nonnormalized percentages of paired samples from macaques with LN collected at day 3 following immunizations using the Wilcoxon signed-rank Test. T cell and B cell levels are shown as means and SEMs. #, P = 0.039 compared to the preimmunization value; Θ, P = 0.073 compared to the preimmunization value.

FIG 5

Evaluation of GC B cell subsets in LNs of rhesus macaques over the course of immunization. (A) Analysis of CCs (defined as CD20⁺ BCL-6⁺ Ki67^low/Int/neg) over the course of immunization. Percentages of cellular populations were normalized to preimmunization frequencies as described in the legend to Fig. 1. Nonnormalized frequencies of CCs were used for paired comparisons at day 3 (B), day 7 (C), and day 14 (D) following each immunization using the Wilcoxon signed-rank test. (E) SIV_M766 Env-specific memory (ESM) B cells were normalized to preimmunization frequencies, and comparisons were performed by the Mann-Whitney test. B cell levels are shown as means and SEMs. #, P values of 0.035, 0.018, and 0.027 at weeks 13, 37, and 38, respectively, compared to the preimmunization levels.

FIG 6

Correlations between T_FH cells and GC B cells. (A to D) GC T_FH and GC B cell correlations assessed 4 weeks prior to 1st prime immunization in macaques with LNs collected 3 days postimmunizations (A), day 3 following the 2nd prime (B), 2 weeks following 2nd prime (week 14) (C), and day 3 following the 2nd boost (D). (E) Correlation between non-GC T_FH cells and GC B cells at day 3 following the 2nd Ad-SIV immunization. The Spearman rank correlation test was used to assess immunological correlates.

FIG 7

Associations of GC T follicular regulatory (T_FR) cells and GC T_FH cells. (A) Representative gating strategy of GC resident T_FR cells, defined as PD-1^hi CXCR5⁺ CD25⁺ FoxP3⁺ CD4⁺ T cells. (B) Correlation between GC T_FH cells and GC B cells at week 13 in macaques with LNs collected 7 days postimmunization. (C) Correlation between GC T_FR cells and GC T_FH cells at week 13. (D) Correlation trend between GC T_FR and GC B cells at week 13. (E) Correlation between GC T_FR cells and CBs at week 13. Control samples (recipients of empty Ad vector) added to obtain statistical power are represented by squares. The Spearman rank correlation test was used to assess immunological correlates.

FIG 8

Correlation analyses of GC T_FH cells and GC B cell subpopulations. Correlations between GC T_FH cells and CBs 4 weeks prior to 1st prime immunization in macaques with LNs collected 3 days postimmunization (A), 2 weeks following 2nd prime (week 14) (B), and 2 weeks following the second boost (week 38) (C). (D and E) Correlation analyses of GC T_FH cells and GC SIV ESM B cells at day 3 following the 2nd prime (D) and 2 weeks following the 2nd boost (week 38) (E). (F and G) Correlations between CCs and ESM B cells 2 weeks following the 2nd prime (week 14) (F) and 2 weeks following 2nd boost (week 38) (G). The Spearman rank correlation test was used to assess immunological correlates.

FIG 9

Analysis of ESM B cells in various tissues. (A and B) Frequency of SIV_M766 gp120 ESM B cells in PBMCs (A) and rectal mucosa (B) over the course of immunization in all immunized animals. (C) Correlation between GC ESM B cells at day 3 after the 2nd Ad5hr-SIV prime and ESM B cells in the rectal mucosa at week 14 after the Ad5hr-SIV prime. (D) Levels of IgG-positive SIV_M766 ESM B cells before and 2 weeks after the 2nd prime and 2nd boost immunizations as assessed by ELISpot assay. Data are means and SEMs.

FIG 10

Immunological correlates of acute viremia control. (A) Peak viral loads (VL) in ALVAC/Env- and DNA&Env-immunized groups and controls post-SIV infection. Bars indicate geometric means with 95% confidence limits (CL). (B and C) Correlation analyses between GC ESM B cells at day 3 following the 2nd boost and peak viral loads (B) and (C) median peak viremia (weeks 1 to 8 postinfection). (D) Magnitudes of GC ESM B cell responses were compared between ALVAC/Env- and DNA&Env-immunized groups post-boost immunization. Bars represent median values.