Vaccine-induction of LN-resident SIV Env-specific T_FH cells in rhesus macaques

Abstract

Measurement of antigen-specific T follicular helper (T_FH) cell activity in rhesus macaques has not previously been reported. Given that rhesus macaques are the animal model of choice for evaluating protective efficacy of HIV/SIV vaccine candidates and that T_FH cells play a pivotal role in aiding B cell maturation, quantifying vaccine-induction of HIV/SIV-specific T_FH cells would greatly benefit vaccine-development. Here we quantified SIV Env-specific IL-21-producing T_FH cells for the first time in a non-human primate vaccine study. Macaques were primed twice mucosally with Adenovirus 5 host range mutant (Ad5hr) recombinants encoding SIV Env, Rev, Gag and Nef followed by two intramuscular boosts with monomeric SIV gp120 or oligomeric SIV gp140 proteins. Two weeks after the second protein boost we obtained lymph node biopsies and quantified the frequency of total and SIV Env-specific IL-21⁺ T_FH cells and total germinal center (GC) B cells, the size and number of GCs, and the frequency of SIV-specific antibody secreting cells in B cell zones. Multiple correlation analyses established the importance of T_FH for development of B cell responses in systemic and mucosally localized compartments including blood, bone marrow, and rectum. Our results suggest that the SIV-specific T_FH cells, initially induced by replicating Ad-recombinant priming, are long-lived. The multiple correlations of SIV Env-specific T_FH cells with systemic and mucosal SIV-specific B cell responses indicate that this cell population should be further investigated in HIV vaccine development as a novel correlate of immunity.

INTRODUCTION

Despite the fact that protective immunity involves the coordinated work of humoral and cellular mechanisms, most functional vaccines available today prevent pathogen acquisition through the induction of antibodies (1, 2). During HIV infection a small fraction of individuals produce broadly neutralizing antibodies (bNAbs), which possess potent cross-clade neutralizing activity, widely considered a necessary component of a protective HIV vaccine (3, 4). A common characteristic of bNAbs is their high degree of somatic hypermutation (5), which typically results from extensive affinity maturation and antigen-specific interaction with T follicular helper (T_FH) cells within the germinal centers (GC) of secondary lymphoid organs (6, 7).

T_FH cells are a highly specialized CD4⁺ T cell subset that provides help to B cells by contact-dependent and independent mechanisms. Phenotypically, human CD4⁺ T_FH cells are characterized by expression of CXCR5, PD-1, CD95, ICOS, and the transcription factor Bcl-6, which mediates their lineage development (8, 9). Although T_FH cells can arise from multiple precursor T helper cell lineages (10-13), their generation is strongly dependent on IL-21, IL-6 and Bcl-6 (14, 15). Localized within immune-protected B cell follicular areas of secondary lymphoid organs, T_FH cells have been identified as the major CD4⁺ T cell compartment for HIV and SIV persistence during chronic infection even under elite controlling conditions (16-20). Nonetheless, T_FH cells increase in both HIV (21, 22) and SIV (23, 24) infection in association with GC expansion (25). Indeed, T_FH dynamics display multiple adverse effects attributed to infection (25).

Rhesus macaques are the animal model of choice for evaluating pre-clinical HIV/SIV vaccine candidates (26). Although several studies have phenotypically and functionally characterized the total population of macaque T_FH cells in naïve and SIV-infected animals (23, 27-31), quantification of vaccine-induced SIV-specific IL-21-producing macaque T_FH cells has not yet been reported. In order to better understand the development of humoral immune responses and the contribution of T_FH to protective efficacy, in the present study we have identified and quantified SIV-specific LN-resident IL-21⁺ T_FH cells for the first time in a pre-clinical non-human primate vaccine trial. Rhesus macaques were initially vaccinated with mucosally-delivered replicating Adenovirus type 5 host-range mutant (Ad5hr)-recombinants expressing SIV Env, Rev, Gag and Nef proteins followed by intramuscular boosting with either monomeric SIV gp120 or oligomeric SIV gp140 proteins as detailed in a previous study (32). At the end of the vaccination regimen LNs were collected and stored. We measured the frequency of SIV-specific IL-21-producing T_FH cells in the LNs together with GC B cells. The results correlated with multiple systemic and mucosal humoral immune responses. Subsequently we analyzed the data with regard to the challenge outcome of the vaccine study, which showed a sex bias in protective efficacy. Namely, the vaccinated female but not male macaques exhibited delayed SIV acquisition associated with vaccine-induced mucosal B cell responses (32). Here we report that the vaccine regimen elicited SIV-specific T_FH cells, critically important for development of B cell immunity, and initially induced by the replicating Ad5hr-SIV-recombinant priming immunizations. Furthermore, elevated T_FH levels were observed in vaccinated females compared to males. Together with correlations obtained in females between T_FH cells and some B cell responses, our data support continued investigation of a potential contribution of T_FH cells to sex-based differences in vaccine-induced immune responses.

MATERIALS AND METHODS

Animals, immunization regimen and sample collection

The rhesus macaques used in this study were housed and cared for at Advanced Bioscience Laboratories, Inc. (Rockville, MD) and at Bioqual, Inc. (Rockville, MD) under the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care and according to the recommendations of the Guide for the Care and Use of Laboratory Animals. Prior to initiation, all protocols and procedures were approved by the Institutional Animal Care and Use Committee of the respective facility. Initially, to develop a protocol for identification of LN-resident T_FH cells (Fig. 1), viably frozen LN cells obtained from inguinal LN biopsies from naïve (n=10) and chronically SIV-infected (n=10) Indian rhesus macaques (33) were used. Subsequently, vaccine-induced SIV-specific T_FH cell function was evaluated in LN biopsies obtained from sixty Indian rhesus macaques vaccinated as previously reported (32) and outlined in Table 1. Antibody and B cell responses used in correlation analyses were also previously reported in the same publication (32). Inguinal LN biopsies were collected from the vaccinated macaques prior to immunization and at week 53 (2 weeks following the second Env protein boost), minced and passed through a 40-μm cell strainer before contaminating RBCs were lysed. Cells were washed and resuspended in R10 medium (RPMI 1640 containing 10% FBS, 2mM L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate and antibiotics). Cells were used fresh for the determination of total and SIV-specific T_FH cell populations and remaining cells were viably frozen (FBS + 10% DMSO) for later GC B cell assays. Additional LN biopsies from a subset of macaques (12 from gp120-immunized, 12 from gp140-immunized and 6 from control macaques) were fixed in SafeFix II (Thermo Fisher Scientific, Waltham, MA) and embedded in paraffin for later sectioning and analysis.

Fig. 1. Phenotypic characterization of macaque IL-21-producing T_FH cells.

Inguinal lymph nodes (LN) were collected, processed into single-cell suspensions, and used fresh for flow cytometry phenotypic analysis. (A) Gating strategy used to define LN-resident T_FH cells as live CD3⁺CD4⁺CCR7⁻ICOS⁺PD-1^hi in naïve (top) and SIV-infected (bottom) rhesus macaques. (B) Percentage of T_FH cells in naïve and SIV-infected macaques. (C) IL-21 production by T_FH cells in response to PMA/Ionomycin treatment. (D) Percent of IL-21-producing T_FH cells in naïve and SIV-infected macaques. (E) CTLA-4, CD40L and CD95 expression in IL-21⁺ T_FH cells (red histograms) compared to non-T_FH cells (naïve CD4⁺ T cells, blue histograms). Data reported are means ± SEM. ***, p<0.001 indicates statistically significant differences between the compared groups by the Mann-Whitney test.

Table 1.

Summary of immunization regimen of macaques used in this study.

Immunization group	Week 0	Week 12	Week 39	Week 51
gp120 group (n = 24)	Ad5hr-SIV recombinants* (IN + O)	Ad5hr-SIV recombinants (IT)	Monomeric SIV gp120 in MF59 (IM)	Monomeric SIV gp120 in MF59 (IM)
gp140 group (n = 24)	Ad5hr-SIV recombinants (IN + O)	Ad5hr-SIV recombinants (IT)	Oligomeric SIV gp140 in MF59 (IM)	Oligomeric SIV gp140 in MF59 (IM)
Controls (n = 12)	Ad5hr empty vector (IN + O)	Ad5hr empty vector (IT)	MF59 only (IM)	MF59 only (IM)

^*Three Ad5hr-SIV recombinants separately encoding SIV_smH4 env(gp140)/rev; SIV_mac239 gag; and SIV_mac239 nef_Δ1-13 were administered by the indicated routes at 5× 10⁸ pfu/recombinant/route. The control macaques received a total dose of 1.5 × 10⁹ pfu/route of the empty Ad vector. SIV_mac239 Env proteins were given at a dose of 100 ug. IN = intranasal; O = oral; IT = intratracheal; IM = intramuscular.

Detection of GCs and T_FH cells in fixed LNs

GCs and GC-resident T_FH cells were detected using immunofluorescence staining. Briefly, 6-μm sections were cut and adhered to silanized slides and stained with the following antibodies: mouse anti-human CD4 (1F6, Leica Microsystems Inc., Buffalo Grove, IL), goat anti-human PD-1 (AF1086, R&D Systems, Minneapolis, MN), or goat anti-human CD20 (MS4A1, Origene, Rockville, MD) and rabbit anti-human Ki67 (SP6, ThermoFisher Scientific). Sections on slides were pretreated in 1 mM EDTA (pH 8.0) in a Presto pressure cooker (National Presto Industries, Eau Claire, WI) at 121°C for 35 seconds to unmask antigens. Sections were blocked with normal horse serum, and incubated with primary antibodies overnight at 4 °C. After washing in PBS, the slides were incubated at room temperature for 2 h with donkey AlexaFluor 647 anti-mouse IgG, AlexaFluor 488 donkey anti-goat IgG and AlexaFluor 594 donkey anti-rabbit IgG and counterstained with DAPI (4',6-diamidino-2-phenylindole), all from Life Technologies (Carlsbad, CA). After washing in PBS, the slides were coverslipped and examined using a Nikon A1 confocal microscope system (Nikon, Melville, NY). GCs were enumerated and size quantified using ScanScope slide scanner and ImageScope software (Aperio-Leica Biosystems, Buffalo Grove, IL).

Quantification of GC-resident T_FH cells in fixed LNs

Expression levels were extracted from confocal images by first using the interactive segmentation software ilastik (v. 1.1.5) to separate cells from the background based on features defined by color/intensity, edges, and texture in all image channels (34). Although the ilastik segmentation efficiently delineated cells from the image background, groups of cells were not adequately separated from each other. Therefore, the segmentation mask, along with the raw confocal images was imported into custom software written in MATLAB (R2015a, Mathworks, Natick, MA), with use of the image processing toolbox. The custom software performed morphological erosion on the segmentation mask to isolate individual cells, which were then each given a unique identification number, and then returned to their original size by use of morphological dilation. Fluorescence levels in each confocal channel were then calculated for the individual cells by summing the pixel intensities within each cell boundary, and dividing by the number of pixels.

Detection of SIV-specific B cells in fixed LNs by inverse immunohistochemistry

SIV-specific B cells were detected in safe fixed tissues using biotin conjugated SIV gp120 or gp140 and inverse immunohistochemical staining. Briefly, sections on slides were pretreated in 10 mM sodium citrate (pH 6.0) in a waterbath (National Presto Industries, Eau Claire, WI) at 98°C for 15 minutes to unmask antigens. Endogenous biotin was blocked using Dako Biotin Blocking System (Dako, Carpinteria, CA). Biotin-SIV gp120 or gp140 was added to sections and allowed to incubate overnight at 4°C. Sections were washed with TBS (pH 8.0), and allowed to incubate with streptavidin-HRP for 1 hr at room temperature and washed with TBS (pH 8.0). Signal was amplified using a Tyramide (TSA) Biotin amplification system following the manufacturer’s instructions (Perkin Elmer, Waltham, MA).

Flow cytometric detection of T_FH cells

Anti-human fluorochrome-conjugated mAbs known to cross-react with rhesus macaque antigens were used in this study, including PE anti-IL-21 (3A3-N2.1), PE-CF594 anti-CD95 (DX2), PE-Cy5 anti-CD154 (TRAP1), APC anti-CD152 (BNI3) and Alexa Fluor 700 anti-CD3 (SP34-2) (all from BD Biosciences, San Jose, CA); PerCP-eFluor710 anti-CD279 (eBioJ105) and APC-eFluor780 anti-CD197 (3D12) (all from eBioscience, San Diego, CA); PE-Cy7 anti-IL-17A (BL168) and Pacific Blue anti-CD278 (C398.4A) (BioLegend, San Diego, CA); and QDot605 anti-CD4 (T4/19Thy5D7) (NIH Nonhuman Primate Reagent Resource, Boston, MA). The Yellow LIVE/DEAD viability dye (Invitrogen, Carlsbad, CA) was used to exclude dead cells. At least 500,000 singlet events were acquired on a SORP LSRII (BD Biosciences and analyzed using FlowJo Software (FlowJo LLC, Ashland, OR). For all samples gating was established using a combination of isotype and fluorescence-minus-one controls.

SIV-specific IL-21-producing T_FH cell detection

SIV-specific production of IL-21 by LN-resident T_FH cells was assayed by stimulating 2 × 10⁶ LN cells with 1 μg/ml SIV_mac239 (NIH AIDS Research and Reference Reagent Program) or SIV_smH4 (Advanced BioScience Laboratories, Inc., Rockville, MD) Env peptide pools (complete sets of 15-mer peptides, overlapping by 11aa) for 6 h. Stimulation was performed in the presence of 2 μg/ml anti-CD49d and anti-CD28 (BD Biosciences), BD Golgi Plug, BD Golgi Stop, and APC-eFluor780 anti-CD197 at the manufacturer’s recommended concentrations. Subsequently, cells were washed and surface stained before fixation and permeabilization with BD Cytofix/Cytoperm and intracellular staining. Stimulation with PMA plus Ionomicyn was used to elicit IL-21 production by T_FH cells.

Detection of GC B cells by flow cytometry

Frozen LN cells were thawed, washed in PBS, and 2x10⁶ cells stained with Aqua LIVE/DEAD viability dye (Invitrogen). Surface staining was performed in PBS/0.5% BSA/2mMEDTA with the following fluorochrome-conjugated Abs known to cross-react with rhesus macaque antigens: Qdot605 anti-CD14 (Tuk4) and anti-CD2 (S5.5) (Invitrogen); eFL650NC anti-CD20 (2H7) (eBioscience); PECy5 anti-CD19 (J3-119) (Beckman Coulter, Brea, CA); and Texas Red goat polyclonal anti-IgD (Southern Biotech, Birmingham, AL). Intra-nuclear staining for Alexa Fluor 700 anti-Ki67 (B56) and Alexa Fluor 647 anti-Bcl6 was performed using the Transcription Factor Buffer Set according to the manufacturer’s recommendations (all reagents from BD Biosciences). At least 250,000 singlet events were acquired on a LSRII (BD Biosciences) and analyzed using FlowJo Software (FlowJo LLC, Ashland, OR). Gating was established using a combination of isotype and fluorescence-minus-one controls.

Statistical analysis

Data were analyzed as described in figure legends using Prism (v6.01, GraphPad Software). A p value ≤ 0.05 was considered statistically significant.

RESULTS

Identification of IL-21-producing LN resident T_FH cells and their correlation with vaccination-induced humoral immune responses

At the time of experimentation the only rhesus macaque anti-CXCR5 antibody available was from the NIH Non-human Primate Reagent Resource; however, this antibody does not specifically label rhesus T_FH cells (23, 24). Therefore we used other available phenotypic markers to identify rhesus macaque LN-resident T_FH cells. Initially, LN from naïve and chronically SIV-infected macaques were used to identify the total population of LN-resident T_FH cells as live CD3⁺CD4⁺CCR7⁻ICOS⁺PD-1^hi cells (Fig. 1A), hereafter termed total T_FH cells. These cells represented ~0.5% of the total LN-resident CCR7⁻CD4⁺ T cell pool present in naïve animals and their proportion was increased approximately 3-fold in chronically SIV-infected macaques (p < 0.001; Fig. 1B). Within the LN T_FH cell population from naïve macaques, approximately 15% were capable of producing IL-21 in response to PMA plus ionomycin stimulation (Fig. 1C, D). Similarly, the proportion of IL-21 producing cells was increased approximately 3-fold in chronically SIV-infected macaques when compared to naïve animals (p < 0.001; Fig. 1D). The IL-21 producing T_FH cells were IL-17 negative (data not shown), and the majority expressed CTLA-4, CD40L and CD95, a further indication that the IL-21-producing cells were indeed T_FH cells (Fig. 1E). Given that no rhesus macaque cross-reacting anti-CXCR5 antibody was available at the time of this study, our phenotypic identification of total T_FH cells may have included some CXCR5^−/lo pre-T_FH cells (35).

Following phenotypic and functional identification of macaque T_FH cells in LN of naïve and SIV-infected animals, we proceeded to measure the frequency of these cells after priming with Ad5hr-SIV recombinants and boosting with monomeric gp120 (gp120 Group) or oligomeric gp140 (gp140 Group) SIV Env proteins (Table 1). Inguinal LN biopsies were taken at week 53, two weeks after the second intramuscular protein immunization, and processed to quantify total T_FH cells. No significant increases in the abundance of these cells were seen in the vaccinated groups compared to the controls (Fig. 2A). This is not surprising as the total population of LN-resident T_FH cells includes cells of many different antigenic specificities. Moreover, the controls received empty Ad vector and adjuvant, only differing from the vaccinated groups in the SIV transgenes and Env protein immunogens (Table 1). Nonetheless, the percentage of LN-resident T_FH cells present in all vaccinated macaques had a significant positive correlation with the percentage of SIV Env-specific memory B cells in the bone marrow at week 53 (Fig. 2B), highlighting the role of T_FH cells in driving B cell responses.

Fig. 2. Correlation between humoral responses and T_FH cell abundance in vaccinated rhesus macaques.

(A) Percentage of T_FH cells in the inguinal LN of vaccinated and control macaques at week 53 of the study. Data reported are means ± SEM. (B) Correlation between abundance of total LN T_FH cells at week 53 and SIV Env-specific memory B cells in the bone marrow at week 53 in vaccinated macaques. Spearman’s correlation analysis was used to determine statistical significance. Env-specific memory B cell data are from ref. 32.

Effects of vaccination on GC and T_FH cells

After initial pre-GC interactions, T_FH are known to bind B cells within the GC regions of secondary lymphoid organs and selectively reinforce a program of proliferation and somatic hypermutation. To more directly assess the role of T_FH within GCs, we measured GC (CD20⁺Ki67⁺) areas and quantified the number of GC-resident T_FH cells (CD4⁺PD-1^hi within Ki67⁺ areas) in vaccinated rhesus macaques by immunofluorescence. Although ICOS expression was not used to identify GC-resident T_FH cells by confocal microscopy, the use of CD4 and PD-1 markers in Ki67 rich areas has previously been described as a valid approach to identify LN-resident T_FH cells (23, 24). LN tissue sections were collected at week 53 and stained for Ki67, CD20 and CD4 to enumerate and measure GCs (Fig. 3A), and with CD4, PD-1 and Ki67 to enumerate T_FH cells within GCs (Fig. 3B). No significant differences were observed in the number (Fig. 3C) or area (Fig. 3D) of GCs between vaccinated and control macaques. Additionally, a software-based approach was used to enumerate T_FH cells (CD4⁺PD-1^hi cells) within Ki67 rich GC areas (Fig. 3E). As shown in Fig. 3F, no significant differences were observed in the abundance of GC-resident T_FH cells between vaccinated and control macaques. These results are again consistent with the fact that both control and vaccinated macaques received equivalent doses of the Ad vector and MF59 adjuvant (Table 1) as discussed above.

Fig. 3. Effect of vaccination on GC size and T_FH cell number.

(A-F) LN biopsies from a subset of vaccinated macaques were collected at week 53 and fixed in 10% formalin prior to paraffin embedding. (A-B) Tissue sections were recovered and stained specifically with Ki67, CD20 and CD4 to identify and measure the size of GCs (A), and with CD4, PD-1 and Ki67 to identify and enumerate GC-resident T_FH cells (B). Bars represent 100 and 50μm in A and B, respectively. (C-D) The number of GCs (C) and the average GC area per animal (D) were measured in LN sections as displayed in A with Aperio ImageScope Quantification Software. (E-F) Confocal images of LN sections stained as in B were analyzed as described in Materials and Methods to determine PD-1 and CD4 expression intensity levels on an individual cell basis. Gates (red line) were drawn to enumerate T_FH (PD-1^hiCD4⁺) cells per GC (E) and to compare between vaccinated and control macaques (F). Data reported are means ± SEM

Within GCs, B cells may cycle through multiple rounds of antigen-specific selection, proliferation, and somatic hypermuation before exiting the GC as memory B cells or plasma cells (PC). GC B cells are classified either as centrocytes, which compete for T_FH binding in the light zone or positively-selected, proliferating centroblasts which reside in the dark zone (36, 37). We measured the abundance of total GC-resident B cells in the LN of control and vaccinated macaques. Fig. 4A shows the gating strategy used to identify total GC B cells (IgD⁻CD20⁺Bcl6⁺), as well as dark zone centroblasts (IgD⁻CD20⁺Bcl6⁺Ki67^hi) and light zone centrocytes (IgD⁻CD20⁺Bcl6⁺Ki67^neg/lo) by flow cytometry. The gate for centrocytes includes both Ki67⁻ and Ki67^lo cells because as depicted in Figure 3A, the light zone likely accommodates both newly-arrived, Ki67⁻ cells as well B cells recently returned from the dark zone, expressing low levels of Ki67. As was noted for T_FH cells, no significant increase in the frequency of total GC B cells (Fig. 4B) or in the centroblast (Fig. 4C) and centrocyte (4D) subpopulations was observed in vaccinated macaques compared to controls.

Fig. 4. Effect of vaccination on GC-resident B cell subpopulations.

(A-D) Frozen inguinal lymph node cells from controls and vaccinated macaques were thawed and stained (A) in order to identify the absolute population of IgD⁻CD20⁺Bcl6⁺ GC cells (B), IgD⁻CD20⁺Bcl6⁺Ki67^hi centroblasts (C) and IgD⁻CD20⁺Bcl6⁺Ki67^neg/lo centrocytes (D) by flow cytometry. Data reported are means ± SEM.

Interestingly, as shown in Fig. 5A, we detected a positive correlation in vaccinated macaques between the numbers of GCs after the last immunization (Fig. 3C) and the abundance of total T_FH cells detected by flow cytometry. Similarly, the number of GCs in vaccinated macaques at week 53 also correlated positively with SIVgp140- and SIVgp120-specific binding titers in serum at week 57 (Fig. 5B) and with the Env-specific IgA secreting plasma cells in the bone marrow at week 53 (Fig. 5C). Although vaccination did not induce a significant increase in the number of GCs or in the number of GC-resident T_FH cells compared to controls (Figs. 3C and 3F), we did observe a direct correlation between the number of GC-resident T_FH cells and the number (Fig. 5D) and area (Fig.5E) of GCs in the LN. Moreover, the number of GC-resident T_FH cells as determined by histological analysis significantly correlated with the abundance of total T_FH cells as determined by flow cytometry at week 53 in vaccinated rhesus macaques (Fig. 5F).

Fig. 5. Correlations between GC parameters and GC B cells with humoral immune responses and total T_FH cells.

Correlations in vaccinated macaques between the number of histologically-determined GCs in the LN at week 53 and the abundance of flow cytometry-determined absolute T_FH cells at week 53 (A), SIV-specific gp120 and gp140 serum binding titers at week 57 (B) and SIV Env-specific IgA activity in bone marrow at week 53 (C). Correlations between the number of histologically-determined T_FH cells per GC at week 53 and the number of GCs per LN (D), the average GC area (E), and the abundance of T_FH cells as determined by flow cytometry (F). (G-H) Correlation analyses of the abundance of flow cytometry-determined T_FH cells and the abundance of IgD⁻CD20⁺Bcl6⁺ GC B cells (G) and IgD⁻CD20⁺Bcl6⁺Ki67^hi GC centroblasts (H) at week 53. Note, histologically-determined GC data were obtained on only half of the vaccinated and control macaques as described in Materials and Methods. Spearman’s correlation analysis was used to determine statistical significance. Humoral immune response data are from ref. 32.

Further validating the role T_FH cells play in helping B cell responses, a significant correlation was observed between total T_FH cells and total GC cells (Fig. 5G). Similarly, we observed a significant positive correlation between the abundance of total T_FH cells and centroblast B cells in the LN (Fig. 5H). No correlation with centrocyte B cells was observed (data not shown). The correlation with centroblasts may be a reflection of the direct stimulation that occurs between GC B cells and T_FH cells which results in their movement to the dark zone and subsequent proliferation as centroblasts. In contrast, depending on their affinity and receptor expression level, not all centrocytes will interact productively with T_FH cells. Moreover, centrocytes that have successfully completed their transit through the GC cycle will mature and migrate out of the light zone, again leading to a lack of correlation. The dynamics of GC B cells and the influence of T_FH cells on GC function are areas in need of further study.

Measurement of SIV-specific IL-21-producing T_FH cells

Given that total T_FH cells represent a population of cells with many different antigenic specificities, we sought to functionally identify the proportion of T_FH cells specific for SIV. We initially evaluated LN cells from chronically SIV_mac251-infected animals by stimulating with SIV_mac239 Env peptides. Upon stimulation a small subset of the total LN T_FH cells produced IL-21 (Fig. 6A). To further identify these cells as SIV Env-specific T_FH cells, we confirmed expression of CTLA-4, CD40L and CD95 within the IL-21⁺ T_FH cells (Fig. 6B). To determine the frequency of SIV Env-specific IL-21⁺ T_FH cells in the vaccinated macaques we split the animals into two groups. Cells from one group were stimulated with SIV_smH4 Env peptides, matching the SIV Env encoded in the Ad5hr recombinant, and cells of the other with SIV_mac239 Env peptides, matching the strain of the Env protein boosts. Interestingly, stimulation with SIV_smH4 Env peptides from the Ad prime elicited significant production of IL-21 by Env-specific LN-resident T_FH cells in all vaccinated macaques (Fig. 6C). When macaques were separated based on the boosting immunogen, we observed a marginally non-significant upregulation of SIV-specific IL-21 in gp120-boosted macaques and a significant upregulation of IL-21 production by gp140-immunized macaques (Fig. 6C). It may be that the T_FH cells from the gp140-immunized macaques received additional stimulation from the gp41 peptides present in the peptide pool, whereas the gp120-immunized macaques did not. Stimulation with SIV_mac239 Env peptides did not induce significant IL-21 production by LN-resident T_FH cells in vaccinated macaques (Fig. 6D).

Fig. 6. SIV-specific production of IL-21 by T_FH cells in vaccinated rhesus macaques.

(A) IL-21 production by LN-resident T_FH cells of vaccinated and SIV⁺ macaques in response to SIV envelope peptide stimulation. (B) CTLA-4, CD40L and CD95 expression by IL-21⁺ SIV-specific T_FH cells. SIV_smH4- (C) and SIV_mac239-specific (D) IL-21 production by T_FH cells at week 53 post-vaccination. Vaccinated data in C and D include macaques of both vaccination groups. Data reported are means ± SEM. **, p<0.01 indicates statistically significant differences between the indicated time points by the Wilcoxon signed rank test.

The identification and quantification of SIV Env-specific IL-21⁺ LN-resident T_FH cells of vaccinated macaques subsequently linked the antigen specific process of B cell maturation in GCs with numerous mucosal and systemic humoral immune responses reported previously (32). SIV_smH4 Env-specific IL-21⁺ T_FH cells in all vaccinated animals (boosted either with gp120 or gp140) correlated significantly with the presence of gp140-specific binding antibodies in serum (Fig. 7A) and with the presence of SIV_251.6-specific neutralizing antibodies (Fig. 7B) both at week 53. Moreover, both gp120- and gp140-specfic IgG1 and IgG3 binding titers were significantly correlated with SIV_smH4 Env-specific IL-21⁺ T_FH cells (Fig. 7C,D). Further, SIV_smH4 Env-specific IL-21⁺ T_FH cells correlated significantly with SIV gp140-specific IgA (Fig. 7E) and IgG (Fig. 7F) present in rectal secretions at week 53. Finally, SIV_smH4 Env-specific IL-21⁺ T_FH cells were also positively correlated with the presence of Env-specific IgG memory B cells in the bone marrow at week 57 (Fig. 7G) and with serum ADCC activity against SIVgp120- and SIVgp140-coated target cells (Fig. 7H) at week 53.

Fig. 7. Correlation between SIV-specific humoral responses and SIV-specific T_FH cell activity.

(A-H) Correlations between SIV_smH4-specific IL-21 production by LN-resident T_FH cells at week 53 post-vaccination and SIVgp140-specific week 53 serum binding titers (A), SIV_251.6 neutralizing antibodies in serum at week 53 (B), gp140- and gp120-specific IgG1 (C) and IgG3 (D) end point titers measured by ELISA, SIVgp140-specific IgA (E) and IgG (F) antibodies in rectal secretions at week 53 as measured by ELISA, IgG Env-specific memory B cell activity in bone marrow at week 57 as measured by ELISPOT (G) and gp140- and gp120-specific 50% maximum killing ADCC titers as measured by the RFADCC assay (H). Spearman’s correlation analysis was used to determine statistical significance. Humoral immune response data are from ref. 32.

Evaluation of GC B cells and T_FH cells in vaccinated male and female macaques

We previously reported that vaccinated female but not male macaques exhibited delayed SIV acquisition correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and total rectal plasma cells (32). This sex bias was most evident in the gp120 group of immunized macaques. Therefore, we asked whether there was a sex bias in vaccine-induced generation of GC B cells and LN-resident T_FH cells. No sex differences were observed in the number or area of GC; SIV Env-specific ASC in LN B cell zones; the abundance of total GC B cells, centroblasts or centrocytes; or the number or percentage of T_FH per GC (data not shown). However, analyses of total and SIV_smH4 Env-specific IL-21⁺ LN-resident T_FH cells revealed differences. Although no differences in pre or post-vaccination (wk 53) levels of total T_FH cells were seen when macaques were stratified by sex, at the end of the vaccine regimen (week 53) female macaques exhibited significantly greater proportions of total T_FH cells compared to males (Fig. 8A). Further, following vaccination, females exhibited significantly elevated levels of SIV_smH4 Env-specific IL-21⁺ T_FH cells compared to pre-immunization levels whereas males did not (Fig. 8B). However, it is possible that because the vaccinated macaques were divided for stimulation with either SIV_smH4 or SIV_mac239 Env peptides, the resulting small number of males available for analysis may have contributed to a lack of significance.

Fig. 8. Total and SIV Env-specific T_FH cell responses in female versus male rhesus macaques.

(A) Abundance of total T_FH cells before and after vaccination in male and female rhesus macaques. *, p<0.05 indicates statistically significant differences between the indicated groups by the Mann-Whitney test. (B) Vaccine-induced SIV_smH4 Env-specific IL-21⁺ T_FH cells in male and female rhesus macaques. **, p<0.01 and *, p<0.05 indicate statistically significant differences between the indicated time points by the Wilcoxon signed rank test. Data reported are means ± SEM. (C-D) Correlations between total T_FH cells at week 53 post-vaccination and gp120-specific serum binding titer at week 57 as measured by ELISA (C), and the abundance of rectal plasmablasts 2 weeks post-infection (D) in vaccinated female macaques. Correlation between SIV_smH4-specific IL-21 production by LN-resident T_FH cells at week 53 post-vaccination and gp120-specific 50% maximum killing ADCC titers as measured by the RFADCC assay in vaccinated female macaques. Spearman’s correlation analysis was used to determine statistical significance. Humoral immune response data are from ref. 32.

We subsequently conducted further correlation analyses by sex. No correlations were observed with vaccinated male macaques. As shown, in females we observed correlations between total T_FH cells and gp120 antibody binding titers (Fig. 8C), as well as with rectal plasmablasts 2 weeks post-infection (Fig. 8D). Note, the correlation with rectal plasmablasts remained significant when apparent outliers were removed (2 outliers removed: r = 0.4825, p = 0.0069; 3 outliers removed, r = 0.4350, p = 0.018). Additionally, a significant correlation between SIV_smH4 Env specific IL-21⁺ T_FH cells and ADCC activity against gp120-coated target cells was obtained. Marginally non-significant correlations were seen between SIV_smH4 Env-specific IL-21⁺ T_FH cells and ADCC activity against gp140-coated target cells (p = 0.061) and neutralizing activity against SIV_251.6 (p = 0.054) (data not shown).

Effect of vaccination on SIV-specific B cell responses

Having shown that the proportion of LN T_FH cells is directly correlated with the proportion of GC B cells and centroblasts (Fig. 5G-H) and that SIV-specific IL-21⁺ T_FH cells are directly correlated with numerous humoral immune responses, we next examined SIV Env-specific B cells in LN of vaccinated macaques. Using reverse immunohistochemistry we identified and enumerated SIV Env-specific antibody secreting cells (ASC) in B cell areas of LN obtained at week 53. As shown in Fig. 9A, equivalent numbers of SIV Env-specific ASC were observed in SIVgp120 and SIVgp140 boosted macaques. Similarly, no difference was observed in numbers of SIV Env-specific ASC between male and female macaques (data not shown). We were not able to determine if the number of SIV Env-specific ASC correlated with site-specific SIV Env-specific IL-21⁺ T_FH cells because the values were obtained from different LN by different techniques (reverse immunohistochemistry and flow cytometry, respectively). Interestingly, however, the abundance of LN-resident SIV Env-specific ASCs inversely correlated with the plasma viral load at week 1 post-infection in vaccinated female but not male macaques (Fig. 9B). As previously reported, the vaccinated macaques in this study modestly controlled acute viremia (32), so such transient control of viremia in the first week following SIV acquisition was not unexpected. This acute control was previously correlated with CD8⁺ T cell responses in all vaccinated macaques and vaccinated males but not females (32). We speculated that the result in females reflected a waning of the cellular response during their significant delay in SIV acquisition. Here our result suggests that SIV Env-specific ASC may contribute to control of viremia in females, but that SIV specific antibody may similarly exhibit a waning effect due to acquisition delay.

Fig. 9. Histological identification of SIV-specific B cells in the LN of vaccinated macaques.

LN biopsies from a subset of vaccinated macaques were collected at week 53 and fixed in 10% formalin prior to paraffin embedding. (A) Tissue sections were recovered, processed and stained for inverse-IHC using biotin-SIVgp120 or gp140. Images were captured using a ScanScope Slide scanner and analyzed using ImageScope software. Graph represents the number of SIV-specific antibody secreting cells (ASC) per mm² of LN B cell zone. Data reported are means ± SEM. (B) Correlation between the number of SIV-specific ASC at week 53 and the plasma viral load at week 1 post-infection in female macaques (from ref. 32). Spearman’s correlation analysis was used to determine statistical significance.

DISCUSSION

A complex and unique mixture of signals, factors and cellular processes are involved in the interaction between T_FH cells and B cells within the GC. These contact-dependent and –independent interactions influence B cell survival, proliferation, hypermutation, and immunoglobulin class switching (38). Among these interactions, T_FH-derived IL-21 together with CD40L and IL-4 play pivotal roles in regulating B cell activation and maturation (39). Although total T_FH cell dynamics have been previously studied in rhesus macaque models of SIV/HIV infection, the quantification of SIV-specific T_FH cell activity induced through vaccination has not been previously shown. To examine this question, we took advantage of a pre-clinical vaccine trial (32) in which LN biopsies were obtained prior to vaccination and 2 weeks following the last immunization (wk 53). Although this sampling time point may have missed peak T_FH responses, we were nevertheless able to detect and quantify LN-resident vaccination-induced SIV-specific IL-21⁺ T_FH cells. We found that total T_FH cells correlated with bone marrow Env-specific memory B cells and with GC B cells, in particular, centroblasts. Further, measurements of SIV Env-specific T_FH activity provided correlations with multiple functional systemic and mucosal humoral responses observed at the time of LN biopsy (wk 53) and/or 4 weeks later (wk 57). Thus, vaccine-induced SIV Env-specific IL-21⁺ T_FH cells were shown to be directly associated with the development of a variety of vaccine-elicited B cell responses. Future studies using flow cytometry to evaluate both SIV Env-specific T_FH and Env-specific GC B cells from the same LN should further support this relationship. Overall, the evaluation of antigen-specific T_FH cells can provide insight into vaccine-induced humoral immune responses and further elucidate potential correlates of protective immunity.

Our data suggest that the initial priming immunization used in our vaccination protocol induced a long-lasting SIV-specific T_FH response, persisting at least 53 weeks past the first immunization. Whether this response was due to memory T_FH or persistent antigen expression is unknown. It is not yet clear whether memory T_FH cells are generated within the GC or from non-GC T_FH cells, or whether they remain in LNs after GC resolution or exit to the blood (40, 41). It is conceivable that the long-lasting SIV-specific T_FH response we observed reflects persistent antigen exposure as opposed to memory since the replication-competent Ad5hr-recombinant priming immunogens we used have been shown to persist at least 25 weeks following administration (42). Further studies are warranted to address these questions. Moreover as all data here were obtained at week 53, a more complete picture of the effects of Ad5hr-recombinant priming and overall kinetics of T_FH cell development could be revealed by studying additional, earlier time points. This approach is being pursued in an ongoing pre-clinical vaccine trial. Regardless, it is evident from this study that Ad5hr-SIV recombinant priming induced SIV_smH4 Env-specific IL-21⁺ T_FH cells that contributed to the development of both systemic and mucosal B cell responses.

While antigen-specific T_FH cells are beneficial for antibody induction, few studies have explored strategies to enhance development of this cell population. We found that induction of SIV_mac239 Env-specific IL-21⁺ T_FH cells was not as robust following the boosting immunizations compared to the induction following the Ad5hr-recombinant priming. Yet MF59, the adjuvant used for protein boosting in this pre-clinical vaccine study, has been shown to promote GC B cell differentiation and T_FH induction (43, 44). It may be that the modest elicitation of SIV_mac239 Env-specific compared to the SIV_smH4 Env-specific IL-21⁺ T_FH cells reflects a difference between persistent antigen exposure from Ad5hr-SIVenv recombinant and short-term antigen exposure from the Env protein boosts. If so, this highlights the potential benefits of replicating vaccine vectors. In any case, additional adjuvant formulations that can enhance T_FH development should be explored. Other approaches directed at T_FH cell induction have included nanoparticle vaccines, shown to expand T_FH cell populations and promote GC development, leading to enhanced humoral immunity (45). Vaccine strategies incorporating DNA priming have facilitated T_FH and GC development along with associated antigen-specific antibody responses compared to immunization with protein alone (46). In view of the strong associations of SIV-specific T_FH cells with multiple systemic and mucosal antibody responses seen here, continued exploration of strategies to enhance antigen-specific T_FH development through the modulation of vaccine regimens is warranted.

The LN studied here were obtained from macaques that exhibited a clear sex bias in vaccine-induced protective efficacy (32). This outcome was consistent with the known sex differences in the pathogenesis of viral diseases including HIV infection, where women initially control the disease better than men (47). This sex bias has been associated with differences in immune responses. Females have been reported to have better antigen recognition by pattern recognition receptors, enhanced induction of innate and adaptive immune responses, and elevated production of inflammatory cytokines compared to men. They exhibit both greater antigen-specific humoral responses and higher basal immunoglobulin levels (48). Having identified and quantified vaccine-induced SIV Env-specific T_FH cells, we asked whether there was a similar sex bias in the induction of these cells, and how these cells might relate to the protective immune responses previously correlated with delayed SIV acquisition in vaccinated females. Initially we noted that females had elevated levels of both total T_FH cells and SIV_smH4-Env-specific IL-21⁺ T_FH cells compared to males (Fig. 8A,B). The origin of T_FH cells is not well defined. Rather than arising from pre-existing T_FH cells, they may develop from memory CD4⁺ T cell populations committed to a T_FH lineage (12, 13). Thus the higher level of total T_FH cells we observed in female macaques may not have significantly influenced the subsequent development of viral-specific T_FH cells; rather it may indicate that females have an enhanced mechanism for generating and maintaining this cell population.

With regard to contributions of T_FH cells in females to B cell responses previously associated with delayed acquisition, the correlation of T_FH cells with rectal plasmablasts (Fig. 8D) is of interest as mucosal B cell responses were a clear factor in the observed sex bias (32). This correlation is presumably linked to the Ad5hr-recombinant priming immunizations administered to the upper respiratory tract and gut to target mucosal inductive sites resulting in both SIV Env-specific IL-21⁺ T_FH cells and rectal plasmablasts. While here we studied inguinal LNs, future examination of mesenteric LN of similarly vaccinated macaques might help identify additional mucosal immune responses correlated with induction of T_FH cells and potentially the female sex bias in protective efficacy.

The previous study also reported that the vaccinated male macaques developed higher antibody titers than the females, yet the females exhibited equivalent antibody-dependent functional activities (32). Thus the correlation here of SIV_smH4 Env-specific T_FH in females with gp120-specific ADCC activity (Fig. 8E) and the marginally non-significant correlations with gp140-specific ADCC and neutralizing antibody activities suggest a hypothesis that elevated T_FH cell levels compensated for lower antibody titers by promoting better quality antibody in the female macaques. This hypothesis can be pursued by in-depth characterization and functional analyses of the antibodies elicited and further investigation in future pre-clinical studies.

Overall this study illustrates the importance of antigen specific T_FH cells for the development of B cell immunity, critical for vaccine-induced protective efficacy. Continued studies are needed to elucidate how best to enhance this population in vaccine protocols. Further, our results suggest that SIV-specific T_FH cells should be routinely investigated as a correlate of protective immunity.