Regulatory and Helper Follicular T cells and antibody avidity to SIV-gp120

Abstract

T follicular regulatory cells (T_FR) are a suppressive CD4⁺ T cell subset that migrates to germinal centers (GC) during antigen presentation by up-regulating the chemokine receptor CXCR5. In the GC, T_FR control T follicular helper cells (T_FH) expansion and modulate the development of high-affinity antigen specific responses. Here we identified and characterized T_FR as CXCR5⁺ CCR7⁻-“follicular” T regulatory cells (T_REG) in lymphoid tissues of healthy rhesus macaques, and we studied their dynamic throughout infection in a well-defined animal model of HIV pathogenesis. T_FR were infected by SIV_mac251 and had comparable levels of SIV-DNA to CXCR5⁻ CCR7⁺-“T-zone” T_REG and T_FH. Contrary to the SIV-associated T_FH expansion in the chronic phase of infection, we observed an apparent reduction of T_FR frequency in cell suspension, as well as a decrease of CD3⁺ Foxp3⁺ cells in the GC of intact lymph nodes. T_FR frequency was inversely associated with the percentage of T_FH and, interestingly, with the avidity of the antibodies that recognize the SIV-gp120 envelope protein. Our findings show changes in the T_FH/T_FR ratio during chronic infection and suggest possible mechanisms for the unchecked expansion of T_FH cells in HIV/SIV infection.

Introduction

The generation of long-lived plasma cells and high affinity antibodies is largely dependent on T-B-cell interaction in the B-follicles of secondary lymphoid organs (1) (2) (3). Antigen-activated B cells making contact with a specialized subset of CD4⁺ T cells, called T follicular helper cells (T_FH), can enter the germinal centers (GCs) to undergo to somatic hypermutation and affinity maturation (4). T_FH home to B follicles and GC (5) (6, 7) (8, 9) by up-regulating the chemokine (C-X-C motif) receptor 5 (CXCR5) and down-regulating the chemokine (C-C motif) receptor 7 (CCR7) (10)(5)(6). T_FH express high levels of programmed death 1 (PD-1), inducible co-stimulator (ICOS), and Bcl-6, a master transcriptional regulator that orchestrates T_FH differentiation (11)(12)(13). In the GC, T_FH provide signals for B-cell survival and differentiation (10)(5) via IL-21 production and CD40L expression, and they promote the generation of antibodies with high affinity (11)(12)(13, 14)(15)(16).

GC reactions are tightly regulated to prevent the emergence of B-cell clones that are specific or cross-reactive against self-antigens, while selecting for high affinity antibodies to microbes (17)(18). The maintenance of the appropriate number of T_FH is crucial (19); the absence of T_FH has a negative impact in the generation of the GC (20)(21), while their excessive accumulation leads to increased GC reactions and the onset of some autoimmune diseases (4)(22)(23)(24).

CD4⁺ T follicular regulatory cells (T_FR) contain T_FH numbers and in doing so, they control the magnitude of GC responses (25) (26). Similarly to T_FH, T_FR migrate to the GC by expressing CXCR5 and down regulating CCR7 during T-cell activation (6)(27)(28)(29)(25). T_FR differentiate from natural CXCR5⁻ Foxp3⁺ CD25⁺-T_REG and express high levels of the typical T_REG markers (i.e. Foxp3, CD25, CTLA-4) and T_FH canonical markers such as ICOS, PD-1 and Bcl-6 (25) (26). While Bcl-6 is essential for CXCR5 expression on B- and T_FH cells and for their localization to the GC (25)(26), T_FR co-express Blimp-1, which is known to repress CXCR5 expression (25)(30). Ablation of the activated T-cell nuclear factor (NFAT)-2 in mice results in reduced expression of CXCR5 on T_FR, but not on T_FH, suggesting that this transcriptional factor may enable the proper localization of T_FR within B-cell follicles, possibly by inhibiting Blimp-1–mediated repression of CXCR5 expression (31). T_FR restrict T_FH numbers, and help to maintain a steady ratio of IgM⁺ to IgM⁻ (switched) B cells (32) via IL-10 production (29); in vivo depletion of CD4⁺ T cells with suppressive activity including T_FR, or in vivo blockade of IL-10 or transforming grow factor –β (TGF-β) receptors results in T_FH expansion, loss of normal proportion of IgM⁻ B cells and in increased levels of high affinity antibodies (26) (29)(33).

A hallmark of HIV and SIV infection is the immune dysfunction of humoral responses characterized by loss of memory B cells and hypergammaglobulinemia (34) (35). T_FH frequency is significantly increased in the lymph nodes of HIV infected individuals and chronically SIV_mac251 infected macaques (8)(36). Production of the IL-21 cytokine by T_FH is significantly reduced during HIV/SIV infection, possibly affecting GC homeostasis and the development of effective humoral responses to the virus (37). The HIV/SIV associated changes in T_FH number and function may contribute to the impairment of B-cell responses (9)(36)(38), however other studies have found associations between the levels of functional T_FH and broadly neutralizing antibodies in chronic HIV patients (39). While the relative role of T_FH in HIV pathogenesis needs further investigation, it would be important to understand the molecular and cellular mechanisms that regulate T_FH expansion.

T_FR dynamic in HIV-infection has not been investigated yet. We identified T_FR as CXCR5⁺-T_REG in the lymph nodes of rhesus macaques, a well-established model of HIV infection. We show that 1) T_FR are infected by SIV_mac251, 2) there is an apparent decrease in T_FR levels, particularly during chronic infection, 3) T_FR levels are associated with the levels of T_FH and the total frequency of IgG⁺ B cells, and 4) T_FR levels are inversely correlated with the avidity of antibodies to SIV-gp120 protein. Taken together these findings suggest a potential role for T_FR in modulating humoral responses against HIV/SIV.

Materials and Methods

Animals and challenge

All of the animals used in this study were colony-bred rhesus macaques (Macaca mulatta) obtained from Covance Research Products (Alice, TX). The animals were housed and maintained in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgery was performed under general anesthesia, and all efforts were made to minimize suffering. All macaques were negative for simian retrovirus, simian T-cell leukemia virus type 1, and herpesvirus B. Macaques were infected with a single high dose (6300 TCID₅₀) (40) or with 10 repeated low doses (120 TCID₅₀) of SIV_mac251 given rectally (41) (Table I).

Table I.

Serological data, Acute and Chronic animals

Acute	Challenge	Weeks p.i	VL	CD4 count
P061	High dose I.R	2	5806601	748
P066	High dose I.R	2	25556960	489
P074	High dose I.R	2	30925780	333
P136	High dose I.R	2	39844500	382
P273	High dose I.R	2	34856014	524
P251	Low dose I.R	3	115008398	647
P434	Low dose I.R	3	215693077	517
P650	Low dose I.R	3	23469446	489
P716	Low dose I.R	3	108209623	454
P712	Low dose I.R	3	49568224	757

Chronic	Challenge	Weeks p.i	VL	CD4 count
P015	High dose I.R	14	121368	485
P061	High dose I.R	14	368663	928
P074	High dose I.R	14	11883300	437
P134	High dose I.R	14	16963740	260
P136	High dose I.R	14	19165270	340
P266	High dose I.R	15	172649	265
P273	High dose I.R	15	163587	360
P274	High dose I.R	15	1297398	265
E060	Low dose I.R	14	4957329	nd
E074	Low dose I.R	14	nd	nd
P632	Low dose I.R	12	7825190	111
P761	Low dose I.R	15	31347	1330
P758	Low dose I.R	15	24934	1431
P760	Low dose I.R	15	1425169	426
R158	Low dose I.R	15	1440103	820
P733	Low dose I.R	15	1358565	212
P746	Low dose I.R	15	143386	618
P754	Low dose I.R	15	2168071	734
P698	Low dose I.R	15	21182252	572
P609	Low dose I.R	15	5462462	239
P251	Low dose I.R	15	111656	406
P633	Low dose I.R	15	1104050	514
P761	Low dose I.R	15	147114	1330

Viral load: SIV/RNA copies/ml plasma Absolute CD4⁺ T number/mm2 of blood

I.R= intrarectal; p.i. post infection; VL= viral load

Cell isolation from lymph nodes and mucosa

Cells were isolated from blood, lymph nodes and spleen by density-gradient centrifugation. Tissues from the rectum, jejunum and colon were treated with 1 mM Ultra Pure DTT (Invitrogen Life Technologies) for 30 min followed by incubation in calcium/magnesium-free HBSS (Invitrogen Life Technologies) for 60 min with stirring at room temperature to remove the epithelial layer. Lamina propria lymphocytes were separated by cutting the tissue into small pieces and incubating in 10% FBS IMDM (Invitrogen Life Technologies) with collagenase D (400 U/ml; Boehringer Mannheim) and DNase (1 μg/ml; Invitrogen Life Technologies) for 2.5 h at 37°C. Mononuclear cells were placed over 42% Percoll (General Electric Healthcare) and centrifuged at 800 × g for 25 min at 4°C. Lamina propria lymphocytes were collected from the cell pellet (42).

Antibodies & staining

We used the following antibodies: CD3-Alexa700 (SP34-2), CD4-PerCP-Cy5.5 (L200), CD95-PeCy5 (DX2), CD197-PeCy7 (CCR7, clone 3D12), CD25-APCcy7 (M-A251), CD195-PE (CCR5, clone 3A9), CD14-Alexa700 (M5E2), CD16-Alexa700 (3G8), CD56-Alexa700 (B159), IgM-PerCP-Cy5.5 (G20-127), IgG-Qdot605 (G18-145), Ki67-PE (B56), CD21-PECy7 (B-ly4), all from BD Biosciences; Bcl-6-PE (IG191E/A8); CD278-PacBlue (ICOS, clone C398.4A), CD25-PacBlue (BC96), PD-1-APC (EH12.2H7), CD39-BV421 (MOCP-21), CD39 (A1) from BioLegend; Foxp3-FITC (PCH101), CXCR5-PE-eFluor610 (MU5UBEE), CD20-Qdot650 (2H7) all from eBioscience; CD103-FITC (αE integrin, clone 2G5), CD19-PE-Cy5 (J3-119), CD127-PE (eBioRDR5) from Beckman Coulter. The α4β7 antibody (Act-1) was obtained through the NIH Nonhuman Primate Reagent Resource Program (AIDS Reagent Program, Division of AIDS, NIAID, NIH). Vivid-amine-reactive dye was used to discriminate live/dead cells (Invitrogen). IgA-Texas Red (polyclonal) was obtained from SouthernBiotech, and CD38-FITC (clone AT-1) from StemCell.

For phenotypic characterization of CD4⁺ T cells subsets, cells were stained with surface markers CD3, CD4, CD95, CD25, CCR7, CXCR5, ICOS and Vivid. Cells were then fixed and permeabilized according to eBioscience’s instructions and stained with anti-Foxp3 and anti-Bcl-6 for 30 min. The appropriate isotype-matched control Ab was used to define positivity. T_FR cells were gated as live CD3⁺ CD4⁺ CD95⁺ T cells, and their percentage was calculated as the frequency of CXCR5⁺ & CCR7⁻ within Foxp3⁺ CD25⁺ cells (% of T_REG) or within CD95⁺ CD4⁺ T cells (% of memory CD4⁺ T cells) (26). Similarly, Double Positive were CXCR5⁺ & CCR7⁺ cells and CCR7⁺-T_REG were CXCR5⁻ & CCR7⁺. Finally, T_FH cells were gated as CXCR5⁺ PD-1^hi cells within the Foxp3 negative region or the memory CD4⁺ T cells population (Figure 1A).

Figure 1. Characterization and distribution of T_FR in SIV-uninfected macaques.

(A) Double positive CD3⁺ (red) and Foxp3⁺ cells (green) are present in B-follicles (CD20 in grey) in lymph nodes from a naïve macaque (blue =DAPI; scale bar: 20μm). (B) Representative flow cytometry plots showing the gating strategy for T_FR, CCR7⁺-T_REG and T_FH cells in lymph nodes. All the subsets were gated on singlet/live/CD3⁺ CD4⁺ CD95⁺. T_FR and CCR7⁺-T_REG were identified as Foxp3⁺ CD25⁺ and CXCR5⁺ CCR7⁻ or CXCR5⁻ CCR7⁺, respectively; T_FH cells as Foxp3⁻ CXCR5⁺ PD-1^high. (C) Geometric mean (MFI) of Foxp3 and (D) CD25 expression. (E) Cell surface expression of PD-1, Bcl-6 ICOS and α4β7 in T_FR (red), T_REG (blue) and T_FH cells (green). Isotype controls are in grey. Frequency of (F) CXCR5⁺ & CCR7⁻ cells and (G) CXCR5⁻ & CCR7⁺ cells within Foxp3⁺ CD25⁺ CD4⁺ T (upper panel) or within memory CD4⁺ T cells (corresponding lower panels) in blood, peripheral lymph nodes and in the GALT (colon, jejunum and rectal mucosa) of naïve animals. The median is shown. (H) T_FH frequency on Foxp3 negative (upper panel) and memory CD4⁺ T cells (lower panel) in different tissues.

B cells were gated as live/lineage negative (lin^neg: CD3⁻ CD14⁻ CD16⁻ CD56⁻) positive for CD20 and or CD19 markers. For plasmablasts, cells were stained with lineage markers, CD20, CD38, CD39, IgM, IgG and IgA. Cells were treated with Cytofix/Cytoperm (BD Biosciences) and stained with Ki67. Plasmablasts (PBs) were gated as lineage negative (lin^neg CD20⁺ and/or CD19⁺ CD21⁻ Ki67⁺ CD38⁺ CD39⁺ (43). Marker expression was analyzed with a LSRII flow cytometer using FACSDiva software (BD Biosciences). FACS analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR). A minimum of 10,000 cells per tube was analyzed.

CD4⁺ T cells counts

The absolute number of CD4⁺ T cells was calculated as previously described (44).

Sorting

To determine the RNA levels for IL-10, TGF-β and SIV-DNA, cells from lymph nodes were stained with Vivid, CD3, CD4, CD25, CCR7 and CXCR5. T_FR cells were defined as live CD3⁺ CD4⁺ CD25⁺ CXCR5⁺ & CCR7⁻, T_REGS as CXCR5⁻ & CCR7⁺. For proliferation, CD25⁺ CD4⁺ T cells (live CD3⁺ CD4⁺ CD25⁺) were sorted. Sorting was performed on a FACS ARIA (BD Biosciences).

Migration assay

Sorted live CD3⁺ CD4⁺ CD25⁺ cells were migrated for 1 hour to 1μg/ml CXCL13 (R&D Systems, Cat no. 801-CX/CF) using 5μm Pore Polycarbonate Membrane inserts (Millipore).

Proliferation

Cell proliferation was determined by dilution of CFSE (Life Technologies). Briefly, CD25-depleted (CD3⁺ CD4⁺) cells were stained with CFSE for 10 minutes and were then placed in a 24-well plate in the presence or absence of CD3 (10 μg/ml; clone FN18) with soluble αCD28 (1 μg/ml; clone CD28.2), in the presence or absence of autologous CD3⁺ CD4⁺ CD25⁺ cells migrated to CXCL13 (10:1 ratio), for 4 days. Cells were then stained and analyzed by FACS as described above.

Cyclosporin A in vitro treatment

Cells from lymph nodes were incubated 30h with 50ug of cyclosporine A (Sigma) and incubated for 6h with or without PMA and in the presence of brefeldin A (Golgi-Plug; BD Biosciences).

RT-PCR

Total RNA was extracted from whole tissue with RNeasy Plus (Qiagen) and reverse transcribed with the High-capacity cDNA reverse transcription kit (Applied Byosistems). After reverse transcription the relative amounts of transcripts were determined by real-time PCR with the SYBR Green qPCR Master Mix (Promega) using 0.2 μM of PCR primers for IL-10 (5′-AGAACCACGACCCAGACATC-3′ (forward), 5′-GGCCTTGCTCTTGTTTTCAC-3′ (reverse)), and transforming growth factor β (TGF-β). The TGF-β was described elsewhere (45). Quantification of cDNA was normalized in each reaction according to the internal β-actin control (5′-GGCACCCAGCACAATGAAG-3′ (forward), and 5′-GCTGATCCACATCTGCTGG-3′ (reverse)). A real-time nucleic acid sequence-based amplification (NASBA) assay was used to quantitative SIV-RNA in plasma (46). SIV-DNA was quantified as previously described (40).

Immunohistochemistry in lymph nodes

The primary antibodies included Anti-Foxp3 (Abcam, Rabbit), Anti-CD20 (DAKO, mouse Ig2a) and Anti-CD3 (UCD Rat). Tris-buffered saline with 0.05% Tween 20 was used for all washes. Antibody diluent (Dako, Inc., Carpinteria CA) was used for all antibody dilutions. For all primary antibodies, slides were subjected to an antigen retrieval step consisting of incubation in AR10 (Biogenex Inc, San Ramon, CA) for 2 min at 125 °C in the Digital Decloaking Chamber (Biocare Medical, Concord, CA) followed by cooling to 90 °C before rinsing in water. Primary antibodies were replaced by normal rabbit IgG, mouse IgG (Invitrogen, Grand Island, NY) and rat IgG (Vector, Burlingame, CA) were included with each staining series as the negative control. Nonspecific binding sites were blocked with 10% goat serum and 5% bovine serum albumin (BSA, Jackson ImmunoResearch, West Grove, PA). Binding of the primary antibodies was detected simultaneously using Alexafluor Goat anti Rat 568, Alex fluor Goat anti Mouse IgG2a 647, and Alex fluor Goat anti Rabbit 488. All slides were coverslipped using ProLong Gold with 4′, 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI, Molecular probes, Grand Island, NY) to stain nuclei. All the control experiments gave appropriate results with minimal nonspecific staining.

Slides were visualized with epi-fluorescent illumination using a Zeiss Axioplan 2 microscope (Carl Zeiss Inc., Thornwood, NY) and appropriate filters. Digital images were captured and analyzed by using Openlab software (Inprovision, Waltham, MA). Alex flour 647 was captured in black and white channel while other fluorescence dyes were pictured in color channels. Five high-power (40x) microscope fields were randomly chosen and captured digitally with the system described above. Each captured field includes an area of approximately 0.04 mm2. Only clearly positive cells with distinctly labeled nuclei (DAPI) and bright staining were considered positive. Individual positive cells in the five captured high-power microscope fields of the immunohistochemical stained tissue sections were counted manually by a single observer. The numbers of positive cells are presented as cells per square millimeter.

Avidity assay

Avidity was analyzed as previously described (41). Briefly, recombinant SIV gp120 protein made from codon-optimized SIV_mac239 gp120 fused to the C-terminal tag of HIV-1 gp120 was used as an antigen for the capture ELISA to detect SIV Abs against conformational epitope. Antibody avidity was determined by parallel ELISA. Heat-inactivated plasma samples were serially diluted and applied to a 96-well plate capturing SIV_mac239 gp120. After 1 h of incubation, the plate was washed and half the samples were treated with Tris-buffered saline (TBS), while the paired samples were treated with 1.5 M sodium thiocyanate (NaSCN; Sigma-Aldrich) for 10 min at room temperature. The plate was washed and a goat anti-monkey IgG-detecting Ab (Fitzgerald) was used. The avidity index (%) was calculated by taking the ratio of the NaSCN-treated plasma dilution giving an OD of 0.5 to the TBS-treated plasma dilution giving an OD of 0.5 and multiplying by 100. Plasma of uninfected normal macaques served as negative controls.

Statistical analysis

Tests of two groups of animals for differences between cell types, tissues or stages of infection were performed using the exact Wilcoxon rank sum test. Differences before and after infection within the same group of animals were assessed using the Wilcoxon signed rank test. Differences across three stages of infection were modeled using repeated measures analysis of variance when distributional assumptions were met. Correlation analyses were performed using the exact Spearman rank correlation method. Trends across three groups were assessed by the Jonckheere-Terpstra test. Due to the exploratory nature of this study, the p values reported were not corrected for multiple comparisons. Values of p ≤ 0.05 were labeled statistically significant, and we note that for outcomes where all pairwise comparisons of three groups are possible, values of p ≤ 0.02 remain significant after correction for the multiple tests.

Results

Characterization and tissue distribution of T_FR in naïve Rhesus macaques

T_FR localize in the GC of mice and humans (29)(25)(26)(47). We confirmed the presence of Foxp3⁺ CD3⁺ T cells in healthy macaque’s B-cell follicles of lymph nodes, by immunohistochemistry (Figure 1A). We then characterized T_FR in cell suspension by flow cytometry, and compared their frequency, phenotype and localization to that of CCR7⁺-T_REG and T_FH cells. The gating strategy used to define these 3 cell subsets is shown in Figure 1B. T_FR and T_REG were gated within the live Foxp3⁺ CD25⁺ CD95⁺ CD4⁺ T cells, and defined as CXCR5 positive and CCR7 negative, consistent with GC location, and as CXCR5 negative and CCR7 positive, consistent with T-zone location, respectively (Figure 1B). Of note, the T_REG population identified by this strategy only includes a specific subset based on the expression or the lack thereof of the two considered chemokines. We will refer to this subset as CCR7⁺-T_REG or T_REG for simplicity.

In agreement with Sage et al. and Linterman et al., we used the Foxp3 marker to distinguish between T_FH and T_FR subsets because both populations express CXCR5, PD-1, ICOS and Bcl-6 (48)(25). T_FH were defined as Foxp3^neg CXCR5⁺ PD-1^high CD4⁺ T cells (Figure 1B).

Consistent with their mouse counterparts, macaque T_FR expressed comparable levels of Foxp3 (Figure 1C), equal intensity and frequency of CD25, and frequency of CD39 to T_REG (Figure 1D and Supplemental Figure 1A and B) (25)(26). T_FR were also negative for CD127, the marker for the IL-7 receptor, and expressed common T_FH markers, such as PD-1, Bcl-6 and ICOS (Supplemental Figure 1C and Figure 1E) (28). Only a subset of T_REG but not of T_FR or T_FH cells was positive for the αE (CD103) and α4β7 integrin (25)(26) (Figure 1E and data not shown).

Within the Foxp3⁺ CD25⁺ population we identified an additional CXCR5⁺ CCR7⁺ double positive (DP) cell subset (Figure 1B). DP cells expressed intermediate levels of PD-1 as compared to T_FR and T_REG and had equal levels of Bcl-6 to T_FR cells (Supplemental Figure 1D).

We looked at the distribution of T_FR, T_REG and T_FH in blood, peripheral lymph nodes, and in the gut-associated lymphoid tissue (GALT; colon, jejunum and rectal mucosa) obtained from 6, 21 and 8 naïve macaques, respectively (Figure 1F–H). Representative flow plots obtained from blood, lymph node and rectal mucosa tissue from one healthy animal are shown in Supplemental Figure 1E. T_FR were mainly in the GALT and lymph nodes, and only a few were detected in blood (Figure 1F and Supplemental Figure 1E). Within the Foxp3⁺ CD25⁺ population, cells that expressed only CXCR5 were less frequent in lymph nodes than in the GALT (p<0.0001 by the Wilcoxon rank sum test; Figure 1F, upper panel), and the opposite was observed for cells that expressed only CCR7 (p<0.0001 by the Wilcoxon rank sum test; Figure 1G, upper panel). The apparent difference in tissue distribution in lymph nodes and GALT was lost when we looked at the frequency of T_FR and T_REG within the memory CD4⁺ T cell population (lower panels Figure 1F and G). DP cells were equally distributed among all the tissues analyzed, including the blood (Supplemental Figure 1F). Finally T_FH frequency was significantly higher in lymph nodes and in the GALT than in the blood, as previously described (PBMCs vs. LN, p<0.0001; PBMCs vs. GALT, p=0.0031) (49) (Figure 1H).

Because we could not determine whether DP cells home exclusively to the B-zone, we excluded them from the rest of the analysis.

Macaque T_FR suppress CD4⁺ T cells and T_FH proliferation of in vitro

In mice, T_FR control T_FH numbers and decrease their proliferation in vivo and in vitro (26). We studied if macaque’s lymph nodes also contained a suppressive CD4⁺ T cell population that homes to the B follicles. We sorted CD4⁺ CD25⁺ live cells from the lymph nodes of 2 naïve animals and isolated those capable of migrating in response to CXCL13, the ligand for CXCR5 (Figure 2). While we could not use Foxp3, an intracellular marker, to discriminate suppressor CD4⁺ T cells, sorted CD25⁺ CD4⁺ T cells from lymph nodes consisted primarily of Foxp3⁺ CD4⁺ T cells (Figure 2A). Regulatory CD4⁺ T cells that migrated to CXCL13 had higher levels of CXCR5, lower levels of CCR7 and expressed higher levels of Bcl-6 than those that did not migrate (Figure 2B). This strategy allowed us to obtain a population of CD4⁺ T cells, highly enriched for T_FR, which could be used in downstream functional assays. Unsorted and sorted cells were stimulated with or without CD3 and CD28, in the presence or absence of migrated CD25⁺ CD4⁺ T cells (follicular-T_REG-enriched population). We assessed proliferation (% of CFSE^dim cells) within CXCR5⁺ PD-1^high CD4⁺ T cells (T_FH gated cells) by FACS analysis. Representative plots of the gated CXCR5⁺ CD4⁺ T cells (Gate 1) are shown in Supplemental Figure 2A. Interestingly, in both the two lymph nodes used for this analysis, T_FH proliferated less in the presence of T_FR-enriched cells following stimulation than in the absence of CD25⁺ cells, as shown in the representative plots in Figure 2C and graphically in Figure 2D. As expected, T_FR also reduced non-T_FH CD4⁺ T cells subsets proliferation (Gate 2), Supplemental Figure 2B).

Figure 2. T_FR suppress in vitro T_FH cell proliferation.

(A) Representative density plot showing unsorted (upper panel), sorted CD25⁺ CD4⁺ T cells (central panel) and CD25⁺ CD4⁺ T-depleted cells (lower panel) from a lymph node of a naïve macaque. (B) Cell surface expression of CXCR5, CCR7 and Bcl-6 on CD4⁺ T cells that migrated (red line) or did not migrate (black line) to CXCL13. (C) Representative density plot showing proliferation (CFSE^dim) of stimulated (CD3, CD28) unsorted, CD25⁺ depleted CD4⁺ T cells alone or co-cultured with CXCL13 migrated-CD25⁺ CD4⁺ T cells. (D) Percentage of proliferating CXCR5⁺ PD1^high CD4⁺ T cells in all conditions. The bars represent the mean ± standard error from 3 lymph nodes. (E) IL-10 and (F) TGF-β mRNA measured by RT-PCR from T_FR and CCR7⁺-T_REG sorted from lymph nodes of 4 naïve animals. The bars represent the mean ± standard error.

Consistent with their regulatory function and similar to T_REG, T_FR produce IL-10 and TGF-β, which together suppress the proliferative potential and function of CD4⁺ T cells in mice (50) (51). Thus, we measured the levels of IL-10 and TGF-β in enriched populations of T_FR (CD3⁺ CD4⁺ CD25⁺ CXCR5⁺ CCR7⁻) and CCR7⁺-T_REG (CD3⁺ CD4⁺ CD25⁺ CXCR5⁻ CCR7⁺) obtained from peripheral lymph nodes of 3 naïve macaques. T_FR had equivalent IL-10 and TGF-β mRNA levels, by RT-PCR, than CCR7⁺-T_REG (Figure 2E and F).

SIV_mac251 infection and T_FR cell frequency

Sorted T_FR and T_REG from lymph nodes of 4 chronically infected macaques expressed comparable levels of CCR5 (Figure 3A), and harbored equivalent levels of SIV-DNA (Figure 3B). Similar SIV-DNA levels were also found in T_FH cells.

Figure 3. T_FR susceptibility and dynamics during SIV chronic infection.

(A) Percentage of CCR5⁺ T_FR, CCR7⁺-T_REG and T_FH. (B) SIV-DNA levels in sorted T_FR CCR7⁺-T_REG and T_FH by PCR. (C) Frequency of T_FR and (D) CCR7⁺-T_REG within Foxp3⁺ CD25⁺ cells, and (E) T_FR and (F) CCR7⁺-T_REG within memory CD4⁺ T cells in lymph nodes of naïve, acutely and chronically SIV infected macaques. (G) Number of Foxp3⁺ CD3⁺ cells in the B-follicles (T_FR) and (H) in the T-zone (T_REG) of intact lymph nodes from naïve, acutely and chronically infected macaques.

We analyzed the effect of SIV_mac251 infection on the frequency of T_FR and CCR7⁺-T_REG in the lymph nodes of 10 acutely (2 or 3 weeks after infection) and 23 chronically infected (12–15 weeks after infection) macaques (Figure 3C and D, Table I). Representative dot-plots for 2 macaques before and after infection are shown in Supplemental Figure 3A. Cells expressing only CXCR5⁺ were significantly reduced within the Foxp3⁺ CD25⁺ population during chronic infection (chronic vs. negative p< 0.0001; chronic vs. acute p< 0.0001) (Figure 3C), while CCR7-single positive cells simultaneously increased (chronic vs. negative p<0.0001; chronic vs. acute p= 0.0003) when compared to non-infected and chronically infected animals (Figure 3D).

We looked at the levels of T_FR and CCR7⁺-T_REG with respect to the memory CD95⁺ CD4⁺ T population. T_FR showed a downward trend from negative to acute to chronic (p=0.0005 by the Jonckheere-Terpstra test for trend), with marginal differences in acute (negative vs. acute: p=0.049), and a significant decrease in chronic infection (negative vs. chronic p=0.0003) (Figure 3E). A trend for increase in CCR7⁺-T_REG levels was also observed and significance was reached between the values detected in acute and in chronic phase (p=0.0018) (Figure 3F).

We then performed immunohistochemistry in lymph nodes, at 3 and 12 weeks after infection from 7 and 8 animals, respectively (Figure 3G and H). The numbers of CD3⁺ cells expressing Foxp3 in the B-cell follicles was significantly reduced in acute infection (p=0.0022, n=7) and contracted even further in chronic infection (p<0.0001, n=8), when compared to naïve (n=8) animals (Figure 3G and Supplemental Figure 3B). The number of Foxp3⁺ CD3⁺ cells in the T-zone did not change, as described by others (Figure 3H) (52).

We did not see any significant correlation between the frequency of T_FR or T_REG and the SIV-RNA plasma levels (data not shown).

To further explore possible mechanisms for the SIV-associated decrease of T_FR, we looked at markers of immune activation. We could not find any association with the frequency of Ki67⁺ CD4⁺ T cells in lymph nodes of 10 chronic animals (data not shown).

In mice NFAT-2 is critical for the up-regulation of CXCR5 on T_FR (31), hence for their migration to the GC. Thus NFAT-2 may be involved in the reduction of T_FR during chronic infection. To determine whether CXCR5 expression on macaque T_FR was also dependent on NFAT activity, we treated lymph nodes cells from 2 naïve animals with cyclosporin A (CsA), and measured changes in the CXCR5 and CCR7 levels within Foxp3⁺ CD25⁺ CD4⁺ T cells (Supplemental Figure 3C and D). While CsA treatment had no effect on CCR7, it decreased CXCR5 expression levels on Foxp3⁺ CD25⁺ CD4⁺ T cells.

Decreased T_FR and increased T_FH frequency during SIV_mac251 infection

The decrease of CXCR5⁺ regulatory T-cells may be associated with T_FH expansion during SIV_mac251 infection. We measured the frequency of T_FH in lymph nodes of infected macaques as shown in Figure 1B. The percentage of T_FH cells within the memory CD4⁺ T cell population did not change during acute infection but this population significantly expanded during chronic infection (week 12–15 after infection), as described by others (8) (negative vs. chronic and acute vs. chronic p<0.0001) (Figure 4A and B). Of note, we found a significant inverse correlation between the levels of T_FR and T_FH on memory CD4⁺ T cells during acute and chronic infection (R=−0.82, p=0.0058 and R=−0.69, p= 0.0010 by the Spearman Rank test) (Figure 4B and 4C).

Figure 4. Association between T_FR and T_FH levels in SIV infection.

(A) Frequency of T_FH on memory CD4⁺ T cells in lymph nodes of naïve, acutely and chronic SIV-infected macaques. (B) Correlation between the percentage of T_FR and T_FH on memory CD4⁺ T cells in acute and (C) chronic infection.

T_FR frequency correlates with decreased avidity of antibodies to the gp120

In mice, T_FR cells play a role in reducing plasma-cell differentiation (26). We measured the frequency of IgM⁺, IgG⁺ or IgA⁺ CD20⁺ B cells and plasmablasts, defined as lineage⁻ (CD3⁻ CD14⁻ CD16⁻ CD56⁻) CD20⁺ and/or CD19⁺ and CD21⁻ Ki67⁺ CD38⁺ CD39⁺ in lymph nodes of SIV_mac251 chronically infected animals (n=14) by flow cytometry. While we did not find any associations with the frequency of total memory B cells or plasmablasts measured in lymph nodes, we found a weak negative correlation between the frequency of IgG⁺ B cells and the frequency of IgM-switched plasmablasts (IgG⁺ and IgA⁺) and the percentage of T_FR within Foxp3⁺ CD25⁺ cells (Figure 5A and B).

Figure 5. T_FR levels and SIV specific antibody avidity.

(A) Correlation between the frequency of T_FR cells on Foxp3⁺ CD25⁺ cells and IgG⁺ B cells or (B) switched IgM⁻ plasmablasts in lymph nodes of chronically infected macaques. (C) Avidity index of plasma SIV-gp120 IgG measured in acutely and chronically infected macaques (the median is shown). (D) Correlation between the frequency of T_FH or (E) T_FR cells and the avidity index in plasma of all the SIV infected macaques and (F) in chronically infected macaques.

T_FH are associated with the avidity to influenza virus and SIV (8)(53). In SIV_mac251 infected macaques, avidity to the gp120 is low during acute infection and increases during chronic infection (Figure 5C). We confirmed that T_FH were positively associated with gp120 avidity when all the infected macaques were considered (R=0.88; p=0.0031; Figure 5D), but not when the acute and chronic phases values were looked at separately. Importantly, T_FR levels on memory CD4⁺ T cells were associated with a reduction of binding high avidity antibodies to SIV-gp120 in all the infected animals (R=−0.85; p=0.0061, and in chronic phase (R=−1.0 and p=0.017), but not during the acute phase (R=−0.80; p=0.33) (Figure 5E and F and data not shown).

Discussion

In this study we identified T_FR in lymphoid tissues of healthy non-vaccinated rhesus macaques. We confirmed the presence of Foxp3⁺ T cells in the B-zone of intact lymph nodes of macaques. Because T_FR share markers of T_FH and T_REG, they have been isolated and functionally characterized in mice as T_FH cells positive for Foxp3 (25), or alternatively, as “follicular” T_REG expressing CXCR5 (26)(47). We opted for the latter identification strategy to characterize macaque T_FR in lymph nodes. In addition, we identified a subset of CCR7-expressing T_REG that are CXCR5 negative and therefore, in principle, unable to enter the GC, and another subset of CXCR5 & CCR7-positive T_REG (DP). Because it is possible that DP may localize at the T-B borders/mantle zone, following gradients of CXCL13 (B-cell follicles-GC) and of CCL21 and CCL19 (T-zone), we excluded this population from our analysis (7).

In accordance with their mouse counterpart, macaque T_FR expressed high levels of PD-1, ICOS and Bcl-6, they were CD39⁺ and CD127⁻, and mainly resided in the GALT and lymph nodes. In a few macaques, T_FR had similar levels of IL-10 and TGF-β mRNA as CCR7⁺-T_REG (25, 26). It is possible that IL-10 and TGF-β may play a role in the T_FR-mediated control of T_FH proliferation, as shown in mice. We did not directly assess the suppressive ability of sorted CD25⁺ CXCR5⁺ CCR7⁻ cells, however lymph nodes of healthy non-infected macaques contained a CXCR5^high CCR7^low Bcl-6^hi CD25⁺ CD4⁺ population displaying in vitro chemotaxis toward CXCL13, and suppressive activity on CD4⁺ T cells and T_FH proliferation. Further characterization is needed to confirm IL-10 and TGF-β production by macaque T_FR and their role in the apparent suppression.

We took advantage of the established similarities between SIV-infection of macaques and HIV-1 infection of humans to study and T_FR susceptibility and dynamic during infection in comparison to CCR7⁺-T_REG. Previous studies on CD4⁺ T susceptibility have shown that CD25⁺ Foxp3⁺ cells are less susceptible than other subsets to HIV/SIV infection, due to their anergic nature and to the Foxp3-mediated inhibition of HIV-1-LTR activation (54)(55)(56)(57). Therefore the relative frequency of CD25⁺ Foxp3⁺ CD4⁺ T increases in acute and chronic HIV/SIV infection, while their absolute number remains the same (58)(59)(56). Similarly, we observed a trend for increased frequency of CCR7⁺-T_REG within the memory CD4⁺ population and no differences in the number of CD3⁺Foxp3⁺ cells in the T-zone of intact lymph nodes in chronic infection.

T_FR from naïve macaques expressed comparable levels of surface CCR5 to T_REG, and had equivalent levels of SIV-DNA following infection. Conversely, we saw a reduction in T_FR frequency and a decrease of CD3⁺ Foxp3⁺ cells in the B-follicles of infected animals, in particular during the chronic phase of infection. While we could not discern between CD8⁺ T cells and CD4⁺ T cells in our immunohistochemical analysis, some chronically infected animals had negligible numbers of Foxp3⁺ T cells in the GC indicating an overall reduction of regulatory cells, likely including T_FR.

We could not determine whether T_FR are more susceptible to SIV_mac251 infection than T_REG, and we did not observe any association between the T_FR levels and viral replication levels or with immune activation.

The reduction in Foxp3⁺ cells in the GC may be driven by SIV-associated changes in the homing patterns of these cells. NFAT-2 is an essential transcriptional factor for CXCR5 expression on mice T_FR (31). HIV-envelope induces NFAT-2 translocation to the nucleus, where it binds to multiple sites within the HIV-LTR (60)(61). HIV/SIV may therefore alter NFAT-dependent expression of CXCR5. We showed that cyclosporin A, a calcineurin inhibitor that blocks NFAT dephosphorylation, decreases CXCR5 levels on macaque CD4⁺CD25⁺Foxp3⁺ cells, but we were unable to test this intriguing hypothesis in our model due to the lack of reagents that cross-react with macaque NFAT proteins.

T_FH numbers expand in acute and chronic viral infections, such as in Influenza A virus (IAV) infection in mice, and in chronic Hepatitis B and HIV/SIV in humans and macaques (62)(63, 64). In particular during acute IAV infection, a temporary T_FH expansion occurs 3 days after challenge (62). Differently, in HIV/SIV infection, a sustained expansion of T_FH is seen in chronic, but not during the acute phase of infection, as we also observed in our study (8).

We found an association between the levels of T_FR and T_FH in acutely and chronic macaques infected with SIV_mac251. It is possible that a reduction or lack of expansion of T_FR may contribute to the increased T_FH number in chronic infection. Alternatively, the persistence of the antigen may lead to the increase in T_FH cells, resulting in higher levels of PD-1 in the GC and in T_FR reduction (47). Additionally, changes in T_FR function, other regulatory subsets in the GC (CD8⁺ and NK T-cells), imbalanced cytokine milieu (i.e. increased IL-6), and immune activation are likely to participate to the HIV-associated increase in T_FH numbers (8)(19).

To our knowledge our study is the first to describe T_FR dynamics and changes in the T_FH/T_FR ratio during SIV infection, together with the accompanied study by Chowdhury and colleagues reporting similar findings in SIV_smE660 infected macaques.

By suppressing T_FH numbers and proliferation, T_FR modulate B-cell responses in mice (26)(29)(47). No correlation was found with the frequency of plasmablasts or with the IgA⁺ B cells. We found an association between the relative frequency of CXCR5⁺ cells within the CD25⁺ Foxp3⁺ population and the frequency of total IgG⁺ B cells and of overall switched IgM⁻ (IgA⁺ IgG⁺) B-cells and plasmablasts in lymph nodes (32).

The accumulation of T_FH in chronic SIV infection is associated with increased titers of higher avidity SIV-specific immunoglobulins (8). Interestingly, we observed an antithetic role of T_FR and T_FH in the avidity of antibodies to the SIV-gp120 protein throughout the infection, and only T_FR levels were strongly correlated with the increased in avidity during chronic infection. It has been proposed that T_FH accumulation, together with HIV-associated changes in cell function, may lead to a reduction in affinity maturation due to a lowered competition for B-cell selection. However, so far, the role of T_FH cells in HIV/SIV pathogenesis has been studied without making a clear distinction between T_FR and T_FH. Thus, the relative contribution of T_FH and T_FR in the impairment in B-cell selection during HIV infection remains to be determined.

In summary, we identified a population of macaques CD4⁺ T cells with a phenotype, function and location consistent with T_FR cells, and we revealed SIV-associated changes in the T_FR and T_FH ratio, adding to the complexity of humoral immunity to HIV.

Nonstandard abbreviations

T_REGS

T regulatory cells

T_FH

T follicular helper

T_FR

T follicular regulatory cells