Codelivery of Envelope Protein in Alum with MVA Vaccine Induces CXCR3-Biased CXCR5+ and CXCR5− CD4 T Cell Responses in Rhesus Macaques This information is current as of September 27, 2021. Smita S. Iyer, Sailaja Gangadhara, Blandine Victor, Rosy Gomez, Rahul Basu, Jung Joo Hong, Celia Labranche, David C. Montefiori, Francois Villinger, Bernard Moss and Rama Rao Amara

J Immunol 2015; 195:994-1005; Prepublished online 26 June Downloaded from 2015; doi: 10.4049/jimmunol.1500083 http://www.jimmunol.org/content/195/3/994 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Codelivery of Envelope Protein in Alum with MVA Vaccine Induces CXCR3-Biased CXCR5+ and CXCR52 CD4 T Cell Responses in Rhesus Macaques

Smita S. Iyer,* Sailaja Gangadhara,* Blandine Victor,* Rosy Gomez,* Rahul Basu,* Jung Joo Hong,* Celia Labranche,† David C. Montefiori,† Francois Villinger,* Bernard Moss,‡ and Rama Rao Amara*,x

The goal of an HIV vaccine is to generate robust and durable protective Ab. Vital to this goal is the induction of CD4+ T follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and quality of vaccine-elicited Ab titers. In this study, we investigated these questions in the context of a DNA/modified vaccinia virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide in rhesus macaques. In addition, we Downloaded from determined the frequency of vaccine-induced CD4+ T cells coexpressing chemokine receptor, CXCR5 (facilitates migration to

B cell follicles) in blood and whether these responses were representative of lymph node TFH responses. We show that booster modified vaccinia virus Ankara immunization induced a distinct and transient accumulation of proliferating CXCR5+ and CXCR52 CD4 T cells in blood at day 7 postimmunization, and the frequency of the former but not the latter correlated with

TFH and B cell responses in germinal centers of the lymph node. Interestingly, gp140 boost induced a skewing toward CXCR3 http://www.jimmunol.org/ expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited Ab response. However, CXCR3+ cells preferentially expressed the HIV coreceptor CCR5, and vaccine-induced CXCR3+CXCR5+ cells showed a moderate positive association with peak viremia following SIV251 infection. Taken together, our

findings demonstrate that vaccine regimens that elicit CXCR3-biased TFH cell responses favor Ab persistence and avidity but may predispose to higher acute viremia in the event of breakthrough infections. The Journal of Immunology, 2015, 195: 994–1005.

he induction of robust and long-lived Ab responses forms arise within B cell follicles typically 2–4 wk after immunization the basis of protective immunity elicited by most vaccines and are composed of Ag-specific B cell clones of varying affinity T (1). Ab dynamics following immunization result from that result from rapid B cell proliferation and receptor diversifi- by guest on September 27, 2021 activation of Ag-specific B cells and their subsequent commitment cation. High-affinity clones that successfully engage TCRs on + into two distinct cell fates—extrafollicular plasmablasts or ger- CD4 T follicular helper (TFH) cells within the GCs receive TFH minal center (GC) B cells. Plasmablasts are rapidly proliferating cell help in the form of cytokines such as IL-21, IL-2, and IL-4 Ab-secreting cells (ASCs) within secondary lymphoid organs that and costimulatory signals such as ICOS and CD40L resulting in mainly contribute to peak Ab titers within the first few weeks after their survival and differentiation to plasma cells or memory immunization (1, 2). Long-lived serological memory is estab- B cells (3, 4). lished by GC-derived bone marrow–resident plasma cells. GCs The vital role of TFH cells in the induction of humoral immunity makes them attractive vaccine targets, and characterizing vaccine elicited T cell responses associated with broad and robust Ab *Division of Microbiology and Immunology, Emory Vaccine Center, Yerkes National FH Primate Research Center, Emory University, Atlanta, GA 30329; †Department of titers will provide valuable information for vaccine design. Until Surgery, Duke University, Durham, NC 27705; ‡LaboratoryofViralDiseases,Na- recently, tracking vaccine-elicited TFH cells in humans represented tional Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and xDepartment of Microbiology and Immunology, Emory a challenge because of the belief that TFH cells are exclusively University, Atlanta, GA 30329 localized to the GCs of secondary lymphoid organs. However, Received for publication January 13, 2015. Accepted for publication May 25, 2015. there is some evidence that TFH cells circulate transiently as + + This work was supported in part by National Institutes of Health Grants PO1 CXCR5 CD4 T cells in blood and, based on the expression AI088575 (to R.R.A.), P51 OD011132 (to Yerkes National Primate Research Center), profile of activation markers, are predictive of Ab responses. For P30 AI50409 (to Emory Center for AIDS Research), and HHSN27201100016C (to instance, HIV+ individuals that respond to H1N1 vaccine show D.C.M.). Partial support was also provided by the Division of Intramural Research, + + National Institute of Allergy and Infectious Diseases, National Institutes of Health. expansion of CXCR5 CD4 T cells in the blood (peripheral [p] + Address correspondence and reprint requests to Dr. Rama Rao Amara, Division of TFH), and the frequency of ICOS pTFH cells correlates with Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate concurrent H1N1 titers (5). Likewise, CCR7loPD-1+ cells within Research Center Room 3024, Emory University, 954 Gatewood Road, Atlanta, GA the pT cell pool are induced after influenza vaccination; this 30329. E-mail address: [email protected] FH subset is overrepresented in patients with autoimmune syndromes The online version of this article contains supplemental material. and highly correlates with anti-dsDNA Abs and disease severity Abbreviations used in this article: alum, aluminum hydroxide; ASC, Ab-secreting cell; DDMM, DNA DNA MVA MVA; GC, germinal center; LN, lymph node; MFI, (6). Taken together, these studies indicate that vaccine-elicited median fluorescence intensity; MVA, modified vaccinia virus Ankara; p, peripheral; TFH cells circulate during the effector response to vaccination, PD, programmed death; SLAM, signaling lymphocyte activation marker; TFH,T these pT cells demonstrate an activated phenotype, and their follicular helper. FH magnitude correlates with vaccine-specific Ab titers generated Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 within a month after vaccination. What is less understood is www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500083 The Journal of Immunology 995

whether pTFH cells are predictive of long-term Ab titers and quality in augmented Ab responses, 14 animals were randomized to receive gp140 Env in alum along with the second MVA (DDMM-Pro). The DNA immu- and how they compare with lymph node (LN) TFH cell responses. Recent studies have underscored the expression of chemokine nogen expressed SIV239 Gag-Pol, Env, Tat, Rev, and coexpressed macaque CD40L and was delivered at 3 mg/dose (10). The MVA immunogen receptors as a key functional attribute of TFH cells (7). Blood expressed SIV239 Gag, Pol, and Env and was delivered at a 108 PFU/dose CXCR5+CD4+ T cells in humans are composed of CXCR3+ and (11, 12). Protein vaccination consisted of 100 mg endotoxin-free SIV239 CXCR32 subsets, which show heterogeneity in B cell helper po- gp140 (Immune Technology), which was premixed with 500 mg alum (2% tential. For instance, induction of CXCR3+ICOS+CXCR5+ pT alhydrogel; InvivoGen) prior to vaccination and was delivered in the thigh FH contralateral to MVA immunization. Briefly, protein and alum were combined cells at day 7 after influenza vaccination predicts increase in Ab in a 1:1 volume ratio and were mixed by rocking at room temperature for 10 titers at day 28 postimmunization (8). In contrast, in HIV-infected min. Prepared inoculum was stored on ice until the time of inoculation. All 2 individuals, frequency of CXCR3 PD-1+CXCR5+ cells is associated immunizations were delivered in PBS i.m. in a single shot in the outer thigh. with the development of neutralizing Abs (9). These data indicate SIV infection that phenotypic characteristics of pTFH cells may be specific to the vaccine/infectious agent and the resulting inflammatory response. In At 6 mo after vaccination, all vaccinated animals were challenged with the context of HIV infection, the data suggest that CXCR3- T cells SIVmac251 intrarectally on a weekly basis for a maximum of 5 wk or until FH detection of plasma viremia above 250 copies/ml for two consecutive may be favorable for induction of Abs. However, this paradigm has weeks. Infection with SIVmac251 (day 8, 7.9.10 virus stock from N. Miller not been explored in the context of HIV vaccination. [National Institutes of Health, Bethesda, MD]) was performed using a 1-cc In the current study, we examined the role of blood and LN slip tip syringe containing 1 ml SIVmac251 at 200 50% tissue culture- CXCR5+CD4+ Th cells in the development of Env-specific Ab infective dose. A syringe was inserted gently ∼4 cm into the rectum, the plunger was depressed to instill the virus, and the animal was returned to Downloaded from responses in the context of a DNA prime, recombinant modified the cage in a prone position. vaccinia virus Ankara (MVA) boost (DNA/MVA) SIV vaccine in the presence and absence of a gp140 protein boost adjuvanted with Sample collection and processing aluminum hydroxide (alum) in rhesus macaques. We found that PBMCs were isolated from whole blood collected in sodium citrate tubes + + + booster immunization with MVA induced Ki-67 CXCR5 CD4 and isolated by density gradient centrifugation, according to standard T cells, which circulated in blood (albeit at low levels) and were procedures as described previously (11). PBMCs were isolated, counted, + + and used for various assays within 6 h of blood collection. LN biopsies heterogeneous for expression of CXCR3. Blood Ki-67 CXCR5 http://www.jimmunol.org/ + were collected in RPMI 1640 medium and 10% FBS. To obtain single-cell CD4 T cells at the peak effector phase were representative of suspensions, LNs were manually processed. Briefly, subsequent to removal GC TFH responses, and CXCR3 expression on GC TFH cells was of fat and connective tissue, biopsies were incised using a scalpel, and a determinant of persistence, neutralization potential, and avidity tissue segments were manually disrupted over a 100-mm cell strainer using of anti-Env Ab response. These data offer an important parameter a 10-cc plunger. Cells were washed and resuspended in complete media with which to identify, track, and characterize CD4+ T cells in Biopsies and resulting suspensions were placed on ice at all times to FH preserve cell viability. After determination of cell counts, cells were used vaccine studies to gain an understanding of the types of TFH cell immediately or cryopreserved using standard techniques. Cell viability in responses that lead to protective Abs. both PBMCs and LN suspensions was determined to be .90%.

ELISPOT assays by guest on September 27, 2021 Materials and Methods Ethics statement ELISPOT assays were performed using fresh PBMCs using a standard ELISPOT assay (13, 14). Prior to use, PBMCs were washed up to five All animal protocols were approved by the Emory University Institutional times in media to remove traces of bound Abs that could contaminate the Animal Care and Use Committee protocol YER-2002343. All experiments assay. ELISPOT plates (Millipore, Billerica, MA) were coated with were conducted in strict accordance with U.S. Department of Agriculture SIV239 gp140 at 1 mg/ml for detecting Ag-specific ASCs or with IgG at 10 regulations and the recommendations for conducting experiments in accord mg/ml (Rockland Immunochemicals, Rockland, PA) for detecting total IgG with the highest scientific, humane, and ethical principles as stated in the ASCs. After coating the plates overnight, PBMCs were incubated for up Guide for the Care and Use of Laboratory Animals. Animals were housed in to 8 h and detected using biotinylated goat anti-monkey IgG (Rockland pairs in standard nonhuman primate cages. Animals received standard pri- Immunochemicals), followed by alkaline phosphatase–conjugated strep- mate feed as well as fresh fruit and enrichment daily and had free access to tavidin (Mabtech). Spots were detected using one-step NBT substrate water. Upon infection, animals were housed singly. Immunizations, infec- (Thermo Scientific). tions, blood draws, and biopsy procedures were performed under anesthesia by trained research staff. All efforts were made to schedule samples on Measurement of Ab titers paired animals concurrently so as to minimize potential distress. Binding Ab titers against SIV239 gp140 were performed on frozen sera that were thawed and heat-inactivated as described previously (10). A NaSCN Animals displacement ELISA as previously described was used for determining Twenty-eight male Indian rhesus macaques were included in this study. avidity against SIV239 gp140 (10). SIV-specific neutralization Ab was Animals ranged in age from 2 to 4 y and in weight from 3 to 6 kg at the start measured as a function of reduction in luciferase reporter gene expression of the study. Animals were STLV2 and SIV2 at study commencement. after a single round of infection in TZM-bl cells as described previously (15). None of the animals expressed Mamu class 1 alleles B08 and B17, and Quantitation of SIV RNA and DNA each experimental group had a total of three Mamu A*O1 animals. Ani- mals from both experimental groups were randomized into four sampling The SIV copy number in plasma was determined using a quantitative real- groups for immunization and sampling of blood and tissue biopsies. time PCR as described previously (10). For viral load determinations in LN, Animals were monitored throughout the study period and, based on clin- total DNA was extracted from ∼50,000 sorted CD4+ subsets, and data were ical parameters and complete blood counts, were reported to be healthy normalized to albumin or GAPDH as depicted. Sorting experiments were and immunocompetent throughout. All animals were housed at the Yerkes performed on cryopreserved LN samples. All PCRs were performed in National Primate Research Center (Atlanta, GA) and treated in accordance duplicates with a limit of detection of 60 copies/reaction. with the Yerkes National Primate Research Center Institutional Animal Care and Use Committee regulations. Flow cytometry Study design and immunizations Staining on whole blood was done at room temperature, whereas PBMCs and LN cell (106 cells) suspensions were stained in PBS containing 2% FBS The vaccine study consisted of two experimental groups; all 28 animals (FACS buffer) for 30 min at 4˚C. Cells were stained with fluorochrome- received two CD40L-adjuvanted DNA primes (0 and 8 wk), followed by two conjugated Abs specific for CD3 (SP34-2), CD4 (OKT4), CD8 (SK1), CD20 MVA boosts (16 and 32 wk; DNA DNA MVA MVA [DDMM] regimen). To (2H7), CD95 (DX2), CXCR3 (1C6), CCR5 (3A9), Bcl-6 (K112-91), Ki-67 determine whether inclusion of protein boost along with second MVA resulted (B56), IFN-g (B27), TNF-a (MAb11), and IL-2 (MQ1-17H12). + 996 VACCINE-INDUCED CD4 TFH CELLS IN MACAQUES

Monoclonal Abs against IL-21 (3A3-N2.1) were obtained from BD pared with that of Ki-67+CD4+ T cells. To enumerate vaccine- Pharmingen (San Jose, CA); IL-4 (7A3-3) was from Miltenyi Biotec (San induced SIV-specific CD4 responses, PBMCs were stimulated Diego, CA); signaling lymphocyte activation marker (SLAM; A12), pro- with overlapping peptide pools (15-mer overlapping by 11 aa) grammed death (PD)-1 (EH12.2H7), T-bet (4B10), and ICOS (C398.4A) were from BioLegend (San Diego, CA); CXCR5 (MU5UBEE) was from derived from SIVmac239 Gag and Env sequences and examined eBioscience (San Diego, CA); CD127 (R34-34) was from Tonbo Bio- for the production of IFN-g and IL-21 using flow cytometry. The sciences; and CCR7 (FAB197A) was from R&D Systems. Dead cells were majority of IL-21–producing CD4 T cells coexpressed IFN-g excluded from analysis based on staining for Live/Dead Near-IR dead cell (data not shown) and peaked at week 1 post-MVA boost (Fig. 1C). stain from Molecular Probes, Invitrogen (Grand Island, NY). Bcl-6, T-bet, + + and Ki-67 stains were performed after cells were stained for surface Ags, The frequency of SIV-specific IFN-g IL-21 CD4 T cells showed + + followed by permeabilization/fixation using the Foxp3 kit, and protocol, a positive association with the frequency of total Ki-67 CD4 followed by intracellular staining. Prior to intracellular staining for cyto- T cells at day 7 following MVA boost (Fig. 1C, right panel), kines, fresh PBMCs were stimulated with peptide pools of Gag and Env for suggesting that proliferating cells were a reasonable indicator of 5 h in the presence of brefeldin A (GolgiPlug; BD Biosciences, San Jose, vaccine-induced CD4+ T cells at the peak of the effector response. CA) and then fixed and permeabilized with CytoFix/CytoPerm (BD Bio- + + sciences), according to the manufacturer’s instructions. Unstimulated cells To more formally identify Ag-specific CXCR5 CD4 T cells in from each animal served as negative control, and PBMCs stimulated with blood, we set out to examine CXCR5 expression on responding phorbol myristic acetetate/ionomycin served as positive controls. Samples cells after stimulation. Downregulation of CXCR5 after 6 h of were acquired on an LSR Fortessa (BD Biosciences), and 500,000 total culture at 37˚C necessitated staining of cells with CXCR5 prior to events were collected for each sample. Cell sorting was performed using + + the Aria II (BD Biosciences). Data were analyzed using FlowJo software stimulation. With this strategy, we observed that CXCR5 CD4 version X.0.7 (Tree Star, Ashland, OR). Data were analyzed after gating T cells made robust amounts of IFN-g and IL-21 in response to + out dead cells and subsequently gating on singlet cell populations. CD3 , PMA/ionomycin stimulation (Fig. 1D). IFN-g and IL-21 respon- Downloaded from 2 CD20 cell subsets were gated, and all analysis with CD4+ T cells was + + 2 ses in CXCR5 cells after Gag peptide stimulation were modest performed on parent CD4 ,CD8 T cell subsets. The minimum number of but discernable. The frequency of Gag-specific CXCR5+ CD4 events required to score a response as positive was set at 20 events after subtraction from background. T cells expressing either IFN-g or IL-21 ranged from 0.004 to 0.15% of total CD4s (Fig. 1E) and correlated positively with the Statistical analysis frequency of Ki-67+CXCR5+ CD4 T cells (r = 0.42; p , 0.05) + + Statistical analysis was performed using Graph Pad Prism version 5.0. A (data not shown). About 0.5 to 20% of IFN-g or IL-21 CD4 cells http://www.jimmunol.org/ two-tailed nonparametric t test was used for all comparisons unless oth- expressed CXCR5 (data not shown). erwise specified. Spearman correlation was used to determine associations Notably, CXCR52CD4+ T cells were the major producers of between variables. Statistical significance was set at p , 0.05. IFN-g and IL-21 in response to PMA/ionomycin and Gag. Al- though this could be due in part to our inability to capture all Results CXCR5-expressing cells, these data reveal that akin to human DNA/MVA vaccine induces transient accumulation of PD-1 + 2 + CD4 T cells IL-21 production is not an exclusive feature of and ICOS expressing CXCR5 and CXCR5 CD4 T cells with CXCR5+ CD4 T cells (17). Taken together, the data point to the B cell helper potential in peripheral blood conclusion that DNA/MVA vaccine–induced Ag-specific CXCR5+ Twenty-eight rhesus macaques were vaccinated i.m. with DNA/ CD4+ T cells circulate, albeit at very low frequencies, in periph- by guest on September 27, 2021 SIV vaccine (10) at 0 and 8 wk and with MVA/SIV vaccine eral blood during the peak effector response. (10) at 16 and 32 wk (DDMM group) (Fig. 1A). From this group, To determine whether these circulating CXCR5+CD4+ T cells 14 macaques also received i.m. SIV gp140 protein boost in alum possessed B cell helper potential, we cocultured FACS-sorted concurrently with the second MVA (DDMM-Pro). Blood was CXCR5+ and CXCR52 memory CD4+ T cells along with autol- sampled at baseline and peak effector and memory time points ogous memory B cells, and cell supernatants were collected at after each immunization. A single LN biopsy was obtained at 2 wk days 3 and 7 to examine secreted IgG (Fig. 1F). At day 7, cells after the second MVA immunization. were also harvested and examined for B cell proliferation. Both To determine whether DNA/MVAvaccine–elicited CD4+ T cells CXCR5+ and CXCR52CD4+ T cells efficiently induced IgG se- in blood have the potential to migrate to B cell follicles, we ex- cretion and B cell proliferation compared with naive CD4+ T cells amined Ki-67+ (marks proliferating cells) CD4+ fraction for ex- indicating that circulating CXCR5+ and CXCR52CD4+ T cells pression of the chemokine receptor CXCR5, a defining TFH cell possess B cell helper potential in vitro. This helper potential is marker that facilitates homing to B cell follicles/GCs (Fig. 1B) consistent with the expression of ICOS by these cells (Fig. 2C). (16). We also monitored the CXCR52Ki-67+CD4+ T cell fraction Taken together, these data indicated that the first MVA booster + (presumably non-TFH fraction). About 0.1–1.7% (mean of 1%) of immunization resulted in the transient accumulation of Ki-67 CD4+ T cells prior to boost were CXCR5+Ki-67+, and this fraction CD4 T cells in blood at the peak of the effector response. These increased by 2-fold at day 7 following the first MVA boost and cells were phenotypically heterogeneous for expression of CXCR5, returned to near baseline levels at 8 wk postimmunization. The but both CXCR52 and CXCR5+ cells demonstrated B cell help frequency of CXCR52Ki-67+CD4+ T cells was ∼3- to 4-fold functionality in vitro. + higher compared with CXCR5 counterparts and expanded with + 2 + + DNA/MVA vaccine induced CXCR5 and CXCR5 Ki-67 similar kinetics. Thus, CXCR5 CD4 T cells constituted a small + proportion (∼15–25%) of total vaccine-elicited CD4 T cell re- CD4 T cells in peripheral blood demonstrate a Th1 propensity sponses in circulation. A similar phenotypic distribution was The cytokine profile of SIV-specific CD4+ T cells indicated the also observed within the Ki-672 memory compartment in blood induction of Th1-like CXCR5+CD4+ T cells. Therefore, we wanted (Supplemental Fig. 1A). This is consistent with data in mice to determine the CXCR3 expression profile of Ki-67+ cells. demonstrating that ∼25% of circulating OT-II effectors represent Flow plots in Fig. 2A show distribution of CXCR3 (X3) and + + TFH cells (6) and with data in humans showing that only a small CXCR5 on Ki-67 CD4 T cells at baseline and at 1 wk after the fraction (5–20%) of total tetanus–specific memory cells in blood is first MVA. Prior to MVA boost, X3+ cells were present in both CXCR5+ (9). CXCR52 (X3 single positive, blue population) and CXCR5+ Next, we examined SIV-specific CD4+ T cell responses to study (double positive, green population) fractions, and the frequency of how kinetics and phenotype of Ag-specific CD4+ T cells com- X3+ cells were lower compared with X32 cells within the respective The Journal of Immunology 997 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 1. DNA/MVA vaccine induces transient accumulation of PD-1 and ICOS expressing CXCR5+ and CXCR5- CD4+ T cells with B cell helper potential in peripheral blood. (A) Rhesus macaques were immunized with plasmid DNA expressing Gag, gp160 Env, Prt, RT, Tat, and Rev (3 mg at 0, 8 wk), followed by boost with recombinant MVA expressing Gag, gp150 Env, and Prt (1 3 108 PFU at 16, 32 wk; DDMM). Fourteen macaques received 100 mg gp140 protein (adsorbed in 500 mg alum) at week 32 concurrent with second MVA immunization (DDMM-Pro). All immunizations were administered i.m. in the thigh. Peripheral blood and LN biopsies were obtained at stated time points. (B) Examination of peripheral blood for the nuclear Ag Ki-67 together with CXCR5 showed that MVA immunization induced a proliferative burst in CXCR5+ (black line) and CXCR52 (gray line) CD4+ T cells at day 7 (two-tailed paired t test showed significantly higher frequencies at week 1 compared with baseline and week 8; *p , 0.05). Data are mean 6 SEM. (C) Kinetics of SIV-specific CD4 T cell response for Gag and Env in 28 rhesus macaques. Data are mean 6 SEM; response for Gag and Env at week 1 was significantly higher compared with baseline and week 8 using a paired two-tailed t test; ****p , 0.0001. Magnitude of total Ki-67+CD4+ T cells correlated with the magnitude of Gag + Env IFNg+IL-21+CD4+ T cell response (Spearman R2 = 0.25 with a two-tailed test; p , 0.01). (D) Shows cytokine profile of CXCR5+ cells in blood under the following conditions: NS, PMA/ionomycin, and Gag at 1 wk after first MVA. Frequencies of Gag-specific CXCR5+ cells were above background for IFN-g and IL-21. (E) Scatter plot shows range of CXCR5+IFNg+ and CXCR5+IL-21+ responses in NS (triangle) and Gag stimulated PBMCs (circle); each dot represents one animal. (F) To determine B cell helper potential of blood CXCR5+ cells, these cells were cocultured with autologous memory B cells obtained at week 1 after first MVA. Blood CXCR52 and CXCR5+ CD4 T cells efficiently induced IgG secretion and B cell proliferation. Data are representative of one of four independent replicates.

CXCR5+ or CXCR52 subsets. However, at 1 wk following the estingly, both CXCR5+ and CXCR52 CD4 T cells expressed ICOS, MVA boost, the frequency of X3+ cells increased significantly in and ICOS expression was higher on CXCR3+ cells compared with both CXCR52 and CXCR5+ subsets indicating generation of Th1 CXCR32 cells. CXCR52CXCR3+ cells expressed the highest levels 2 + biased non-TFH and TFH cell subsets (Fig. 2B). Noncycling (Ki-67 ) of ICOS. This phenotype contrasts with blood CD4 effectors in memory CD4+ T cells showed a variable composition and phe- humans where CXCR52 CD4 T cells do not express ICOS (8). notype of these subsets likely a result of diverse antigenic history Expression of the activation receptor associated with IFN-g pro- and quiescent phenotype (Supplemental Fig. 1B). duction, SLAM was lower on CXCR5+Ki-67+CD4+ T cells relative To better understand the nature of cycling blood CXCR5+ and to CXCR52 counterparts, reflective of the SLAMlo phenotype of CXCR52CD4+ T cells in the context of CXCR3, we performed LN CXCR5+ cells (18, 19). Similar to ICOS expression, SLAM was + a phenotypic characterization of these cells with respect to expressed at higher levels on X3 cells. Unlike LN-resident TFH markers associated with LN-resident TFH cells (Fig. 2C). A ma- cells, which downregulate CCR7 to exit the T cell zone and gain jority of blood Ki-67+CD4+ T cells within both the CXCR52 and access to the GCs (20), blood CXCR5+CD4+ T cells were positive CXCR5+ compartments were positive for PD-1 with CXCR5+ for CCR7 irrespective of X3 expression. In contrast, a majority of CD4+ T cells expressing relatively higher levels of PD-1. Inter- X32CXCR52CD4+ T cells had downregulated CCR7 (20). + 998 VACCINE-INDUCED CD4 TFH CELLS IN MACAQUES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. DNA/MVA vaccine induced CXCR5+CD4+ T cells in peripheral blood demonstrate a Th1 propensity. (A) Ki-67+CD4+ T cells were dis- tinguished into four subsets based on expression of CXCR5 (X5) and CXCR3 (X3). (B) Proportion of Ki-67+CD4+ subsets for expression X5 and X3 at stated time points (****p , 0.00001, ***p , 0.001, **p , 0.01 computed using a paired two-tailed t test). (C) Histograms show relative expression of phenotypic markers in naive (gray) X5+X32 (red), X5+X3+ (green), X52X32 (tungsten), and X52X3+ (blue) subsets in blood at week 1 after first MVA. Box plots show MFI of respective markers in Ki-67+ CD4 T cells for several markers; PD-1 (X5+ subsets showed significantly higher expression compared with X3 SP cells; *p , 0.05, **p , 0.01), X3+ cells expressed significantly higher levels of ICOS and SLAM and did not express CCR7. Data shown are from four animals and are representative of three independent replicates. (D and E) expression of CD40L and IFN-g on FACS-sorted cell subsets memory cells after stimulation with PMA/ionomycin.

To further corroborate the Th1-like functionality of double pos- a higher MFI of CD40L, whereas CXCR52,X3+ subset showed itive CD4+ cells in blood, we wanted to examine cytokine pro- highest expression of IFN-g. Taken together, the data demonstrate duction upon stimulation. Because dynamic changes in expression that DNA/MVA-induced blood CXCR5+CD4+ T cells show phe- of CXCR3 and CXCR5 occur upon stimulation, we sorted CXCR5+ notypic and functional heterogeneity based on CXCR3 expression, and CXCR52 cells based on expression CXCR3 to accurately and vaccination significantly enhances the frequency of Th1-like discern differences in cytokine profile among these subsets. Sorted CXCR5+ and CXCR52 cells during the effector phase in blood. subsets were stimulated with PMA/ionomycin and examined for + + expression of CD40L and IFN-g. Consistent with their Th1 phe- Blood CXCR5 CD4 T cells predict GC TFH cell and B cell notype, X3+ cells showed higher frequency of CD40L+IFN-g+ cells responses in LNs within both CXCR52 and CXCR5+ compartments (Fig. 2D). In We next determined the relationship between CXCR5+CD4+ terms of median fluorescence intensity (MFI), CXCR5+ cells had T cells in blood and LN and examined how inclusion of alum- The Journal of Immunology 999 adjuvanted gp140 protein along with second MVA boost impacted not significantly increase the frequency of CXCR5+Ki-67+ cells the magnitude of these CD4+ T cell responses. Examination of beyond that induced by second MVA alone. Interestingly, blood Ki-67+CD4+ T cells at week 1 following the second MVA however, gp140 protein boost resulted in a significant induction + + boost showed that MVA+gp140 immunization resulted in ∼2-fold of X3 expression on GC TFH cells and CXCR5 PD-1 cells in higher frequencies of Ki-67+ CD4 T cells in circulation. The the LN (Fig. 4A). Although the relative contribution of gp140 frequency of Ki-67+CD4+ T cells ranged from 5 to 13% of total Ag versus alum or a combination thereof to this phenotype CD4s in MVA only compared with 15–33% of CD4s in MVA+ cannot be discerned, these results demonstrate that alum did not gp140 group (Fig. 3A). We also observed an increase for SIV skew against a Th1 CD4+ helper recall response established by Env–specific IFNg+, IL-21+, and IL-4+CD4+ T cells (Fig. 3B) in the preceding DNA/MVA immunizations. Interestingly, we ob- the protein group, and as expected, this increase was not observed served a direct positive association between the magnitude of + + for Gag-specific CD4 T cells (Fig. 3C). Taken together, the data GC B cell responses and proportion CXCR3 GC TFH cells showed that concurrent gp140 immunization augmented magni- within LNs in the DDMM-Pro group (Fig. 4A, right panel). As + 2 tude of Env-specific CD4 T cell responses. anticipated, CXCR3 GC TFH cells showed an inverse corre- Having identified that DNA/MVA induced CXCR5+ cells cir- lation (Supplemental Fig. 2A). culate in blood during the effector response, we next examined TheincreaseinX3expressiononCXCR5+ cells following + TFH cells in the LNs and compared them with blood CXCR5 MVA boost led us to examine phenotypic differences among cells. We chose to biopsy the right LN, which was nondraining to X32 and X3+ subsets in the LN to understand possible functional the second MVA immunization so as not to interfere with Ag differences among these two subsets. We started by examining complexes sequestered within the draining inguinal LN after expression of the transcription factor Bcl-6, a unique and char- Downloaded from ++ immunization. It is conceivable that virus-like particles derived acteristic marker of GC cells (Fig. 4B). PD-1 GC TFH cells from MVA-infected cells in vivo could engage memory cells in exclusively expressed Bcl-6 albeit at lower levels compared with + distal lymphoid organs including the right LN, which was the GC B cells (data not shown). Within GC TFH cells, the CXCR3 draining LN for the preceding DNA primes and for the first MVA subset expressed lower levels of Bcl-6 on a per cell basis in most boost. of the animals. As expected, decreased levels of Bcl-6 were as- + Examination of LN sections by immunohistochemistry showed sociated with higher relative expression of T-bet in CXCR3 GC http://www.jimmunol.org/ presence of distinct clusters of proliferating B cells within B cell TFH cells. This dichotomy in T-bet expression was true for all follicles (data not shown). Single-cell suspensions stained for CXCR5+ and CXCR52PD-1+ subsets. These data suggest that X3 flow cytometry showed presence of three distinct populations of expression identifies Th1-like TFH cells in GCs and extrafollicular CXCR5+CD4+ T cells showing graded levels of PD-1 expression: regions. + ++ + + + GC TFH cells (CXCR5 PD-1 ), CXCR5 PD-1 , and CXCR5 Because Bcl-6 positively regulates expression of PD-1 (9), we 2 + PD-1 cell subsets (Fig. 3D). The frequency of GC TFH cells next examined PD-1 expression and found that X3 cells also correlated directly with GC B cells identified by expression of expressed lower amounts of PD-1 on a per cell basis. This dif- + + Bcl-6 as shown and with CXCR5 PD-1 cells but not with ference was only observed within the GC TFH subset. We asked CXCR52PD-1+ cells or with CXCR5+PD-12 cells (Supplemental whether the same was true for expression of another activation/ by guest on September 27, 2021 Fig. 2A). Examination of the frequency of GC responses revealed costimulation marker, ICOS. Unlike PD-1, ICOS was expressed at + that the gp140 boost did not alter either the frequency of total GC higher levels by CXCR3 GC TFH cells reflecting expression + 2 + + TFH cells or GC B cells (Fig. 3E). This is not surprising because profile of X3 and X3 Ki-67 CXCR5 cells in blood. Higher + total GC TFH cells and GC B cells are composed of polyclonal T magnitude expression of ICOS on CXCR3 GC TFH cells is and B cell responses directed against epitopes of different anti- supportive of enhanced B cell helper potential. PD-1+ and PD-12 genic specificities including MVA and SIV Gag; a modest increase CXCR5+ cells expressed lower levels of ICOS compared with GC in Env-specific GC responses elicited by gp140 protein boost TFH cells and demonstrated no difference in expression in the would be difficult to discern. context of X3. Expression of an IFN-g–related marker SLAM was + + 2 We next evaluated possible associations between blood CXCR5 consistently higher on X3 subsets; X3 GC TFH cells did not cells and PD-1–expressing GC TFH cells and GC B cells within the express SLAM showing expression levels comparable to naive LN and found a positive correlation between Ki-67+CXCR5+ cells cells. X3+ subsets showed higher levels of CD95. Frequency of in blood and all three LN subsets (Fig. 3F). We next asked whether Ki-67 among X3+ and X32CXCR5+PD-1+ subsets was compa- the Ki-67+CXCR52 CD4 T cell subset was also positively associ- rable, indicating that the phenotypic differences were not due to ated with GC responses. The data showed that CXCR52 cells did cell cycle status of these subsets. not correlate with any of the measured GC responses (Supplemental Levels of another memory marker, CD127 were low on GC TFH Fig. 2B). Therefore, the magnitude of responding blood CXCR5+ cells but relatively higher in the X3 compartment and the same CD4+ T cells but not CXCR52CD4+ T at peak postvaccination was true for the CXCR5+PD-1+ compartment, raising the possi- + was predictive of subsequent GC responses. Studies showing that bility that X3 GC TFH cells represent memory precursors that gene expression profile of blood PD-1+CXCR5+ cells but not of differentiate into CXCR5+PD-12 cells after Ag clearance. 2 CXCR5 cells closely matches that of GC TFH cells are consistent To determine whether these phenotypic differences resulted with this possibility (9). in differential ability to provide B cell help, we sorted GC TFH and CXCR5+PD-1+ cells from the LN based on X3 expression Alum-adjuvanted gp140 protein boost enhances the frequency + + (Supplemental Fig. 2C). Sorted subsets were cocultured with of CXCR3 CD4 TFH cells in LN autologous memory B cells in the presence of staphylococcal Next, we wanted to ascertain whether inclusion of gp140+Alum enterotoxin B. Determination of IgG in supernatant after 7 d + + 2 resulted in discernable effects on phenotype of CXCR5 cells in revealed that both X3 and X3 GC TFH subsets were able to blood and LN. Consistent with the first MVA boost, the majority of provide B cell help. The relative ability was variable; two animals Ki-67+ cells after the second MVA boost were composed of X3 SP RGf14 and RVz12 showed 1.5- to 8-fold higher IgG in X3+ GC + 2 CD4 T cells, and the inclusion of protein boost further enhanced TFH B cell cocultures compared with X3 counterparts, whereas 2 the frequency of these cells (data not shown). The protein boost did in two animals RPq13 and RWf114, the X3 GC TFH subset + 1000 VACCINE-INDUCED CD4 TFH CELLS IN MACAQUES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

+ + + + FIGURE 3. Blood CXCR5 CD4 T cells predict GC TFH cell and B cell responses. (A) Higher frequency of Ki-67 CD4 T cells after 2nd MVA in DDMM-Pro group (****p , 0.0001 at week 1 relative to baseline with a two-tailed, nonparametric t test). (B) To determine whether gp140 boost resulted in higher Env-specific CD4+ T cells, responses were compared using a one-tailed t test, which showed increased Env IFN-g+, IL-21+, and IL-4+ frequencies in DDMMgp140 group at week 1 after second MVA immunization (*p , 0.05, **p , 0.01) and no change in Gag responses (C). (D) Inguinal LNs were biopsied from the LN nondraining to immunization site at 2 wk after second MVA immunization. (E) Flow plot shows presence of distinct subsets of + + ++ + 2 CXCR5 CD4 T cells in LN; PD-1 GC TFH (red), PD-1 (blue), and PD-1 cells. Middle panel shows GC B cells identified by Bcl-6, and scatter plot + + shows correlation between GC B cells and GC TFH cells. (D) Inclusion of gp140 boost did not alter frequencies of GC TFH cells, CXCR5 PD-1 , and GC B cells. (F) To determine whether responses in blood (at week 1 after second MVA) were positively correlated with magnitude of GC responses, these indices + + 2 + + were correlated using a one-tailed Spearman correlation (p , 0.05). Blood Ki-67 CXCR5 cells correlated with GC TFH cells (R = 0.21), CXCR5 PD-1 2 2 TFH cells (R = 0.24), and GC B cells (R = 0.20) in LN.

+ + + showed higher B cell helper potential. CXCR5 PD-1 cell subsets CXCR3 GC TFH cells correlate with longevity and avidity of also showed similar variability among X3+ and X32 compart- gp140 Ab titers + ments. In all, the data indicate that LN CXCR3 GC TFH cells are Among the first B cell effectors are plasmablasts/ASCs, derived 2 phenotypically distinct from CXCR3 GC TFH cells, but both either from activation of naive B cells during a primary response or subsets demonstrate potential to help B cells in vitro. from memory B cells during Ag re-exposure (21, 22). After the The Journal of Immunology 1001 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

+ + FIGURE 4. Alum-adjuvanted gp140 protein boost enhances the frequency of CXCR3 CD4 TFH cells in LN. Inclusion of protein boost in alum skewed + + + the phenotype of responding CD4 T cells toward CXCR3 in LN GC TFH and CXCR5 PD1 cells (A) examined at 2 wk after second MVA boost. Clear bars, DDMM group; filled bars, DDMM-Pro group. Comparisons were made using a two-tailed nonparametric t test: **p , 0.001, *p , 0.01. Scatter plot + shows association of percentage of CXCR3 GC TFH cells with magnitude of GC B cell response. (B) Histograms show expression of markers on GC TFH cells (red), CXCR5+ PD1+ cells (blue), and CXCR5+PD12 cells (green) in LN. Solid lines denote expression in CXCR3+ subsets, dotted line CXCR32 2 + subset and whisker plots show MFI for X3 (empty plots) and X3 (hashed plots) TFH subsets for the transcription factors Bcl-6, T-bet; activation markers ICOS, PD-1, SLAM, CD95, and memory marker CD127 (#p = 0.08; ****p , 0.0001, ***p , 0.001, *p , 0.05 computed using a paired two-tailed t test). second MVA boost, gp140-specific ASC responses were observed ond MVA increased the magnitude of ASC response by 3-fold, in 100% of immunized macaques (Fig. 5A). These responses which ranged from 30 to 400 (mean 200) ASCs per million ranged from 30 to 160 (mean 63) ASCs per million PBMCs in the PBMCs. MVA-specific ASC responses were comparable between DDMM group. Concurrent immunization of gp140 with the sec- the two groups and were not inhibited as a result of gp140 im- + 1002 VACCINE-INDUCED CD4 TFH CELLS IN MACAQUES munization. To explore the role of CD4+ T cells in fostering SIVE660 pseudovirus isolates. Although no detectable responses plasmablast responses, we examined peripheral Ki-67+CD4+ against tier 2 isolates was observed, coimmunization with alum in T cell responses at day 7 as a surrogate for T cell responses in the gp140 resulted in stronger neutralization titers against tier 1 isolates LN on or prior to day 5. After the second MVA boost, the mag- at weeks 2 and 20 following the second MVA boost (Fig. 5D). As nitude of total Ki-67+CD4+ T cells (Fig. 5B) in the DDMM-Pro a measure of Ab maturation, we monitored relative Ab avidity group correlated with higher gp140 ASC responses. Correspond- following first and second MVA immunizations. Our analyses ingly, Env IFNg+CD4+ T cells were also positively correlated with using an ELISA against SIV239 gp140 with a 1.5 M sodium ASC responses (Supplemental Fig. 3A). The gp140 ASC response thiocyanate wash showed that gp140 avidity increased over time at day 5 was a key determinant of peak gp140 titers at 2 wk and reached a maximal response at week 20 after the second MVA following the second MVA boost. boost (Fig. 5E). To ascertain whether inclusion of gp140 protein To delineate CD4 determinants of peak and memory anti-gp140 boost during the second MVA increased avidity of anti-Env Ab, Ab responses, we first examined the kinetics of Ab by ELISA we examined relative increase in avidity at week 20 versus week 2 (Fig. 5C). Anti-gp140 Ab titers were below the limit of detection and observed greater increase in avidity with inclusion of gp140 in 25 of 28 animals after the second DNA immunization. The first protein boost (Fig. 5F). MVA immunization induced titers ranging from 5 to 200 mg/ml at Next, we wanted to investigate how the GC TFH response in the week 2, which contracted by week 8 and were potently recalled LN at week 2 after the second MVA boost influenced the magnitude after the second MVA resulting in higher magnitude gp140 titers and longevity of anti-Env Ab titers. The frequency of CXCR3+ GC at memory. TFH cells showed a trend for correlation with the binding titers at

We next determined the neutralization potential of anti-Env Ab week 20 (p = 0.05) or at week 10 (data not shown) and directly Downloaded from against tier 1 (easy to neutralize) and tier 2 (harder to neutralize) correlated with neutralizing activity and avidity of anti-Env Ab. http://www.jimmunol.org/ by guest on September 27, 2021

+ FIGURE 5. CXCR3 GC TFH cells correlate with longevity and avidity of gp140 Ab titers. ELISPOT assays were used to quantify gp140 and MVA ASCs (153 magnification shown). ELISPOT image for gp140 and MVA (A) at day 5 after second MVA boost in DDMM (top) and DDMM-Pro (bottom) groups. Spots shown are for an input of 100,000 PBMCs per well. Graph in (A) shows concurrent immunization of gp140 with second MVA increases gp140 IgG ASC responses (***p , 0.001 with a nonparametric two-tailed t test). MVA IgG ASCs comparable in DDMM (s) and DDMM-Pro groups (d). (B) Correlation between gp140 ASCs and magnitude of Ki-67+ CD4 T cells (Spearman R2 = 0.38). Magnitude of gp140 ASC response correlates with scale of gp140 Ab titers at week 2 after second MVA (Spearman R2 = 0.24). (C) Kinetics of gp140 Ab responses after first MVA boost showed significant increase with gp140 boost at week 2 and week 10 after second MVA (**p , 0.001, *p , 0.01 using a nonparametric two-tailed t test). (D) Neutralization titers against tier 1 SIV E660 also increased with gp140 boost. **p , 0.01. (E) Kinetics of Ab avidity against SIV239 gp140. (F) Computation of increase in avidity (week 20 to week 2) after second MVA immunization revealed greater increase with gp140 boost (*p , 0.05 with a nonparametric one-tailed t test). + # (G) shows a trend for association between CXCR3 GC TFH and gp140 titers at memory measured at week 20 ( p = 0.05); shows inverse correlation + 2 between contraction of gp140 titers and percentage of CXCR3 GC TFH cells in DDMMgp140 group (Spearman R = 0.33; p , 0.05). CXCR3 expression on 2 2 GC TFH cells directly correlated with neutralization titers (Spearman R = 0.12; p , 0.05) and avidity index (Spearman R = 0.62; p , 0.01). The Journal of Immunology 1003

We also observed an inverse correlation between the frequency prompted us to test associations between CD4+ T cell response + of CXCR3 GC TFH andfoldcontractioninpeakAbtiters induced at peak after second MVA boost and peak viremia at 3 wk + (Fig. 5G). These results demonstrated that CXCR3 GC TFH post-SIV challenge. We found a moderate but significant direct cells contribute significantly for the generation of long-lived Ab correlation with Ki-67+CXCR5+X3+CD4+ T cells and peak vire- responses. mia (Fig. 6C, last panel). Interestingly, this association was not + + + 2 + + + observed with either total Ki-67 CD4 T cells or CXCR5 X3 Vaccine elicited induction of CXCR5 CXCR3 CD4 T cells CD4+ T cells. associates with peak viral load postinfection with SIV251 + To determine whether permissiveness of X3 TFH cell subsets Studies in humans have shown that blood X3+CCR6+CD4+ T cells to SIV infection facilitated seeding of reservoirs, we sorted LN express high levels of the HIV coreceptor CCR5 (23), raising the CXCR5+ cells (PD-1++ and PD-1+) based on X3 expression at 3 possibility that vaccine-mediated generation of CXCR3+CXCR5+ wk postinfection from several animals with a log-fold difference cells could favor viral replication upon infection. To address this in viral RNA copies in plasma; the data showed that in five of the + +/++ possibility, we first examined CCR5 expression in LN and blood six animals sampled X3 PD-1 TFH cells harbored significantly X3+ CD4+ T cells in SIV naive animals (Fig. 6A, 6B) and found higher levels of proviral DNA compared with X32 counterparts that a significantly higher fraction of X3+CD4+ T cells express (p , 0.05) (Fig. 6D). CCR5 compared with X32 cells. This was true both in LN We do not, at present, fully understand the relationship between (Fig. 6A) and blood compartments (Fig. 6B) and within CXCR5+ the SIV-specific CD8 T cell responses postinfection and the in- 2 + and CXCR5 subsets. Following five weekly intrarectal chal- fection status of CXCR3 TFH cells. However, we observed that lenges with SIVmac251, all vaccinated animals became infected the frequency of Ki-67+ CD8 T cells associated with peak viral Downloaded from but showed a 2-log-fold decrease (p , 0.01) in peak viremia load and positively correlated with frequency of GC TFH cells + compared with unvaccinated controls (data not shown). The in- and proportion of CXCR3 GC TFH cells, consistent with Ag or + + duction of CXCR3 CXCR5 CD4 TFH cells following vaccina- inflammation-driven immune activation of T cells (Supplemental tion and the higher expression of CCR5 on CXCR3+ CD4 T cells Fig. 4). More detailed studies are needed to investigate the dynamics http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Vaccine elicited induction of CXCR3+CXCR5+CD4+ T cells associates with peak viral load postinfection with SIV251. Expression of CCR5 on CXCR5+ and CXCR52 CD4 T cells in SIV naive LN (A) and blood (B). **p , 0.01, ****p , 0.0001. (C) Vaccine-induced CXCR3+CXCR5+ CD4 T cells at week 1 post-second MVA associate with peak viremia at 3 wk post-SIV infection (Spearman R2 = 0.2; p , 0.01 with a nonparametric two-tailed t + + +/++ 2 test). (D) CXCR3 CXCR5 PD-1 TFH cells in LN at 3 wk post-SIV infection harbor higher levels of proviral SIV DNA compared with X3 subsets. Values in parentheses are plasma viral RNA copies at 3 wk postinfection for each animal. + 1004 VACCINE-INDUCED CD4 TFH CELLS IN MACAQUES of Ag-specific CD8 T cell responses in peripheral blood and of T-bet in CD4 T cells, is consistent with the proposed model + lymphoid compartments in controlling frequency and infection of TFH and Th1 CD4 helper differentiation during recall re- 2 + status of CXCR3 and CXCR3 TFH cell subsets. sponses (30). + In summary, our data raise the possibility that vaccination The mechanisms by which CXCR3 GC TFH cells promote Ab regimens that skew toward generation of X3+CXCR5+ cells, while longevity and avidity remain to be understood. Our in vitro assays favoring durable, higher-avidity Ab, potentially could facilitate indicate that both X32 and X3+ subsets were effective at helping viral replication and seeding of reservoirs by generating target B cells. The dynamics of X3+ cells in vivo in fostering Ab cells that are efficiently recalled upon Ag re-exposure. responses could be due to differential localization and/or due to enhanced expression of B cell helper cytokines and costimulatory Discussion signals in the context of an IFN-g–rich environment. With regards Post-RV144, HIV vaccine strategies to augment anti-Env Ab to HIV, there is evidence that CXCR32CXCR5+ cells possess responses have generated immense interest and concurrent im- superior B cell help potential. Recently, Locci et al. (9) reported munization of Env subunit proteins with recombinant viral vectors higher percentage of blood PD-1+CXCR32CD4+ cells (within expressing HIV immunogens is emerging as a promising HIV total CXCR5+ cells) at 4 and 40 mo postinfection in HIV+ indi- vaccine modality (24). Although a great deal of research has fo- viduals that develop broadly neutralizing Abs. Furthermore, cused on Ab responses following Env subunit immunization, CXCR3+ but not CXCR3- cells were effective at providing B cell + relatively little is known about the CD4 helper response and, in help in vitro. It is possible that phenotypic attributes of TFH cells + particular, the B cell helper CD4 TFH cell response resulting from that foster Ab responses during vaccination and during infection these immunization regimens. A better understanding of CD4 are very different especially during chronic Ag stimulation where Downloaded from helper correlates of Ab responses will, undoubtedly, yield useful CD4+ T cells are viral targets. This is particularly relevant as insights for rational vaccine design (25). CCR5 expression is largely confined to CXCR3+CD4+ subsets. In Using the best available experimental model for HIV vaccines— our study, we found that the magnitude of vaccine-induced X3+ the rhesus macaque—we conducted an in-depth characteriza- CXCR5+CD4+ T cells was significantly associated with peak viral tion of the CD4+ T cell response and examined its effects on load postinfection. This would suggest that rapid recall of vaccine + + + + Env B cell responses in the context of a DNA/MVA gp140 induced X3 CXCR5 CD4 T cells upon Ag re-exposure results in http://www.jimmunol.org/ vaccine regimen. Our data allow for three main conclusions; first, generation of target cells for viral replication (31). These data DNA/MVA vaccine induces CXCR5+CD4+ T cells with B cell indicate that in the context of a Th1-skewed immunization regi- + helper potential, which circulate at the peak of immune response. men, CXCR3 TFH cells favor Ab responses but could skew to- Second, vaccine-induced CXCR5+CD4+ T cells are skewed toward ward higher peak viremia. expression of CXCR3, which is augmented by coimmunization Our findings underscore a pressing need to better understand with alum-adjuvanted gp140 protein. Third, CXCR3 expression on the effect of adjuvanted Env subunit protein immunizations on CXCR5+ LN-resident CD4+ T cells is strongly associated with Ab CD4+ T cell helper responses in humans. Protein immunizations persistence and with Ab avidity. The data offer an important pa- augment CD4+ responses and typically do not raise/boost CD8 rameter with which to predict Env memory responses and sug- responses (32). The generation of CD4 viral target cells that are by guest on September 27, 2021 gest that strategies to target the CXCR5+CXCR3+ subset could efficiently recalled upon Ag re-exposure in absence of rapid CD8 provide a means to enhance Env titers. However, an association recall responses could favor initial viral replication. There is ev- between vaccine-raised CXCR5+CXCR3+ cells and peak viral idence supporting this paradigm—a previous macaque study by load, albeit modest, suggests that these cells could represent Buge et al. (33) demonstrated that inclusion of alum-adjuvanted targets for viral replication and predispose to higher acute viremia gp120 boost to a DDM vaccination regimen resulted higher peak upon infection. Taken together, these results provide a strong viremia following a simian HIV challenge compared with DDM rationale to investigate and delineate protein-adjuvant driven alone. Several studies have shown that a combination regimen of CXCR5+CXCR3+CD4+ T cell responses in HIV vaccine studies to Env subunit proteins and viral vectored vaccines does not improve better understand CD4 helper correlates of vaccine efficacy and control of viremia compared with subunit protein immunization viral control. alone (32, 34). In light of the study by Buge et al. and the present The chemokine expression profile of CD4+ T cells typifies findings, it would be important to include a viral vector alone differentiation status and is regulated by the antigenic stimulus vaccine arm to determine whether of addition of adjuvanted pro- and the resulting inflammatory response (7). MVA is a potent tein boost results in higher peak viremia. This is critical as ex- inducer of IFN-g production in DCs (26), and significant systemic tended boosting regimens with subunit proteins are being tested in induction of IFN-g–induced protein 10, one of the ligands of RV144 vaccinees as a means to overcome waning humoral im- CXCR3, is observed after primary and booster MVA immuniza- mune responses over time. Although multiple booster immuni- tion in rhesus macaques (27). Because CXCR3 is induced by zations offer the advantage of augmenting magnitude and quality IFNs, the generation of CXCR3 skewed CD4 response after MVA of Ab responses, their potential to engender target CD4+ T cells immunization is anticipated. Notably, this phenotype was aug- without boosting CD8 responses could tip the balance in favor of mented by addition of alum-adjuvanted gp140 and was accom- viral replication. In this context, characterizing and understanding panied by an increase in Env-specific IFNg+,IL-4+ responses. CD4 responses in terms of CXCR5 and CXCR3 expression within Induction of a Th1 bias with alum, which is considered largely genital and rectal mucosa, the major portals of HIV entry could as a Th2 adjuvant, is surprising but is not without precedence. provide significant insights into their roles in acquisition of in- Studies in mice show that OVA in alum immunization results fection. in CXCR3 expression on ASCs and GC B cells, which is me- In conclusion, our data present an integrated view of CD4 helper diated by IFNg+ Ag-specific CD8+ T cells (28, 29). Our data determinants of Env Ab responses in a multicomponent HIV show that Th1 and Th1-skewed CXCR5+ recall responses primed vaccine in rhesus macaques. The study adds to our understanding of by preceding DNA/MVA immunizations were reacquired after vaccine-induced immune responses; further research is needed to the second MVA boost and were not inhibited by alum adju- determine the application of our findings to other leading poxvirus vant. This phenotype, likely driven by vaccine-mediated induction vectors and to HIV vaccine outcomes in humans. The Journal of Immunology 1005

Acknowledgments Vaccine-induced plasmablast responses in rhesus macaques: phenotypic char- acterization and a source for generating antigen-specific monoclonal antibodies. We thank the Yerkes Division of Research Resources, Stephanie Ehnert, J. Immunol. Methods 416: 69–83. Christopher Souder, Robert Sheffield, and all the animal care staff for 15. Montefiori, D. C. 2005. Evaluating neutralizing antibodies against HIV, SIV, and immunizations, infections sample collections, and animal care. We thank SHIV in luciferase reporter gene assays. Curr. Protoc. Immunol. 64: 12.11: Drs. Elizabeth Strobert, Sherrie Jean, and veterinary staff for veterinary 12.11.1–12.11.17. 16. Schaerli, P., K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, and B. Moser. counsel. We also thank the Emory Flow Cytometry Core, Barbara Cervasi, 2000. CXC chemokine receptor 5 expression defines follicular homing T cells and Kiran Gill for cell sorting. We thank Nancy Miller for SIV251chal- with B cell helper function. J. Exp. Med. 192: 1553–1562. lenge stock; Patricia Earl and Jeffrey Americo for providing the MVA 17. Chtanova, T., S. G. Tangye, R. Newton, N. Frank, M. R. Hodge, M. S. Rolph, virus; the National Institutes of Health AIDS Research and Reference and C. R. Mackay. 2004. T follicular helper cells express a distinctive tran- scriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide Reagent Program for the provision of peptides; the Emory University Cen- help for B cells. J. Immunol. 173: 68–78. ter for AIDS Research Virology Core, Benton Lawson, and Melon T. Nega 18. Yusuf, I., R. Kageyama, L. Monticelli, R. J. Johnston, D. Ditoro, K. Hansen, for assays on viral load; Shane Crotty and Colin Havenar-Daughton for B. Barnett, and S. Crotty. 2010. Germinal center T follicular helper cell IL-4 critical input regarding CXCR5 staining in rhesus samples; Chris Ibegbu, production is dependent on signaling lymphocytic activation molecule receptor (CD150). J. Immunol. 185: 190–202. Rama S. Akondy, and Vijayakumar Velu for suggestions on Abs for flow 19. Petrovas, C., T. Yamamoto, M. Y. Gerner, K. L. Boswell, K. Wloka, E. C. Smith, cytometry; Michael J. Sabula for technical help; and Reben Rahman for D. R. Ambrozak, N. G. Sandler, K. J. Timmer, X. Sun, et al. 2012. CD4 T assistance with figures. follicular helper cell dynamics during SIV infection. J. Clin. Invest. 122: 3281– 3294. 20. Haynes, N. M., C. D. Allen, R. Lesley, K. M. Ansel, N. Killeen, and J. G. Cyster. Disclosures 2007. Role of CXCR5 and CCR7 in follicular Th cell positioning and appear- ance of a programmed cell death gene-1high germinal center-associated sub- R.R.A. is the coinventor of DNA/MVA technology that has been licensed population. J. Immunol. 179: 5099–5108. to Geovax, Inc. by Emory University. The other authors have no financial 21. Wrammert, J., K. Smith, J. Miller, W. A. Langley, K. Kokko, C. Larsen, Downloaded from conflicts of interest. N. Y. Zheng, I. Mays, L. Garman, C. Helms, et al. 2008. Rapid cloning of high- affinity human monoclonal antibodies against influenza virus. Nature 453: 667– 671. 22. Wrammert, J., N. Onlamoon, R. S. Akondy, G. C. Perng, K. Polsrila, References A. Chandele, M. Kwissa, B. Pulendran, P. C. Wilson, O. Wittawatmongkol, et al. 1. Slifka, M. K., M. Matloubian, and R. Ahmed. 1995. Bone marrow is a major site 2012. Rapid and massive virus-specific plasmablast responses during acute of long-term antibody production after acute viral infection. J. Virol. 69: 1895– dengue virus infection in humans. J. Virol. 86: 2911–2918.

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A 100 ***

13% 31% 80 + 60

40 % CD95 FSC-A FSC-A 30% 20 CD4 Ki-67 CD3 0 - + FSC-H CD20 CD8 CD95 CXCR5 CXCR5 CXCR5 B Ki-67-, CD95+ CD4+ *** 80 31% 25% + 60 CD4 + 40 **

20 20% 22% % CD95 CXCR3 0 CXCR5 X3- X3+ X3- X3+ CXCR5+ CXCR5-

Supplemental Figure 1: Distribution of CXCR5 subsets on memory CD4 T cells (A) Gating strategy to examine CD4+ T cells and distribution of CXCR5 on quiescent memory CD4 T cells (B) Distribution of X5 and X3 subsets among non-cycling memory cells and frequencies shown in scatter plot.

Supplemental Figure 2

DDMM A 8 15 8 DDMM-Pro 6 10 2 4 r = -0.6 (% CD4) (% CD4) (% * FH FH 2 0.5 5 GC T GC r = 0.52 T GC

** 0 ns CD20) (% cells B GC 0 0 5 10 15 0 5 10 15 40 60 80 100 PD-1+ T (% CD4) CXCR5-, PD-1+ (% CD4) CXCR5+ PD-1+FH (% CD4) - CXCR3 (% GC TFH)

B

8 15 15 ns ns ns DDMM

6 DDMM-Pro

(% CD4) (% 10 10 +

(% CD4) (% 4

FH 5 5

2 CXCR5 + GC B (% CD20) (% B GC GC T GC 0 0 0 1 10 PD-1 1 10 1 10 Ki-67+ CXCR5-

+ + C GC TFH X5 PD-1 250 1500 X5+ PD-1+ RPq13 (0.3) 1250 RVz12 (0.7) RWf14 (5.2) CD4 subsets co-cultured 1000 naive GC TFH with autologous RGf14 (9) memory B cells + SEB 125 750 40 7 days 20 CXCR3 IgG (fold over naive) over (fold IgG PD-1 0 0 CXCR5+ CD4+, naive CD4+ shown X3- X3+ X3- X3+ as overlay in grey

+ - Supplemental Figure 2: Association between lymph node CXCR5 and CXCR5 subsets. GC TFH cells positively correlate with (A) CXCR5+ PD1+ cells but do not associate with CXCR5-, PD-1+ CD4+ T - cells. Right panel in A shows inverse association between CXCR3 GC TFH cells and GC B cell + - nd frequencies. (B) Ki-67 CXCR5 CD4 T cells at peak post 2 MVA do not correlate with GC TFH cells, CXCR5+ PD-1+ cells, and with GC B cells. (C) Indicated CD4 subsets from lymph node at 2 weeks post 2nd MVA were FACS sorted and co-cultured with autologous memory B cells in the presence of SEB. - + + + IgG in supernatant is expressed in X3 and X3 GC TFH and CXCR5 PD1 subsets as fold increase over IgG in naive-B cell co-cultures. Data are shown for 4 animals assayed independently and values in parenthesis indicate IgG in supernatant (pg/mL) in naive-B cell co-cultures. Supplemental Figure 3

A B C DDMM DDMM-Pro 500 100 r = 0.6 100 p < 0.001 ) 80 FH 10 250 60 (% T (%

+ 40 per 10 ^6 PBMCs ) DDMM-Pro ( r= 0.68 gp140 IgG ASCs 1 CXCR3 20 p < 0.05

0 IgG gp140 in change Fold 0 1 2 0 Contraction Maintenance Env IFN- + CD4) 60 70 80 90 100 γ (% Wk 2/Wk 10 Wk 10/Wk 20 Avidity Index (M2 Wk 20)

Supplemental Figure 3: Determinants of vaccine-induced B cell responses. (A) Gp140 ASC response correlate with Env IFNγ+ CD4 responses (B) Antibody titers undergo contraction between week 2 to week 10 after 2nd MVA boost and are largely maintained from week 10 to week 20 (C)

CXCR3 expression on TFH cells directly correlates with antibody avidity. Supplemental Figure 4 Week 3 post-infection A B C

107 15 r = 0.47 ) 100 r = 0.55

r = 0.43 * FH ** CD4 ) * + 80 106 10 60

5

10 (% GC T + 40 5

104 cells (% Fas 20 FH SIV RNA SIVcopies/ml RNA 103 0 CXCR3 0 0 20 40 60 GC T 0 20 40 60 0 20 40 60 Ki-67+(% CD8)

+ Supplemental Figure 4: Association between Ki-67 CD8 T cell responses and GC TFH cell responses at 3 weeks post-infection. Frequency of Ki-67+ CD8 T cells in blood correlate with + concurrent measurements of (A) peak plasma viral load (B) GC TFH cells (C) CXCR3 GC TFH cells.