Detection of Ag-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of Ag-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be stingy cytokine producers, fundamentally different from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation-induced marker (AIM) methodology to identify Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus–specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4+ T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine–specific CD4+ T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers.

This article is featured in In This Issue, p.679

Germinal center (GC) T follicular helper (Tfh) cells are key drivers needed to generate a GC response (1). Within the GC are resident GC B cells, which have the capacity to become memory B cells and plasma cells with proper instruction (2). GC Tfh cells instruct neighboring GC B cells to undergo class-switch recombination and affinity maturation. These cells can then differentiate into memory B cells and plasma cells with the capacity to produce affinity-matured class-switched Igs. The instruction received by the GC B cells arises from interactions with receptors on Ag-specific GC Tfh cells and cytokines produced by these cells. Receptors for cognate GC Tfh:GC B cell interactions include programmed death 1/programmed death ligand 1 (PD-1/PD-L1), ICOS/ICOSL, CD40/CD40L, SLAM family receptors, and OX40/OX40L (3). IL-21, IL-4, and CXCL13 are the canonical secreted molecules of Tfh help to B cells (49).

Tfh cells have been associated with protective roles in human infectious disease (9, 10), vaccines (11, 12), and cancer (13, 14). Thus, quantifying and understanding these cells is important for biomedical research. In infections, Ag-specific GC Tfh cells are necessary to provide appropriate instruction to GC B cells for the development of T-dependent neutralizing or opsonizing Abs. However, detection of Ag-specific GC Tfh cells has been very difficult (15). This appears to be related to GC Tfh cells producing little cytokine. This problem likely stems from the intrinsic biology of a GC Tfh cell, which is to instruct GC B cells in direct physical contact, therefore not requiring large amounts of cytokine production. Repeated and cyclical interaction with Ag-specific GC Tfh fuels the selection of GC B cells with affinity-matured B cell receptors, but this evolutionary selection process can occur only if the GC Tfh cell help is selective, and thus a GC Tfh cell bathing an entire GC in cytokines would likely be counterproductive.

GCs only exist in lymphoid tissues and tertiary lymphoid structures. GC B cells and GC Tfh cells are not present in peripheral blood. Accordingly, GC biology must be studied using lymphoid tissue. Human tonsil serves as an accessible lymphoid tissue to study human Tfh and GC responses. We therefore explored approaches to identify human tonsillar Ag-specific GC Tfh cells. In doing so, we developed a cytokine-independent method (AIM) for detection of Ag-specific GC Tfh cells. Using the AIM methodology, we determined that conventional cytokine staining missed 98% of human Ag-specific GC Tfh cells. We further determined that AIM is a highly sensitive technique valuable for detecting human CD4+ T cells specific for a range of viral and bacterial Ags.

Fresh tonsils were obtained from pediatric donors undergoing tonsillectomy at Rady Children’s Hospital or the Naval Medical Center. Informed consent was obtained from all donors under protocols approved by the institutional review boards (IRBs) of the University of California, San Diego, the La Jolla Institute for Allergy and Immunology (LJI), and the Naval Medical Center. Tonsillar mononuclear cells were obtained by homogenizing the tissue using a wire mesh, passage through a cell strainer, and isolation via Ficoll density gradient using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). For the dengue virus (DENV) studies, peripheral blood was obtained from the National Blood Center and approved by the IRBs of both LJI and the Medical Faculty, University of Colombo (16). For the Mycobacterium tuberculosis studies, healthy control subjects or individuals with latent tuberculosis infection (LTBI) were obtained from the University of California, San Diego Antiviral Research Center. LTBI status was confirmed by a positive IFN-γ release assay (IGRA; QuantiFERON-TB Gold In-Tube [Cellestis] or T-SPOT.TB [Oxford Immunotec]), and healthy controls all had a negative IGRA. None of the subjects had received a Bacillus Calmette-Guérin vaccination. For the pertussis studies, individuals who were originally primed with either acellular pertussis (aP) or whole cell pertussis vaccine were from San Diego, California. A subset of these donors was boosted with aP within 3 mo of donation. Informed consent was obtained from all donors and approved by the IRB at LJI. All individuals included in the EBV/CMV studies were assumed to have been exposed to one or both of these viruses.

PBMCs were isolated by density gradient centrifugation, according to manufacturer’s instructions. Cells were suspended in FBS containing 10% dimethyl sulfoxide and cryopreserved in liquid nitrogen.

DENV seropositivity was determined by dengue IgG ELISA as previously described (17). Flow cytometry–based neutralization assays were performed for further characterization of seropositive donors, as previously described (18). Neutralization assays determined that all DENV donors included in this study have experienced infection with more than one serotype.

Peptides were synthesized by A and A (San Diego) as crude material on a 1-mg scale. Individual peptides were resuspended in DMSO, and equal amounts of each peptide were pooled to construct peptide pools. The pools used in this study were DENV [32 peptides, previously defined epitopes (16)], ESAT-6 [22 peptides, 15-mers overlapping by 10 and optimal epitopes (19)], CFP10 [21 peptides, 15-mers overlapping by 10 and optimal epitopes (19)], EBV/CMV [122 previously defined epitopes (20)], and pertussis [132 previously defined epitopes (21)]. ESAT-6 and CFP10 proteins are specific for the M. tuberculosis complex, which includes M. bovis but not the Bacillus Calmette-Guérin vaccine that has the region encoding ESAT-6 and CFP10 deleted (19). The resulting peptide pools of >100 peptides were then lyophilized and resuspended in DMSO to minimize DMSO concentrations.

Cryopreserved tonsillar cells were thawed and cultured in serum-free AIM-V media (Life Technologies, Grand Island, NY) overnight for 18 h. Cells were stimulated with 10 μg/ml heat-inactivated antibiotic-killed Group A Streptococcus, strain 5448. Four hours before staining, cells were incubated with 10 μg/ml brefeldin A. As a positive control, cells were stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin in the presence of brefeldin A for 4 h.

Cells were labeled with fixable viability dye eFluor 780 or eFluor 506 (eBioscience). FACS panels used for the AIM assay are detailed in Supplemental Table I. Anti-human Abs for surface staining, by company, are as follows: eBioscience (San Diego, CA): CD19 e780 (clone HIB19), CD14 e780 (clone 61D3), CD16 e780 (clone eBioCB16), CD8α e780 (clone RPA-T8), CD3 AF700 (clone UCHT1), CD4 allophycocyanin, FITC or e780 (clone RPA-T4), CXCR5 allophycocyanin (clone MU5UBEE), CD25 PE and PE-Cyanine 7 (clone BC96), PD-1 PE-Cyanine 7 (clone eBioJ105), CD45RA e450 (clone HI100); Biolegend (San Diego, CA): CD4 BV650 (clone OKT4), CD8a BV650 (clone RPA-T8), OX40 PECy7 (clone Ber-ACT35), CD45RA BV570 (clone HI100), PD-1 BV785 (clone EH12.2H7), PD-L1 PE (clone 29E.2A3), CCR7 PerCPCy5.5 (clone G043H7); BD Biosciences (Franklin Lake, NJ): CD8a V500 (clone RPA-T8), CD45RA PE-CF594 (clone HI100), CXCR5 BV421 (clone RF8B2), CD19 V500 (clone HIB19), CD14 V500 (clone M5E2), CD25 FITC (clone M-A251). Intracellular cytokine Abs used included: TNF AF488 (clone MAb11), CD40L PerCP-e710 (clone 24-31), IFN-γ PE-Cyanine7 (clone 4S.B3), IL-21 PE (clone eBio3A3-N2), IL-10 PE (clone JES3-9D7), IL-4 PE-Cyanine7 (clone 8D4-8), IL-17F PerCP-eFluor 710 (clone SHLR17) (eBioscience), and IL-13 allophycocyanin (clone JES10-5A2) (Biolegend). The tetramer used was DRB5*01:01 CFP1052–66–conjugated using PE-labeled streptavidin (Tetramer Core Laboratory, Benaroya Research Institute, Seattle WA). Cells were acquired on a BD Fortessa or BD LSRII and analyzed using FlowJo Software, version 9.8 for tonsils and FlowJo Software, version 10 for PBMCs.

Cryopreserved tonsillar cells were thawed and cultured in RPMI 1640 medium containing 10% Human AB serum (Gemini Bio-Products, West Sacramento, CA). As a positive control, cells were stimulated with 100 ng/ml staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL). For Ag-specific proliferation, cells were stimulated with Streptolysin O, which had been previously heat inactivated at 65°C for 20 min (Sigma, St. Louis, MO). Cells were labeled with Cell Trace Violet (CTV; Thermo Scientific, Waltham, MA) and cultured for 96 h in medium supplemented with 4 ng/ml IL-7 (Peprotech, Rocky Hill, NJ). Cells were labeled with fixable viability dye eFluor 780 (eBioscience). Tonsils were sorted using BD FACSAria III to isolate GC Tfh cells (Live CD19, CD14, CD16, CD8a, CD4+CD45RACXCR5hiPD-1hi), mTfh cells (Live CD19, CD14, CD16, CD8a, CD4+CD45RACXCR5+PD-1+), and non-Tfh cells (Live CD19, CD14, CD16, CD8a, CD4+CD45RACXCR5). Sorted cells were incubated at a 1 CD4+ T cell:1 APC ratio with irradiated autologous lymphoblastoid cell lines (LCLs) created from each donor tonsil as APCs. Cells were cultured for 96 h and acquired on BD Fortessa.

Cryopreserved tonsillar cells were thawed and cultured in serum-free AIM-V media (Life Technologies, Grand Island, NY) overnight for 18 h. Cells were stimulated with 10 μg/ml heat-inactivated antibiotic-killed Group A Streptococcus, strain 5448. As a positive control, cells were stimulated with 1 μg/ml SEB (Toxin Technology, Sarasota, FL). Cells were cultured for 18 h and acquired on BD Fortessa. Brefeldin A or monensin or other golgi inhibitors (e.g. Golgi Stop) must not be added to the culture. PBMCs were thawed and cultured in complete RPMI 1640 with 5% human AB serum (Gemini Bio-Products) for 24 h. Cells were stimulated with 2 μg/ml peptide pools or 10 μg/ml PHA.

Ex vivo IFN-γ ELISPOT assays were used for M. tuberculosis peptide pools as previously described (22). Responses were considered positive if the net spot-forming cells per 106 were ≥20, the stimulation index ≥2, and p ≤ 0.05 (Student t test, mean of triplicate values of the response against relevant pools versus the DMSO control). For pertussis, PBMCs were cultured for in vitro expansion by incubating in RPMI 1640 supplemented with 5% human AB serum (Gemini Bioscience), GlutaMAX (Life Technologies), and penicillin/streptomycin (Omega Scientific) at 2 × 106/ml in the presence of individual pertussis Ags; filamentous hemagglutinin (Reagent Proteins), pertactin (Reagent Proteins), formaldehyde fixed pertussis toxin (‘PT’, Reagent Proteins), and fimbriae 2/3 (List Biological Labs), each at 5 μg/ml. Every 3 d, 10 U/ml IL-2 in media were added to the cultures. After 14 d of culture, responses to peptides were measured by IFN-γ and IL-5 dual ELISPOT as previously described (23). Response had to fulfill all three criteria described earlier to be considered positive.

Comparisons between groups were made using the Mann–Whitney U test. Prism 5.0 (GraphPad) was used for all of these calculations.

To identify Ag-specific GC Tfh cells within tonsils, we tested for expression of different cytokines by intracellular cytokine staining (ICS). Ag-experienced CD4+ T cells within lymphoid tissue were categorized as GC Tfh (CD4+CD45RACXCR5hiPD-1hi), follicular mantle Tfh (mTfh, also known as Tfh or pre-Tfh; CD4+CD45RACXCR5+PD-1+), and non-Tfh or effector cells (CD4+CD45RACXCR5) (Fig. 1A). One ubiquitous pathogen is Group A Streptococcus, which is the causative agent for strep throat. Given the prevalence of strep throat within the pediatric population and ∼10–15% asymptomatic carriage rate in healthy children, we reasoned that essentially all individuals have been exposed to this pathogen (2427). We thus tested for production of IL-21 after stimulation with whole heat-inactivated antibiotic-killed Group A Streptococcus. Very few cells produced detectable IL-21 after stimulation with Group A Streptococcus (median <0.1%, Fig. 1A), and the IL-21 mean fluorescence intensity was close to the limit of detection, which was insufficient to allow for confident identification of Streptococcus-specific GC Tfh cells. This is similar to other studies in which Ag-specific GC Tfh responses have been difficult to identify via IL-21 production (15, 28). Streptococcus-specific TCR stimulation did not increase IL-4 production (data not shown). We then tested other cytokines, including TNF, CD40L, IFN-γ, IL-13, IL-17, and IL-10 (Fig. 1B, 1C). There was minimal IFN-γ, IL-13, IL-17, and IL-10 expression by GC Tfh cells in response to Ag stimulation (Supplemental Fig. 1), consistent with previous observations that GC Tfh cells produced minimal Th1, Th2, or Th17 cytokines upon strong PMA/ionomycin stimulation (8, 29, 30). Basal levels of CD40L were high and heterogenous from donor to donor, making identification of Ag-specific cells based on CD40L alone infeasible. Intracellular costaining for TNF and CD40L upregulation identified Streptococcus-specific GC Tfh cells better than any other cytokine or combination of cytokine plus intracellular CD40L (Fig. 1B). Nevertheless, only 0.053% of GC Tfh cells were Streptococcus specific based on TNF and CD40L upregulation. These data prompted us to conclude that cytokine production may significantly underestimate Ag-specific GC Tfh cell frequencies and prompted us to explore cytokine-independent approaches to identify Ag-specific human GC Tfh cells.

FIGURE 1.

Limited cytokine production by GC Tfh cells after Ag stimulation. (A) Representative flow cytometry plot of GC Tfh (CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh (CXCR5CD45RACD4+) cell gating with representative flow cytometry plots of IL-21 production by live GC Tfh cells from three different tonsils. (B) Median intracellular cytokine production of TNF+IL-21+, TNF+IFN-γ+, and TNF+CD40L+ by Streptococcus-specific GC Tfh, mTfh, and non-Tfh cells. Representative FACS plots after 18-h culture of tonsil cells with 10 μg/ml Group A Streptococcus or PMA/ionomycin, as a positive control. Limit of detection denoted by the gray dotted line. Data are from nine donors. The response by Ag-specific cells was background subtracted for each donor. (C) Median intracellular cytokine production of IL-4+IL-13+, IL-10+, and IL-17+ by Streptococcus-specific GC Tfh, mTfh, and non-Tfh cells. Limit of detection denoted by the gray dotted line. Data are from 19 donors.

FIGURE 1.

Limited cytokine production by GC Tfh cells after Ag stimulation. (A) Representative flow cytometry plot of GC Tfh (CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh (CXCR5CD45RACD4+) cell gating with representative flow cytometry plots of IL-21 production by live GC Tfh cells from three different tonsils. (B) Median intracellular cytokine production of TNF+IL-21+, TNF+IFN-γ+, and TNF+CD40L+ by Streptococcus-specific GC Tfh, mTfh, and non-Tfh cells. Representative FACS plots after 18-h culture of tonsil cells with 10 μg/ml Group A Streptococcus or PMA/ionomycin, as a positive control. Limit of detection denoted by the gray dotted line. Data are from nine donors. The response by Ag-specific cells was background subtracted for each donor. (C) Median intracellular cytokine production of IL-4+IL-13+, IL-10+, and IL-17+ by Streptococcus-specific GC Tfh, mTfh, and non-Tfh cells. Limit of detection denoted by the gray dotted line. Data are from 19 donors.

Close modal

We next sought to identify Ag-specific GC Tfh cells by proliferation. GC Tfh cells are not end-stage differentiated cells. GC Tfh cells can migrate within and between GCs (31), proliferate (3234), and become memory cells (1, 33, 3537). Maintenance in the GC Tfh state requires TCR triggering by surrounding GC B cells, as well as costimulation by other activation markers. We therefore assessed GC Tfh proliferation after a 96-h culture of CTV-labeled tonsillar cells in the presence or absence of SEB. We were able to identify SEB-responsive cells based on proliferative responses within the GC Tfh (CD4+CD45RACXCR5hiPD-1hi), mTfh (CD4+CD45RACXCR5intPD-1int), and non-Tfh populations (CD4+CD45RACXCR5) (Fig. 2A). Interestingly, proliferating SEB-responsive cells upregulated CD25 (IL-2Rα) and OX40 (Fig. 2B). OX40 participates in GC Tfh:GC B cell cognate interactions, and thus may be expected to be upregulated in response to Ag stimulation in GCs. However, CD25 upregulation was surprising because CD25 expression has been shown to be quite low on differentiating Tfh cells (38, 39) and IL-2 has been shown to inhibit Tfh differentiation both in mice and in humans (4043). Human GC Tfh cells express low amounts of CD25 ex vivo (Supplemental Fig. 2). We speculate that either supraphysiological quantities of peptide:MHC (p:MHC) complex in vitro drive CD25 expression in these restimulation conditions that would not be induced in vivo, or negative feedback loops exist in vivo to prevent high CD25 expression but those mechanisms are not present in vitro.

FIGURE 2.

Proliferation of GC Tfh cells upon restimulation. (A) Representative histograms of proliferating GC Tfh, mTfh, and non-Tfh cells from two different tonsils after stimulation of whole tonsil cells with 100 ng/ml SEB. (B) Representative flow cytometry plots of proliferating GC Tfh, mTfh, and non-Tfh cells from a donor after stimulation of whole tonsil cells with 100 ng/ml SEB. Cells were gated on CTV+CD25+ or CTV+OX40+. FACS plots are representative of a total of 11 samples from 2 independent experiments. (C) Representative flow cytometry plots of sorted GC Tfh (live CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh cells (CXCR5CD45RACD4+) from two different tonsils. Cells were cocultured with autologous irradiated EBV-transformed LCLs for 96 h, stimulated with 100 ng/ml SEB, and analyzed for CTV+CD25+ or CTV+OX40+ expression. FACS plots show only 20% of collected events, for easier visualization. FACS plots are representative of a total of 21 samples from 6 independent experiments.

FIGURE 2.

Proliferation of GC Tfh cells upon restimulation. (A) Representative histograms of proliferating GC Tfh, mTfh, and non-Tfh cells from two different tonsils after stimulation of whole tonsil cells with 100 ng/ml SEB. (B) Representative flow cytometry plots of proliferating GC Tfh, mTfh, and non-Tfh cells from a donor after stimulation of whole tonsil cells with 100 ng/ml SEB. Cells were gated on CTV+CD25+ or CTV+OX40+. FACS plots are representative of a total of 11 samples from 2 independent experiments. (C) Representative flow cytometry plots of sorted GC Tfh (live CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh cells (CXCR5CD45RACD4+) from two different tonsils. Cells were cocultured with autologous irradiated EBV-transformed LCLs for 96 h, stimulated with 100 ng/ml SEB, and analyzed for CTV+CD25+ or CTV+OX40+ expression. FACS plots show only 20% of collected events, for easier visualization. FACS plots are representative of a total of 21 samples from 6 independent experiments.

Close modal

Because CD4+ T cells could potentially change phenotype during the 18-h in vitro culture, we sought to determine whether the proliferating SEB-responsive cells identified as CXCR5hiPD-1hi after proliferation were truly GC Tfh cells. To establish that we were observing proliferating GC Tfh cells after 96 h in culture, separate GC Tfh, mTfh, and non-Tfh cell cultures were required. We created autologous LCLs for each tonsil donor as APCs. GC Tfh, mTfh, and non-Tfh cells were FACS sorted from donors and cultured with autologous LCL in the presence or absence of SEB. Proliferating GC Tfh cells highly upregulated both CD25 and OX40 in response to SEB, a potent TCR stimulus (Fig. 2C). CD25 and OX40 were also upregulated on SEB-responsive mTfh and non-Tfh cells (Fig. 2C). Thus, Ag-specific GC Tfh could potentially be identified by proliferation in response to Ag. We observed extensive SEB-responsive proliferation in the GC Tfh sorted populations, as the LCLs had much improved Ag-presenting capacity over primary B cells. Moreover, small quantities of IL-7 were added to enhance survival during culture, at a concentration that does not promote T cell proliferation. Subsequent CTV proliferation experiments with whole Group A Streptococcus, heat-inactivated Streptolysin O (a serodiagnostic marker for recent Group A Streptococcus infection), or diphtheria CRM197 were unable to consistently identify GC Tfh cells by proliferation because of variable low-level nonspecific proliferation (data not shown).

Tfh help is the primary positive selection step in a GC (1, 2). Tfh cells selectively provide help to the B cells with the most antigenic peptides, which are the high-affinity B cells that have bound and endocytosed the most Ag (44, 45). The amount of help provided by the Tfh cell directly translates to the number of cell divisions and mutations a GC B cell will undergo in the dark zone in a single selection cycle (46, 47). One of the functional challenges for Tfh cells in GCs is that they are constantly exposed to Ag. They must therefore be able to distinguish between GC B cells with modest differences in their numbers of p:MHC and respond by providing help preferentially to the cognate B cells presenting more p:MHC complexes. Thus, we hypothesized that although GC Tfh cells retain sensitive TCR signaling, the outcome of the TCR detection of cognate Ag is not reflected by cytokine production, as GC Tfh cells may produce only infinitesimal quantities of cytokine necessary to signal the GC B cell in synaptic contact. We therefore explored whether changes in expression of proteins other than cytokines might be more consistent indicators of TCR signaling by GC Tfh cells. Based on our observation that proliferating GC Tfh cells upregulate OX40 and CD25 in vitro, we sought to determine whether CD25 and OX40 were consistently upregulated on Ag-specific GC Tfh cells in response to TCR stimulation at more proximal time points. After an 18-h SEB stimulation, we observed a clear and robust population of CD25+OX40+ GC Tfh cells, consistent with the expected frequency of cells expressing SEB-reactive TCRβ-chains (Fig. 3B). Each tonsil varied in the frequency of CD25+OX40+ GC Tfh cells observed after 18 h in vitro in the absence of exogenous Ag, with a mean of 4.1%. We speculated that the CD25+OX40+ GC Tfh cells observed in the absence of exogenous Ag were responding to Ag presented by GC B cells and other APCs in the in vitro culture, as each tonsil contains Ag (Supplemental Fig. 3A). SEB stimulation of GC Tfh cells from 14 donors yielded a mean frequency rate of 44% CD25+OX40+ SEB-responsive GC Tfh cells (Fig 3C) [At a concentration of 1 μg/ml, SEB stimulates proliferation of lower-affinity TCR Vβ families (48)]. Thus, CD25 and OX40 were reproducible indicators of GC Tfh cell TCR stimulation.

FIGURE 3.

AIM induction by GC Tfh cells. (A) Experimental design for human tonsillar AIM assay. (B) Representative flow cytometry plots of CD25+OX40+ upregulation by live GC Tfh cells (CXCR5hiPD-1hiCD45RACD4+) after 18-h stimulation with 1 μg/ml SEB. (C) Median CD25+OX40+ expression by live GC Tfh cells after stimulation with SEB. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (D) Comparison of Streptococcus-specific GC Tfh by ICS (TNF+CD40L+) and AIM (CD25+OX40+). The percentage Streptococcus-specific GC Tfh responses were background subtracted. Data are from nine samples from two independent experiments. The response by Ag-specific cells was background subtracted for each donor. (E) CD25+OX40+ GC Tfh cells are also PD-L1+. Representative FACS plots of CD25+PD-L1+ coexpression after stimulation with either Group A Streptococcus or SEB. Streptococcus-specific PD-L1+CD4+ GC Tfh cells (black dots) were overlaid onto Streptococcus-specific CD25+OX40+ GC Tfh cells (gray contour plot). SEB-responsive PD-L1+CD4+ GC Tfh cells (black contour plot) were overlaid onto SEB-responsive CD25+OX40+ GC Tfh cells (gray dots). FACS plots shown are representative of 14 samples from 2 independent experiments. (F) Representative flow cytometry overlay plots demonstrating CD25+OX40+ cells (black dots) within each sorted population (gray dots) for each condition (Streptococcus-stimulated and SEB-stimulated). This is representative of three independent experiments consisting of seven donors.

FIGURE 3.

AIM induction by GC Tfh cells. (A) Experimental design for human tonsillar AIM assay. (B) Representative flow cytometry plots of CD25+OX40+ upregulation by live GC Tfh cells (CXCR5hiPD-1hiCD45RACD4+) after 18-h stimulation with 1 μg/ml SEB. (C) Median CD25+OX40+ expression by live GC Tfh cells after stimulation with SEB. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (D) Comparison of Streptococcus-specific GC Tfh by ICS (TNF+CD40L+) and AIM (CD25+OX40+). The percentage Streptococcus-specific GC Tfh responses were background subtracted. Data are from nine samples from two independent experiments. The response by Ag-specific cells was background subtracted for each donor. (E) CD25+OX40+ GC Tfh cells are also PD-L1+. Representative FACS plots of CD25+PD-L1+ coexpression after stimulation with either Group A Streptococcus or SEB. Streptococcus-specific PD-L1+CD4+ GC Tfh cells (black dots) were overlaid onto Streptococcus-specific CD25+OX40+ GC Tfh cells (gray contour plot). SEB-responsive PD-L1+CD4+ GC Tfh cells (black contour plot) were overlaid onto SEB-responsive CD25+OX40+ GC Tfh cells (gray dots). FACS plots shown are representative of 14 samples from 2 independent experiments. (F) Representative flow cytometry overlay plots demonstrating CD25+OX40+ cells (black dots) within each sorted population (gray dots) for each condition (Streptococcus-stimulated and SEB-stimulated). This is representative of three independent experiments consisting of seven donors.

Close modal

We next tested whether Streptococcus-specific GC Tfh cells could be detected by CD25 and OX40 expression after stimulation with Group A Streptococcus Ag. Upon stimulation, we observed a robust Streptococcus-specific GC Tfh cell population based on CD25+OX40+ coexpression (Fig. 3D). This activation-induced marker (AIM) assay detected Streptococcus-specific GC Tfh cells at a mean frequency rate of 4.53% (Fig. 3D). In contrast, ICS yielded a mean frequency rate of only 0.053% Streptococcus-specific GC Tfh cells (TNF+CD40L+; Fig. 3D). Background signal present in unstimulated samples was subtracted in all cases. The CXCR5hiPD-1hi cells detected by AIM were GC Tfh cells, as sorting of GC Tfh cells and mTfh cells followed by Group A Streptococcus stimulations showed that the CXCR5 and PD-1 expression profiles of each cell type were maintained after activation (Fig. 3F). The AIM method provided, on average, an 85-fold improvement in sensitivity for quantifying Streptococcus-specific GC Tfh cells. Thus, cytokine-dependent identification missed >98% of Ag-specific GC Tfh cells.

In a separate study, RNAseq analysis of rhesus macaque (Macaca mulatta) lymph node GC Tfh cells revealed that PD-L1 was robustly induced upon TCR stimulation (49). We therefore examined whether PD-L1 was upregulated on Ag-specific GC Tfh cells upon Streptococcus Ag stimulation or SEB stimulation. Similar to CD25+OX40+ coexpression, we observed a population of CD25+PD-L1+ GC Tfh cells after either Group A Streptococcus or SEB stimulation (Fig. 3E). PD-L1 expression was present on 95.7% of Streptococcus-specific GC Tfh cells expressing CD25+OX40+ (Fig. 3E). PD-L1 thus functions as a TCR signaling output after Ag-specific GC Tfh cells stimulation, and PD-L1, OX40, and CD25 are interchangeable for identification of responding GC Tfh cells.

After establishing a highly sensitive method to detect Ag-specific GC Tfh cells, we next wanted to assess the ability of the AIM assay to identify Streptococcus-specific mTfh and non-Tfh. As with GC Tfh cells, we found CD25, OX40, and PD-L1 expression on Streptococcus-specific mTfh and non-Tfh cells upon stimulation with Group A Streptococcus Ag (Fig. 4). Streptococcus-specific non-Tfh cells or effector cells (CD4+CD45RACXCR5) could be identified by the AIM assay as either CD25+OX40+ (Fig. 4A, 4C) or CD25+PD-L1+ (Fig. 4B, 4D). Thus, the cell-surface proteins CD25, OX40, and PD-L1 appear to be activation markers expressed by Ag-specific human CD4+ T cells, independent of whether the cells were GC Tfh cells. Therefore, the AIM assay may be of practical utility for identifying Ag-specific CD4+ T cells in lymphoid tissue, independent of abundant production of a given cytokine.

FIGURE 4.

AIM induction by mTfh and non-Tfh cells. (A) Representative flow cytometry plots of CD25+OX40+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) after stimulation with Group A Streptococcus or 1 μg/ml SEB. Median CD25+OX40+ expression by live mTfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (B) Representative flow cytometry plots of and CD25+PD-L1+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) after stimulation with Group A Streptococcus or 1 μg/ml SEB. Median CD25+PD-L1+ expression by live mTfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (C) Representative flow cytometry plots of CD25+OX40+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+OX40+ expression by live non-Tfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (D). Representative flow cytometry plots of CD25+PD-L1+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+PD-L1+ expression by live non-Tfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor.

FIGURE 4.

AIM induction by mTfh and non-Tfh cells. (A) Representative flow cytometry plots of CD25+OX40+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) after stimulation with Group A Streptococcus or 1 μg/ml SEB. Median CD25+OX40+ expression by live mTfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (B) Representative flow cytometry plots of and CD25+PD-L1+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) after stimulation with Group A Streptococcus or 1 μg/ml SEB. Median CD25+PD-L1+ expression by live mTfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (C) Representative flow cytometry plots of CD25+OX40+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+OX40+ expression by live non-Tfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor. (D). Representative flow cytometry plots of CD25+PD-L1+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+PD-L1+ expression by live non-Tfh after stimulation with Group A Streptococcus. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by Ag-specific cells was background subtracted for each donor.

Close modal

We identified CD25, OX40, and PD-L1 as TCR activation-dependent markers of Ag-specific human GC Tfh cells. Independently, CD25+OX40+ coexpression was explored for detection of memory CD4 T cells to EBV/CMV and Mycobacterium tuberculosis in human peripheral blood (50, 51). Those studies frequently used ∼48-h stimulation periods, which may allow sufficient time for significant T cell proliferation or death, thus skewing the quantitation of responding T cells. In addition, 50% of LTBI cases, which are known to respond to M. tuberculosis Ags in the QuantiFERON-TB Gold assay, failed to be detected upon stimulation with IGRA Ags in one study (50). In this study, using AIM, we identified EBV/CMV-specific CD4+ T cells in PBMCs from healthy individuals presumed seropositive for either virus using previously defined EBV and CMV class II epitopes (16) (Fig. 5A). We assessed the ability of AIM to detect tuberculosis-specific CD4+ T cells using M. tuberculosis–specific proteins CFP10 and ESAT-6. ESAT-6 and CFP10 are M. tuberculosis–specific proteins used in the QuantiFERON-TB Gold clinical test to determine previous exposure to M. tuberculosis (36). Healthy control subjects (QuantiFERON-TB Gold negative) were compared with individuals with LTBI who are by definition QuantiFERON-TB Gold+. In an IFN-γ ELISPOT assay, PBMCs from most LTBI individuals produced IFN-γ in response to both ESAT-6 and CFP10 (Fig. 6A). In the AIM assay, PBMCs from LTBI individuals had a 14.5-fold increase in the percentage of CD25+OX40+ memory CD4+ T cells after stimulation with ESAT-6, and a nearly 40-fold increase after stimulation with CFP10 compared with healthy control subjects (p = 0.027 and p < 0.0001, respectively) (Fig. 6B). Of interest, 100% of LTBI donors were detected as positive by AIM using CFP10, in contrast with ELISPOT (Fig. 6B). Identification of M. tuberculosis–specific and EBV/CMV-specific CD4+ T cells by AIM was specific for Ag-experienced CD4+ T cells (Figs. 5A, 6B). Notably, background noise in the AIM assay was very low when using PBMCs (Figs. 5A, 6B), in contrast with Ag-containing tonsillar tissue (Figs. 2B, 3B), consistent with the background signal in tonsils being due to an Ag-specific CD4+ T cell responding to Ags in tonsil, given that tonsils are a sentinel tissue. There was also undetectable bystander activation in the PBMCs, as there was no upregulation of CD25+OX40+ on CD8+ T cells or naive CD4+ T cells (Figs. 5A, 7, 8A).

FIGURE 5.

Detection of EBV/CMV-specific CD4+ T cells in peripheral blood. (A) Three individuals were tested by AIM for response to a class II EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RO+CD4+), naive CD4+ T cells (CD45ROCD4+), memory CD8+ T cells (CD45RO+CD8+), and naive CD8+ T cells (CD45ROCD8+) were quantified. **p = 0.0079. (B) An LTBI DRB5*01:01 donor with defined DRB5*01:01 M. tuberculosis epitope was stimulated with M. tuberculosis and EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells were quantified. (C) Representative FACS plot demonstrates that EBV/CMV-specific CD4+ T cells (CD25+OX40+) were not positive for M. tuberculosis-tetramer (black dots).

FIGURE 5.

Detection of EBV/CMV-specific CD4+ T cells in peripheral blood. (A) Three individuals were tested by AIM for response to a class II EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RO+CD4+), naive CD4+ T cells (CD45ROCD4+), memory CD8+ T cells (CD45RO+CD8+), and naive CD8+ T cells (CD45ROCD8+) were quantified. **p = 0.0079. (B) An LTBI DRB5*01:01 donor with defined DRB5*01:01 M. tuberculosis epitope was stimulated with M. tuberculosis and EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells were quantified. (C) Representative FACS plot demonstrates that EBV/CMV-specific CD4+ T cells (CD25+OX40+) were not positive for M. tuberculosis-tetramer (black dots).

Close modal
FIGURE 6.

Detection of M. tuberculosis–specific CD4+ T cells in peripheral blood. Eight QuantiFERON-TB Gold+ patients with LTBI and eight QuantiFERON-TB Gold healthy control subjects (HC) were tested for response to a pool of ESAT-6 or CFP10 epitopes. (A) IFN-γ ELISPOT of PBMCs from LTBI patients and HC (**p = 0.0035 for ESAT-6, *p = 0.013 for CFP10). Limit of detection denoted by the gray dotted line. (B) AIM assay (CD25+OX40+) of PBMCs from LTBI patients and HC similarly had higher CD25+OX40+ expression by AIM assay (*p = 0.027 for ESAT-6, ****p < 0.0001 for CFP10). Limit of detection denoted by the gray dotted line. Representative flow cytometry plots of CD25+OX40+ by an LTBI patient and an HC patient.

FIGURE 6.

Detection of M. tuberculosis–specific CD4+ T cells in peripheral blood. Eight QuantiFERON-TB Gold+ patients with LTBI and eight QuantiFERON-TB Gold healthy control subjects (HC) were tested for response to a pool of ESAT-6 or CFP10 epitopes. (A) IFN-γ ELISPOT of PBMCs from LTBI patients and HC (**p = 0.0035 for ESAT-6, *p = 0.013 for CFP10). Limit of detection denoted by the gray dotted line. (B) AIM assay (CD25+OX40+) of PBMCs from LTBI patients and HC similarly had higher CD25+OX40+ expression by AIM assay (*p = 0.027 for ESAT-6, ****p < 0.0001 for CFP10). Limit of detection denoted by the gray dotted line. Representative flow cytometry plots of CD25+OX40+ by an LTBI patient and an HC patient.

Close modal
FIGURE 7.

Detection of DENV-specific CD4+ T cells in peripheral blood. Representative FACS plots of CD4+ and CD8+ T cells after stimulation with DENV peptides or PHA, as a positive control. Cumulative data from five DENV+ and five DENV patients with known expression of the HLA DRB1*0401 molecule. PBMCs were stimulated with dengue DRB1*0401-restricted peptide for 24 h. *p = 0.016, **p = 0.0079.

FIGURE 7.

Detection of DENV-specific CD4+ T cells in peripheral blood. Representative FACS plots of CD4+ and CD8+ T cells after stimulation with DENV peptides or PHA, as a positive control. Cumulative data from five DENV+ and five DENV patients with known expression of the HLA DRB1*0401 molecule. PBMCs were stimulated with dengue DRB1*0401-restricted peptide for 24 h. *p = 0.016, **p = 0.0079.

Close modal
FIGURE 8.

Detection of pertussis-specific CD4+ T cells in peripheral blood using a pertussis peptide megapool. (A) AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RACCR7+CD4+), naive CD4+ T cells (CD45RA+CCR7+CD4+), memory CD8+ T cells (CD45RACCR7+CD8+), and naive CD8+ T cells (CD45RA+CCR7+CD8+) were quantified. ****p < 0.0001. (B) CD25+PD-L1+ memory CD4+ T cells were quantified (****p < 0.0001) in 10 individuals. (C) Quantification of AIM+ (CD25+OX40+) pertussis-specific within CD4+ T cell subsets. Pertussis-specific cells were predominantly in the CD45RACCR7 (**p = 0.0079). (D) Comparison of AIM+ (CD25+OX40+) pertussis-specific and ELISPOT pertussis-specific responses in individuals before and after aP booster vaccination.

FIGURE 8.

Detection of pertussis-specific CD4+ T cells in peripheral blood using a pertussis peptide megapool. (A) AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RACCR7+CD4+), naive CD4+ T cells (CD45RA+CCR7+CD4+), memory CD8+ T cells (CD45RACCR7+CD8+), and naive CD8+ T cells (CD45RA+CCR7+CD8+) were quantified. ****p < 0.0001. (B) CD25+PD-L1+ memory CD4+ T cells were quantified (****p < 0.0001) in 10 individuals. (C) Quantification of AIM+ (CD25+OX40+) pertussis-specific within CD4+ T cell subsets. Pertussis-specific cells were predominantly in the CD45RACCR7 (**p = 0.0079). (D) Comparison of AIM+ (CD25+OX40+) pertussis-specific and ELISPOT pertussis-specific responses in individuals before and after aP booster vaccination.

Close modal

We performed a test of AIM specificity using an M. tuberculosis–specific class II tetramer (HLA DRB5*01:01 CFP1052–66 tetramer). The HLA DRB5*01:01 LTBI donor selected was found to have a very strong M. tuberculosis–specific CD4+ T cell response by AIM (17%; Fig. 6B) and was also EBV/CMV+ (4% EBV/CMV+ CD4+ T cells; Fig. 5A). When PBMCs from the HLA DRB5*01:01 M. tuberculosis+EBV/CMV+ donor were stimulated with the EBV/CMV peptide pool and then stimulated with the M. tuberculosis tetramer, the M. tuberculosis-tetramer–specific CD4+ T cells showed no evidence of bystander activation (Fig. 5B, 5C). Thus, the AIM method specifically detects Ag-specific CD4+ T cells.

We then sought to assess the applicability of AIM for identifying DENV-specific CD4+ T cells or rare pertussis vaccine–specific memory CD4+ T cells in peripheral blood. We first focused on identifying DENV-specific CD4+ T cell responses using PBMCs from healthy individuals from a highly endemic area who were either seropositive (DENV+) or seronegative (DENV) for prior DENV infection. High frequencies of DENV-specific CD4+ T cells have been correlated with a decreased likelihood of developing DENV hemorrhagic fever (16, 52). We were able to discern DENV-specific memory CD4+ T cell responses using the AIM assay (Fig. 7). Using PBMCs from five DENV+ individuals bearing the HLA DRB1*04:01 allele, we were able to detect 0.08–0.94% DENV-specific memory CD4+ T cells after stimulation with dengue DRB1*04:01-restricted epitopes after 24 h in culture, p = 0.016 (16). CD8+ T cells did not respond in the AIM assay to the dengue class II peptides, as expected (Fig. 7).

Pertussis-specific CD4+ T cells have been detected in most individuals only after 14-d expansion of cells in vitro followed by cytokine ELISPOT. We examined whether pertussis-specific CD4+ T cells could be detected directly ex vivo by the AIM assay, as a stringent test of assay sensitivity. Ten donors were tested, and pertussis-specific CD4+ T cells in all 10 donors were detected by AIM (CD25+OX40+: mean = 0.97, range 0.45–2.32%, Fig. 8A; CD25+PD-L1+: mean = 0.36, range 0.0134–2.13%, Fig. 8B). These donors were healthy individuals who had no recent pertussis immunizations. Pertussis-specific CD4+ T cells were memory cells (Fig. 8C) and were found to predominantly (80%) have an effector memory phenotype (CD45RACCR7) (Fig. 8C). Two donors received an aP vaccine boost within a 3-mo period from their pre-aP boost PBMC specimen. The AIM assay detected an increase in pertussis-specific CD4+ T cell responses after the aP vaccination (Fig. 8D). In comparison with a 14-d restimulation ELISPOT assay, the 24-h AIM assay followed the same data trends as the 14-d restimulation ELISPOT assay (Fig. 8D). The AIM assay was simpler and shorter, and allowed for extensive phenotypic cellular characterization at the single-cell level by multiparameter flow cytometry.

We have shown that it is extremely difficult to quantify Ag-specific GC Tfh cells within human tonsils using the traditional ICS method because GC Tfh cells are inherently stingy cytokine producers and make little to no detectable cytokine upon Ag stimulation. However, we determined that Streptococcus-specific GC Tfh cells do retain sensitive TCR activation in response to Ag, resulting in upregulation of CD25, OX40, and PD-L1. We exploited this biology to identify Ag-specific GC Tfh cells with 85-fold greater sensitivity than ICS. Furthermore, we observed that the utility of this approach is not limited to GC Tfh cells but can be extrapolated to other CD4+ T cells within human lymphoid tissue and peripheral blood.

Upon TCR triggering of an Ag-experienced CD4+ T cell, costimulatory molecules and coinhibitory molecules are upregulated (53). The interplay of costimulatory and coinhibitory molecules and which signals dominate are part of Ag-specific CD4+ T cell development (54). OX40 is one such receptor, upregulated on Ag-experienced CD4+ T cells as early 1–4 h upon TCR stimulation, depending on the type of CD4+ T cell and the strength of the p:MHC II complex (3). OX40 on Ag-specific CD4+ T cells interacts with OX40L on APCs to promote CD4+ T cell survival (3). Similarly, CD25 is another activation marker quickly upregulated upon TCR stimulation and sustained after expression of IL-2 in an autocrine or paracrine manner (55). For GC Tfh cells, OX40 upregulation upon Ag stimulation by GC B cells in the follicle was not unexpected as OX40:OX40L constitutes part of the GC Tfh:GC B cell cognate interaction (1). However, CD25 upregulation on GC Tfh cells within the GC follicle is counterintuitive because IL-2 is known to inhibit Tfh differentiation (40, 41). GC Tfh cells expressed low amounts of CD25 directly ex vivo (Supplemental Fig. 2C), even though most GC Tfh cells experience frequent TCR stimulation in GCs (56, 57). Thus, a negative feedback mechanism to limit CD25 expression appears to be engaged in vivo but not in vitro. An alternative explanation is that stimulation with a small amount of Ag in vitro is supraphysiologic and does not recapitulate what is seen in vivo, permitting CD25 upregulation. Many receptors that inhibit GC Tfh cells are expressed by GC Tfh cells (58, 59), pointing to a central role of inhibitory pathways in maintaining appropriate Tfh biology in GCs.

PD-L1 upregulation on Streptococcus-specific GC Tfh was also unexpected. A role of PD-L1 on GC Tfh cells has not been described. PD-L1 expression is not restricted to myeloid cells and nonhematopoietic cells; PD-L1 expression has previously been observed on both CD4+ and CD8+ T cells in certain conditions (60). PD-L1 has been found on activated CD4+ T cells in rheumatoid arthritis patients (61, 62). For GC Tfh cells, interpretation of the function of PD-L1 is complicated because these cells also express PD-1. The PD-1/PD-L1 axis is involved in GC Tfh interactions with GC B cells, with PD-1 highly expressed by GC Tfh and PD-L1 expressed by GC B cells (63). High PD-1 expression prevents GC Tfh cell proliferation, allowing the GC to function properly, as the purpose of the GC is to drive GC B cell proliferation and selection while maintaining a relatively constant number of GC Tfh cells (34, 58, 64). Expression of PD-L1 by an activated GC Tfh cell may inhibit neighboring PD-1hi GC Tfh cells in a bystander manner, as an additional mechanism to limit excessive GC Tfh cell activation and/or proliferation.

The AIM method exhibited greatly increased sensitivity for detecting Streptococcus-specific GC Tfh cells in comparison with ICS. IL-21 protein production was rarely detected by ICS of GC Tfh cells after Ag stimulation. Detection of IL-21 protein from Ag-specific human GC Tfh cells within lymphoid tissue has been difficult, with perhaps only one reported success (15). In macaque studies, there has been little to no detection by ICS of IL-21 protein induction in Ag-specific GC Tfh cells (28, 65). IL-21 RNA can be detected in stimulated human GC Tfh cells, and an IL-21 fluorescent reporter mouse detects shifts in fluorescent reporter protein expression after Ag stimulation in vivo (56), consistent with Il21 mRNA induction. Thus, it appears that GC Tfh cells are intrinsically stingy producers of IL-21 protein. Note that this cytokine biology of GC Tfh cells within lymphoid tissue does not extend to circulating resting memory Tfh cells or circulating recently activated Tfh cells in blood (9, 11, 66, 67), which are not GC Tfh cells. GC Tfh cells are not in peripheral blood. CD4+ T cells in peripheral blood much more readily produce cytokine than do bona fide GC Tfh cells in lymphoid tissues. Peripheral Tfh cells consist of multiple populations, but the vast majority (∼99%) (9) can be categorized as resting central memory Tfh cells (based on their resting phenotype), and a small population (∼1–5%) can be characterized as recently activated Tfh-like cells, based on their activated phenotype (ICOS+PD-1hiKi67+, but no detectable Bcl6 protein, or Maf, or CD200). A significant fraction of the resting central memory Tfh cells circulating through blood readily produce cytokines, including IL-21, as shown by multiple laboratories. GC Tfh cells are quite different, as they are more differentiated cells, in a very different environment, and have much more restricted cytokine production.

As with ICS, the AIM technique is also subject to the potential concern of possible bystander activation. We have demonstrated a lack of bystander activation by several independent tests. Class II peptide pools DENV specific for HLA DRB1*04:01 did not induce CD25+OX40+ expression on CD8+ T cells. Second, minimal background was observed in PBMCs. Finally, we demonstrated a lack of M. tuberculosis-tetramer+ cells among the CD25+OX40+ CD4+ T cells stimulated with an unrelated Ag.

Although identification of Ag-specific GC Tfh cells by CD25, OX40, and PD-L1 coexpression is novel, CD25+OX40+ coexpression has previously been studied in peripheral blood (50, 51, 68, 69). We show in this study that DENV-specific and pertussis-specific memory CD4+ T cells are sensitively detected in peripheral blood by this approach, and we show controls for the specificity of the methodology. For DENV infections, the AIM assay could potentially be useful in detecting Ag-specific responses irrespective of the functional cytokine specificity. Indeed it has been reported that severe DENV infection–associated pathology is linked to complex patterns of cytokine production, and the AIM assay will ensure detection regardless of the effector specificity of the DENV-specific T cells (52). In addition, given the resurgence of pertussis in the United States, the AIM assay could potentially identify children with waning vaccine responses who may warrant more frequent booster vaccinations via ex vivo analysis of their pertussis-specific CD4+ T cell responses (7072).

In summary, Ag-specific GC Tfh, mTfh, and non-Tfh cells can be detected with great sensitivity within secondary lymphoid tissue using AIM. This method will likely be valuable for understanding GC biology as it applies to infections, cancer, and autoimmune disease. The wide applicability of this assay also makes this ideal for detecting rare ex vivo human Ag-specific CD4+ T cell responses. Moreover, because AIM is a live cell assay, it is well suited in combination for downstream applications.

We thank Ericka Anderson and Victor Nizet (University of California, San Diego) for providing the initial Group A Streptococcus stock, and the members of the Flow Cytometry core at LJI for technical support. We thank Daniel Kaufmann (University of Montreal) for valuable discussions.

This work was supported by the Thrasher Research Fund for an Early Career Award, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant 1UM1AI100663, La Jolla Institute for Allergy and Immunology Institutional Funds, and by National Institutes of Health Training Grants 5T32AI007036-35 and 5T32AI007384-25.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AIM

activation-induced marker

aP

acellular pertussis

CTV

Cell Trace Violet

DENV

dengue virus

GC

germinal center

ICS

intracellular cytokine staining

IGRA

IFN-γ release assay

IRB

institutional review board

LCL

lymphoblastoid cell line

LJI

La Jolla Institute for Allergy and Immunology

LTBI

latent tuberculosis infection

mTfh

mantle Tfh

PD-1

programmed death 1

PD-L1

programmed death ligand 1

p:MHC

peptide:MHC

SEB

staphylococcal enterotoxin B

Tfh

T follicular helper.

1
Crotty
S.
2014
.
T follicular helper cell differentiation, function, and roles in disease.
Immunity
41
:
529
542
.
2
Victora
G. D.
,
Nussenzweig
M. C.
.
2012
.
Germinal centers.
Annu. Rev. Immunol.
30
:
429
457
.
3
Croft
M.
2010
.
Control of immunity by the TNFR-related molecule OX40 (CD134).
Annu. Rev. Immunol.
28
:
57
78
.
4
Havenar-Daughton
C.
,
Lindqvist
M.
,
Heit
A.
,
Wu
J. E.
,
Reiss
S. M.
,
Kendric
K.
,
Bélanger
S.
,
Kasturi
S. P.
,
Landais
E.
,
Akondy
R. S.
, et al
IAVI Protocol C Principal Investigators
.
2016
.
CXCL13 is a plasma biomarker of germinal center activity.
Proc. Natl. Acad. Sci. USA
113
:
2702
2707
.
5
Linterman
M. A.
,
Beaton
L.
,
Yu
D.
,
Ramiscal
R. R.
,
Srivastava
M.
,
Hogan
J. J.
,
Verma
N. K.
,
Smyth
M. J.
,
Rigby
R. J.
,
Vinuesa
C. G.
.
2010
.
IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses.
J. Exp. Med.
207
:
353
363
.
6
Yusuf
I.
,
Kageyama
R.
,
Monticelli
L.
,
Johnston
R. J.
,
Ditoro
D.
,
Hansen
K.
,
Barnett
B.
,
Crotty
S.
.
2010
.
Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).
J. Immunol.
185
:
190
202
.
7
Reinhardt
R. L.
,
Liang
H. E.
,
Locksley
R. M.
.
2009
.
Cytokine-secreting follicular T cells shape the antibody repertoire.
Nat. Immunol.
10
:
385
393
.
8
Ma
C. S.
,
Suryani
S.
,
Avery
D. T.
,
Chan
A.
,
Nanan
R.
,
Santner-Nanan
B.
,
Deenick
E. K.
,
Tangye
S. G.
.
2009
.
Early commitment of naïve human CD4(+) T cells to the T follicular helper (T(FH)) cell lineage is induced by IL-12.
Immunol. Cell Biol.
87
:
590
600
.
9
Locci
M.
,
Havenar-Daughton
C.
,
Landais
E.
,
Wu
J.
,
Kroenke
M. A.
,
Arlehamn
C. L.
,
Su
L. F.
,
Cubas
R.
,
Davis
M. M.
,
Sette
A.
, et al
International AIDS Vaccine Initiative Protocol C Principal Investigators
.
2013
.
Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.
Immunity
39
:
758
769
.
10
Obeng-Adjei
N.
,
Portugal
S.
,
Tran
T. M.
,
Yazew
T. B.
,
Skinner
J.
,
Li
S.
,
Jain
A.
,
Felgner
P. L.
,
Doumbo
O. K.
,
Kayentao
K.
, et al
.
2015
.
Circulating Th1-cell-type Tfh cells that exhibit impaired B cell help are preferentially activated during acute malaria in children.
Cell Reports
13
:
425
439
.
11
Bentebibel
S. E.
,
Lopez
S.
,
Obermoser
G.
,
Schmitt
N.
,
Mueller
C.
,
Harrod
C.
,
Flano
E.
,
Mejias
A.
,
Albrecht
R. A.
,
Blankenship
D.
, et al
.
2013
.
Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination.
Sci. Transl. Med.
5
:
176ra32
.
12
Vargas-Inchaustegui
D. A.
,
Demers
A.
,
Shaw
J. M.
,
Kang
G.
,
Ball
D.
,
Tuero
I.
,
Musich
T.
,
Mohanram
V.
,
Demberg
T.
,
Karpova
T. S.
, et al
.
2016
.
Vaccine induction of lymph node-resident simian immunodeficiency virus Env-specific T follicular helper cells in rhesus macaques.
J. Immunol.
196
:
1700
1710
.
13
Gu-Trantien
C.
,
Loi
S.
,
Garaud
S.
,
Equeter
C.
,
Libin
M.
,
de Wind
A.
,
Ravoet
M.
,
Le Buanec
H.
,
Sibille
C.
,
Manfouo-Foutsop
G.
, et al
.
2013
.
CD4⁺ follicular helper T cell infiltration predicts breast cancer survival.
J. Clin. Invest.
123
:
2873
2892
.
14
Bindea
G.
,
Mlecnik
B.
,
Tosolini
M.
,
Kirilovsky
A.
,
Waldner
M.
,
Obenauf
A. C.
,
Angell
H.
,
Fredriksen
T.
,
Lafontaine
L.
,
Berger
A.
, et al
.
2013
.
Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.
Immunity
39
:
782
795
.
15
Lindqvist
M.
,
van Lunzen
J.
,
Soghoian
D. Z.
,
Kuhl
B. D.
,
Ranasinghe
S.
,
Kranias
G.
,
Flanders
M. D.
,
Cutler
S.
,
Yudanin
N.
,
Muller
M. I.
, et al
.
2012
.
Expansion of HIV-specific T follicular helper cells in chronic HIV infection.
J. Clin. Invest.
122
:
3271
3280
.
16
Weiskopf
D.
,
Bangs
D. J.
,
Sidney
J.
,
Kolla
R. V.
,
De Silva
A. D.
,
de Silva
A. M.
,
Crotty
S.
,
Peters
B.
,
Sette
A.
.
2015
.
Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity.
Proc. Natl. Acad. Sci. USA
112
:
E4256
E4263
.
17
Kanakaratne
N.
,
Wahala
W. M.
,
Messer
W. B.
,
Tissera
H. A.
,
Shahani
A.
,
Abeysinghe
N.
,
de-Silva
A. M.
,
Gunasekera
M.
.
2009
.
Severe dengue epidemics in Sri Lanka, 2003-2006.
Emerg. Infect. Dis.
15
:
192
199
.
18
Kraus
A. A.
,
Messer
W.
,
Haymore
L. B.
,
de Silva
A. M.
.
2007
.
Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization.
J. Clin. Microbiol.
45
:
3777
3780
.
19
Arlehamn
C. S.
,
Sidney
J.
,
Henderson
R.
,
Greenbaum
J. A.
,
James
E. A.
,
Moutaftsi
M.
,
Coler
R.
,
McKinney
D. M.
,
Park
D.
,
Taplitz
R.
, et al
.
2012
.
Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ).
J. Immunol.
188
:
5020
5031
.
20
Carrasco Pro
S.
,
Sidney
J.
,
Paul
S.
,
Lindestam Arlehamn
C.
,
Weiskopf
D.
,
Peters
B.
,
Sette
A.
.
2015
.
Automatic generation of validated specific epitope sets.
J. Immunol. Res.
2015
:
763461
.
21
Bancroft
T.
,
Dillon
M. B. C.
,
da Silva Antunes
R.
,
Paul
S.
,
Peters
B.
,
Crotty
S.
,
Lindestam Arlehamn
C. S.
,
Sette
A.
.
2016
.
Th1 versus Th2 T cell polarization by whole-cell and acellular childhood pertussis vaccines persists upon re-immunization in adolescence and adulthood
.
Cell. Immunol
.
304–305
:
35
43
.
22
Carpenter
C.
,
Sidney
J.
,
Kolla
R.
,
Nayak
K.
,
Tomiyama
H.
,
Tomiyama
C.
,
Padilla
O. A.
,
Rozot
V.
,
Ahamed
S. F.
,
Ponte
C.
, et al
.
2015
.
A side-by-side comparison of T cell reactivity to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five continents.
Tuberculosis (Edinb.)
95
:
713
721
.
23
Oseroff
C.
,
Sidney
J.
,
Vita
R.
,
Tripple
V.
,
McKinney
D. M.
,
Southwood
S.
,
Brodie
T. M.
,
Sallusto
F.
,
Grey
H.
,
Alam
R.
, et al
.
2012
.
T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts.
J. Immunol.
189
:
1800
1811
.
24
Roberts
A. L.
,
Connolly
K. L.
,
Kirse
D. J.
,
Evans
A. K.
,
Poehling
K. A.
,
Peters
T. R.
,
Reid
S. D.
.
2012
.
Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage.
BMC Pediatr.
12
:
3
.
25
Ebell
M. H.
,
Smith
M. A.
,
Barry
H. C.
,
Ives
K.
,
Carey
M.
.
2000
.
The rational clinical examination. Does this patient have strep throat?
JAMA
284
:
2912
2918
.
26
Shaikh
N.
,
Leonard
E.
,
Martin
J. M.
.
2010
.
Prevalence of streptococcal pharyngitis and streptococcal carriage in children: a meta-analysis.
Pediatrics
126
:
e557
e564
.
27
DeMuri
G. P.
,
Wald
E. R.
.
2014
.
The group A streptococcal carrier state reviewed: still an enigma.
J. Pediatric Infect. Dis. Soc.
3
:
336
342
.
28
Hessell
A. J.
,
Malherbe
D. C.
,
Pissani
F.
,
McBurney
S.
,
Krebs
S. J.
,
Gomes
M.
,
Pandey
S.
,
Sutton
W. F.
,
Burwitz
B. J.
,
Gray
M.
, et al
.
2016
.
Achieving potent autologous neutralizing antibody responses against tier 2 HIV-1 viruses by strategic selection of envelope immunogens.
J. Immunol.
196
:
3064
3078
.
29
Kroenke
M. A.
,
Eto
D.
,
Locci
M.
,
Cho
M.
,
Davidson
T.
,
Haddad
E. K.
,
Crotty
S.
.
2012
.
Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation.
J. Immunol.
188
:
3734
3744
.
30
Yu
D.
,
Rao
S.
,
Tsai
L. M.
,
Lee
S. K.
,
He
Y.
,
Sutcliffe
E. L.
,
Srivastava
M.
,
Linterman
M.
,
Zheng
L.
,
Simpson
N.
, et al
.
2009
.
The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.
Immunity
31
:
457
468
.
31
Shulman
Z.
,
Gitlin
A. D.
,
Targ
S.
,
Jankovic
M.
,
Pasqual
G.
,
Nussenzweig
M. C.
,
Victora
G. D.
.
2013
.
T follicular helper cell dynamics in germinal centers.
Science
341
:
673
677
.
32
Cubas
R. A.
,
Mudd
J. C.
,
Savoye
A. L.
,
Perreau
M.
,
van Grevenynghe
J.
,
Metcalf
T.
,
Connick
E.
,
Meditz
A.
,
Freeman
G. J.
,
Abesada-Terk
G.
 Jr.
, et al
.
2013
.
Inadequate T follicular cell help impairs B cell immunity during HIV infection.
Nat. Med.
19
:
494
499
.
33
Lüthje
K.
,
Kallies
A.
,
Shimohakamada
Y.
,
Belz
G. T.
,
Light
A.
,
Tarlinton
D. M.
,
Nutt
S. L.
.
2012
.
The development and fate of follicular helper T cells defined by an IL-21 reporter mouse.
Nat. Immunol.
13
:
491
498
.
34
Wang
C.
,
Hillsamer
P.
,
Kim
C. H.
.
2011
.
Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets.
BMC Immunol.
12
:
53
.
35
Kitano
M.
,
Moriyama
S.
,
Ando
Y.
,
Hikida
M.
,
Mori
Y.
,
Kurosaki
T.
,
Okada
T.
.
2011
.
Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.
Immunity
34
:
961
972
.
36
Hale
J. S.
,
Youngblood
B.
,
Latner
D. R.
,
Mohammed
A. U.
,
Ye
L.
,
Akondy
R. S.
,
Wu
T.
,
Iyer
S. S.
,
Ahmed
R.
.
2013
.
Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.
Immunity
38
:
805
817
.
37
Tubo
N. J.
,
Fife
B. T.
,
Pagan
A. J.
,
Kotov
D. I.
,
Goldberg
M. F.
,
Jenkins
M. K.
.
2016
.
Most microbe-specific naïve CD4⁺ T cells produce memory cells during infection.
Science
351
:
511
514
.
38
Pepper
M.
,
Pagán
A. J.
,
Igyártó
B. Z.
,
Taylor
J. J.
,
Jenkins
M. K.
.
2011
.
Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells.
Immunity
35
:
583
595
.
39
Choi
Y. S.
,
Kageyama
R.
,
Eto
D.
,
Escobar
T. C.
,
Johnston
R. J.
,
Monticelli
L.
,
Lao
C.
,
Crotty
S.
.
2011
.
ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6.
Immunity
34
:
932
946
.
40
Johnston
R. J.
,
Choi
Y. S.
,
Diamond
J. A.
,
Yang
J. A.
,
Crotty
S.
.
2012
.
STAT5 is a potent negative regulator of TFH cell differentiation.
J. Exp. Med.
209
:
243
250
.
41
Ballesteros-Tato
A.
,
León
B.
,
Graf
B. A.
,
Moquin
A.
,
Adams
P. S.
,
Lund
F. E.
,
Randall
T. D.
.
2012
.
Interleukin-2 inhibits germinal center formation by limiting T follicular helper cell differentiation.
Immunity
36
:
847
856
.
42
Kenefeck
R.
,
Wang
C. J.
,
Kapadi
T.
,
Wardzinski
L.
,
Attridge
K.
,
Clough
L. E.
,
Heuts
F.
,
Kogimtzis
A.
,
Patel
S.
,
Rosenthal
M.
, et al
.
2015
.
Follicular helper T cell signature in type 1 diabetes.
J. Clin. Invest.
125
:
292
303
.
43
Cubas
R.
,
van Grevenynghe
J.
,
Wills
S.
,
Kardava
L.
,
Santich
B. H.
,
Buckner
C. M.
,
Muir
R.
,
Tardif
V.
,
Nichols
C.
,
Procopio
F.
, et al
.
2015
.
Reversible reprogramming of circulating memory T follicular helper cell function during chronic HIV infection.
J. Immunol.
195
:
5625
5636
.
44
Tangye
S. G.
,
Ma
C. S.
,
Brink
R.
,
Deenick
E. K.
.
2013
.
The good, the bad and the ugly - TFH cells in human health and disease.
Nat. Rev. Immunol.
13
:
412
426
.
45
De Silva
N. S.
,
Klein
U.
.
2015
.
Dynamics of B cells in germinal centres.
Nat. Rev. Immunol.
15
:
137
148
.
46
Gitlin
A. D.
,
Shulman
Z.
,
Nussenzweig
M. C.
.
2014
.
Clonal selection in the germinal centre by regulated proliferation and hypermutation.
Nature
509
:
637
640
.
47
Gitlin
A. D.
,
Mayer
C. T.
,
Oliveira
T. Y.
,
Shulman
Z.
,
Jones
M. J.
,
Koren
A.
,
Nussenzweig
M. C.
.
2015
.
T cell help controls the speed of the cell cycle in germinal center B cells.
Science
349
:
643
646
.
48
Llewelyn
M.
,
Sriskandan
S.
,
Terrazzini
N.
,
Cohen
J.
,
Altmann
D. M.
.
2006
.
The TCR Vbeta signature of bacterial superantigens spreads with stimulus strength.
Int. Immunol.
18
:
1433
1441
.
49
Havenar-Daughton, C., S. M. Reiss, D. G. Carnathan, J. E. Wu, K. Kendric, A. T. de la Peña, S. P. Kasturi, J. M. Dan, M. Bothwell, R. W. Sanders, et al. 2016. Cytokine-independent detection of antigen-specific germinal center T follicular helper cells in immunized nonhuman primates using a live cell activation-induced marker technique. J. Immunol. 197: 994–1002
.
50
Escalante
P.
,
Peikert
T.
,
Van Keulen
V. P.
,
Erskine
C. L.
,
Bornhorst
C. L.
,
Andrist
B. R.
,
McCoy
K.
,
Pease
L. R.
,
Abraham
R. S.
,
Knutson
K. L.
, et al
.
2015
.
Combinatorial immunoprofiling in latent tuberculosis infection. Toward better risk stratification.
Am. J. Respir. Crit. Care Med.
192
:
605
617
.
51
Zaunders
J. J.
,
Munier
M. L.
,
Seddiki
N.
,
Pett
S.
,
Ip
S.
,
Bailey
M.
,
Xu
Y.
,
Brown
K.
,
Dyer
W. B.
,
Kim
M.
, et al
.
2009
.
High levels of human antigen-specific CD4+ T cells in peripheral blood revealed by stimulated coexpression of CD25 and CD134 (OX40).
J. Immunol.
183
:
2827
2836
.
52
Weiskopf
D.
,
Sette
A.
.
2014
.
T-cell immunity to infection with dengue virus in humans.
Front. Immunol.
5
:
93
.
53
Chen
L.
,
Flies
D. B.
.
2013
.
Molecular mechanisms of T cell co-stimulation and co-inhibition.
Nat. Rev. Immunol.
13
:
227
242
.
54
Zhu
Y.
,
Yao
S.
,
Chen
L.
.
2011
.
Cell surface signaling molecules in the control of immune responses: a tide model.
Immunity
34
:
466
478
.
55
Malek
T. R.
,
Castro
I.
.
2010
.
Interleukin-2 receptor signaling: at the interface between tolerance and immunity.
Immunity
33
:
153
165
.
56
Shulman
Z.
,
Gitlin
A. D.
,
Weinstein
J. S.
,
Lainez
B.
,
Esplugues
E.
,
Flavell
R. A.
,
Craft
J. E.
,
Nussenzweig
M. C.
.
2014
.
Dynamic signaling by T follicular helper cells during germinal center B cell selection.
Science
345
:
1058
1062
.
57
Liu
D.
,
Xu
H.
,
Shih
C.
,
Wan
Z.
,
Ma
X.
,
Ma
W.
,
Luo
D.
,
Qi
H.
.
2015
.
T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction.
Nature
517
:
214
218
.
58
Crotty
S.
2011
.
Follicular helper CD4 T cells (TFH).
Annu. Rev. Immunol.
29
:
621
663
.
59
Proietti
M.
,
Cornacchione
V.
,
Rezzonico Jost
T.
,
Romagnani
A.
,
Faliti
C. E.
,
Perruzza
L.
,
Rigoni
R.
,
Radaelli
E.
,
Caprioli
F.
,
Preziuso
S.
, et al
.
2014
.
ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer’s patches to promote host-microbiota mutualism.
Immunity
41
:
789
801
.
60
Rosignoli
G.
,
Lim
C. H.
,
Bower
M.
,
Gotch
F.
,
Imami
N.
.
2009
.
Programmed death (PD)-1 molecule and its ligand PD-L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus-1-infected individuals.
Clin. Exp. Immunol.
157
:
90
97
.
61
Dong
H.
,
Zhu
G.
,
Tamada
K.
,
Chen
L.
.
1999
.
B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.
Nat. Med.
5
:
1365
1369
.
62
Dong
H.
,
Strome
S. E.
,
Matteson
E. L.
,
Moder
K. G.
,
Flies
D. B.
,
Zhu
G.
,
Tamura
H.
,
Driscoll
C. L.
,
Chen
L.
.
2003
.
Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis.
J. Clin. Invest.
111
:
363
370
.
63
Perreau
M.
,
Savoye
A. L.
,
De Crignis
E.
,
Corpataux
J. M.
,
Cubas
R.
,
Haddad
E. K.
,
De Leval
L.
,
Graziosi
C.
,
Pantaleo
G.
.
2013
.
Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production.
J. Exp. Med.
210
:
143
156
.
64
Hams
E.
,
McCarron
M. J.
,
Amu
S.
,
Yagita
H.
,
Azuma
M.
,
Chen
L.
,
Fallon
P. G.
.
2011
.
Blockade of B7-H1 (programmed death ligand 1) enhances humoral immunity by positively regulating the generation of T follicular helper cells.
J. Immunol.
186
:
5648
5655
.
65
Yamamoto
T.
,
Lynch
R. M.
,
Gautam
R.
,
Matus-Nicodemos
R.
,
Schmidt
S. D.
,
Boswell
K. L.
,
Darko
S.
,
Wong
P.
,
Sheng
Z.
,
Petrovas
C.
, et al
.
2015
.
Quality and quantity of TFH cells are critical for broad antibody development in SHIVAD8 infection.
Sci. Transl. Med.
7
:
298ra120
.
66
Schultz
B. T.
,
Teigler
J. E.
,
Pissani
F.
,
Oster
A. F.
,
Kranias
G.
,
Alter
G.
,
Marovich
M.
,
Eller
M. A.
,
Dittmer
U.
,
Robb
M. L.
, et al
.
2016
.
Circulating HIV-specific interleukin-21(+)CD4(+) T cells represent peripheral Tfh cells with antigen-dependent helper functions.
Immunity
44
:
167
178
.
67
Chahroudi
A.
,
Silvestri
G.
.
2016
.
HIV and Tfh cells: circulating new ideas to identify and protect.
Immunity
44
:
16
18
.
68
Keoshkerian
E.
,
Helbig
K.
,
Beard
M.
,
Zaunders
J.
,
Seddiki
N.
,
Kelleher
A.
,
Hampartzoumian
T.
,
Zekry
A.
,
Lloyd
A. R.
.
2012
.
A novel assay for detection of hepatitis C virus-specific effector CD4(+) T cells via co-expression of CD25 and CD134.
J. Immunol. Methods
375
:
148
158
.
69
Sadler
R.
,
Bateman
E. A.
,
Heath
V.
,
Patel
S. Y.
,
Schwingshackl
P. P.
,
Cullinane
A. C.
,
Ayers
L.
,
Ferry
B. L.
.
2014
.
Establishment of a healthy human range for the whole blood “OX40” assay for the detection of antigen-specific CD4+ T cells by flow cytometry.
Cytometry B Clin. Cytom.
86
:
350
361
.
70
Tartof
S. Y.
,
Lewis
M.
,
Kenyon
C.
,
White
K.
,
Osborn
A.
,
Liko
J.
,
Zell
E.
,
Martin
S.
,
Messonnier
N. E.
,
Clark
T. A.
,
Skoff
T. H.
.
2013
.
Waning immunity to pertussis following 5 doses of DTaP.
Pediatrics
131
:
e1047
e1052
.
71
Quinn
H. E.
,
Snelling
T. L.
,
Macartney
K. K.
,
McIntyre
P. B.
.
2014
.
Duration of protection after first dose of acellular pertussis vaccine in infants.
Pediatrics
133
:
e513
e519
.
72
Matthias
J.
,
Pritchard
P. S.
,
Martin
S. W.
,
Dusek
C.
,
Cathey
E.
,
D’Alessio
R.
,
Kirsch
M.
.
2016
.
Sustained transmission of pertussis in vaccinated, 1-5-year-old children in a preschool, Florida, USA.
Emerg. Infect. Dis.
22
:
242
246
.

The authors have no financial conflicts of interest.

Supplementary data