T follicular helper (Tfh) cells are a subset of CD4+ T lymphocytes that promote the development of humoral immunity. Although the triggers required for the differentiation of the other major Th subsets are well defined, those responsible for Tfh cell responses are still poorly understood. We determined that mice immunized with peptide or protein Ags emulsified in IFA or related water-in-oil adjuvants develop a highly polarized response in which the majority of the Ag-specific CD4+ T cells are germinal center–homing CXCR5+Bcl6+ Tfh cells. Despite the absence of exogenous microbial pathogen-associated molecular patterns, the Tfh cell responses observed were dependent, in part, on MyD88. Importantly, in addition to IL-6, T cell–intrinsic type I IFN signaling is required for optimal Tfh cell polarization. These findings suggest that water-in-oil adjuvants promote Tfh cell–dominated responses by triggering endogenous alarm signals that, in turn, induce type I IFN–dependent differentiation pathway functioning in T cells.
A major challenge of vaccine research is to induce high-titer durable Ab responses against protein Ags that represent major targets for disease prevention. T follicular helper (Tfh) CD4+ T lymphocytes are well recognized as specialized providers of T cell help to B cells within germinal centers (GCs) of secondary lymphoid organs. B lymphocyte responses are dramatically impaired in the absence of Tfh cells (1), and boosting Tfh cell function was shown to promote long-lived humoral immunity by amplifying Ab production and class-switch recombination (2). Therefore, an understanding of the mechanisms underlying the induction and regulation of Tfh cells could provide important clues for the rational design and development of more durable and effective Ab-based vaccines.
In comparison with other Th cell subsets, Tfh cell differentiation in vivo is poorly understood and, importantly, cannot be fully recapitulated in vitro. Tfh cells express the lineage-defining transcription repressor Bcl6 (3–5), which is expressed by naive T cells early after encounter with Ag-presenting dendritic cells (6, 7) in the presence of IL-6 (1). This leads to upregulation of the chemokine receptor CXCR5 and cell migration from the cortex to the B cell follicles of secondary lymphoid organs. At the T–B border, pre-Tfh cells interact with cognate B cells, resulting in their differentiation into short-lived extrafollicular plasmablasts or their comigration with pre-Tfh cells into B cell follicles to form GCs. The proliferation and B cell helper activities of Tfh cells are controlled, in turn, by a subset of Foxp3+ T follicular regulatory (Tfr) cells that also express Bcl6 and CXCR5 and arise from thymically derived natural regulatory T cells or from naive cells in the periphery (8, 9).
Numerous studies investigated Tfh cell differentiation in the context of infection and/or by the use of adoptive-transfer experiments with TCR-transgenic T cells. In an attempt to better characterize the minimal endogenous signals required for Tfh cell differentiation, we used a basic model of immunization with peptide or protein Ag emulsified in water-in-oil (W/O) adjuvants in the absence of microbial immunostimulants. The primary adjuvant chosen for these studies was IFA, which consists of paraffin oil and mannide monooleate as a surfactant. Although less immunostimulatory than CFA, which is supplemented with heat-killed Mycobacterium tuberculosis extracts, IFA was shown to promote substantial T cell–dependent Ab production associated with Tfh cell activity (10). In performing these studies, we also compared IFA with other more chemically defined, clinically acceptable, W/O adjuvants.
We found that, in contrast to other adjuvant preparations, W/O-only adjuvants appear to selectively trigger the polarization of naive T cells into GC-associated Tfh cells. In addition, we uncovered unexpected roles for MyD88 and type I IFN signaling in driving this preferential Tfh cell response. Together, these findings establish a convenient model for studying the signals involved in Tfh cell differentiation and support a major role for nonmicrobial stimuli in the development of adjuvants designed to promote Tfh cell activity.
Materials and Methods
Male or female (7–14 wk old) C57BL/6 (CD45.2/2), B6.SJL-Cd45a(Ly5a)/Nai (CD45.1/1), and C57BL/6J × B6.SJL-CD45a(Ly5a)/Nai F1 (CD45.1/2) mice were used alternatively as wild-type (WT) animals and were supplied by Taconic Farms via a contract with the National Institute of Allergy and Infectious Diseases (NIAID). Il12p40−/−, Il1r1−/−, Tlr3−/−, Ifnar−/−, Ifih1−/− (MDA5), muMT−/−, and Tcra−/− mice were also obtained from Taconic Farms. Il18−/− (stock #004130) and Sting−/− (Tmem−/−, strain #17537) were from the Jackson Laboratory. Myd88−/− and Trif−/− mice, backcrossed to B6 mice for 10 generations, were originally obtained from S. Akira (Osaka University, Osaka, Japan). Stat1fl/fl CD4 CRE and Stat3fl/fl CD4 CRE mice were a gift from J. O’Shea (National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health [NIH]), Tlr4−/−, Tlr5−/−, Tlr9−/− mice were a gift from G. Trinchieri (National Cancer Institute, NIH), and Unc92b−/− animals were a gift from E. Latz and D. Golenbock (University of Massachusetts, Worcester, MA). All mice were maintained in an American Association for Laboratory Animal Care–accredited animal facility at the NIAID, NIH. Mice were used according to animal study proposals approved by the NIAID Animal Care and Use Committee.
Bone marrow reconstitution and chimeras
Congenically marked recipient mice were lethally irradiated (950 rad) and reconstituted (later on the same day) with a total of 107 donor bone marrow (BM) cells isolated from WT, Myd88−/−, or Ifnar−/− animals or with a 50/50 mixture of congenically marked BM cells from WT and Myd88−/− animals or from WT and Ifnar−/− animals. Mice were allowed to reconstitute for 8–10 wk before immunizations.
Alum hydroxide (Alhydrogel 2%) was from Brenntag (Baltimore, MD), IFA and CFA were from Sigma-Aldrich (St. Louis, MO), and Montanide ISA 51 VG and Montanide ISA 720 VG were from SEPPIC (Fairfield, NJ). The adjuvant/Ag emulsions were created using two silicone and latex-free syringes connected with a Double Female Luer Lock Adapter (both from Air-Tite, Virginia Beach, VA) and mixed for up to 8 min.
AS15 (AVEIHRPVPGTAPPS), ESAT6 (QQWNFAGIEAAASA), or Ag85B (FQDAYNAAGGHNAVF) peptides were provided by Jan Lukszo at the Research Technologies Branch, NIAID. Ag85B protein from M. tuberculosis was obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA) and from Aeras (Rockville, MD).
Immunization and harvest
Unless otherwise indicated, immunizations were performed by s.c. injection at four sites along the back (50 μl/site) with alum, IFA, or CFA containing a total of 100 μg (or less if indicated) of AS15, ESAT6, or Ag85B peptides or a total of 4 μg of Ag85B protein. Unless otherwise stated, the draining (inguinal, axillary, and brachial) lymph nodes (LNs) were harvested 10–14 d after immunization. LNs were dissociated through 100-μm cell strainers, and 2 × 106 LN cells were plated in a round-bottom 96-well plate and stained for flow cytometry. To measure Ab responses, WT and Tcra−/− mice were immunized twice with Ag85B in IFA, first with s.c. priming immunization and then boosted 21 d later by i.p. immunization.
I-A(b) Toxoplasma gondii TGME49_012300 605–619 AVEIHRPVPGTAPPS (AS15), I-A(b) M. tuberculosis ESAT6 4–17 QQWNFAGIEAAASA, and I-A(b) M. tuberculosis Ag85B 280–294 FQDAYNAAGGHNAVF tetramers were obtained from the NIH Tetramer Core Facility. Tetramer staining (15–30 μg/ml) was performed at 37°C for 1 h. Abs against the following molecules were purchased from eBioscience (San Diego, CA), BioLegend (San Diego, CA), or BD Biosciences (San Jose, CA): CD4 (RM4-4), CD45.1 (A20), CD45.2 (104), CD44 (IM7), CD62L (MEL-14), SLAM (TC15-12F12.2), CXCR5 (SPRCL5), PD-1 (29F.1A12), ICOS (7E.17G9), GL7 (GL7), Foxp3 (FJK-16s), Tbet (4B10), RORγt (B2D), and Bcl6 (K112-91). Fixable Viability Dye eFluor 780 was from eBioscience. All samples were acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).
Immunofluorescence and confocal microscopy
Draining LNs (dLNs) were harvested and fixed with PLP buffer (0.05 M phosphate buffer containing 0.1 M l-lysine [pH 7.4], 2 mg/ml NaIO4, and 10 mg/ml paraformaldehyde) for 12 h. Following fixation, dLNs were incubated in 30% sucrose for 6 h before embedding in OCT compound (Tissue-Tek). Thirty-micrometer sections were cut on a CM3050S cryostat (Leica) and adhered to Super Frost Plus Gold slides (Electron Microscopy Services). Frozen sections were permeabilized and blocked for 1–2 h in PBS containing 0.3% Triton X-100 (Sigma-Aldrich), 1% normal mouse serum, 1% BSA, and 10% normal goat serum. Sections were stained for a minimum of 12 h at 4°C with the following directly conjugated Abs from BioLegend, unless stated otherwise: anti-CD4 (GK1.5), anti-B220 (RA3-6B2), anti-IgD (11-26c.2a), anti–PD-1 (29F.1A12), and anti-Bcl6 (K112-91; BD Biosciences). Stained slides were mounted with Fluoromount G (eBioscience) and sealed with a glass coverslip. Each section was visually inspected by epifluorescent light microscopy, and several representative sections from different dLNs were acquired using an SP8 confocal microscope (Leica) with 40× (NA 1.3) or 63× (NA 1.4) objectives. Fluorophore emission was collected on separate detectors, and sequential laser excitation was used to minimize spectral spillover. To calculate the total number of GCs per dLN, whole dLNs were serially sectioned, and GCs were scored for each section. GC area was calculated using the surface creation module in Imaris (Bitplane). Graph plots depict the widest area for each GC present in the dLNs examined.
Total anti-Ag85B–specific IgM, IgA, and IgG Abs were detected in the serum. In brief, 96-well plates were coated with 1 μg/ml Ag85B and incubated with 3-fold serial dilutions of the sera, followed by incubation with HRP-coupled anti-IgM, anti-IgA, and anti-IgG Abs. ABTS was added as a substrate, and OD was measured at 405 nm.
Total RNA from dLNs was isolated using the MagMAX-96 Total RNA Isolation Kit, according to the manufacturer’s protocol, and cDNA was reverse transcribed with Superscript III reverse transcriptase and random primers (all from Thermo Fisher, Waltham, MA). Quantitative PCR was performed on an ABI Prism 7900 HT Sequence Detection System using Power SYBR Green Master Mix (Applied Biosystems/Life Technologies) for detection. Fold induction of gene expression was calculated by normalizing mRNA levels for each sample to levels of hypoxanthine guanine phosphoribosyltransferase. The following primer pairs were used: Hprt forward, 5′-GCCCTTGACTATAATGAGTACTTCAGG-3′ and reverse, 5′-TTCAACTTGCGCTCATCTTAGG-3′; pan-ifna forward 5′-SAWCYCTCCYAGACTCMTTCTGCA-3′ and reverse 5′-TATDTCCTCACAGCCAGCAG-3′; and ifnb forward 5′-CCCTATGGAGATGACGGAGA-3′ and reverse 5′-ACCCAGTGCTGGAGAAATTG-3′.
In vivo treatment
In vivo cytokine blockade was performed by i.p. injections of 500 μg Ig MP5-20F3 mAb (anti-mouse IL-6R) or Ig MAR1-5A3 mAb (anti-mouse IFNAR-1; both from Bio X Cell, West Lebanon, NH) for three consecutive days at the time of immunization, followed by 250 μg once every 3 d for the remainder of the experiment. WT mice were provided antibiotic-treated water (containing ampicillin [1 g/l], vancomycin [500 mg/l], neomycin trisulfate [1 g/l], and metronidazole [1 g/l]; Sigma-Aldrich) ad libitum for a month prior to immunization to partially deplete gut microbiota.
The statistical significance of differences between groups was analyzed via the unpaired Mann–Whitney t test or paired t test (when comparing groups in the mixed BM chimera settings) using GraphPad Prism software version 5.0c (GraphPad).
W/O adjuvants in the absence of microbial components promote Tfh cell polarization in response to peptide immunization
IFA and CFA contain nonmetabolizable paraffin oil, resulting in W/O emulsions, and form long-lasting depots at sites of injection. CFA is IFA supplemented with heat-killed M. tuberculosis, and it induces a strong proinflammatory response associated with Th1, Th17, and Tfh cell development, along with B cell maturation (10). However, oil and surfactant-only adjuvants, such as IFA, are also known to promote Ab responses (11). This led us to address the question of whether these adjuvants promote Tfh cell responses in the total absence of additional microbial stimuli.
We first performed kinetic studies comparing the responses of mice following s.c. immunization with Ag admixed with alum or emulsified in IFA or CFA. The immunogen chosen for our initial studies was a 15mer peptide (AS15) representing a highly immunodominant T cell epitope present in an unidentified protein from T. gondii (12). We found that IFA was able to promote a substantial AS15-specific CD4+ T cell response in dLNs (Supplemental Fig. 1A, 1B) that includes Tfh cells, as defined by the coexpression of CXCR5 and Bcl6 (Supplemental Fig. 1C, 1D). At the peak of the response, at approximately day 12 postimmunization, the frequency (Fig. 1A, 1B) and total number (Fig. 1C) of Ag-specific CD4+ T cells were significantly increased after IFA immunization compared with alum immunization, although this response was lower than that observed with CFA-emulsified Ag. Interestingly, a significantly higher percentage of Ag-specific Tfh cells was induced following IFA immunization compared with CFA immunization (Fig. 1A, 1D), although the overall number of Ag-specific Tfh cells was lower (Fig. 1E). In contrast, Ag incorporated in alum induced only low-level Tfh cell responses in this model (Fig. 1A, 1D, 1E). The high frequency of Tfh cells observed following immunization with IFA was not due to the choice of Ag, because similar results were seen following administration of immunogenic peptides from M. tuberculosis proteins (Supplemental Fig. 1E–H).
Importantly, in terms of the total response, IFA immunization resulted in the selective polarization of CD4+ T cells toward the Tfh subset, with nearly half of the tetramer+ cells expressing the Tfh lineage–defining transcription factor Bcl6 compared with only 14% that expressed the T-bet or RORγt canonical transcription factors for the Th1 and Th17 subsets (Fig. 1F). In contrast, CFA immunization induced a less Tfh-polarized T lymphocyte response, with higher percentages of Th1 and Th17 cells. This dominance of Tfh cells over Th1 and Th17 cells following IFA immunization was also apparent when the total numbers of each subset were calculated (Fig. 1G). The Tfh cells induced by both adjuvants were indistinguishable with regard to their increased expression of CXCR5 and PD1 and reduced expression of SLAM (Fig. 1H), arguing that both protocols trigger Tfh cells that are at the same state of differentiation.
It was reported that the Ag dose can critically impact the extent of Th1 cell versus Tfh cell differentiation (13). However, under the experimental conditions that we used, no significant dose-dependent differences were observed in the Ag-specific T cell expansion (Supplemental Fig. 2A) or the frequency of Tfh cells within the Ag-specific population (Supplemental Fig. 2B).
Although IFA is not approved for human use, other better-defined oil- and surfactant-only–containing adjuvants have been used clinically (14). To determine whether the ability to induce polarized Tfh cell responses is shared by these adjuvants, we tested two of them, Montanide ISA 51 VG and Montanide ISA 720 VG, which have been used in numerous clinical trials related to therapeutic vaccines (14, 15). Montanide ISA 51 VG, similar to IFA, contains a nonmetabolizable mineral oil as its main component, whereas Montanide ISA 720 VG incorporates squalene as a metabolizable oil. We found that ISA 51 VG and ISA 720 VG, like IFA, were able to drive AS15-specific T cell expansion (Fig. 2A) and promote the generation of high percentages of tetramer+ CXCR5+ Bcl6+ Tfh cells (Fig. 2B, 2C). Similarly to IFA, immunization with ISA 51 VG or ISA 720 VG resulted in a Tfh subset–dominated profile (Fig. 2D).
Having shown that W/O adjuvants are preferential stimulators of Tfh cell responses, we performed imaging studies to visualize the location of these Tfh cells within dLNs. We observed equivalent total numbers of GCs, defined as IgD− Bcl6+ structures, in naive and immunized mice maintained in specific pathogen–free conditions (Fig. 3A). This background level of GC likely reflects the steady-state response to commensals and/or self-antigens. In contrast, the administration of AS15 peptide in IFA or CFA induced a significant increase in the GC size (area) (Fig. 3B). As expected, GC formation in mice immunized with IFA in the absence of peptide Ag was comparable to that observed in naive animals. Importantly, the vast majority of the CD4+ PD1+ Bcl6+ Tfh cells induced by peptide immunization in IFA were localized within these structures and, hence, were considered classical GC Tfh cells (Fig. 3C, 3D). Together, the above experiments performed with peptide-immunization models indicated that W/O-only adjuvants preferentially trigger Ag-specific GC Tfh cells.
W/O-only adjuvants also promote Tfh cell–dominated T cell responses and Ab production during immunization with protein Ag
We next asked whether proteins and not just peptide Ags also trigger polarized Tfh cell responses when emulsified in oil- and surfactant-only adjuvants. For these experiments we used Ag85B, a mycobacterial protein for which class II tetramers are available. We found that, when administered in IFA, ISA 51 VG, or ISA 720 VG, Ag85B stimulates an Ag-specific T cell response (Supplemental Fig. 2C, 2D) containing a high proportion of CXCR5+ Bcl6+ Tfh cells (Supplemental Fig. 2C, 2E). As predicted from previous studies, protein immunization in IFA in this setting resulted in the production of a substantial Ag85B-specific Ab response in C57BL/6 mice, but not C57BL/6 TCRα−/− mice, arguing that the Ag-specific Tfh cells induced in our IFA-immunization protocol have functional B cell helper activity (Supplemental Fig. 2F).
The Tfh cell response induced by immunization with Ag in IFA depends on B cells and IL-6 upstream of STAT1 and STAT3 signaling
The differentiation of conventional GC Tfh cells is known to require Ag presentation and costimulation from B cells (1). Consistent with this property, B cell–deficient μMT mice immunized with peptide in IFA displayed a profound impairment in total CD4+ T cell responses (Supplemental Fig. 3A) and, more importantly, in the Ag-specific Tfh cell population (Supplemental Fig. 3B).
IL-6 also was shown to play a role in Tfh cell differentiation by triggering Bcl6 (4) and CXCR5 (7, 16) expression in the precursors of these cells. We confirmed that the Tfh cells induced following peptide immunization in IFA display a similar IL-6 dependency. Thus, immunized Il6−/− mice, while possessing indistinguishable levels of AS15 tetramer+ lymphocytes compared with immunized WT mice (Supplemental Fig. 3C), exhibited a significant reduction in the frequency of Tfh cells within this Ag-specific population (Supplemental Fig. 3D). Although IL-12 was reported to be required for human (17) and, in some cases, murine Tfh cell (18) differentiation in vitro, no reduction in the generation of these cells was observed when Il12p40−/− mice were immunized with peptide in IFA (Supplemental Fig. 3E, 3F).
IL-6 is thought to mediate its effects on infection-induced Tfh cell differentiation through STAT1 and STAT3 (19). Consistent with these findings, when immunized with peptide in IFA, mice with a T cell–intrinsic deficiency in STAT1 or STAT3 display reduced frequencies of Ag-specific Tfh cells (Supplemental Fig. 3G) while exhibiting normal levels of tetramer+ cells (Supplemental Fig. 3H) after immunization with peptide in IFA. Nevertheless, the reduction in the Tfh cell response was not as profound as that observed in the prior studies involving lymphocytic choriomeningitis virus infection (20).
IFA-induced Tfh cell responses are dependent on MyD88
MyD88 is a major downstream signaling adaptor for most TLRs, as well as IL-1 and IL-18 receptors. We and other investigators showed that MyD88 is critical for the Th1 and Th17 responses triggered by the heat-killed M. tuberculosis in CFA (21) or by an analog of the mycobacterial cord factor trehalose-6,6-dimycolate (22). Similarly, we found that MyD88-deficient mice immunized with peptide in CFA display reduced frequencies (Fig. 4A) and total numbers (Supplemental Fig. 3I) of Ag-specific cells. Interestingly, although the total number of tetramer+ Tfh cells was also reduced in these animals (Supplemental Fig. 3J), their frequency was not (Fig. 4B, 4C).
Unexpectedly, we observed that the response to peptide immunization in IFA, which does not carry exogenous pathogen-associated molecular patterns (PAMPs), also requires MyD88 signaling. We found that MyD88-deficient mice display significant decreases in the percentages (Fig. 4A) and total numbers (Supplemental Fig. 3I) of Ag-specific cells, as well as in the frequency of Tfh cells within the Ag-specific population (Fig. 4B, 4C, Supplemental Fig. 3J). Of note, the requirement for MyD88 appears early because a significant defect in the frequency of Ag-specific Tfh cells was already present as soon as 6 d post-IFA immunization (Supplemental Fig. 3K). Importantly, the effect of MyD88 deficiency on IFA-adjuvanted Tfh responses was also observed in mice immunized with protein Ag (Ag85B) (Supplemental Fig. 3L). In addition to dampening Tfh cell responses, the absence of MyD88 impaired the frequency of GC B cells (Fig. 4D, 4E).
We next used reciprocal BM chimeric mice to address the cellular basis for the role of MyD88 in Tfh cell generation in mice immunized with IFA plus peptide. These experiments showed that the requirement for MyD88 expression is restricted to the hematopoietic compartment (Fig. 4F) but is not T cell intrinsic, because no difference was observed in MyD88-sufficient versus MyD88-deficient T cells found in the same irradiated host following reconstitution with a 50/50 mixture of WT and MyD88−/− BM cells (Fig. 4G).
One hypothesis to explain the unexpected involvement of MyD88 in the IFA-induced Tfh cell response is that the oil component and/or the surfactant contained in IFA leads to local cell death and/or tissue damage that could provide endogenous danger signals for MyD88-dependent triggering of Tfh cell differentiation (23). However, no difference in the frequency of Tfh cells within the Ag-specific population was observed in the absence of TLR2, TLR4, or IL-1R (Supplemental Fig. 3M), three receptors that were implicated in danger-induced signaling. In related experiments, normal Tfh cell responses were also observed in IFA-immunized IL-18–deficient mice (Supplemental Fig. 3M). A second hypothesis we considered is that the requirement for MyD88 reflects the involvement of microbiota-derived signals. In this regard, it was shown recently that sensing of the gut microbiota through TLR5 is necessary for the Ab response observed after s.c. immunization with an unadjuvanted influenza vaccine (24). Nevertheless, no impairment of the Tfh cell response was observed in IFA-immunized Tlr5−/− mice or mice treated with antibiotics using the identical protocol from the latter study (Supplemental Fig. 3M). Similarly, no reduction in Tfh cell response was observed in the absence of the microbial (or self) DNA sensor TLR9 (Supplemental Fig. 3M). Together, these experiments demonstrated a contribution of MyD88-dependent signaling to Tfh cell induction but did not identify an individual TLR or IL-1R family member responsible for this effect.
Optimal W/O adjuvant Tfh cell induction depends on T cell–intrinsic type I IFN signaling
Type I IFN was reported to promote (18, 25) or inhibit (20) Tfh cell responses. We observed that WT IFA-immunized mice, as well as MyD88-deficient mice, displayed a marked upregulation of Ifna and Ifnb mRNA in total LNs compared with unimmunized animals (Supplemental Fig. 4A). To examine the possible function of this IFN production in Tfh cell induction, we compared the responses of WT and IFN receptor–deficient mice (Ifnar−/−) following IFA or CFA immunization. IFNAR-deficient mice displayed unaltered levels of Ag-specific cells (Fig. 5A) but had significantly reduced frequencies of Tfh cells within this population at day 12 (Fig. 5B, 5C, Supplemental Fig. 4B) but not at day 6 postimmunization (Supplemental Fig. 4B). In addition, the absence of IFNAR led to a decreased frequency of GC B cells (Fig. 5D, 5E). Importantly, a role for type I IFN signaling in Tfh cell induction was also observed with two other nonmicrobial-containing W/O-only–containing adjuvants, Montanide ISA 51 VG and Montanide ISA 720 VG (Fig. 5F).
To examine the cellular basis of the contribution of type I IFN signaling to Tfh cell differentiation, we used mixed BM chimera experiments in which sublethally irradiated mice were reconstituted with equivalent numbers of CD45.1 WT and CD45.2 IFNAR-deficient BM cells and then animals were immunized with IFA plus AS15 peptide 8 wk after reconstitution (Fig. 5G). In this system, WT and IFNAR-deficient T cells are readily distinguishable by their different congenic marker expression. Because each population receives the same environmental signals, the intrinsic requirement of IFNAR can be addressed. Although the WT and IFNAR-deficient CD4+ T lymphocytes recovered from the dLN contained comparable frequencies of activated CD44hi cells (Supplemental Fig. 4C), T cell–intrinsic IFNAR expression was required to obtain full expansion of tetramer+ cells (Supplemental Fig. 4D) as well as the Tfh cells within this population (Fig. 5G). In these IFA-immunized animals, the decreased frequency of Tfh cells that developed in the absence of IFNAR was not associated with a concomitant increase in the other T cell subsets (data not shown).
Because W/O-only adjuvants preferentially induce Tfh cells, it was possible that the role of type I IFN in this system reflects a general effect not restricted to that subset. To address this question, we immunized the same mixed BM chimeric mice with peptide and CFA, a protocol that simultaneously induces Th1/Th17 and Tfh subsets (Fig. 1F). With this type of immunization, the absence of type I IFN receptors did not alter the overall frequency of Ag-specific cells (Supplemental Fig. 4E); instead, it again resulted in a marked reduction in the Tfh cell response (Supplemental Fig. 4F), with an accompanying increase in the percentages of T-bet– and RORγt-expressing cells (Supplemental Fig. 4G).
Type I IFN is induced through a variety of innate receptors that include the cytoplasmic and endosomal sensors STING, MDA5, AIM2, TLR3, TLR7/8, and TLR9. The experiments described above argued against a role for TLR9 in Tfh cell induction (Supplemental Fig. 3M), and parallel experiments involving TLR3-, STING-, and MDA5-deficient mice failed to reveal a function for these pattern recognition receptors or the TRIF adaptor molecule in IFA-induced Tfh cell responses (Supplemental Fig. 4H). However, we found that IFA-immunized UNC93b1-deficient mice, in which all endosomal TLRs, including the type I IFN–inducing receptors TLR3, TLR7/8, and TLR9 are nonfunctional, exhibit a significant impairment in Tfh cell responses (Fig. 5H, 5I). Taken together with the other data on pattern recognition receptor involvement shown above, this finding suggests a possible role for TLR7/8 or a combination of multiple endosomal TLRs in W/O adjuvant–promoted Tfh cell induction. In conclusion, the experiments described above identified important roles for MyD88 and type I IFN receptor signaling in the Tfh cell–polarized responses promoted by W/O adjuvants, with IFNAR, but not MyD88, having a T cell–intrinsic function.
The roles of MyD88 and IFNAR in Tfh cell generation are IL-6 independent
As discussed above, IL-6 in our experiments and in other studies (4) was shown to play a major function in Tfh cell generation. To investigate whether the influence of MyD88 and IFNAR on Tfh cell responses in IFA-immunized mice relates to their involvement in IL-6 induction and/or signaling, we simultaneously treated MyD88- or IFNAR-deficient mice with an anti–IL-6 mAb. Interestingly, IL-6 neutralization in either of these deficient settings caused a marked reduction in the frequencies (Fig. 6A), as well as the total numbers (Fig. 6B), of Ag-specific Tfh cells that exceeded in magnitude the decreased response seen in anti-IL-6–treated WT mice. In contrast, treatment of immunized Myd88−/− mice with an IFNAR-blocking mAb caused only a minor, statistically insignificant decrease in the magnitude of the Tfh cell response (Fig. 6B). Together, these findings are consistent with the existence of a common pathway for MyD88 and IFNAR in the Tfh cell response, which is likely to be unrelated to that involved to IL-6 induction and function.
The stimulation of Tfh cell development and function is a logical approach toward the development of improved vaccines based on humoral immunity. Nevertheless, Tfh cells are distinct from other CD4+ T cell subsets in that relatively little is known about the signals that guide their selective differentiation. In this article, we described a model using W/O adjuvants that can be used to study the polarization and immunologic activity of Tfh cells in vivo, and we used this system to reveal MyD88 and type I IFN signaling as two innate determinants involved in their induction.
Most previous studies on the mechanism of Tfh cell differentiation used infectious challenge and/or adoptive transfer of transgenic T cells to dissect this process. In contrast, the IFA-immunization model used in this study offers the ability to assess Tfh cell induction endogenously and in the absence of added microbial stimuli. Initially, we examined the response to an immunogenic peptide (AS15) with a high precursor frequency (26) that is readily detected by tetramer staining. This allowed us to focus on endogenous Tfh cell development driven solely by a single epitope. Although a previous study in an infection model indicated that the choice of peptide epitope can influence the degree of Tfh cell polarization, we confirmed that the Tfh cell induction observed following immunization with AS15 in W/O adjuvant can be reproduced with other structurally unrelated peptides. Similarly, in contrast to what was observed in other models (13), the capacity of IFA immunization to trigger polarized Tfh cell responses appeared to be independent of the dose of peptide used.
In the current study, immunization with each of the three W/O-only adjuvants tested consistently resulted in strong Tfh cell polarization. The oils incorporated in each adjuvant differ in their degree of refinement and biodegradability; however, all three share the same surfactant, mannide monooleate, at different degrees of purity, raising the question of its direct contribution to Tfh cell induction. Because the presence of a surfactant is essential for the generation of W/O emulsions, this possibility cannot be formally ruled out. Nevertheless, immunization with peptide plus mannide monooleate (the same preparation used in the manufacture of IFA) in PBS in the absence of oil induced the same basal Tfh cell response observed with peptide alone (data not shown). Regardless, the presence of microbial PAMPs in the adjuvant(s) clearly was not required for Tfh cell induction, and the response was not altered in antibiotic-treated animals. The latter observation argues against a significant role for the endogenous microbiota as was documented previously for the B cell response observed following immunization with a nonadjuvanted flu vaccine (24).
W/O-only adjuvants induced a frequency of Tfh cells that was substantially higher than that promoted by alum or CFA, although the latter adjuvant stimulated a greater total number of these cells. This finding is consistent with the differences in Ab response typically triggered by the above adjuvants (11). Indeed, compared with CFA, W/O-only adjuvants barely stimulated Th1, Th17, and Th2 (data not shown) induction, supporting their preferential capacity to trigger Tfh cell responses and, as also shown in this study, GC formation.
The central question raised by this study concerns the mechanism underlying the Tfh cell polarization observed following immunization with W/O-only adjuvants. Because these formulations do not contain any microbial components that are able to drive the differentiation of other CD4+ T cell subsets (e.g., Th1 or Th17), one possibility is that the Tfh cell polarization occurring in this system represents a default pathway seen when T cell responses are induced in the absence of exogenous PAMPs. However, this explanation seems unlikely, given the failure of anti-CD3/CD28–activated purified CD4+ T cells to default to a CXCR5- and Bcl6-expressing Tfh cell–like phenotype, even in the presence of IL-6 or IL-21 (27). Nevertheless, in contrast to the in vitro situation, Bcl6-expressing CD4+ T cells are readily induced during early immune responses in vivo (6, 28) and appear to be followed by a second wave of differentiated Tfh cells or are replaced by T cells that belong to other subsets, depending on whether the appropriate transcriptional activators are present. Thus, the induction of Tfh cell responses may require a positive-differentiation signal that promotes the development of early Bcl6 and CXCR5 cells into mature Tfh cell and the concomitant absence of polarizing stimuli for the other major CD4+ T cell subsets.
In our experiments using W/O-only adjuvant immunization, we were able to identify two innate immune system components (in addition to IL-6) that influence Tfh cell polarization and that may provide clues as to the nature of the positive-differentiation signal referred to above. The first, the adaptor protein MyD88, has been studied extensively in the context of Ab responses where its role is controversial (11, 29). In the case of immunization with protein Ags in oil adjuvants not containing PAMPs, MyD88-deficient mice displayed normal or defective Ab production (11, 30). Although the effects of MyD88 deficiency on Tfh cell induction were reported (24, 31), they involved experimental models in which direct microbial stimulation from the gut flora was proposed to play a major contribution. In the studies reported in this article, MyD88 dependence was observed in a system in which exogenous microbial PAMPs should be absent, and oral antibiotic treatment failed to impair the response. Although we were unable to formally identify the ligands and host receptors involved, the reduction in the Tfh cell response seen in UNC93b1–deficient mice implicates a role for TLR3/7, TLR9, or a combination of these receptors in this pathway. Moreover, based on our BM-reconstitution experiments, these receptors appear to be functioning exclusively in the hematopoietic compartment.
In addition to MyD88, type I IFN signaling was implicated in our experiments as an innate immune component participating in adjuvant-induced Tfh cell induction. The role of this pathway was investigated previously but with differing outcomes: it promoted (18, 25) or inhibited (20) Tfh cell responses depending on the system studied. In our model, which does not use adoptive transfer and/or viral infection, we observed significant IFNAR involvement in the Tfh cell responses following immunization with all three W/O-only adjuvants tested. Additionally, our mixed BM chimera experiments revealed a major CD4+ T cell–intrinsic component for this function. Further analysis suggested that MyD88 and type I IFN signaling may be part of a common pathway, because, in contrast to IL-6 blockade, Ab-mediated IFNAR blockade failed to significantly diminish the Tfh cell responses observed in immunized MyD88-deficient mice. However, at day 10 postimmunization and in total LNs, we found a similar upregulation of Ifna and Ifnb mRNA in WT and Myd88−/− mice, revealing a more complex relationship between MyD88 and type I IFN for which in-depth kinetic and anatomic (e.g., LNs versus immunization sites) studies are required. Interestingly, although the requirement for MyD88 to generate Ag-specific Tfh cells appears early after immunization (day 6), presumably acting during the APC-activation and/or T cell–priming steps, that of IFNAR is important between days 6 and 12 postimmunization, when GC B cells play a prominent role in the persistence of Tfh cells. Of note, Myd88−/− and Ifnar−/− mice exhibited a significantly lower frequency of GC B cells at day 12 postimmunization, but not at day 6, compared with WT mice. Based on the above series of observations, we speculate that the adjuvant-stimulated Tfh cell polarization occurring in our model may involve the production of type I IFN from a hematopoietically derived cell population (e.g., B lymphocytes, plasmacytoid dendritic cells, myeloid cells), which, in turn, triggers the induction of a T cell–intrinsic type I IFN–dependent signal important for the differentiation and persistence of this subset. Further work is necessary to formally confirm the central features and, in particular, the role of MyD88 in this pathway.
The finding that W/O-only adjuvants preferentially drive Tfh cell responses in the absence of exogenous microbial PAMPs suggests an important role for endogenous danger signals or damage-associated molecular patterns in the mechanism underlying this process. Although the ligands involved were not identified in the current study, the dual dependency on MyD88 and type I IFN signaling raises the question of a possible role for self–nucleic acids derived from stressed or damaged cells (32).
Once delineated, these signals could potentially be used to engineer enhanced vaccine-induced Ab production. Nevertheless, the simple induction of higher frequencies of Tfh cells may not necessarily lead to improved B cell maturation and humoral immunity. In this regard, it is becoming clear that Foxp3+ Tfr cells, which balance the function of Tfh cells in GCs, can also strongly influence the outcome of immunization. Based on previous studies (9) and our own preliminary observations (data not shown), major increases in this cell population appear to be strongly promoted by W/O-only adjuvants and could contribute positively or negatively to the overall functional outcome on humoral immunity. Further studies on the role of adjuvants in regulating the Tfh/Tfr cell balance offer an approach for addressing this question.
We thank the NIAID Flow Cytometry and Tetramer Facilities and Animal Care Branch for major contributions to this work. We thank Daniel L. Barber (NIAID, NIH) for helpful discussions and Ronald N. Germain (NIAID, NIH) for thoughtful input and support. We also acknowledge Marie-Eve Koziol (SEPPIC Inc./Air Liquide Healthcare North America) for the generous gift of Montanide ISA 51 VG and Montanide ISA 720 VG adjuvants and Paul Herzog (Hudson Institute of Medical Research) for IFN primer sequences.
This work was supported by the Intramural Research and the Intramural Vaccine Adjuvant Programs of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.