The aryl hydrocarbon receptor modulates T follicular helper cell responses to influenza virus infection in mice

Abstract

T follicular helper (Tfh) cells support antibody responses and are a critical component of adaptive immune responses to respiratory viral infections. Tfh cells are regulated by a network of signaling pathways that are controlled, in part, by transcription factors. The aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that modulates many aspects of adaptive immunity by binding of a range of small molecules. However, the contribution of AHR signaling to Tfh cell differentiation and function is not known. Herein, we report that AHR activation by three different agonists reduced the frequency of Tfh cells during primary infection of C57Bl/6 mice with influenza A virus (IAV). Further, using the high affinity and AHR-specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we show that AHR activation reduced Tfh cell differentiation and T-cell dependent B cell responses. Using conditional AHR knockout mice, we demonstrated that alterations of Tfh cells and T-cell dependent B cell responses after AHR activation required the AHR in T cells. AHR activation reduced the number of T follicular regulatory (Tfr) cells; however, the ratio of Tfr to Tfh cells was amplified. These alterations to Tfh and Tfr cells during IAV infection corresponded with differences in expression of BCL6 and FOXP3 in CD4⁺ T cells and required AHR to have a functional DNA-binding domain. Overall, these novel findings support that the AHR modulates Tfh cells during viral infection, which has broad reaching consequences for understanding how environmental factors contribute to variation in immune defenses against infectious pathogens, such as influenza and SARS-CoV-2.

Introduction

During the past several decades, there have been major advances in the knowledge of immune responses to viral infection and ways to reduce the impact of pathogenic viruses on human health. In spite of these developments, respiratory viral infections remain among the major causes of illness and death (1). The SARS-CoV-2 pandemic has brought respiratory viruses to the forefront; yet, even prior to the emergence of this new threat, other viruses such as influenza viruses, have been persistent causes of epidemics and pandemics (2). Among key unanswered questions about pathogenic viruses are uncertainties about the drivers of disparities in responses to infection at the individual and population level (3, 4). It is largely acknowledged that environment factors, such as contaminated water, affect the emergence and spread of communicable diseases. Yet, the impact of chemicals from our environment on immune responses to infection is often overlooked. In part, this reflects the complex connections between the environment, host, and pathogens (3). Environmental factors, ranging from pollutants to nutritional sources, can modify immune responses in a manner that can be protective or detrimental (4). However, the causal relationships and cellular mechanisms are poorly understood. Despite these uncertainties, there is mounting evidence that environmental exposures are important contributors to differences in antiviral immune responses (3, 5).

Ultimately, the adaptive immune response is responsible for resolving infection and building long-term immunity (6). Thus, environmental agents that influence the function of the adaptive immune system have the potential to modulate disease severity. Several epidemiological studies have reported associations between exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) and increased severity of respiratory infections, as well as dampened responses to vaccination (7–9). The AHR is an environmental-sensing transcription factor that was initially discovered as an intracellular receptor for a broad class of persistent pollutants, which includes dioxins and polychlorinated biphenyls (10). In addition to pollutants, AHR ligands include small molecules derived from commonly consumed foods, metabolites from microorganisms, and several pharmaceuticals (10, 11). Thus, the AHR is a conduit for environmental signals to modulate the magnitude and nature of immune responses (3, 11). Indeed, in animal models, changes to adaptive immunity are among the most consistently observed consequences of AHR activation (10–12). CD4⁺ T cells, for example, play a pivotal role in adaptive immune responses to influenza and other respiratory viruses (13, 14). During acute infection with influenza A virus (IAV), CD4⁺ T cells clonally expand and differentiate into functionally distinct effector subsets, which include T helper 1 (Th1), Th17, T follicular helper (Tfh), and regulatory T (Tregs) cells (14). During primary IAV infection in mice, AHR activation reduces the frequency of Th1 cells and increases the frequency of Tregs (15). In other disease models, AHR activation alters the frequency of Th1, Th2, Th17, and Treg cells, although the affected subset(s) and direction of change varies depending on experimental system (10, 11). Moreover, AHR modulation of CD4⁺ T cells has been associated with changes in disease progression and outcome (16–22).

In contrast, relatively little research has examined the impact of AHR activation on Tfh cells. A recent report of IAV infection in mice, suggested that in addition to modulating other T cell populations, AHR activation likely also affects Tfh cells (15). This is intriguing for two reasons. First, Tfh cells are essential to establish and support germinal centers, which are the main sites in which B cells produce high-affinity, isotype switched antibodies and generate antibody-secreting plasma cells (23). Second, activation of the AHR perturbs antibody responses to immune challenges, including IAV infection (15, 24, 25). However, the impact of AHR activation on Tfh cells and the contribution of AHR signaling in CD4⁺ T cells on the germinal center response during viral infection is unclear. To address this knowledge gap, we determined the impact of AHR activation on Tfh cell differentiation and proliferation over the course of acute IAV infection. We also evaluated the consequences of AHR activation on key Tfh cell functions. Using conditional AHR knockout mice, we determined whether the AHR within the CD4⁺ cell lineage directly contributes to observed changes in Tfh cells, germinal center B cells, plasma cells, and virus-specific antibodies.

Methods and Methods

Mice and Treatments.

C57Bl/6 (B6) mice (age 5–6 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME). Initial breeding stocks of B6 AHR^fx/fx and AHR^dbd/dbd (26, 27) mice were provided by Christopher Bradfield (University of Wisconsin, Madison, WI), and B6.Cg-Tg(CD4-cre)1Cwi (CD4^Cre) mice were purchased from the Jackson Laboratory (28). DNA isolated from tail biopsies was used to genotype AHR^fx/fx, AHR^dbd/dbd, CD4^Cre, and CD4^CreAHR^fx/fx mice by PCR (26, 27, 29). All primers were purchased from Integrated DNA Technologies (San Diego, CA). The AHR^fx allele was detected using the primers OL4064 (5′-CAGTGGGAATAAGGCAAGAGTGA-3′) and OL4088 (5′-GGTACAAGTGCACATGCCTGC-3′) (26). The following primers were used to detect the AHR^dbd allele: OL941 (5′-CTGAGGGGACGTTTTAATG-3′) and OL942 (5′-AACATTTGCACTCATGGATAG-3′) (27). For some experiments, CD4^Cre mice were crossed with AHR^fx/fx mice. To verify Ahr excision from CD4⁺ T cells in CD4^CreAHR^fx/fx mice, DNA was isolated from purified CD4⁺ T cells using DirectPCR Lysis Reagent (Viagen Biotech, Los Angeles, CA) with 150 μg of Proteinase K (Invitrogen), and PCR was performed using the primers OL4062 (5′-GTCACTCAGCATTACACTTTCTA-3′), OL4064 (5′-CAGTGGGAATAAGGCAAGAGTGA-3′), and OL4088 (5′-GGTACAAGTGCACATGCCTGC-3′) (26). The CD4^Cre transgene was detected using the primers cd4cremu (5′-TTAGGGTGGGGCTCAGAAGG-3′), cd4creco (5′-AACTTCACAGC TCAGATGC-3′), and cd4crewt (5′-ACCTGGAT CC ACAAA CTTG-3′) (29). Mice were maintained at the University of Rochester Medical Center (URMC). All mice were housed in microisolator cages in a specific-pathogen free facility, and were provided food (LabDiet 5010, St. Louis MO) and water ad libitum. The animals were housed on a 12 hr light/dark cycle, and ambient temperatures were between 20–22°C. Commercially obtained mice were randomly housed with 3–5 mice per cage. Mice bred on-site were housed with same sex litter mates at weaning, and then randomly assigned to treatment groups at 6–12 weeks of age, with 3–5 mice per cage. All data presented are from female mice that were 6–12 weeks of age at the time of experiments, and each point in time relative to infection included 3–8 mice in each treatment group.

To activate the AHR, mice were administered the prototype AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; ≥99% purity, Cambridge Isotope Laboratories, Tewksbury, MA). TCDD was dissolved in anisole and diluted in peanut oil. B6 mice were randomly assigned to a group and administered a single dose orally of either TCDD (10 μg/kg body weight) or the vehicle control (peanut oil-anisole). AHR^dbd/dbd mice express the Ahr^d/d allele, which encodes a protein with 10-fold lower binding affinity for TCDD compared to the Ahr^b/b allele expressed by B6 mice (30). Thus, AHR^dbd/dbd mice were treated with 100 μg TCDD/kg body weight (31). For some experiments, mice were administered a different AHR agonist. Specifically, either 2-(1H-Indol-3-ylcarbonyl)-4-thiazolcarboxylic acid methyl ester (ITE; Tocris Bioscience, Minneapolis, MN) or Kynurenic acid (KYNA; Tocris Bioscience, Minneapolis, MN). ITE was dissolved in acetone and diluted in peanut oil. Mice were injected i.p. with 10 mg ITE/kg body weight/day, or the peanut oil acetone vehicle control, starting one day prior to infection (15). KYNA was dissolved in autoclaved drinking water at 250 mg/L, and provided to mice via the drinking water at a concentration of 25 mg/L (32). Mice were given the KYNA-containing water starting 14 days prior to infection and maintained on this water until they were sacrificed. The KYNA solution was made and replenished every 3–5 days. Another group of mice was given sterile, vivarium drinking water as controls for KYNA. For some experiments, mice were injected i.p. with 100 μg of BrdU 3 h prior to sacrifice (33, 34). All animal treatments had prior approval of the Institutional Animal Care and Use Committee of the University of Rochester. The University is accredited through the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were treated humanely and with due consideration to alleviation of any distress and discomfort. All U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals guidelines for the handling of vertebrate animals were followed.

Influenza A virus.

Influenza A virus (IAV) strain A/HKx31 (HKx31; H3N2) was propagated according to previously described methods (25, 35). Briefly, fertilized chicken eggs (Charles River, Wilmington, MA) were inoculated with 0.05 hemagglutinating units (HAU) influenza virus in 100 μL of Hank’s balanced salt solution (HBSS), containing 10 mM HEPES. Inoculated eggs were incubated for 48 h at 37°C, followed by refrigeration overnight at 4°C. Allantoic fluid was harvested under aseptic conditions, centrifuged, and frozen at −80°C. The titer of the allantoic fluid was determined by hemagglutination of avian erythrocytes (35). Mice were anesthetized by i.p. injection of avertin (2,2,2-tribromoethanol; Sigma Aldrich, St. Louis, MO) and infected intranasally (i.n.) with a sublethal dose (120 HAU virus in 25 μL) diluted in endotoxin-tested PBS (15, 25). Infections were performed in the morning (between 8:00 and 10:00 AM). All work with infectious agents was conducted with prior approval of the Institutional Biosafety Committee of the University of Rochester, following guidelines of the NIH/CDC.

Tissue collection and preparation of immune cells.

Tissue collection was initiated in the morning. Mice were sacrificed using lethal dose of anesthetic (e.g., Euthasol, Virbac, Greeley, CO), and either cervical dislocation or exsanguination. Blood was collected by cardiac puncture, with syringes containing 100 μL of 250 U/mL of heparin sodium salt (Grade I-A, ≥180 USP units/mg; Sigma Aldrich), and plasma was stored at −80 °C. Immune cells were isolated from the mediastinal lymph nodes (MLN), as previously described (36, 37). Briefly, single-cell suspensions were prepared by pressing lymph nodes from a single mouse between the frosted ends of two microscope slides. MLN cells were suspended in cold HBSS containing 2.5% FBS (Hyclone, Logan, UT). Erythrocytes were removed by incubating pelleted cells in a solution containing 0.15 M NH₄Cl, 10 mM NaHCO₃, and 1 mM EDTA for 5 min at room temperature. Erythrocyte lysis was terminated by adding equal volume of cold HBSS containing 2.5% FBS. Cell suspensions were then washed and passed through a 70μM cell strainer. After centrifugation (200 × g; 6 min), cell pellets were re-suspended in cold HBSS, 2.5% FBS for enumeration using a TC10 automated cell counter (BioRad, Hercules, CA), or a hemocytometer and Trypan blue exclusion. Cells were maintained at 4°C until labeling with fluorescent-conjugated antibodies, which was performed the same day.

Flow cytometry.

Single-cell suspensions containing 2 × 10⁶ lymph node cells were incubated with anti-mouse CD16/32 monoclonal antibody (mAb; clone93) for 10 min at 4° C to block non-specific staining. Cells were then incubated for 20 min at 4° C with previously determined optimal concentrations of fluorochrome-conjugated monoclonal antibodies against extracellular antigens (Supplemental Table I). After cell surface labeling, cells were fixed using 2% formaldehyde in PBS for 20 min, and analyzed directly by flow cytometry, or permeabilized for labeling of intracellular constituents (Supplemental Table I). Specifically, to detect BCL6 and FOXP3, cells were fixed and permeabilized using the FOXP3 Staining Kit (eBioscience, San Diego, CA) followed by incubation with anti-mouse CD16/32 mAb to block intracellular non-specific staining for 10 min at room temperature. Cells were then incubated with antibodies to BCL6 and FOXP3 for 45 min at room temperature. The BrdU Flow Kit (BD Biosciences, Franklin Lakes, NJ) was used to detect BrdU, and staining was performed following the manufacturer’s instructions. Cells were then stained with intracellular antibodies to BrdU for 20 min at room temperature. To identify live cells, prior to extracellular staining, cells were incubated for 30 min in PBS at 4°C with fixable viability dye eFluor 506 (eBioscience). Tfh cells were defined as CD4⁺ T cells that were CD44^hiCXCR5^hiPD1^hi (15, 38, 39). Tfr cells were defined as CD4⁺CD44^hiCXCR5^hiPD1^hiFOXP3⁺ cells (40). Germinal center (GC) B cells were defined as CD3⁻B220⁺CD95⁺GL7⁺ cells (41). Plasma cells were identified as CD3⁻B220^intCD138^hi cells (42). Fluorescence minus one (FMO) controls were used to determine non-specific fluorescence and define gating parameters. Data (500,000 events per sample) were collected using an LSRII flow cytometer (BD Biosciences). Data were analyzed using the FlowJo software program (Version 10; Tree Star, Ashland, OR).

Anti-influenza virus antibody ELISA.

Relative levels of influenza virus-specific antibodies in plasma were measured using enzyme linked immunosorbent assays (ELISA) as previously described (15, 24). In summary, 96-well ELISA microplates (MICROLON®, flat-bottom, Greiner Bio-one, Monroe, NC) were coated with purified HKx31 IAV (5 μg/mL, Charles River, Wilmington, MA) at 37°C overnight. Plates were washed with PBS containing 0.05% Tween 20 in between each step. To reduce non-specific binding, PBS containing 5% BSA was added to all wells, and plates incubated at 4°C for 2 h. Plasma samples were serially diluted in PBS 5% BSA from 1:100 to 1:25600. Diluted plasma was added to the plates and incubated overnight (16–20 h) at 4°C. Biotinylated goat anti-mouse IgG2a antibodies (Southern Biotechnology) were diluted to 1:5000 from commercial stock in PBS 5% BSA, and added to the plates, which were incubated at room temperature for 45 min. Avidin-peroxidase (1:400 dilution in PBS 5% BSA; Sigma Aldrich) was added directly to the plates and incubated at room temperature for 30 min. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) with 0.03% H₂0₂ was used to induce colorimetric change. Absorbance values were read at 405 nm using a SpectraMax Plate reader (Molecular Devices) To measure antibody affinity, PBS containing 1.0M guanidine hydrochloride (GuHCL; Sigma Aldrich), 0.2%Tween20 and 10 mg/mL BSA was added to the plates for 15 min at room temperature prior to adding biotinylated isotype-specific antibodies (43).

Statistical analysis.

Data were analyzed using JMP Pro 15 software (SAS Institute Inc.). Differences between means of two treatment groups at a single point in time were evaluated using a Student’s t-test. A two-way ANOVA followed by a Tukey honest significant difference (HSD) test was used to analyze means between treatment groups across time or mouse genotype. A two-way ANOVA followed by a Dunnett’s post hoc test was used to compare means within a treatment group across time. The specific statistical test used is indicated in each figure legend. Differences were considered statistically significant when p-values were ≤ 0.05. Error bars on all graphs and tables represent standard error of mean (SEM).

Results

AHR activation modulates Tfh cells over the course of IAV infection.

The AHR can be activated by small molecule agonists derived from a range of sources (44). For example, some AHR ligands, such as kynurenic acid (KYNA), are products of tryptophan metabolism and can arise in vivo. Other small molecules that bind AHR, such as 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE), are derived from natural substances and are produced synthetically (45). Xenobiotics, including industrial chemicals and pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also activate the AHR (44). We compared the impact of these three different AHR-binding molecules on Tfh cells during primary IAV infection. Tfh cells can be identified by flow cytometry as CD4⁺ T cells that express high levels of CD44, PD1, and CXCR5 (Fig. 1A) (15, 23, 38, 39). Treatment of mice with KYNA (Fig. 1B), ITE (Fig. 1C), and TCDD (Fig. 1D) significantly reduced the percentage of Tfh cells compared to infected mice treated with vehicle (control). Similarly, exposure to each AHR agonist significantly reduced the number of Tfh cells in infected mice (Fig. 1E–G). To further interrogate the effects of AHR activation on Tfh cells, we selected TCDD for three key reasons: it has a well-defined in vivo distribution and metabolism (46–48), it binds the AHR with high affinity and specificity (49, 50), and there is evidence that AHR-binding dioxin-like pollutants, affect humoral immune defenses in human populations (7, 51, 52). Therefore, TCDD is an excellent compound to study the AHR because it does not act through other receptors and pathways, maintains AHR activation for the duration of acute primary infection, and is relevant to human health.

FIGURE 1. AHR activation modulates Tfh cells during IAV infection.

C57BL/6 mice (6 weeks old, female) were administered the indicated AHR ligands as follows: TCDD (10 μg/kg body weight) was administered once by gavage one day prior to infection, ITE (10 mg/kg body weight) was given i.p. daily on days −1 to +8 relative to infection, and KYNA (25 mg/L) was administered via drinking water, starting 14 days prior to infection, and continuing until termination of the experiment. Control mice received the appropriate vehicle following the same treatment route and dosing schedule. Mice were infected intranasally (i.n.) with 120 hemagglutinating units (HAU) influenza A virus (IAV, strain HKx31; H3N2). Mediastinal lymph nodes (MLN) were harvested on day 9 post-infection for analysis by flow cytometry. (A) FACS plots depict gating strategy for identifying Tfh cells (CD4⁺CD44^hiCXCR5^hiPD1^hi). Prior gating excluded doublets and non-viable cells, and gated regions were defined using FMO controls. (B-D) Bar graphs indicate the percent of CD4⁺ T cells that were Tfh cells in mice treated with (B) KYNA, (C) ITE, and (D) TCDD on day 9 of infection. Bar graphs show the number of Tfh cells in (E) KYNA, (F) ITE, and (G) TCDD treated mice. *Denotes p≤ 0.05 compared to vehicle treatment group (two-sided Student’s t-test). All experiments had 6–8 mice in each treatment group. Experiments using ITE and TCDD were performed, independently, at least twice, whereas the effects of KYNA on Tfh cells were measured once.

The activation and differentiation of naïve CD4⁺ T cells to Tfh cells is a multi-factorial process that occurs over time (23). AHR activation could modulate Tfh cells at several different phases; thus, we evaluated the impact of AHR activation on Tfh cells over the course of acute primary IAV infection. In the control group, Tfh cells became clearly discernable by the 5^th day of infection, peaked around the 7–9^th day after infection, and then contracted (Fig. 2A). For instance, 5 days post-infection the percent of Tfh cells was over 55-fold higher than uninfected mice (Fig. 2A) and remained significantly higher than naïve mice till day 14 (Supplemental Table II). The number of Tfh cells showed a similar pattern (Fig. 2B). Compared to infected controls, AHR activation by the prototype AHR agonist, TCDD significantly reduced the percent (Fig. 2A) and number (Fig. 2B) of Tfh cells over the course of infection. For example, on day 5 post-infection, the percent of Tfh cells in TCDD treated mice was 70% less than in vehicle-treated infected mice and was not different from naïve mice (Fig. 2A and Supplemental Table II). Moreover, at the peak of the Tfh cell response, on day 9 post-infection, the number of Tfh cells was 15-fold lower in TCDD-treated mice compared to infected mice given the vehicle control (Fig. 2B). Thus, AHR activation suppressed the generation of Tfh cells over the course of infection.

FIGURE 2. AHR activation by TCDD reduces the percentage and number of Tfh cells over the course of IAV infection.

C57BL/6 mice were given TCDD or vehicle by gavage one day prior to infection with IAV. MLN were harvested on the indicated days relative to infection for analysis by flow cytometry. (A) FACS plots depict the percent of Tfh cells (CD4⁺CD44^hiCXCR5^hiPD1^hi cells) on the indicated days relative to infection. The number on each plot denotes the mean percentage of CD4⁺ T cells (± SEM) that were defined as Tfh cells. (B) Line graph shows the number of Tfh cells over time. Closed circles indicate the vehicle group and open squares indicate the TCDD treatment group. Error bars indicate the SEM. The p-value of the 2-way ANOVA is shown on the graph. *Denotes p≤ 0.05 compared to vehicle treatment group on the same day relative to infection (2-way ANOVA, Tukey HSD). A complete list of p-values from the ANOVA and post-hoc tests is in Supplemental Table II. Data are representative of one independent time-course experiment with 48 mice in each treatment group. All mice were infected at the same time, and each point in time relative to infection represents 6–8 mice in each group.

AHR activation by TCDD suppresses the germinal center response during infection.

Given that a major function of Tfh cells is helping B cells in germinal centers (GC) (23), we determined whether reduction in Tfh cells observed when the AHR was activated correlated with diminished GC B cells. In vehicle treated mice, the percent (Fig. 3A) and number (Fig. 3B) of GC B cells were significantly greater than naive mice by the 9^th day after infection (Supplemental Table II). On day 14 post-IAV infection, the number of GC B cells was over 500-fold greater than uninfected mice (Fig. 3B). However, AHR activation significantly reduced the percent (Fig. 3A) and number (Fig. 3B) of GC B cells in IAV infected mice. For instance, the percent of GC B cells in the TCDD treatment group did not significantly increase above what was observed in naïve mice until the 14^th day after infection (Fig. 3A, Supplemental Table II). Moreover, the number of GC B cells in mice treated with TCDD never exceeded the number detected in uninfected mice at any point in time examined (Fig. 3B, Supplemental Table II). When integrated with examination of Tfh cells, there was a strong correlation between AHR-driven reductions in the number of GC B cells and Tfh cells (Fig. 3C).

FIGURE 3. AHR activation by TCDD suppresses the germinal center response during viral infection.

C57BL/6 mice were given TCDD or vehicle by gavage one day prior to infection with IAV. MLN and blood (plasma) were harvested on the indicated days relative to infection for analysis by flow cytometry and ELISA. (A-B) Line graphs show (A) the percentage of live cells defined as germinal center (GC) B cells (CD3⁻B220⁺CD95⁺GL7⁺), and the (B) number of GC B cells. The p-value of the 2-way ANOVA is shown on the graph. *Denotes p≤ 0.05 for treatment groups compared on the same day after infection (2-way ANOVA, Tukey HSD). (C) The scatter plot depicts the number of Tfh cells versus the number of GC B cells on days 9 and 14 post-infection. The best-fit line was determined by linear regression. Open squares denote TCDD and closed circles indicate vehicle treated group. (D) Representative plots show the mean percentage of live cells defined as plasma cells (CD3-B220^intCD138^hi) in the MLN on day 14 of infection. (E) The bar graph shows the number of plasma cells. (F,G) Anti-IAV antibody levels in serially-diluted plasma (1:100 to 1:25600) were determined using ELISA. (F) Graph shows the mean circulating levels of IAV-specific IgG2a (1:1600 dilution). *Denotes p≤ 0.05 compared to vehicle (two-sided Student’s t-test). (G) Line graph depicts levels IAV-specific IgG2a in serially diluted plasma that was either untreated or treated with 1M GuHCL. Samples from vehicle treated mice are denoted with circles, and samples from TCDD treated mice with squares. Open symbols denote samples that were not treated with GuHCl (control), while closed symbols indicate treatment with 1M GuHCl. Samples from uninfected (naïve) mice are denoted using triangles. (H) Bar graphs show the median area under the curve for all plasma dilutions. * p≤ 0.05 for treatment groups that are significantly different from on another (2-way ANOVA, Tukey HSD). A compilation of p-values from the 2-way ANOVA and post-hoc tests are in Supplemental Table II. All data are shown as mean ± SEM, and are representative of two independent assessments with 6–8 mice per treatment group at each point in time.

Other B cell functions that depend on Tfh cells are the generation of plasma cells and high-affinity antibodies (23). AHR activation reduced the percentage (Fig. 3D) and number (Fig. 3E) of plasma cells by >80% compared to infected mice in the control group. Consistent with the diminished number of GC B cells and plasma cells, AHR activation reduced the levels of IAV-specific IgG2a by 7-fold (Fig. 3F). Antibody avidity can be measured using a modified ELISA that incorporates a chaotropic agent, such as guanidine hydrochloride (GuHCL), which disrupts low avidity antigen-antibody interactions (43). In plasma from control-treated infected mice, treatment with GuHCL significantly reduced levels of anti-IAV specific antibody by about 30%, such that the area under the curve (AUC) declined from a mean AUC of 3.65 to 2.45 (Fig. 3G–H). In contrast, treatment with GuHCL did not cause a significant change in IAV-specific antibodies in plasma from infected mice in the TCDD treatment group (Fig. 3G–H). At first glance, this might suggest greater antibody avidity compared to vehicle; however, the level of anti-IAV antibodies in the TCDD treated group was not different from plasma of naïve animals (Fig 3G–H). When integrated together, these results show that AHR activation reduced the generation of Tfh cells and attenuated key Tfh cell functions. The generation of Tfh cells involves CD4⁺ T cell proliferation and the upregulation of key molecules that drive Tfh cell fate commitment. Therefore, the reduced frequency and function of Tfh cells observed could result from AHR-mediated impact on proliferation, or the expression of critical transcriptional regulators that modulate Tfh cells, such as BCL6, and these are not mutually exclusive.

AHR activation attenuates CD4⁺ T cell proliferation in response to viral infection

To determine whether AHR activation affects Tfh cell proliferation, we used BrdU to label dividing cells in vivo (34). In mice treated with vehicle, BrdU⁺ Tfh cells were discernable 3 days after infection (Fig. 4A). The percent (Fig. 4A) and number (Fig. 4B) of BrdU⁺ Tfh cells peaked on the 5^th day and steadily declined thereafter in infected controls. This pattern was similar in mice in which the AHR was activated (Fig. 4A); however, the number of BrdU⁺ Tfh cells was significantly reduced (Fig. 4B). For instance, on day 5 post-infection, which was the peak day of Tfh cell proliferation in vehicle controls, there were >3 times fewer BrdU⁺ Tfh cells in the TCDD-treated group (Fig. 4B). When assessing the entire CD4⁺ T cell population, CD4⁺ T cells proliferated with similar kinetics as Tfh cells after infection (Fig. 4C–D). Consistent with prior reports using other antigenic challenges (53, 54), AHR activation blunted CD4⁺ T cell proliferation over the course of IAV infection (Fig. 4C–D). These findings suggest that one means by which AHR activation dampens the frequency of Tfh cells is, in part, by diminishing CD4⁺ T cell proliferation.

FIGURE 4. AHR activation attenuates CD4⁺ T cell proliferation in response to viral infection.

C57BL/6 mice were given TCDD or vehicle control one day prior to infection with IAV. Mice were administered (i.p.) 100 μg BrdU 3 hr prior to tissue collection. MLN were obtained on the indicated days relative to infection for analysis by flow cytometry. (A-B) The graphs indicate the (A) percent and (B) number of Tfh cells (CD4⁺CD44^hiCXCR5^hiPD1^hi cells) that were BrdU⁺ prior to and up to 14 days following IAV infection. Closed circles indicate mean values from vehicle group and open squares indicate the TCDD treatment group. (C-D) Line graphs show the (C) percent of CD4⁺ T cells that were BrdU⁺ and (D) number of BrdU⁺CD4⁺ T cells. The p-values on graphs were derived from the 2-way ANOVA. *Denotes p≤ 0.05 compared vehicle treated mice on the same day post-infection (2-way ANOVA, followed by Tukey HSD). Data are presented as mean ± SEM, and are from time-course experiment, in which all mice were infected at the same time. Each point in time relative to infection included 6–8 mice from each treatment group. A complete list of p-values from the 2-way ANOVA and post-hoc tests are in Supplemental Table III.

Fewer Tfh cells correlates with diminished levels of BCL6 in CD4⁺ T cells and alterations to T follicular regulatory (Tfr) cells

The transcriptional regulator BCL6 is required for Tfh cell differentiation (55). In response to IAV infection, CD4⁺ T cells significantly upregulated BCL6, which was 2.6-fold greater on day 5 post-infection compared to uninfected mice (Fig. 5A). Compared to day 5 post-infection, BCL6 levels in CD4⁺ T cells were about 2-fold lower on day 7, but were still significantly elevated compared to naïve mice. Moreover, 9 days after infection, BCL6 levels were higher than on day 7, but not as high as on day 5. By 14 days post-infection, BCL6 levels were similar to CD4⁺ T cells from uninfected mice (Fig. 5A). However, this biphasic expression pattern was not observed after AHR activation (Fig. 5B). Compared to vehicle treated mice, AHR activation reduced overall levels of BCL6 in CD4⁺ T cells (p=0.0008; 2-way ANOVA). Similar to the vehicle group, infection triggered an initial elevation in BCL6 (e.g., 5 days after infection compared to naïve); although the level of BCL6 was significantly lower than it was in CD4⁺ T cells from vehicle treated mice at this point in time (Fig. 5B). Moreover, 7 and 9 days after infection, BCL6 levels in CD4⁺ T cells from TCDD treated mice were not different from uninfected mice (Fig. 5B). Regression analysis showed a significant correlation between the level of BCL6 expression in CD4⁺ T cells and percentage of Tfh cells in vehicle and TCDD treated mice on day 9 post-infection (Fig. 5C). Thus, AHR-driven reduction of BCL6 levels in CD4⁺ T cells correlates with decreased Tfh cells.

FIGURE 5. BCL6 expression is diminished in CD4⁺ T cells after AHR activation.

C57BL/6 mice were administered TCDD or vehicle control one day prior to infection. MLN were harvested on the indicated days relative to infection for analysis by flow cytometry. (A-B) Histogram shows BCL6 expression in CD4⁺CD44^hi T cells from (A) vehicle and (B) TCDD treated mice over the course of infection. The number on each histogram denotes the mean fluorescence intensity (MFI) of BCL6 in CD4⁺CD44^hi cells. The fluorescence minus one (FMO) control is denoted with light grey. Asterisks (*) indicate p ≤ 0.05 compared to vehicle (2-way ANOVA, Tukey HSD) at that specific point in time. The hashtags (#) specify p ≤ 0.05 compared to naïve mice (day 0) in the same treatment group (2-way ANOVA, Dunnett’s test). A complete set of p-values from 2-way ANOVA and post-hoc tests are in Supplemental Table IV. (C) Regression analysis of the percentage of Tfh cells and BCL6 MFI in CD4⁺CD44^hi T cells on day 9 post-infection. Best-fit line was determined by linear regression. Closed circles denote vehicle and open squares denote TCDD treated groups. Data are mean ± SEM and are from a time course experiment. There were 6–8 mice/treatment group at each point in time.

Another category of CXCR5^hiPD1^hiCD4⁺ T cells are T follicular regulatory (Tfr) cells, which can be distinguished from Tfh cells based on the expression of the transcription factor FOXP3 (Fig. 6A) (40, 56). Whether AHR activation affects Tfr cells has not been investigated. During IAV infection, AHR activation significantly reduced the number of Tfr cells (Fig. 6B). However, the percentage CD4⁺CD44^hiCXCR5^hiPD1^hi cells that expressed FOXP3⁺ was elevated after AHR activation (Fig. 6C), such that the ratio of Tfr to Tfh cells was increased by 2.6-fold compared to infected mice given vehicle control (Fig. 6D). These results suggest that AHR activation shifts the balance of immunostimulatory and immunosuppressive follicular CD4⁺ T cells during IAV infection.

FIGURE 6. AHR activation modifies the balance of Tfh cells and T follicular regulatory (Tfr) cells.

C57BL/6 mice were given TCDD or vehicle control one day prior to infection. MLN were harvested on 9 days after infection for analysis by flow cytometry. (A) Representative flow plots show the gating strategy to identify Tfr cells (CD4⁺CD44^hiCXCR5^hiPD1^hiFOXP3⁺). (B) The bar graph shows the number of Tfr cells in the MLN. (C) Graph depicts the percent of follicular CD4⁺ T cells (CD4⁺CD44^hiCXCR5^hiPD1^hi) that were FOXP3⁺. (D) Stacked bar graph shows the ratio of Tfr to Tfh cells. The number beside each bar indicates the percentage of Tfr cells. *Denotes p≤ 0.05 compared to vehicle (two-sided Student’s t-test). Data are mean ± SEM and are representative of two independent experiments with 3–7 mice/group in each experiment.

The AHR modulates Tfh cells via a mechanism that requires AHR’s intrinsic DNA binding domain.

After ligand binding and translocation into the nucleus, the activated AHR-ligand complex binds a highly conserved core DNA sequence called an aryl hydrocarbon response element (AHRE) to modulate gene expression (57). There is also some evidence that the AHR may influence gene expression via alternative pathways that are independent of AHR binding to AHREs (58, 59). To determine whether AHR activation affects Tfh cells through a mechanism that requires AHR’s DNA binding domain (DBD), we used AHR DBD mutant (AHR^dbd/dbd) mice. These mice express an AHR protein that binds ligands, and translocates to the nucleus, but lacks its intrinsic DBD and cannot bind AHREs (27). In contrast to wild-type mice, exposure of AHR^dbd/dbd mice to TCDD no longer reduced the percent (Fig. 7A) or number (Fig. 7B) of Tfh cells during infection with IAV. Correspondingly, AHR activation significantly reduced the MFI of BCL6 in CD4⁺ T cells of wild-type, but not mutant mice (Fig. 7C). Furthermore, unlike in wild-type animals, AHR activation did not alter the percent (Fig. 6D) and number (Fig. 6E) of Tfr cells in AHR^dbd/dbd mice. In addition, the MFI of FOXP3 was significantly increased in CD4⁺ T cells from wild-type, but not mutant mice (Fig. 7F). Therefore, the AHR requires its ability to interact with DNA via its cognate DBD in order to alter Tfh, as well as Tfr cells during IAV infection.

FIGURE 7. The AHR modulates Tfh cells via a mechanism that requires AHR’s intrinsic DNA binding domain.

C57BL/6 (AHR^wt/wt) and DNA binding mutant (AHR^dbd/dbd) mice were administered vehicle or TCDD once by gavage one day prior to infection. 9 days after infection, mice were given 100 μg BrdU i.p. 3 hr prior to tissue collection. (A) Representative flow plots show the percentage of Tfh cells in the MLN. (B) Bar graph shows the number of Tfh cells. (C) Histograms show BCL6 expression in CD4⁺CD44^hi T cells from vehicle (solid, grey line) and TCDD (dotted, black line) treated AHR^wt/wt and AHR^dbd/dbd mice. (D-E) Bar graphs show the (D) percent of CD4⁺CD44^hiCXCR5^hiPD1^hi cells that are Tfr cells and the (E) number of Tfr cells. (F) Histograms show FOXP3 expression in CD4⁺CD44^hi T cells from vehicle (solid, grey line) and TCDD (dotted, black line) treated AHR^wt/wt and AHR^dbd/dbd mice. The numbers on the histograms denote the MFI. *Signifies p≤ 0.05 compared to vehicle in treatment group (2-way ANOVA, Tukey HSD). Data are mean ± SEM and are representative of two independent experiments, each of which had 6–8 mice/group.

AHR modulation of Tfh cells and T cell-dependent B cell responses to IAV infection requires the AHR in CD4⁺ cells.

The AHR is broadly expressed in immune cells, including dendritic cells, B cells and CD4⁺ T cells, including Tregs and Tfh cells (10, 22, 60–66). To determine whether changes in Tfh cell differentiation and function observed upon AHR activation are intrinsic to CD4⁺ T cells, we crossed CD4^Cre mice with AHR^fx/fx mice to ablate AHR from CD4⁺ lineage cells. Similar to wild-type mice, AHR activation reduced the percent (Fig. 8A) and number (Fig. 8B) of Tfh cells AHR^fx/fx mice. In addition, in AHR^fx/fx mice, AHR activation reduced the number of Tfr cells (Fig. 8C), but significantly increased the percent (Fig. 8D) of Tfr cells. In contrast, infected CD4^CreAHR^fx/fx mice treated with TCDD no longer exhibited a change in the percent (Fig. 8A) or number (Fig. 8B) of Tfh cells. The percent (Fig. 8D) and number (Fig. 8C) of Tfr cells was also unchanged by AHR activation in CD4^CreAHR^fx/fx mice. To examine Tfh cell function, we assessed key B cell responses during infection that rely on Tfh cells. While there were fewer GC B cells in AHR^fx/fx mice treated with TCDD (Fig 8E–F), in CD4^CreAHR^fx/fx mice, AHR activation did not affect the percent (Fig. 8E) or number (Fig. 8F) of GC B cells. Similarly, the percent (Fig. 8G) and number (Fig. 8H) of plasma cells in CD4^CreAHR^fx/fx were no longer repressed by AHR activation. In AHR^fx/fx mice, TCDD treatment reduced levels of IAV-specific IgG2a compared to vehicle (Fig. 8I,8K), while vehicle and TCDD treatment groups generated comparable levels of IAV-specific IgG2a in CD4^CreAHR^fx/fx mice (Fig. 8J, 8K). Thus, in the absence of AHR expression in CD4⁺ T cells, the antibody response is protected from the suppressive effects of TCDD. Another observation from these studies was that, compared to infected AHR^fx/fx mice, CD4^CreAHR^fx/fx mice produced significantly less IAV-specific antibodies (Fig. 8K). This suggests an intrinsic role of AHR in CD4⁺ T cells for optimal virus-specific antibody responses during acute primary infection. More broadly, these studies using CD4^CreAHR^fx/fx mice, indicate that AHR mediated events in CD4⁺ T cells modulate the generation and function of Tfh cells during IAV infection.

FIGURE 8. AHR modulation of Tfh cells and T cell-dependent B cell responses requires the AHR in CD4⁺ cells.

AHR^fx/fx and CD4^CreAHR^fx/fx mice were administered vehicle or TCDD once by gavage one day prior to i.n infection with IAV. MLN were harvested 9 and 14 days after infection, and plasma was collected 14 days after infection. (A-D) Tfh and Tfr cells were assessed in MLN 9 days after infection by flow cytometry. (A) The percentage of all CD4⁺ T cells that were Tfh cells (CD4⁺CD44^hiCXCR5^hiPD1^hi cells) in AHR^fx/fx and CD4^CreAHR^fx/fx mice. (B) The number of Tfh cells in AHR^fx/fx and CD4^CreAHR^fx/fx mice. (C-D) The (C) percent and (D) number of Tfr cells, defined as CD4⁺CD44^hiCXCR5^hiPD1^hiFOXP3⁺ cells. GC B cells, plasma cells and IAV-specific antibody levels were measured 14 days after infection using flow cytometry and ELISA. (E) Representative flow plots show the percent of live cells defined as GC B cells (CD3⁻B220⁺CD95⁺GL7⁺) in each group day 14 post-infection. The number on each plot indicates the mean for each group of mice. (F) Bar graph shows the number of GC B cells in the MLN from AHR^fx/fx and CD4^CreAHR^fx/fx mice. (G-H) Bar graphs show the (G) percent and (H) number of live cells defined as plasma cells (CD3⁻B220^intCD138^hi cells) in the MLN. (I-J) IAV-specific antibody levels in serially-diluted plasma were determined by ELISA. Line graphs show the relative level of IAV-specific IgG2a in (I) AHR^fx/fx and (J) CD4^CreAHR^fx/fx mice treated with vehicle (black circles) and TCDD (open squares). The p-values of the 2-way ANOVA between vehicle and TCDD are shown on the graphs. (K) Bar graph shows the median AUC for each group. *Signifies p≤ 0.05 compared to vehicle in treatment group (2-way ANOVA, Tukey HSD). Data are mean ± SEM with 6–8 mice/treatment group, and are representative of two independent experiments.

Discussion

While there is a growing appreciation that environmental exposures contribute to the variation observed in immune responses to infection in the human population (3, 4), it is unclear how external factors influence antiviral immune defenses. The work reported expands our understanding of how the AHR influences adaptive immune responses, showing that in addition to other T cell subsets, the AHR modulates Tfh cells. Using a common respiratory virus, IAV, and the prototype agonist, TCDD, we demonstrated that triggering the AHR affected several cellular processes critical for generating Tfh cells. These included CD4⁺ T cell proliferation and differentiation, which directly correlated with reduced metrics of antiviral humoral immune defenses, such as fewer germinal center B cells, plasma cells, and virus-specific antibodies. Given that Tfh cells are gatekeepers of humoral immunity and play a critical role in the production of high-affinity antibodies, these new findings have broad implications for host defenses against viral pathogens. Moreover, multiple studies have reported associations between exposure to chemicals that bind the AHR and increased severity of respiratory illness and diminished immune responses to vaccination (7–9). In animal models, prior studies have linked AHR ligands to alterations in cell-mediated antiviral T cell responses (15); however, less is known about how AHR alters T cell responses related to the humoral arm of adaptive responses to viral infection. Given that antibody responses are essential for long-lasting immunity, this current study provides new insight into how AHR signaling in T cells contributes directly to antiviral humoral immune defenses.

One of the key discoveries reported herein was that AHR activation modulated the proliferation and differentiation of CD4⁺ T cells into Tfh cells, as well as attenuated key Tfh cell-dependent functions over the course of infection. Moreover, AHR within T cells is a central driver of AHR-mediated influence on humoral immunity to respiratory infection. Although AHR modulation of Tfh cells has not been explored in other disease contexts, this finding is consistent with studies that have pointed to CD4⁺ T cells as central to AHR-mediated changes in non-infectious disease models in mice (20, 67–69). For example, transfer of CD4⁺ T cells treated with the AHR ligand ITE into mice with colitis significantly improved disease score and reduced colonic inflammation (67). In another mouse colitis model, transfer of AHR deficient Tregs into mice worsened gut inflammation and histopathological changes (69). The AHR was also required in CD4⁺ T cells in order for AHR agonist treatment to increase pathogenic Th17 cells in a mouse model of rheumatoid arthritis (68). Other than autoimmune diseases, AHR activation by TCDD also dampens T cell function in a graft-vs-host model in a manner that depends on the AHR in CD4⁺ T cells (20). Importantly, in CD4^CreAHR^fx/fx mice, the AHR is also excised from other cell types that express CD4 during development, including CD8⁺ T cells and a small subset of dendritic cells (70, 71). Prior studies have shown that AHR activation with TCDD and ITE suppresses CD8⁺ T cell responses during IAV infection (15). As such, one explanation that Tfh cells are unaffected by TCDD in CD4^CreAHR^fx/fx mice is that CD8⁺ T cells are able to control infection. However, vehicle and TCDD treated mice have similar lung viral burdens over the course of IAV infection (15, 24, 25, 72); thus, it is less likely that CD8⁺ T cells have a role in AHR-driven alterations to Tfh cells. Yet, the observation that only the second phase of elevated BCL6 expression in CD4⁺ T cells was attenuated by AHR activation suggests that AHR signaling in other cell types may also contribute to modulation of Tfh cell responses to infection. For example, dendritic cells play a particularly important role in early T cell activation, and are also modulated by AHR activation (31, 37, 73, 74). As such, even though events in CD4⁺ T cells were clearly affected by the AHR, the overall mechanism by which the AHR regulates CD4⁺ T cell activation and their differentiation to Tfh cells may be multi-faceted and involve more than one immune cell type.

In addition to demonstrating that AHR in CD4⁺ T cells is necessary for in vivo modulation of Tfh cells by an exogenous AHR ligand, the current study strongly connects AHR in CD4⁺ T cells to regulation of humoral immune responses to viral infection more broadly. That is, key B cell responses facilitated by Tfh cells were no longer affected by AHR ligand when CD4⁺ T cells lacked the AHR. This finding suggests that AHR signaling in CD4⁺ T cells not only regulates CD4⁺ T cell differentiation, but also modulates T cell cues to B cells. Further support that AHR in CD4⁺ T cells provides signals to B cells comes from the overall lower level of IAV-specific antibodies when CD4⁺ T cells lacked the AHR. This suggests that even in the absence of an exogenously added agonist, endogenous AHR signaling regulates key CD4⁺ T cell functions that influence antibody production or isotype switching. Other studies have reported that AHR activation affects CD4⁺ T cell expression of numerous cytokines, such as IFNγ and IL-10, suggesting one possible means to influence antibody isotype switching (75–77). Thus, the AHR likely regulates multiple pathways in CD4⁺ T cells. These include those involved in differentiation to functionally distinct subsets, including Tfh cells, and others involved in promoting optimal antibody production and isotype switching.

Another important finding from these current studies was that AHR activation blunted Tfh cell differentiation and proliferation starting during initial stages of viral infection. Although the percentage of BrdU⁺ Tfh cells unaffected by AHR activation, the entire pool of proliferating Tfh cells was significantly smaller in mice given TCDD. When integrated with AHR-mediated changes to Tfh cells occurring in a CD4⁺ T cell intrinsic manner, these results indicate that the AHR influences multiple early events in CD4⁺ T cells, including activation, proliferation, and differentiation. This idea aligns well with and extends prior observations from a graft-vs-host model, which showed that AHR activation induced transcriptional and functional changes in CD4⁺ T cells as early as 2 days into the response (19). Other prior studies have shown that AHR activation modulates CD4⁺ T cell proliferation, primarily in the context of non-infectious antigenic challenges, such as ovalbumin and alloantigens (17, 53, 54). Given that AHR activation also reduced the proliferation of the entire CD4⁺ T cell population in IAV-infected mice, the AHR may regulate some pathways that are generalizable to CD4⁺ T cells, rather than specific to Tfh cells. This seems likely because during IAV infection, AHR activation also reduced the frequency of Th1 cells, while increasing the percentage of Tregs (15). Furthermore, in response to a range of immune challenges, AHR activation can elicit distinct effects on CD4⁺ T cell subsets, such that there are more or less of a particular subset (15, 22). For instance, in contrast to TCDD, treatment of mice with FICZ increased the frequency of Tfh and Th1 cells during IAV infection (15). TCDD and FICZ also had opposite effects on Th17 cells and Tregs in a mouse model experimental autoimmune encephalomyelitis (22). Thus, it is likely that the AHR modulates CD4⁺ T cell proliferation and differentiation, but the specific consequences are dependent upon the antigenic challenge, AHR ligand, and also potentially microenvironmental differences in organs and tissues.

Tfr cells are a subset of follicular CD4⁺ T cells that are unique among FOXP3⁺ Treg cells in that they gain access to germinal centers and can suppress Tfh and B cell functions (56, 78, 79). Similar to Tfh cells, the impact of AHR activation on Tfr cells is poorly characterized. Considering that AHR activation increases Tregs during IAV infection (15) and modulates Tregs in other model systems (17, 22, 75), we postulated that Tfr cells may also be affected. Our observations suggest a nuanced role of AHR in modulating regulatory T cell subsets. Similar to Tfh cells, the number of Tfr cells was significantly reduced by AHR activation during IAV infection, which is consistent with, and perhaps a consequence of, diminution of CD4⁺ T cell responses overall. However, the relative ratio of Tfr to Tfh cells was almost tripled in TCDD treated mice. Thus, within the pool of follicular CD4⁺ T cells, AHR activation affects the balance of helper vs regulatory cells. One mechanism by which the AHR may regulate the proportion of Tfr and Tfh cells is by altering expression of key signaling molecules, such as the transcription factors BCL6 and FOXP3. BCL6 expression in CD4⁺ T cells is required for Tfh cell differentiation (55), and both BCL6 and FOXP3 are required to generate Tfr cells (79, 80). During IAV infection, AHR activation concurrently reduced BCL6 and increased FOXP3 levels in CD4⁺ T cells. One way in which AHR could affect these transcriptional regulators is by regulating gene expression. Consistent with this idea, changes in FOXP3 and BCL6 levels in CD4⁺ T cells from infected mice treated with TCDD required the AHR to have a functional DBD. Other studies have shown that the AHR regulates FOXP3 expression in CD4⁺ T cells by directly binding AHREs in the Foxp3 promoter region (22, 81). In addition to IAV infection, treatment with TCDD or ITE reduced BCL6 levels in CD4⁺ T cells in other model systems (67, 82); however, the mechanism by which the AHR regulates BCL6 is not yet certain. The AHR may directly regulate BCL6 gene transcription, as genomic analyses show that the Bcl6 promoter region harbors AHREs (83). Importantly, another explanation for the changes in protein expression of BCL6 and FOXP3 in CD4⁺ T cells is that AHR activation is changing the proportion of BCL6 and FOXP3-expressing cells, which could in turn affect the percentage of cells expressing these markers, or the overall level of expression of these transcription factors detected. Nonetheless, one potential mechanism by which the AHR modulates the balance between Tfh and Tfr cells may be via divergent regulation of Bcl6 and Foxp3 expression.

Demonstrating that the AHR modulates the differentiation and expansion of Tfh cells provides deeper understanding of how AHR agonists modulate humoral immune defenses during acute primary respiratory viral infection. This is important because humans are regularly exposed to AHR ligands from a variety of anthropogenic and natural sources (10, 84, 85). Thus, exposure to AHR ligands is one source of variability in responses to infection that are observed even in populations exposed to the same or similar pathogen (3, 5, 86). Indeed, exposure to AHR ligands from the environment, such as polyaromatic hydrocarbons in air pollution and cigarette smoke, as well as dioxins and PCBs in foods and other substances, has been associated with increased incidence and severity of infection and reduced antibody responses (7–9). Thus, via binding the AHR, environmental factors may contribute to variable responses to infection, and also potentially to vaccination. Furthermore, the importance of the AHR as a modulator of Tfh cells extends beyond understanding how pollutants impact humoral immunity in two key ways. There are myriad AHR agonists, which are not pollutants, that may be useful for attenuating antibody-mediated diseases (10, 11, 87).

Indeed, there is evidence that engaging the AHR may not necessarily always result in suppression of a beneficial immune response. For instance, in some instances AHR activation, even with TCDD, leads to a favorable outcome (22, 88). Also, some AHR agonists, particularly ones that transiently activate the AHR, may be able to boost Tfh cell responses which could, in turn, bolster antibody responses (15, 89). For example, during IAV infection, mice given the short-lived AHR ligand FICZ had a greater frequency of Tfh cells compared to control mice (15). This may relate to differences in ligand metabolism; however, the molecular reasons for differential immunological effects of distinct AHR ligands is not completely understood (15, 90, 91). In addition to boosting the number of Tfh cells, another possible avenue to elevate Tfh cell responses is screening for AHR ligands that modulate Tfr cells (89). However, approaches to bolster Tfh cells need to be viewed with caution as excessive Tfh cells can promote spontaneous germinal center formation, autoantibody production, and impair affinity maturation (23, 92, 93). Hence, there are scenarios in which modulating Tfh cell responses are beneficial in one setting and deleterious in another, and the AHR provides a potential targetable means to modulate Tfh cell responses. In summary, AHR modulation of Tfh cells and humoral immunity expands our understanding of how environmental contaminants can impact human health and disease, and also provides new perspectives on how we can improve vaccine efforts to pathogenic viruses that impact humans, such as IAV and SARS-CoV-2.

Key Points.

• AHR modulates Tfh cell responses during infection in a CD4⁺ T cell intrinsic manner

• AHR alters Tfr:Tfh cell ratio and levels of BCL6 and FOXP3 in CD4⁺ T cells

• AHR requires a functional DBD to modulate Tfh and Tfr cells during IAV infection

Abbreviations used in this article:

MLN

mediastinal lymph node

IAV

influenza A virus

AHR

aryl hydrocarbon receptor

Tfh

T follicular helper

Treg

regulatory T cells

Tfr

T follicular regulatory

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

GuHCL

guanidine hydrochloride

GC

germinal center

DBD

DNA-binding domain

AHRE

aryl hydrocarbon response element

i.n.

intranasally

ITE

2-(1H-Indol-3-ylcarbonyl)-4-thiazolcarboxylic acid methyl ester

KYNA

Kynurenic acid