Follicular helper T cells mediate IgE antibody response to airborne allergens

Abstract

Background: T_H2 cells have long been believed to play a pivotal role in allergic immune responses, including IgE antibody production and type 2 cytokine-mediated inflammation and pathology. A new T-cell subset, follicular helper T (T_FH) cells, is specialized in supporting B-cell maturation and antibody production.

Objective: We sought to investigate the roles of T_FH cells in allergic immune responses.

Methods: Naive mice were exposed to cytokines or natural allergens through the airways. Development of allergic immune responses was analyzed by collecting draining lymph nodes and sera and by challenging the animals. Cytokine reporter mice and gene-deficient mice were used to dissect the immunologic mechanisms.

Results: We observed the development of IL-4-producing T_FH cells and T_H2 cells in draining lymph nodes after airway exposure to IL-1 family cytokines or natural allergens. T_FH and T_H2 cells demonstrated unique phenotypes, tissue localization, and cytokine responses. T_FH cells supported the sustained production of IgE antibody in vivo in the absence of other T-cell subsets or even when T_H2 cell functions were severely compromised. Conversely, conditional deficiency of the master regulator Bcl6 in CD4⁺ T cells resulted in a marked reduction in T_FH cell numbers and IgE antibody levels, but type 2 cytokine responses and eosinophilic inflammation in the airways remained unaffected.

Conclusion: T_FH cells play critical roles in the regulation of IgE antibody production. Allergic immune responses to airborne allergens likely involve 2 distinct subsets of IL-4-producing CD4⁺ T cells, namely T_FH and Th2 cells.

Keywords: Follicular T cells; IL-4; IgE; T(H)2 cells; allergens; allergy.

Figure 1

IL-33 promotes the development of two distinct subsets of IL-4-competent CD4⁺ T cells. (A) A schematic representation of a mouse airway sensitization model. Naïve 4get mice (BALB/c background) were exposed i.n. to OVA with or without IL-1β or IL-33. (B) On day 14, plasma was analyzed for anti-OVA antibodies by ELISA. * = p<0.05 and ** = p<0.01 compared to mice exposed to OVA alone. # = p<0.05 compared to mice exposed to OVA plus IL-33. Data are presented as the mean ± SEM (n = 4–5 in each group). (C) Day 11 mLN cells were cultured with or without OVA. The cytokine levels in the supernatants were analyzed by ELISA. # = p<0.05 compared to OVA plus IL-33. Data are presented as the mean ± SEM (n = 4–5 in each group). (D–G) Day 11 mLN cells were gated on lymphocytes, and the expression of CD4, IL-4eGFP, ST2, and CXCR5 was analyzed by FACS. (D) Representative scattergrams showing the expression of CD4 and IL-4eGFP in a total lymphocyte population, and the expression of ST2 and CXCR5 in CD4⁺IL-4eGFP⁺ lymphocytes. (E) Numbers of each cell population are presented as the mean ± SEM (n = 4 in each group). * = p<0.05 between the groups indicated by horizontal lines. (F) Naïve 4get mice were exposed i.n. to OVA with different doses of IL-33 as described in Figure 1A, and day 11 mLN cells were analyzed. (G) Numbers and the ratio of each cell population are presented as the mean ± SEM (n = 4 in each group). ** = p<0.01 between the groups indicated by horizontal lines or as compared to 1 ng IL-33. (C, E, G) Data are a pool of two separate experiments.

Figure 2

ST2^-CXCR5⁺ CD4⁺ T cells are bona fide Tfh cells. (A–D) Naïve 4get mice (BALB/c background) were exposed i.n. to OVA plus IL-33 as described in Figure 1A. The cell populations indicated (CD4⁺IL-4eGFP⁻, CD4⁺IL-4eGFP⁺ST2⁺CXCR5⁻, and CD4⁺IL-4eGFP⁺ST2⁻CXCR5⁺) were sorted from day 11 mLN cells, and mRNA was analyzed by microarray and qRT-PCR. (A) The heat map shows microarray gene expression data (genes selected for fold difference >10) based on cells isolated from two separate experiments. (B) The Venn diagram shows the number of gene probes in each gene expression group. (C) Representative genes in each group are presented using the criteria described in the Table. (D) mRNA expression of select genes was analyzed by qRT-PCR, and the results were normalized to 18s RNA levels and expressed as a ratio to the CD4⁺IL-4eGFP⁻ population (mean ± SEM, n = 4). * = p<0.05 and ** = p<0.01 between the groups indicated by horizontal lines. (E, F) Naïve BALB/c mice were exposed i.n. to OVA plus IL-33, as described in Figure 1A. Day 11 mLN cells were sorted into CD3⁺CD4⁺ST2^-CXCR5⁻, CD3⁺CD4⁺ST2⁺CXCR5⁻, and CD3⁺CD4⁺ST2⁻CXCR5⁺ cell populations and cultured with B cells isolated from mLNs in the presence of OVA. (F) Cytokine levels in the supernatants were analyzed by ELISA. Data are presented as the mean ± range from duplicate cultures. The figure is representative of two separate experiments.

Figure 3

Th2 and Tfh cells preferentially mediate allergic airway inflammation and IgE antibody production, respectively. (A) A schematic representation of an adoptive transfer model to Tcrb^−/− mice (C57BL/6 background) (B) The plasma antibody levels were analyzed by ELISA. Results are the mean ± SEM (n = 3 in each group). ** = p<0.01 compared to mice that received Th2 cells. (C) A schematic representation of an adoptive transfer model to Il7^−/− mice (C57BL/6 background). BAL fluids were analyzed for cytokine levels (D) and inflammatory cell numbers (E). Results are the mean ± SEM (n = 3 in each group). ** = p<0.01 between the groups indicated by horizontal lines. (B, D, E) Each figure is representative of two separate experiments.

Figure 4

Th2 and Tfh cells localize differently during allergic immune responses. (A) A schematic representation of a mouse airway sensitization and challenge model. (B) Representative scattergrams show the expression of ST2 and CXCR5 in the CD4⁺IL-4eGFP⁺ population. (C) Percentages of each cell population are presented as the mean ± SEM (n = 8 in each group). ** = p<0.01 between the groups indicated by horizontal lines. Data are a pool of two separate experiments.

Figure 5

Airway exposure to natural allergens induces Tfh and Th2 cells. (A) A schematic representation of the airway allergen exposure model. Naïve 4get mice (BALB/c background) were exposed i.n. to allergen extracts without any adjuvants. Day 11 mLN cells were gated on lymphocytes, and the expression of CD4, IL-4eGFP, ST2, and CXCR5 was analyzed by FACS. (B) Representative scattergrams are shown. (C) Numbers of each cell population are presented as the mean ± SEM (n = 3 in each group). Data are representative of two separate experiments, except for HDM, where the experiment was performed only once.

Figure 6

Tfh cells are diminished by depletion of Bcl6 in CD4⁺ T cells (A) A schematic representation of a mouse airway sensitization model. (B) Representative scattergrams show the expression of ST2, CXCR5, and PD-1 in the CD4⁺ T cell population. mLN cells were analyzed by gating on CD4⁺ T cells in the lymphocyte population. (C) Cell numbers in each cell population are presented as the mean ± SEM (n = 5–7 in each group). * = p<0.05 and ** = p<0.01 between the groups indicated by horizontal lines. Data are a pool of three separate experiments. (D, E) Expression of B220, PNA, and FAS in mLN lymphocytes was analyzed by FACS. (D) Representative scattergrams show the expression of B220 in the lymphocyte population and the expression of PNA and FAS in the B220⁺ cell population. (E) Percentages and cell numbers in each cell population are presented as the mean ± SEM (n = 4 in each group). * = p<0.05 and ** = p<0.01 between the groups indicated by horizontal lines. (F) Day 11 mLN cells were cultured with or without OVA, and the cytokine levels in the supernatants were analyzed by ELISA. Results are shown as the mean ± SEM (n = 4 in each group). * = p<0.05 between the groups indicated by horizontal lines.

Figure 7

Tfh cells are necessary for the production of allergen-specific IgE antibody. (A) A schematic representation of a mouse airway sensitization and challenge model. (B) Plasma levels of anti-OVA IgE and IgG1 antibodies and total IgE, IgG1, IgM, and IgA were analyzed by ELISA. ** = p<0.01 compared to Bcl6 ^fl/fl mice previously exposed to OVA alone. ## = p<0.01 compared to Bcl6 ^fl/fl mice previously exposed to Alternaria extract plus OVA. Data are presented as the mean ± SEM (n = 5 in each group). The cytokine levels in lung homogenates (C) and the numbers of inflammatory cells in BAL fluids (D) were analyzed on day 45. (C, D) Results are presented as the mean ± SEM (n = 5 in each group). * = p<0.05 and ** = p<0.01 between the groups indicated by horizontal lines. Data are representative of two separate experiments.

Figure 8

OX40L contributes to type 2 cytokine responses and airway inflammation but does not affect the development of Tfh cells or IgE antibody production. (A–C) Naïve C57BL/6 (WT) and OX40L knockout (Tnfsf4^−/−) mice were exposed i.n. to OVA and Alternaria extract on days 0 and 7, and mLN cells were analyzed on day 11. (A) Representative scattergrams show the expression of ST2 and CXCR5 in the CD4⁺ T cell population. (B) Numbers of each cell population are presented as the mean ± SEM (n = 4 in each group). (C) mLN cells were cultured with or without OVA, and the cytokine levels in the supernatants were analyzed by ELISA. Results are the mean ± SEM (n = 5–7 in each group). * = p<0.05 between the groups indicated by horizontal lines. Data are representative of two separate experiments. (D) A schematic representation of a mouse airway sensitization and challenge model. The cytokine levels in lung homogenates (E) and the numbers of inflammatory cells in BAL fluids (F) were analyzed on day 25. Results are the mean ± SEM (n=5–7 in each group). * = p<0.05 and ** = p<0.01 between the groups indicated by the horizontal lines. (G) Plasma levels of anti-OVA IgE, IgG1, and IgG2a antibodies were analyzed by ELISA. * = p<0.05 and ** p<0.01 between the groups indicated by horizontal lines. Data are presented as the mean ± SEM (n = 5–7 in each group). (C, E, F, G) Data are a pool of two separate experiments.