Interleukin 9 mediates T follicular helper cell activation to promote antibody responses

Abstract

Antigen-specific humoral responses are orchestrated through complex interactions among immune cells in lymphoid tissues, including the collaboration between B cells and T follicular helper (Tfh) cells. Accumulating evidence indicates a crucial role for interleukin-9 (IL-9) in the formation of germinal centers (GCs), enhancing the generation of class-switched high-affinity antibodies. However, the exact function of IL-9 in Tfh cell regulation remains unclear. In this study, we examined the humoral immune responses of CD4^Cre/+Il9ra^fl/fl mice, which lack an IL-9-specific receptor in Tfh cells. Upon intraperitoneal immunization with sheep red blood cells (SRBCs), CD4^Cre/+Il9ra^fl/fl mice displayed diminished levels of SRBC-specific IgG antibodies in their sera, along with reduced levels of GC B cells and plasma cells. Notably, Il9ra-deficient Tfh cells in the spleen exhibited decreased expression of their signature molecules such as B-cell lymphoma 6, C-X-C chemokine receptor 5, IL-4, and IL-21 compared to control mice. In models of allergic asthma induced by house dust mite (HDM) inhalation, CD4^Cre/+Il9ra^fl/fl mice failed to elevate serum levels of HDM-specific IgE and IgG. This was accompanied by reductions in Tfh cells, GC B cells, and plasma cells in mediastinal lymph nodes. Furthermore, group 2 innate lymphoid cells (ILC2s) were identified as producers of IL-9 under immunizing conditions, possibly induced by leukotrienes released by activated IgD⁺ B cells around the T-B border. These observations may indicate the critical role of IL-9 receptor signaling in the activation of Tfh cells, with ILC2s potentially capable of supplying IL-9 in organized lymphoid tissues.

Keywords: IL-9; ILC2; T follicular helper cells; follicular mantle zone B cells; germinal center response; leukotrienes.

Figure 1

Tfh cells express IL-9 receptor. (A) Gating strategy for CD4⁺ T cell populations (CD3⁺CD4⁺) derived from human tonsillar lymphocytes, including Tfh cells (CD45RA^-CXCR5⁺PD-1⁺), naive CD4⁺ T cells (Tnaiv cells; CD45RA⁺CXCR5^-PD-1^-), and conventional CD4⁺ T cells (Tconv cells; CD45RA^-CXCR5^-PD-1^-). Histograms indicate the surface expression of IL-9RA on these CD4⁺ T cell populations. (B) Expression levels of IL-9RA on Tfh cells, Tnaiv cells, and Tconv cells in A using the geometric mean fluorescence intensity index (gMFI). ***p = 0.0002 (Tnaiv cells vs. Tfh cells), ***p = 0.0006 (Tconv cells vs. Tfh cells). Each dot represents an individual result from a tonsil, with a total of six tonsils analyzed. Data represent the mean ± SD. (C) Experimental protocol for the immunization of FoxP3^GFP/DTR mice. SRBCs were administered intraperitoneally, followed by the isolation and sorting of CD4⁺ T cell populations from splenocytes. (D) Sorting strategy for CD4⁺ T cell populations (B220^-CD4⁺CD8^-) from the splenocytes of FoxP3^GFP/DTR mice in C, including Tfh cells (GFP^-CXCR5⁺PD-1⁺), Tfr cells (GFP⁺CXCR5⁺PD-1⁺), Treg cells (GFP⁺CXCR5^-PD-1^-), and Tconv cells (GFP^-CXCR5^-PD-1^-). (E) Expression levels of Il9ra transcripts in CD4⁺ T cell subsets as measured using RT-qPCR. Each dot represents the average value of a group of mice (n = 8-10) in a single experiment. Data from 4-5 experiments are presented as the mean ± SD. **p = 0.0024 (Tconv cells vs. Tfh cells), **p = 0.0051 (Treg cells vs. Tfh cells), *p = 0.0135 (Tfr cells vs. Tfh cells). Similar results were obtained across three independent experiments. (F) Experimental protocol for in vitro culture of CD4⁺ T cell populations from FoxP3^GFP/DTR mice. SRBCs were administered intraperitoneally, and CD4⁺ T cell populations were isolated from splenocytes. Subsequently, CD4⁺ T cell populations were cultured with anti-CD3/CD28 mAbs in the presence or absence of IL-9. (G) Sorting strategy for CD4⁺ T cell populations (B220^-CD4⁺) from the splenocytes of FoxP3^GFP/DTR mice in F, including Tfh cells (GFP^-CD44⁺CXCR5⁺PD-1⁺), Tnaiv cells (GFP^-CD44^-CXCR5^-PD-1^-), and Tconv cells (GFP^-CD44⁺CXCR5^-PD-1^-). (H) Percentages of each CD4⁺ T cell population per total live cells, as assessed using flow cytometry with 7-AAD. ****p < 0.0001 (without IL-9 vs. with IL-9 in Tfh cells). (I) Expression levels of Bcl6 in each CD4⁺ T cell population, as analyzed using intracellular flow cytometry with histgrams and gMFI values. **p = 0.0024 (without vs. with IL-9 in Tfh cells). Each dot represents the average of triplicates in one experiment, and the data indicate the mean value. Similar results were obtained in two independent experiments. Statistical significance was analyzed using Tukey’s multiple comparisons test (B, E) and the unpaired t-test (H, I).

Figure 2

IL-9 assists Tfh cells in promoting GC formation. (A) Experimental protocol for the immunization of CD4^Cre/+Il9ra^fl/fl and control CD4^+/+Il9ra^fl/fl mice. Sera were collected from the tail vein. (B) Serum levels of SRBC-specific IgM and IgG. *p = 0.0476. (C) Representative confocal images of spleen sections, with B cells (CD19⁺) in white and GCs (PNA⁺) in red. Graphs show individual values of PNA⁺ GC areas in a tissue section, calculated using Zen software. *p = 0.0491. (D–F) Cell populations in the spleen ( Supplementary Figures S3A, B ). The percentages and cell numbers of each cell population are shown. (D) GC B cells (percentages, **p = 0.0078, *p = 0.0132; cell numbers, p = 0.0651, *p = 0.0173) and IgG1⁺ GC cells (percentages, **p = 0.0070; cell numbers, **p = 0.0098). (E) Plasma cells (percentages, **p = 0.0027, *p = 0.0293; cell numbers, ***p = 0.0003, *p = 0.0212) and IgG1⁺ plasma cells (percentages, *p = 0.0119; cell numbers, *p = 0.0251). (F) Tfh cells (percentages, *p = 0.0332, *p = 0.0202; cell numbers, **p = 0.0011, p = 0.0713). (G) Representative confocal images of spleen sections visualized for CD4 (green) and PNA (red). Graphs show the percentages of CD4⁺ areas localized within the PNA⁺ GC areas measured in CD4^Cre/+Il9ra^fl/fl and control CD4^+/+Il9ra^fl/fl mice 7 days after immunization with SRBCs. Imaging data were analyzed using ImageJ software. *p = 0.0162. Data represent the mean ± SD of 7-11 mice per group. Statistical significance was analyzed using the unpaired t-test (B–G). Similar results were obtained in two-four independent experiments.

Figure 3

IL-9 induces Tfh cells to express Bcl6, IL-4, and IL-21. (A) Experimental protocol for the immunization and analysis of FoxP3^GFP/DTRCD4^Cre/+Il9ra^fl/fl and control FoxP3^GFP/DTRCD4^+/+Il9ra^fl/fl mice. (B) Tfh cells (GFP^-B220^-CD4⁺CD44⁺CXCR5⁺PD-1⁺) and Tfr cells (GFP⁺B220^-CD4⁺CD44⁺CXCR5⁺PD-1⁺) in the spleen, assessed using flow cytometry. Tfh cell percentages, *p = 0.0492 on day 7, *p = 0.0350 on day 35. Tfh cell numbers, *p = 0.0484 on day 7. (C) Expression levels of Bcl6 in Tfh cells. Histograms show the results obtained using intracellular flow cytometry. Graphs depict gMFI values as the levels of Bcl6 expression. ****p < 0.0001 on day 7, p = 0.0575 on day 35. (D, E) Analysis of IL-4⁺ Tfh cells and IL-21⁺ Tfh cells in the spleen cells. After stimulation with PMA and ionomycin for 6 hr, cells were examined using intracellular flow cytometry ( Supplementary Figure S5A ). (D) IL-4⁺ Tfh cells. Percentages, ****p < 0.0001; cell numbers, **p = 0.0050. (E) IL-21⁺ Tfh cells. Percentages, ***p = 0.0005; cell numbers, *p = 0.0162. (F) Analysis of Tfh cell populations. Tfh cells were grouped into a series of four populations (1-4 in the panels) based on the levels of PD-1 and CXCR5 expression in flow cytometric profiles. Graphs of %Tfh cells in each population are presented in the panels. From left to right, p = 0.0523 and **p = 0.0084 on day 7; p = 0.0962, *p = 0.0158, *p = 0.0311, and p = 0.0812 on day 35. Data represent the mean ± SD of 5-13 mice per group. Statistical significance was analyzed by the unpaired t-test. Similar results were obtained in three independent experiments.

Figure 4

ILC2s produce IL-9 in secondary lymphoid tissues. (A) Experimental protocol for the immunization and analysis of FoxP3^GFP/DTR mice. Spleen cells were cultured with or without PMA and ionomycin (P/I) for 6 hr. (B, C) Analysis of ILCs (Lin^-CD45⁺CD127⁺) and ILC2s (GATA3⁺ ILCs) in the spleen cells before and after immunization (day 0 and day 7, respectively), as measured using intracellular flow cytometry ( Supplementary Figures S6A–C ). (B) Ratios of ILC2s in ILCs (GATA3⁺/isotype control), **p = 0.0040. Cell numbers of ILC2s, *p = 0.0320. (C) Ratios of IL-9⁺ ILC2s in ILC2s (IL-9⁺/isotype control), *p = 0.0404 (none in day 0 vs. P/I in day 7), **p = 0.0048 (P/I of day 0 vs. P/I in day 7), *p = 0.0193 (none in day 7 vs. P/I in day 7). Cell numbers of IL-9⁺ ILC2s, *p = 0.0267. P/I, PMA and ionomycin. (D–F) Ratios (IL-9⁺/isotype control) of immune cells expressing IL-9, as measured using intracellular flow cytometry ( Supplementary Figures S7A–C ). (D) CD4⁺ T cells (CD3⁺CD4⁺) and CD4⁺ T subsets including Tconv cells (CD3⁺GFP^-CD4⁺CXCR5^-PD-1^-), Tfh cells (CD3⁺GFP^-CD4⁺CXCR5⁺PD-1⁺), and CD8⁺ T cells (CD3⁺CD8⁺). (E) B cells (B220⁺) and B cell subsets including IgD⁺ B cells (B220⁺IgD⁺), GC B cells (B220⁺IgD^-FAS⁺GL7⁺), plasma cells (B220^-IgD^-CD138⁺), memory B cells (B220⁺IgD^-FAS⁺GL7^-CD38⁺). (F) Dendritic cells (CD11c⁺MHC-II⁺). Data represent the mean ± SD of 5-7 mice per group. Statistical significance was analyzed using Tukey’s multiple comparisons test (ratios in C) and the unpaired t-test (B, cell numbers in C, D–F). Similar results were obtained in three independent experiments.

Figure 5

Follicular mantle zone B cells can activate ILC2s. (A) Experimental protocol for B cell analysis of wild-type (WT) C57BL/6 mice. After the immunization, IgD⁺ and IgD^- B cells were sorted from the spleen cells. These B cells were cultured with or without anti-IgM (20 μg/ml) for 24 hr. (B) Leukotrienes in the culture supernatants of B cells. The graphs for LTB4; p = 0.0740 (none vs. anti-IgM in IgD⁺ B cells), p = 0.0723 (IgD⁺ vs. IgD^- B cells with anti-IgM), p = 0.0771 (medium vs. none in IgD⁺ B cells), **p = 0.0015 (medium vs. none in IgD^- B cells), ****p < 0.0001 (medium vs. anti-IgM in IgD⁺ B cells). The graphs for LTC4; **p = 0.0071 (none vs. anti-IgM in IgD⁺ B cells), *p = 0.0493 (IgD⁺ vs. IgD^- B cells with anti-IgM), **p = 0.0068 (medium vs. anti-IgM in IgD⁺ B cells). (C) Experimental protocol for ILC analysis of the WT mice. After the immunization, spleen cells were cultured with or without leukotriene (LTB4 or LTC4) and PMA and ionomycin (P/I) for 6 hr. (D) Ratios of ILC2s in ILCs (GATA3⁺/isotype control), ****p < 0.0001 (none vs. LTC4), ****p < 0.0001 (LTB4 vs. LTC4). Cell numbers of ILC2s, p = 0.0797 (none vs. LTC4). (E) Ratios of IL-9⁺ ILC2s in ILC2s (IL-9⁺/isotype control), **p = 0.0025 (none vs. LTC4), *p = 0.0152 (LTB4 vs. LTC4). Cell numbers of IL-9⁺ ILC2s, **p = 0.0020 (none vs. LTC4), *p = 0.0345 (LTB4 vs. LTC4). (F) Experimental protocol for ILC2 analysis of the WT mice. After the immunization, ILC2s were isolated from spleen cells and cultured with or without LTC4 for 6 hr. (G) Ratios of IL-9⁺ ILC2s in ILC2s (IL-9⁺/isotype control), *p = 0.0094. Cell numbers of IL-9⁺ ILC2s, **p = 0.0044. Data represent the mean ± SD of 3-7 mice per group. Statistical significance was analyzed using Tukey’s multiple comparisons test (B, D, E) and the unpaired t-test (G). Similar results were obtained in two independent experiments.

Figure 6

IL-9 promotes Tfh cells to generate allergen-specific antibodies. (A) Experimental protocol for HDM exposure in CD4^Cre/+Il9ra^fl/fl and control CD4^+/+Il9ra^fl/fl mice. Mice were sensitized by intranasal instillation of 25 μg HDM in 50 μl PBS or 50 μl PBS alone as a control for three days per week for 5 consecutive weeks. The mice were analyzed on day 37. (B) Representative microscopy images of formalin-fixed paraffin-embedded tissue sections of the lung stained with HE and PAS. (C) Quantification of PAS-positive mucous cells in airways. Graphs depict the ratio of PAS⁺ areas within the airway area for each group. Imaging data were analyzed using ImageJ software. ****p < 0.0001. (D) Serum levels of HDM-specific antibodies. Anti-HDM-specific IgE, ***p = 0.0005; anti-HDM-specific IgG1, *p = 0.0181. (E–G) Representative flow cytometric profiles and graphs of lymphocyte subsets within the mediastinal lymph nodes ( Supplementary Figures S3A, B ). (E) IgG1⁺ GC B cells (B220⁺IgD^-FAS⁺GL7⁺). Percentages, **p = 0.0055; cell numbers, *p = 0.0167. (F) IgG1⁺ plasma cells (B220^loIgD^-CD138^hi). Percentages, *p = 0.0205. (G) Tfh cells (B220^-CD4⁺CD44⁺CXCR5⁺PD-1⁺). Percentages, *p = 0.0446; cell numbers, p = 0.0796. Data represent the mean ± SD of 5-10 mice per group. Statistical significance was analyzed using the unpaired t-test (C–G). Similar results were obtained in three independent experiments.