A dominant, coordinated T regulatory cell-IgA response to the intestinal microbiota

Abstract

A T cell receptor transgenic mouse line reactive to a microbiota flagellin, CBir1, was used to define mechanisms of host microbiota homeostasis. Intestinal IgA, but not serum IgA, was found to block mucosal flagellin uptake and systemic T cell activation in mice. Depletion of CD4(+)CD25(+) Tregs decreased IgA(+) B cells, total IgA, and CBir1-specific IgA in gut within days. Repletion of T cell-deficient mice with either CD4(+)CD25(+) or CD4(+)foxp3(+) Tregs restored intestinal IgA to a much greater extent than their reciprocal CD4(+) subsets, indicating that Tregs are the major helper cells for IgA responses to microbiota antigens such as flagellin. We propose that the major role of this coordinated Treg-IgA response is to maintain commensalism with the microbiota.

Fig. 1.

CBir1 Tg T cells proliferated in response to systemic stimulation, but not to mucosal stimulation with CBir1 flagellin in B6.WT mice (A). CFSE-labeled CD4⁺ T cells (1 × 10⁶) from Thy1.2. CBir1 Tg mice and 1 × 10⁶ CFSE-labeled CD4⁺ T cells from Thy1.1. OT-II mice were co-transferred into wild-type Thy1.2. B6 mice. The recipient mice were injected i.p. with 50 μg CBir1 and 100 μg OVA or gavaged with 100 μg CBir1 and 100 μg OVA the next day respectively. Spleen CD4⁺ T cell proliferation was analyzed 48 h later from the i.p. injected recipients and 72 h later from the gavaged recipient mice. The percentage of divided Thy1.1⁺ OT II T cells and Thy1.1⁻ CBir1 Tg T cells was calculated by flow cytometry by gating on Thy1.1⁺ and Thy1.1⁻ cells. *, P ≤ 0.01 compared to divided CBir1 Tg T cells in response to CBir1 flagellin given i.p. (B and C) CFSE-labeled CD45.1⁺ CBir1 Tg T cells (1 × 10⁶) were transferred into wild-type B6 or B6.IgA KO mice. (D and E) CFSE-labeled TCRVß8.3⁺ CBir1 Tg T cells (1 × 10⁶) were transferred into wild-type B6 or B6.pIgR KO mice. (F and G) CFSE-labeled TCRVß8.3⁺ CBir1 Tg T cells (1 × 10⁶) were transferred into wild-type B6 or and B6.TCRβ×δ KO mice. The recipient mice were gavaged with CBir1 the next day. Splenic CD4⁺ T cell proliferation was analyzed 72 h later. CFSE-CBir1 Tg T cells proliferated in response to gavaged CBir1 Flagellin in B6.IgA KO mice (C), B6.pIgR KO mice (E), and B6.TCRβxδ KO mice (G), but not in WT B6 mice (B, D, and F). Representative data of three experiments is shown. (H) The percentage of divided CBir1 Tg T cells from six recipient mice of each group was shown as mean ± SE. *, P ≤ 0.01 compared to divided cells in B6 recipients.

Fig. 2.

Depletion of CD4⁺CD25⁺ Treg cells decreased intestinal IgA responses. B6 mice were injected with 100 μg anti-CD25 mAb twice at days 0 and 3. Stool pellets were collected at days −3, 0, 3, 5, and 7. LP IgA B cells were determined by flow cytometry at days 5 and 7. (A) CD4⁺CD25⁺ T cells were depleted in LP of the mice treated with anti-CD25 but not control mAb. (B) IgA⁺ B220⁻ B cells were decreased in LP of the mice treated with anti-CD25 but not control mAb. (C) B cell numbers from four mice of each group. *, P ≤ 0.05 compared to control mAb treated mice. (D) Total pellet IgA production, assessed by ELISA. (E) Pellet anti-CBir1 IgA production, assessed by ELISA. *, P ≤ 0.05 compared to control Ab-treated group. (F) CFSE-labeled CD4⁺ T cells (1 × 10⁶) from CBir1 Tg mice were transferred into CD25-depleted or control B6 mice at day 3, the same day that recipient mice were administered the second mAb injection. Recipient mice were gavaged with CBir1 flagellin the next day (day 4) and CBir1 Tg CD4⁺ T cell proliferation was determined 3 days later (day 7). One representative of three experiments is shown.

Fig. 3.

Adoptive transfer of CD4⁺foxp3⁺ Treg cells restored intestinal IgA in B6.TCRβ×δ^−/− mice. CD4⁺ foxp3⁺ Treg cells and CD4⁺ foxp3⁻ T cells from B6.GFP-foxp3 mice were transferred into B6.TCRβ×δ^−/− mice at 1 × 10⁶/mouse. (A) LP IgA⁺ B cells were analyzed by flow cytometry at day 21. (B) B cell numbers from four mice of each group. *, P ≤ 0.05 compared to the recipient mice of CD4⁺ foxp3⁻ T cells. (C and D) Stool pellets were collected at days 0, 4, 7, 14, and 21. Total IgA (C) and anti-CBir1 IgA (D) were assessed by ELISA. *, P ≤ 0.05 compared to foxp3⁻ T cell group.

Fig. 4.

CD25⁺ foxp3⁺ Treg cells provided help to IgA B cells via TGFβ. Splenic IgD⁺ B cells (2.5 × 10⁶) from B6 mice were cultured with 1 × 10⁶ flow sorted CD4⁺foxp3⁺ T cells or 1 × 10⁶ CD4⁺foxp3⁻ T cells in the presence of 20 ng/mL TGFβ or 10 μg/mL anti-TGFβ mAb in the plate coated with 5 μg/mL of anti-CD3 and 10 μg/mL soluble anti-CD28 mAb. B cells were stimulated only by the activated T cells. (A) Supernatant was collected at day 5 and IgA was measured by ELISA. (B) B cells were re-isolated at day 3 and RNA was isolated from B cells for determination of AID using RT-PCR. One representative of three experiments is shown.

Fig. 5.

Treg cells promoted B cell IgA production in an antigen-specific manner. (A) PP B220⁺ B cells (2.5 × 10⁶) from B6 mice were cultured with 1 × 10⁶ CD4⁺foxp3⁺ T cells in the presence of 1 × 10⁶ CBir1-pulsed BMDCs or rIB9-pulsed BMDCs. Culture supernatant was collected at day 5 and IgA was measured by ELISA. (B) PP B220⁺ B cells (2.5 × 10⁶) from B6 mice were cultured with 1 × 10⁶ CD4⁺CD25⁺ Treg cells from CBir1 Tg mice in the presence of 1 × 10⁶ CBir1-pulsed BMDCs or rIB9-pulsed BMDCs. Culture supernatant was collected at day 5 and IgA was measured by ELISA. *, P ≤ 0.05 compared to B cells cultured with antigen-pulsed DC alone; **, P ≤ 0.01 compared to B cells cultured with DCs without antigen.