Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

Abstract

Immunity to intestinal helminth infections requires the rapid activation of T helper 2 cells (Th2 cells). However, simultaneous expansion of CD4⁺Foxp3⁺ regulatory T cells (T reg cells) impedes protective responses, resulting in chronic infections. The ratio between T reg and effector T cells can therefore determine the outcome of infection. The redifferentiation of T reg cells into Th cells has been identified in hyperinflammatory diseases. In this study, we asked whether ex-T reg Th2 cells develop and contribute to type-2 immunity. Using multigene reporter and fate-reporter systems, we demonstrate that a significant proportion of Th2 cells derive from Foxp3⁺ cells after Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus Through selective deletion of Il4ra on Foxp3⁺ cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting T reg cells into Th2 cells could concomitantly enhance Th2 cells and limit T reg cell-mediated suppression.

Figure 1.

A shift in the ratio of T reg to Th2 cells correlates with the functional expulsion of H. polygyrus. (A) Experimental model. Female Il4^GFPFoxp3^RFP mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on days 14–15 and reinfected on day 28. Hp 1° mice were given a primary infection at the same time point. (B) Intestinal worm burden at day 14 after infection. Data are representative of at least three experiments with three mice per group. (C) Hematoxylin and eosin (H&E)– and Alcian blue–periodic acid–Schiff (AB-PAS)–stained sections of the small intestine of Hp 1° and Hp 2° mice day 7 after infection, depicting H. polygyrus larvae. (D) Gene expression of Arg1, Retnla, Retnlb, and Chil3 in the small intestine of Hp 1° and Hp 2° mice day 7 after infection, expressed as fold-change relative (rel.) to naive small intestine. Data represent six to seven mice per group pooled from two independent experiments. (E) Representative FACS plots of Il4^GFP and Foxp3^RFP expression in CD4⁺TCRβ⁺ MLN cells of naive, Hp 1°, and Hp 2° mice day 7 after infection. (F–I) Proportion (F) and absolute number (G) of Il4^GFP+ cells and Foxp3^RFP+ cells (H and I) in the MLN and PPs of naive, Hp 1°, and Hp 2° mice day 7 after infection. (J and K) Ratio of Il4^GFP+ cells over and Foxp3^RFP+ cells in the MLN (J) and PPs (K). Data are representative of three experiments with three to five mice per group. *, P ≤ 0.05; one-way ANOVA or Mann-Whitney test. Error bars represent SEM.

Figure 2.

Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4-expressing cells after secondary infection with H. polygyrus. (A) Experimental model. T reg cells (HpTR; CD4⁺TCRβ⁺Il4^GFP–Foxp3^RFP+CD25^high) were sort purified from male Il4^GFPFoxp3^RFP mice infected with H. polygyrus for 14 d. nT cells (CD4⁺TCRβ⁺CD25^–CD44^lowIl4^GFP–Foxp3^RFP–) were sort purified from the spleen and MLN of naive male Il4^GFPFoxp3^RFP mice. HpTR or nT cells were transferred to male Tcra^−/− mice further subjected to a secondary infection with H. polygyrus. (B) Representative FACS plots of Foxp3^RFP, Il4^GFP, and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3^RFP+CD25^high cells in HpTR recipients day 42 after transfer and relative to Foxp3^RFP expression before transfer (C) and proportion of CD4⁺TCRβ⁺Il4^GFP+Foxp3^RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→Il4^GFP+, HpTR→Il4^GFP+, or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4⁺CD44^highIL-4⁺ cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4⁺TCRβ⁺Foxp3^RFP+CD25^high or CD25^low cells were sort purified from Il4^GFPFoxp3^RFP mice infected with H. polygyrus for 14 d and transferred to Tcra^−/− mice subjected to a secondary infection with H. polygyrus. (G) Representative FACS plots of Foxp3^RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4⁺TCRβ⁺Il4^GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.

Figure 3.

Gene expression profiling reveals ex-Foxp3 cells express characteristic Th2 cell–related genes. (A) Representative FACS plots of Il4^GFP and Foxp3^RFP expression in sort-purified Il4^GFP+ cells originating from transferred HpTR cells (HpTR→Il4^GFP+). Gene expression profiling was performed using a microarray. (B) Venn diagram showing comparative analysis of common and uniquely expressed genes relative to nT cells between donor HpTR cells, HpTR→Il4^GFP+ cells, and Il4^GFP+ cells originating from transferred nT cells (nT→Il4^GFP+). (C) Pathway analyses of differentially expressed genes in HpTR→Il4^GFP+ cells compared with nT→Il4^GFP+ and HpTR cells. For each biological replicate, cells were sorted from three to four mice. This figure represents a mean gene expression for three biological replicates (D) Genes involved in proximal TCR signaling, uniquely expressed in HpTR→Il4^GFP+ cells. (E) Expression of Th2 and T reg cell–related genes in HpTR cells, HpTR→Il4^GFP+, or nT→Il4^GFP+ relative to nT cells. Data are representative of one experiment with three biological replicates per group. For each biological replicate, cells were sorted from three to four mice. T reg cells (HpTR; CD4⁺TCRβ⁺Il4^GFP–Foxp3^RFP+CD25^high), Th2 (CD4⁺TCRβ⁺Il4^GFP+Foxp3^RFP–) cells, and double-positive cells (CD4⁺TCRβ⁺Il4^GFP+Foxp3^RFP+) were sort purified from male Il4^GFPFoxp3^RFP mice infected with H. polygyrus for 14 d. nT cells (CD4⁺TCRβ⁺CD25^–CD44^lowIl4^GFP–Foxp3^RFP–) were sort purified from the spleen and MLN of naive male Il4^GFPFoxp3^RFP mice. (F) Expression of Th2 and T reg cell–related genes, relative to Hprt as assessed by qRT-PCR. Data are representative of two experiments with cells pooled from four to six donor mice in each experiment. DP, double positive.

Figure 4.

A proportion of Th2 cells originate from a Foxp3-expressing past and expand after secondary infection with H. polygyrus. (A) Experimental model. Female Il4^GFPFoxp3^YFP/CreR26R^FP635 mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Hp 1° mice were given a primary infection at the same time point. (B) Representative FACS plots of FP635 expression (Foxp3^FATE) within CD4⁺TCRβ⁺Il4^GFP–Foxp3^YFP+ (Foxp3^YFP+) and CD4⁺TCRβ⁺Il4^GFP+Foxp3^YFP– (Il4^GFP+) cells. (C–E) Proportion (C) and absolute number (D) of CD4⁺TCRβ⁺Il4^GFP+Foxp3^FATE+ cells and absolute number of total CD4⁺TCRβ⁺Il4^GFP+ Th2 cells (E) in the spleen of Hp 1° and Hp 2° mice day 7 after infection. Data represent 8–14 mice/group pooled from three independent experiments. (F) Intracellular Foxp3 protein expression in sort-purified T reg (Foxp3^YFP+Foxp3^FATE+), Th2 (Il4^GFP+Foxp3^YFP–Foxp3^FATE–), and ex-Foxp3 Th2 (Il4^GFP+Foxp3^YFP–Foxp3^FATE+) cells. Two mice per group were used. (G and H) CD25, GITR, CD103 (G), and intracellular CTLA-4 (H) protein expression in sort-purified T reg, Th2, and ex-Foxp3 Th2 cells. Data represent two to three independent experiments (with two to five mice per experiment). Bisulfite modification of DNA was performed followed by the amplification, cloning, and sequencing of the T reg cell–specific demethylated region of the Foxp3 locus (CNS2). (I and J) Pictorial (I) and graphical (J) representation of the frequency of methylated cytosines in the Foxp3 CNS2 and control (Ctrl) region of sort-purified T reg, Th2, and ex-Foxp3 Th2 cells. Data are pooled from two independent experiments with five mice per experiment and represent 8–27 clones per region. (K) Il4^GFP+ or Il4^GFP+Foxp3^FATE+ cells were sort purified from Il4ra^wt/wt or Il4ra^fl/fl Hp 2° fate-reporter mice day 7 after infection and cultured in T reg cell–polarizing conditions for 7 d. (L) Proportion of Foxp3^YFP+ cells at day 7. Data are representative of two independent experiments. Sorted T cells were pooled from four to five mice per experiment. *, P ≤ 0.05; Mann-Whitney test or one-way ANOVA. Error bars represent SEM.

Figure 5.

IL-4 is sufficient to promote T reg to Th2 cell conversion in vitro. (A–G) HpTR cells and nT cells were sort purified from Il4^GFPFoxp3^RFP reporter mice as described in Fig. 2. Sorted cells were stimulated with recombinant IL-4 at 37°C for 15 min or with media as a control. (A) Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells. Data are representative of three independent experiments. Sorted T cells were pooled from three to four mice. mW, molecular weight. (B and C) nT or HpTR cells were cultured with anti-CD3/CD28 and IL-2, with and without the addition of IL-4. Representative FACS plots (B) and graph (C) showing the frequency of CD4⁺TCRβ⁺Foxp3^RFP+ and CD4⁺TCRβ⁺Il4^GFP+ cells in day 7 cultures are shown. HpTR cells were cultured with anti-CD3/CD28 and IL-2, with increasing concentrations of IL-4, and cells were harvested for FACS or qRT-PCR at day 7. (D and E) Frequency of CD4⁺TCRβ⁺Il4^GFP+ (D) and CD4⁺TCRβ⁺Foxp3^RFP+ (E) cells in day 7 cultures. (F and G) Gene expression of Gata3 (relative [rel] to Hprt; F) and mir182 (relative to rnu6b; G) in cultured cells at day 7. Data are representative of two independent experiments. Cells were sort purified from four to six mice. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

Figure 6.

IL-4 signaling in T reg cells is required for the development of ex-Foxp3 Th2 cells in vivo. (A and B) nT, Il4ra^fl/fl, Il4ra^fl/wt, or Il4ra^wt/wt HpTR cells were sort purified from naive (A) or Hp 1° (B) Il4ra^wt/wt or Il4ra^fl/fl fate-reporter mice day 14 after infection and stimulated with recombinant IL-4 at 37°C for 15 min or media as a control. Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells are shown. Data are representative of two independent experiments. Sorted T cells were pooled from two to three mice. mW, molecular weight. (C) Proportion and absolute number of Foxp3^YFP+ cells in the spleen of naive Il4ra^fl/fl, Il4ra^fl/wt, or Il4ra^wt/wt fate-reporter mice. Data are representative of two independent experiments. (D) Experimental model. Il4^GFPFoxp3^YFP/CreR26R^FP635Il4ra^fl/fl mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Mice were harvested at day 7 after infection. (E) Representative FACS plots of Foxp3^FATE expression within CD4⁺TCRβ⁺Il4^GFP+ cells in Il4ra^fl/fl or Il4ra^wt/wt Hp 2° mice day 7 after infection. (F and G) Proportion (F) and absolute number (G) of CD4⁺TCRβ⁺Il4^GFP+Foxp3^FATE+ cells in the spleen. Data represent two independent experiments with three to four mice per group. (H) Experimental model. T reg cells were sort purified from Il4ra^wt/wt or Il4ra^fl/fl Hp 1° mice day 14 after infection. Il4ra^wt/wt or Il4ra^fl/fl T reg cells were transferred to Tcra^−/− mice. Recipient mice were infected with H. polygyrus, treated with pyrantel embonate at days 14–15, and then infected with H. polygyrus at day 35 and harvested at day 42 after transfer (see experimental model in Fig. 2 A). (I and J) Representative FACS plots of CD4⁺TCRβ⁺Il4^GFP+ cells (I) and frequency of Il4^GFP+ cells (J) in the spleen and MLN of Il4ra^fl/fl or Il4ra^wt/wt HpTR-recipient mice day 14 after infection. Data are representative of two experiments with three to five mice per group. Adopt., adoptively. (K) Experimental model. In brief, Il4ra^fl/fl or Il4ra^wt/wt mice were subjected to a model of HDM/alum sensitization. i.t., intratracheally. (L) Frequency of CD4⁺TCRβ⁺Il4^GFP+Foxp3^FATE+ cells in mediastinal LNs (medLN) and lungs 24 h after challenge. (M) Proportion of eosinophils in the BAL fluid. Data represent two independent experiments with four to nine mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

Figure 7.

Ex-Foxp3 Th2 cells secrete type-2 cytokines, promote AAMφ in vitro, and are sufficient to drive the expulsion of H. polygyrus. (A–J) T reg, Th2, and ex-Foxp3 Th2 cells were sort purified from Hp 1° and Hp 2° Il4^GFPFoxp3^YFP/CreR26R^FP635 mice and stimulated with PMA/ionomycin for 24 h. (A) Concentration of IL-4, IL-13, IL-5, and IL-2 in the supernatant of restimulated cells. Three technical replicates were used. (B) BMDMs were cultured with FACS-purified T cells for 24 h or with media and recombinant IL-4 + IL-13. (C) Expression of Arg1 and Retnla in stimulated BMDMs. Data are representative of two to three independent experiments with three technical replicates. Sort-purified T cells were pooled from three to four donor mice. rel., relative. (D) Th2 and ex-Foxp3 Th2 cells were sort purified from Hp 2° Il4^GFPFoxp3^YFP/CreR26R^FP635 mice at day 14 after infection and transferred to Hp 1°C57BL/6 recipients 2 d after infection. (E) Intestinal worm burden at day 21 after infection. Data represent two pooled experiments with 6–10 mice per group. (F) Experimental model (see model in Fig. 6 D). Absolute number of CD4⁺TCRβ⁺Il4^GFP+Foxp3^FATE– and CD4⁺TCRβ⁺Il4^GFP+Foxp3^FATE+ cells in the spleens is shown. Four mice per group were used. (G) Small intestine expression of key type-2 response genes, expressed relative (rel.) to Hprt. Four mice per group were used. (H and I) IgE (H)- and H. polygyrus (I)–specific IgG1 levels in the serum. Data represent two independent experiments with three to four mice per group. a.u., arbitrary units. (J) Intestinal worm count day 14 after infection. Data represent two independent experiments with five to seven mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.