PMC

(a) Naïve CD4 T cells were sorted from OT-II–4C13R reporter mice and cultured under T_H2 conditions for 3 rounds. Cells were cultured in IL-2-containing medium for 10 days and then injected i.v. into C57BL/6 recipient mice. 24h later, mice were intratracheally challenged with PBS, or indicated cytokines (150 ng IL-33 and/or 100 ng IL-7 each mouse) for 3 consecutive days, or OVA (100 μg endotoxin-free OVA in PBS) once. Lungs were collected and cytokine production was examined 24h after last cytokine administration (72h after OVA challenge). Cells shown were gated on transferred OT-II T_H2 cells. (b) Statistical analysis of the cytokine production. Error bars represent standard deviation from the mean. ****, P <0.0001 by two tailed student’s t-test. Data are representative of three independent experiments with 3–6 mice in each group (a, b).

(a, b) 0.5×10⁶ sorted naïve CD4 T cells from OT-II–4C13R reporter mice were injected i.v. into C57BL/6 mice. One day after cell transfer, mice were immunized subcutaneously with a mixture of 500 N. brasiliensis (N.b.) infective larvae L3 and 100 μg endotoxin-free OVA and then boosted intratracheally 5 days later with OVA (100 μg) in PBS. Twenty-five days after N. brasiliensis infection, mice were challenged intratracheally with PBS, OVA (100 μg, endotoxin-free) once, or papain (25 μg) for 3 consecutive days with or without an anti-MHCII antibody. 500 μg anti-MHCII antibody was administered i.v. on day 1 and day 3 of papain challenge. Lungs were collected 24h after last papain challenge; DsRed and AmCyan expression by transferred OT-II T_H2 cells were analyzed. Statistical analysis of the cytokine production by OT-II T_H2 cells. **, P<0.01; ns, not significant, P>0.05; ****, P<0.0001. Data are representative of three independent experiments with 2–4 mice in each group.

(a) Lung T_H2 cells expanded dramatically upon N. brasiliensis infection. Lungs were harvested from mice either unimmunized or 10 days after N. brasiliensis infection. T_H2 cells were identified as CD4⁺CD44⁺Foxp3⁻GATA3⁺IL-33R⁺. Markers for ILC2 cells were Lin⁻CD44⁺GATA3⁺IL-33R⁺CD127⁺Thy1⁺. Data are representative of at least three independent experiments with 3 mice in each group. (b) Number of lung-resident T_H2 and ILC2 cells in individual mice that were unimmunized or 13 days or 21–31 days post-N. brasiliensis infection. ****, P<0.0001; ***, P<0.001 by paired t-test. Data are compiled from multiple independent experiments. (c, d) Twenty-five days after N. brasiliensis infection, 4C13R reporter mice were challenged intratracheally with PBS, or papain (25 μg in PBS) for 3 consecutive days with or without anti-MHCII antibody. 500 μg anti-MHCII antibody was administered i.v. on day 1 and day 3 of papain challenge. DsRed and AmCyan expression by lung-resident T_H2 and ILC2 cells were analyzed 24h after last papain challenge. (d) Comparison of percent of DsRed⁺ T_H2 and ILC2 cells among total live lymphocytes. ns, not significant, P>0.05; **, P<0.01; ***, P<0.001. Data are representative of three independent experiments with 4 mice in each group (c–d).

(a, b) C57BL/6 mice were uninfected or infected with N. brasiliensis. Twenty-three days later, mice were challenged intratracheally with PBS or HDM (25 μg in PBS) daily for 3 consecutive days. 24h after the last challenge, BAL fluids and lungs were collected and analyzed. N.b., N. brasiliensis only; HDM, HDM challenge in uninfected mice; N.b.+HDM, HDM challenge 23 days after N. brasiliensis infection; N.b.+HDM+anti-MHCII, 500 μg anti-MHCII antibody was injected i.v. on day 1 and day 3 of HDM challenge. Percentages shown were calculated as a percent of the live cells. ****P<0.0001; **, P<0.01; ***, P<0.001; *, P<0.05; ns, not significant, P>0.05. (c) Lung histology analysis. Top 3 panels, lung sections were stained with H&E; left panel arrow - mucus metaplasia; center panel arrow - perivascular infiltration; right panel arrow - alveolar infiltration. Lower left panel, PAS staining arrow - mucus metaplasia; lower middle, Luna staining arrow - eosinophils; and lower right, Ym1 staining arrow – Ym1⁺ macrophages. Data are representative of two (a, c) or three (b) independent experiments with 3–4 mice in each group.

(a) Mice were infected with N. brasiliensis and challenged with HDM as described in . Anti-MHCII antibodies were injected i.v. at the time of 1^st and 3^rd HDM challenge. BAL fluid was collected for cellular constituents analysis. Lungs were harvested and lung sections were analyzed by H&E staining and lunar staining. ns, not significant, P>0.05. (b) Mice were infected and challenged as in . 500 μg of anti-CD4 neutralizing antibody was injected i.v. on day 1 and day 3 of HDM challenge. *, P<0.05. (c) Wild-type mice or IL-33-deficient (Il33^−/−) mice were infected with N. brasiliensis and challenged with HDM as described in . 150 ng IL-33 was injected i.v. into IL-33-deficient mice on day 1 and day 5 of N. brasiliensis infection. One group of IL-33-deficient mice infected with N. brasiliensis was challenged with HDM without supplemental IL-33 while a second group received IL-33 supplementation (100 ng per mouse on day 1 of HDM challenge). BAL cellular constituents were analyzed. ***, P<0.001; ns, not significant, P>0.05. (d) Left, 5×10⁶ in vitro cultured OT-II T_H2 cells were injected i.v. into Rag2^−/−Il2rg^−/− recipient mice. Some groups of mice were challenged intratracheally with HDM (25 μg in PBS) daily for 4 weeks. 500 μg anti-CD4 antibody was injected into one group on days 2, 8 and 15. BAL cellular constituents were analyzed 24h after the last challenge. Right, 0.5×10⁶ ILC2 derived from IL-25-injected mice and cultured for 3 days in IL-7 plus IL-33 were injected i.v. into Rag2^−/−Il2rg^−/− recipient mice. Mice were similarly treated and analyzed as those receiving primed OT-II T_H2 cells. ns, P>0.05; ***, P<0.001. Data are representative of three (a–c) or two (d) independent experiments with 3–7 mice in each group.

(a, b) 0.5×10⁶ naïve CD4 cells from OT-II–4C13R reporter mice were transferred i.v. into wild-type (WT) recipient mice or IL-33-deficient (Il33^−/−) recipient mice. Mice were immunized and challenged as described in 2a. 150 ng IL-33 was administered twice i.v. on Day 1 and Day 5 of infection. Lungs were collected 24h after last papain challenge; DsRed and AmCyan expression by transferred OT-II T_H2 cells were analyzed. Statistical analysis of the cytokine production by the transferred OT-II T_H2 cells. ns, not significant, P>0.05; **, P<0.01. (c–e) Naïve CD4 T cells were sorted from wild-type (WT) CD90.1⁺CD90.2⁺ DO11.10 mice or Il1rl1^−/−CD90.1⁻CD90.2⁺ DO11.10 mice. Cells were cultured under T_H2 conditions with OVA peptide and APC for three rounds. After being cultured in IL-7-containing medium for 7 days, wild-type T_H2 cells and Il1rl1^−/− T_H2 cells were mixed at 1:1 ratio and i.v. injected into wild-type CD90.1⁻CD90.2⁺ BALB/c recipients. 24h later, mice were intratracheally challenged with IL-33 (150 ng), IL-7 (100 ng), papain (25 μg), heat-inactivated papain (25 μg) or PBS for 3 consecutive days, or with endotoxin-free OVA (100 μg) once. 24h after IL-33 plus IL-7, papain or PBS challenge or 4h after OVA challenge, lungs were isolated and single cell suspensions were prepared. Transferred WT and Il1rl1^−/−DO11.10 T_H2 cells were sorted from lung cell suspensions. The amounts of Il13 mRNA and Il4 mRNA were measured by real time PCR. Il13 (d) and Il4 (e) mRNA amount in wild-type cells in PBS-treated recipients were set as 1. Data are representative of two (a, b) or one (c–e) independent experiments with 2–5 mice in each group.

(a) Mice were inoculated with A. summ eggs orally and 11 days later received a subsequent subcutaneous inoculation with 500 N. brasiliensis L3. The mice in control groups were only inoculated subcutaneously with 500 N. brasiliensis L3 or only A. suum eggs. (b) N. brasiliensis adult worms in the small intestine and eggs in the fecal pellets were determined on day 8 post N. brasiliensis inoculation. ***, P<0.001; *, P<0.05. (c) Total number of lung-resident T_H2 and ILC2 cells. (d) Lungs were harvested on day 6 post N. brasiliensis inoculation. DsRed and AmCyan expression by lung-resident T_H2 and ILC2 cells were analyzed by flow cytometry. (e) Statistics of cell number of DsRed-expressing T_H2 and ILC2 cells. *, P<0.05. (f) 5×10⁶ in vitro cultured OT-II T_H2 cells were injected i.v. into Rag2^−/−Il2rg^−/− recipient mice. 24h later, mice were inoculated with N. brasiliensis and adult worms in the small intestine were determined on day 8 after N. brasiliensis inoculation. Data were compiled from two independent experiments. **, P<0.01. (g) BM chimeras were generated by injecting wild-type or RORα-deficient BM cells into Rag2^−/−Il2rg^−/− recipient mice. 8 weeks after reconstitution, chimeras were inoculated with N. brasiliensis L3 alone or with A. summ eggs followed by N. brasiliensis as described in Fig. 7a. Adult worms in small intestine were determined on day 8 after N. brasiliensis inoculation. Some mice received 500 μg anti-CD4 antibodies i.v. on day 0 and day 4 after inoculation with N. brasiliensis. RORα-deficient BM chimeras that have less than 3% ILC2 cells in lung with and without injection of anti-CD4 antibody are shown. Data were compiled from three independent experiments with 4–5 mice in each group. *, P<0.05; **, P<0.01. (h) Mice were inoculated with N. brasiliensis L3 and 12 days later with A. summ eggs. The mice in the control group were only inoculated with A. suum eggs. The parasitic third-stage larvae in lung were determined 8 days after A. summ inoculation. ***, P<0.001. Data are representative of five (b) or two (c–e, h) independent experiments with 3–5 mice in each group.