Fig 2: IL-33-Induced IL-13 Production by Th2 Cells Is Dependent on a Signaling Complex between T1/ST2 and EGFRWT and Egfrfl/flxCd4-cre (EGFRΔCD4) mice were infected with H. polygyrus larvae, and on day 14 post infection, mLN were harvested.(A–C) Cells were stimulated with rIL-33, anti-CD3, or media in the presence of gefitinib, marimastat, or vehicle, and p-ERK (A and C) or IL-13 (B) expression was determined by intra-cellular staining and flow cytometry analysis.(D and E) Cells were stimulated with rHB-EGF, anti-CD3, or media, and IL-13 (D) and p-ERK (E) expression was determined by intra-cellular staining and flow cytometry analysis.(F) EGFR expression on stimulated mLN WT CD4+ T cells in the presence of monensin was analyzed by flow cytometry.(G) HEK293T cells were transfected as indicated with T1/ST2, the IL-1RacP, or the EGFR alone or in combination. Subsequently, the cells lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot.(H) mLN (upper) or HaCaT cells (lower) were stimulated with rIL-33, rHB-EGF, or media, and the EGFR phosphorylation at position Y1068 was determined by intra-cellular staining and flow cytometry analysis (upper) or immunoblot (lower).All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S6.

Fig 3: Transient EGFR Expression Licenses Th2 Cells to Express IL-13 in Response to IL-33(A) Splenocytes were isolated from H. polygyrus-infected WT and Egfrfl/flxCd4-cre (EGFRΔCD4) mice, and expression of EGFR on T cells was determined by flow cytometry analysis after overnight incubation with H. polygyrus excretory-secretory products (HES), adult worm extract (HEX), anti-CD3, vehicle (C), rIL-7, rIL-2, rTSLP, or rIL-33 (upper). Alternatively (lower), splenocytes were stimulated overnight with rTSLP, and subsequently, IL-13 expression by T cells was analyzed by intra-cellular cytokine staining and flow cytometry analysis following re-stimulation with rIL-33 or anti-CD3 antibody.(B) Ly5.2+ WT mice were infected with H. polygyrus, and 2 weeks after infection, mLN cells were transferred into naive Ly5.1+ hosts. Spleens of recipient mice were harvested after 0–3.5 days post transfer, and the expression of EGFR (upper) and T1/ST2 (lower) on transferred Ly5.2+ CD4+ T cells was determined by flow cytometry analysis. Expression of IL-13 (upper) and IL-5 (lower) upon exposure to rIL-33 was determined by intra-cellular cytokine expression and flow cytometry analysis (n = 3 mice).(C) WT H. polygyrus-infected mice were treated with pyrantel embonate, and mLN were harvested after 0–6 days post treatment. EGFR (upper) and T1/ST2 (lower) expression on CD4+ T cells was determined by flow cytometry, and expression of IL-13 (upper) and IL-5 (lower) upon exposure to IL-33 was determined by intra-cellular cytokine staining and flow cytometry analysis (n = 3 mice).Data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. Data in (B) and (C) are presented as percentage of expression on day 0.

Fig 4: IL-33-Induced IL-13 Production by Th2 Cells Is Dependent on EGFR ExpressionWT, Egfrfl/fl, and Egfrfl/flxCd4-cre (EGFRΔCD4) mice were infected with H. polygyrus, and on day 14 post infection, mLN were harvested.(A) Comparison of T1/ST2 expression on mLN CD4+ T cells from H. polygyrus-infected Egfrfl/fl and EGFRΔCD4 mice.(B) mLN cells were stimulated with rIL-33, anti-CD3, or media only, and expression of IL-13 and IL-5 was determined by intra-cellular cytokine staining and flow cytometry analysis.(C) mLN CD4+ T cells from H. polygyrus-infected WT mice were flow cytometry-sorted based on T1/ST2 and/or EGFR expression, and T cell populations were stimulated with rIL-33 for 4 hr. Subsequently, Il13 and Il5 mRNA was analyzed by RT-PCR.(D) MHCII-deficient mice were infected with H. polygyrus and 7 days post infection received CD4+ T cells derived from mLN of naive or H. polygyrus-infected WT or EGFRΔCD4 mice. Worm burden and egg counts were determined 2 weeks post infection.(E–G) MHCII-deficient mice were infected with N. brasiliensis and simultaneously received CD4+ T cells from mLN of naive (WT) or H. polygyrus-infected WT, EGFRΔCD4, or T1/ST2-deficient mice, collected 2 weeks after infection. At day 6 post transfer, worm burden and eggs counts (E) were determined, IL-13 and IL-5 (F) expression in BAL was evaluated by ELISA, and expression of Ym1 in alveolar macrophages and total number of eosinophils in the BAL (G) were analyzed by flow cytometry.Data are representative of two independent experiments (mean ± SEM); results for individual mice are shown as dots.

Fig 5: EGFR Expression on Th2 Cells Is Induced by TCR Activation and Maintained by Cytokines(A–E) WT H. polygyrus-infected mice were either treated with pyrantel embonate or left untreated; mLN were collected 14 days after treatment.(A) EGFR expression on activated (CD69+, red bars) or memory (CD25–CD45RBlowCD44hi, blue bars) CD4+ T cells was analyzed by flow cytometry.(B) Activated CD4+ T cells were flow cytometry-sorted from infected mice into ST2+ EGFR+ (red) and ST2+ EGFR– (orange) populations. Memory CD4+ T cells (CD25–CD45RBlowCD44hi) were flow cytometry-sorted from dewormed mice.(C) After sorting, activated EGFR+ and memory cells were stimulated with rIL-33 or media only, and expression of IL-13 was determined by flow cytometry analysis.(D–E) Activated EGFR– and memory cells were cultured overnight with rTSLP or anti-CD3 antibodies, and expression of EGFR on CD4+ T cells was determined by flow cytometry analysis (D) or were re-stimulated with rIL-33 and expression of IL-13 determined by intra-cellular cytokine staining and flow cytometry analysis (E).(F–G) Egfrfl/flxCd4-cre (EGFRΔCD4) mice were infected with H. polygyrus and received either flow cytometry-sorted activated CD4 T cells (red) (F) or flow cytometry-sorted naive or memory T cells (blue) (G) from OVA-challenged mice. Worm burden and egg counts in feces were determined 2 weeks post transfer.Data in (A)–(E) are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S5.