Fig 2: Long-lived memory CD4+ T cells, without uropodia, have a lower threshold for re-stimulation, when they make further, independent fate decisions to develop uropodia or re-express CD62L. (A–C) Cell trace violet (CTV) labeled female A1RAG CD4+ T cells were stimulated with bone marrow-derived dendritic cells (bmDC) + 100 nM Dby for 3 days in the presence of IL2 (50 U/ml) without TGFß (A), IL2 (50 U/ml) plus TGFß (2 ng/ml) (B), or TGFß plus anti-IL2 (clone S4B6, 50 µg/ml) (C). Cells were “in situ” labeled with live/dead aqua, fixed and permeabilized, then 7AAD. Histograms show the absolute frequencies of CTV dilution, with gray dashed lines for all images (both live and dead), while filled red (cells with uropodia >10 mm2) and blue line (cells without uropodia) histograms are gated for live cells only (live/dead aqua negative, bright field contrast low). Total numbers of cells in each histogram are indicated. One of two similar experiments is shown. (D–F) CTV labeled female MARKI CD4+ T cells were stimulated with bmDC + 10 nM Dby peptide with IL2 (50 U/ml) either with (E,F) or without (D) TGFß (2 ng/ml) for 10 days. Cells were “in situ” labeled with live/dead aqua and Mitotracker DR (not shown), fixed and permeabilized, then Tbet-Alexa488, foxp3-PE-Cy7, (GATA3-PE, ROR?t-PE-CF594, CD4-APC-Cy7, all not shown), and 7AAD. Histograms of absolute frequencies of CTV dilution for total live and dead singlet cells (dashed gray), live cells with (filled red), and without (blue lines) uropodia, together with total numbers of cells are shown (D,E). The intensities of Tbet and foxp3 staining within the nucleus of the live cells without uropodia are plotted in (F). A similar result was also observed using female A1RAG CD4+ T cell 10 day cultures (not shown). (G–I) Female A1RAG CD4+ T cells (not CTV labeled) were stimulated in the presence of IL-2, TGFß, and ATRA with bmDC + 100 nM Dby peptide (G,H) or CD3/CD28 beads (I) for 6 days. An aliquot of each sample was analyzed as above for uropodia area, CD62L, and nuclear foxp3 expression (summarized in orange panels). Cells were harvested, Ficoll–Hypaque separated, labeled with CTV and Mitotracker DR, then re-stimulated with either CD3/CD28 beads (G) or bmDC + 100 nM Dby peptide (H,I) for 3 days. Cultures were labeled “in situ” with CD4-PE-CF594, CD44-APC-eflour780, CD62L-BV605, and live/dead aqua, fixed and permeabilized, and intracellularly stained for foxp3-PE-Cy7 (Mito-ID-Red, pAKTS473-PE, not shown) and Sytox Green for DNA. Plots show the intensity of CD44 and CD62L staining, with % in each quadrant indicated, after gating for live singlet cells with G0/G1 DNA content, and nfoxp3- cells with uropodia (area >10 µm2: percentage and median area shown in panel above) in red, without uropodia in blue, and nuclear foxp3+ cells (% of all cells shown in panel above) with or without uropodia shown in orange and yellow, respectively. One of three similar experiments is shown.

Fig 3: ILC2s are altered in infected mice after DSS treatment. (A) Gating strategy for ILCs. ILCs are identified by gating on CD45+ live cells, and then looking for a population positive for CD127 and negative for lineage (Lin) markers CD11c dendritic cells (DCs), CD11b (myeloid cells), Ter-119 (granulocytes), B220 (B cells), Ly-6g, and CD3 (lymphocytes). From the CD127+ Lin- population, we gated on GATA3+ cells (ILC2), ROR?T+ cells (ILC3), and cells negative for GATA3 and ROR?T that were positive for T-bet (ILC1). Dot plots are representative of ILCs in the mesenteric lymph nodes. (B) T-bet+ ILC1s do not display significant differences between the 2 groups (unpaired t test, 99.55 ± 1.96 vs 88.90 ± 2.10; P = NS; N = 4). (C) GATA3+ ILC2s (gated on CD127+ Lin-) of the infected mice show a significant increase compared with the uninfected group (unpaired t test, 10.28 ± 2.28 vs 2.08 ± 0.34; P < .05). (D) ROR?T+ ILC3s show no significant differences between the 2 groups (unpaired t test, 0.85 ± 0.44 vs 2.35 ± 1.27; P = NS). (E) T-bet+ ILC1s do not display significant differences in the colonic mucosa between the 2 groups (unpaired t test, 43.33 ± 5.97 vs 36.45 ± 7.13; P = NS; N = 4). (F) GATA3+ ILC2s do not display significant differences in the colonic mucosa between the 2 groups (unpaired t test, 43.82 ± 9.14 vs 33.90 ± 6.42; P = NS; N = 4). (G) ROR?T+ ILC3s show no significant difference in the colonic mucosa between the 2 groups (unpaired t test, 15.30 ± 7.70 vs 9.45 ± 2.65; P = NS). Data are presented as means ± SEM, and are representative of 3 independent experiments.

Fig 4: TNF-a, via TNFR2 Signaling, Enhances ILC2 Survival and Activation In Vivo(A) Rag2-/- mice received i.v. injections of 100 µg aTNFR2 or an isotype control and 0.5 µg rmIL-33 or PBS i.n. on days 1–3. On day 4, lung ILC2 numbers, proliferation, and apoptosis were measured.(B) Representative flow cytometry plots of lung ILC2s in isotype control- or aTNFR2-treated mice. ILC2s were gated as lineage- CD45+ ST2+ CD127+.(C) Total number of lung ILC2s, presented as mean numbers ± SEM.(D and E) Representative flow cytometry plots of AnnexinV/DAPI (D) and intranuclear Ki67 expression (E) with corresponding quantitation, presented as frequency of Ki67+ ILC2s ± SEM (D) and AnV+DAPI+ ILC2s ± SEM (E).(F) Representative flow cytometry plots of intracellular IL-5 and IL-13 expression by lung ILC2s cultured for 4 h with phorbol 12-myristate 13-acetate (PMA), ionomycin, and GolgiPlug and corresponding quantitation, presented as mean frequency ± SEM.(G) Representative flow cytometry plots of intranuclear GATA-3 in lung ILC2s (CD45+Lin-ST2+CD127+) and corresponding quantitation, presented as MFI ± SEM.(H) Representative flow cytometry plots of ST2 expression in GATA-3+ ILC2s and corresponding quantitation, presented as mean MFI ± SEM.(I) C57BL/6 and TNFR2-/- mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured in vitro for 3 days with rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 µg/mL) prior to transfer of 50 × 103 aILC2s in 2 separate cohorts of Rag-/- Il2rg-/- mice, followed by i.n. administration of 50 ng rmTNF-a on days 1–3. On day 4, lung function, BAL, and lung ILC2s were analyzed.(J) Lung resistance in response to increasing doses of methacholine.(K) Total number of eosinophils in the BAL fluid, presented as mean numbers ± SEM.(L) Total number of ILC2s in the lungs, presented as mean numbers ± SEM.Data are representative of 3 individual experiments (n = 4–5). *p < 0.05, **p < 0.01. See also Figures S4–S6.

Fig 5: ATP-Linked OXPHOS Controls BATF Induction by Regulating TCR Signaling(A and B) Naive CD4 T cells were differentiated under Th1 or Th2 polarizing conditions in the presence of vehicle or oligomycin for 24 h, and expression of (A) BATF, IRF4, and (B) GATA3 was determined by flow cytometry (4 to 5 independent experiments).(C and D) Naive CD4 T cells from Nur77/GFP spleens were activated under Th17 conditions in the presence of vehicle or oligomycin for indicated period of time.(C) Graph shows the expression of Nur77/GFP. (D) Representative plots are gated on live CD4 T cells (representative of 3 independent experiments).(E) Naive CD4 T cells were activated under Th17 conditions with vehicle or oligomycin. Representative histogram overlay shows the phosphorylation of ZAP70 at 24 h (representative of 5 independent experiments).(F) Naive CD4 T cells from Nur77/GFP reporter spleens were stimulated with Th17 cytokines for 6 h. BATF expression in GFP+ and GFP- cells were determined by flow cytometry (3 independent experiments).Graphs show the average ± SD; (A and C) two-way ANOVA; (B, E and F) unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001.