** p0.01 and *** p0.001, ??? indicates p0.001 for H2-Ab1fl/fl comparisons versus matched media control. fig. from your mLN (top panel) and cLPL (bottom panel). Gate shows frequency of MHCII expression amongst gated ILC3s (blue). All data representative of at Formononetin (Formononetol) least 3 impartial experiments with at least n=3 mice per group. SPF, specific pathogen free. Results are shown as the mean +/- s.e.m.fig. S2. MHCII and co-stimulatory molecule expression of mLN ILC3s is usually unaffected by TLR ligands or pro-inflammatory cytokines. A) Mean fluorescent intensity of ILC3 MHCII, CD80 and CD86 from mLN or cLPL cells stimulated with media alone, TLR ligands (LPS, Poly I:C) or pro-inflammatory cytokines (IL-23, IL-1, IFN-). B) Frequency of MHCII+ (Lin- CD127+ CCR6+) ILC3s in the mLN (top panel) and cLPL (bottom panel) of Capase 1/11-/- and MyD88-/- mice. C) Representative histograms depicting expression of MHCII, CD80 and CD86 on WT C57BL/6 DCs (black collection), WT ILC3s (blue collection) or Capase 1/11-/- ILC3s (reddish collection) in the mLN (top panel) or cLPL (bottom panel). All data representative of at least 3 impartial experiments with 3-4 mice per group or 3 biological replicates. Results are shown as the mean +/- s.e.m. fig. S3. CIITA transcriptional control of MHCII expression on B cells, DCs and TECs and IFN- dependence of MHCII expression in colonic ILC3s. Expression of MHCII was decided on B220+ CD11c- B cells or CD11b+ CD11chi DCs from your mLN or CD45- EpCAM+ Ly51-/low mTECs or CD45- EpCAM+ Ly51+ cTECs from your thymus of mice deficient in A) CIITA and B) CIITA-specific promoters (pIII/pIV, pIV). MHCII expression on C) mLN CCR6+ ILC3s from mice deficient CIITA in promoter regions (pIII/pIV, pIV) D) cLPL CCR6+ ILC3s from IFN- or IFN-R1-deficient mice and E) mLN and cLPL CCR6+ ILC3s from STAT-1 deficient mice. All data representative of at least 3 impartial experiments with n=2-3 mice per group. Results are shown as the mean +/- s.e.m. fig. S4. ILC3-intrinsic MHCII selectively controls commensal bacteria-specific CD4+ T effector cells in the intestine. A) Relative frequencies and B) total cell numbers of na?ve (CD44lo), Teff (CD44hi) and Treg (FoxP3+) CD4+ T cells in the colonic lamina propria of MHCIIILC3 mice or H2-Ab1fl/fl littermate controls. C) Analysis of the frequencies of na?ve (grey), Teff (blue) and Treg (green) amongst CD4+ T cells expressing commonly utilized TCR V Formononetin (Formononetol) chains in the thymus and colonic lamina propria of MHCIIILC3 mice or H2-Ab1fl/fl littermate controls. D) Frequency of proliferating cells (CFSEdim) in CD4+ T cells derived from MHCIIILC3 mice or H2-Ab1fl/fl littermate controls and stimulated with fecal and tissue-derived homogenate antigens in vitro for 72 h. All data representative of at least 2 impartial experiments with 3 biological replicates or n=3 mice per group. Results are shown as the mean +/- s.e.m. Data was analyzed by student’s t-test (B) or one-way ANOVA (D). ** p0.01 and *** p0.001, ??? indicates p0.001 for H2-Ab1fl/fl comparisons versus matched media control. fig. S5. ILC3-intrinsic MHCII selectively controls CBir1 CD4+ T effector cells in the intestine. OT-II or CBir1 TCR transgenic mice were crossed with either MHCIIILC3 mice or H2-Ab1fl/fl littermate controls and total V5+ (OT-II) or V8.3+ (CBir1) CD4+ T cell figures were determined. A) CBir1 CD4+ T cell figures in the GluN1 mLN of standard or ABX-treated CBir1 transgenic mice crossed with either MHCIIILC3 mice or H2-Ab1fl/fl littermate controls. B-C) Frequencies of IFN-+ and/or TNF-+ T cells following activation with cognate antigen, OVA peptide (OT-II) or CBir1 peptide (CBir1), for 5 h in the presence of Brefeldin A. D) Frequencies of CD45+ CD3- B220- Ly6C+ Ly6G+ neutrophils in the cLPL of Rag1-/- MHCIIILC3 Formononetin (Formononetol) mice or Rag1-/- H2-Ab1fl/fl littermate controls. E) Quantity of CD4+ Teff or Treg in the colonic lamina propria of CBir1 transgenic mice crossed with either MHCIIILC3 mice or.