reagent (Life Technologies). For combined mucus staining, anti-MUC2 antibody (1: 100 15334, Santa Cruz Biotechnology) was applied for 15 min at RT, after a first wash step in hybridization buffer. Secondary antibody incubation(1: 200, Al1008, Life Technologies including DAPI counterstain was performed for 15 min at RT. Tissue sections were washed in washing buffer for 5 min at RT and covered with antifade mounting medium. Microscopic analysis was performed by using the Axio Imager ZI microscope and Axiovision software(all Zeiss) Flow cytometry analyses of LP cell subsets. Isolation of lamina propria cells from colon. The whole colon was harvested and Peyers patches were removed, as well as all fat residues and feces. Colons were cut longitudinally and then transversally into pieces cm length. After removing the intra-epithelial lymphocytes (IELs), the colon pieces further cut into approximately Imm squares, and incubated with 0.25 mg/ml collagenase VIll and 10 U/ml DNase I for 40 min at 37C with shaking, in order to isolate lamina propria cells(LPCs). After digestion, intestinal pieces were passaged through a 100um cell strainer. For flow cytometry analysis, cell suspensions were subjected to a percoll gradient for 20 min, centrifuged at 2100 RPMI. Anti-mouse antibodies for CD45.2(104), CD3(145-2CI1), CD4(GK1.5), IFN (XMG1. 2), RORyt(AFKJS-9), anti-ICOS(7E17G9)were obtained from BioLegend, eBioscience and R&D. CTLA4 staining on lamina propria cell subsets. Large intestines longitudinally, washed from feces and incubated on ice in PBS/EDTA(25 without Ca /Mg, gibco). Epithelial cells were removed by repeated rounds of shaking in PBS Subsequently, the intestine was cut into small pieces and digested with Liberase TL(Roche)/ DNasel( Sigma) mix in DMEM(Gibco) at 37 C Tissue was disrupted by pipetting and passing over a 100 um mesh(BD). Homogenates were subjected to a 40/80 Percoll (GE Healthcare) gradient. Lymphocytes were harvested from the interphase and stained with fixable Live/Dead stain Blue(Invitrogen) according to the manufacturers instruction. Surface staining was performed with anti-CD3-PE-CF594(145-2Cll, BD), anti-CD4-Horizon V500(RM4-5, BD), anti-CD19-Brilliant Violet650 (6D5, BioLegend), anti-Thy 1. 2-PerCp-eFluor710 (30-H12 eBioscience), anti-NKp46-biotin (polycle (BioLegend) and anti-CTLA-4-APC (UC10-4B9, eBioscience) or Armenian Hamster IgG type Control APC(eBio299Arm, eBioscience) Cells were fixed with 4% PFA(Sigma)and10 reagent (Life Technologies). For combined mucus staining, anti-MUC2 antibody (1:100, sc15334, Santa Cruz Biotechnology) was applied for 15 min at RT, after a first wash step in hybridization buffer. Secondary antibody incubation (1:200, A11008, Life Technologies) including DAPI counterstain was performed for 15 min at RT. Tissue sections were washed in washing buffer for 5 min at RT and covered with antifade mounting medium. Microscopic analysis was performed by using the Axio Imager Z1 microscope and Axiovision software (all Zeiss). Flow cytometry analyses of LP cell subsets. Isolation of lamina propria cells from colon. The whole colon was harvested and Peyer’s patches were removed, as well as all fat residues and feces. Colons were cut longitudinally and then transversally into pieces of 1-2 cm length. After removing the intra-epithelial lymphocytes (IELs), the colon pieces were further cut into approximately 1mm squares, and incubated with 0.25 mg/ml collagenase VIII and 10 U/ml DNase I for 40 min at 37 °C with shaking, in order to isolate lamina propria cells (LPCs). After digestion, intestinal pieces were passaged through a 100μm cell strainer. For flow cytometry analysis, cell suspensions were subjected to a percoll gradient for 20 min, centrifuged at 2100 RPMI. Anti-mouse antibodies for CD45.2 (104), CD3 (145-2C11), CD4 (GK1.5),  Ǧγ (XMG1.2), γ (AFKJS-9), anti-ICOS (7E17G9) were obtained from BioLegend, eBioscience and R&D. CTLA4 staining on lamina propria cell subsets. Large intestines were opened longitudinally, washed from feces and incubated on ice in PBS/EDTA (25 mM, without Ca2+/Mg2+, Gibco). Epithelial cells were removed by repeated rounds of shaking in PBS. Subsequently, the intestine was cut into small pieces and digested with Liberase TL (Roche) / DNaseI (Sigma) mix in DMEM (Gibco) at 37o C. Tissue was disrupted by pipetting and passing over a 100 μm mesh (BD). Homogenates were subjected to a 40/80 % Percoll (GE Healthcare) gradient. Lymphocytes were harvested from the interphase and stained with fixable Live/Dead stain Blue (Invitrogen) according to the manufacturer’s instruction. Surface staining was performed with anti-CD3-PE-CF594 (145-2C11, BD), anti-CD4-HorizonV500 (RM4-5, BD), anti-CD19-BrilliantViolet650 (6D5, BioLegend), anti-Thy1.2-PerCp-eFluor710 (30-H12, eBioscience), anti-NKp46-biotin (polyclonal, R&D), Streptavidin-BrilliantViolet421 (BioLegend) and anti-CTLA-4-APC (UC10-4B9, eBioscience) or Armenian Hamster IgG Isotype Control APC (eBio299Arm, eBioscience). Cells were fixed with 4% PFA (Sigma) and