α4β7 apc

γδ T cells subsets were identified using a fixable aqua blue dead cell stain (Life Technologies), and a combination of antibodies including CD3-APC-Cy7 (SP34-2), pan γδ TCR-PE (B1), CD4-Pac blue (L200) (all from BD Bioscience); Vδ2-FITC (15D;Thermofisher); and CD8-Ax700 (RPA-T8;eBioscience). CD3⁺ T cells were divided into Vδ1⁺ and Vδ2⁺ populations as described by Harris et al. (8 (link)) and as illustrated for a mucosal sample (Suppl Fig. 1), and were further subdivided by their CD4 and CD8 expression patterns. NKG2 receptor expression was assessed using combinations of anti-NKG2A-APC (Z99; Beckman Coulter) and anti-NKG2D-PE-Cy7 (1D11; BioLegend). For homing receptor expression and activation markers the following antibodies were used: α4β7-APC (NIH NHP Reagent Resource) and CCR7-PE-Cy7 (3D12 (BD BioScience) and CD69-Pac blue (FN50; BioLegend). For representative staining see Suppl Fig.2). For intracellular staining cells were fixed and permeabilized using a Perm/fix solution (BD Biosciences) prior to incubation with IFN-γ- PE-Cy7 (4S.B3; BDBioscience), TNF-α- PerCpCy5.5 (Mab11; BioLegend), CD107-PE-Cy5 (H4A3; BD BioScience) and Granzyme B-APC (GB12; Invitrogen). At least 50,000 CD3⁺ T cell events were acquired on a LSRII (BD Biosciences) and analyzed using FlowJo software version 9.8.5 (TreeStar Inc).

Antibodies used in this study were CD3-APC (145-2C11), CD4-APC H7 (GK1.5), Vα2-PE (B20.1), CD45.1-PerCP Cy5.5 (A20), CD69-AlexaFluor700 (H1.2F3), CD8-APC (53.6-7), CD11c-BV421 (HL3), MHC Class II-FITC (2G9), α4β7-APC (DATK32), CD103-biotin (M290), CD11b-PE Cy7 (M1/70), CD80-V450 (16-10A1), CD86-AF700 (GL1), OX40L-PE (RM134L), CD40-PE (3/23) purchased from BD Biosciences, and Foxp3-CF594 (MF23), GATA-3-eFluor710 (TWAJ), RORγt-APC (B2D), T-bet-PE Cy7 (eBio4B10), CCR9-PE Cy7 (CW-1.2) purchased from eBioscience. Staining was performed in 1×PBS supplemented with 2% FCS and 2 mM sodium azide, and anti-CD16/32 (24G2). Intracellular staining for transcription factors was performed using the Foxp3 staining kit from eBioscience, as per manufacturer’s instructions. Samples were acquired on the FACS LSR II cytometer (BD Biosciences). FlowJo software (Tree Star, Inc.) was used for analysis.