Bv605 ccr6

Flow cytometry was used to measure the expression of cell markers (LSR II, BD Biosciences). Quantification of α4β7 levels was performed by gating β7⁺CD45RA⁻ within the population of CD4⁺ T cells to identify α4β7^hi populations based on previously published data that demonstrated that αEβ7, the only other form of β7 found on human T cells, is rarely found in the blood [2 (link),13 16 (link),17 ]. PBMCs were stained with the following combination of antibodies from BD Biosciences: APC-H7-CD3 (560176), PerCP-Cy5.5-CD45RA (563429), BV421-CCR5 (562576), PE-Integrin β7 (555945), BB515-CD25 (564467), PE-Cy7-CD27 (560609), PE-CF594-HLA-DR (562304), BUV395-CD8 (563795), BUV496-CD38 (564657); and the following antibodies from Biolegend: BV605-CCR6 (353420), AlexaFluor 700-CD4 (344622), BV510-CCR7 (353232), AlexaFluor 647-CD127 (351318) (Supplemental Figure S1).

Cells were stained with fluorochrome-conjugated monoclonal antibodies and 2.4G2 blocking antibody and incubated in the dark for 30 min on ice. For CD1d tetramer staining, cells were incubated in the dark for 15 min at room temperature followed by 15 min on ice. Flow cytometric analysis was performed on a FACSAria (BD Biosciences) or a MACSQuant (Miltenyi Biotec). Data were analyzed using FlowJo (Tree Star).
TCRβ-FITC, HSA-FITC, CD4-FITC, TCRβ-PE, Ly49C/I/F/H-PE, TCRβ-PErCPCy5.5, Ly49G2-PerCPeFluor710, CD62L-PE-Cy6, NK1.1-PE-Cy7, KLRG1-APC, CD25-APC, CD19-PE, CD44-APCeFluor780, CD8α-eFluor450, and CD3-eFluor450 were purchased from ebioscience. Ly49G2-FITC, Ly49C/I-FITC, Ly49A/D-PE, and CD4-PerCP were purchased from BD Pharmingen. BV421-CD127, BV510-TCRβ, BV570-CD45, BV605-CD3, BV605-CCR6, and BV785-NK1.1 were purchased from Biolegend. PE- and APC-conjugated α-GalCer loaded CD1d tetramer was prepared in our laboratory. CD1d, M45 and M57 tetramers were kindly provided by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility at Emory University (Atlanta, GA, USA).

After stimulation, cells were washed, stained with LIVE/DEAD^® Fixable Near-IR Stain (Invitrogen) and, subsequently, surface stained with the following antibodies: CD14-APC/Alexa Fluor 750 (Invitrogen) and CD19-APC/Alexa Fluor 750 (Invitrogen), CD4-FITC (BD), CD27-BV711 (BD), CCR4-BV510 (Biolegend), CCR6-BV605 (Biolegend), CXCR3-PE-Cy7 (BD Biosciences), KLRG1-PerCP/eFluor 710 (eBioscience), and HLA-DR-PE (BD). Cells were then fixed and permeabilized using Cytofix/Cytoperm buffer (BD) and stained with CD3-BV650 (BD), IL-2-PE/Dazzle™ (Biolegend), TNFα-eFluor 450 (eBioscience), and IFNγ-Alexa Fluor 700 (BD). Finally, cells were washed and fixed in 1% formaldehyde in PBS. Samples were acquired on a LSR-II (BD) and analyses were performed using FlowJo (Treestar). A positive IFNγ response was defined as at least twice the background measured in the presence of co-stimulatory antibodies without antigen. Cell polyfunctionality was analyzed using Pestle and Spice software (19 (link)). The gating strategy is presented in Figure S1 in Supplementary Material.