Anti tcrβ h57 597

Isotype control antibodies and blockade of Fc receptor Abs were provided from BioLegend (San Diego, CA, USA). Anti-CD3 (17A2), anti-CD11c (N418), anti-CD40 (HM40-3), anti-CD49b (DX5), anti-CD80 (16-10A1), anti-CD69 (H1.2F3), anti-CD86 (GL-1), anti-CD253 (TRAIL, N2B2), anti-granzyme B (GB11), anti-IFN-γ (R4-6A2), anti- IL-6 (MP5-20F3), anti-IL-12/23p40 (C17.8), anti- MHC class I (AF6-88.5), anti-MHC class II (M5/114.15.2), anti-NK1.1 (PK136), anti-perforin (S16009A), anti-TCR-β (H57-597) and anti-TNF-α (MP6-XT22) were purchased from BioLegend (San Diego, CA, USA). Anti- PD-L1 Abs were purchased from BioXcell (Lebanon, NH, USA). LPS (O111:B4) was purchased from Sigma-Aldrich.

Single cell suspensions obtained from the intestinal mucosa or MLNs were stained and analyzed on FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo software v10.8.2 (TreeStar, Ashland, OR, USA). The following antibodies were used: anti-B220 (RA3-6B2; BioLegend), anti-CD38 (90; BioLegend), anti-CD45 (30-F11; BioLegend), anti-GL7 (GL7; BioLegend), anti-CD138 (281-2; BioLegend), anti-CD4 (GK1.5; BioLegend), anti-CXCR5 (L138D7, BioLegend), anti-PD-1 (29F.1A12, BioLegend), anti-Siglec-F (E50-2440, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD11c (N418, BioLegend), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-IgE (RME-1, BioLegend), anti-CD103 (W19396D, BioLegend), anti-CD11b (M1/70, BioLegend), anti-IL-7Rα (A7R34, BioLegend), anti-CCR6 (29-2L17, BioLegend), anti-TCRβ (H57-597, BioLegend), and anti-TCRγδ (UC7-13D5, BioLegend). Dead cells were gated out using a Zombie NIR Fixable Viability Kit (BioLegend). Before staining, Fc receptors were blocked with an anti-CD16/32 antibody (2.4G2, BioLegend). Negative controls consisted of isotype-matched, directly conjugated, nonspecific antibodies (BD Biosciences). Intracellular staining was performed using anti-Foxp3 antibody (FJK-16s; Thermo Fisher Scientific) and anti-RORγt antibody (Q31-378; BD Biosciences), as described previously [29 (link)].

For cell surface staining, cells were pre-incubated with 2.4G2 Fcγ RII/RIII blocking mAb for 15 min and then stained with the appropriate combinations of mAbs diluted in HBSS + 2% FBS for 30 min at 4 °C. Cells were analyzed on a BD FACS CantoII, or Cytek Aurora Spectral Cytometer, and data were processed with FlowJo software (TreeStar). The complete list of antibodies use is as follows: anti-human Vβ5.1 (LC4, Invitrogen), anti-TCRβ (H57-597, BioLegend), anti-CD44 (IM7, BioLegend), anti-Vβ11 (KT11, BioLegend), anti-CD45.2 (104, BioLegend), anti-CD45.1 (A20, BioLegend), anti-SiglecF (E50-2440, BD Bioscience), anti-CD45 (30-F11, BioLegend), anti-CD11c (N418, BioLegend), anti-CD11b (M1/70, BioLegend), anti-Ly6G (1A8, BioLegend), anti-CD19 (6D5, BioLegend), anti-NK1.1 (PK136, BioLegend), anti-CD3 (17A2, BioLegend), Zombie Red (BioLegend), anti-mouse CD185/CXCR5 (L138D7, Biolegend), anti-mouse CD279/PD-1 (29 F.1A12, Biolegend), and anti-Ki67 (SolA15, Invitrogen).

After surface staining, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) and permeabilized with PBS buffer containing 0.1% saponin (Sigma-Aldrich, Munich, Germany) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich, Munich, Germany). Then, cells were stained with antibodies against intracellular cytokines or cAMP. Anti-TCRβ (H57-597), anti-IFNγ (XMG1.2), and anti-IL4 (11B11) were purchased from Biolegend (San Diego, California). Anti-cAMP was purchased from Abcam (Cambridge, England). PBS57-CD1d tetramer was provided by the NIH Tetramer Core Facility. Cells were analyzed with a FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ) and data was analyzed with FlowJo 7.6 software (Tree star, Ashland, Oregon).