Tcrγδ gl3

Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols^. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 10⁶) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).

For staining of murine samples, monoclonal antibodies specific for BCL6 (REA373 and K112-91), IRF4 (3E4), CD4 (GK1.5, RM4-8, and S11), CD3e (145-2C11, 17A2, and REA641), CD8 (53-6.7 and YTS156.7.7), PD-1 (RMP1-30, REA802, and 29F.1A12), CXCR5 (2G8 and REA215), CD44 (IM7), CD62L (MEL-14), CD19 (1D3 and 6D5), B220 (RA3-6B2), GL7 (GL7), CD95 (JO2 and REA453), CCR6 (REA277 and 29-2L17), CD38 (90), CD138 (REA104 and 281–2), TACI (8F10), CD86 (GL1), CXCR4 (2B11), IgM (REA979 and RMM-1), IgD (11-26c2a), IgG1 (REA1017 and RMG1-1), Igλ (R26-46 and JC5-1), CD21/CD35 (7E9), CD45.1 (A20 and REA1179), CD45.2 (104 and 104-2), Igκ (187.1), Gr-1 (RB6-8C5), F4/80 (BM8), CD11c (HL3 and N418), CD11b (M1/70), Ly6G (1A8), NK1.1 (PK136), TCRβ (H57-597 and REA318), TCRγδ (GL3), and Ter119 (Ter119) were purchased from BioLegend, BD Biosciences, Miltenyi Biotec, or Thermo Fisher Scientific and were used at dilutions specified by the manufacturer or determined experimentally. For staining for TCRβ subtypes in Bhlhe40^−/− tumor mice, the mouse Vβ TCR screening panel (BD Bioscience) was used.