Anti cd19 clone 1d3

The following monoclonal antibodies (eBiosciences) were used in appropriate combinations: anti-CD3 (clone 145–2C11), anti-CD4 (clone RM 4–5), anti-TCR γδ (clone GL3), anti-CD19 (clone 1D3), anti-CD11b (clone M1/70), anti-Gr1 (clone-RB6-8C5), eFlour 780 anti-CD90 (clone53-2.1) and anti-CD16/CD32 (clone 2.4G2). The cells were preincubated for 20 minutes with anti-CD16/CD32 to block Fc receptors to avoid nonspecific binding. Cells were then washed and labeled with the appropriate mixture of antibodies or isotype matched controls for 30 minutes, centrifuged at 650 g, and resuspended in flow cytometry staining buffer (FACS) buffer. To exclude dead/dying cells and therefore nonspecific antibody-binding cells, leukocytes were gated according to forward and side scatter. The percentages of CD4⁺, CD8⁺, CD4⁺ CD8⁺ and γδ T cell subsets were calculated on a CD19⁻ CD3⁺ gate. For intracellular cytokine staining, cells were restimulated for 4 h with 50 ng/ml PMA and 1 µg/ml Ionomycin (Sigma) and Golgi Stop and block (BD biosciences) were added for the last two hours. The cells were fixed and permeabilized using fixation and permeabilization solution (eBiosciences). Staining was performed for IL-4 (clone 11B11) and IFN-γ (clone XMG1.2) antibodies, and the cells were analyzed on a LSRII flow cytometer (BD Biosciences) using FlowJo software (Tree Star).

Cells from the spleen and lymph nodes of Actb^ECFP reporter or Ccr9^−/− mice were isolated by being passed through a steel wire mesh, filtered on a 40-μm cell strainer and after red blood cells lysis using ACK buffer. To enrich for T cells, single-cell suspensions were first incubated with biotin-conjugated anti-B220 (clone RA3-6B2; eBioscience), anti-CD19 (clone 1D3; eBioscience), anti-CD11b (clone M1/70; BD Biosciences), anti-CD11c (clone N418; eBioscience), anti-Gr1 (clone RB6-8C5; eBioscience); anti-NK1.1 (clone PK136; BD Biosciences) and anti-Ter119 (clone TER-119; BD Biosciences) antibodies followed by antibiotin microbeads (Miltenyi Biotec) and negatively selected by magnetic cell separation with magnetic-activated cell sorting technology (Miltenyi Biotec); 5 × 10⁶ enriched T cells were intravenously transferred to Rag2^−/−Rorc^GFPIl22^TdT (RR22) mice; recipient mice were analyzed at day 14 post-transfer.