Cd3 pb

Spleens and thymuses were removed from Itpkb^+/+ and Itpkb^fl/fl mice, pretreated with tamoxifen for 5 days and allowed to rest for 3–7 days. Following RBC lysis, cells (1-5x10e⁵ cells/well) were washed in cold FACS buffer (1% FBS/2mM EDTA/PBS), blocked with 50 μl Fc Block (BD) for 5–10 min on ice, then surface stained with either CD3-PB (BD), CD8-FITC (BD), B220-PE (BD), IgM-APC (BD) and CD4-APC-Cy7 (BioLegend) for splenocyte staining or CD3-PB (BD), CD69-FITC (BD), TCR-beta (BD), CD4-APC (eBioscience) and CD8-APC-Cy7 (BioLegend) for thymocyte staining. Cells were stained for 30–60 minutes on ice, protected from light. Cells were washed twice and resuspended in either 150 μl FACS buffer to read immediately or BD stabilizer Fixative (BD) for later analysis on the BD LSR II flow cytometer.

Splenocytes were isolated under aseptic conditions from all mice groups and layered on Ficoll Hypaque for density gradient separation of the splenic lymphocytes. These splenic lymphocytes were stained for various cell surface and intracellular markers such as CD3, CD4, CD8, CD19, CD11b, F4/80, and RANKL. Briefly, Aliquots of 1 × 10⁶ splenic lymphocytes were washed with ice-cold 1XPBS and fixed with 1% paraformaldehyde for 15 min at 4°C. The cells were washed with FACS buffer (1XPBS + 2% FBS + 0.001% sodium azide) and then labeled with fluorophore tagged antibodies CD3-PB, CD4-PECF594, CD8-PE, CD19-FITC, CD11b-Percp cy5.5 (BD Biosciences, USA), and F4/80 APC-cy7, RANKL-PE (BioLegend, San Diego, USA). Further, the cells were washed with FACS buffer and acquired on FACS Aria (BD Biosciences San Jose, USA). Data analysis was done using FlowJo software (Tree Star, Ashland, USA).

ErcDCs and macrophages were sorted from ccRCC tissue-cell-suspensions stained with CD45-PeCy7, CD11c-APC, CD3-PB, CD209-PE (all BD Biosciences), CD14-PerCPCy5.5 (eBioscience, San Diego, CA, USA) and LIVE/DEAD^® Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Sorting gates were set on CD209⁺CD14⁺ cells (ercDCs) and CD209⁻CD14⁺ cells (macrophages), among pre-gated CD45⁺, live, single CD11c⁺ CD3⁻ cells. CD1c⁺ DC and slanDCs were sorted from B- and NK-depleted PBMCs of healthy donors (HD) using anti-CD11c-PE, anti-CD3-PB (all BD Biosciences), anti-CD56-APC (Beckman Coulter, Brea, CA, USA), anti-CD19-PB (Dako by Agilent, Santa Clara, CA, USA), anti-CD1c-PeCy7 (Biolegend, San Diego, CA, USA), anti-slan-FITC (Miltenyi Biotec) and LIVE/DEAD^® Fixable Near-IR Dead Cell Stain Kit. The gating strategy and instrument parameters are in Supplemental Figure S1. Gates were set very strictly, not covering the whole population, to avoid contamination with other cell populations. Cell population purity varied between 98–100%. Cells were directly sorted into 250 µL of RLT lysis buffer with ß-mercaptoethanol (RNeasy Micro Kit by Qiagen, Venlo, The Netherlands) using FACSAria IIIu (BD Biosciences), then homogenized (QIAshredder by Qiagen) and stored at −80 °C. Details about sorted cell types and numbers of biological replicates are listed in Table S2.