Anti cd45.2 104

Mouse splenocytes and thymocytes were prepared from pooled thymus or spleen. Thymic and splenic cells were pre-incubated with Fc-block before staining with other antibodies. The mouse antibodies used included anti-CD4 (RM4–5, eBioscience); anti-CD8 (53–6.7, eBioscience); anti-CD45.1 (A20, BioLegend), anti-CD45.2 (104, eBioscience); anti-CD25 (PC61.5, eBioscience); anti-CD69 (H1.2F3, eBioscience); anti-CD24 (M1/69, eBioscience); anti-Helios (22F6, eBioscience) before flow cytometry analysis. For intracellular staining of Ki-67 (B56, BD), pSTAT5 (47/Stat5(pY694), BD) and Foxp3 (NRRF-30, eBioscience), cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation / Permeabilization Solution Kit (554,714, BD) and stained according to the manufacturer’s protocols. The CD4⁺ T cells from the thymus and spleen of WT or RelB^−/− mice were analyzed by flow cytometry and gated as shown in the Supplementary Figure 1. The samples were analyzed using a BD LSRFortessa flow cytometer and FlowJo software (Tree Star Inc). All single-cell suspensions from the tissues were stained with Abs diluted in PBS containing 2% FCS for 30 min on ice.

Thymus tissues were freshly embedded in OCT compound and snap frozen in liquid nitrogen. Cryosections that were 6 μm thick were air-dried and fixed for 10 min in cold acetone. For fixation by IC buffer or paraformaldehyde, tissues were first fixed and then dehydrated before embedded in OCT compound. Cryosections were air-dried and used directly. Cryosections were blocked for 1 h in PBS containing 2% FBS and 1 mg/ml anti-FcγRII/FcγRIII (2.4G2) (in-house production). Cryosections were incubated overnight at 4°C with the following antibodies: anti-CD31 (MEC13.3) (eBioscience), anti-Ly6C (HK1.4) (BioLegend), anti-collagen IV (LSL), anti-GFP, anti-BST-1 (BP-3) (BioLegend), and anti-CD45.2 (104) (eBioscience). Unconjugated antibodies were detected with the following secondary antibodies: AlexaFluor®549-conjugated anti-rabbit IgG (Jackson) and TRITC-conjugated streptavidin (Jackson). Microscopic analysis was performed using a confocal microscope (Zeiss LSM-710), and the images were processed with ZEN 2010 software (Carl Zeiss, Inc.).

Cells from murine and human cell lines were plated and treated as above, and were collected and washed in PBS with 1% BSA and 0.1% NaN₃ followed by staining with fluorochrome-conjugated antibodies. Murine lines were stained with conjugated anti-CD74 (In-1) and anti- I-A/I-E (M5/114) (BD Biosciences, San Jose, CA). Human cell lines were stained with conjugated anti-CD74 (LN2) and anti-HLA-DR, DP (Tu36) (BioLegend, San Diego, CA). Fixation and permeabilization were performed with BD Cytofix/Cytoperm (BD Biosciences). Murine MISIIR-Tag tissues were stained with conjugated anti-CD45.2 (104) (eBioscience, San Diego, CA) anti-IA/IE (IgG2b, κ) (BioLegend) and anti-CD74 (WF) (BD Biosciences). Fixation and permeabilization were performed with the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience). MHC II and CD74 expression were evaluated on gated CD45-negative single cells within dissociated tumors. All samples were evaluated using a BD FACS CANTO II (BD Biosciences). FlowJo (FlowJo, Ashland, OR) v10.0.8r1 was used to analyze flow cytometry data.

The following mAbs were used: anti-CD3 (17A2 and 145-2C11), anti-TCRβ (H57-597), anti-CD49b (DX5), anti-CD43 (1B11), anti-Ly49H (3D10), anti-CD25 (PC61), CellTrace Violet stain, anti-phospho-S6 (S235/S236) (CUPK43K), anti-CD45.1 (A20), and anti-CD45.2 (104) from eBioscience; anti-NK1.1 (PK136), anti-Ki-67 (B56), anti-CD69 (H1.2F3), anti-CD120a (55R-286), anti-CD120b (TR75-89), anti-IFN-γ (HXG1.2), anti-CD107a (1D4B), anti-NFκB-p65 (pS529), and anti-BrdU (3D4) from BD Biosciences; anti-CD71 (RT7217), anti-CD98 (RL388) from BioLegend; and Live/Dead Fixable Yellow Dead Functional grade TNFα (MP6-XT22) and NKp46 (29A1.4) monoclonal antibodies were obtained from eBioscience. Intracellular staining of Ki-67 was carried out using a Foxp3 staining kit (eBioscience). Cells were acquired using BD LSRFortessa or ThermoFisher Attune NxT and analyzed using Kaluza 1.3 Analysis software (Beckman Coulter) or FlowJo (V10). Control anti-mouse IgG2a kappa isotype antibodies or unstained cells for a particular antibody were used as FMO. Intracellular staining with anti-phospho-S6 (S235/S236), anti-CD107a, and anti-IFN-γ antibodies was performed using BD Cytofix/Cytoperm protocols (BD Biosciences).