Cd3 17a2

For immunofluorescence, spleens were embedded and frozen in OCT, sectioned at 15 μm and mounted on SuperFrostPlus Adhesion glass (Thermo Fisher Scientific). Sections were dehydrated using silica beads, fixed with 4% paraformaldehyde for 10 min and washed with PBS. Samples were blocked using 5% normal goat serum for 2 h before staining. Samples were incubated with antibodies against B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience) and F4/80 (BM8, Biolegend) diluted in 5% NGS for 2 h at room temperature in the dark. After staining, samples were washed with PBS at least three times. Samples were then mounted using ProLong Gold Antifade Mountant (Invitrogen) and imaged using an inverted LSM780 microscope (Carl Zeiss) and a plan apochromat 63× NA 1.40 oil-immersion objective (Carl Zeiss). For haematoxylin and eosin (H&E) staining, organs were collected and fixed in 10% formalin. Fixed samples were embedded in paraffin and sectioned at 10 μm, mounted on SuperFrostPlus Adhesion glass and stained using H&E. Mounted samples were imaged using a Nikon SMZ1270 Stereo Microscope. Imaging data were analysed using Fiji (ImageJ) software (NIH).

Human single cell suspensions were surface stained with fluorescence conjugated mAb to CD134 (L106, BD Bioscience), CD137 (4B4-1, BD Bioscience), CD69 (FN50, eBioscience), CD56 (N901, Beckman Coulter), CD3 (SK7, eBioscience) and CD5 (OKT1, in house). Murine cells were first FcγR-blocked (2.4G2, in-house) and then stained with CD49b (DX5, eBioscience), NKp46 (29A1.4, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4–5, eBioscience), CD8 (53–6.7, eBioscience) or CD25 (PC61.5, eBioscience). For intracellular FOXP3 (NRRF-30, eBioscience) staining, cells were fixed and permeabilised as per manufacturer’s protocol (eBioscience). Acquisition was performed using FACSCalibur (BD Biosciences) and analysed using Cytobank (Cytobank).

Single-cell suspensions of bone marrow, spleen, and lymph nodes were incubated with 1 x ACK solution (for red blood cell lysis) to deplete erythrocytes. Staining was performed with the following antibodies (conjugated with FITC, PE, APC, eFI450, BV786, AF700, PE-Cy7, PerCP Cy5.5, APC-eFI780 or biotin): anti–Siglec-H (551.3D3; Biolegend), anti-mPDCA1 (JF05-1C24.1; Biolegend), anti-CD11c (N418; Biolegend), anti-B220 (RA3-6B2; Biolegend), anti-CCR9 (242503; R&D Systems), anti-FcgR2b (own hybridoma), anti-IL2r (PC61; Biolegend), CD19 (eBio1D3, eBioscience), CD3 (17A2, eBioscience), CD5 (53-7.3, eBioscience), CD4 (GK1.5, eBioscience), CD138 (281-2, BD), CD95 (Jo2, BD), IgD (11-26c.2a, BD), CD80 (16-10A1, eBioscience), PD-L2 (TY25, BD), IL-4 (11B11, BD), IL-6 (MP5-20F3, BD), IL-10 (JES5-16E3, eBioscience), IFN-g (XMG1.2, eBioscience) and Fc-block (2.4G2; own hybridoma). Biotinylated antibodies were detected using streptavidin PerCPCy5.5 or streptavidin PECy7 (Biolegend). Cells were analysed using either Cytoflex (Beckmann Coultier) and FlowJo software (Tree Star).

Single cells suspensions were prepared from mesenteric lymph nodes (MLN) by forcing the organs through an 80µm mesh (Sigma-Aldrich). Cells were washed twice with PBS and then suspended in Flow Cytometry Staining Buffer (eBioscience). To block unspecific binding of antibodies, cell suspensions were incubated with an anti‐CD16/32 mAb (2.4G2, eBioscience) for 15 min on ice and then stained with combinations of the following fluorochrome-labeled antibodies against surface markers for 30 min on ice: CD3 (17A2, eBioscience), CD4 (GK1.5, eBioscience), CD69 (H1.2F3, Abcam), and CD25 (PC61.5, eBioscience). Intracellular staining for FoxP3 (FJK-16s, eBioscience) was performed using the FoxP3/Transcription factor staining buffer set (eBioscience) according to the manufacturer’s instructions. Flow cytometric analysis was performed using a FACSCalibur based on CellQuest software (BD-Becton Dickinson, Franklin Lakes, USA). T cell effectors (Teff) were defined by: CD3, CD4, CD69+. T cell regulators (Treg) were defined by: CD3, CD4, CD25, FoxP3+.

Flow cytometry data were acquired on a FACSCanto (BD Biosciences, San Diego, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR). To determine expression of cell surface proteins, mAb were incubated at 4°C for 20–30 min and cells were fixed using Cytofix/Cytoperm Solution (BD Biosciences) and, in some instances followed by mAb incubation to detect intracellular proteins. The following mAb clones were used: NK1.1 (PK136, eBioscience), CD3 (17A2, eBioscience), Ly49H (3D10, eBioscience), Ly49D (4E5, eBioscience), NKp46 (29A1.4, eBioscience), CD27 (LG.7F9, eBioscience), CD11b (M1/70, eBioscience), IFN-γ (XMG1.2; eBioscience), Granzyme B (MHGB04, Invitrogen), CD107a (1D4B, BD Pharmingen), AKT1 (55/PKBa/AKT, BD Pharmigen), pS473 (M89-61, BD Pharmigen).
Intracellular cytokine staining: For direct ex vivo, staining cells were incubated for 1 additional hour in the presence of Brefeldin A (BFA) before surface and intracellular IFN-γ staining. For cytokine staining following in vitro stimulation BFA was added during the last hour of stimulation. Intracellular signaling staining: For detection of intracellular AKT and pAKT (pS473) cells were methanol fixed and permeabilized according to BD protocol. Apoptosis was evaluated using Vybrant FAM Caspase-3/7 Assay Kit (Invitrogen) according to manufacturer’s protocol.