Ctla4 pecy7

Multicolor flow cytometric analysis was performed using the following fluorochrome-labeled monoclonal anti-human antibodies: TIGIT-FITC (MBSA43, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), PD1-PE (PD1.3.1.3, Miltenyi Biotec, Bergisch Gladbach, Germany), CD56-PerCPCy5.5 (5.1H11, BioLegend, San Diego, CA, USA), CTLA4-PeCy7 (14D3, Invitrogen, Thermo Fisher Scientific), CD3-APC (OKT3, BioLegend), CD4-APCCy7 (REA623, Miltenyi Biotec) and CD8-BV421 (REA734, Miltenyi Biotec). Cells were stained for 30 min at 4°C, measured with a Facs CantoII flow cytometer and analyzed using Kaluza Analysis 2.1 (Beckman and Coulter Life Sciences, Brea, CA, USA). The gating strategy is reported in Figure 4.

Draining s.c. LNs (axillary and popliteal) and the spleen were isolated from mice. Spleens were first mashed into a single cell suspension with plain DMEM media (Gibco 11966025), filtered through 70 μM cell strainers, and lysed with ACK lysis buffer (Gibco A1049201). LNs were digested with 1 mg/mL Ca²⁺ supplemented Collagenase D (Roche 11088866001) for 45 min at 37°C and gently mashed into a single cell suspension, also with DMEM and through 70 μM cell strainers. The cells were resuspended in IMDM media (Gibco 12440053), supplemented with 10% FBS and 1% Penicillin-Streptomycin (Gibco 15140122), and counted using a LUNA automated fluorescent cell counter (Logos biosystems). Cells were seeded at a count of 1x10⁶ - 3x10⁶ per well in 96-well round-bottom plates for subsequent antibody staining for flow cytometry. Antibodies against the following markers were used: CD3 – BUV395 (BD Biosciences 563565), CD8-BUV737 (BD Biosciences 612759), CD4 – BUV496 (BD Biosciences 612952), Foxp3 – FITC (BD Biosciences 560403), CD25 – BV605 (BioLegend 120235), ST2 – BV421 (BD Biosciences 566309), Lag3 – PerCP-Cy5.5 (BD Biosciences 564673), CTLA4 – PE-Cy7 (eBioScience 17-1522-82), IFNγ – APC (BioLegend 505810), TNFα – BV605 (BioLegend 506329), IL-2 – FITC (BioLegend 503806), IL-10 – APC-Cy7 (BioLegend 505036), PD-1 – BV711 (BioLegend 135231), Tim3 – PE (BD Biosciences 566346).