Ctla4 apc

MLs phenotypes from patients’ samples of the ELYP cohort at different timepoints were performed using CD103-FITC, CD73-PE, CTLA4-APC, CD39-APC-Vio770, CCR6-PE, PD1-PE-Vio770, NKG2D-APC, CD45RO-APC-Vio770 (Miltenyi), CD3-AF700, CD161-PE-Cy5, CD8-BV605 and CD4-BV711 (BD Biosciences), . MLs phenotypes from patients’ samples of the REMIND and IMCO cohort at different timepoints were performed using CD69-FITC, CD45RO-PerCP, CD5-AF700, CD8-BV605, CD4-BV711 (BD Biosciences), NKG2D-APC and CD103-APC-Vio770 (Miltenyi). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in FACS buffer (Miltenyi).
For the allogenic cocultures, cells were then stained using the following antibodies: Annexin V-FITC, E-Cadherin-PE-Vio770 HLA-E-APC, MICA/MICB-APC-Vio770 (Miltenyi), HLA-ABC-AF700 (BioLegend), HLA-DPDQDR-BV510, CD45-BV605 (BD Biosciences) and ULBP 2/5/6-PE (RD). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in Annexin V binding buffer (Miltenyi).

Splenocytes were thawed, washed with 10 mL of flow cytometry buffer (PBS + 5% FBS + 2 mM ethylenediaminetetraacetic acid), and counted with a hemocytometer. Per panel, 1×10⁶ cells were labelled with fluorescent antibodies diluted in 100 µL of flow cytometry buffer according to the manufacturer’s protocol. Panel 1: CD3-FITC (Miltenyi Biotec), CD279 (PD-1)-PE (Miltenyi Biotec), CD49b-PE-Vio 770 (Miltenyi Biotec), CD152 (Cytotoxic T-Lymphocyte Associated Protein 4, [CTLA-4])-APC (Miltenyi Biotec), and CD366 (T-cell Immunoglobulin and Mucin-Domain Containing-3 [Tim-3])-Brilliant Violet (BioLegend). Panel 2: CD11b-PE (Miltenyi Biotec), CD274 (PD-L1)-PE/Cyanine7 (BioLegend), CD273 (PD-L2)-APC (Miltenyi Biotec), and CD366 (Tim-3)-Brilliant Violet (BioLegend). After labelling, cells were washed, suspended in 300 µL flow cytometry buffer, and transferred to 5-mL round bottom tubes (Falcon). Fifty thousand viable singlets, as determined by light scatter properties, were analyzed with a Gallios flow cytometer (Beckman Coulter). The percentages of PD-1⁺, CTLA-4⁺, and Tim-3⁺ cells among CD3⁺CD49b^- T cells and CD3^-CD49b⁺ natural killer (NK) cells as well as the percentages of PD-L1⁺, PD-L2⁺, and Tim-3⁺ cells among CD11b⁺ macrophages were determined using FlowJo v10.7.2 (FlowJo LLC).