Ccr7 apc cy7

Following 4-day co-cultures of CAR T cells and SKBR3 cells at a ratio of 4:1, cells were extracted by centrifugation and prepared for flow cytometry analysis of immunological markers. First, cells were stained for viability (Zombie Aqua, Biolegend) in PBS, washed and stained in FACS buffer (2% FBS, 0.1% NaN3 in PBS) for the following markers: HLA-DR-Alexa Fluor 647 (L243), CD69-Pacific Blue (FN50), CD25-PE/Cy7 (M-A251), CD137/4-1BB-PE/Dazzle 594 (4B4-1), CD45RA- PE/Dazzle 594 (HI100), CCR7-APC/Cy7 (3D12), CD27-BV570 (O323), CD39-FITC (A1), CD127-PE (A019D5), CTLA-4-BV785 (L3D10), LAG-3-BV711 (11C3C65), TIGIT-BV421 (A15153G) from Biolegend; and CD3-BUV395 (UCHT1), CD4-BUV496 (SK3), CD8-BUV805 (SK1), CD62L-BV650 (SK11), PD-1-BB700 (EH12.1), TIM3-BV480 (7D3) from BD Biosciences (Supplementary Table 3). After washing, cells were analyzed using the Cytek Aurora full-spectrum flow cytometry technology. Data were further processed with FlowJo 10 software (BD Biosciences; Supplementary Fig. 12).

Subsets of CD8⁺ T-cells were distinguished on the basis of cell surface receptor expression as described previously (28 (link)), using antibodies specific for CD45RA-ECD (clone 2H4LDH11LDB9, Beckman Coulter), CCR7-APC/CY7 (Clone G043H7, BioLegend, San Diego, CA, USA), and CD27-PC5 (clone 1A4CD27, Beckman Coulter. The following subsets were analyzed in this study: Naïve (CD45RA⁺CCR7⁺CD27^+/−), Effector (E, CD45RA⁺CCR7⁻CD27^+/−), Early Effector Memory (e-EM, CD45RA⁻CCR7⁻CD27⁺), Late Effector Memory (l-EM⁻, CD45RA^+/−CCR7⁻CD27⁻), and Central Memory (CM, CD45RA⁻CCR7⁺CD27^+/−). Cell subsets were assessed by flow cytometry using a FC500 machine (Beckman Coulter, Marseille, France). This began by gating on the lymphocyte population based on FSC/SSC, followed by doublet-exclusion and live/dead staining using the Live-Dead Fixable Stain Kit (Molecular Probes, Eugene, Oregon, USA). Cells were evaluated by applying the fluorescence minus-one color compensation strategy followed by gating on CD45RA⁺ and CD45RA⁻ subsets, followed by subset determination based on CCR7 and CD27 expression on the latter subsets. Data were analyzed using FlowJo software (FLOWJO, LLC, Ashland, Oregon).

Lysed cells were acquired and subsequently stained for 30 min at 4 °C with the following fluorescein-conjugated monoclonal antibodies: CD3-PE (Bio- Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CCR7-APC/Cy7 (BioLegend). The T cell subsets were defined as previous study reported [10 (link)]: Naive T cells being CCR7⁺ and CD45RO⁻; central memory T cells as CD45RO⁺ and CCR7⁺; effector memory T cells as CD45RO⁺ and CCR7⁻, and effector memory RA (EMRA) T cells as CD45RO⁻ and CCR7⁻ (Figure S1). A total of 200,000 events were acquired by the BD LSRFortessa™ flow cytometer (BD Bioscience, San Jose, CA, USA). The data analysis was carried out with Flowjo v10.1 Software (Tree Star, Ashland, OR). The absolute number of each T cell subset was calculated as follows: (percentage of each cell population among total lymphocytes) × (total lymphocytes count)/100.

The phenotypes of CD8⁺ T-cells in whole blood (using Optylyse, Beckman Coulter, Marseille, France) or isolated cells were distinguished by flow cytometry using multiple antibodies: CD127-PE (5μl, clone R34.34, AB_131301, Beckman Coulter), CD8-FITC/PeCy5 (5μl, clone HIT8a, AB_395996 and AB_395998), CD45RA-APC/PECy5 (3μl, clone HI100, AB_398468 and AB_395881, BD Pharmingen, BD Bioscience, San Jose, CA, USA) and CCR7-APCCy7 (5μl, clone G043H7, AB_10916390, Biolegend, San Diego, CA, USA). Freshly isolated cells (1x10⁵ lymphocytes per sample) were incubated in 1% BSA-PBS (100μl) for 30 minutes on ice, followed by 2 washes with 1% BSA-PBS, protocol adapted from Nascimbeni and Rehermann [37 (link)]. Cell subsets were distinguished as follows: naïve (T_N, CD45RA⁺CCR7⁺), central memory (T_CM, CD45RA^-CCR7⁺), effector memory (T_EM, CD45RA^-CCR7^-), and terminally differentiated effector memory (T_EMRA, CD45RA⁺CCR7^-). When analyzing IH-CD8⁺ T-cells, the flow cytometer was calibrated using blood CD8⁺ T-cells to conserve the number of IH-CD8⁺ T-cells available for study, which revealed a higher degree of autofluorescence in IH-CD8⁺ T-cells compared to blood-derived cells, as previously reported by others [38 (link)]. All flow cytometry analyses excluded dead cells, on the basis of forward and side scatter profiles, and gates were set using the principle of fluorescence minus one (FMO).