Pd1 apc

Peripheral blood of all participates was extracted using heparin anti-coagulation venipuncture tubes. Blood (200 μL) was incubated with mouse mAbs against human CD56 (FITC), CD3 (PerCP) (BD Biosciences, San Jose, CA, USA), and PD-1 (APC) (BioLegend, San Diego, CA, USA) for 15 min at 23 °C. The control groups were stained with isotype-matched antibodies. Red blood cell lysis buffer was added to the tubes containing red blood cells to lyse them. After incubating at 23 °C for 5 min and one wash with PBS, cells were detected using a BD FACSCalibur cell analyzer (BD Biosciences). Data was then analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Eight days after activation, CAR T cells were either maintained alone or cocultured with SKBR3 tumor cells for 8 and 24 h under control and pro-oxidative conditions. Then, CAR T cells were collected, fixed, and stained to assess: i) CAR construct expression, ii) exhaustion state, and iii) memory state. The following antibodies were used for staining: i) CAR construct expression, IgG-PE (Southern Biotech, 2042-09); ii) exhaustion state CD3-PE (BD Biosciences, 345765), CD4-V450 (BD Biosciences Biosciences, 560345), CD8-FITC (BD Biosciences, 555366), PD1-APC (BioLegend, 367406), TIM3-APC-Cy7 (BioLegend, 345026), and LAG3-PE-Cy7 (BioLegend, 369310); and iii) memory state CD4-AF700 (BioLegend, 317426), CD8-APC-Cy7 (BioLegend, 344714), CD45RA-APC (BioLegend, 304112), CD45RO-PerCP-Cy5.5 (BioLegend, 304222), CCR7-PE-Cy7 (BioLegend, G043H7), CD62L-PE-Cy5 (BioLegend, 304808), Granzyme B-FITC (BD, 558132), IFN-γ-BV621 (BioLegend, 502536), CD127-BV421(BioLegend, 351310), and CD57-PE (BioLegend, 359612) according to the manufacturer’s instructions.

For splenic, PP or MLN T cell flow cytometry, positively selected CD4⁺ cells were stained with combinations of antibodies (0.5 µg/ml per) to Tim3-APC (BioLegend, San Diego, CA) or Lag3-APC (BD Bioscience), PD1-APC (BioLegend, San Diego, CA), CTLA4-APC (BioLegend, San Diego, CA), Ly6A/E-APC (BD Biosciences, San Jose, CA), and GITR-APC (BD Biosciences, San Jose, CA). Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before analysis using Attune NxT (ThermoFisher, Waltham, MA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo (Tree Star, Inc., Ashland, OR).

Cryopreserved PBMCs were thawed and left for 1 h at 37 °C in the CO₂ incubator. Subsequently the cells were collected and stained with BD Horizon™ Fixable Viability Stain 510 to identify live cells, as per manufacture protocol. The cells were then washed, and surface stained for IL-21R PE expression on, (i) CD4, CD8 and Tfh (CD4/CD45RA -/CXCR5⁺/PD1⁺); (ii) B cell CD20, B1(CD20⁺CD27⁺CD43⁺), Plasma blast (CD20⁺CD38⁺) and iii) monocytes CD14⁺HLADR⁺.
After staining, the cells were washed and fixed using 2% PFA. Acquisition was done on BD FACSCelesta (Becton-Dickenson, San Jose, CA). Forward and side scatters and singlets were used to gate and exclude cellular debris. The flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences, Ashland, OR). The details of the antibodies used are as follows: Fixable Viability stain510, CD4 FITC (clone: RPA-T4) from BD Bioscience (San Jose, CA), IL-21R PE (clone: 2G1-K12), CD8 PerCP (clone: SK1), PD1 APC (clone: EH12.2H7), CXCR5 BV421 (clone: J252D4), CD45RA BV605 (clone: HI100), CD38 BV421 (clone: HB-7), CD16 AF700 (clone: B73.1), CD14 BV650 (clone: M5E2), HLADR BV605 (clone: L243), CD20 PerCP (clone: 2H7), CD27 FITC (clone: M-T271), CD43 APC (clone: CD43-10G7) from BioLegend (San Diego, CA).