Cd8 bv510 clone sk1

Staining of unfractionated fresh blood was performed according to the lyse and stain approach using monoclonal antibodies: CD45 FITC (clone 2D1), CD4 PE/CF594 (clone RPA-T4, BD Biosciences, San Jose, CA, USA), CD3 PE-Cy7 (clone UCHT1), CD8 BV510 (clone SK1), CD16 PE (clone B73.1), HLADR BV786 (clone L243, BioLegend, San Diego, CA, USA) and the results read in the Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Living cells (stained with LIVE/DEAD Fixable Dead Cell Stain Kit with BV421 fluorochrome, Life Technologies Carlsbad, CA, USA) were evaluated. The gating was done using both CD45 and side scatter signals. NovoExpress Software (Agilent Santa Clara, CA, USA) was used for subpopulation analysis.

The following fluorochrome-conjugated antibodies were used to identify and characterize MAIT cell populations by multi-color flow cytometry: Vα7.2 FITC (clone REA179, Miltenyi Biotec), NKG2A PE (clone REA110, Miltenyi Biotec), CD3 PerCP (clone BW264/56, Miltenyi Biotec), NKG2D PeVio770 (clone BAT221, Miltenyi Biotec), CD161 Brilliant Violet (BV)-421 (clone HP-3G10, BioLegend), CD8 BV510 (clone SK1, BioLegend), PD-1/CD279 APC-Cy7 (clone EH12.2H7, BioLegend). MAIT cells were defined as CD3⁺/Vα7.2⁺/CD161^high lymphogated cells. Cells were acquired on a CyAn ADP cytometer (Beckman Coulter) using Summit software (Version 4.0). Gate limits were determined by using fluorescence minus one (FMO) controls. Data was analyzed using FlowJo software, Version 10 (FlowJo, LLC).

Human HER2 and PD-L1 expression on tumor cells was detected using human HER2-PE-CY7 (clone 24D2, Biolegend) and PD-L1-APC (clone MIH1, eBioscience). HER2 4D5 CAR was detected using the anti-trastuzumab idiotype Alexa Fluor 647-conjugated antibody (clone 2661E, R&D system). HER2 4D5 CAR was also detected using human recombinant HER2 protein conjugated with Alex Fluor 647. Human T cells surface phenotype and transduction efficiency were assessed using the following antibodies: NGFR-FITC (clone ME20.4, Biolegend), Q8 (clone QBEND/10, ThermoFisher), CD45-AF700 or CD45-BV605 (clone HI30, Biolegend), CD3-APC (clone SK7, Biolegend), CD4-PerCP (clone SK3, Biolegend), CD4-BB700 (clone SK3, BD Bioscience), CD8-BV510 (clone SK1, Biolegend), CD27-PE-CY7 (clone M-T271, Biolegend), CD28-BV605 (clone CD28.2, Biolegend. Expression of T cell inhibitory receptors was analyzed using PD-1-BV421 or PD-1-BV605 (clone EH12.2H7, Biolegend), TIM-3-BV605 or TIM-3-PE-CY-7 (clone F38-2E2, Biolegend), CD39 (clone A1, Biolegend), LAG-3-BV711 (clone 11C3C65, Biolegend). Live/dead discrimination was determined using LIVE/DEAD fixable Near-IR dead cell stain kit (ThermoFisher). Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).