Cd8 bv711

Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).

For direct multi-colour flow cytometry (LRS Fortessa X-20; BD Bioscience), cells were incubated for 30 min at 4 °C with the antibodies mentioned below. Live/dead cell discrimination was performed by staining with the Zombie Aqua Fixable Viability Kit (BioLegend, catalogue number: 423101). To block Fc receptors, a purified anti-mouse CD16/CD32 (eBioscience, catalogue number: 14-0161-86) was used.
CD19 APCCy7 (clone 6D5, Biolegend, catalogue number: 115530, 1:300), Ly6G PerCPCy5.5 (clone 1A8, Biolegend, catalogue number: 127616, 1:300), CD45 PEDazzle (clone 30-F11, Biolegend, catalogue number: 103146, 1:400), F4/80PECy7 (clone BM8, eBioscience, catalogue number:254801-82, 1:200), CD3 APC (clone 145-2c11, Biolegend, catalogue number: 100312, 1:100), CD11b Alexa700 (clone M170, Biolegend, catalogue number: 101222, 1:400), TCγδV421 (clone GL3, Biolegend, catalogue number: 118120, 1:100), CD4 BV605 (clone RM4-5, Biolegend, catalogue number: 100548, 1:300), CD8 BV711 (clone 53-6.7, Biolegend, catalogue number: 100748, 1:300) and NK1.1 PE (clone PK136, Biolegend, catalogue number: 557391, 1:300). IL-10^GFP production was measured in the FITC channel.

Surface markers and intracellular Ki67were stained for phenotyping and measuring in vivo adoptively transferred and proliferating anti-SHIV T Cells. After PBMCs were isolated from the blood and tissue samples, cells were washed twice with PBS and stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher SCIENTIFIC) for 20 min at room temperature followed by staining with the following antibodies: CD3-Cy7-APC (Clone: SP34.2, BD Biosciences), CD4-BV605 (Clone: OKT4, BioLegend), CD8-BV711 (Clone: RPA-T8, BioLegend), CD28-Cy5PE (Clone: 28.2, BD Biosciences), CD45RA-Cy7-PE (Clone: L48, BD Pharmingen), CD69-ECD (Clone: TP1.55.3, Beckman Coulter), CCR7-Alexa Flour 680 (Clone: 150503, BD Pharmingen) and HLA-DR-BV421 (Clone: LN3, Biolegend). After surface staining, cells were permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (eBiosciences) for 20 min at room temperature, then intracellular stained with anti-Ki67-PE (Clone: B56, BD Pharmingen).

T cells were stained with CD8-BV711, CD4-BV786, and ZombieUV (BioLegend) according to the manufacturer’s protocol and analyzed on the same day with an LSR Fortessa X20 using mCherry fluorescence as a marker of CAR expression.

Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).

At 20 weeks post-transplantation cells from the hematopoietic organs of recipient mice were processed as described above and analyzed for multilineage repopulation. Cells were dissociated in 1mg/ml Collagenase-Dispase (Roche) at 37°C for 40 min and filtered through a 35μm nylong mesh. Cells were washed in FACS buffer (PBS without Ca²⁺ and Mg²⁺, 2% Foetal Calf Serum (FCS), and 50 U/ml P/S) and centrifuged for 5 min at 300 g. Cells were stained for 1 hour in CD45_1-V450 (A20, BD Horizon, RRID:AB_1727490), CD45_2-V500 (104, Biolegend, RRID:AB_10897142), b220-APC-cy7 (RA3-6B2, eBioscience, RRID:AB_394335), CD11c-PE-cy7 (N418, BioLegend, RRID:AB_493569), Ter119-FITC (Thermo Fisher, RRID:AB_465311), Gr1-PE (RB6-8C5, Thermo Fisher, RRID:AB_466044), CD335-BV711 (29A1.4, BioLegend, RRID:AB_2563289), CD3e-APC (145-2C11, Thermo Fisher, RRID:AB_11153519), CD4-APC (GK1.5, Thermo Fisher, RRID:AB_11152647), CD8-BV711 (53.67, BioLegend, RRID:AB_11219594). Cells were washed in FACS buffer and 7AAD was added as live/dead marker. Cells were then analyzed on a BD LSR Fortessa (BD). In all cases, FMOs were used to gate negative populations. OneComp and UltraComp beads (both ThermoFisher) were stained with single antibodies for automatic compensation by the Fortessa. All data was analyzed on FLOWJo software (BD).