Cd8 53 6

Cell surface and intracellular stainings were performed using antibodies as follows: CD4 (RM 4–5; BioLegend), CD8 (53–6.7; eBioscience), CD43 (1B11; BioLegend), CD62L (MEL-14; BioLegend), CD11b (M1/70; BioLegend), KLRG1 (2F1/KLRG1; BioLegend), Ki67 (16A8; BioLegend), FasL (MFL3; BD Biosciences), TRAIL (N2B2; eBioscience), Foxp3 (FJK-16s; eBioscience), GzmB (GB12; ThermoFisher Scientific), GzmA (GzA-368.5; eBioscience) and Gzm K (Orb102688; Biorbyt). Dead cells were excluded by fixable viability dye (eBioscience) staining. For FasL staining, lymphocytes were isolated and restimulated with anti-CD3 (145-2C11, eBioscience) and anti-CD28 (37.51, eBioscience) antibodies for 5 hours at 37 °C. BD Cytofix/Cytoperm Fixation/Permeabilization kit was used for intracellular staining following the manufacturer’s instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences). Analyses were done using FlowJo 5.0 software (Tree Star Inc., Ashland, OR).

For HSC (LK⁺S⁺ cells) isolation, BM cells were firstly enriched for c-Kit expression by immuno-selection with CD117 conjugated micro-magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. Enriched cells were then stained with PE-cy7 conjugated with a mixture of lineage antibodies (anti-CD3 145-2C11, Mac-1 M1/70, Gr-1 RB6-8C5, CD4 GK1.5, B220 RA3-6B2, CD8 53-6.7, Ter-119 TER119; all were purchased from e-Bioscience), PE conjugated Sca-1 (D7, e-Bioscience), APC conjugated c-Kit (2B8, e-Bioscience) and Percp-cy5.5 conjugated CD45.2 (104, e-Bioscience). Normal hematopoietic and leukemic cells were sorted by CD45.2 and GFP expression, respectively; and 4′,6-diamidino-2-phenylindole (DAPI) was used to exclude dead cells during the sorting procedure.

Human single cell suspensions were surface stained with fluorescence conjugated mAb to CD134 (L106, BD Bioscience), CD137 (4B4-1, BD Bioscience), CD69 (FN50, eBioscience), CD56 (N901, Beckman Coulter), CD3 (SK7, eBioscience) and CD5 (OKT1, in house). Murine cells were first FcγR-blocked (2.4G2, in-house) and then stained with CD49b (DX5, eBioscience), NKp46 (29A1.4, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4–5, eBioscience), CD8 (53–6.7, eBioscience) or CD25 (PC61.5, eBioscience). For intracellular FOXP3 (NRRF-30, eBioscience) staining, cells were fixed and permeabilised as per manufacturer’s protocol (eBioscience). Acquisition was performed using FACSCalibur (BD Biosciences) and analysed using Cytobank (Cytobank).

Single cell suspensions were prepared as previously described 15 (link), and contaminating erythrocytes were lysed using ammonium chloride buffer where appropriate. Cell suspensions were stained with optimal dilutions of antibodies directly conjugated to either Biotin, FITC, PE, PE-Texas Red, PE-Cy5.5, PerCP-Cy5.5, PerCP-eFluor710, PE-Cy7, Pacific Blue, eFluor 450, allophycocyanin, Alexa Fluor 647, or allophycocyanin-eFluor 780. Biotinylated antibodies were visualized using streptavidin PE-Texas Red (BD Bioscience). Lineage positive cells were gated out of whole BM suspensions using mouse hematopoietic lineage eFluor450 cocktail (eBioscience).The following specific antibodies were used to detect cell surface antigens: CD117/c-Kit (2B8), Sca-1/Ly6A/E (D7), CD127/IL-7Rα (A7R34), CD34 (RAM34), CD135/Flt3 (A2F10), CD25 (PC61.5), AA4.1 (AA4.1), CD27 (LG.7F9), CD150 (mShad150), CD48 (HM48-1), CD244.2 (eBio244F4), CD45.1 (A20), CD45.2 (104), GR-1/Ly-6G (RB6-8C5), CD11b (M1/70), CD11c (N418), CD45R/B220 (RA3-6B2), CD19 (eBio1D3), CD3 (145-2C11), CD4 (GK1.5), and CD8 (53-6.7). All antibodies were obtained from eBioscience. The CD1d PBS-57 tetramer was obtained from the NIH Tetramer Core Facility (Emory University, Atlanta, USA).

For T cell immune responses specific for virus antigens, the splenocytes were harvested from boost immunized mice and were in vitro stimulated with pooled S peptides and full-length S protein for 24 h. The lymphocytes were stained with anti-mouse CD4 (RM4-5, eBioscience), CD8 (53-6.7, eBioscience), and CD3 (17A2, BioLegend) monoclonal antibodies. A BD Cytofix/Cytoperm^TM Plus kit was used to fix and permeabilize cells prior to staining with anti-mouse IFN-γ (XMG1.2, eBioscience) monoclonal antibody. All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences) and analyzed using Flowjo software (Tree Star Inc., Ashland, OR, USA).