Klrg1

Fluorescent-dye-labeled antibodies against cell-surface markers CD4, CD8, CD44, CD62L, Gr1, CD19, CD45.1, CD45.2, CD127, CD27, and KLRG1 were purchased from eBioscience (San Diego, CA, USA). FITC- or PE-conjugated antibodies against cytokines were from BioLegend (San Diego, CA, USA). APC- or PE-conjugated K^b-ova+ tetramer was obtained from QuantoBio (Beijing, China). Splenic cells were depleted of erythrocytes by hypotonic lysis. The cells were washed with FACS washing buffer (2% FBS, 0.1% NaN₃ in PBS) twice and were then incubated with fluorescence-conjugated antibodies against cell-surface molecules for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. Isotype antibodies were included as negative controls. For intracellular cytokine staining, single-cell suspensions were stimulated with 10 nM SIINFEKL peptide in the presence of Brefeldin A solution (eBioscience) for 6 h at 37 °C. After stimulation, cells were stained with fluorescence-conjugated antibodies against cell-surface markers, fixed, and permeabilized using a fixation/permeabilization kit (eBioscience) and stained with fluorescence-conjugated specific antibodies against Smad4 (Santa Cruz, Santa Cruz, CA, USA), IFN-γ, TNF-α, and GzmB in accordance with the manufacturer's instructions. Flow cytometry was performed using a Becton Dickinson FACSCalibur machine.

Tumors or lymph nodes were excised, weighed, crushed, strained through a 0.45 micron filter and re-suspended in FACS buffer (PBS, 1% FCS, 5 mM EDTA) and surface stained with anti-CD8, CD45.2, H2DB, H2KB, KLRG1, PD1, CD95, Tim3, CD62L or CD44 (eBioscience) A full list of all antibodies is provided in Supplementary Table 4. For tetramer staining, singly suspended cells were incubated with tetramer-gp33 or tetramer-np396 (CD8) for 15 min at 37 °C. After incubation, surface antibodies (anti-CD8, anti-PD1, anti-KLRG1) were added for 30 min at 4 °C. For intracellular cytokine re-stimulation following in vivo LCMV infection, singly suspended cells were stimulated with LCMV specific peptide gp33, for 1 h after which Brefeldin A (eBiocience) was added for another 5 h incubation at 37 °C followed by staining with anti-Granzyme B, anti-IFNγ and anti-TNF-α. Staining of CD8⁺ T cells for Granzyme B and IFNγ within tumors, lymph nodes and for ex vivo co-culture systems was performed using the Foxp3 mouse Treg cell staining buffer kit (eBioscience). Experiments were performed with the BD Fortessa Cell Analyzer (BD Biosciences) or CytoFLEX (Beckman Coulter) and analyzed using FlowJo software.

Blood samples (100 µl) collected from the participants were incubated for 25 min at 25 °C in a flow cytometry tube with the following specific antibodies (5 µl each): CD3 (563219, BD), CD16 (302018, Biolegend), CD56 (555518, BD), TIGIT (12-9500-42, eBioscience), DNAM-1 (559788, BD), NKG2A (130-113-565, Miltenyi Biotec), LAIR-1 (ab27744, Abcam), Siglec-7 (ab200558, Abcam), and KLRG1 (25–9488-42, eBioscience). In addition, an isotype control was prepared for each sample. After incubation, the reaction mixture in each tube was mixed thoroughly with 2 ml of hemolysin. The samples were then washed for flow cytometry analysis (BD FACSCanto™ Flow Cytometer) within 4 h.

Cells were washed with FACS buffer [phosphate buffered saline (PBS) and 2% FCS] and stained with anti-Fc II/III receptor monoclonal antibody 2.4G2 for 15 min at 4°C. After an additional FACS buffer wash, the following antibodies were incubated with the cells for 30 min at 4°C: CD4 (RM4-5, eBioscience), CD8α (53-6.7, eBioscience), CD3 (145-2C11, eBioscience), CD44 (IM7, eBiosience), and B8R tetramer (NIH tetramer facility) were used to determine total T cell and VacV-specific CD8 T cell subsets. Isolated spleen and lung B8R⁺ CD8 T cells from WT and Bcl11b^−/− mice were also stained for their surface expression of KLRG-1 (eBioscience) and IL-7Ra (eBioscience). All samples were acquired on a FACS LSR II or Canto II (BD Bioscience) and analyzed using FlowJo (Tree Star).

For surface staining, cells were labeled with mAbs against various targets including CD8, CD62L, CCR7, CD45RA, CD45RO, CD25, CD44, OX40, PD-1, CTLA-4, and KLRG-1 (Ebiosciences), incubated at 4 °C for 15 minutes, then washed twice with PBS 1% FBS (FACS buffer), and finally fixed in PBS containing 1% paraformaldehyde (Fix buffer). For T-bet (4B10) and Eomes (Dan11mag), and Foxp1 intracellular staining, cells were first labeled with surface markers CD8, CD62L, OX40 (Ebiosciences) for T-bet and Eomes, or CD25 (BD Biosciences), CD8, CD27, and CD44 (Ebiosciences) for Foxp1 detection, respectively, and then fixed and permeabilized with Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Ebiosciences) in a 96-round-well plate. Intracellular labeling for T-bet and Eomes (Ebiosciences), and Foxp1 (LifeSpan Technologies) was performed according to manufacturer’s instruction. For evaluation of Perforin, Granzyme B, and IFN-ɤ cytokine secretion, effector cells were incubated with brefeldin A for 4 hours at 37 °C to disrupt Golgi-mediated transport and accumulate cytokines. Cells were then surface stained, permeabilized with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Biosciences), and intracellularly stained as recommended. Flow cytometry data was analyzed using FlowJo Version 10.0.7 software.