Tcrβ clone h57 597

The following antibodies from BD, eBioscience and BioLegend were used from cell surface antigen staining: CD8α (clone 53-6.7; eBioscience and BD), CD5 (clone 53-7.3; eBioscience), CD25 (clone 7D4; BD), CD44 (clone IM7; eBioscience), CD69 (clone H1.2F3; BioLegend and eBioscience), CD62L (clone MEL-14; eBioscience and BD), CD98 (clone RL388; BioLegend), CD122 (clone 5H4; eBioscience), Vα2 (clone B20.1; eBioscience), CD107a (clone 1D4B; BD), and TCRβ (clone H57-597; BD).
For intracellular staining, the following antibodies from eBioscience were used: TNF (clone MP6-XT22), IFNγ (clone XMG1.2), and Themis (clone 1TYMS).
For nuclear staining, the following antibodies were used: Foxp3 (clone MF-14; BioLegend), GATA3 (clone TWAJ, eBioscience), and Eomes (clone Dan11mag; eBioscience).
For metabolic marker analysis, the following primary antibodies from Cell Signaling were used: c-Myc (clone D3N8F), pAkt T308 (clone D25E6), and p-S6 ribosomal protein S235/236 (clone D57.2.2E). Goat anti-rabbit F(ab’)2 fragment Alexa Fluor 488 (Invitrogen) was used as secondary antibody.

Lymphocytes were counted and subjected to viability staining (Fixable Aqua Dead Cell staining, Life Technologies, #L34957) and subsequently to receptor Fc blockade (BD #553142). Staining was performed using the following antibodies: CD4 (clone RM4-4, BD), CD45 (clone 30-F11 eBioscience), CD45.1 (clone A20, BD), CD45.2 (clone 104, BD) CD8a (clone 53-6.7, eBioscience or BD), CD8b (clone YTS156.7.7, BioLegend), CD11b (clone RM2817, Thermo Fisher), Ly6c (clone AL-21, BD), CD3ε (clone 145-2C11, BD), CD19 (clone 1D3, BD), CD90.2 (53-2.1, BD), CD127 (clone A7R34, BD), CX3CR1 (clone SA011F11, BioLegend), CXCR3 (clone CXCR3-173, Biolegend), CD103 (clone 2E7, eBioscience or Biolegend), CD69 (clone H1.2F3, BD), TCR-β (clone H57-597, BD, TCR-γδ (clone eBio-GL3, eBioscience). H2-K^b:SIINFEKL or H2-M3:fMIGWII tetramers were either produced in house or, for the former, obtained thorugh the NIH tetramer core. Samples were fixed (IC Fixation Buffer, eBioscience), washed, resuspended in FACS buffer and acquired with a LSRII flow cytometer (BD) either immediately or on the following day. For intracellular staining, the following antibodies were used: IFN-ϒ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) in Permeabilization buffer (eBioscience).

Organs from vaccinated mice were collected 10 days post vaccination and placed on ice in DMEM supplemented with 10% FBS. Harvested spleens were forced through a 70 μm nylon filter (Fisher, Hampton, NH, USA), which was then rinsed three times with collection media and pelleted at 800×g for 5 min. Supernatant was decanted and pellet was resuspended in collection media and red blood cell lysis buffer (Sigma, St. Louis, MO, USA) for 1 min at room temperature. Collection tubes were then filled with 50 mL of FACS wash buffer (0.05% BSA in PBS) and spun as before, cells were then resuspended in FACS staining buffer (1% BSA in PBS) and enumerated. A total of 1 × 10⁶ cells in 50 μL were stained with either MHC Tetramer, H-2 Kb OVA (MBL: T03000); TCRβ clone H57-597 (BD Pharmingen: 553174); CD8α clone 53-6.7 (BD Pharmingen: 553030 or BD Pharmingen: 553033) for depletion analysis, all at 1:500 in FACS tubes for 30 min on ice. After 30 minutes, FACS tubes were filled with FACS wash buffer and spun as before. Supernatant was then decanted and cells were resuspended in 10% neutral buffered formalin and analyzed on a BD LSR-II (Franklin Lakes, NJ, USA) at the UTHSCSA flow cytometry core.