Anti thy1

Cells isolated from spleens and graft-draining axillary and brachial lymph nodes (dLN) were stained with anti-CD4 (BD Biosciences), anti-CD8 (Invitrogen) and anti-Thy1.1 (BD Biosciences). For phenotypic analysis cells were also surface-stained with anti-PD-1 (BioLegend), anti-2B4 (BD Biosciences or eBioSciences), anti-Thy1.1 (BD Biosciences), anti-LAG-3 (BioLegend), anti-CD127 (BioLegend), anti-KLRG-1 (eBioSciences), anti-CD44 (BioLegend or BD Biosciences), and anti-CD48 (BioLegend). Absolute numbers of lymphocytes from the spleen and draining lymph nodes were calculated using a Cellometer Auto T4 Cell Viability Counter (Nexcelom) according to the manufacturer’s instructions. Samples were analyzed on an LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.). For intracellular cytokine staining, lymphocytes were restimulated ex vivo with 1 μg/mL phorbol 12-myristate 13-acetate (PMA) (Sigma Life Sciences) and 1 μg/mL ionomycin (Sigma Life Sciences) where indicated, in the presence of 1 μg/mL Brefeldin A (BD Biosciences) for 4 hours. The Fix/Perm intracellular staining kit (BD Pharmingen) was used to detect IL-2 (BD Biosciences), TNF (BioLegend), and IFN-γ (BD Biosciences), according to manufacturer’s instructions.

Splenocytes were isolated and prepared as a single-cell suspension. 3×10⁶ WT and 2B4KO OT-I T cells, respectively, were resuspended in 1.5 mL of complete media (glucose-free RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 0.5 mM 2-mercaptoethanol) in a 24-well flat-bottomed plate at 37°C in a 5% CO₂, humidified atmosphere. A D-glucose solution of 200g/L (Gibco by Life Technologies) was titered into the glucose-free complete media and diluted on a half-log scale. Cells were then stimulated with SIINFEKL N4 peptide at 1 nM for 5 days. After 5 days in culture cells were harvested and stained with anti-CD8 (BD Horizon), anti-Thy1.1 (BD Biosciences or BioLegend), anti-CD244 (eBioSciences), anti-Va2 (BD), anti-CD127 (BioLegend), anti-CD62L (BD), anti-KLRG-1, anti-Vb5 (BD), anti-CD44 (BD), 7AAD (BD) and AnnexinV (BioLegend), anti-IL-2 (BD), anti-IFN-g (BD) as described by the manufacturer. As described above, samples were analyzed on an LSRII flow cytometer (BD Biosciences) and data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.).

To assess the ability of 2B4-deficient OT-I T cells to take up glucose, 10⁴ WT and 2B4KO OT-I T cells were transferred into naïve C57BL/6 hosts and infected them with 10⁴ colony forming units (CFU) of OVA-expressing Listeria monocytogenes (LM-OVA) two days. 14 days after infection, the animals were sacrificed and spleens were harvested. The cells were isolated and resuspended in a single cell solution in PBS. 2×10⁶ splenocytes were stained with anti-CD4 (BD Biosciences), anti-CD8 (Invitrogen) and anti-Thy1.1 (BD Biosciences), anti-CD44 (eBioSciences), anti-CD44 (BD Biosciences) for 30 minutes at 4°C. Cells were washed twice in 250 μl of PBS and then resuspended in 200 μl of 50 μM 2-NBDG (Thermofisher) and incubated at 37°C for 30 minutes. Cells were washed with PBS and then analyzed on an LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo 9 software (Treestar) and Prism 6 software (GraphPad Software Inc.).