Kavynfa

Spleens were mechanically dissociated using a 40 μm filter. RBCs were lysed by resuspension in 1 ml ACK lysis buffer for 2 min. Lysis was quenched by addition of 10 mls of T cell media. T cells were centrifuged at 350 × g for 5 min at 4 °C and resuspended in 10 ml of T cell media containing 10 ng/μl recombinant human IL-2 (rhIL-2, Peprotech), 5 ng/μl recombinant murine IL-7 (rmIL-7, R&D Systems), and 1 μg/ml anti-CD3ε (clone 145-2C11) and 1 μg/ml anti-CD28 (clone 37.51) (BD Biosciences). Alternatively, we used 10 ng/μl recombinant human IL-2 (rhIL-2, Peprotech) and 10 μg/ml Msln_406-414 peptide (GQKMNAQAI, Genscript) or 10 μg/ml GP33 peptide (KAVYNFATM, Genscript) for studies evaluating antigen-specific T cell activation. Splenocytes were cultured in T25 flask for overnight at 37 °C, 5% CO₂. Cells were counted using a hemocytometer and Trypan blue exclusion. A total of 5  × 10⁵ cells were transferred into a 12 well plates at and incubated at 37 °C, 5% CO₂ for 24 h prior to editing, or for longer periods of time as indicated.

Adult mice were infected with 200,000 pfu LCMV-Armstrong intraperitoneally. Peripheral blood was acquired by retro-orbital bleeds on indicated days. Red blood cells were lysed in RBC lysis buffer (eBiosciences) and blood was analyzed by flow cytometry as described above. Mice were euthanized after 60 days post infection (DPI) and splenocytes were isolated as described above. One million splenocytes per sample were stimulated with 30 ng/mL exogenous gp_33–41 peptide (KAVYNFATM, GenScript) for approximately 5 h in the presence of GolgiPlug (BD), after which surface staining, tetramer staining, and intracellular cytokine staining were performed as described above. Alternatively, mice were euthanized at three and eight DPI, and liver and spleens were removed. Eight DPI spleens were sorted for CD8+ T cells which were used for RNA-seq and metabolomics as described below. Liver was homogenized and used to determine viral load by plaque assay as described in detail by others [28 ]. Briefly, serial dilutions of liver homogenate were added to a monolayer of Vero cells and overlayed with 0.5% agarose for 5 days, stained with neutral red for 1 day, and PFUs were counted.