Private specificities of CD8 T cell responses control patterns of heterologous immunity

Kim, Sung-Kwon; Cornberg, Markus; Wang, Xiaoting Z.; Chen, Hong D.; Selin, Liisa K.; Welsh, Raymond M.

Dual tetramer staining of T cell culture. 10 7 splenocytes from LCMV-immune mice were cocultured with VV A11R 198 peptide-pulsed 10 6 RMA-S cells in RPMI 1640 supplemented with 100 U/ml penicillin G, 100 g/ml streptomycin sulfate, 2 M L-glutamine, 10 M Hepes, 1 M sodium pyruvate, 0.1 M MEM nonessential amino acids, 0.05 M ␤-mercaptoethanol, and 10% FBS for 5 d at 37ЊC at 5% CO 2 . The IL-2 culture supplement BD T-Stim (BD Biosciences) was added after 5 d of culture. After 10 d of stimulation, T cell lines were analyzed by tetramer staining. Cells were incubated in FACS buffer with streptavidin and Fc block to prevent nonspecific binding, washed, and stained with phycoerythrin-and/or allophycocyanin-labeled tetramers for 60 min. After 40 min of tetramer incubation, PerCP anti-mouse CD8␣ was added. Thereafter, cells were washed twice with FACS buffer and fixed in Cytofix (BD Biosciences). All staining was performed on ice. Samples were analyzed with FlowJo software (Tree Star, Inc.). MHC class I K b tetramers specific for VV A11R 198 and LCMV GP118 were generated as described previously (41) .