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.

Synthetic peptides. Several previously defined T cell epitopes encoded by LCMV were used in this study (38, 39) . LCMV-specific epitopes include NP396-404 (FQPQNGQFI), GP33-41 (KAVYNFATC), GP276-286 (SGVENPGGYCL), NP205-212 (YTVKYPNL), and GP118-125 (ISHN-FCNL). We identified one VV-specific K b -restricted epitope from VV protein A11R 198-205 (AIVNYANL). Synthetic peptides listed here were purchased from American Peptide and were purified with reverse phase-HPLC to 90% purity. CDR3 length spectratyping analysis and sequencing. CDR3 length "spectratype" analysis was performed as described previously with modification (16, 40) . RNA samples were isolated from 5,000-60,000 MHC dimer-or MHC-tetramer-sorted epitope-specific CD8 T cells or from an NP205specific cell line derived from an LCMV-immune mouse. For NP396-404and GP33-41-specific cells, RNA samples were amplified with primers for C␤ and for V␤8.1, using a GeneAmp RNA kit (PerkinElmer). The PCR products were subjected to five cycles of runoff reaction with six fluorophorelabeled J␤ primers (J␤ 1.1, 1.2, 1.3, 1.4, 1.5, and 2.1) . The runoff products were mixed with gel-loading buffer (five parts of 100% formamide and one part of 2.5% blue dextran/50 M EDTA), loaded onto a 4.75% acrylamide sequencing gel, and analyzed on an automated DNA sequencer using Gene-Scan software (Applied Biosystems). For NP205-specific T cells, TCR V␤ analysis was performed by a qualitative PCR with specific primers for V␤ 1-18. The dominant V␤16 PCR products were subcloned and sequenced across the CDR3 region according to the method of Naumov et al. (11) .