Induction and Exhaustion of Lymphocytic Choriomeningitis Virus–specific Cytotoxic T Lymphocytes Visualized Using Soluble Tetrameric Major Histocompatibility Complex Class I–Peptide Complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2D(b), chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8(+) cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2D(b) in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer(+) during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8(+) T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.