An improved protocol for measuring cytotoxic T cell activity in anatomic compartments with low cell numbers.

The study of target cell lysis and cytokine production are valuable tools to characterize antigen-specific T and NK cell function during virus infections. After localized infections in compartments such as the lung or the brain, however, cell numbers isolated from these organs are too low to perform standard assays with individual mice. Here, we report a few simple modifications of the classical 51Cr release assay allowing reduction of the number of required effector cells by a factor of 10 without loosing sensitivity or specificity. Using not more than 4x10(5) effector cells, we were able to study ex vivo virus-specific CTL or NK activity from the lungs of individual mice after infection with respiratory syncytial virus (RSV) and from the brains of mice infected with Borna disease virus (BDV). Flow cytometric analysis of interferon-gamma production by virus-specific T cells including appropriate controls was achieved with as few as 10(5) effector cells.

CD8(+) T lymphocytes mediate Borna disease virus-induced immunopathology independently of perforin.

Perforin-mediated lysis of target cells is the major antiviral effector mechanism of CD8(+) T lymphocytes. We have analyzed the role of perforin in a mouse model for CD8(+) T-cell-mediated central nervous system (CNS) immunopathology induced by Borna disease virus. When a defective perforin gene was introduced into the genetic background of the Borna disease-susceptible mouse strain MRL, the resulting perforin-deficient mice developed strong neurological disease in response to infection indistinguishable from that of their perforin-expressing littermates. The onset of disease was slightly delayed. Brains of diseased perforin-deficient mice showed similar amounts and a similar distribution of CD8(+) T cells as wild-type animals. Perforin deficiency had no impact on the kinetics of viral spread through the CNS. Unlike brain lymphocytes from diseased wild-type mice, lymphocytes from perforin-deficient MRL mice showed no in vitro cytolytic activity towards target cells expressing the nucleoprotein of Borna disease virus. Taken together, these results demonstrate that CD8(+) T cells mediate Borna disease independent of perforin. They further suggest that the pathogenic potential of CNS-infiltrating CD8(+) T cells does not primarily reside in their lytic activity but rather in other functions.

Identification of the immunodominant H-2K(k)-restricted cytotoxic T-cell epitope in the Borna disease virus nucleoprotein.

Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8(+) T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2K(k)-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensitivity to lysis by CTLs isolated from brains of BDV-infected MRL mice with acute neurological disease. Vaccinia virus-mediated expression of a p40 variant with mutations in the two K(k)-specific anchor residues of the TELEISSI peptide (p40(E130K,I136T)) did not sensitize L929 target cells for lysis by BDV-specific CTLs, whereas expression of wild-type p40 did. Furthermore, unlike vaccination with wild-type p40, vaccination of persistently infected symptomless B10.BR mice with p40(E130K,I136T) did not result in central nervous system inflammation and neurological disease. These results demonstrate that TELEISSI is the immunodominant CTL epitope of BDV p40 in H-2k mice.