Analysis of Anti-Reovirus Cytotoxic T Lymphocytes (CTL) Using the H-2^dml Mutant

Abstract

The murine cytotoxic T lymphocyte response to reovirus -- a double-stranded, segmented RNA virus -- has been analyzed using the dm1 mutant and its d parent strain. Reovirus stimulates a strong CTL response in mice of both haplotypes. The H-2D/L^dm1, H-2D^d and H-2L^d antigens allserveas. anti-reovirus CTL restricting elements. These CTL most likely recognize nonmembrane structures of reovirus because this “naked” virus is nonenveloped and nonbudding with no known membrane maturation stage in its replication cycle. The fine-specificity of the H-2 restriction imposed by the individual class I molecules of these haplotypes has been examined by analyzing the crossreactivity patterns of parent and mutant CTL tested with L cells transformed with various cloned H-2 genes. Preliminary data indicates that d anti-reovirus CTL generally do not crossreact with infected targets expressing the mutant H-2D/L^dml antigen whereas dm1 antireovirus CTL frequently cross-lyse reovirus infected targets expressing either the H-D^d or H-L^d antigens. The nonreciprocity of the d and dm1 CTL response suggests that fundamentally different immunodominant determinants are selected for CTL recognition in d as compared to dm1 mice although substantial sequence homology exists between the H-2D/L^dml hybrid antigen and the two parent antigens from which the mutant class I molecule arose. Based on the unique hybrid nature of the H-2D/L^dml antigen, additional hybrid class I molecules have been constructed in collaboration with Drs. Iwona Stroynowski and Lee Hood, using the mutant and parent genes for exonshuffled gene constructs. Preliminary data is presented showing that dm1 CTL can recognize reovirus infected L cell transformants expressing the class I variant, [D^d]-[D/L^dml]-[D^d], with the brackets denotingtheboundaries between the [a1]-[m2]-[a3] extracellular domains. Eventually additional variants of this type will be constructed and expressed in L cells. In conjunction with the recently-isolated cloned reovirus genes, these novel class I genes will provide further insight into the structural elements of these molecules which govern anti-viral CTL recognition.