Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: Functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(α₁) and C1(α₂) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2L(d) has been replaced by that of H-2D(d). Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2D(d) replaced by that of H-2L(d)) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2L(d) and H-2D(d) or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2L(d) and H-2D(d) to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2L(d) substituted for that of H-2D(d)), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2L(d) or anti-H-2D(d) N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of β₂M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2D(d) and anti-H-2L(d) CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2L(d)-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens. We conclude that T cell recognition of cells expressing DL antigen occurs because this antigen closely resembles both H-2L(d) and H-2D(d) and preserves many of the fine structural determinants present on both parental antigens. In contrast, the hybrid LD antigen has lost all or most of the determinants recognized by T cells specific for either parental antigen. A structural model to explain these findings is present.