M F Luciani

Mechanisms of T cell-mediated cytotoxicity remain poorly defined at the molecular level. To investigate some of these mechanisms, we used as target cells, on the one hand, thymocytes from lpr and gld mouse mutants, and on the other hand, L1210 cells transfected or not with the apoptosis-inducing Fas molecule. These independent mutant or transfectant-based approaches both led to the conclusion that Fas was involved in the Ca(2+)-independent component of cytotoxicity mediated by at least two sources of T cells, namely nonantigen-specific in vitro activated hybridoma cells, and antigen-specific in vivo raised peritoneal exudate lymphocytes. Thus, in these cases, T cell-mediated cytotoxicity involved transduction via Fas of the target cell death signal.

To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

CD28, initially detected on human T lymphocytes with the help of antibodies, and CTLA-4, obtained by reverse genetics through its preferential expression in mouse activated T cells, are both single-V domain members of the Ig superfamily. Early work showed a relationship between these two molecules, which we wished to further document, in particular because of the growing realization of the functional importance of CD28 in some T cell activation pathways. Isolation and analysis of the mouse CTLA-4 gene and further analysis of the human CTLA-4 gene showed that both of these and the human CD28 gene share the same overall intron/exon organization. The nucleic acid sequence homology of the exons was found to extend across both molecules and species, whereas the 5' and 3' flanking regions exhibited homology across species but not between molecules. Message expression of human CTLA-4 was only detected in activated T cells and, thus, shares with that of mouse CTLA-4 and of mouse and human CD28 a lymphoid tissue distribution, although apparently broader for the latter. Two main human CTLA-4 transcripts of about 1.8 and 0.8 kb were detected, the smaller of which may derive, as reported for human CD28, from the use of an alternate degenerated polyadenylation signal sequence. The nucleic acid sequence data allowed a direct comparison of the four putative complete protein sequences of CD28 and CTLA-4 in the mouse and the human, showing striking homologies, especially in some stretches (such as a MYPPPY hexamer in the hinge region) conserved across molecules and across species. The mouse CD28 gene was localized to chromosome 1 band C by in situ hybridization with three different radioactive probes, indicating, together with previous data, that the CD28 and CTLA-4 genes map to the same chromosomal region in both the mouse and the human. Thus, CD28 and CTLA-4 were found to be strikingly similar in most respects, in terms of structure, sequence, expression, and gene location, furthermore in two species, strongly suggesting that their genes are the direct products of a duplication event and raising the possibility of functional homologies between the corresponding proteins.