CLONING THE RAT HOMOLOG OF THE CD28/CTLA-4-LIGAND B7-1 - STRUCTURAL AND FUNCTIONAL-ANALYSIS

T cell activation involves the delivery of two independent signals to the naive T cell. The first signal occurs with engagement of the TCR, One of the best characterized second signals is ligation of CD28 on the surface of T cells by B7 molecules (B7-1, B7-2) present on the surface of activated antigen presenting cells (APCs). Recent studies have demonstrated that injection of a human fusion protein, CTLA-4-lg, which in humans binds to both B7-1 and B7-2, prevents cardiac allograft rejection in a rat transplantation model when given 48 h after engraftment. In order to better characterize the role of B7-1 (which is maximally expressed 48 h after activation of APCs) in this model, as well as in models of tumor-induced immune responses, we have cloned the rat homolog of B7-1, and now report on its structure and function. A 1030 bp cDNA containing the entire coding sequence of the rat B7-1 was cloned with a polymerase chain reaction strategy utilizing degenerate primers derived from published murine and human B7-1 sequences. The rat B7-1 coding sequence is 67 and 81% homologous to human and murine B7-1 cDNAs, and the predicted peptide sequence is likewise 57 and 66% identical to the peptide sequences of human and murine B7-1 respectively. The greatest area of identity occurs in the extracellular portion of the molecule, particularly the lg-C like domain. Rat B7-1 shares only similar to 25% overall peptide homology with the published peptide sequences for human and murine B7-2 but retains a region of similarity in the lgC domain that is present in all 87 molecules sequenced to date. RNA blot hybridization of lipopolysaccharide-stimulated splenocytes demonstrates six distinct bands ranging from 1.2 to 6.3 kb. Rat B7-1 transfected CHO cells specifically bind human CTLA-4-lg as assessed by flow cytometry. Furthermore, CHO-rat B7-1(+) cells co-stimulate highly purified T cells activated with phytohemagglutinin or bacterial superantigen, and this effect is virtually completely blocked by human CTLA-4-lg. Thus, the immunosuppressive effect of human CTLA-4-lg in rat transplantation models correlates with functional blockade of rat B7-1.