Ex vivo stimulation and expansion of both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells of human cytomegalovirus-seropositive blood donors by using a soluble recombinant chimeric protein, IE1-pp65

The transfer of anti-human cytomegalovirus (HCMV) effector T cells to allog eneic bone marrow recipients results in protection from HCMV disease associ ated with transplantation, suggesting the direct control of CMV replication by T cells. IE1 and pp65 proteins, both targets of CD4(+) and CD8(+) T cel ls, are considered the best candidates for immunotherapy and vaccine design against HCMV. In this report, we describe the purification of a 165-kDa ch imeric protein, IE1-pp65, and its use for in vitro stimulation and expansio n of anti-HCMV CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMC) of HCMV-seropositive donors. We demonstrate that an important proportion of anti-HCMV CD4(+) T cells was directed against IE1-pp65 in HCM V-seropositive donors and that the protein induced activation of HLA-DR3-re stricted anti-IE1 CD4(+) T-cell clones, as assessed by gamma interferon (IF N-gamma) secretion and cytotoxicity. Moreover, soluble IE1-pp65 stimulated and expanded anti-pp65 CD8(+) T cells from PBMC of HLA-A2, HLA-B35, and HLA -B7 HCMV-seropositive blood donors, as demonstrated by cytotoxicity, intrac ellular IFN-gamma labeling, and quantitation of peptide-specific CD8(+) cel ls using an HLA-A2-peptide tetramer and staining of intracellular IFN-gamma . These results suggest that soluble IE1-pp65 may provide an alternative to infectious viruses used in current adoptive strategies of immunotherapy.