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

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Abstract
The transfer of anti-human cytomegalovirus (HCMV) effector T cells to allogeneic bone marrow recipients results in protection from HCMV disease associated with transplantation, suggesting the direct control of CMV replication by T cells. IE1 and pp65 proteins, both targets of CD4+and CD8+T cells, are considered the best candidates for immunotherapy and vaccine design against HCMV. In this report, we describe the purification of a 165-kDa chimeric protein, IE1-pp65, and its use for in vitro stimulation and expansion 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 HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4+T-cell clones, as assessed by gamma interferon (IFN-γ) 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, intracellular IFN-γ labeling, and quantitation of peptide-specific CD8+cells using an HLA-A2–peptide tetramer and staining of intracellular IFN-γ. These results suggest that soluble IE1-pp65 may provide an alternative to infectious viruses used in current adoptive strategies of immunotherapy.

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