Cytomegalovirus (CMV) infections remain a serious problem in hematopoietic stem cell transplant patients. Following transplantation CMV infections can cause peumonititis, hepatitis, enteritis, and marrow failure. CMV seropositive transplant recipients can be treated with antiviral agents such as ganciclovir at the onset of infection or at the time of stem cell engraphment, but ganciclovir therapy is associated with renal toxicity and suppression of neutrophil counts. Preliminary studies have found that adoptive immunetherapy using CMV-reactive cytoxic T lymphocytes (CTL) may be an effective and less toxic alternative to prevent CMV infection in seropositive recipients of marrow transplants. The purpose of this study is to develop new treatment strategies for producing CMV-reactive CTLs that can be used for adoptive immunetherapy and to better understand the cellular immune response to CMV. CMV contains over 200 proteins, but several investigators have shown that the CTLs in most HLA-A*0201 CMV seropositive healthy donors reacted most intensely with a single CMV protein, pp65 a lower matrix protein. Furthermore within pp65, HLA-A*0201 CTLs from most people recognize only a single peptide, a nanomer pp65 495-503. Currently, most protocols being developed to treat CMV with donor-derived CTLs are using pp65 or pp65 495-503 to expand CMV-reactive CTLs from HLA-A*0201 donors in vitro. We used peripheral blood mononuclear cells collected by apheresis from HLA-A*0201 CMV seropositive donors and cell culture techniques to confirm that pp65 495-503 can be used to expand CMV reactive CTLs. We also developed a method to detect the activation of CMV-reactive CTL by CMV peptides without using long-term cultures. This technique detects lymphocyte activation by measuring interferon gamma production using quantitative real time PCR. Within 3 to 6 hours of adding CMV pp65 495-503 to lymphocytes from CMV seropositive HLA-A*0201 donors, elevations in interferon-g mRNA levels can be detected using real time quantitative PCR. This technique allows the rapid assessment of other peptides within CMV protein pp65 for their ability to stimulated lymphocytes from seropositive donors with HLA-A*0201 genotypes and other HLA types. This technique has been used to identify a new CMV pp65 epitope recognized by HLA-A*24 CTLs. This technique is also being use to identify epitopes within other CMV proteins recognized by CTLs.
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