Volume 43, November 2003 TRANSFUSION 1567 Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322003 American Association of Blood BanksNovember 20034311Original Article A UNIQUE PEPTIDE pp65341-350 FOR HLA-A24/A1PROVENZANO ET AL. ABBREVIATIONS: BLC = B-lymphoblastoid cell; CTL = cytotoxic T lymphocyte; DC = dendritic cell; E:T = effector:target; PBL = peripheral blood lymphocytes; pp65 = phosphorylated matrix protein 65; qRT-PCR = quantitative real-time PCR. From the Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland; and the Surgery Branch, Department of Oncological and Surgical Science, University of Padova, Italy. Address reprint requests to: Maurizio Provenzano, MD, Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Building 10/1C711, Bethesda, MD 20892; e-mail: mprovenzano@mail.cc.nih.gov. Received for publication March 25, 2003; revision received June 13, 2003, and accepted June 23, 2003. TRANSFUSION 2003;43:1567-1574. TRANSPLANTATION AND CELLULAR ENGINEERING The matrix protein pp65 341-350 : a peptide that induces ex vivo stimulation and in vitro expansion of CMV-specific CD8+ T cells in subjects bearing either HLA-A*2402 or A*0101 allele Maurizio Provenzano, Jong-Baeck Lim, Simone Mocellin, Vladia Monsurro, Maria Bettinotti, Francesco M. Marincola, and David F. Stroncek BACKGROUND: The stimulation of PBMNCs with HLA Class I restricted synthetic peptides derived from CMV phosphorylated matrix protein 65 (pp65) evokes CMV- specific cytotoxic T lymphocyte (CTL) activity, a neces- sary condition for initiating adoptive immunotherapy against CMV-related diseases in immune-compromised patients. It was previously demonstrated that the CMV decamer (10-mer) peptide pp65 341-350, QYDPVAALFF, was able to induce CMV-specific CTLs in HLA-A*2402 CMV- seropositive individuals. STUDY DESIGN AND METHOD: We investigated the ability of the peptide pp65 341-350 to reactivate memory CD8+ T cells in CMV-seropositive subjects bearing either the HLA-A24 or A1 allele. CTL responses were measured by IFN-g mRNA expression and IFN-g protein production as well as cytotoxic activity. RESULTS: In this study it was found that peptide pp65 341-350 induced a specific reactivation of memory CD8+ T cells from CMV-seropositive donors expressing either HLA-A*2402 and/or HLA-A*0101. Moreover, a pp65 341-350 -specific selection and expansion using PBMNCs of CMV-seropositive donors bearing both HLA- A*2402 and HLA-A*0101 alleles produced cytotoxic CTLs to both HLA-A24 and A1 peptide-pulsed and autologous CMV-infected target cells. CONCLUSION: The results demonstrate that pp65 341-350 induced a specific CTL activity at both molecular and protein levels and that the peptide is specifically pro- cessed, presented, and recognized by subjects bearing HLA-A*2402 and/or A*0101. These findings suggest that it may be possible to use this single immune dominant peptide to induce and expand CMV-reactive CTLs for the treatment of individuals with both HLA-A24 and A1 types. he development of a protective cellular immune response against CMV is the most important determinant of recovery from CMV infection after allogeneic PBPC or marrow and solid-organ transplantation. 1 There is a growing interest in developing adoptive therapies using peptide-stimulated CMV- specific cytotoxic T lymphocytes (CTLs) to prevent CMV disease in immune-compromised hosts. 2,3 Among the 200 proteins encoded by CMV, phosphorylated matrix protein 65 (pp65) is the most immunogenic. 4 The identification of new candidate HLA Class I restricted peptide sequences within this protein is important for vaccination, for adop- tive immune therapy, and for monitoring of patients’ immune response. Grefte et al. first demonstrated that, more than any other protein, CMV pp65 is involved in CTL responses to CMV-infected cells. They found that 15-mer regions within this protein are capable of stimulating CMV-specific reactive CTLs through HLA Class I path- ways. 5 After CMV-specific CTL activity in HLA-A*0201 individuals was found to be caused by a single peptide, pp65 495-503 , 6 many other investigators began to study HLA- T