Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific cytotoxic T cell (CTL) clones can restore immunity in allogeneic stem cell transplant recipients, providing protection against CMV disease. Current methods for selecting and expanding CMV-specific T cell clones are technically difficult, making adoptive T cell therapy impractical for routine clinical use. In this study, we describe a method for ex vivo generation and expansion of high-purity CMV-specific CTL using peptide-pulsed dendritic cells as antigen-presenting cells. Generation of CMV-specific CTL in numbers sufficient for clinical use in the time span of 4 weeks was accomplished in 6 of 8 CMV-seropositive donors. Examination of pp65 specificity by HLA/peptide tetramer staining demonstrated that a purity of greater than 95% peptide-specific cells could be obtained after two weekly stimulations and retained after further expansion for 3-4 weeks. Median expansion of total cell number was greater than 500-fold and expansion of peptide-specific CTL by tetramer staining was greater than 1.7 x 10(5)-fold. Four weeks after initiating CTL culture, we were able to generate greater than 10(9) total cells that specifically lysed target cells loaded with CMV peptide and cells infected with CMV. This simple and rapid method for generating high-purity CMV-specific CTL for adoptive immunotherapy is currently being examined for routine clinical use for allogeneic stem cell transplantation.
Dendritic cells have been used effectively to select for human cytomegalovirus (CMV)-specific T cells for immunotherapy applications. The ability to process and present relevant major histocompatibility complex class I and II peptides to T cells makes them ideal for selecting CD4+ and CD8+ T cells regardless of HLA tissue type. This study compared the generation of CMV-specific T cells by using dendritic cells loaded with either CMV pp65495-503 peptide or CMV lysate or transduced with adenovirus encoding the pp65 gene (Ad5pp65GFP) for the generation of CD4+ and CD8+ CMV-specific T cells in HLA-A2+ and HLA-A2 - donors. In HLA-A2+ donors, CD8+ tetramer+ T cells increased with all antigens but were greatest in peptide- and Ad5pp65GFP-stimulated T cells. The CD4+ /CD8+ ratio in the stimulated T-cell cultures proved to be dependent on the antigen used. CMV lysate-stimulated cells were primarily CD4+, whereas peptide- and Ad5pp65GFP-stimulated cultures were mostly CD8+. Analysis of cells from lysate-stimulated or gene-transduced-stimulated cultures showed expansion of CMV-specific CD4+ T cells, indicating that major histocompatibility complex class II peptides were present in both antigens. Furthermore, CMV-specific T cells were generated from HLA-A2 - donors by using Ad5pp65GFP transduction or CMV lysate stimulation and were able to recognize a pp65 peptide restricted to the HLA-B35 allele. These data indicate that either CMV lysate or adenovirus encoding CMV antigenic genes may be useful for the generation of both CD4+ and CD8+ CMV-specific T cells in donors irrespective of HLA tissue type and may be applicable to clinical immunotherapy.
Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I-and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLAidentical siblings. CD4 þ clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-g in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferong after melanoma stimulation. No CD4 þ clones responded to stimulation with recipient haemopoietic cells. The majority of CD8 þ clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4 þ clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma.
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