8513449

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1

To study in vivo activated cytolytic T cells, CD8+ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8+ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8+ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell line, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8+ and 2 CD4+ CD8+ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V alpha and V beta gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V alpha 17/V beta 7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V alpha 17/V beta 7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8+ CTL. CTL with a range of melanoma specificities and different TCR alpha beta dimers were encountered in this patient, perhaps as a result of hyperimmunization.(ABSTRACT TRUNCATED AT 400 WORDS)

eng

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MEDLINE

0340-7004

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15-25

pubmed-meshheading:8513449-Adult, pubmed-meshheading:8513449-Antigens, CD, pubmed-meshheading:8513449-Antigens, CD8, pubmed-meshheading:8513449-Base Sequence, pubmed-meshheading:8513449-Cell Separation, pubmed-meshheading:8513449-Clone Cells, pubmed-meshheading:8513449-Cytotoxicity, Immunologic, pubmed-meshheading:8513449-DNA, Neoplasm, pubmed-meshheading:8513449-Female, pubmed-meshheading:8513449-Flow Cytometry, pubmed-meshheading:8513449-Histocompatibility Antigens Class I, pubmed-meshheading:8513449-Histocompatibility Antigens Class II, pubmed-meshheading:8513449-Humans, pubmed-meshheading:8513449-Immunophenotyping, pubmed-meshheading:8513449-Immunotherapy, Active, pubmed-meshheading:8513449-Melanoma, pubmed-meshheading:8513449-Molecular Sequence Data, pubmed-meshheading:8513449-Receptors, Antigen, T-Cell, alpha-beta, pubmed-meshheading:8513449-T-Lymphocytes, Cytotoxic, pubmed-meshheading:8513449-Tumor Cells, Cultured, pubmed-meshheading:8513449-Vaccines, Synthetic

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy.

Journal Article, Research Support, U.S. Gov't, P.H.S., Case Reports, Research Support, Non-U.S. Gov't