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

Abstract

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)

• Authors: J Kan-Mitchell, X Q Huang, L Steinman, J R Oksenberg, W Harel, J W Parker, P S Goedegebuure, T L Darrow, M S Mitchell
• Journal: Cancer immunology, immunotherapy : CII (Cancer Immunol Immunother) Vol. 37 Issue 1 Pg. 15-25 (Jul 1993) ISSN: 0340-7004 [Print] Germany
• PMID: 8513449 (Publication Type: Case Reports, Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antigens, CD
• CD8 Antigens
• DNA, Neoplasm
• Histocompatibility Antigens Class I
• Histocompatibility Antigens Class II
• Receptors, Antigen, T-Cell, alpha-beta
• Vaccines, Synthetic

Topics

• Adult
• Antigens, CD (analysis)
• Base Sequence
• CD8 Antigens (immunology)
• Cell Separation
• Clone Cells
• Cytotoxicity, Immunologic
• DNA, Neoplasm (analysis, chemistry)
• Female
• Flow Cytometry
• Histocompatibility Antigens Class I (analysis)
• Histocompatibility Antigens Class II (analysis)
• Humans
• Immunophenotyping
• Immunotherapy, Active
• Melanoma (immunology, therapy)
• Molecular Sequence Data
• Receptors, Antigen, T-Cell, alpha-beta (genetics)
• T-Lymphocytes, Cytotoxic (immunology)
• Tumor Cells, Cultured
• Vaccines, Synthetic (therapeutic use)