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

  • Original Articles
  • Cytotoxic T Lymphocytes, Human Melanoma, T Cell Receptor, Active Specific Immunotherapy
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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 lines, 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) Vα and Vβ 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 Vα17/Vβ7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the Vα17/Vβ7 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 αβ dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8+ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.

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Authors and Affiliations

  1. Department of Pathology, University of Southern California School of Medicine, Norris 710, 2025 Zonal Avenue, Los Angeles, Calif., USA

    June Kan-Mitchell, Xiu Qing Huang & John W. Parker

  2. Department of Medicine, University of Southern California School of Medicine, Los Angeles, Calif., USA

    William Harel & Malcolm S. Mitchell

  3. Department of Neurology and Pediatries, Stanford University, Stanford, Calif., USA

    Lawrence Steinman & Jorge R. Oksenberg

  4. Department of Surgery, Duke University Medical Center, Durham, N. C., USA

    Peter S. Goedegebuure & Timothy L. Darrow

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  1. June Kan-Mitchell
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  2. Xiu Qing Huang
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  3. Lawrence Steinman
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  4. Jorge R. Oksenberg
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  5. William Harel
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  7. Peter S. Goedegebuure
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  8. Timothy L. Darrow
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  9. Malcolm S. Mitchell
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Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation

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Kan-Mitchell, J., Huang, X.Q., Steinman, L. et al. Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy. Cancer Immunol Immunother 37, 15–25 (1993). https://doi.org/10.1007/BF01516937

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  • DOI: https://doi.org/10.1007/BF01516937

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