Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy.
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)
Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033.
J
J
Kan-Mitchell
X Q
XQ
Huang
L
L
Steinman
J R
JR
Oksenberg
W
W
Harel
J W
JW
Parker
P S
PS
Goedegebuure
T L
TL
Darrow
M S
MS
Mitchell
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D001483:Base Sequence; D016827:CD8 Antigens / Q000276:immunology*; D002469:Cell Separation; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic; D004273:DNA, Neoplasm / Q000032:analysis / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping; D016233:Immunotherapy, Active; D008545:Melanoma / Q000276:immunology* / Q000628:therapy; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured; D014614:Vaccines, Synthetic / Q000627:therapeutic use
D015703:Antigens, CD; D016827:CD8 Antigens; D004273:DNA, Neoplasm; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014614:Vaccines, Synthetic