CLONAL ANALYSIS OF INVIVO ACTIVATED CD8-LYMPHOCYTES FROM A MELANOMA PATIENT RESPONSIVE TO ACTIVE SPECIFIC IMMUNOTHERAPY( CYTOTOXIC T)

To study in vivo activated cytolytic T cells, CD8+ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active sp ecific immunotherapy for 5 years. CD8+ T lymphocytes, purified by fluo rescence-activated cell sorting, were cloned directly from the periphe ral blood without antigen-presenting cells in the presence of irradiat ed autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunizatio n. Of the 28 cytolytic CD8+ T cell clones, 21 lysed the autologous mel anoma 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 m elanoma-specific, although their reactivities were broader, lysing sev eral melanoma cell lines but not HLA-matched lymphoblastoid cells. Eig ht clones from the first group, ostensibly self-MHC-restricted, were e xpanded for further analysis. All expressed cluster determinants chara cteristic of mature, activated T cells, but not those of thymocytes, n aive T cells, B cells or natural killer (NK) cells. They also expresse d CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD 8, but dual expression was not correlated with specificity of lysis. T wo CD8+ and 2 CD4+ CD8+ clones were specific for the autologous melano ma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones wa s restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) Valpha and Vbeta gene seg ments revealed heterogeneous usage by the A2-restricted clones and, pe rhaps, also by the broadly melanoma-specific clones. Apparent TCR-rest ricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the Valpha17/Vbeta7 dimer. Since the T cell clones were deri ved from separate precursors of circulating cytotoxic T lymphocytes (C TL), the Valpha17/Vbeta7 TCR was well represented in the peripheral bl ood lymphocytes of this patient. In summary, we show that melanoma cel ls presented their own antigens to stimulate the proliferation of mela noma-reactive CD8+ CTL. CTL with a range of melanoma specificities and different TCR alphabeta dimers were encountered in this patient, perh aps as a result of hyperimmunization. Restricted TCR gene usage was no ted only for classical self-MHC-restricted CD8+ T cell clones, althoug h lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-cha racterized T cell clones in this and other patients should provide new insight into the human anti-tumor immune response.