SUMMARYMelanoma-speci®c cytotoxic T lymphocytes (CTL) can be generated from peripheral blood lymphocytes (PBL) by mixed lymphocyte±tumour cell cultures. Analysis of CTL precursor frequencies in peripheral blood of melanoma patients is generally used for immunomonitoring purposes to evaluate vaccination ef®cacy. At present, it is unclear whether PBL-derived CTL generated in vitro are indicative of an anti-tumour immune response in vivo. Three tumour-speci®c human leucocyte antigen (HLA)-B/C-restricted CTL clones were derived from peripheral blood of a melanoma patient immunized with interleukin-7 (IL-7) gene-modi®ed tumour cells. CTL clones differing in their T-cell receptor-c (TCRc) rearrangement produced interferon-c, IL-4 and/or IL-10. On the basis of their unique TCRc gene rearrangements clone-speci®c primers were generated for detection of clone-speci®c DNA by polymerase chain reaction. One CTL clone (E5) of the three was found to be selectively expanded in one of seven metastases obtained at autopsy, as determined by Southern blot hybridization. However, the presence of E5 in only one of seven metastases at death indicates that the in vivo accumulation of the speci®c CTL clone was not suf®cient to contain tumour progression. Nevertheless, our data support the proposition that analysis of anti-tumour activity of PBL-derived CTLs may re¯ect an anti-tumour immune response in vivo.
A phase I clinical trial using autologous, IL -7 gene -modified tumor cells in patients with disseminated melanoma has been recently completed. Although no major clinical responses were observed, increased antitumor cytotoxicity was measured in postvaccine peripheral blood lymphocytes in a subset of treated patients. To analyze the in situ immune response, the T cell receptor -chain variable region ( BV ) repertoire of T cells infiltrating postvaccine lesions was studied in two patients, and compared with that of T cells present in prevaccine ones, in peripheral blood lymphocytes, and, in one patient, in delayed type hypersensitivity ( DTH ) sites of autologous melanoma inoculum. A relative expansion of T cells expressing few BVs was observed in all postvaccine metastases, and their intratumoral presence was confirmed by immunohistochemistry. Length pattern analysis of the complementarity determining region 3 ( CDR3 ) indicated that the repertoire of T cells expressing some of these BVs was heterogeneous. At difference, CDR3, -chain joining region usage, and sequence analysis enabled us to demonstrate, within a T -cell subpopulation commonly expanded at DTH sites and at the postvaccine lesion of patient 1, that both DTH sites contained identical dominant T -cell clonotypes. One of them was also expressed at increased relative frequency in the postvaccine lesion compared to prevaccine specimens. These results provide evidence for immunological changes, including in situ clonally expanded T cells, in metastases of patients vaccinated with IL -7 gene -transduced cells.
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