In vivoaccumulation of the same anti-melanoma T cell clone in two different metastatic sites

Abstract

In a patient with progressing metastatic melanoma, we showed that the same autologous tumorcytolytic CD8 ؉ tumor infiltrating lymphocyte (TIL) clone accumulated in two separate metastatic sites. This clone, which represented three of eight independently derived clones from a tumor deposit on the skin of the abdomen, also represented two of eight clones derived from a skin lesion on the shoulder. This clone could be identified by its use of a unique TCRBV2-nD1n-J1S6 sequence, and could also be detected by single-stranded conformational polymorphism (SSCP) as the dominant TCRBV2-expressing clone among CD8 ؉ TILs propagated from both shoulder and abdominal lesions. Using SSCP analysis, we also demonstrated that this clone was dominant in the fresh tumor tissue and in all TILs in which CD8 ؉ were strongly represented, including several separate but parallel cultures. The SSCP pattern for this clone was not apparent among CD4 ؉ TILs or CD8؉ peripheral blood mononuclear cells. The SSCP analysis of the tumor tissue prior to in vitro culture is an indication that the selection for this anti-tumor cytotoxic T cell clone was a ref lection of its in vivo accumulation. Thus, we provide evidence that melanomas are immunogenic and able to select for cytotoxic antitumor-specific TIL clones that are expanded in vivo and can circulate to accumulate in different tumor sites. However, because these clones were isolated from progressing tumor metastases, the accumulation of these specific cytotoxic T cells was not sufficient to contain tumor growth.It has been shown that the immune response to human melanoma includes cytotoxic T lymphocytes capable of lysing autologous tumor cells in vitro (1-15). Some of the melanoma tumor antigens recognized have been identified and cloned (16)(17)(18)(19)(20)(21)(22). Despite the presence of these cytotoxic lymphocytes, it is evident that the immune response fails to eradicate clinically apparent tumors. Several mechanisms have been proposed to explain tumor escape from immune surveillance, including the loss of HLA alleles needed for T cell recognition (23, 24), as well as other cell surface molecules necessary for induction of T cell activation and proliferation (25).Several laboratories have shown that the tumor infiltrating lymphocytes (TILs) propagated from human melanoma often show restricted T cell antigen receptor (TCR) usage (1,4,12,(26)(27)(28)(29)(30)(31)(32)(33)(34). In some cases it has also been possible to demonstrate that the dominant T cells among the TILs are able to lyse autologous tumors (3,12,35). Still, an unresolved concern has been the lack of unequivocal evidence that the dominant clones isolated in vitro were representative of the in vivo accumulation of these cells within the tumor, rather than a reflection of an in vitro selection. By comparing the TCR usage among the freshly isolated TILs with the TCR which could be sequenced from anti-tumor cytotoxic clones, we addressed the question of whether significant accumulations of melanoma-specific cytotoxic T cells...

Selective Growth, In Vitro and In Vivo, of Individual T Cell Clones from Tumor-Infiltrating Lymphocytes Obtained from Patients with Melanoma

“…In these reports, however, the tumor reactivity of dominantspecific TRBV-expressing T cells was not evaluated. Singlestranded conformational polymorphism analysis indicated that a TCR V␤2-expressing antitumor cytotoxic T cell clone was dominant in the fresh tumor tissue, but these cells were incapable of preventing tumor cell progression (22). RT-PCR analysis conducted using TRBV primers capable of amplifying each of the individual TRBV genes indicated that relatively few TRBV-gene families were expressed at significant levels in both the regressive and progressive regions of the same primary human malignant melanoma (23).…”

Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes

“…For example, our observation of a predominant tumour-specific cytotoxic T lymphocyte (CTL) cell clone in multiple metastatic lesions from the same patient indicated that such cells were able to propagate, circulate, home and accumulate within tumour deposits. However, these tumour-specific CTL clones were found in a patient with progressive melanoma, indicating that they were not sufficient to contain tumour growth [32]. Thus, the discrepancy between a demonstrated in vitro tumour lytic activity, and the in vivo tumour progression in the presence of this lymphoid infiltrate, remains to be clarified.…”

A Novel Autocrine Pathway of Tumor Escape from Immune Recognition: Melanoma Cell Lines Produce a Soluble Protein That Diminishes Expression of the Gene Encoding the Melanocyte Lineage Melan-A/MART-1 Antigen Through Down-Modulation of Its Promoter

“…Melanoma cells and TIL from the melanoma tissues were obtained according to approved Massachusetts General Hospital guidelines, and propagated in vitro, as previously described (1). Briefly, tumors were cultured in DMEM medium supplemented with 10% FBS, and TIL were propagated in RPMI 1640 supplemented with 5% human serum containing human rIL-2 at 100 U/ml (Cetus, Emoryville, CA).…”

“…T he cellular immune response against human melanomas is often characterized by the accumulation of tumor-infiltrating lymphocytes (TIL), 2 which include tumor-specific cytotoxic T cells (1)(2)(3). In some patients, TIL show strong in vitro lytic activity that is often directed against HLA-A2 ϩ targets presenting the immunodominant peptide of the melanocyte lineage Ag Melan-A/MART-1 (aa 27-35; AAGIGILTV) (4 -7).…”