Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells

Abstract

Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells.

IL-12 stimulation but not B7 expression increases melanoma killing by patient cytotoxic T lymphocytes (CTL)

“…1c. One peripheral blood lymphocyte in 21 000 could respond to autologous Mel 30; this frequency estimation is within the range previously obtained for autologous melanoma cells (1/900 to 1/ 33 000) [6,7]. Addition of IL-12 did not increase the number of anti-melanoma CTL precursors of this patient (1/23 000 with Uninfected melanoma cells did not show any B7 expression on their surface.…”

“…We therefore optimized CTL generation by the addition of hydrocortisone [48], which decreased the generation of non-specific LAK and NK cells during in vitro, allogeneic PBL stimulation (data not shown). Furthermore, killing assays were performed in the presence of an excess of unlabelled K562 cells (a LAK-specific target) [7]. This inhibited the nonspecific killing of melanoma targets by LAK cells generated by incubation of PBL in a high concentration of IL-2 (data not shown).…”

“…Both shared and unique determinants can be recognized by such CTL in an MHCrestricted fashion, either with HLA-A1 or with HLA-A2 [5]. The finding that specific CTL precursors could be detected in the peripheral blood of most melanoma patients suggested that vaccination to stimulate an immune response could be used to treat melanoma [6,7].…”

Gene therapy study of cytokine-transfected xenogeneic cells (Vero-interleukin-2) in patients with metastatic solid tumors

“…There was also no sign of a dose response effect of any of the biological and clinical endpoints of this study. Because of the heterogenous group of patients treated in this phase I study, the absence of known tumor-specific Ags in most malignancies, and the lack of sufficient tumor material from the majority of our patients, more specific tests such as cytotoxic T-lymphocyte precursor 28 or peptide recognition assays 29 could not be performed. Although tumor response was not a primary endpoint of this study and the selection of highly pretreated patients (see Table 1) made responses to any therapy unlikely, our clinical observations deserve some comments.…”

IL2 treatment for cancer: from biology to gene therapy

“…The results so far obtained in experimental models and, more recently, also with human neoplastic cells, suggest that transfer of cytokine/growth factor genes into neoplastic cells is no longer only a theoretical consideration, but rather it represents a reality which opens potential new prospects in released by the injected cells may elicit an anti-tumour immune response possibly contributed by the generation of cytotoxic cells specifically directed against the tumour, gains further strength by the recent evidence that in the peripheral blood of melanoma patients specific anti-tumour cytotoxic lymphocyte precursors have been described (Coulie et al, 1992). It is conceivable that these cytotoxic cells may be amplified in vivo following repeated boosting with IL2-gene transduced tumour cells.…”