Clonal analysis of cytotoxic T‐lymphocyte response to autologous human metastatic melanoma

Abstract

Peripheral blood lymphocytes (PBL) from a melanoma (Me) patient, previously shown to be unable to react against the autologous tumor (Me 28) after mixed lymphocyte-tumor culture (MLTC), were cultured in vitro with the autologous tumor in MLTC and/or with IL-2-containing supernatants. T-cell clones were then obtained by limiting dilution and by micromanipulation. Eleven clones, selected for autologous tumor (Auto-Tu) cytotoxicity, were tested for specificity on a panel of 17 cell cultures of normal and neoplastic origin, revealing a complex spectrum of lytic activities. Three groups of clones could be identified depending on the patterns of cytotoxicity. One clone (B11.12) lysed Me28 and expressed a borderline reactivity against one allogeneic Me. A second group of clones (A4, A4.18, H10, E12, C9) lysed the Auto-Tu and allogeneic Me. The last group of clones (A4.2, A4.3, A4.4, A7, B7) expressed a broader pattern of reactivity with significant cytotoxicity against targets of different histologic origin. Furthermore, the second and third groups of clones lysed K562 while B11.12 did not. The Auto-Tu-restricted reactivity of clone B11.12, confirmed by a further test on 13 allogeneic Me and on autologous IL-2 cultured lymphocytes, suggests the recognition of antigenic structures preferentially expressed on Me28. Blocking studies, performed with monoclonal antibodies (MAb), revealed that an anti-HLA class 1 MAb (w6/32), but not two anti-DR MAbs (L243, D1.12), could inhibit the cytotoxic activity of clones B11.12 on Me28. A significant blocking effect by w6/32 on Me28 was achieved also with clones A4.4 and H10 but not with clones A4.2, A4.3 and A7. The phenotype of all clones was T3⁺, T4⁻, T8⁺, HNK-1⁻, suggesting that the anti-tumor effectors were of the T-cell lineage. Taken together, these data indicate that it is possible to isolate anti-tumor CTL-clones after MLTC from a PBL population of a metastatic melanoma patient. Furthermore, we present evidence suggesting a role of class-1 antigens in the interaction of some cloned effectors with the autologous tumor target.