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

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.

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