Generation of cytotoxic‐T‐lymphocyte (CTL) responses against mutated ras peptides from peripheral‐blood mononuclear cells (PBMC) was attempted in a group of HLA‐A2.1+ healthy donors. Bulk PBMC cultures were stimulated in vitro with a mixture of peptides encompassing 12 Gly → Val, 61 Gln → Lys or 61 Gln → Leu ras mutations and displaying HLA‐A2.1 binding motifs, selected by a computer program. A promiscuous tetanus toxoid peptide was also added. Weekly thereafter, PBMC were re‐stimulated with peptide pulsed autologous Epstein‐Barr‐virus (EBV)‐transformed B cells. After 8 rounds of re‐stimulation, reproducible cytotoxic activity against peptide‐pulsed target cells was detectable in one donor. The CTL line recognized 2 nonamers encompassing ras 61 Gln → Leu mutation. Killing was mediated by CD8+ T cells displaying αβ TCR and was inhibited by anti‐HLA‐A2.1 monoclonal antibodies. No killing of tumor cells expressing the specific mutation could be observed. More than 60 CTL clones were generated. Fine specificity studies revealed effective, though differing cytotoxic activity against both 53‐LDILDTAGL‐61 and 55‐ILDTAGLEE‐63, but not against 54‐DILDTAGLE‐62 mutated peptides, in all but one of the clones. None was able to exert effective cytotoxic activity against tumor cells expressing the specific mutation. T‐cell‐receptor (TCR) usage was then analyzed phenotypically, by reverse‐transcription‐polymerase‐chain‐reaction (RT‐PCR) and by sequence analysis. This study revealed the monoclonal nature of the CTL response against mutated nonamers, with TCR expressing Vβ14 gene product in combination with, Jβ2.7 and Cβ2. © 1996 Wiley‐Liss, Inc.

Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).

For a better understanding of the processes that lead to the activation or inhibition of type VI collagen synthesis, we have identified and characterized the cis-acting elements of the chicken α2 (VI) collagen promoter. This promoter encompasses four sites, termed S1, S2, X and S3, which interact with nuclear factors. Sites S1, S2 and S3 are each recognized by the same two proteins that belong to the Sp 1 multigene family. Site X appears to interact with a novel transcription factor of 43 kDa. When a fragment containing all four of the elements is placed in front of a reporter gene, the resulting construct is able to induce a high level of transcription in chicken fibroblasts. As soon as a single element is deleted from this construct, the activity decreases drastically. Thus, the four elements are essential for the transcriptional activation of the α2 (VI) collagen gene.