Peptide‐specific ctl in tumor‐infiltrating lymphocytes from metastatic melanomas expressing mart‐1/melan‐a, gp100 and tyrosinase genes: A study in an unselected group of hla‐a2.1‐positive patients

Abstract

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).

High density oligonucleotide array analysis of interferon-α2a sensitivity and transcriptional response in melanoma cells

“…1-restricted CTL lines recognizing epitopes derived from MART-1/Melan-A, pmel-17/gp 100, tyrosinase or TRP-2 proteins (Spagnoli et al, 1995;Noppen et al, 2000). In contrast, ME59 HLA-A2.1 positive cells, that did not express the genes under investigation failed to be killed by the specific CTL (Spagnoli et al, 1995). Remarkably, IFN-α treatment did not appear to influence the expression of the genes encoding these TAA.…”

“…These results were consistent with our previously published data, showing that D10, HLA-A2.1-positive, melanoma cells were effectively killed by HLA-A2. 1-restricted CTL lines recognizing epitopes derived from MART-1/Melan-A, pmel-17/gp 100, tyrosinase or TRP-2 proteins (Spagnoli et al, 1995;Noppen et al, 2000). In contrast, ME59 HLA-A2.1 positive cells, that did not express the genes under investigation failed to be killed by the specific CTL (Spagnoli et al, 1995).…”

Selective responsiveness to common gamma chain cytokines in peripheral blood‐derived cytotoxic T lymphocytes induced by Melan‐A/MART‐127–35targeted active specific immunotherapy

“…13,14 Briefly, cells were cultured in 60-well Terasaki plates (Nunc, Glostrup, Denmark) at 0.3 cells per well in 20 ml volumes, in the presence of 10,000 irradiated allogenic PBMC/well, in RPMI 1640 medium, supplemented with 1 mM sodium pyruvate, 2 mM nonessential amino-acids, 2 mM L-glutamine, 10 mM HEPES buffer and kanamycin (all from Gibco BRL, Paisley, UK) and 5% pooled human serum (RPMI-HS), to which rIL-2 (200 units/ml) and purified phytohemagglutinin (PHA, 0.5 mg/ml, Remel, Dartford, UK) were added. After 10-14 days wells where cell growth was microscopically detectable were expanded in RPMI-HS supplemented with 100 units/ml rIL-2 and screened for antigen-specific cytotoxic activity (see below).…”

“…15 The first 2 express Melan-A/MART-1 gene (HBL high expressors, D10 intermediate expressors 13 ), whereas NA8 does not express it. One thousand 51 Cr-labeled cells were added to different numbers of effector cells together with 10 5 unlabeled K562 cells (decoy target for NK-like nonspecific lytic activity).…”

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen

“…Synthetic peptides of HLA -restricted epitopes, derived from tumor-associated antigens, have been used in vitro and in vivo and have indeed demonstrated their capacity to elicit specific responses. 9,19 Modification of peptide sequences also led to the definition of more immunogenic reagents. 15,18 Recently, HLA -A201 MART-1/Melan -A 27-35 peptide analogues or the corresponding mutated complete antigen were compared using vaccinia virus expression vectors.…”