Comprehensive analysis of the frequency of recognition of melanoma-associated antigen (MAA) by CD8 melanoma infiltrating lymphocytes (TIL): implications for immunotherapy

Abstract

Fifty-nine tumor-infiltrating lymphocyte (TIL) cultures established from melanoma-invaded lymph nodes were screened for recognition of 28 melanoma-associated antigens (MAA) in association with31 HLA molecules. Twenty-three (39%) TIL lines reacted to at least one melanoma antigen. Melanosomal proteins were recognized by 19 TIL populations and the most prominent responses against these proteins were directed against Melan-A/MART-1 (mainly in association with HLA-A*0201) and gp100 (in association with diverse HLA contexts). Ten TIL populations reacted against 10 tumor-specific antigens, in association with 8 different HLA molecules. HLA-A*0201 and B*3501-restricted responses were the most frequent with, respectively, 17 and 7 responses directed against 5 distinct antigens. Unexpectedly, the recognition by TIL of different MAA was frequently restricted by a single HLA in individual tumors, and there was no evidence for the existence of dominant MAA epitopes between tumors,except for Melan-A/MART-1 antigen. This analysis also led to the detection of 21 new HLA-peptide complexes recognized by melanoma TIL. This study, which is to our knowledge the most comprehensive analysis of TIL specificity to tumor antigens, has several implications for the design of immunotherapeutic strategies based on immunization against selected tumor epitopes.

Identification of Five New HLA-B*3501-Restricted Epitopes Derived from Common Melanoma-Associated Antigens, Spontaneously Recognized by Tumor-Infiltrating Lymphocytes

“…Melanoma TIL, specific for Melan-A, gp100, tyrosinase, NY-ESO-1, and MAGE-A3/A6 Ags in the HLA-B35 context, had been evidenced in a previous study (11). We now used TIL-derived clones to identify the recognized epitopes.…”

“…Mouse fibrosarcoma WEHI 164 clone 13, used for TNF production assays and COS-7 cells were obtained from T. Boon (Ludwig Institute for Cancer Research (LICR), Brussels, Belgium). COS-7 cells, WEHI 164 clone 13, and melanoma cells lines were cultured, as described before (11). The EBV-B-transformed cell line LAZ 338 was a gift from T. Hercend (Vertex Pharmaceutical, Abingdon, U.K.).…”

“…Reverse transcription was performed, as previously described (11). PCR amplification was then performed on 50 ng of the cDNA with PCR buffer, …”

“…Through a systematic screening of the specificity of 60 melanoma tumor-infiltrating lymphocyte (TIL) populations, we reported previously the recognition of ϳ20 new HLA-class I/peptide complexes, among which 6 were restricted by the HLA-B*3501 allele (Melan-A/MART-1, tyrosinase, gp-100, NY-ESO-1, and MAGE-A3/A6) (11). Because this HLA is one of the most frequently expressed HLA-B alleles (12), we sought to identify the B*3501-restricted epitopes recognized by these TIL populations and to look for the efficacy of their presentation by HLA-B35 melanoma cells.…”

Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTL

“…From the same biopsy, a CTL clone (M4.48) was isolated from a tumor-infiltrating lymphocyte population by limiting dilution, as described previously (16). Abs and reagents to the following human targets were used: anti-HLA class I (W6/32) and anti-HLA-A2.1 (MA2.1) mAb (produced in our laboratory); Von Willebrand factor and angiotensin-converting enzyme (Tebu-bio); Melan-A/MART-1 Ab (DakoCytomation); CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) and Orange 5-(and -6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) probes (Molecular Probes); calcein-acetoxymethyl ester (AM) (used at 5 g/ml) (Molecular Probes); Alexa Fluor 488 goat anti-mouse IgG (H and L chains) F(abЈ) 2 and Alexa Fluor 633 goat anti-rabbit IgG (H and L chains) F(abЈ) 2 (Invitrogen); antiphosphotyrosine 4G10 clone (Upstate Biotechnology); FITC-goat antimouse IgG F(abЈ) 2 (Beckman Coulter); anti-connexin-43 (Sigma-Aldrich),18␣-glycyrrhetinic acid (18GA; Sigma-Aldrich); and InSolution Epoxomicin (Calbiochem).…”