A constructed HLA-A2-restricted pMAGE-A1278–286 tetramer detects specific cytotoxic T lymphocytes in tumour tissues in situ

Abstract

Objective To construct a human leucocyte antigen (HLA)-A2-restricted peptide 278–286 of melanoma-associated antigen family A, 1 (pMAGE-A1278–286) tetramer to analyse the distribution of cytotoxic T lymphocytes (CTLs) in tumour tissue and tumour-adjacent normal tissue. Methods A HLA-A2-pMAGE-A1278–286 tetramer was constructed. The distribution of pMAGE-A1278–286-specific CTLs was investigated in tumour tissues and tumour-adjacent normal tissues from patients with hepatocellular carcinoma using in situ HLA-A2-pMAGE-A1278–286 tetramer staining. Results Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis indicated that HLA-A2 heavy and light chain proteins were successfully obtained. The successful construction of the HLA-A2-pMAGE-A1278–286 monomer was confirmed with Western blot analysis using W6/32 antibody. Flow cytometry confirmed the specific binding of HLA-A2-pMAGE-A1278–286 tetramer to pMAGE-A1278–286-specific CTLs. In situ HLA-A2-pMAGE-A1278–286 tetramer staining demonstrated that the number of pMAGE-A1278–286-specific CTLs in tumour tissues was significantly higher than in tumour-adjacent normal tissues. Conclusions The HLA-A2-pMAGE-A1278–286 tetramer was useful for the detection of pMAGE-A1278–286-specific CTLs in both tumour tissues and tumour-adjacent normal tissues. In situ tetramer staining is a powerful tool for investigating the distribution of pMAGE-A1278–286-specific CTLs in the tumour microenvironment.