Western Blot Analysis of Antigens on Melanoma Cells Recognized by Cytotoxic T Cells

Abstract

Antigens recognized by cloned cytotoxic T lymphocytes (CTLs) from patients with melanoma were examined by methods based on the ability of antigens immobilized on nitrocellulose paper to stimulate proliferation of the CTLs. The proliferative response depended on the presence of histocompatible antigen-presenting cells (APCs) in the cultures in the form of either autologous lymphoid cell Lines (Epstein-Barr virus-transforrned B cells) or histocompatible peripheral blood lymphocytes and was maximal at 3 days. Presentation appeared to be via class II major histocormpatibility complex antigens, in that monoclonal antibodies (MAbs) against the class II antigens, but not the class I antigens, on the APCs inhibited the proliferative responses. The response the CTLs appeared to be mediated by interaction with the αβ3 CD3 T-cell receptor complex, in that pretreatment of the CTL clones with MAbs against CD3 inhibited the response of these clones to the antigen extracts irrespective of the phenotypes of the clones. Extracts from several nonmelanoma cells did not stimulate CTL clones specific for melanoma. At least two different specificities were detected in extracts from autologous and allogeneic tumor cells. The specificity of proliferative responses by CD3+ CD4+ and CD3+ CD8+ CTLs appeared to be similar to their cytotoxic activity, but CTLs with the CD3+ CD16+ CD8+− phenotype had wider cytotoxic activity against target cells not stimulating proliferative responses. The antigen(s) responsible for the stimulation were shown in all instances to have a mdecular mass of 48 kilodaltons. Preliminary anatysis suggested that the antigen(s) have both protein and glycolipid (ganglioside) components.