Figure 2. CD8+ TIL isolated from an MCC patient specifically recognize an MCPyV-derived peptide and endogenously processed large T-Ag (LT).
(A) Serial sections from w447 MCC tumor. From left: H&E stain; T-Ag expression (CM2B4 antibody); CD8 lymphocyte staining; HLA-I staining (EMR8-5 clone). Scale bar = 200μm. (B) CD8 IFN-γ response to LT-derived peptides in an ICC assay. From left: TIL stimulated with DMSO; pool 2; 13-mer peptide LT-89-101 from pool 2; and minimal 10-mer epitope LT-92-101. Autologous PBMC were used as APCs. (C) TIL have high functional avidity for peptide LT-92-101. (D) TIL recognize MCPyV LT-89-101 but not homologous peptides from indicated polyomaviruses. Y-axis represents flow cytometry data gated as in 2B. (E) Western blot primary bands after probe with anti-MCPyV LT (CM2B4) or anti-alpha-actin loading control in MKL-1 (MCPyV positive MCC cell line) and Cos7 cells transfected as indicated. (*) and (**) indicate distinct truncation isoforms. (F) Flow cytometry analysis of HLA-A24 expression on Cos7 cells transfected as indicated. Data represent percent cells above PE-fluorescence intensity threshold. (G) TIL IFN-γ responses to Cos7 cells transfected as indicated. Numbers shown in plots are the percentage of CD8-gated TIL.