Figure 4. Characterization and cloning of MCPyV-specific CD4 TIL from an MCC patient.
(A) MCC tumor from subject w347. From Left: Primary tumor arising on forearm; MCPyV T-Ag expression (CM2B4); CD4 and FoxP3 (236A/E7 clone) staining. Scale bar = 200μm. (B) Identification of IFN-γ CD4 T-cell responses to CT domain peptides using ICC. From left: TIL stimulated with DMSO; CT pool 1; representative non-reactive pool 1 peptide; reactive pool 1 peptide CT-57-69. PBMC were used as APCs. Gating as in Figure 2. (C) TIL cytokine response to MCPyV peptide or its homologs as measured by ELISA. Left: Bulk CD4 TIL IFN-γ response to CT-57-69 or homologous peptides from the indicated polyomaviruses. Right: Bulk CD4 TIL secretes IL-5 and IL-10. Error bars represent standard error between triplicates. (D) CD4 clone isolated from primary tumor TIL secretes IFN-γ and IL-10 in response to CT-57-69. Data represent single measurements at two time points. (E) CD4 TIL from recurrent lesion recognize the same peptide as TIL from primary lesion. From left: Recurrent w347 MCC (red outline); representative non-reactive T-cell clone; T-cell clone reactive to peptide CT-57-69.