Specificity of protein-specific CD4 T-cell lines raised against KLH or human Epo. Expanded unsorted CD4 T cells were obtained after repeated weekly stimulations of CD4 T lymphocytes purified from healthy donors by autologous mature DCs loaded with KLH or Epo. Reactivity of all the CD4 T-cell lines was assessed by interferon-γ ELISpot assay in the presence and in absence of KLH or Epo. Briefly 10⁴ CD4 T cells were incubated in triplicate with 5 × 10³ autologous unloaded DCs or DCs loaded with the appropriate antigen. Spot staining was done after 24-hour incubation at 37°C. (A) Examples of ELISpot wells from 2 Epo-specific T-cell lines. (B) KLH-specific T-cell lines of donor #483 and Epo-specific T-cell lines derived in this study. *P < .05. †P < .005. ‡P < .0005.