Monitoring CD4⁺ T cell responses against viral and tumor antigens using T cells as novel target APC

CD4⁺ T cells play an important role in the induction and maintenance of an effective antiviral and antitumor immune response. However, standardized monitoring of antigen-specific CD4⁺ T cells has not been established at the single-cell level. We now present a sensitive, specific, and simple methodology in which purified memory CD4⁺ T cells are expanded from PBMC in a single cycle of antigen-driven stimulation and quantitatively assayed by interferon-γ ELISPOT. Issues of nonspecific background in assays were resolved with the use of innovative target cells, autologous PHA-expanded CD4⁺ T cells (T-APC). Remarkably, T-APC could not only present peptide epitopes from model antigens NY-ESO-1 and influenza nucleoprotein, but could also process full-length antigen endogenously expressed from recombinant fowlpox vector. This approach makes it possible to monitor CD4⁺ T cells in large series of patients, regardless of HLA haplotype, against the full peptide repertoire of a given antigen.