Simultaneous Identification of Functional Antigen-Specific CD8⁺ and CD4⁺ Cells after In Vitro Expansion Using Elongated Peptides

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8⁺ and CD4⁺ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8⁺ T cells was dependent on the elongation site of the EP, whereas CD4⁺ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8⁺ and CD4⁺ T cell reactivities.