Ex Vivo Monitoring of Antigen-Specific CD4+T Cells after Recall Immunization with Tetanus Toxoid

To monitor antigen-specific CD4+T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4+T cells, was performed. Grossly, twice as many TT-specific CD4+T-cell clones, ex vivo derived from the CCR7+/−CD69+interleukin-2-positive (IL-2+) CD4+subsets, belonged to the central memory (TCM; CD62L+CD27+CCR7+) compared to the effector memory population (TEM; CD62L−CD27−CCR7−). After the boost, a predominant expansion of the TCMpopulation was observed with more limited variations of the TEMpopulation. TCR beta-chain-variable region (BV) spectratyping and sequencing confirmed a large concordance between most frequently expressed BV TCR-CDR3 from ex vivo-sorted CCR7+/−CD69+IL-2+CD4+subsets and BV usage of in vitro-derived TT-specific CD4+T-cell clones, further demonstrating the highly polyclonal but stable character of the specific recall response to TT. Taken together, ex vivo flow cytometry analysis focused on the CCR7+/−CD69+IL-2+CD4+subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.