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 (T(CM); CD62L+ CD27+ CCR7+) compared to the effector memory population (T(EM); CD62L- CD27- CCR7-). After the boost, a predominant expansion of the T(CM) population was observed with more limited variations of the T(EM) population. 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.