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

Abstract

ABSTRACT 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.