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

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_CMpopulation was observed with more limited variations of the T_EMpopulation. 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.