A minority of proliferating human CD4⁺ T cells in antigen-driven proliferation assays are antigen specific

Antigen-driven T-cell proliferation is often measured using fluorescent dye dilution assays, such as the CFSE-based proliferation assay. Dye dilution assays have been powerful tools to detect human CD4⁺ T-cell responses, particularly against autoantigens. However, it is not known how many cells within the proliferating population are specific for the stimulating antigen. Here we determined the frequency of CD4⁺ T cells specific for the stimulating antigen within the antigen-responsive population of CFSE-based proliferation assays. We compared CD4⁺ T-cell responses to a type 1 diabetes autoantigen (proinsulin C-peptide) and to a vaccine antigen (tetanus toxoid). The TCRs expressed by antigen-responsive CD4⁺ T cells were sequenced, and their antigen specificity was tested functionally by expressing them in a reporter T-cell line. Responses to C-peptide were weak, but detectable, in PBMC from individuals with T1D, whereas responses to tetanus toxoid were much stronger. The frequency of antigen-specific CD4⁺ T cells correlated with the strength of the response to antigen in the proliferation assay. However, antigen-specific CD4⁺ T cells were rare among antigen-responsive CD4⁺ T cells. For C-peptide, an average frequency of 7.5% (1%-11%, n = 4) of antigen-responsive CD4⁺ T cells were confirmed to be antigen specific. In the tetanus-toxoid-stimulated cultures, on average, 45% (16%-78%, n = 5) of the antigen-responsive CD4⁺ T cells were tetanus toxoid specific. These data show that antigen-specific CD4⁺ T cells are a minority of the cells that proliferate in response to antigen and have important implications for in vitro CD4⁺ T-cell proliferation assays.