Single cell gene expression signatures of in vitro stimulated antigen-responsive regulatory and conventional CD4+ T cells

Human ex vivo regulatory T cells (Treg) are commonly distinguished from conventional T cells (Tconv) by their expression of CD4⁺ CD25⁺, FOXP3⁺ and the lack of CD127. However, upon stimulation Tconv can up- or downregulate Treg characteristic markers making it difficult to distinguish effector and regulatory responder cells.

We aimed to determine the features that distinguish Treg responding cells from Tconv responding cells after antigen-specific stimulation. A 5-day proliferation assay was used in which separately labelled Treg and Tconv were stimulated with tetanus toxoid (TT), Influenza vaccine (Flu) or the autoantigens proinsulin and GAD65 (AA), followed by FACS and single cell sorting of responding and non-responding Treg and Tconv for analysis by multiplex qPCR (Biomark).

Both Treg and Tconv had marked changes in their expression profiles following activation and proliferation with both cell types exhibiting mainly upregulation of genes. There were, however, clear differences between responding Treg and Tconv. Although there was overlap between cell types, responding Treg had little or no expression of cytokine genes, which in contrast were markedly elevated in responding Tconv. The expression of Treg markers FOXP3, HELIOS, CD127 and CTLA-4, and number of transcription factors and activation markers were also quantitatively different between responding Treg and Tconv. Antigen-related differences were observed in the responding Tconv cells. The findings provide a template for defining antigen-responsive Treg and Tconv cells using in vitro methods and may be helpful in monitoring therapies that target these cell types.