BLIMP-1 and CD39 expression define human effector memory regulatory T cells in blood and recall responses to viruses

BLIMP-1, a transcriptional repressor initially identified in plasma B-cell differentiation, also regulates CD8⁺ T cells and is differentially expressed between naïve and effector/memory CD4⁺ T cells. However, the functions of BLIMP-1 in human CD4⁺ regulatory T cells (Tregs) remain unclear. We found BLIMP-1 mRNA levels were low in naïve Tregs from thymus and cord blood, but high in effector/memory Tregs from adult peripheral blood. We confirmed the expression of BLIMP-1 mRNA by microarray of healthy T-cell populations and single-cell RNA-sequencing of SARS-CoV-2 spike-specific CD4⁺ T cells from convalescent COVID-19 patients. We found activated memory Tregs expressed the highest levels of BLIMP-1, FOXP3, and CD39 transcripts and protein. We show that CD39⁺ Tregs, regardless of in vitro or in vivo activation, had the highest levels of BLIMP-1 protein expression and significantly more IL-10 mRNA than CD39^neg cells. The same result was found through reanalysis of public single cell RNA-sequencing data from lung (tissue and lymph node) SARS-CoV-2 spike-specific Tregs, where cells with high levels of BLIMP-1 mRNA had significantly more IL-10 mRNA than cells with low BLIMP-1 mRNA. Single-cell RNA-sequencing identified an extended transcriptomic profile of these CD39⁺ Tregs that includes the genes encoding HPGD, CTLA-4, HELIOS, TIGIT, CXCR6, CCR8, and GARP. Together, these data indicate that both circulating and lung resident effector memory Tregs maintain a conserved phenotype following stimulation by cognate antigen. Our data provide further insight into the functions and potential therapeutic targets of the large regulatory T-cell component of CD4⁺ T-cell responses to pathogens.