Single-cell analysis of CCR6+ human Th cells reveals varying degrees of the type-17 phenotype, ranging from cells co-expressing to those fully suppressing opposing pathways

Abstract

Previously, we found that human type-17 Th cells from blood are fully contained within the CCR6-expressing subset. We also found positive correlations between levels of expression of CCR6 and levels of other type-17 proteins such as IL-17A/F, IL-22, and CCL20, □□□ negative correlations of CCR6 expression with that of genes/proteins associated with non-type-17 (Th1, Th2, TFH) pathways. In the present study, analysis of single Th cells expressing various levels of CCR6 using cell sorting followed by RT-PCR revealed that 1) patterns of expression of individual type-17 genes differed, being either binary (all-or-none-like) or graded, 2) expression levels of type-17 genes were positively correlated with each other and negatively correlated with genes associated with non-type-17 lineages, 3) cells could be identified that co-expressed genes for both type-17 and non-type-17 lineages, and 4) these “multi-lineage” cells expressed intermediate/low as opposed to high levels of the lineage-specific genes. Our data suggest that acquisition of the type-17 (and non-type-17) phenotype(s) is not all or none, but rather that cells occupy many intermediate states in forming a highly heterogeneous memory population, that genes defining “opposing” lineages can be co-expressed, and that expression of genes associated with opposing lineages is extinguished in cells at the far end of the type-17 spectrum. These findings are consistent with pathways of human Th differentiation in vivo that allow for the co-expression of multiple potentials, each to a limited degree, early on, but that subsequently narrow as a single phenotype becomes fully expressed.