A transcriptomics approach to study the molecular mechanisms regulating interleukin-10 gene expression in T helper cells

Interleukin-10 (IL-10) is an immunoregulatory cytokine that has a vital role in maintaining a balanced and appropriate immune response. CD4+ T helper (Th) cells are important in regulating an effective immune response, and are a dominant source of IL-10. Despite the different signalling pathways that result in the polarisation of each Th subset and lead to the expression of their hallmark cytokines, IL-10 is expressed by all of the different Th subsets. We show that Th1 cells cultured with IL-12 produce IFNγ and small amounts of IL-10, while Th1 cells cultured with IL-12 and IL-27 produce large amounts of IL-10 and IFNγ. Furthermore, we show that Th17 cells can produce IL-10, or not, depending on the presence of IL-2. However, these Th cell populations are phenotypically heterogeneous, particularly with respect production of IL-10 protein. Less than half of each of these in vitro cultured Th cell populations expresses IL-10 or the hallmark cytokine, and co-expression of IL-10 and the hallmark cytokine is also heterogeneous. Therefore we devised and implemented an innovative new technique that enables RNA-Seq analysis of different intracellular cytokine producing cell subpopulations from within Th subsets. We find that it is possible to extract high quality mRNA from Th samples, even though they have been fixed and stained for intracellular cytokines, and that the data from these samples is replicable. Using this technique we have identified potential molecules and pathways by which (I) IL-27 drives IFNγ and IL-10 production by Th1 cells, and (II) IL-2 drives IL-10 production by Th17 cells. Furthermore, by separating different intracellular cytokine producing subpopulations within different Th1 and Th17 cell subsets we have found that cytokine producing and non-cytokine producing subpopulations within heterogeneous Th subsets have significantly different transcriptional profiles and that some pathways and molecules are enriched in IL-10 producing cells