A MOLECULAR AND IMMUNOLOGICAL SIGNATURE OF IL-10 PRODUCING CD4+ T CELLS IN VISCERAL LEISHMANIASIS

Abstract

Intro: Control of the intracellular parasites causing visceral leishmaniasis (VL) is dependent on IFNγ+ Tbet+ CD4+ T (Th1) cells generated by infected hosts. IL- 10 produced by Th1 cells mitigates subsequent inflammation and related pathology. However, IL-10- producing Th1 (Tr1) cells can also promote parasite persistence, as well as impair immunity following re-infection or vaccination. Here, we identify molecular and phenotypic signatures that distinguish Th1 cells from Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6 mice. Methods: To identify molecules that demarcate Th1 and Tr1 cells and contribute to their distinct functions, we first used cytokine capture based on IFNγ and IL-10 expression to sort Th1 (CD4+ IFNγ+ IL-10-) and Tr1 (CD4+ IFNγ+ IL-10+) cells from the spleens of C57BL/6 mice infected with L. donovani at day 14 p.i. RNA isolated, libraries were prepared from cDNA and 50bp single end mRNA- sequencing was performed on the Illumina HiSeq. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples (in some cases CD4+ cells from PBMCs) and used for gene expression analysis. Whole blood assays were performed on fresh VL patient blood to measure antigen-specific IFNγ production. Findings: We identify LAG3 as an immune checkpoint target in VL, describe heterogeneity of co-inhibitory receptor expression by Tr1 cells and discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. Conclusion: In summary, we have demonstrated that Tr1 cells in VL comprise heterogeneous cell populations, based on their expression of CIRs, chemokine receptors and transcription factors. Furthermore, we have identified unique molecular signatures that distinguish these important regulatory cells from the effector (Th1) cell population they emerge from. This information can be used to identify appropriate molecular targets to manipulate Tr1 cells not only in parasitic disease, but also other conditions where these cells can influence disease outcome.