Differentiation of pathogenic granzyme A-producing CD4+ T cells in graft-versus-host-disease

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PROJECT SUMMARY/ABSTRACT. In graft-versus-host disease (GVHD), a detrimental allo-immune response that occurs after hematopoietic cell transplant (HCT), donor CD4+ T helper (Th) cells traffic to multiple organs and their presence in the intestines correlates with worsened clinical prognosis. Despite their known role in GVHD, how Th cells contribute to intestinal disease remains unclear. My laboratory has recently identified a novel population of intestinal Th cells that produced the serine protease granzyme A (GrA) which were critical for intestinal damage and GVHD-associated mortality. However, GrA+ Th cells were dispensable for the beneficial anti-cancer effects of HCT and therefore represent a promising therapeutic target in GVHD patients. As effective GrA inhibitors are currently unavailable, defining the molecular mechanisms that lead to GrA+ Th cell differentiation will uncover novel drug targets for GVHD and provide insight into how pathogenic Th cells differentiate within the intestines. Here we show that GrA+ Th cells differentiate independently from other Th lineages and require the integration of both STAT3-dependent and -independent cytokine signaling pathways for their optimal differentiation during disease. GrA+ Th cells expressed high levels of the STAT3- induced transcription factors (TFs) BATF and Aiolos that were also required for their development. However, the STAT-activating cytokines that initiate GrA+ Th cell differentiation and how STAT3-induced TFs guide this process during GVHD remain undefined. As STAT3 is required for optimal GrA+ Th cell differentiation, we hypothesize that the STAT3-activating cytokines IL-6 and IL-21 function in parallel with other cytokine/STAT pathways to initiate GrA+ Th cell differentiation and that a STAT3/BATF/Aiolos network cooperatively promotes the GrA+ Th cell lineage during aGVHD. To test this hypothesis we will 1) Define the cytokine/STAT signaling requirements that regulate pathogenic GrA+ Th cell differentiation during GVHD and 2) We will determine how STAT3/BATF/Aiolos TF network and its downstream effector genes regulate the GrA+ Th cell differentiation program using combined gene expression, chromatin occupancy analysis and a novel genome-wide CRISPR screen. Upon completion of these studies we will have defined the inflammatory and transcription factor requirements that are required for the development of GrA+ Th cells in GVHD and validated these factors as therapeutic targets in pre-clinical models of disease. This work is highly significant as GrA+ Th cells are a major driver of intestinal pathology and lethal disease. Therefore, the signaling pathways and genes involved in the GrA+ Th cell transcriptional signature that we identify may be potential druggable targets for GVHD patients. These experiments are also highly innovative as we will leverage a novel Gzma-GFP reporter mouse with cutting-edge sequencing technologies (RNA-Seq, CUT&RUN) to uncover how a STAT3-driven TF network drives pathogenic Th cell differentiation outside of the traditional Th17/T follicular helper cell paradigm.