Long noncoding RNA-mediated regulation of T-cell alloimmunity

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Project Abstract The goal of this K08 proposal is to provide didactic and experiential training in new techniques needed to achieve my long-term career goal of developing an independent research program exploring the novel intersection of long noncoding RNAs (lncRNA) and alloimmunity. The proposed training will allow me to develop expertise in animal models of hematopoietic stem cell transplantation (HSCT), T cell functional assays, bioinformatics analysis of next-generation sequencing data sets, techniques to identify lncRNA-protein interactions, grant writing, mentorship, and laboratory management. This 5-year training plan will be directed by my primary mentor, Dr. Pavan Reddy, who is an accomplished transplant immunology physician-scientist with a track record of successfully mentoring junior faculty. I will also be advised by a committee with expertise in bioinformatics, RNA biology, molecular genetics, and HSCT immunology. The didactic training program covers bioinformatics, grant writing, advanced immunology, mentorship, and laboratory management. Allogeneic (allo) HSCT is a curative treatment for high-risk hematologic disorders. Acute graft-versus-host disease (GVHD) is an allo-T cell-driven major complication of allo-HSCT, for which improved treatments are needed. LncRNAs control gene expression with tissue specificity and fine-tune immune responses. To identify potential lncRNA regulators of allo-T cells, we recently performed RNA-sequencing on well-annotated clinical HSCT samples. This work identified LINC00402 as a novel, conserved, T cell-enriched lncRNA that was differentially expressed by allo-T cells. Functionally, LINC00402 promoted allo-T cell proliferation. However, it is unknown if LINC00402 exacerbates acute GVHD in vivo and what molecular mechanisms are responsible for its effects on allo-T cell function. Based on these prior data and additional new observations, this proposal will test the overall hypothesis that LINC00402 regulates acute GVHD in vivo by enhancing ERK-c-FOS signaling in CD4 type 1 (Th1) helper cells. This will be tested using complementary murine and human in vitro and in vivo systems. Aim 1 will determine the cellular mechanisms of LINC00402 in T cell responses and will test the specific hypothesis that LINC00402 augments Th1 differentiation and the production of Th1 cytokines by allo-T cells. Aim 2 will define the molecular mechanisms mediating LINC00402’s regulation of T cells and will test the hypothesis that LINC00402 directly enhances ERK-c-FOS-dependent T cell receptor signaling. It will also serve as a training platform to learn bioinformatics analysis of next-generation sequencing datasets and methods to identify lncRNA binding partners. Aim 3 will elucidate the role of LINC00402 in experimental acute gastrointestinal GVHD, and will explore the hypothesis that LINC00402 enhances acute gastrointestinal GVHD by promoting accumulation of Th1 cells. This will be tested using complementary xenogeneic and allogeneic murine models. Altogether, these studies will explore a unique area of alloimmunity and determine if LINC00402 is a target for improving allo-HSCT outcomes.