Lymphocyte signaling and immune cell response

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Abstract

The study of chromatin landscape has shown us how genomic architecture can control immune response. We established that genomic accessibility plays a factor in the differential rate of response of innate versus adaptive immune cells. Super-enhancers (SEs) are another distinct feature of the chromatin landscape where multiple enhancers, transcription factors (TFs), RNA polymerase II and transcriptional co-activators conjugate to regulate the expression of specific genes. Genes regulated by SEs are highly linked to the cell identity, but also denote functional hubs. It has been shown that SEs in adaptive immune cells are linked to genes that encode cytokines, cytokine receptors and transcription factors, which are vital for their normal development and function. In addition, SE architecture is also enriched for loci encoding long noncoding RNAs, which often flank key protein coding genes. The goal of our research is to understand how the chromatin landscape regulates immune cell development and function. To do so, we identified SEs in natural killer (NK) cells to determine if there are genes within these regions with unknown relevance to NK cell biology. We hypothesize that genes transcribed from SEs in NK cells are likely to play critical roles in NK cell development and effector function and may provide clues to mechanism in removal of virally infected and cancer cells. We anticipated that this SE-based approach has the potential to identify key NK genes that were previously overlooked but likely to have critical functions. Within the top-ranking SE of NK cells was Ugcg, a gene that encodes UDP-glucose ceramide glucotransferase (UGCG), which is an enzyme that catalyzes the first glycosylation step in the synthesis of glycosphingolipids. Along with Ugcg, a putative long noncoding RNA (lncRNA), Gm12596 also lies within this SE. Our approach in understanding if Ugcg is required for NK cell biology is two-fold. We first developed a conditional mouse model to determine if any aspect of NK cell development will be influenced. This Vav1cre-Ugcgloxp mouse model will help us to determine if NK cells and other hematopoietic cells require Ugcg. For the second arm of the study, we investigated whether NK cells with loss of UGCG activity have the ability to respond to infection and tumor cells. We utilized the drug ibiglustat, which is an inhibitor of UGCG activity. Using flow cytometry, high-throughput sequencing, and in vitro and vivo models, we have found that loss of Ugcg in the Vav1cre-Ugcgloxp mouse model and inhibition of UGCG activity with ibiglustat treatment, significantly reduced the number of NK cells in various tissues, while other immune cell subsets remained intact. The very few NK cells that remained were mostly immature cells (CD27+CD11b-KLRG1-). We are currently exploring the mechanism by which the loss of UGCG leads to reduced NK cell frequency. Additionally, we have found that CD8+ T cells require Ugcg for activation. After MCMV infection, mice treated with ibiglustat do not generate effector CD8+ T cells. Also, CD8+ and CD4+ T cells with loss of Ugcg have higher expression of CCR7a chemokine receptor that causes T cells to remain in secondary lymphoid tissues. We have created a CD4cre-Ugcgloxp mouse model, which we will use to determine the reason why effector memory CD8+ T cells do not expand after infection, and whether the upregulation of CCR7 impedes the ability of T cells to exit secondary lymphoid tissues to clear infection and target tumor cells. We have also created a knockout (KO) mouse model for Gm12596. LncRNAs are noncoding RNAs that are at least 200 bp and control multiple aspects of cell biology. One function of lncRNAs is to control expression of neighboring genes. Therefore, we plan to determine if the Gm12596-KO mice have lower expression of Ugcg, and if we see the similar effects on NK cells that we have found in the Ugcg conditional knockout models. Within the second top-ranking SE of NK cells is Mirt1(myocardial infarction associated transcript 1). Mirt1 is another lncRNA located on chromosome 19. Studies of Mirt1 has only been published in six articles, none of which are studies surrounding immune cells. These studies found that inhibition of Mirt1 improves symptoms related to myocardial ischemia. The expression pattern of Mirt1 among hematopoietic cells is highly selectivewith NK cells expressing higher levels compared to other hematopoietic cells. This data suggests that Mirt1 plays a critical role in the development and/or function of NK cells. We have created a Mirt1-KO mouse, which will be used to explore if Mirt1 is required for NK cell biology.