The immune system is complex, dynamic, and absolutely critical to maintain human health. The immune system comprises an enormous breadth of cell types, each able to respond to numerous extracellular environments. We have lacked adequate tools to dissect the mechanisms maintaining immune homeostasis through regulation of diverse cellular identities and stimulation responses. Human genetics has revealed key genes required to prevent immune dysfunction and many genomic loci associated with immune disease1,2. However, natural constraint, statistical power limitations, and lack of causal data hinder genetic association studies, preventing the identification of all key regulators and limiting conclusions about the relationships between regulatory genes 3–5. Experimental manipulation of gene expression with CRISPR provides the ability to investigate the structure and function of immune regulatory systems without the inherent limitations of association studies6.

In the T cell compartment, distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis, but the dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. To reveal the regulators of a core immune gene, IL2RA, expressed dynamically across the T cell compartment and required to prevent immune disease, we applied pooled CRISPR KO screens across cellular contexts7–10. We defined critical context specific regulators of IL2RA expression as well as regulators that affect the overall rest and activation state of the cell. One regulator in particular, MED12, coordinated gene regulatory networks required to maintain both T cell rest and activation. CRISPR ablation of MED12 blunted the cell state transitions between rest and activation and protected from activation-induced cell death, revealing a previously unappreciated gene regulatory mechanism governing T cell activity.

The effects of genetic variation on complex traits act mainly through changes in gene regulation. Although many genetic variants have been linked to target genes in cis, the trans-regulatory cascade mediating their effects remains largely uncharacterized. In a separate study, we investigated the function of and relationship between transcription factors associated with immune disease which are categorized as inborn errors of immunity (IEI) genes. We formed a large regulatory network consisting of regulators of IL2RA, IEI genes, and transcription factors without known immune disease associations1,11. These connections revealed shared paths and novel regulatory nodes that enable control over specific immune traits.

As a whole, this thesis work uses gene perturbation in distinct human T cell populations under different conditions to discover critical mechanisms regulating cell type and state specific gene expression. Detailed gene regulatory maps resulting from these studies, coupled with functional immune assays and biochemical data, provide new insights into human genetic variants linked to immune dysfunction and have potential to predict new modification strategies that enhance immunotherapies for the betterment of human health.

Inflammation early in life can prime the local immune milieu of peripheral tissues, causing lasting changes in immunologic tone that confer disease protection or susceptibility. The cellular and molecular mechanisms that incite changes in immune tone in many nonlymphoid tissues remain largely unknown. We find that time-limited neonatal inflammation induced by transient reduction of neonatal regulatory T cells (Tregs) causes a dramatic dysregulation of subcutaneous tissue in murine skin, accompanied by the selective accumulation of Th2 cells within a distinct microanatomic niche. Th2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as Th2-interacting fascial fibroblasts (TIFFs), which expand in response to Th2 cytokines to form subcutaneous fibrous bands. Activation of the Th2-TIFF niche by neonatal inflammation primes skin for altered reparative responses to wounding. We further identify fibroblasts in healthy human skin expressing the TIFF transcriptional signature and find these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a novel Th2 niche in skin, functionally characterize a disease-associated fibroblast population, and suggest a mechanism of immunologic priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells, establishing an immunological set-point in tissues that is maintained throughout life.

MicroRNAs are important mediators in the control of helper T cell differentiation, where small perturbations in responses to extracellular signals leads to early polarization and specialization. Identifying gene targets of highly expressed helper T cell miRNAs can lead to identification of novel players in these networks. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a powerful tool for identifying these gene targets by pulling down the miRNA binding protein argonaute (Ago) and sequencing the co-immunoprecipitated miRNA and targeted cognate mRNA fragment.

We performed HITS-CLIP on mouse CD4+ helper T cells and identified miRNA binding sites. While a majority of these sites were in well annotated 3’ untranslated regions (UTRs), we also identified a number of miRNA sites in coding regions, downstream of annotated 3’UTRs and in difficult to annotate regions of the genome. We identified hundreds of binding sites that were dependent of the presence of miR-29a, a miRNA highly expressed in CD4 T cells and implicated for its roles in T cell differentiation. We also observed a role for miR-29 in IL-17 production, which led us to identify a number of miR-29 targeted genes with roles in Th17 differentiation, one of which, ICOS, was a new target that we found to be directly regulated by miR-29. Our study identifies Ago dependent miRNA binding sites important in Th17 biology and identifies a role for miR-29 in IL17 production.

Additionally, our lab has generated a tool that aims to create an easy to use interface for labs working with CLIP-Seq data to create high quality graphics that are easily edited in a vector based graphics program. ClipPlot is a webapp that can be run on a local server and used by all members of a lab simultaneously, requiring only the technical expertise of a single user. While this is not a replacement for sophisticated visualization tools like IGV, ClipPlot provides an easy to use platform for researchers working with CLIP-Seq or RNA-Seq data to quickly create presentable and easily manipulated graphics, visualizing the shape of sequencing data to defined regions.

MicroRNAs (miRNAs) are emerging as important components of the epigenetic landscape that shape immune cell differentiation and effector functions. MiRNA-deficient T helper (Th) and natural killer (NK) lymphocytes show severe defects in differentiation, survival, proliferation, and effector functions, suggesting that miRNAs can modulate all of these processes. The study of individual miRNAs as well as global miRNA regulation are important steps in understanding the contribution miRNAs make to establishing lineage-specific gene expression programs.

The first part of this dissertation describes a method for profiling small RNAs in lymphocytes by high-throughput sequencing. We then apply this technique to defining the miRNA repertoire in Th cells actively differentiating into Th1 or Th2 subsets. Consistent with profiling experiments from fully polarized Th1 and Th2 cells, differentiating Th1 and Th2 cells had highly similar miRNA expression profiles as determined by both miRNA microarray and deep sequencing. There was, however, a subtle enrichment of miR-146a, miR-155, and miR-31 in differentiating Th1 cell cultures, and members of the 23b~27b~24-1 cluster were more highly expressed in Th2 cell cultures. These profiling studies served as the basis for a screen of abundantly expressed Th cell miRNAs that influence cytokine production in miRNA-deficient T cells.

We next sought to determine if the conserved 3'-to-5' exoribonuclease Eri1 regulates miRNA abundance and effector functions in lymphocytes. Eri1-deficient mice surprisingly showed cell-intrinsic defects in the development, receptor acquisition, and maturation of NK cells, which play a critical role in early host defense to infected and transformed cells. Furthermore, Eri1 was required for immune-mediated control of mouse cytomegalovirus (MCMV) infection. Antigen-specific Ly49H+ NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1-deficient T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Taken together, these studies demonstrate an important role for Eri1 in posttranscriptional RNA regulation and in lymphocyte development and antiviral immunity.