Transcriptional Regulation of Immune Cell Development, Activation and Functions

National Institute Of Allergy And Infectious Diseases

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Abstract

CD4 T helper (Th) cells play a central role in orchestrating adaptive immune responses both in mice and in humans. After T cell activation, naive CD4 T cells differentiate into distinct T helper (Th) lineages, including Th1, Th2 and Th17 cells that produce interferon (IFN)-gamma, interleukin (IL)-4 and IL-17, respectively. Through the production of these signature cytokines, specific Th subsets mediate crucial functions during different types of protective immune responses to various microorganisms. Th1 cells are important for host defense against intracellular bacteria and viruses; Th2 cells for expelling extracellular parasites such as helminths; and Th17 cells for controlling extracellular bacteria and fungi. Inappropriate Th responses to pathogens may lead to chronic infection and/or tissue damage to the host, whereas aberrant Th cell differentiation may result in many inflammatory allergic or autoimmune diseases including asthma, inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) and multiple sclerosis (MS). Innate lymphoid cells (ILCs), which lack expression of antigen receptors, mirror different Th cell subsets in their cytokine production. Therefore, ILCs are classified into group 1 innate lymphoid cells (ILC1s) that produce IFNgamma, group 2 innate lymphoid cells (ILC2s) that produce IL-5 and IL-13, and group 3 innate lymphoid cells (ILC3s) that produce IL-17 and IL-22. Although some ILCs, such as lymphoid tissue inducer (LTi) cells, are specifically critical for lymphoid organogenesis, most ILCs, like Th cells, are important for protective immune responses to infections and contribute to the pathogenesis of many inflammatory diseases in human patients. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors that are induced and/or activated by a combination of cytokines and TCR-mediate signaling. Our major research goal is to better understand the transcriptional regulatory networks and mechanisms that control differentiation processes leading to the distinct Th and ILC lineages. We have chosen to focus on the master regulators (also known as lineage-determining transcription factors, LDTFs) including T-bet, GATA3, and RORgammat (for type 1, type 2 and type 3 lymphocytes, respectively), because we hypothesize that they are the major nodes in these networks. By comparing the regulation and actions of T-bet, GATA3 and RORgammat in distinct Th and ILC lineages, we aim to identify new components and/or connections of these complex networks controlling Th cell differentiation and ILC development. Comparing gene regulation at the transcriptomic and epigenomic level between these two cell types will allow us to identify the core elements that determine their shared functionality and unique molecules/pathways that control their specialized functions. During the past fiscal year, we continued to investigate the role of the transcription factor pair Bhlhe40-Pou2af1 in regulating Tfh cell maturation. The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. We recently uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 is specifically expressed in Tfh cells whereas Bhlhe40 expression is found high in non-Tfh cells. Pou2af1 mainly induces optimal expression of CXCR5 and migration of Tfh cells into GC without affecting Bcl6 expression whereas Bhlhe40 represses this process. Pou2af1 directly binds to the Cxcr5 locus and overexpression of CXCR5 in Pou2af1 deficient cells partially rescues the generation of GC-Tfh cells. Therefore, a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1 for regulating CXCR5 expression operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate (manuscript under minor revision). We continued to study the function of a FOXP3 mutant, which was identified from studying IPEX (Immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. The transcription factor Foxp3 dictates the development and function of regulatory T cells (Tregs) to maintain self-tolerance. The mutation in the 370th amino acid (M to I) of FOXP3 protein had been identified in IPEX patients with type-2 autoimmunity. By generating Foxp3-M370I-Knock-in (KI) mice via CRISPR-Cas9, we have found that homozygous female KI (Foxp3KI/KI) mice and the hemizygous male KI (Foxp3KI/Y) mice display dramatic T cell activation with multi-organ inflammation. The KI Tregs are capable of producing effector T cell cytokines including IL-2, IFN-γ, IL-4, IL-13, and IL-17A. We also generated heterozygous KI female (Foxp3KI/GFP) mice which turn out to be healthy in general. However, the KI Tregs still maintain their ability to produce IL-2 but not type 2 cytokines. AlphaFold structure prediction (in collaboration with Dr. David Margulies) indicates that the M370I mutation favors the Foxp3 swap domain dimer over the functional head-to-head Foxp3 dimer which is important for interacting with the transcription factor Runx1 and thus for silencing IL-2 production in Tregs. Indeed, mass spectrometry analysis (in collaboration with Dr. Aleksandra Nita-Lazar’s group) of samples from Foxp3 immunoprecipitation shows that the mutant has reduced interaction with Runx1. During the past fiscal year, we also continued to use CRISPR-Cas9 technology to screen for transcription factors that are functionally involved in regulating the expression of lineage-specific genes. We have previously found that hundreds of transcription factors are dynamically regulated during T cell differentiation, however, the functions of most of these transcription factors during this process are largely unknown. In collaboration with Dr. Pamela Schwartzberg’s lab in our department and Dr. Ken Cheng’s group at the NCATS, we have constructed a retroviral library containing sgRNAs against all mouse transcription factors and cofactors (~2,400 genes with 6 sgRNAs for each). We used this library in three different screening through high throughput sequencing and obtained several important results for follow up studies. 1. To identify transcription factors that regulate Foxp3 induction. In this screening, we identified over one hundred genes that are differentially enriched or depleted in Foxp3+ cells. One of the top hits identified, Bhlhe40, was enriched in Foxp3+ cells. In vitro culture of Bhlhe40fl/fl-CD4-Cre (cKO) naïve CD4+ T cells under iTreg differentiation conditions resulted in a significant increase in CD25+Foxp3+ Treg cells compared to WT control. By contrast, overexpression of Bhlhe40 expression suppressed Foxp3 induction. Furthermore, transfer of naïve CD4+ cKO cells to Rag1-deficient mice resulted in increased Foxp3+ cells in the absence of Bhlhe40. Consistent with the notion that Bhlhe40 suppresses Foxp3 induction, Bhlhe40 expression was found higher in CD25+Foxp3- than in CD25+Foxp3+ cells. We are currently focusing on the importance of this regulation in vivo. 2. To identify transcription factors that are involved in Th1, Th2, Th17 and Treg induction. In this screening, we infected activated CD4 T cells from T-bet/RORgt/Foxp3 triple reporter mice that also carry Cas9 transgene with the retroviral sgRNA library. After infection, cells were separated into Th1, Th2, Th17 and Treg conditions and cultured for 3 days. Enrichment or depletion of each sgRNA was analyzed by comparing sgRNA sequences recovered from Th1 (T-bet reporter positive), Th2 (triple negative), Th17 (RORgt reporter positive) and Treg (Foxp3 reporter positive) cells. Several dozens of genes have been identified to be specifically associated with the differentiation of one lineage but not others. These hits are being currently verified individually and/or with a small library prepared for single cell analysis. 3. To identify transcription factors that differentially regulate type 2 cytokines. Although GATA3 is the critical transcription factor for type 2 immune response and dispensable for type 2 cytokine expression, GATA3 expression itself cannot explain the heterogeneity of type 2 lymphocytes in producing different type 2 cytokines such as IL-4 and IL-13. To identify transcription factors that are uniquely involved in individual cytokine expression, we compared the sequences of sgRNAs recovered from GATA3-expressing Th2 cells that also express either IL-4 or IL-13. Through initial screening and subsequent validation, we found that Runx1-CBFβ complex is critical for IL-13 production whereas NFATC2 is important for inducing IL-4 in Th2 cells. CBFβ binds to the promoter of Il13 and modulates its accessibility whereas NFATC2 binds to DNase I hypersensitivity (HS) site Va at the Il4 gene. Chromatin interaction assay indicates that CBFβ is required for Il13 promoter interacting with multiple enhancers. We are currently investigating whether a heighted activity of CBFβ in ILC2s may offer a potential explanation for ILC2s being superior in IL-13 production than Th2 cells. Overall, our study indicates that a combination of the activity of multiple transcription factors together with GATA3 determines the heterogeneity of type 2 lymphocytes. In addition, in collaboration with Dr. Warren Leonard's lab at NHLBI, we found that thymic stromal lymphopoietin (TSLP) differentially acts on effector T cells versus regulatory T cells to balance type 2 immune responses. While TSLP is critical for Th2 cell differentiation, its signaling in Tregs limits type 2 responses. Therefore, TSLP not only is a driver of Th2 effector cells but also acts in a negative feedback loop to limit allergic inflammation via activating Tregs. This work has been published in Science Immunology (2025).