Dan Littman's laboratory investigates how T lymphocytes acquire functional properties during development in the thymus and upon receiving signals initiated by distinct commensal microbes in the intestine. These studies are aimed at better understanding how alterations in the intestinal microbiota influence systemic inflammatory processes, such as T cell-mediated autoimmune diseases and responses to infection with HIV and other pathogenic microbes.
The vertebrate immune response to both invasive pathogenic microbes and commensal microbiota requires coordination between early sensing of microbial products by innate immune cells and subsequent activation of antigen-specific lymphocytes that make up the adaptive immune system. Our laboratory studies the regulatory mechanisms that govern the development of distinct classes of T lymphocytes within the thymus, their subsequent differentiation into cells with specialized effector functions, dictated by innate immune system cells, and their migration to tissues during steady state and inflammation. We are particularly interested in elucidating how the commensal microbiota influence the differentiation of T cells and innate lymphoid cells and contribute to systemic inflammatory diseases.
The majority of mature T lymphocytes are either CD4+ helper cells or CD8+ cytotoxic cells, which are derived from double-positive (DP) thymocytes that express both CD4 and CD8 coreceptors. The mechanism by which this binary cell fate decision is achieved, particularly how T cell antigen receptor (TCR) signals specify alternate cell fates, remains poorly understood. To elucidate the mechanism of lineage specification, which is coupled to positive selection of peptide-MHC-specific T cells, we have studied the transcriptional regulation of the coreceptor genes. We identified a lineage-specific transcriptional silencer that heritably shuts off CD4 expression in CD8-lineage cytotoxic cells following binding of the transcription factor Runx3 and its partner protein, CBFβ. Another transcription factor, ThPOK, directs positively selected cells to the CD4 lineage and prevents specification of CD8-lineage T cells by blocking expression of Runx3 in MHC II-selected thymocytes. We also found an enhancer that is required to initiate CD4 expression in DP thymocytes and to establish a heritable state of active gene expression in T helper cells that are mature. Thus, cd4 is regulated by epigenetic heritable ON and OFF marks, insofar as deletion of either enhancer or silencer sequences in mature CD4+ and CD8+ cells does not affect expression. We seek to identify the signaling pathways and trans-acting factors involved in initiation and maintenance of these heritable states. In recent studies, we have found that cd4 DNA methylation and demethylation govern the silencing and stable expression in the CD8 and CD4 lineages, respectively. We are using gene-editing technology to determine the roles of specific methylcytosine marks and of the enzymes that establish or remove them during distinct stages of T cell development. Results from these studies will provide insight into how binary decisions are made during development and will potentially direct us toward the signaling pathways distinguishing the fates of helper and cytotoxic T cells.
Littman Research Abstract Slideshow
We are also studying the function of the transcription factor RORγt, a member of the nuclear receptor family that regulates the life span of DP cells and the development of lymphoid tissue inducer (LTi) cells required for morphogenesis of lymph nodes and tertiary intestinal lymphoid tissues. We found that RORγt is required for the differentiation of Th17 cells that are abundant in the mucosa of the small intestine, where they protect the epithelial barrier but also contribute to multiple systemic autoimmune diseases. In mice defective for RORγt, Th17 cells and type 3 innate lymphoid cells (ILC3s) that produce interleukin-22 (IL-22) are absent, and the animals are resistant to multiple models of autoimmune disease, including autoimmune encephalomyelitis (EAE), a mouse disease that resembles multiple sclerosis, and inflammatory bowel disease.
We have used genetic and proteomic approaches to screen for molecules required for RORγt transcriptional activity. This has led to the identification of multiple RORγt-associated complexes that have critical roles in Th17 cell differentiation. These include nuclear pore components and the RNA helicase DDX5, whose binding to RORγt requires it to first interact with the long noncoding RNA Rmrp. Binding of DDX5/Rmrp to RORγt at distinct sites throughout the genome is required for induction of genes that contribute to the pro-inflammatory activity of Th17 cells. We are also studying RORγt posttranslational modifications that are critical for the induction of Th17 cytokines. The genetic screen was used to discover compounds that specifically inhibit RORγt, prevent mouse and human Th17 cell differentiation and EAE in mice, and are being developed to treat human autoimmune disease. We are using expression profiling and genome-wide chromatin immunoprecipitation of RORγt and other key transcription factors to characterize transcriptional and posttranscriptional regulatory networks involved in Th17 cell differentiation. Together, these studies may help identify additional targets for modulation of inflammatory lymphocyte function.
Germ-free mice lack Th17 cells in the intestinal mucosa, but they acquire such cells when they are colonized with segmented filamentous bacteria (SFB), a species of gram-positive bacteria. Mice colonized with SFB have enhanced protection from Citrobacter rodentium, an enteropathogenic bacterium, but they are also more susceptible to a model of Th17-dependent spontaneous arthritis. Thus, specific bacteria such as SFB contribute to maintaining a balance between protective and pathogenic activities of proinflammatory T helper cells. We have been investigating the mechanism by which Th17 cell induction by intestinal bacteria can trigger systemic autoimmunity. We recently found that the majority of Th17 cells in SFB-colonized mice are specific for SFB antigens and that different gut-resident bacteria employ distinct mechanisms to selectively induce T cells with diverse effector programs. We are currently investigating how SFB and other bacteria that elicit polarized intestinal T cell responses (e.g., Th1, Th17, and regulatory T cells) communicate with cells of the mononuclear phagocyte network (e.g., dendritic cells and macrophages) to achieve their specific outcomes. We found that SFB acts through monocyte-derived dendritic cells to induce RORγt expression in SFB antigen-specific CD4+ T cells in the mesenteric lymph nodes draining the small intestine. These poised cells then distribute widely, but express IL-17 only in the small intestine upon exposure to epithelium-derived serum amyloid A proteins (SAA1 and SAA2), which are produced locally following stimulation of ILC3 by SFB. These studies may provide insights into how SFB-induced Th17 cells contribute to systemic autoimmunity (e.g., autoimmune arthritis). They may also contribute to a better understanding of pathogenesis in a mouse model for autism spectrum disorder (ASD), in which innate immune system activation during pregnancy results in maternal Th17-mediated growth defects of the fetal brain as well as behavioral abnormalities in the offspring. Factors that affect IL-17a production in the mother may thus influence the risk for ASD in the offspring. This autism model is dependent on expression of the IL-17 receptor in the fetus, and we are investigating which cells are involved and how their gene expression profiles are affected by maternal IL-17a.
Based on the results with SFB-induced arthritis, we have undertaken a study of human rheumatoid arthritis patients, examining their fecal microbiota by shotgun sequencing. We found that 75 percent of untreated patients are colonized with the gram-negative anaerobe Prevotella copri, in contrast to 15 percent of healthy subjects. To determine if this bacterium contributes to disease onset, as SFB does in mice, we are culturing patient-derived bacteria and assessing their effect on the immune system of colonized germ-free mice. Results from such studies may help identify commensal bacteria that contribute to either protective or pathogenic outcomes in the host.
Our studies have shown that the commensal microbiota can shape the effector T cell repertoire, suggesting that some of the microbial constituents may have been selected for their ability to elicit pathogen-specific immune responses. In this context, microbiota that communicate with the host immune system may be harnessed to prime responses against pathogenic viruses, such as HIV, which replicates extensively in the intestinal mucosa following transmission. We are seeking to identify such bacteria that could potentially be used for protective or tolerogenic mucosal vaccination. We are also investigating how dendritic cells contribute to infection of CD4+ T cells with HIV, as this is a process likely to be influenced by the composition of the microbiota. We found that dendritic cells carry HIV on actin-dependent dendrites that, upon interacting with T cells, facilitate infection. One of our goals is to determine how the host immune system can be harnessed to block this process while permitting activation of an antiviral interferon response to limit HIV replication.
Grants from the National Institutes of Health provided support for the work on HIV pathogenesis, the Th17 cell transcriptional regulatory network, and the RORγt-associated molecules. Grants from the Simons Foundation Autism Research Initiative, NYSTEM (New York State Stem Cell Science, New York State Department of Health), and the Kenneth Rainin Foundation are supporting studies on the mouse ASD model, homeostasis of intestinal epithelial stem cells, and diversity of intestinal epithelial cell responses to microbiota, respectively.
As of March 14, 2016