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Pathways of Lymphoid Cell and Organ Development and Mechanisms of HIV Transmission and Pathogenesis
Summary: Dan Littman's laboratory studies signaling pathways involved in T lymphocyte development, migration, and activation and in lymphoid organogenesis, as well as the mechanism of HIV infection and pathogenesis.
The vertebrate immune system is composed of diverse cell types that cooperate to mount responses against invading microorganisms. Our laboratory is interested in understanding the regulatory mechanisms that govern the development of distinct classes of T lymphocytes, their migration to lymphoid organs and sites of inflammation, and their activation following interaction with cells specialized to present microbial antigens. Microbial pathogens have in turn adopted numerous strategies designed to circumvent immune responses. We study mechanisms by which the human immunodeficiency virus (HIV) subverts normal immune defenses and destroys the pivotal regulatory subset among T lymphocytes, the helper cells.
The majority of mature T lymphocytes fall into one of two functional categories: helper cells, which react with peptides complexed to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells, and cytotoxic cells, which recognize peptides bound to MHC class I molecules. These cells are distinguished on the basis of surface expression of the CD4 or CD8 coreceptors, which are coexpressed on immature double-positive (DP) thymocytes but are singly expressed upon maturation on thymocytes with T cell antigen receptors (TCRs) specific for class II and class I, respectively.
In the thymus, DP cells with TCRs of desired affinity for self-antigen undergo "positive selection" and migration to secondary lymphoid organs. The selection is coupled to transcriptional shutoff of CD4 or CD8 and to commitment to the cytotoxic or helper lineage, respectively. To understand the mechanism of lineage specification, we study signals involved in transcriptional regulation of coreceptor genes. Other groups recently showed that the transcription factor ThPOK directs positively selected cells to the CD4 lineage and prevents specification of CD8-lineage T cells. Our efforts are aimed at understanding how CD8-lineage specification is achieved: Is this a default function, when ThPOK is not expressed, or is there a factor that, similarly to ThPOK, directs cells to this lineage? To identify transcription factors involved in CD8-lineage specification, we have focused on characterizing a region within the Cd4 gene that is required for silencing expression in immature thymocytes and in CD8+, but not in CD4+, T cells. We found that the transcription factors Runx1 and Runx3, as well as their common partner protein, CBFβ, bind to motifs in the silencer and are required to shut off CD4 expression at the two stages of thymocyte development. The Runx3-CBFβ complex contributes to the activation or repression of a number of genes in CD8-lineage cells, but is not required for cells to differentiate down this pathway, and hence does not perform a function in this lineage analogous to that of ThPOK in the CD4 lineage. We are using several approaches to identify additional direct targets of the Runx complexes.
If the silencer sequence or Runx-CBFβ complex is inactivated early in lymphocyte development, CD4 is expressed at all stages, including mature CD8+ cytotoxic T cells. In contrast, their inactivation in mature CD8+ cells does not result in derepression, indicating that silencing is maintained by a heritable epigenetic imprint. This system provides an ideal opportunity to characterize general mechanisms involved in initiating an epigenetic mark during vertebrate development. We are attempting to identify other factors that bind to the silencer and determine if they differ in expression or function at different stages of thymocyte development. Because the RNA interference machinery has been shown to be important in formation of silent heterochromatin in yeast, we are also exploring the potential role of small RNAs in Cd4 silencing and in T cell differentiation.
In our studies of thymocyte development, we identified another transcription factor, RORγt, that is expressed in DP cells and prolongs their survival, thus assuring selection of a broad repertoire of T cells. RORγt, an orphan nuclear hormone receptor, is also required for the development of all secondary lymphoid organs, with the exception of the spleen. In the fetus, it is expressed in lymphoid tissue inducer (LTi) cells, a small migratory population of cells that induce mesenchymal cells to differentiate and attract the cells that form organized lymph nodes and Peyer's patches. In the adult, RORγt is expressed in organized lymphoid structures in the intestinal lamina propria, including in cryptopatches, cell clusters that are surrounded by dendritic cells at the base of crypts. In mice lacking RORγt, neither LTi cells nor the cryptopatch cells develop. In addition, isolated lymphoid B cell follicles, which require intestinal flora for their development, are absent in mice lacking RORγt. We have hypothesized that these follicles differentiate from cryptopatches upon their exposure to gut commensal flora and that the RORγt+ cryptopatch cells have an inductive function in the adult that is similar to that of LTi cells in the fetus. We are exploring the potential role of RORγt+ cells in development of tertiary lymphoid tissues that may protect the gut mucosa from bacteria and that may also contribute to inflammatory bowel diseases. We have recently found expression of RORγt in a small population of T cells that is present constitutively in the small intestine and that is induced at other sites upon inflammatory stimuli. We are investigating the possibility that interfering with RORγt function may reduce the differentiation of these cells and the severity of inflammation in autoimmune diseases.
Another specialized subset of T cells, the regulatory T cells, is required to prevent autoimmune activation of naïve and resting effector T cells. To study the relationship between regulatory T cell and effector T cell functions in vivo, we have developed mice in which the regulatory T cells, which express the transcription factor Foxp3, can be selectively eliminated at different times during immune responses. These mice will be useful to examine the role of regulatory T cells in antitumor T cell responses, in primary and secondary responses to microbial pathogens, and in maintenance of immunological tolerance. Our recent studies suggest that the regulatory T cells keep in check potentially harmful self-reactive T cells that express RORγt. Disruption of this homeostasis can result in autoimmune encephalomyelitis, a mouse disease that resembles multiple sclerosis.
Dendritic cells (DCs) form a dense network in the intestinal lamina propria and directly communicate with the intestinal lumen through processes that extend between epithelial cells. In normal conditions, these processes may sample the lumen for commensal microorganisms, and may thus communicate with cryptopatches to regulate follicle differentiation and with T cells to present antigen. Although DCs mediate innate immune defenses against mucosal pathogens, they are also likely to be exploited by pathogens during infection and dissemination in humans. For example, infection of T cells with HIV is substantially enhanced if the virus first interacts with DCs. HIV infects helper T cells and monocytes through the interaction of its envelope glycoprotein with CD4 and one of several chemokine receptors, particularly CCR5 and CXCR4. When DCs are present, there is dramatic enhancement of viral infectivity in the absence of replication in the DCs. The enhanced infectivity requires the internalization of HIV into a specialized recycling vesicular compartment in DCs. We have proposed that HIV exploits DCs to ensure DC-mediated delivery to sites in lymphoid organs rich in T cells, to maximize efficiency of transmission to T cells, and to "hide out" in a nonreplicating virion form in vesicles within DCs, where it is resistant to antiretroviral therapies. Our current work is aimed at identifying DC host genes required for enhanced delivery of HIV to T cells and at testing the hypothesis that DCs can serve as a reservoir for nonreplicating virus in vivo. Characterization of the molecular mechanism by which DCs enhance HIV infectivity may result in new therapies to block viral transmission and latency.
In vivo studies of HIV pathogenesis require the availability of a good small-animal model. We have prepared transgenic mice that express human CD4, CXCR4, CCR5, and cyclin T (which enhances HIV gene expression) in their T cells and macrophages, as well as human DC-SIGN in their DCs. Because T cells and macrophages from these mice will only support partial replication of HIV, we are using genetic and biochemical approaches to identify additional species-specific host cell genes required for productive replication in murine cells.
Grants from the National Institutes of Health provided support for the work on HIV entry and pathogenesis. A grant from the Sandler Program in Asthma Research is supporting our studies on the role of regulatory T cells and dendritic cells in lung inflammation.
Last updated: May 15, 2006
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