DAN LITTMAN
littman@saturn.med.nyu.edu
Summary: Our laboratory studies signaling pathways involved in T lymphocyte development, migration, and activation and 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. Cells that have T cell antigen receptors (TCRs) for self-MHC class I molecules express CD8, and cells with receptors for MHC class II express CD4. CD4 and CD8 bind to nonpolymorphic regions of class II and class I, respectively, and signal through their association with the cytoplasmic protein-tyrosine kinase Lck.
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 signaling pathways initiated by TCR interaction with MHC/peptide complexes, particularly those involved in transcriptional regulation of coreceptor genes. Our major effort has been 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. In mice lacking this silencer, CD4 is expressed at all stages of T cell development and in mature CD8+ cytotoxic T cells. In contrast, deletion of the silencer after CD8+ cells have achieved maturity does not result in expression of CD4, indicating that silencing is maintained by a heritable epigenetic imprint. Targeted mutagenesis of the murine silencer has revealed several motifs whose mutation results in derepression or variegation of CD4 expression in cytotoxic T cells. We have shown that members of the Runt family of transcription factors bind to two of the silencer motifs and that Runx1 and Runx3 are differentially required for silencing in immature double-negative thymocytes versus mature CD8-lineage T cells. We are characterizing histone modifications within the Cd4 locus at different developmental stages and are identifying targets of the Runx transcription factors in thymocytes and mature T cells.
Only a small fraction of DP thymocytes is subject to positive selection, as most cells undergo "death by neglect" because their TCRs do not interact with host MHC with sufficiently high avidity. We have identified an orphan nuclear hormone receptor, RORg, that directs expression of the anti-apoptotic protein Bcl-xL in DP thymocytes, thus prolonging the temporal window for positive selection. RORg is also required in the embryo for the development of lymph nodes and Peyer's patches. We have shown that RORg expression defines a small population of lymphotoxin-producing cells that induce expression of the integrin ligands ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) in mesenchyme and that are required for lymphoid organogenesis. RORg is required for the development of these lymphoid tissue - inducer (LTi) cells in the fetus and in the postnatal intestinal lamina propria, where they reside in cryptopatches. We are exploring the potential role of LTi cells in development of tertiary lymphoid tissues in gut inflammatory diseases and are employing expression arrays to identify transcriptional targets of RORg.
To dissect signaling pathways in thymocytes and T cells following engagement of TCR and the costimulatory receptor CD28, we are studying mice with mutations in genes encoding key signaling molecules involved in the activation of the transcription factor NF-kB. Of particular interest are PKC-q (protein kinase C-q), which translocates to the TCR complex after recognition of antigen, and CARMA1, a membrane-associated adapter protein. Mature T cells from mice lacking either of these proteins have defective NF-kB activation and interleukin-2 (IL-2) production, and they have defects in the balance of helper T cell responses. PKC-q-deficient mice exhibit reduced Th2 responses and have exaggerated expression of interferon-g. We are seeking to characterize the relationship between PKC-q and CARMA1 and to determine the function of the TCR-initiated NF-kB signaling pathway in differentiation of diverse subsets of T cells that regulate immune responses.
CD4 functions not only in TCR signaling but also as a requisite component of the HIV receptor on helper T cells and monocytes. Several chemokine receptors, particularly CCR5 and CXCR4, are required along with CD4 for HIV entry. These G protein-coupled receptors have key roles in leukocyte migration and lymph node homeostasis. CXCR4 has additional key functions in development of the central nervous system and other tissues and in germ cell migration. We are studying the myriad functions of CXCR4 in mice in which the gene can be inactivated in specific embryonic or adult tissues. We are also using mutant mice to study the function in inflammation and immune homeostasis of several other chemokine receptors that are used by HIV for entry, including CX3CR1 and CXCR6. Using mice in which the gene encoding green fluorescent protein (GFP) was used to replace the coding sequence for CX3CR1, we have shown that the level of chemokine receptor expression defines two distinct populations of blood monocytes that differ in their homing properties.CX3CR1loCCR2hi cells home to sites of inflammation, while CX3CR1hiCCR2- cells are precursors for tissue-resident macrophages and dendritic cells, and are dependent on CX3CR1 for efficient migration into tissues.
Mice in which the gene encoding CXCR6 was replaced with gfp have been used to study migration of a specialized subset of T cells (NKT cells) with semi-invariant TCRs that recognize lipid antigen and the class IŠlike molecule CD1d. These cells are abundant in liver, but they are dramatically reduced in CXCR6-deficient mice. We found that the interaction of CXCR6 on NKT cells with its membrane-bound chemokine ligand, CXCL16, on sinusoidal endothelium, is required for arrest of circulating NKT cells and their subsequent survival in liver. In collaboration with the laboratory of Michael Dustin (Skirball Institute, New York University), we have used intravital microscopy to show that arrested liver NKT cells initiate amoeboid movement within sinusoids, and that their subsequent activation with lipid antigen results in cessation of this movement. Future intravital microscopic studies will explore NKT cell behavior after induction of damage to hepatocytes.
Transmission of HIV is likely to be facilitated by the interaction of the virus with dendritic cells (DCs), which are abundant at mucosal surfaces and traffic to draining lymph nodes. We have shown that HIV binds to DCs, through a specific interaction of the viral envelope glycoprotein with the C-type lectin DC-SIGN, and that this results in dramatic enhancement of viral infectivity in the absence of replication in the DCs. We have proposed that HIV exploits DCs to ensure delivery to sites in lymphoid organs rich in T cells and to maximize efficiency of transmission to T cells. The enhanced infectivity requires the internalization of HIV into a specialized recycling vesicular compartment in DCs. We are employing RNA interference technology to examine the mechanism by which viral internalization results in increased infectivity of target T cells, and we are focusing on the role of specific intracellular compartments and on the components of the immunological synapse formed between T cells and DCs.
To study the function of DCs in vivo, we have prepared transgenic mice whose DCs express diphtheria toxin receptor, allowing their specific elimination within hours after treatment with diphtheria toxin. Studies with these mice have demonstrated that responses of naive CD8+ T cells against cell-associated antigen or pathogens such as Listeria and the malaria parasite require the presence of DCs. This model will be used to validate the importance of DCs in infection of the host with HIV. To this end, we have focused on generating mice that can provide a model for studying HIV pathogenesis and for testing potential therapeutic compounds and candidate vaccines. 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 cannot sustain infection with HIV-1, we are also using genetic and biochemical approaches to identify additional species-specific host cell genes required for productive HIV replication in murine cells, particularly for release of HIV particles. We are also inactivating the murine cytidine deaminase APOBEC3G/CEM15, which is incorporated into virions and blocks the viral life cycle after reverse transcription of the HIV genome in infected cells. Characterization of key host cell factors not only will facilitate studies of HIV in vivo but also may allow development of novel antiviral drugs.
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