Research Interests

Molecular approaches are being applied to address three basic problems. The first is the molecular basis of the activation and differentiation of T-lymphocytes. Upon activation, precursor (naive) T-cells undergo numerous cell divisions and differentiate into effector cells; some of the products of this activation become what are known as long term memory T-lymphocytes, which upon restimulation, even up to many years after the initial activation, can respond more rapidly and effectively to the immune insult. We have used a variety of gene subtraction and functional genomic techniques to identify the genes that are involved in directing the processes of differentiation of these T-lymphocytes along these various pathways. We have used gene targeting to identify a number of signaling molecules that play a critical role in this differentiation process. We have focused on the stress activated MAP kinases c- JunN terminal (JNK) and P38 MAP kinases. Jnk1 appears to play a critical role in suppressing the Th2 response in developing Th1 cells. In its absence, exaggerated Th2 response occurs which render animals more susceptible to infection with Leishmania and render the animals hypersensitive to asthmatic insults. Conversely, the P38 MAP kinase pathway conversely plays an important role in IFN gamma production in Th1 cells and in T cell death.

We also use similar approaches to elucidate the mechanisms of CD4 T cell differentiation to effector cells. Transcription factors selectively expressed in developing Th1 or Th2 cells have been identified and their roles assessed. Thus, GATA3 and JunB play a causal role in Th2 cell differentiation, while Rac2 and GADD45g play important roles in Th1 cell development through activation of the NFkB, Jnk, and p38 pathways in the case of Rac2 and the Jnk and p38 pathways in the case of GADD45g. We study the epigenetic events occurring in the chromosome which regulate gene expression. We are investigating the TH2 cytokine locus (genes for IL-4, IL-5 and IL-13 are clustered on Chromosome 11) for mechanisms which underlie coordinated expression and we have studied global chromatin changes in this locus upon commitment to the TH2 program of T cell development. These changes include Histone modification and regulation of cell fate by Notch.

We have found that notch signaling regulate TH1 and TH2 differentiation. Specifically, TH2 differentiation derives from the induction of the jagged family of ligands on dendritic cells by TH2 inducing stimuli. Jagged in turn stimulates the notch receptors on CD4 T cells and induces their differentiation into TH2 cells through the induction of GATA 3 and the direct action on the R04 locus.

Our studies on the organization of the TH2 locus in the cell nucleus have focused on interactions between enhancers, promoters and the locus control region. These results have shown that the locus is tightly folded in a poised confirmation in which the promoters are juxtaposed to the enhancers in LCR. In addition we have found that the IL4 locus and the interferon gamma locus which are on different chromosomes are physically associated in the cell nucleus. Thus, interchromosomal complexes exist which may have many implications for gene expression. First regulatory elements on one chromosome can regulate genes on others. Second, this intimate association between chromosomes might lead to abnormal recombination in certain circumstances such as is seen in tumors.

Following the activation of T-lymphocytes and the development of effector cells, the majority of these effector cells will eventually die by the process of apoptosis. Our laboratory has studied the molecular mechanisms that underlie apoptosis by eliminating the function of key enzymes that direct these processes known as caspases. In this way we have shown that caspases 9 and 3 play a critical role in apoptosis in certain cell types and that apoptosis through this (apoptosome) pathway plays a critical role in cell death under numerous stimuli. Our laboratory has eliminated the function of several other caspases and their function is currently being studied. It has become apparent that the caspase family can be subdivided into several categories. Initiator caspases begin the cascade and are responsible for cleavage of other caspases. Activation of 'effector' caspases are the penultimate step committing the cell to apoptosis. A final grouping of caspases includes Caspase 1 and 11 which seem not to be directly involved in cell death but rather in inflammatory responses. The signals that lead to apoptosis itself are of considerable interest and we have shown that the Jnk pathway plays an important role in the induction of apoptosis in a variety of cell types, including cell death in the thymus, the death of fibroblasts in response to radiation, chemotherapeutic agents and other stimuli, and during the development of the central nervous system. It is of considerable interest to determine how Jnk achieves these aims and what the target molecules are that mediate these effects.

Finally, our laboratory integrates the molecular approaches that we use to understand molecular mechanisms of differentiation and death into the study of critical autoimmune diseases. Activation of the immune system and inflammatory responses are controlled by the innate arm of the immune system. The Toll-like receptors (TLR) described by Medzhitov and Janeway appear key in this activation process. Our laboratory has knocked out several of these TLRs in mice and are dissecting what these receptors recognize and the downstream factor involved in their function. We have also identified several intracellular molecules that will help connect signals at the cell surface with final activation of cells. The mammalian genome incodes approximately twenty such genes and we have targeted the genes in several of these in mice. These demonstrate interesting phenotypes. Notably this year we have published that the molecule Nod 2 enhances susceptibility of mice to intestinal infection. Since this molecule predisposes humans to development of Chrohn’s Disease this is an interesting correlation which may teach us much about how inflammatory bowel disease is develops.

The way in which these molecules direct the immune response to self tissues (autoimmunity), infectious agents and tumors will be studied in the coming years.

Autoimmune pathologies lead to substantial morbidity and mortality in the human population and result from the disregulation of the immune response. Our laboratory has elucidated the role of inflammatory responses in the initiation of autoimmune diabetes in a variety of mouse models. We have shown that inflammation promotes the apoptosis of islet cells, their uptake by antigen presenting cells and the presentation of islet antigen to autoreactive T-lymphocytes. In recent studies we have found that this process is regulated by other T-lymphocytes which dampen this response in an attempt to prevent the development of autoimmunity. We are currently studying the cellular and molecular mechanisms that underlie this regulatory process. One of these regulatory events is mediated by the anti-inflammatory cytokine TGF beta that inhibits the function of T-cells and antigen presenting cells. We have found that in the absence of the inhibition of T-lymphocytes by TGF beta, immune responses go out of control and animals succumb readily to self reactive T cells that do not affect their normal littermates. We are currently studying the relationship between this form of regulation and other regulatory mechanisms that seem to act at different levels of the immune response. Another cytokine involved in this regulatory pathway is IL-10. We examine which cells express IL-10 using a knockin reporter construct which will produce green fluorescence in cells expressing IL-10 as well as generating mice which cannot respond to Il-10 by expression of a dominant negative form of the IL-10 receptor. In this way we can determine when and where IL-10 is expressed in real time in vivo and in vitro as well as elucidate the consequences of the lack of IL10 signaling.