Adrian C. Hayday - US grants

DESCRIPTION (Adapted from Investigator's abstract): Mice congenitally lacking alpha/beta T-cells frequently display high levels of serum immunoglobulins (Igs). These Igs are of all isotypes which is surprising given the established dependence of IgG, and particularly IgE synthesis, on T-cells. Therefore, a novel pathway of B-cell maturation would appear to be active in alpha/beta T-cell deficient mice. This idea is supported by our finding that such mice also develop germinal centers, anatomical sites associated with T-cell-dependent B-cell maturation. The investigators, therefore, hypothesize that these mice display an heretofore uncharacterized capacity to T-B collaboration between B-cells and non alpha/beta T-cells. Notable among such cells are gamma/delta T-cells, that they have recently shown to "help" B-cells, by demonstrating that mice in which the only T-cells are gamma/delta T-cells, also display germinal centers. Gamma/delta T-cells have not heretofore been ascribed an effector function in B-cell maturation. To test their hypothesis, they plan to characterize B-cell biology in alpha/beta T-cell deficient mice. Interestingly, the antibodies formed in alpha/beta T-cell deficient mice are largely auto-reactive, targeting the same set of antigens as are targeted in human lupus patients. Other recently-derived data demonstrate that alpha/beta T-cell deficient mice indeed develop a lupus-like disease. The investigators, therefore, believe that characterization of the pathway can shed light on immuno-deficiency-associated autoimmune syndromes, such as have been reported in individuals with AIDS, and such as may be operative in one or more cohorts of lupus patients.

Type 1 diabetes or 'IDDM' is a severe autommune disease afflicting hundreds of thousands of Americans. It is characterized by an attack on the pancreas, that abrogates insulin production. While the attacking infiltrate comprises multiple cell types, T cells are critical. The precise target of the T cells may also be manifold, but among them, central importance is attached to glutamic acid decarboxylase (GAD), particularly the 65kD form, GAD65. Experimental administration of recombinant GAD to non obese diabetic (NOD) mice that are highly predisposed to IDDM, ameliorated the attack on the pancreas, and subsequent disease development. These and other results have prompted the Food and Drug Administration to review the idea of administering GAD to individuals at high risk for developing IDDM. This notwithstanding, there is currently very little understanding of autoreactivity to GAD. In particular, it remains unclear why NOD mice and (by extrapolation) humans predisposed to IDDM fail to develop tolerance to GAD naturally. The characterization of the autoreactivity to GAD65 can be facilitated by the development of novel strains of NOD mouse in which GAD65 and/or T cell antigen T cell antigen receptors to GAD65, are expressed in unusual patterns. Such mice also facilitate the study of how T cells autoreactive to GAD can be regulated by other lymphocytes, and by the microbial environment. The development of such mice is described in the proposal and the case made for their detailed study.

DESCRIPTION: (Adapted from the applicant's abstract) All known vertebrates harbor three sets of lymphocytes, B cells, alpha/beta (ab) T cells, and gamma/delta (gd) T cells, defined by their respective antigen receptors. The role of gd T-cells in host protection and maintenance is poorly understood, and many studies aimed at resolving this have compared and contrasted gd T cells with the other two types of lymphocytes. Interestingly, emerging functional properties of gd T-cells suggest that they may share features hitherto regarded as B-cell-specific or ab T-cell-specific. In this application, studies are described that will continue our examination of gd T-cell development. Again, emerging information indicates that some gd T cells are highly similar to ab T cells, being derived from the same lineage, whereas others are B-cell like, even being extrathymically derived. The application lists seven specific aims, the success of which can confirm this "dual pathway" view of gd T-cell development and provide important mechanistic insight into how development is regulated. Such knowledge can significantly improve our basic depiction of T-cell development. In addition, it provides guidance as to how T-cell development might be experimentally manipulated in cases of autoimmunity, immunodeficiency, or transplant.

DESCRIPTION (Adapted from the Investigator's abstract): So far as is known, all vertebrates contain two types of T-cells, an alpha/beta and a gamma/delta T-cell, distinguished by their alpha/beta and gamma/delta T-cell antigen receptors. The role of alpha/beta T-cells is reasonably well understood, but this is not the case for gamma/delta T-cells. Studies from many groups suggest that gamma/delta T-cells are quite different from alpha/beta T-cells in terms of antigen recognition and anatomical localization. But, there has been a longstanding need for a bioassay, by which to assess the function of gamma/delta T-cells and the effector mechanisms(s) that underlie it. In part, the problem has been the scarcity of gamma/delta T-cells, that in mice and humans are much less abundant than alpha/beta T-cells. To tackle these issues, the investigators' laboratory has developed mice that lack alpha/beta T-cells, and have challenged those, together with mice that lack gamma/delta T-cells with a natural protozoan parasite that targets the intestinal epithelium, a resident site of gamma/delta T-cells. The investigators found that the normal response of mice to this parasite is significantly affected by the deletion of either alpha/beta T-cells or gamma/delta T-cells, but in different ways. Whereas alpha/beta T-cells are essential for protection of the host, gamma/delta T-cell deficiency is associated with severe pathology. Thus the investigators believe that alpha/beta and gamma/delta T-cells indeed represent very different components of the immune system, and that an understanding of gamma/delta T-cell functions is therefore essential for our understanding of immune-related pathologies, particularly of the gut, e.g., inflammatory bowel disease. The bioassay for gamma/delta T-cells provided by the investigators' system should facilitate such an improved understanding. Both cellular and genetic approaches will be adopted. The investigators shall undertake the analysis of another bioassay for gamma/delta T-cells, for which the investigators have recently developed convincing evidence. In this assay, gamma/delta T-cells offer a significant level of anti-microbial protection without establishing immunity. Together the study of these assays may reveal why gamma/delta T-cells are so highly conserved.

The use of transgenic mice has been instrumental in the characterization and understanding of the genetic basis of mammalian development and physiology. The genetics group at Yale University in particular has utilized transgenic mice to study the evolution of the HOX genes that are essential for normal development, the role of the transcription factor AP-2 in vertebrate morphogenesis, and the role of T-cells in immune system function and development, among other projects. This proposal requests funds to augment the transgenic mouse facility at Yale University. The facility presently has one old transgene injection set-up and one modern set-up for blastocyst injection of stem cells. The transgene apparatus is now 20 years old and despite mechanical wear and tear is used at capacity. This project proposes to add a new transgene injection station, to repair and upgrade the existing apparatus, and to add ancillary equipment that will generally support the overall program in transgene research and education. As transgene techniques continue to expand, the facility comes under even greater use by the faculty and students at Yale. The sheer volume of experimental studies necessitates the acquisition of new instrumentation. New instruments will enhance the efficiency of both research and teaching activities. Additional equipment will also make possible the development of new techniques such as the controlled expression of transgenes, fluorigenic reporter constructs, and the modification of endogenous genes by "knock-in" procedures. The requested equipment will be accessible to a broad spectrum of investigators and students both within and outside of Yale University. Access if facilitated by housing the equipment in an Animal Research Facility managed by a consortium of faculty investigators rather than within a single investigator's group. The facility is self- contained and integrates both animal care and housing and transgene laboratory space.

DESCRIPTION (provided by applicant): There is a strong body of evidence that the sensitivity to allergies, such as asthma, results from a dysregulation of immune responses in early life. This obliges us to better understand early life immune responses, and in particular, the expression of interferon gamma (IFN gamma), a cytokine with the capacity to down-regulate allergic T cell responses. We have recently shown that in early life, unconventional T cells, in particular gamma delta cells, are an important source of IFN gamma, and that young mice depend on such cells for primary protection against mucosal infection. Therefore, it is logical to ask whether the experimental up-regulation of unconventional T cells in early life will promote IFN gamma production that will alter the susceptibility to allergy. To accomplish this, mice will be infected with a natural gut pathogen that activates gamma delta cells, and their susceptibility then assessed to food allergy, and to airway hypersensitivity. The gut, rather than the lung, has been chosen as the primary target of unconventional T cell activation because the appropriate natural infection system is in place. In short, the first aim of the application is a proof of principle. Nevertheless, should the protocol successfully regulate allergy, it will lay an important foundation to attempt to regulate unconventional T cells directly in the lung (which is beyond the scope of this application) or to use non-infectious protocols to regulate unconventional T cells more globally, which is the subject of Aims 2 and 3. Although the immune systems of young mice and human children differ in their states of maturity, data are presented that show the relevance of human gamma delta cells to the production of IFN gamma in early life. Thus, Aim 4 will examine the degree to which the strategies that we develop in the animal model may be practically applied to humans.