Immunobiology of Normal T Cells

(Askenase, Bothwell, Bottomly, Craft, Flavell,* Girardi, Hayday, *Herrick, Janeway, Redlich, Shlomchik, Tigelaar)

Dr. Philip Askenase's lab is interested in the pathogenesis and regulation of T cell mediated immunity in vivo, as exemplified by contact hypersensitivity. His laboratory found an early initiating phase of these responses (occurring within hours after antigen challenge) and requiring activation of local mast cells and platelets to release mediators, prominently serotonin, as well as transcription of genes and release of cytokines such as TNF-a. More recently his group has discovered an involvement of the complement system, in particular C5a, that most likely activates C5a receptors, on mast cells and possibly platelets, to activate mediator release. Even more recently, their studies have shown that Vg5Vd4+ cells, a newly recognized subset ofgd T cells, have a positive role in assisting the second acting ab T cell transfers, by blocking endogenous suppressive cells. Other studies from his group are examining the regulation of these responses with positive-acting ab T cells, possibly releasing soluble T cell receptors binding to receptors on APC to release IL-12 to make the effector T cells resistant to suppression. There also are assisting gd T cells that may interact with activation/heat shock protein antigens on the T cells, to further strengthen their signaling. Studies conducted by Dr. Askenase and Dr. Robert Tigelaar describing and partially characterizing T cell-derived, IgE-like factors, whose participation in the early phases of contact sensitivity are crucial for full expression of these reactions, continue to force the re-examination of paradigms which simplisticly divide immunologically mediated cutaneous inflammatory reactions into IgE-mediated "immediate" and T cell-mediated "delayed" types. Ongoing collaborative studies with Dr. Robert Tigelaar and Dr. Adrian Hayday are examining the role(s) in contact hypersensitivity of the specific subset of dendritic gd cells which populate the epidermis of all normal mice. Finally, additional studies with Dr. Hayday recently uncovered down-regulatory (suppressive) gd T cells in tolerized TCR a- "knock-out" mice; these suppressor cells are active in strikingly small numbers (2,500/ mouse). The molecular details are now being investigated through the development of gd suppressive T cell clones. All of the above studies have involved extensive use of the flow cytometry facilities of the Immunobiology Section, priority access to which had been guaranteed and supported by a collaborative arrangement between the YSDRCC Tissue Acquisition and Analysis Core and the Immunobiology Section. Dr. Askenase's collaboration with Dr. Jordan Pober (Pathology) in P/F #6 study are described below (see Endothelial Cell:T Cell Interactions).

A major focus of Dr. Alfred Bothwell's research has been to investigate control of hematopoietic cell lineage development via Ly-6 antigens; this work focuses on the potential role(s) of Ly-6A/E in murine T cell development and peripheral T cell function. Studies of a human structural homologue of Ly-6, CD59, has extended these investigations into studies of human cells. The goal of these studies is to elucidate the role of Ly-6A in signal transduction in responses to antigen. The murine Ly-6A antigen on the murine Th2-type T cell line, D10, has been studied extensively using antisense RNA expression in stably transfected D10 cells. These studies have shown that this antigen is critical to antigen specific responses in D10 cells and indicated a functional association with the fyn tyrosine kinase. Efforts are currently directed toward molecular cloning of the ligand. Modifications of Ly-6 expression in vivo are also being examined via transgenic mice and homologous recombination to delete the Ly-6 gene. Antisense transfectants of additional molecules that affect Ly-6 expression or signaling properties are being generated and studied including fyn, lck, c-yes, zap70 and csk. Finally, the primary cytokine that regulates Ly-6 expression is interferon; characterization of tissue specific regulation of Ly-6 by interferon in fibroblasts, T cells and B cells and macrophages is in progress.

The structural homologue of Ly6 in humans is the GPI-anchored cell surface molecule CD59, a multifunctional molecule possessing complement regulatory activity and both adhesion and costimulatory activity for human T cells via the CD2 antigen on T cells. Dr. Bothwell, in collaborative studies with Dr. Jordan Pober, has focused on the structure/function properties of CD59 in regulating the formation of the complement membrane attack complex and the development of systems to study the costimulatory properties on purified human T cell populations. Several projects are directed toward a detailed structure/function analysis of CD2/CD58 and CD59 interactions that result in signaling. (Additional collaborative studies with Drs. Pober, Kluger and Schechner are described below in the Endothelial Cell: T Cell Interactions cluster).

A final area of research activity in Dr. Bothwell's lab involves the direct evaluation of the recognition properties of T cell receptor (TCR) and MHC antigens. Soluble forms of a MHC class I -restricted TCR specific for L(d) plus peptide alloantigen (from the 2C T cell line) as well as of a MHC class II- restricted TCR specific for a peptide of conalbumin plus I-A(k) (from the D10 T cell line) have been generated. The affinity of the 2C TcR for L(d) plus peptide has also been determined. Studies of recognition of variant peptides have given insight into differences in functional activation of T cells using different assays. A new collaborative association between Drs. Bothwell, Hayday, and Tigelaar will take advantage of Dr. Bothwell's molecular expertise in engineering soluble TCRs to attempt to make a soluble form of the unique Vg5/Vd1 expressed on dendritic epidermal T cells which can then be used to purify the currently unidentified ligand (antigen) for this homogeneous TCR

Dr. Kim Bottomly (Immunobiology and Dermatology) is an internationally recognized cellular immunologist whose research efforts have focused on understanding the factors which contribute to the selective activation of Thl or Th2 effector CD4 T cells. Effective clearance of pathogens depends on the presence of the appropriate effector CD4 T cell, and exaggerated Th2 responses are characteristic the atopic state. Dr. Bottomly and her coworkers have shown that selective activation of Th2 cells occurs when the antigen concentration is low or when the number of peptide:MHC class II complexes is limiting, suggesting that signals through the T cell receptor influence the differentiation of CD4 T cells into specific types of effector cells. They have also shown that the presence of costimulatory molecules, B7-1 and B7-2 also modulate the type of effector CD4 T cell response elicited. Both B7-1 and B7-2 contribute to the differentiation of Th2 cells, whereas little or no such co-stimulation is required for Thl cell generation. These data suggest that CD4 T cells, activated by antigen presenting cells such as antigen-specific B cells or mature Langerhans cells which express abundant B7-2 upon contact with specific antigen and , may selectively develop into Th2 cells. A new collaborative association with Christina Herrick., M.D., PhD., who is currently completing a postdoctoral fellowship (supported by the Department of Dermatology's Research Fellowship Training Grant) in her lab, has produced results which have formed the basis for a P/F project (#27) being submitted with this application. These studies involve using a variety of knockout and transgenic mice (and extensive utilization of the services of the Tissue Acquistion and Analysis Core) to investigate the basis for the extreme Th2 bias seen in responses in both the lung and in the skin following epicutaneous sensitization with protein antigens; such studies should enhance our understanding of a variety of important human diseases, including contact urticaria, latex hypersensitivity and atopic dermatitis. The above studies will also utilize mice previously characterized (in collaborative studies involving the laboratories of Drs. Bottomly, Flavell, Longley, and Tigelaar and the services of both the Center's Tissue Analysis Core and its (former) Molecular Analysis Core), in whom expression of an MHC class II I-E transgene is differentially expressed on different subpopulations of APCs, including differential expression on Langerhans cells vs B cells or splenic dendritic cells.

Dr. Bottomly collaborates extensively with other YSDRCC investigators based not only in the Immunobiology Section (most notably Drs. Janeway and Flavell), but also with Dr. Carrie Redlich (Occupational Medicine) in ongoing studies aimed at understanding how the pathology associated with asthma can be elicited and regulated by activated T cells. Dr. Redlich and her coworkers have also very recently initiated a new project with Drs Bottomly and Herrick to investigate the skin of patients with isothiocyanate-induced asthma, both because many of these patients exhibit not only asthma but also a positive reaction following patch testing with this antigen (suggesting the possibility that the skin may actually be the initial site of sensitization), and because such patch test sites appear to be an extremely convenient source from which to clone antigen-specific T cells which can be used to define the immunodominant epitopes of this particular antigen. Finally, Dr. Tigelaar also is collaborating with Dr. Bottomly to investigate the expression of different CD45 isoforms on dendritic epidermal T cells (DETC) and the capacities of these isoforms to function as accessory molecules in TCR-mediated activation of DETC.

Dr. Joseph Craft's (Rheumatology) research focus is on systemic lupus erythematosus, an illness characterized by activation of autoreactive T and B cells, with resultant tissue injury in the skin, kidneys and other organs. Using murine models, his laboratory seeks to define the antigenic specificity and mechanisms of activation of autoreactive T cells in lupus and mechanisms of their loss of self tolerance, and their capacity (both ab and gd T cells) to propagate and to regulate autoreactive B cell help for pathogenic autoantibody production. Other recent studies have also focused upon the need for, and characterization of, T cell regulation of B cell tumor development in the setting of Fas deficiency. Collaborative studies utilizing both in vitro approaches, as well as in vivo ones in transgenic and in T cell deficient ( ab, gd, and ab & gd "knockout"), lupus-prone (e.g, MRL/lpr) mice. These studies, carried out in collaboration with Adrian Hayday, Ph.D (Biology)., Richard Flavell, Ph.D. (Immunobiology), Mark Shlomchik, M.D., Ph.D. (Laboratory Medicine) and Jennifer Madison McNiff, M.D. (Dermatology), have defined a role for autoreactive ab T cells in promotion of disease in MRL mice, with and without intact Fas molecules, with ongoing efforts devoted to identifying the role of gd T cells, if any, in regulation of pathogenic ab cells. Until about two years ago, both Dr. Craft's and Dr. Shlomchik's attentions had been focused on the serum autoantibody responses and renal immunopathology seen in "normal" and the various T cell-deficient MRL/lpr mice. However, a discussion early in 1996 following a YSDRCC seminar involving Drs. Craft, Shlomchik, Madison and Tigelaar convinced them to direct more attention to the skin lesions these mice develop. This redirection of focus resulted in a new P/F study which investigates the role(s) of ab and/or gd T cells as well as of B cells and autoantibody production in the immunopathogenesis of the lupus-like skin lesions seen in MRL/lpr mice.

Dr. Richard Flavell (Immunobiology), is an internationally recognized molecular immunologist who has several research activities in the immune response as it relates to skin biology and pathology. First, Dr. Flavell has used gene targeted mice lacking L-selectin to analyze the limiting steps in the activation of leukocytes and their migration to inflammatory sites in the skin. These results have shown that in the absence of L-selectin this response is significantly retarded. In collaboration with Dr. Greg Geba (Pulmonary Medicine), Dr. Flavell has also used a similar system to study the role of CD40 ligand in DTH; in CD40 ligand knockout mice, the DTH deficit is demonstrable, but considerably less dramatic than the deficit seen in systemic immune responses. Dr Flavell's laboratory has also collaborated with Drs. Longley., Bottomly and Tigelaar in elucidating the molecular basis of the expression of MHC class II molecules in Langerhans cells. Dr. Flavell's very extensive and productive collaborative associations with Drs. Barthold, Feng, Fikrig, Malawista and Montgomery in studies of human Lyme disease, the murine model of Lyme disease, and the development of immunoprotective vaccines against B. burgdorferi are described below in the Infectious Disease subgroup.

Dr. Michael Girardi* was recruited to the Dermatology Department in 1997 as an Instructor after completing his postdoctoral fellowship here at Yale. As a Yale medical student already clearly committed to a career in investigative dermatology, Dr. Girardi conducted his M.D. thesis research in Dr. Tigelaar's laboratory, developing an animal model in which the autoimmune manifestations of graft-versus-host disease could be ameliorated by prior immunization of the recipients with 8-MOP/UVA-inactivated allogeneic effector cells. As a postdoctoral fellow he spent two years working in the laboratory of Dr. Adrian Hayday. This collaborative association with Dr. Hayday focused on the lineage relationships of developing ab and gd T cells in the thymus, and involved population analyses of PCR products of TCR d and b gene junctional (CDR3) rearrangements derived from T cell-rich tissues in normal adult mice as well as in TCR a- and TCR d-deficient (knockout) mice. RFLP patterns distinguished productive vs nonproductive rearrangements, consistent with a model of T cell development where ab and gd T cells share a late common precursor.

Dr. Adrian Hayday (Biology) has had long-standing collaborative associations with a large number of YSDRCC investigators, including Drs. Askenase, Bottomly, Craft, Girardi, Janeway, and Tigelaar. As an internationally respected molecular immunologist, Dr. Hayday has been very recently recruited to become the K. Glendenning Professor of Immunobiology at the University of London. However, because of his desire to maintain his close scientific relationships with the above individuals here at Yale, as well as Yale's desire to see these productive collaborations continue, he will simultaneously hold an appointment in Yale's Biology Department as Senior Research Scientist, where he will maintain an active research operation. Dr. Hayday is interested in the molecular processes involved in the development of immune responses and immunological diseases. To accomplish this, his laboratory combines a variety of approaches. The major read-out is the response of mice to a reproducible, natural system of infection; the major input are lines of novel mutant mice which they and others develop by use of homologous recombination in embryonic stem (ES) cells, followed by blastocyst injection and transfer to term in pseudopregnant foster mothers. As an example of how these approaches are combined, they are studying the functions of gd T cells which are conserved in all vertebrates, but currently are of unknown function. Dr. Hayday and coworkers have recently shown that after infection of mice with Eimeria, a naturally-occurring parasite of the gut, gd T cells reduce the pathology without affecting the virulence of the pathogen, and ongoing studies are aimed at elucidating the mechanisms by which the gd T cells down-regulate tissue-specific damage.

Highly productive collaborations involving Dr. Hayday and Dr. Joseph Craft (Rheumatology) and others have revealed that knockout mice lacking ab T cells develop normally, but go on to display substantial antibody production, directed not against microbes, but primarily against self. Furthermore, knockout mice congenitally deficient in gd T cells exhibit significantly elevated levels of immunoglobulin synthesis as well as evidence of generalized autoimmunity, and when this gd T cell absence is introduced onto the genetic background of MRL/lpr mice, there is a striking acceleration of their lupus-like autoimmune disease (see also Dr. Craft above).

Dr. Hayday also has had a long-standing collaborative association with Dr. Robert Tigelaar, stemming from their shared and complementary interests in epithelial gd T cells. A P/F study conducted during the 01-02 years of the YSDRCC to characterizes the myosin isoforms unexpectedly found in a long-term line of dendritic epidermal gd T cells (DETC), provided the preliminary data for the ongoing studies of the roles of intermediate filaments in the activation of both ab and gd T cells. Another recent productive collaboration between their labs has shown that chimeric mice in which lymphocytes are deficient in the syk protein tyrosine kinase, display a striking depletion of intraepithelial gd cells in the skin and in the gut; in contrast, splenic gd cells developed normally. Additional collaborative associations with Dr. Tigelaar are described below. Finally, Dr. Hayday's laboratory has also had a major interest in dissecting the complexities of T cell development in the thymus, particularly in the relationship(s) between ab and gd cells and the interplay of differentiation and cell cycle control. These studies have involved another YSDRCC (junior) investigator, Dr. Michael Girardi (see immediately above).

Dr. Charles Janeway (Immunobiology and Dermatology), is an internationally recognized authority in T cell immunobiology, whose broad research interests are aimed at understanding the process of antigen recognition by T cells in normal immune responses, in autoimmune diseases, and more recently in cutaneous T cell lymphoma. His laboratory has been at the forefront of the research to elucidate the co-receptor roles of accessory molecules, such as CD4, in T cell activation. In his long-standing efforts to elucidate TCR interactions with MHC/peptides, and antigen processing/presentation, he has also become a recognized authority in the arena of bacterial exoprotein and endogenous retroviral "superantigen" responses. While Dr. Suguru Imaeda was in his laboratory as a dermatology postdoctoral fellow, studies were initiated to analyze the peptides in the clefts of MHC class I molecules purified from both normal T cell lines as well as B and T cell lymphomas, studies that were subsequently funded as a YSDRCC P/F study during the 02-03 years of this Center, and which Dr. Imaeda is currently continuing as a Dermatology Department faculty member (see Immunobiology of CTCL below). Currently, work in Dr. Janeway's lab is focused on three large, and in some ways, interactive themes. The first of these is the mechanism of antigen recognition by CD4 T cells. They are using mutation of all three elements in the tremendously complex inaction of TCR:MHC:peptide to develop a consistent picture of how this recognition occurs. The second area is the regulation of expression of costimulatory molecules by various microorganisms. In the process of conducting these studies in collaboration with Dr. Diane McMahon-Pratt (Epidemiology/Public Health), they have recently cloned and expressed a Leishmania donovani gene encoding for a peptide isolated from the MHC class II molecules of infected macrophages. The third major area of research in Dr. Janeway's lab is the analysis of two experimental autoimmune diseases, insulin-dependent diabetes and experimental allergic encephalomyelitis (EAE). In a collaborative association between Dr. Janeway and Dr. Tigelaar, a Yale medical student, Susan Wolf, examined the specificity of the down-regulatory T cells arising in response to injection of 8-MOP/UVA inactivated, EAE-inducing T cell clones.

Dr. Mark Shlomchik (Laboratory Medicine) is using transgenic mice developed in collaboration with Dr. Richard Flavell (Immunobiology) to determine the fate of pathologic autoantibody-secreting B cells as they develop in a normal host. Other studies in Dr. Shlomchik's lab are designed to test the role of exogenous antigen that is retained on follicular dendritic cells in the form of immune complexes after a primary immune response. Another knockout mouse created in collaboration with Dr. Flavell has B cells but cannot secrete antibody; they are currently determining the capacity of this mouse to make primary and secondary (memory) immune responses. A related project is examining the role of an autoimmune genetic background (e.g., MRL/lpr) in disregulating or inducing these autoreactive B cell clones. A second area of interest is the role of B cells in spontaneous autoimmune induction and pathogenesis; collaboration with Drs. Flavell and Craft (Rheumatology), has resulted in the creation and ongoing investigation of autoimmune prone (MRL/lpr) mice which lack B cells (by virtue of having a mutation in their Ig H chain locus). These mice do not get nephritis or vasculitis, and have dramatically improved survival compared to wild type MRL/lpr mice. Current studies are also aimed at determining the relative roles of B cells per se versus the autoantibodies such cell normally secrete in causing nephritis and vasculitis. Skin disease is another prominent pathologic manifestation previously reported by others in MRL/lpr murine lupus. However, whether such skin lesions actually depend on the presence of B cells or the autoantibodies they can produce and/or whether they are dependent on ab or gd T cells are questions which have never been definitively addressed. The proposal to investigate these specific issues utilizing MRL/lpr mice genetically deficient in B cells, ab T cells or gd T cells is included as a P/F study included with this application and involves the collaboration of Drs. Shlomchik, and Jennifer Madison (Dermatology) and extensive use of the Tissue Acquisition and Analysis Core. Dr. Shlomchik is also beginning to investigate immunologic mechanisms of graft vs leukemia (GVL) in which a murine model will be used to investigate cell types responsible for GVL and GVH and, in particular, to develop and test strategies for immunotherapy of leukemia using genetically altered T cells.

Dr. Robert Tigelaar's major research focus continues to be the dendritic epidermal T cells (DETC) populating the skin of all normal strains of mice. DETC have been shown to express unique and strikingly homogeneous Vg5/Vd1 TCRs devoid of junctional heterogeneity identical to those expressed by early fetal thymocytes, to preferentially localize and/or proliferate in skin which is in active hair development or growth, and to recognize a ligand expressed by stressed epidermal cells. Studies examining a variety of mutant and transgenic mice with defects in hair growth/cycling (including a collaboration with investigators in the keratinocyte biology cluster (Arthur Broadus and John Wysolmerski) have verified a critical role for growing/cycling hair follicles for the postnatal intraepidermal proliferation of DETC. Extensive collaborations between Dr. Tigelaar's lab and that of Dr. Adrian Hayday include: 1) studies of the myosin isoforms expressed by DETC (a P/F funded in the YSDRCC's 01-02 years) and the very recent demonstration of the syk tyrosine kinase-dependence of epithelial gd T cells (contrasting with the syk-independence of recirculating gd T cells); 2) the recently successful creation of a Vg5 -/- knockout mouse selectively deficient in DETCs expressing the TCR prototypically seen on >95% of such cells. Studies of such mice unexpectedly showed that in the absence of Vg5, the replacement population of gd DETC includes a significant number of cells expressing a Vg1/Vd1 TCR which has a similar 3-D conformation as the conventional Vg5/Vd1 TCR (as defined by an anti-clonotypic antibody to the Vg5/Vd1 TCR), consistent with the possibility that both TCRs recognize the same ligand/antigen; and 3) in collaboration with Dr Al Bothwell they are creating a soluble version of the Vg5/Vd1 TCR which could then be used to identify the elusive ligand for this unusual subset of T cells.

A collaboration involving Dr. Tigelaar and the laboratory of Dr. Alan Houghton (Sloan-Kettering Memorial Institute), to investigate the roles of gd cells (and specifically, of DETC) in the progressive depigmentation observed in sites of active growth in mice injected with monoclonal anti-p75 (brown locus) antibodies was the direct outgrowth of Dr. Houghton's YSDRCC-sponsored seminar during his visit as a member of the Center's External Advisory Board. Another collaboration involving Drs. Tigelaar, Ruth Halaban (Dermatology), and Jack Longley (now at Columbia) followed the discovery that long-term DETC lines express large amounts of c-kit (by flow cytometry), a receptor previously known to be expressed on pre-T cells but not on CD3+ TCR+ peripheral T cells; very recently they have shown, using SGF (MGF), that this receptor is functional, and studies are now underway to examine the state of c-kit phosphorylation in freshly isolated as well as cultured (both resting and activated) DETC. Dr. Tigelaar's ongoing collaborations with Dr. Bottomly regarding CD45 isoforms on DETC has been mentioned previously. Finally, two other active collaborations involving Dr. Tigelaar with other YSDRCC investigators are described elsewhere in this section (see also Dr. Michael Girardi in the CTCL cluster and Dr. Janet Brandsma in the Infectious Disease subcluster.)