Steven J. Burakoff - US grants

The general aim of my research is to develop the capacity to regulate or manipulate the cytolytic T lymphocyte (CTL) response. This interest extends to understanding the CTL response to viral infections, malignancies and also to alloantigens (because of the evidence for the involvement of CTLs in renal and bone marrow transplantation). In order to accomplish my more general goal I feel that is necessary to define on a molecular level, the antigenic requirements for triggering pre-CTLs and the antigens recognized by CTLs. Recent methodological advances have made it possible (1) to clone major histocompatibility complex (MHC) genes; (2) to isolate and purify MHC and viral antigens that retain CTL stimulating activity when inserted into artificial membranes or liposomes and (3) to clone helper T cells and CTLs. These monoclonal T cell lines provide a powerful probe to define the T cell recognition site(s) on MHC and viral antigens. Therefore, the specific aims of my research are as follows: (1) To utilize cloned MHC genes to define the antigenic sites on H-2 molecules recognized by allospecific and virus specific CTLs. Since there are likely to be multiple CTL recognition sites on these molecules, cloned allospecific and virus specific CTLs will be generated. (2) Cloned viral genes will be used to define the antigenic sites on viral proteins recognized by virus specific CTLs. Specifically, a cloned Mooney murine leukemia virus gp70 gene and a cloned influenza virus hemagglutinin (HA) gene will be manipulated to assess serological and CTL recognition sites. (3) Influenza viral proteins and H-2 proteins will be isolated and introduced into liposomes to study the antigenic requirements for triggering influenza virus specific CTLs. (4) Isolated H-2K/D and Ia antigens will be isolated and introduced into liposomes to define the antigenic requirements for triggering cloned T helper and T proliferating cells.

The ultimate goal of this research is to define the human cytolytic T lymphocyte (CTL) response to leukemia by utilizing purified and well-characterized antigens, cloned CTL lines, and monoclonal antibodies to functional T-cell subsets. In order to better understand the CTL response, it is necessary to define the antigens that trigger this response and to define the cell interactiona that regulate this response. Since CTLs appear in all cases studied to respond to integral membrane proteins (whether they be foreign MHC antigens or autologous MHC antigens in association with foreign antigens such as a virus), it has been difficult to define the antigens involved in CTL triggering. Initial studies in murine xenogeneic, allogeneic, and syngeneic CTL model systems demonstrate that H-2 and HLA antigens can be isolated from the cell surface and retain CTL stimulating activity. These results suggest that the antigenic requirements for the stimulation of human CTLs can be approached in a similar manner. The ability to obtain cloned CTL lines should allow one to define more precisely the specificity of CTLs. Thus, one can hope to determine what target antigens are recognized by monoclonal CTLs when triggered by well-defined antigens, i.e., purified MHC antigens or purified viral antigens plus MHC antigens inserted into artificial membranes or liposomes. The ability to obtain cloned helper T cells or suppressor T cells should also allow a more careful delineation of the cell-cell interactions that regulate the CTL response. These cloned functional T-cell lines may provide the appropriate reagents to raise antibodies to antigens specific for functional subsets of T cells. The availability of antibodies to human CTLs should subdivide the T-cell subset defined by OKT5 and OKT8 and allow a separation of CTLs from suppressor T cells. These new reagents may also allow us to isolate biochemically and define these cell surface antigens, possibly even resulting in the isolation and characterization of functional T-cell receptors. (LB)

The general aim of this proposal is to characterize the biological role played by CD4 and CD8 in T cell activation and to further define the biological effects of HIV binding to CD4. The approaches planned rely on recent methodological developments in cell biology, protein chemistry, and molecular biology. In order to accomplish these aims we have developed a model system in which cDNAs encoding human T cell surface molecules can be readily expressed in murine T cell hybridomas that are specifically stimulated by human class I or Class II MHC antigens. This system enables us to analyse the functional effects of various mutations of the cDNAs encoding CD4 and CD8 expressed in an antigne reactive T cell. Detailed structure/function studies can then be performed. The specific aims of the proposal are as follows: (1) Determine whether the ligands for CD4 and CD8 are the class II and class I MHC antigens, respectively; (2) Determine the role of CD4 and CD8 in cell adhesion; (3) Determine the role of CD4 and CD8 in antigen-activated signal transduction; (4) Determine the role of MAbs directed against CD4 and CD8 in signal transduction; (5) Determine the role of anti-CD4 and anti-CD8 MAb in negative signaling; (6) Use CD4 and CD8 mutants to define the relationship between structure and function; (7) Determine the effects ofthe binding of HIV gp120 to CD4.

The ultimate goal of this research is to define the human cytolytic T lymphocyte (CTL) response to leukemia by utilizing purified and well-characterized antigens, cloned CTL lines, and monoclonal antibodies to functional T-cell subsets. In order to better understand the CTL response, it is necessary to define the antigens that trigger this response and to define the cell interactiona that regulate this response. Since CTLs appear in all cases studied to respond to integral membrane proteins (whether they be foreign MHC antigens or autologous MHC antigens in association with foreign antigens such as a virus), it has been difficult to define the antigens involved in CTL triggering. Initial studies in murine xenogeneic, allogeneic, and syngeneic CTL model systems demonstrate that H-2 and HLA antigens can be isolated from the cell surface and retain CTL stimulating activity. These results suggest that the antigenic requirements for the stimulation of human CTLs can be approached in a similar manner. The ability to obtain cloned CTL lines should allow one to define more precisely the specificity of CTLs. Thus, one can hope to determine what target antigens are recognized by monoclonal CTLs when triggered by well-defined antigens, i.e., purified MHC antigens or purified viral antigens plus MHC antigens inserted into artificial membranes or liposomes. The ability to obtain cloned helper T cells or suppressor T cells should also allow a more careful delineation of the cell-cell interactions that regulate the CTL response. These cloned functional T-cell lines may provide the appropriate reagents to raise antibodies to antigens specific for functional subsets of T cells. The availability of antibodies to human CTLs should subdivide the T-cell subset defined by OKT5 and OKT8 and allow a separation of CTLs from suppressor T cells. These new reagents may also allow us to isolate biochemically and define these cell surface antigens, possibly even resulting in the isolation and characterization of functional T-cell receptors. (LB)

OVERALL DESCRIPTION (Applicant's Description) The overall goal of this program project grant is to understand the mechanisms of the major BMT toxicities, in particular graft versus host disease (GVHD), in order to develop novel approaches to their prevention while enhancing the therapeutic aspects of this treatment modality. In order to achieve our goal we have assembled experts in the areas of cellular, biochemical and molecular immunology and cutaneous biology from the research community at Harvard Medical School. The research focus of this grant renewal has been enhanced by increasing its emphasis on a traditional strength of the program in BMT models. Preliminary data in these models demonstrate that the severity and tempo of acute GVHD is determined by the intensity of the BMT condition regimen that regulates the magnitude of the immune response by donor T-cells to host tissues by cytokines. This strategy to separate the toxicities of GVHD from GVL through the control of the cytokine cascade emerges as a central theme of this proposal. There are four projects proposed in this program renewal together with two core sections (administrative (A) and mouse BMT (B)). The four projects are: 1)The roles of BMT conditioning and cytokines in GVHD and GVL. 2)The effects of GVHD on thymocyte development. 3)Tumor vaccines and BMT. 4)T-cell homing and GVHD.

The ultimate goal of this research is to define the human cytolytic T lymphocyte (CTL) response to leukemia by utilizing purified and well-characterized antigens, cloned CTL lines, and monoclonal antibodies to functional T-cell subsets. In order to better understand the CTL response, it is necessary to define the antigens that trigger this response and to define the cell interactiona that regulate this response. Since CTLs appear in all cases studied to respond to integral membrane proteins (whether they be foreign MHC antigens or autologous MHC antigens in association with foreign antigens such as a virus), it has been difficult to define the antigens involved in CTL triggering. Initial studies in murine xenogeneic, allogeneic, and syngeneic CTL model systems demonstrate that H-2 and HLA antigens can be isolated from the cell surface and retain CTL stimulating activity. These results suggest that the antigenic requirements for the stimulation of human CTLs can be approached in a similar manner. The ability to obtain cloned CTL lines should allow one to define more precisely the specificity of CTLs. Thus, one can hope to determine what target antigens are recognized by monoclonal CTLs when triggered by well-defined antigens, i.e., purified MHC antigens or purified viral antigens plus MHC antigens inserted into artificial membranes or liposomes. The ability to obtain cloned helper T cells or suppressor T cells should also allow a more careful delineation of the cell-cell interactions that regulate the CTL response. These cloned functional T-cell lines may provide the appropriate reagents to raise antibodies to antigens specific for functional subsets of T cells. The availability of antibodies to human CTLs should subdivide the T-cell subset defined by OKT5 and OKT8 and allow a separation of CTLs from suppressor T cells. These new reagents may also allow us to isolate biochemically and define these cell surface antigens, possibly even resulting in the isolation and characterization of functional T-cell receptors. (LB)

DESCRIPTION: (Adapted from the Applicant's abstract): The goal of this application is to understand the role played by the CD4 and CD8 coreceptors in T cell activation. Furthermore, the effect of HIV-gp 120 binding to CD4 will be studied on T cell activation. Studies from many laboratories have definitively shown that the ligands for CD4 and CD8 are on class II and class I MHC molecules, respectively. Conclusive evidence has been provided that CD4 and CD8 are signaling molecules that require association with the lymphoid specific tyrosine kinase, p56lck, for their function. In many of the studies, these studies have expressed wild-type or mutant human CD4 and CD8 molecules by DNA mediated gene transfer in an antigen reactive murine T cell hybridoma. This model system has allowed the study of the human CD4 and CD8 coreceptors in an antigen reactive hybridoma. BY hybridomas have been selected that lack the murine homologues of CD4 and CD8 and structure/function analyses have been performed by assessing the effect of expression of wild-type or mutant human CD4 or CD8 molecules on T cell responses. This renewal application proposes to continue to study the role of CD4 and CD8 in T cell activation. Specific Aim 1 proposes to evaluate the signaling pathways in CD4 and CD8 mediated T cell activation. "Double positive" (CD4+CD8+) T cell hybridomas have been recently generated which suggest that CD4 versus CD8 mediated T cell activation involve different signaling pathways. Experiments are proposed to delineate the signaling molecules used in each of these pathways. Specific Aim 2 proposes to further delineate the role of p56lck in T cell signaling. The data regarding the signaling activity of lck may not depend on its kinase activity. Experiments in which mutants of lck are expressed in these T cell hybridoma are proposed to explore the biochemical role of lck in signaling. Specific Aim 3 proposes to define the role played by the cytoplasmic domains of each of the CD3 chains in T cell signaling. Specifically, chimeric molecules of CD16 and the cytoplasmic domains of gamma, delta, epsilon and zeta will be expressed in TcR, CD4+ hybridomas to investigate their role. Specific Aim 4 proposes to study regulated adhesion of CD4 and CD8. It has now been demonstrated that the avidity of several T cell molecules, including CD8, can be regulated by cellular activation. Specific Aim 5 proposes to study the in vivo function of human CD4 and CD8 molecules. CD4 and CD8 will be expressed by retroviral mediated transfer in murine stem cells which will in turn be used to reconstitute lethally irradiated mice. These experiments will allow study of the CD4 and CD8 function in normal thymocytes and peripheral T cells. Specific Aim 6 proposes to delineate mechanism of gp 120 inhibition of T cell activation. Gp 120 can inhibit CD4-dependent T cell activation by competing for the ligand of CD4 on MHC class II molecules. Preliminary data suggest that gp 120 can also suppress T cell activation by inducing a "negative" signal.

The objectives of this proposal are to study the effects of GVHD, CsA and FK506 on T cell ontogeny and to determine whether this results in aberrant T cell function causing susceptibility to autoimmunity. I. Thymic dysfunction in animals treated with CsA or FK506 as determined by Vbeta usage. The effects of CsA and FK506 on positive and negative thymic selection will be studied. Cells that have escaped selection will be analyzed for their functional capacity including their ability to cause autoimmune disease. II. Thymic dysfunction in GVHD as determined by Vbeta usage. The effects of GVHD on positive and negative selection will be explored. furthermore, we will assess whether cells have escaped selection are functional and whether they confer susceptibility to autoimmune disease. III. Immunological consequences of the treatment of GVHD with CsA and FK506. The consequence of GVHD together with CsA or FK506 treatment will be assessed for their combined effect on thymic ontogeny. IV. Influence of GVHD, CsA or FK506 on TcR-Valpha usage. The effect on TcR-Valpha usage subsequent to GVHD or the treatment with either CsA or FK506 will be studied. Whether the function of these cells are changed under these experimental conditions will be assessed.

In this proposal we plan to further define the roles of the adapter proteins Shc and Cbl, and their associated molecules, in T cell signaling. They plan to develop a better understanding of these proteins by employing biochemical approaches, studying the function of these proteins in transformed T cells and finally studying their function in animals. I. The role of Shc in T cell activation and T cell development. They have observed that dominant negative mutants of Shc, when introduced into the murine T cell hybridoma DO. I 1, can disrupt T cell receptor (TCR) mediated IL-2 production and antigen induced cell death (AICD). They will define the Shc signaling-pathwgys by defining the proteins upstream and downstream of Shc. They will also introduce the dominant negative forms of Shc as transgenes into bone marrow stem cells to determine their effect on T cell development and T cell effector function in vivo. II. The role of Cbl in T cell activation and T cell development. Cbl appears to play a role as a negative regulator of T cell signaling. They will use a variant Jurkat cell, Jl16, which lacks ZAP-70 and Syk to define the mechanism by which Cbl inhibits T cell signaling. They will introduce mutant forms of Cbl into c-Cbl-/- mice to define its role in vivo. III. The role of SLAP and TSAD in T cell signaling. Usin2 Cbl-N as bait in the modified yeast two hybrid system they have isolated CDNA clones encoding two adapter proteins, SLAP and TSAD. They propose to study their role in T cell signaling.

DESCRIPTION: (Applicant's Description) Our proposal tests the hypothesis that host-reactive, bone marrow derived T-cells contribute significantly to acute GVHD by disruption of the thymic environment. We will employ approaches to distinguish mature donor inoculum T-cells from donor bone marrow derived T-cells and assess the differential effect of these cell populations. Our proposed studies will also determine whether novel cytokine antagonists positively or negatively impact on thymocyte development in the GVHD model. A future therapeutic emphasis in bone marrow transplantation will be directed toward cytokine antagonists as agents to prevent GVHD. Our studies may yield a better understanding of the cellular, cytokine and biochemical events that occur in the thymus during GVHD. Specific Aim 1: We will test the hypothesis that specific T-cell clones are important in the initiation and maintenance of GVHD. To test our hypothesis, we will utilize T-cell clones with specific Vbeta TCR expression (Vbeta3 and Vbeta6) which can react with host MIs antigens in the initiation and continuation of GVHD in a minor histocompatibility-mismatched murine bone marrow transplantation (BMT) model. The role of each of these T-cell populations as GVHD effectors will be assessed in target organ damage by positive and negative selection of the donor T-cell inoculum, in situ immunofluorescence staining, in vivo depletion with neutralizing antibodies and by adoptive transfer experiments. These studies will also be performed in a model system where GVHD occurs to minor H antigens. Specific Aim 2: We will test the hypothesis that antagonism of inflammatory cytokines, which have been shown to have an ameliorating effect on GVHD, could improve the thymic damage associated with GVHD. There is, however, evidence that IL-1 and TNF may have a physiological role in thymocyte development. Therefore, the effects of anti-IL-1R antibody and TNFR:Fc on thymic damage during GVHD, as well as their effect on normal thymocyte development, will be assessed. We will also determine whether the cytokines IL-11 and keratinocyte growth factor (KGF), known to protect the GI tract and epithelial cells, will indirectly influence the cytokine cascade and its impact on the thymus. Finally, the total body irradiation (TBI) dose can markedly increase GVHD. We also plan to assess in this section the impact of TBI dose on thymic function. Specific Aim 3: We will test the hypothesis that specific cellular and biochemical events account for damage to the thymus in GVHD. We will analyze the effects of GVHD on thymic differentiation, loss of negative selection, decrease in thymic cellularity, and the effects of cytokine cascades on thymic function. The thymic cellular events associated with GVHD will be correlated to the activity of mitogen-activated protein (MAP) kinases and critical DNA binding proteins that are thought to regulate thymocyte development.

The CD4 and CD8 co-receptors play important functions in both thymocyte development as well as in cellular functions such as lymphokine production, cell-mediated cytotoxicity and proliferation. We, as well as others, have observed that the level of Lck kinase activity induced upon CD4 or CD8 co-receptor crosslinking does not correlate with the ability of the co-receptor to enhance IL-2 production. These data question the importance of the Lck kinase activity associated with CD4 and CD8. We have studied the role of Lck kinase activity associated with the CD8 co- receptor in thymocyte development. Mice with a "knock-out" of the CD8beta chain have >80% fewer single positive (SP) CD8 thymocytes and peripheral CD8 T cells. When we studied the Lck kinase activity induced upon CD9 crosslinking in these mice we observed that their Lck activity was markedly reduced. In fact, the level of CD8 associated Lck kinase activity was proportional to the percent of CD8 SP thymocytes that developed. Thus it appears that the CD8beta chain is required for Lck kinase activity and this kinase activity is required for efficient differentiation from double positive (DP) to SP CD8 thymocytes. To test the role of the Lck kinase activity associated with CD8 we have generated a transgene with the cDNA encoding the extracellular and transmembrane domains of murine CD8alpha ligated to Lck (CD8alpha-Lck). This chimera was inserted into the CD2 minigene and it has been expressed in sv129 mice and the following specific aims are proposed. Mice expressing this transgene will be bred to CD8beta-/- mice to determine whether expression of the transgene will reconstitute SP CD8 development. If differentiation is reconstituted then we will express a transgene with a kinase dead Lck, CD8alpha-Lck(R273), to determine whether the kinase activity is critical. If the CD8alpha-Lck transgene fails to reconstitute beta-/- mice then they will be bred to CD8alpha-/- mice, which have endogenous CD8beta to determine if the transgene can reconstitute development in the presence of the beta chain. If CD8alpha- Lck reconstitutes SP CD8 development then we will analyze the biochemical and cellular functions of the cells expressing this transgene. Similar experiments are planned to determine if a CD4-lck transgene expressed in CD4-/- mice can reconstitute SP CD4 thymocyte differentiation. Because of the limited number of cells available to perform biochemical studies from mice expressing the transgene we will express CD8alpha-Lck in a T cell hybridoma and identify tyrosine phosphorylated substrates in this model system. This approach should allow more targeted biochemical experiments using thymocytes from transgenic mice.

The Kaplan Comprehensive Cancer Center (KCCC) is the organization unit of NYU Medical Medical Center bringing together basic, epidemiologic, and clinical scientists to further the progress against cancer. Created in 1975, and now in its 23rd year of uninterrupted NCI core grant funding, the Center seeks five additional years of support. Recent changes in leadership have engendered the strengthening of the Center's programmatic structure, shared resource units and overall commitment by NYU Medical Center. Renewed institutional support to the KCCC is manifested in additional research and clinical space, and financial and organization status comparable to other academic departments; in addition, the new director, Franco M. Muggia, M.D., holds a concomitant appointment as Associate Dean of the medical school. Since assuming the director's position on July 1, 1997, Dr. Muggia has promoted a further integration of clinical and laboratory investigators within the outstanding basic science programs and has sought to strengthen translational research in environmental carcinogenesis and population science, based at the Nelson Institute of Environmental Medicine-a unique feature of this cancer center. Future plans embrace the vision of greater organ-oriented research through new programs in Breast and Genitourinary Cancer Research; another such study, Neuro-oncology, is presented as a developing program. Further reflecting the cross-fertilization of scientific disciplines, two new shared resources have been created: Population Genetics and Molecular Diagnostics. Blending the Center's overall expansion with selected organizational consolidation, we envision continued significant accomplishment through the 9 programs and 13 shared resources which foster and facilitate interchange among its 187 members. Their productivity promises to be reflected in cancer prevention and treatment efforts within the population we serve, and in research advances felt nationally and internationally.

DESCRIPTION (provided by applicant): The NYU Cancer Institute (NYUCI), previously known as the Rita J. and Stanley H. Kaplan Comprehensive Cancer Center (KCCC), is a university based matrix organization, located principally on the main campus of the NYU Medical Center in mid-town Manhattan. In the last three years, the NYUCI has received greatly increased support from its parent organizations, the NYU School of Medicine and the NYU Hospitals Center and has gone through intensive strategic planning to achieve its present integration of basic, translational, clinical, and population science. With this expansion of its role, the KCCC was accorded "Institute" status. Five formal Basic Science Programs are presented. Three of these, Environmental & Molecular Carcinogenesis, Growth Control & Tumor Immunology, have been included in prior CCSG application; however, each has added new areas to complement previous strengths and, in the case of the latter two, are either being led by or have added new members who have revitalized the Programs. Cancer Neurobiology and Stem Cell Biology represent the culmination of new initiatives resulting from the bringing together new members of the NYUCI into collaborations with senior scientists of the NYU School of Medicine and its Skirball Institute of Biomolecular Medicine. One additional Basic Science Program, in the area of Endothelial Research, is in development. Two multi-disciplinary, Disease-Oriented Programs, in Breast & Genitourinary Cancer, represent our emphasis on therapeutic and translational research. Four others, Neuro-oncology, Gynecologic Oncology, Gastrointestinal Oncology & Cutaneous Malignancies, are in development. Finally, one Population Science Program, Molecular Epidemiology & Prevention, demonstrates a broad range of interests in the identification of cancer risk factors and the translation of such knowledge into preventive and clinical interventions at the population level. In addition, nine Shared Resources are presented, with two others in development. Developmental funds for pilot studies, recruitment of new investigators and the establishment of new shared resources are also requested.