Harley Y. Tse - US grants

The objective of this proposal is to develop a clearer understanding of the roles of T lymphocytes in the induction of the autoimmune disease, experimental allergic encephalomyelitis (EAE). EAE is a paralytic and inflammatory disease of the central nervous system (CNS) and in many respects resembles the pathology of the most common human demyelinating disease, multiple sclerosis (MS). Recent development of an adoptive transfer system makes it possible now to induce chronic relapsing phases of the disease in mice. This study will use genetic, immunochemical and T lymphocyte cloning techniques to establish identity or lineage relationship between the transferred cells and the T lymphocytes localized at the CNS lesion sites. This is significant because while T lymphocytes have been implied in the induction of EAE, the mechanisms of EAE induction remain unclear. The design of the experiments takes advantage of the fact that murine T lymphocytes characteristically express a cell surface antigen, Thy-l. Although this antigen is also expressed on some neural cells, the existence of allelic forms of this antigen still makes it a useful marker for cell trafficking studies. By adoptively transferring Thy-1.2+ MBP- specific T lymphocyte clones into naive Thy-1.1+ congenic recipients, the migratory pattern of the transferred cells in the recipients can be followed. Frozen CNS sections of recipients before, during, and after the paralytic attack will be examined for the presence of the transferred cell by immunoperoxidase staining techniques. Factors such as irradiation and anti-la treatment will be examine, to determine their effects on the migratory pattern of donor cells. Recent studies show that lymphocytes localized at CNS lesion sites can be cultured and cloned in vitro. Characterization of these clones in terms of the Thy-l marker, antigen specificity, lymphokine production and encephalitogenicity will reveal whether these cells are related to the donor cells and the extent of the host's T lymphocyte participation in the manifestation of the disease. These experiments are even more important when applied to the chronic relapsing phase of the disease. The migratory pattern and functions of both the donor and host cells during the remission-relapse cycles will be studied. Experiments are designed to determine whether donor T lymphocytes migrate in and out of the CNS during the cycles and the possibility of a host-derived suppressor T lymphocyte dampening the inflammatory response during temporary remission. Taken together, these studies will provide a better appreciation of the underlying mechanisms of MS.

This candidate joined the faculty of Department of Immunology and Microbiology at Wayne State University in 1986 after six years at Merck & Co. Wayne State University is particularly strong in Neuroimmunology research. The candidate quickly seized the opportunity and had since developed a research program aiming at analyzing the T cell receptor gene expression as well as in vivo trafficking of encephalitogenic cells in the murine experimental autoimmune encephalomyelitis (EAE) model. A part of the trafficking study is currently funded by NIH. The study on T cell receptor heterogeneity is currently partly funded by a grant from the National Multiple Sclerosis Society. In addition, an overlapping application is under review at NIH. The immediate objective of the research program is to use a T cell surface antigen as a genetic marker to follow the in vivo trafficking of encephalitogenic cells leading to the development of EAE. Specifically, myelin basic protein (MBP)-specific donor Thy-1.2+ donor T cells will be injected into naive Thy-1.1+ recipients. The fate of the donor cells in the CNS and lymphoid tissues of the recipients will be followed by virtue of the Thy-1 marker which can be detected with monoclonal anti-Thy-1.2 antibodies. This approach is especially important for the investigation of the effector cell mechanisms in the spontaneously relapsing phase of the disease. The stable genetic marker, unlike radioisotope-labeled cells, can be traced over a long period of time. Recently, the candidate's laboratory made the discovery that additional antigenic challenge of cell transfer recipients can lead to disease development in many reputed 'resistant' strains of mice such as C57BI/6 and BALB/C. This experimental system opens up new avenues for studying the genetic basis of susceptibility to EAE induction. One approach is to examine the T cell receptor (TCR) gene usage by encephalitogenic cells from these two strains. The concept of limited heterogeneity in TCR gene usage has raised hope that perhaps modulation of autoimmune diseases can be achieved through manipulation of the TCR itself. It will be very important to confirm these findings in additional mouse strains other than PL and B10.PL, especially in mice that are normally resistant to disease induction. Eventually, this knowledge of disease resistance will be utilized to design therapeutic approaches to human demyelinating diseases. This research program is both challenging and rewarding. The award of a RCDA will enable the candidate to more aggressively pursue the objectives of the program and to foster closer interactions with neuroimmunology experts within and outside of the Department. As noted in the application, the candidate has the full support of the Department and the School.

The objective of this proposal is to develop a clearer understanding of the underlying mechanisms of the autoimmune disease, experimental allergic encephalomyelitis (EAE). EAE is used as a model with the hope that the knowledge gained can be applied to other autoimmune diseases. EAE is a paralytic and inflammatory disease of the central nervous system and in many respects resembles the pathology of the most common human demyelinating disease, multiple sclerosis (MS). Recent development in the analysis of the T cell receptor (TCR) at the molecular level has revealed that clones of autoreactive T cells only express a restrict number of the available T cell receptor specificities. This raises the hope that perhaps modulation of certain autoimmune diseases can be achieved through manipulation of the TCR itself. However, in the murine model, the analysis has so far been made only in three strains or strictly speaking, in two haplotypes because two of the three strains carry identical major histocompatibility complex genes and recognize the same myelin basic protein (MBP) T cell epitope. In rat, only the Lewis strain has been analysed in detail. Human study is still rather preliminary. To generalize the concept of limited heterogeneity in TCR gene usage by autoreactive T cells, this study will analyse the TCR specificities of two genetically diverse strains of mice, C57BI/6 and BALB/C. These two strains are unique in that induction of EAE by adoptive transfer fail to generate signs of disease in the recipient mice. EAE is induced only after the recipients are challenged at a later stage with MBP. The investigation of the nature of the TCR genes expressed in these "relatively resistant" strains will be of value to correlate immune mechanisms with TCR gene usage. To this end, MBP-specific T cell clones from B6 and BALB/C will be generated. Clones will be tested for encephalitogenicity and the MBP epitope they recognize. Cell surface expresion of TCR gene products will be monitored with specific monoclonal antibodies. At the molecular level, DNA probes specific for known V-region genes will be used to quantify RNA expression in encephalitogenic clones. Such analysis will also provide information regarding the prevalence of a specific V-region gene, V-beta- 8.2, in disease expression. Since B6 recipient mice do not develop disease until after challenged with MBP, this provides an ideal system to study the in vivo trafficking patterns of the transferred cells in relation to their "relative resistance" in EAE induction. By using the expression of the Thy-1 genetic marker, the presence of donor cells can be traced in various tissues of the recipient, especially in the brain and spinal cord. Taken together, these studies will provide a better appreciation of the underlying mechanisms of EAE and of MS.

The objective of this proposal is to develop a clearer understanding of the underlying mechanisms of the autoimmune disease, experimental allergic encephalomyelitis (EAE). EAE is used as a model with the hope that the knowledge gained can be applied to other autoimmune diseases. EAE is a paralytic and inflammatory disease of the central nervous system and in many respects resembles the pathology of the most common human demyelinating disease, multiple sclerosis (MS). Recent development in the analysis of the T cell receptor (TCR) at the molecular level has revealed that clones of autoreactive T cells only express a restrict number of the available T cell receptor specificities. This raises the hope that perhaps modulation of certain autoimmune diseases can be achieved through manipulation of the TCR itself. However, in the murine model, the analysis has so far been made only in three strains or strictly speaking, in two haplotypes because two of the three strains carry identical major histocompatibility complex genes and recognize the same myelin basic protein (MBP) T cell epitope. In rat, only the Lewis strain has been analysed in detail. Human study is still rather preliminary. To generalize the concept of limited heterogeneity in TCR gene usage by autoreactive T cells, this study will analyse the TCR specificities of two genetically diverse strains of mice, C57BI/6 and BALB/C. These two strains are unique in that induction of EAE by adoptive transfer fail to generate signs of disease in the recipient mice. EAE is induced only after the recipients are challenged at a later stage with MBP. The investigation of the nature of the TCR genes expressed in these "relatively resistant" strains will be of value to correlate immune mechanisms with TCR gene usage. To this end, MBP-specific T cell clones from B6 and BALB/C will be generated. Clones will be tested for encephalitogenicity and the MBP epitope they recognize. Cell surface expresion of TCR gene products will be monitored with specific monoclonal antibodies. At the molecular level, DNA probes specific for known V-region genes will be used to quantify RNA expression in encephalitogenic clones. Such analysis will also provide information regarding the prevalence of a specific V-region gene, V-beta- 8.2, in disease expression. Since B6 recipient mice do not develop disease until after challenged with MBP, this provides an ideal system to study the in vivo trafficking patterns of the transferred cells in relation to their "relative resistance" in EAE induction. By using the expression of the Thy-1 genetic marker, the presence of donor cells can be traced in various tissues of the recipient, especially in the brain and spinal cord. Taken together, these studies will provide a better appreciation of the underlying mechanisms of EAE and of MS.

DESCRIPTION: This application sets forth studies to examine the chronic relapsing EAE model in the mouse. The principal investigator has discovered that the SJL.B mouse, unlike the SJL, does nor develop a chronic relapsing EAE (CREAE) disease as does the SJL. This is reversed in the B10 mouse in which the B10.S gets CREAE while the normal B10 does not. This indicates to the principal investigator that in the H-2b there is a gene which might encode for resistance to relapses in CNS inflammation. In this set of studies he sets forth to discover the explanation for this phenomenon. His project is divided into three Specific Aims, some of which have subaims. In the first Specific Aim he will investigate recombinants of the B10 background in which the MHC is partially present. In this manner he hopes to narrow the area containing the locus that might control the CREAE phenotype. While a limited number of congenics exist, he will utilize those available and only in the extreme try to generate new ones of his own. The second Specific Aim is large an has five subaims. all are directed toward defining the cellular basis of the non-relapsing phenotype of the SJL.B. In (A) he will investigate the ability of the SJL.B to respond to PLP peptides and whole molecule. This might be relevant if the lack of relapses is due to failure of epitope spreading. In (B) he will further investigate if the non-relapsing phenotype is a lack of epitope spreading to other areas of the initiating antigen, MBP. In this he will use his thy1 congenic mice immunized with MBP and he will test proliferation and ELISPOT assays to define the ability of this mouse to respond to different areas of the MBP molecule first using cleavage fragments followed by synthetic peptides). In (C) he will determine if the absence of CREAE in SJL.B is because there is loss of the initial encephalitogenic (donor) cells or if these donor cells become inactivated or shift to Th2. The fourth subaim (D) will seek to learn if the SJL.B's resistance to CREAE can be corrected by manipulations of cytokines afforded by giving superantigens or antibodies against cytokines. In (E) he hopes to define if the level of resistance to CREAE resides in the encephalitogenic cells themselves or in the recipients tissues. He will do this by reciprocal T cell transfers. In the third and final aim, he hopes to determine if the initial administration of donor (encephalitogenic) T cells is responsible for or related to the CREAE phenomenon by remaining persistent and/or activated in the host. In subaims of this part he will (A) give the normal SJL Thy1 tagged pathogenic cells from an animal transgenic for an anti-MBP peptide TcR. These cells will be encephalitogenic, and also continuously detectable because of their distinct Thy1 molecule. In (B) he will as if these cells retain their pathogenicity and Th1 phenotype during active recurrence of EAE or remission. Part (C) are the donor encephalitogenic cells actually needed for relapses. He will delete them with antibodies against the encephalitogenic cells initially infused, thus eliminating them. Further relapses could not be due to their pathogenic influence. Finally (D) he will seek to discover if the initially infused T cells are expand or if their trafficking patterns (CNS vs. periphery -- spleen) change between remission and relapse. In this he will give BrdU to mice in remission and seek to discover if the cells found in the CNS during relapse are positive for the marker.

DESCRIPTION (provided by applicant): Experimental autoimmune encephalomyelitis (EAE) is a central nervous system (CMS) autoimmune disease that resembles the clinical and pathological features of human multiple sclerosis (MS). In both cases, there are clearly regulatory mechanisms as an individual can be susceptible or resistant to the disease. In addition, individuals recovering from the initial attack may fall victim to subsequent spontaneous cycles of remission/relapses relapses. This laboratory's long-term goals are to understand the mechanisms underlying EAE resistance and development remitting/relapsing disease. Recently, regulatory T cells (Treg) have been shown to inhibit the activation of immune effector cells. Of particular relevance is the finding that depleting natural Treg cells (nTreg) renders mice susceptible to EAE induction. We find that this loss of resistance occurs in one strain of mice (SJL.B) but not in another (B6). Specific aim 1 will investigate if this Treg mechanism is universally utilized by EAE resistant mouse strains to maintain resistance of disease induction. A hypothesis to account for the insensitivity of B6 mice to anti-CD25 treatment is also proposed. Specific aim 2 explores the possibility that antigen-induced Treg cells (Treg) may have differential characteristics from nTreg cells. By using Thy-1 congenic mouse strains developed in this laboratory, the trafficking patterns of Treg cells from the lymphoid tissues to the CNS will be tracked. Of particular interest is the tissue locations whereby Treg cells most efficiently inhibit encephalitogenic effector cells to prevent disease. A possible link between Treg cells and remitting/relapsing EAE is made in specific aim 3. Here, the mechanisms of remitting/relapsing EAE will be investigated from the prospects of epitope spreading, Treg cells and the roles of the acute disease-initiating T cells, using Thy-1 congenic mice. In summary, this project will provide a better understanding of how regulatory T cells help to resist development of EAE and MS. The project also studies the mechanisms of how disease relapses occurs, with the ultimate aim of designing better therapeutic approaches toward management of this autoimmune disease.