The investigators in this program are Dr. Ramesh Ganju (HIV and HCV/HIV co-infection), Dr. Xue-Feng Bai (autoimmunity, cancer immunotherapy), and Dr. Larry Lasky (ex vivo blood cell production).
HIV and HCV/HIV Pathogenesis
Project 1: Worldwide about 40 million people are infected with human immunodeficiency virus type 1 (HIV-1). The depletion of CD4+ T lymphocytes is a central pathogenic feature of HIV-1 infection and is largely responsible for the profound immunodeficiency characteristic of the late stages of HIV disease. Dr. Ganju’s laboratory is analyzing the molecular mechanism of T-cell loss during HIV infection. They have shown that cross-talk between the chemokine receptor CXCR4/CCR5 and immune signaling components modulates the activities of novel apoptotic effector molecules. Dr. Ganju’s laboratory is further defining and elucidating HIV-induced apoptotic signaling mechanism in primary cells isolated from healthy and HIV-infected individuals, and determining how these signaling cascades could be altered to prevent the loss of immune cells during HIV infection. Project 2: Dr. Ganju’s laboratory is also exploring novel molecules to inhibit HIV infection by blocking CXCR4/CCR5 function. In this regard, they have recently shown that a neuronal guidance cue, Slit2 blocks CXCL12/CXCR4-mediated functional effects in T-cells. Since the chemokine receptors, CXCR4, along with CCR5 are important co-receptors for HIV-1 virus and attractive targets for development of anti-HIV therapies, they have explored the potential role of Slit2 in HIV infection. They have documented for the first time an important role for Slit2 as an inhibitor of HIV-1 replication. They have shown that Slit2 attenuated the replication of diverse clinical isolates and drug-resistant strains of HIV-1. Dr. Ganju’s laboratory has also performed studies to identify the mechanism of Slit2-mediated inhibition of HIV-infection. The above studies could facilitate the development of innovative therapeutic strategies against HIV-1 infection. Project 3: HIV-1 infection in T-cells is regulated by T-cell receptor (TCR) activation. However, the cellular proteins of the TCR pathway that regulate HIV-1 infection are poorly characterized. Dr. Ganju’s laboratory has elucidated the role of SLP-76, a key adaptor protein in the TCR signaling complex, in HIV-1 replication. They have demonstrated a novel function of the adaptor molecule, SLP-76, in regulating HIV-1 replication in T-cells with potential to develop innovative strategies against HIV-1. They are further pursuing studies on the role of T-cell receptor signaling components in modulating HIV infection in T-cells. Project 4: Dr. Ganju’s laboratory is also elucidating the molecular mechanism of hepatic injury in Hepatitis C virus (HCV) or HCV/HIV co-infection. Worldwide about 200 million people are infected with HCV and about 40 million are infected with HIV. HCV infects approximately 40% of patients with HIV. HCV/HIV co-infected patients have progressive liver disease that leads to cirrhosis and death. However, the molecular mechanism of enhanced cirrhosis and inflammation observed in these patients is not known yet. Dr. Ganju’s laboratory observed that HCV and HIV envelope proteins induce apoptosis and inflammatory responses in hepatocytes via a novel “innocent bystander” mechanism. They are further elucidating the molecular mechanisms of liver fibrosis caused by HCV/HIV co-infection. In this regard, they have shown that HCV-infected hepatocytes induce the pro-fibrogenic cytokine, connective tissue growth factor through TGF-beta-dependent molecular pathways that in turn mediates liver fibrosis. They are further analyzing the role of these cytokines in HIV/HCV-mediated liver fibrosis. These studies may help in the development of novel anti-fibrotic strategies in HCV/HIV co-infected patients.
Immune Regulation in Experimental Models of Multiple Sclerosis
Multiple sclerosis (MS) is a neurological disorder affecting about 1/1000 Caucasians. It is generally agreed that MS is driven by autoimmune mechanisms. However, very little is known about the pathogenesis of MS, therefore animal models for MS are useful tools for studying disease pathogenesis. The most widely used animal model for MS is experimental autoimmune encephalomyelitis (EAE). In the development of EAE, myelin antigen specific CD4 T cells must be activated, clonally expanded in the lymphoid organs, and must migrate into the central nervous system (CNS) where they undergo further activation. Thus, molecular interactions involved in T cell activation, T cell homing to the CNS, T cell reactivation and survival in the CNS can affect EAE development. Dr. Bai’s studies have revealed that CD24, which was originally described as a co-stimulatory molecule, is required for the pathogenesis of EAE (Journal Clinical Investigation 2000). Further studies suggest that CD24 on the local antigen presenting cells in the CNS can optimally stimulate encephalitogenic T cells in the CNS and makes T cells more aggressive (Journal of Experimental Medicine 2004; Journal of Immunology 2007). His lab’s recent studies suggest that CD24 is also involved in the thymic generation of myelin antigen specific T lymphocytes (Journal of Immunology 2008). Dr. Bai’s future work is to investigate if the involvement of CD24 in thymic generation of myelin antigen specific T cells is a general phenomenon, if so, what are the mechanisms.
Cancer antigen-specific cytotoxic T lymphocytes (CTL) are the major effectors against cancer cells. However, large established tumors are usually not fully controlled by CTL for at least two reasons. First, large established tumors have immune suppressive networks that not only suppress CTL effector function but also permit tumor progression. Second, the genetic instability of cancer cells often results in the selection of antigenic variants by CTL, which allow cancer cells to escape destruction. Simply enhancing T cell capacity may not fully control large established tumors. Other measures such as targeting tumor microenvironment should also be considered. Using transgenic T cells specific for a natural tumor rejection antigen P1A (P1CTL) to treat mice with large established plasmacytoma J558 tumors, Dr. Bai’s laboratory has found that P1CTL selected high numbers of mutations in the P1A antigenic epitope, a phenomena known as antigenic drift. Antigenic drift is a mechanism used by viruses to evade CTL responses, they have shown that cancer cells, like virus, can also use this mechanism to evade CTL destruction (Journal of Clinical Investigation 2003). Moreover, they have shows that this mechanism, although not restricted to B cell tumors, mainly occurs in plasmacytoma cells (Cancer Research 2006). Dr. Bai’s group will study strategies to overcome immune evasion during T cell therapy of cancer by targeting activation-induced cytidine deaminase (AID) and B7 family molecule CD200.
Ex vivo Blood Cell Production
Dr. Lasky’s research involves in vitro production of blood cells. The current project involves the production of platelets for transfusion. Within this project several lines of research are being pursued. Dr. Lasky and colleagues have developed a modular 3D perfusion bioreactor system that allows gas and medium support of a 3D scaffold while allowing collection of newly produced cells on a continuous basis, mimicking the environment found in the marrow. Dr. Lasky and colleagues have succeeded in production of large quantities of platelets in the system, discovering that in the right conditions the system can continuously produce functional platelets for more than a month. The laboratory is currently seeking ways to increase the number and quality of platelets produced. This includes manipulation of the gas mixture provided to the cultures, utilizing reduced oxygen concentration early in the in vitro platelet production process. Studies of the use of varied amounts of shear stress to promote proplatelet release of increased numbers of platelets, along with manipulation of media flow along the surface and through the cell-scaffold constructs, are being carried out. Variations in the media used in the system will also be studied, including the addition of thrombopoietin mimetics to enhance platelet production.
The scaffold used in this bioreactor to date has been polyester fibrous fabric. One line of research is the development of a purpose-built 3D scaffold for use in the bioreactor system. Dr. Lasky is collaborating with Professor Nicholas Kotov at the University of Michigan on the development of a hydrogel scaffold with structure and coating that promote platelet production. Currently, cord blood is being used as the starting material for platelet production in the bioreactor system. Because cord blood contains a fixed number of hematopoietic cells, Dr. Lasky is pursuing two lines of research to maximize the number of early hematoietic cells in the system. The first is to make use of HOXB4 fusion proteins to promote early cell self-renewal in the first stages of platelet production. The second is to study the use of embryonic stem cells and induced pluripotent stem cells, rather than cord blood cells, to produce early hematopoietic cells in large quantities that can then be used to produce platelets.