a. Identification of the mitochondrial effects of GzmA and GzmB

b. Identification of the role of DNA repair pathways in GzmA-mediated cell death and in other forms of apoptosis

c. Mechanism of perforin action

d. Identification of the SNARE complex components involved in cytolytic granule exocytosis

We are interested in how CTL induce apoptosis in their target cells. In particular, we are studying the mechanism of action of granzymes A and B, the major proteases contained in the cytotoxic granules of CTL.

CTLs induce apoptosis of virus-infected or transformed cells primarily by the release of granules that contain a pore-forming molecule perforin and a family of serine protease granzymes. The two predominant granzymes (GzmA and GzmB) independently with perforin induce target cell death. We opened up research possibilities for studying these enzymes by figuring out how to make recombinant enzymes. Our focus has primarily been to understand the mechanism of action of GzmA. Although GzmB activates the caspase pathway of cell death, we have found that either enzyme can activate caspase-independent pathways of cell death, which are significant since many viruses or tumors have devised strategies for evading caspase-activated apoptosis. Others and we found that GzmB directly cleaves critical downstream caspase substrates such as lamin B and the inhibitor of the caspase-activated DNase. We found that the GzmA pathway is completely caspase-independent and active in caspase-resistant cells. This is the first detailed description of a caspase-independent cell death pathway.

a. Effect of overexpression or gene silencing of pp32, SET and APE on cell viability, cell cycle progression and response to stress and apoptosis

b. Identification of molecules that interact with SET and pp32

c. Study of regulation of SET complex nuclear translocation

GzmA-activated cell death has all the characteristic features of apoptosis, except DNA damage is uniquely single-stranded. GzmA targets the SET complex, an endoplasmic reticulum-associated complex we discovered, which contains the GzmA pathway DNase, a nucleosome assembly protein SET, a tumor suppressor protein pp32, the rate limiting base excision repair enzyme Ape1, and HMG-2, the DNA bending protein. The functions of the SET complex are not known, but there is some evidence that it is involved in chromatin remodeling to facilitate transcription, perhaps in response to stress. GzmA cleaves SET, Ape1 and HMG-2 (but not pp32) and in so doing disrupts their known functions and the ability of the cell to repair, forcing it to undergo apoptosis. We have also found that this novel pathway targets key nuclear substrates, including the histones and lamins.

We have recently described the novel pathway used by GzmA to induce DNA damage. The GzmA activated DNase was identified as another component of the SET complex, NM23-H1, a nucleoside diphosphate kinase implicated as a suppressor of tumor metastasis. GzmA activates NM23-H1 by cleaving its specific inhibitor, SET. Upon GzmA loading, NM23-H1 moves into the nucleus and is freed of inhibition by SET. Understanding how this pathway is regulated and understanding the normal functions of the SET complex in response to stress and in oncogenesis are our current focus.

a. 1. Examination of the role of inhibitory receptors on CD8 T cell function

We study the role of cytotoxic T lymphocytes (CTL) in antiviral immunity. A major focus of the laboratory centers around studies of the specific CTL response to HIV-1. HIV-specific CTL are key to the protective immune response to HIV-1 infection. Recently we have been trying to understand why the strong antiviral CTL response fails to control HIV infection. We have found that freshly isolated lymphocytes from HIV-infected individuals are not cytotoxic against HIV-presenting cells and especially in more advanced patients do not produce IFN-gamma.

In looking for the molecular basis for lack of CD8 T cell function, we have uncovered a number of possible mechanisms, some of which have turned out to be part of the normal regulation of CD8 T cell differentiation. Although regulation of CD4 T cells to prevent autoimmunity has been extensively studied, much less is known about how CD8 T cells might be regulated. Antigen-specific CD8 T cells have a higher threshold for signaling, in part because they have down-modulated cell surface expression of CD3zeta, the signaling chain of the T cell receptor, and the costimulatory molecule CD28. Lack of expression of these key signaling molecules leads to reduced expression of IL-2 and the high affinity IL-2 receptor after CTL activation.

We also found that HIV-specific CD8 T cells also have low levels of expression of perforin, the key molecule for lysis of targeted cells. We also found that they lack the homing molecules for trafficking efficiently to lymphoid sites of infection. These properties seem not to be unique to HIV infection; they are shared by cells specific for other persistent infections, like EBV and CMV. How CD8 T cell function is regulated in the setting of persistent infection continues to be a major focus.

Our laboratory was the first to show that the recognition of HIV by CD8 T cells is focused on the recognition of a small number of immunodominant epitopes. I also showed that the dominant epitopes recognized in a genetically diverse population are highly variable and cannot be predicted on the basis of HLA expression. Moreover recognition was less often restricted by the most commonly expressed HLA alleles. This has significant implications for vaccine development and suggests that epitope-based vaccines are unlikely to be useful. Our laboratory developed methods for expanding large numbers of HIV-specific CD8 T cells with potent lytic ability against HIV-infected cells. We showed that the frequency of HIV-specific CD8 T cells in the circulation of HIV-infected donors was substantially higher than the estimates from limiting dilution analysis, and this was later borne out as newer technologies to label antigen-specific cells became available.

We pioneered the use of immune-based therapy to treat HIV infection in pilot trials of infusion of billions of HIV-specific CD8 T cells in the era before effective antiretroviral drugs became available. Although we demonstrated that the infusions were feasible and safe, whatever improvements were seen, and the responses were heterogeneous, were short-lived. We investigated therapeutic immunization to treat HIV infection using the Salk Remune vaccine. Since 1990, Judy Lieberman has played a leadership role in advocating and developing immune-based therapies to treat HIV infection, serving on a number of private and NIH-sponsored groups to organize efforts in this field.

Our lab was the first to show that HIV-specific CD8 T cells have impaired function during chronic infection. Since then, a number of murine and human studies have identified functional defects in cytotoxicity or cytokine secretion by antiviral CD8 T cells, and in conjunction with our studies of chronic EBV, CMV and HIV infection, we have found that these defects are not unique to HIV but are integral to regulation of CD8 T cell function in the setting of chronic antigenic exposure. We were the first to show that impaired cytotoxic and cytokine production could be reversed by exposure to high concentrations of IL-2, suggesting a possible therapeutic benefit of cytokine therapy. In the past five years we have identified several novel molecular mechanisms that regulate CD8 T cell function in the setting of chronic infection, including down-modulation of CD3zeta and CD28, key signaling molecules, inability of antiviral CD8 T cells to express the high affinity IL-2R or produce IL-2 after stimulation, lack of perforin expression, and lack of expression of receptors that direct lymphocyte trafficking to lymphoid sites of infection. Our plan is to continue to study CD8 T cell differentiation and regulation of function in human in vitro studies and in a cleaner murine system.

a. Murine and macaque studies of immune response to attenuated Listeria monocytogenes expressing HIV proteins

b. Effects of route and dose on generating mucosal immunity

We have helped develop two candidate vaccines designed to elicit T cell immunity to prevent HIV infection, using an attenuated Listeria monocytogenes expressing HIV gene products and delivering HIV proteins using proteins engineered with detoxified anthrax toxin. We have been involved in the immunological evaluation of these vaccines in vitro and in mice and in developing the strategic approach to translating the preclinical work into proof of concept clinical trials.

The Listeria vaccine is particularly attractive because it can be administered orally and because it is an incredibly potent inducer of CTL and TH1 type responses as well as of antibodies. Moreover in mice it has been demonstrated to provide protective immunity against a variety of viruses. In a vaccinia-gag challenge model, it provides protection against mucosal (rectal) or systemic challenge. We have recently begun primate studies of the Listeria vaccine and have safely administered 10¹² bacteria orally with no safety issues and with preliminary evidence of immunogenicity in macaques.

5. Translational studies of RNA interference

a. Using RNAi as a therapy to silence apoptotic or proinflammatory genes

b. Using RNAi to treat or prevent viral infection

c. Developing methods for delivering siRNAs for systemic therapy, including targeted delivery to specific cell types, and for mucosal delivery

d. Developing a topical RNAi-based microbicide to prevent sexually transmitted diseases

Recently we have been investigating the translation of the technology of RNA interference for prevention or therapy. In collaboration with the laboratories of P. Sharp and P. Shankar, we showed that this technology can be harnessed to suppress HIV replication in uninfected or already infected cells in vitro by targeting HIV genes as well as host receptors or coreceptors. We have been able to extend this to show complete in vitro suppression lasting for weeks in primary macrophages and T cells. More recently we are investigating delivery methods that should be applicable in vivo. We have also shown that it is possible to inject duplex short interfering RNAs intravenously to prevent and treat autoimmune hepatitis in mice. This was the first demonstration of the efficacy of RNA interference in vivo. We are currently working to study the best delivery methods to target different cell types and tissues as well as the application of RNAi to target viral infections as well as stereotypical immune responses that lead to morbidity and mortality in the setting of diverse noxious insults.

We are also working to develop siRNA into a therapeutic modality.

a. Role of microRNAs in self renewal and tumorigenicity of breast cancer stem cells

b. Role of microRNAs in breast cancer metastasis

c. Role of host microRNAs in HIV infection

d. Role of microRNAs in hematopoietic cell differentiation

e. Developing methods to identify microRNA targets