Eckhard R. Podack, M.D., Ph.D.

Professor and Chair of Microbiology and Immunology
Room 3045, Rosenstiel Medical Sciences Building
1600 NW 10th Avenue
Telephone: 305-243-6694
Fax: 305-243-5522
Email: epodack@med.miami.edu

Research Interests:

Induction of Immunity by heat shock protein gp96-Ig

The function of heat shock proteins is to chaperone intracellular oligo-peptides generated during protein synthesis and degradation, including peptides encoded and generated by infectious agents and tumor antigens. The immune system via heat shock protein receptors (CD91) on APC uses a sophisticated system to detect cell damage and monitor peptides that are associated with liberated heat shock proteins by cross presentation to CD8 cells.

By replacing the KDEL retention signal of human gp96 with the IgG1-Fc portion we generated a secretory form of gp96. Gp96-Ig secreted from gp96-Ig-transfected tumor cells in vivo mediated strong, cognate CD8-CTL expansion, caused tumor rejection and generated long term anti-tumor immunity. Secreted gp96-Ig-associated peptides are cross presented to cognate CD8 cells in vivo with about ten thousand fold increased activity when compared to cross presentation of the native protein alone. Our data suggest that tumor secreted gp96-Ig triggers activation of dendritic cells that subsequently recruit and activate NK cells and cause clonal expansion of cognate CD8 CTL. The molecular and cellular mechanism of gp96-triggered DC-NK-CD8 interaction is studied and the influence of established tumors evaluated.

Graph of Enhanced Antigen Cross Presentation by gp96-Ig

Immunotherapy for Advanced Non Small Cell Lung Carcinoma (NSCLC)

We have generated data and the conceptual frame work to support a paradigm-shift in immunotherapy of cancer: Non-immunogenic tumors such as non-small cell lung carcinoma (NSCLC) are ideal targets for vaccine therapy. Data: In a phase I vaccine study nineteen patients with NSCLC, stages IIIB/IV (6-8 months expected median survival), were vaccinated with an allogeneic NSCLC line transfected with both B7.1 (CD80) and HLA A1 or A2 (B7-vaccine). The B7-vaccine was safe and very efficient in generating statistically highly significant increases in the frequency of NSCLC-specific CD8 CTL detected by IFN-gamma ELI-spots and showed a strong trend for clinical efficacy. Six patients achieved stable disease or partial responses, five of these are alive, three in remission for more than three years.

Concept and hypothesis: We propose to further test the hypothesis that non-immunogenic tumors are susceptible to active immunotherapy with allogeneic cell based vaccines. Non-immunogenic tumors such as NSCLC do not prime a spontaneous immune response and do not spontaneously generate cytotoxic T cells (CTL) and therefore, too, have not been able to evolve resistance mechanisms against cytotoxicity. They may thus be susceptible to rejection by CTL generated by vaccination.

It is our hypothesis that whole-cell, allogeneic vaccines are superior to single antigen vaccines or even to vaccines combining several antigens, because they generate a multivalent CTL-attack forestalling immune evasion of the tumor. It has been shown that allogeneic NSCLC tumors from different individuals share tumor antigens, justifying the deployment of allogeneic cell based vaccines. Allogenicity insures early recruitment of NK and T cells by the vaccine which is further enhanced by B7 and HLA A transfection.

We plan to determine the safety and effectiveness of the B7 vaccine strategy in NSCLC-patients (stage IIIB/IV) who have responded to initial chemotherapy. In an innovative approach, we will vaccinate patients early after chemotherapy. We hypothesize and will determine that during the phase of homeostatic leukocyte expansion post-chemotherapy, immune responses to vaccination may be heightened. In a placebo controlled study we will determine safety, clinical response and immune response to the vaccine cells and determine whether the patients' autologous tumor is recognized by elicited CTL.

Immune response and clinical response to immunotherapy of advanced non small cell lung cancer patients with B7 vaccine

TNFR SF#25 (Death Receptor 3) and its ligand TL1A (TNF SF#15) mediate TH2 bias, contribute to asthma and colitis, control Tregs and support CD8 CTL expansion

The biological function of TNFR-SF25 (TNFR25) is not known. TNFR25-transgenes expressed on T cells were used to determine the physiological function of TNFR25, a member of the TNF-receptor family expressing an intracellular death domain. The full length form of TNFR25, a dominant negative form of TNFR25 (DN-TNFR25) lacking the intracellular death domain and an alternatively spliced form of TNFR25 (delta 5,6-TNFR25) lacking exon 5 and 6 encoding the fourth extracellular cysteine rich domain were analyzed. Transgenic expression of TNFR25 on T cells mediated TH2 polarization of cytokine and antibody production upon T cell activation and antigen exposure. In addition transgenic TNFR25 partially inhibited TCR driven proliferation of CD4 and CD8 cells and reduced total T cell numbers in lymphoid organs without inducing apoptosis. CD8 cells were more affected by TNFR25 than CD4 cells. We conclude that TNFR25 signals may be important in effector responses to pathogens by shaping the ensuing polarization towards TH2 or towards a mixed TH1/TH2 response.

TNFR25 transgenic mice were highly susceptible to antigen induced airway inflammation in an asthma model in mice and produced increased quantities of IL-13 and eosinophils in the lung upon antigen exposure by inhalation. Transgenic mice expressing a dominant negative form of TNFR25 showed increased resistance to airway hyper reactivity when compared to w.t. mice. Similarly, a blocking anti TL1A antibody was able to ameliorate asthma in wild type mice indicating that TNFR25 and TL1a are involved in the pathogenesis of asthma.

Asthma model showing TL1A and TNFR25 interactions among pulmonary DCs, NKT cells, and CD4 T cells in the draining lymph nodes.

CD30 - Governor of T cells?

CD30-L k.o. mice when challenged with tumor secreted gp96-Ig exhibit severely diminished CD8 CTL expansion. When used as allogeneic bone marrow graft recipients, Dr. Levy's laboratory showed that CD30-L k.o. exhibit diminished graft versus host disease in a MHC II mismatch. CD30 is highly expressed on CD45-RO memory cells and serves as T cell costimulator and as regulator of trafficking molecules and of pro and anti apoptotic molecules. CD30 signals lead to IL-13 and IFN-gamma production. The function of CD30 and its ligand in tumor rejection following vaccination is studied.

Macrophage-perforin, a new member of the Perforin/C9 family of proteins

Searching the genomic data base with perforin as the query sequence we found two novel members of the perforin family. Structure analysis suggests that the novel members have a typical pore forming domain but that the proteins themselves are membrane anchored. EST analysis suggests that one new perforin member is expressed in trophoblast cells while the second member is expressed in macrophages. We have cloned the macrophage-perforin (macrophage-Pf = MAPF) and fused a gfp tag to it for ease of detection. Expression of MAPF-gfp in NIH 3T3 cells and in 293T cells results in fluorescence in the nucleus and in the cytoplasm. However, fluorescent cells subsequently round up and die and after several days no fluorescence is detected. The data suggest that expression of macrophage-Pf leads to cell death, putatively by ectopic expression of a pore former. The data further suggest that macrophage-perforin and trophoblast-perforin have essential lytic functions that need to be carefully regulated for expression. We are in the process of deleting Macrophage-Pf and Trophoblast-Pf (TROPF) in order to discover their physiological function. The 5'-untranslated sequence of MAPF mRNA regulates MAPF translation. It contains three short open reading frames suggesting translational control via translation initiation factors.

Proteins with Membrane Attack Complex/Perforin Domains

Highlights

• Lung tumor vaccine (B7-vaccine) is safe and appears to be active in advanced cancer patients
• Discovery of a novel pore-former, Macrophage-Perforin (MAPF), a new member of the Perforin family
• TNFR SF25 does not kill, but produces IL-13 which is immuno suppressive and mediates asthma
• Ten thousand fold enhancement of antigen cross presentation by heat shock protein gp96
• April k.o. mice show increased incidence of spontaneous myositis
• CD30 signals induce c-IAP2 expression and MMP9 and IL-13 production in lymphocytes triggering TGF-B mediated immune suppression

Selected Publications:

Blazar, B. R., R. B. Levy, T. W. Mak, A. Panoskaltsis-Mortari, H. Muta, M. Jones, M. Roskos, J. S. Serody, H. Yagita, E. R. Podack, and P. A. Taylor. 2004. CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease. J Immunol 173:2933.

Raez, L. E., P. A. Cassileth, J. J. Schlesselman, K. Sridhar, S. Padmanabhan, E. Z. Fisher, P. A. Baldie, and E. R. Podack. 2004. Allogeneic vaccination with a B7.1 HLA-A gene-modified adenocarcinoma cell line in patients with advanced non-small-cell lung cancer. J Clin Oncol 22:2800.

Marks, L., N. H. Altman, E. R. Podack, and R. B. Levy. 2004. Donor T cells lacking Fas ligand and perforin retain the capacity to induce severe GvHD in minor histocompatibility antigen mismatched bone-marrow transplantation recipients. Transplantation 77:804.

Raez, L. E., P. A. Cassileth, J. J. Schlesselman, S. Padmanabhan, E. Z. Fisher, P. A. Baldie, K. Sridhar, and E. R. Podack. 2003. Induction of CD8 T-cell-Ifn-gamma response and positive clinical outcome after immunization with gene-modified allogeneic tumor cells in advanced non-small-cell lung carcinoma. Cancer Gene Ther 10:850.

Dix, R. D., Podack, E. R., and Cousins, S. W. Murine cytomegalovirus retinitis during retrovirus-induced immunodeficiency (MAIDS) in mice: interleukin-2 immunotherapy correlates with increased intraocular levels of perforin mRNA. Antiviral Res, 59: 111-119., 2003.

Dai, J., Liu, B., Caudill, M. M., Zheng, H., Qiao, Y., Podack, E. R., and Li, Z. Cell surface expression of heat shock protein gp96 enhances cross-presentation of cellular antigens and the generation of tumor-specific T cell memory. Cancer Immun, 3: 1., 2003.

Gulan, G., Ravlic-Gulan, J., Strbo, N., Sotosek, V., Nemec, B., Matovinovic, D., Rubinic, D., Podack, E. R., and Rukavina, D. Systemic and local expression of perforin in lymphocyte subsets in acute and chronic rheumatoid arthritis. J Rheumatol, 30: 660-670., 2003.

Strbo, N., Oizumi, S., Sotosek-Tokmadzic, V., and Podack, E. R. Perforin is required for innate and adaptive immunity induced by heat shock protein gp96. Immunity, 18: 381-390., 2003.

Dix, R. D., Podack, E. R., and Cousins, S. W. Loss of the perforin cytotoxic pathway predisposes mice to experimental cytomegalovirus retinitis. J Virol, 77: 3402-3408., 2003.

Podack, E. R., Strbo, N., Sotosec, V., and Muta, H. CD30-governor of memory T cells? Ann N Y Acad Sci, 975: 101-113., 2002.

Strbo, N., Yamazaki, K., Lee, K., Rukavina, D., and Podack, E. R. Heat shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo. Am J Reprod Immunol, 48: 220-225., 2002.

Laskarin, G., Tokmadzic, V. S., Strbo, N., Bogovic, T., Szekeres-Bartho, J., Randic, L., Podack, E. R., and Rukavina, D. Progesterone induced blocking factor (PIBF) mediates progesterone induced suppression of decidual lymphocyte cytotoxicity. Am J Reprod Immunol, 48: 201-209., 2002.

Tokmadzic, V. S., Tsuji, Y., Bogovic, T., Laskarin, G., Cupurdija, K., Strbo, N., Koyama, K., Okamura, H., Podack, E. R., and Rukavina, D. IL-18 is present at the maternal-fetal interface and enhances cytotoxic activity of decidual lymphocytes. Am J Reprod Immunol, 48: 191-200., 2002.

Nam, S. Y., Cho, K. S., Heo, Y. M., Ha, J. C., Kim, Y. H., Keun Yi, H., Han Hwang, P., Kim, H. M., and Podack, E. R. Regulation of lymphocyte clustering by CD30-mediated ICAM-1 up-regulation. Cell Immunol, 219: 38-47., 2002.

Raja, S. M., Wang, B., Dantuluri, M., Desai, U. R., Demeler, B., Spiegel, K., Metkar, S. S., and Froelich, C. J. Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin. J Biol Chem, 277: 49523-49530., 2002.

Par, G., Rukavina, D., Podack, E. R., Horanyi, M., Szekeres-Bartho, J., Hegedus, G., Paal, M., Szereday, L., Mozsik, G., and Par, A. Decrease in CD3-negative-CD8dim(+) and Vdelta2/Vgamma9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection. J Hepatol, 37: 514-522., 2002.

Harlin, H., Podack, E., Boothby, M., and Alegre, M. L. TCR-independent CD30 signaling selectively induces IL-13 production via a TNF receptor-associated factor/p38 mitogen-activated protein kinase-dependent mechanism. J Immunol, 169: 2451-2459., 2002.

Kawasaki, C., Ohshima, K., Muta, H., Muta, K., Deyev, V., Podack, E. R., and Kikuchi, M. Prognostic value of Bcl 10 rearrangement in diffuse large B-cell lymphoma. Leuk Lymphoma, 43: 823-826., 2002.

Merger, M., Viney, J. L., Borojevic, R., Steele-Norwood, D., Zhou, P., Clark, D. A., Riddell, R., Maric, R., Podack, E. R., and Croitoru, K. Defining the roles of perforin, Fas/FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine. Gut, 51: 155-163., 2002.

Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity, 16: 417-428., 2002.