Mario Roederer, Ph.D.Vaccine Research CenterBuilding 40, Room 550940 Convent DriveBethesda, MD 20892-3015Phone: 301-594-8491Fax: email@example.com
The ImmunoTechnology Section (ITS) is dedicated to understanding the roles and interactions of the individual components of the mature central immune system, with a particular eye toward the changes occurring during acute or chronic antigenic challenge. Understanding and quantifying these interactions and responses will be crucial to the evaluation of potential vaccine and immunotherapeutic strategies for treatment or prevention of HIV. In general, we are looking for immunological correlates of protection (for vaccines), and correlates of pathogenesis (in disease).
We are also actively involved in the development and advancement of cytometric technology: instrumentation, reagents, analysis, and assays. We carry out a wide range of collaborations to bring our unique and advanced technology (18 color flow cytometry) to other laboratories at the Vaccine Research Center (VRC), the National Institutes of Health, and around the world.
The major animal model for HIV disease is SIV infection in macaques. Our focus in this model is to understand the dynamics of lymphocytes and virus throughout the body. A complete understanding can only be achieved by examining multiple tissues (gut mucosa, lymph nodes, as well as blood). We recently demonstrated that acute SIV infection is accompanied by a massive infection and destruction of CD4 memory cells (Mattapallil et al., Nature, 2005)—and hypothesize that the extent of this destruction predicts subsequent progression and disease. We are now trying to understand the role of prior vaccination in ameliorating this destruction: what functions of lymphocytes impede the viral proliferation during the acute stage? As well, our study raises many interesting questions; for example, why, during chronic infection, is such a small fraction of memory CD4 T cells infected? Using other SIV-related models (thymectomy, drug treatment), we are trying to understand the complex lymphocyte dynamics that accompany SIV and HIV disease.
Mucosal immune responses are undoubtedly critical for successful HIV vaccines. However, measuring these responses on a large scale is impractical; our approach is to identify mucosal-homing T cells in peripheral blood and establish whether their function predicts mucosal immune responses. As well, we are studying the mucosal immune system after SIV/HIV infection to understand the role of this compartment in acute and chronic pathogenesis.
Using 18-color flow cytometry, we can identify hundreds of phenotypically distinct subsets of lymphocytes in peripheral blood. Most of these have unique functional profiles, as determined by cytokine production, proliferative capacity, or apoptotic potential. By examining the representation of antigen-specific responses for different pathogens among these subsets, we can gain an understanding of the roles of each of these subsets in disease.
We characterize the T cells that are induced by vaccination or natural infection both in terms of phenotype—differentiation markers—as well as function. In these experiments, we will make 4 to 6 distinct functional measurements on each cell (for example, production of multiple cytokines, cytotoxic activity, helper activity, proliferation), and use the remaining colors to identify the phenotypic identity of the cells. This gives us a detailed picture of the functional response as well as the differentiation stage of the antigen-specific T cells— measurements that may ultimately provide us with clinical correlates.
Using this technology, we showed that vaccination induces a broad range of T cell function in humans (De Rosa et al., J. Immunol., 2004). Importantly, we demonstrate that such measurements are necessary to give a much more complete picture of the response to vaccines, as no single functional measurement (like gIFN production) assesses the majority of antigen-specific cells for all vaccines.
The ImmunoTechnology Laboratory and Flow Cytometry Core at the VRC is the world-leader in multicolor flow cytometry. We continue to actively develop this technology on a number of different fronts; our focus has shifted from hardware development to reagent and analysis development. For several years, we have collaborated to develop Quantum Dots for use in immunophenotyping experiments. The advent of these fluorochromes provided an enormous advance in multicolor technology, allowing us to proceed from 12-color to 18-color very quickly. As a result, the complexity of the datasets have increased dramatically. We are actively working on new data analysis techniques—one major focus is the analysis and presentation of meta-data (for example, summarizing our functional analysis in which we have broken down each single response into hundreds of categories defined by the expression patterns of individual cytokines or other functional measurements).
We are also actively developing new techniques to assess immune function. A few years ago, we helped develop a viable assay to identify antigen-specific CD8 T cells (Betts et al., J. Immuno. Meth., 2003); more recently, we developed a similar assay to identify antigen specific CD4 T cells (Chattopadhyay et al., Nature Medicine, 2005). These are important assays because they not only identify a broad range of antigen-specific T cells, they do so in a fashion that does not kill the cells; hence, the cells can be recovered for further culture or other analysis.
Other projects in the laboratory focus on particular aspects of immune function or viral dynamics, within the context of the major research efforts. (i) Detailed characterization of cytotoxic T cell functions. We developed a flow cytometry-based assay to quantify both CTL activity as well as target susceptibility to quantitatively determine the dynamics of effector killing in vitro. In the future, we plan to take these assays to an in vivo system to explore the role of CTL in lymphocyte dynamics during viral replication. (ii) Identification of viral reservoirs in CD4 T cell subsets. Sequence analysis of viruses isolated from specific CD4 T cell subsets give us an understanding of the spread of virus through the CD4 compartment, and the contribution of different CD4 subsets to both active viral production and latent reservoirs.
Joanne Yu, Mitzi Donaldson, Kathy Foulds, Carl Hogerkorp, Tess Brodie, Yolanda Mahnke, Stephen Perfetto, Mario Roederer, Richard Nguyen, Pratip Chattopadhyay, Kaimei Song, and Diane Bolton.
Kern, F., LiPira, G., Gratama, J., Manca, F. & Roederer, M. Measuring antigen specific immune responses: Understanding immunopathogenesis and improving diagnostics in infectious disease, autoimmunity and cancer. Trends in Immunology 26, 477-484 (2005).
Chattopadhyay, P. K., Yu, J. & Roederer, M. A live-cell assay to detect antigen-specific CD4 T cells with diverse cytokine profiles. Nature Medicine 11, 1113-1117 (2005).
Mattapallil, J. M., Douek, D. C., Hill, B., Nishimura, Y., Martin, M. & Roederer, M. Massive infection and loss of memory CD4 T cells in multiple tissues during acute SIV infection. Nature 434, 1093-1097 (2005).
Chattopadhyay, P. K., Perfetto, S. P. & Roederer, M. The colorful future of cell analysis by flow cytometry. Discovery Medicine 4, 255-262 (2004).
De Rosa, S. C., Lu, F. X., Perfetto, S. P., Yu, J., Moser, S., Miller, C. J., Evans, T. G. & Roederer, M. Vaccination in humans generates broad T cell cytokine responses. J. Immunol. 173, 5372-5380 (2004).
Perfetto, S. P., Chattopadhyay, P. K. & Roederer, M. 17-Color Flow Cytometry: Unraveling the Immune System. Nature Reviews Immunology 4, 648-655 (2004).
De Rosa, S. C., Andrus, J. P., Perfetto, S. P., Mantovani, J. J., Herzenberg, L. A., Herzenberg, L. A. & Roederer, M. Ontogeny of gamma-delta T cells in man. J. Immunol. 172, 1637-45 (2004).
Berhanu, D., Mortari, F. & Roederer, M. Optimized lymphocyte isolation methods for analysis of chemokine receptor expression. J. Immunol. Methods 279, 199-207 (2003).
De Rosa, S. C., Brenchley, J. M. & Roederer, M. Beyond six colors: A new era in flow cytometry. Nature Medicine 9, 112-117 (2003).
De Rosa, S. C., Herzenberg, L. A. & Roederer, M. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nature Medicine 7, 245-8. (2001).
Wille-Reece, U., Flynn, B. J., Lore, K., Koup, R. A., Kedl, R. M., Mattapallil, J. M., Weiss, W. R., Roederer, M. & Seder, R. A. HIV Gag protein conjugated to a TLR7/8 agonist influences the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates. Proc Natl Acad Sci (USA), in press (2005).
Nishimura, Y., Brown, C. R., Mattapallil, J. M., Igarashi, T., Buckler-White, A., Lafont, B. A. P., Hirsch, V. M., Roederer, M. & Martin, M. Non-activated naive CD4 T cells are the principal source of progeny virus during acute infections of monkeys with highly pathogenic X4-tropic SHIVs. Proc Natl Acad Sci (USA) 102, 8000-8005 (2005).
Song, K., Rabin, R. L., Hill, B. J., De Rosa, S., Perfetto, S. P., Zhang, H. H., Foley, J. F., Reiner, J. S., Douek, D. C., Roederer, M. & Farber, J. M. CXCR3 and CCR4 Reveal Polarized Early Central Memory CD4+ T Cells in Humans. Proc Natl Acad Sci USA 102, 7916-7921 (2005).
Betts, M. R., Exley, B., Price, D. A., Bansal, A., Camacho, Z. T., Teaberry, V., West, S. M., Ambrozak, D. R., Tomaras, G., Roederer, M., Kilby, J. M., Tartaglia, J., Belshe, R., Gao, F., Douek, D. C., Weinhold, K. J., Koup, R. A., Goepfert, P. & Ferrari, G. Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection. Proc Natl Acad Sci (USA) 102, 4512-7 (2005).
Stuge, T., Holmes, S. P., Saharan, S., Rubio, V., Roederer, M., Weber, J. & Lee, P. P. Complexity of endogenous versus vaccine-elicited T cell responses to cancer. PLoS Medicine 1, e28 (2004).
Betts, M. R., Price, D. A., Brenchley, J. M., Lore, K., Guenaga, F., Smed-Sorensen, A., Ambrozak, D. R., Migueles, S. A., Connors, M., Roederer, M., Douek, D. C. & Koup, R. The functional profile of of primary human antiviral CD8(+) T cell effector activity is dictated by cognate peptide concentration. J. Immunol. 10, 6407-6417 (2004).
Sullivan, N. J., Geisbert, T. W., Geisbert, J. B., Xu, L., Yang, Z.-y., Roederer, M., Koup, R. A., Jahrling, P. B. & Nabel, G. Accelerated vaccination for Ebola virus hemorrhagic fever in nonhuman primates. Nature 424, 681-684 (2004).
Rubio, V., Stuge, T., Betts, M. R., Weber, J., Roederer, M. & Lee, P. P. Ex vivo identification, isolation, and analysis of tumor-cytolytic T cells. Nature Medicine 9, 1377-1382 (2003).
Brenchley, J. M., Karandikar, N. J., Betts, M. R., Ambrozak, D. R., Hill, B. J., Crotty, L. E., Casazza, J. P., Kuruppu, J., Migueles, S. A., Connors, M., Roederer, M., Douek, D. C. & Koup, R. A. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T-cells. Blood 101, 2711-2720 (2003).
Yang Shih, T. A., Meffre, E., Roederer, M. & Nussenzweig, M. C. Role of BCR affinity in T cell dependent antibody responses in vivo. Nature Immunology 3, 570-5. (2002).
Shih, T. A., Roederer, M. & Nussenzweig, M. C. Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nature Immunology 3, 399-406. (2002).
Dr. Mario Roederer
NIH/Vaccine Research Center
40 Convent Drive
Bldg. 40, Room 5509
Bethesda, MD 20892-3015
Last Updated March 29, 2013