We have initiated studies to characterize the role of CD8+ T cells in immunity to the intracellular bacterial pathogen, Shigella flexneri. During infection, S. flexneri enter cells and escape into the host cell cytosol. We expected that proteins excreted from S. flexneri would be proteolytically degraded by the MHC-I processing machinery, and that the resulting peptides would be presented to CD8+ T cells in the context of surface MHC-I molecules. During infection with Listeria monocytogenes, another intracellular bacterial pathogen that escapes into the host cell cytosol, Listeria-derived peptides, in complex with host MHC-I, are recognized by CD8+ T cells and contribute to the generation of protective immunity. While CD8+ T cells have been shown to be required for protective immunity to L. monocytogenes, we have found no evidence to suggest that CD8+ T cells play a role in protective immunity to S. flexneri. Even when we engineered S. flexneri to constitutively secrete heterologous epitopes known to stimulate potent CD8+ T cell responses, those responses were not detected. Furthermore, we found that when cultured cells were infected with epitope-tagged S. flexneri, these cells were not recognized by established T cell clones specific for the epitope tag. Our results over the previous award period suggest that S. flexneri inhibit the presentation of MHC-I restricted antigens to CD8+ T cells, and it is this lack of antigen presentation that results in the failure of CD8+ T cells to respond to antigens secreted into the host cell cytosol by S. flexneri. The experiments in this proposal seek to identify and characterize the defect in MHC-I processing and/or presentation that occurs during S. flexneri infection. Specifically: 1) we will use genetic screens to identify S. flexneri gene product(s) responsible for the inhibition;and 2) we will use biochemical assays to determine the step or steps in the MHC-I processing pathway that are altered during infection with S. flexneri and that result in decreased antigen presentation on the host cell surface. Through these experiments, we expect to identify and describe the activity of a bacterial inhibitor of antigen processing and presentation. Such an inhibitor might represent a novel class of virulence determinants specifically able to interfere with development of protective T cell-mediated adaptive immunity. Understanding how this inhibition affects the balance between bacterial virulence and immune clearance will further our understanding of the complex interaction of this bacterial pathogen and its mammalian host.

Public Health Relevance

Shigella flexneri is responsible for serious gastrointestinal disease throughout the world. We have found that this organism has the capacity to inhibit the immune response directed against it by the host. The work proposed in this application is directed at understanding the mechanisms through which the organism is able to avoid detection and identifying which genes in the organism are responsible for immune system inhibition. Our work may lead to therapeutic strategies that might undermine the ability of this organism to hide from the immune system, thereby allowing more effective clearance of the bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Mills, Melody
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Harvard University
Schools of Medicine
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Zhang, Xuqing; Starnbach, Michael N (2015) An Excess of the Proinflammatory Cytokines IFN-? and IL-12 Impairs the Development of the Memory CD8+ T Cell Response to Chlamydia trachomatis. J Immunol 195:1665-75
Nogueira, Catarina V; Zhang, Xuqing; Giovannone, Nicholas et al. (2015) Protective immunity against Chlamydia trachomatis can engage both CD4+ and CD8+ T cells and bridge the respiratory and genital mucosae. J Immunol 194:2319-29
Fankhauser, Sarah C; Starnbach, Michael N (2014) PD-L1 limits the mucosal CD8+ T cell response to Chlamydia trachomatis. J Immunol 192:1079-90
Jehl, Stephanie P; Nogueira, Catarina V; Zhang, Xuqing et al. (2012) IFN? inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I. PLoS Pathog 8:e1002809
Jehl, Stephanie P; Doling, Amy M; Giddings, Kara S et al. (2011) Antigen-specific CD8(+) T cells fail to respond to Shigella flexneri. Infect Immun 79:2021-30
van der Velden, Adrianus W M; Dougherty, Jeffrey T; Starnbach, Michael N (2008) Down-modulation of TCR expression by Salmonella enterica serovar Typhimurium. J Immunol 180:5569-74
Shaw, Christine A; Starnbach, Michael N (2008) Both CD4+ and CD8+ T cells respond to antigens fused to anthrax lethal toxin. Infect Immun 76:2603-11
Shaw, Christine A; Starnbach, Michael N (2008) Antigen delivered by anthrax lethal toxin induces the development of memory CD8+ T cells that can be rapidly boosted and display effector functions. Infect Immun 76:1214-22
D'Orazio, Sarah E F; Shaw, Christine A; Starnbach, Michael N (2006) H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes. J Exp Med 203:383-91
Hang, Howard C; Loureiro, Joana; Spooner, Eric et al. (2006) Mechanism-based probe for the analysis of cathepsin cysteine proteases in living cells. ACS Chem Biol 1:713-23

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