Rapid Analysis of Single T Cell Immunity Signatures in Tuberculosis

Summary

Principal Investigator: Sanjay Tyagi
Abstract: DESCRIPTION (provided by applicant): Classifying disease stage and elucidating a prognosis, which allow the most effective medical intervention, are unattainable when the underlying pathological changes, or the correlates of stage and prognosis, are associated with heterogeneous cells states within a larger population. Single cell analyses can provide an unsurpassed means to measure and unravel heterogeneity in complex biological systems, and thereby to understand the basis for (or to identify correlates of) changes in biological function and disease processes. Here we propose to develop a rapid assay for detection of antigen-specific responses in single T cells from small amounts of blood, based on state-of-the-art techniques that are sensitive and robust. Our proposal aims at developing a test that provides tuberculosis (TB) diagnosticians with the critical ability to distinguish stable latent Mycobacterium tuberculosis infection (when the asymptomatic subject is not progressing to disease, is not infectious, and does not require treatment) from preclinical disease (when the asymptomatic subject is developing disease, is still not infectious, and requires early treatment to block progression of disease and drastically curb transmission of infection). Our multidisciplinary team includes expertise in development of novel single cell analysis methodology, cellular immunology, and biomarker research for TB, which still causes millions of cases of disease and death worldwide every year. Our assay is expected to yield multi-parameter measurements of single T cell functional states by integrating (i) use of artificial Ag-presenting cells (aAPC) to activate T cell receptor signaling and stimulation of gene expression, with (ii) measurement of inducible tell-tale markers of T cell activation and function by quantitative flow cytometry. Induced gene expression will be detected by mRNA enumeration using single molecule fluorescence in situ hybridization (smFISH). The research plan is articulated in four aims, each focused on the development of a specific aspect of the assay: (1) read-out: detection of activation markers in single T cells by smFISH and flow cytometry following conventional stimulation;(2) stimulation: response to aAPC assessed by detection of activation markers in single T cells;(3) response to infection-stage-specific Ag: association of single T cell responses with disease vs asymptomatic infection;(4) infection-stage-specific functional T cell signatures: multi-parameter characterization of single T cell responses and association with disease vs asymptomatic infection. The proposed plan should lead to recognizing and treating active TB prior to the appearance of microbiological and clinical signs and symptoms of disease. This is the current holy grail in TB diagnosis as it is considered to be critical to TB elimination efforts. The new assay principles will be translatable for diagnosis and disease staging of any pathology with T cell involvement, including other infectious diseases, cancer, autoimmunity, and transplantation. PUBLIC HEALTH RELEVANCE: Successful intervention in many diseases is often severely limited by insufficient understanding of the complex underlying pathology. In particular, the ability to classify the disease stage and to elucidate a prognosis may be poor or even impossible when pathological changes are identifiable only in a small number of cells within a larger population. Here we propose to develop a new immunological test that can identify, from among persons carrying an asymptomatic infection with Mycobacterium tuberculosis (the bacteria that cause tuberculosis), those individuals progressing toward disease and becoming infectious. Two billion people (about one-third of the world's population) are currently infected with the TB bacteria, with the highest concentration of new cases in resource-poor areas of South-East Asia and sub-Saharan Africa. Even though 90-95% of infected persons do not become sick, the number of infected individuals is high enough to give rise to 8 million new cases of TB and almost 2 million deaths each year worldwide. Existing blood-based clinical immunodiagnostic assays recognize TB infection but are not geared to distinguish between asymptomatic infection and active disease. The latter is currently identified only when patients excrete TB bacilli while coughing or sneezing (it is estimated that, by the time of diagnosis, a person with active TB has already infected up to twenty contacts). Thus, for public health purposes, it is critical to diagnose active disease when the patient is still asymptomatic and non-infectious. We propose to develop a new, rapid test requiring small amounts of blood that detects tell-tale immune cell subpopulations distinguishing the relevant groups. The detection method in this test requires basic flow cytometry, a technique routinely utilized in the clinical diagnosis of immunopathologies and fully accessible to district/peripheral level clinical laboratories that currently operate a fluorescence microscope in resource-poor regions. Translation of the proposed diagnostic methodology to clinical practice will allow identification and treatment of early cases of TB, thus impeding transmission and helping eliminate TB. While the present proposal is directed to a specific set of end-users in TB clinics, the proposed assay has broad public health relevance, because the same principles and methodologies are directly applicable to any infectious and non-infectious disease that can be characterized based on the properties of single immune cells involved in the body's recognition of or response to a particular condition.
Funding Period: 2012-09-07 - 2017-08-31
more information: NIH RePORT

Top Publications

  1. pmc Barriers to transmission of transcriptional noise in a c-fos c-jun pathway
    Khyati Shah
    1 Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ, USA 2 Department of Biological Chemistry, New Jersey Institute of Technology, Newark, NJ, USA
    Mol Syst Biol 9:687. 2013
  2. pmc Fusion FISH imaging: single-molecule detection of gene fusion transcripts in situ
    Fatu Badiane Markey
    Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America
    PLoS ONE 9:e93488. 2014
  3. pmc Spermatid cyst polarization in Drosophila depends upon apkc and the CPEB family translational regulator orb2
    Shuwa Xu
    Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
    PLoS Genet 10:e1004380. 2014

Detail Information

Publications3

  1. pmc Barriers to transmission of transcriptional noise in a c-fos c-jun pathway
    Khyati Shah
    1 Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ, USA 2 Department of Biological Chemistry, New Jersey Institute of Technology, Newark, NJ, USA
    Mol Syst Biol 9:687. 2013
    ..Sequestration of promoters of the downstream genes within compact chromatin is a likely cause of this insensitivity. These barriers to the propagation and amplification of noise are likely to be commonplace in higher eukaryotes. ..
  2. pmc Fusion FISH imaging: single-molecule detection of gene fusion transcripts in situ
    Fatu Badiane Markey
    Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America
    PLoS ONE 9:e93488. 2014
    ..This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts. ..
  3. pmc Spermatid cyst polarization in Drosophila depends upon apkc and the CPEB family translational regulator orb2
    Shuwa Xu
    Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
    PLoS Genet 10:e1004380. 2014
    ..One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc...

Research Grants30

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    Arshed A Quyyumi; Fiscal Year: 2013
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  2. Shaping Antiviral Immunity by the Inflammatory, Regulatory and Tissue Environment
    E John Wherry; Fiscal Year: 2013
    ..Our goal is to define how unrelated infections impact immunological memory in hopes to improve vaccines. ..
  3. Vaccination and infection: indicators of immunological health and responsiveness
    MARK MORRIS DAVIS; Fiscal Year: 2013
    ..abstract_text> ..
  4. SLAM Gene Family Controlled Pathways to SLE
    CORNELIS P TERHORST; Fiscal Year: 2013
    ..Core A Genetic Mouse Core.PL: Ninghai Wang, Beth Israel Deaconess Medical. Center Core B Administrative Core.PL Cox Terhorst, Beth Israel Deaconess Medical Center. ..
  5. INNATE IMMUNE RESPONSE TO MICROBIAL INFECTION
    William M Nauseef; Fiscal Year: 2013
    ....
  6. B-cell Biology of Mucosal Immune Protection from SIV Challenge
    Eric Hunter; Fiscal Year: 2013
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  7. Molecular and Immunologic Analysis of the Pathobiology of Human Anthrax
    KENNETH MARK COGGESHALL; Fiscal Year: 2013
    ..Overall, we are confident that the four years of funding puts us in a competitive position to exploit the findings of our first cycle to make substantive advances in the next five years. ..
  8. Host Defense Against HIV-related Pulmonary Infections
    Judd E Shellito; Fiscal Year: 2013
    ..abstract_text> ..
  9. TIM recognition of phosphatidylserine regulates innate and adaptive immunity
    Dale T Umetsu; Fiscal Year: 2013
    ..We believe that further study of these molecules will greatly improve our understanding of disease mechanisms, and are likely to lead to novel therapies for asthma and food allergy. ..
  10. Heterogeneity of T-cells in M. tuberculosis Infection
    HENRY BOOM; Fiscal Year: 2013
    ..Aim 3. To determine the ability of ManLAM to inhibit T cell responses to MTB in vivo using the murine MTB infection model and determining if there is variable sensitivity to ManLAM among humans. ..
  11. BEHAVIORAL GENOMICS OF ALCOHOL NEUROADAPTATION
    John C Crabbe; Fiscal Year: 2013
    ..An Education and Outreach component trains pre- and post-doctoral students in alcohol research, disseminates research findings to the public, and engages in a range of activities with elementary-to-high school students. ..
  12. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  13. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  14. Therapeutic Opportunities for Pediatric Astrocytoma
    Rosalind A Segal; Fiscal Year: 2013
    ..The three projects interact with one another and are further unified by economies of scale enabled by an Innovative Neuropathology (INP) core. ..
  15. VACCINE INDUCED IMMUNITY IN THE YOUNG AND AGED
    Rafi Ahmed; Fiscal Year: 2013
    ....
  16. Directing Tumor-specific T cells to Tumors
    Pawel Kalinski; Fiscal Year: 2013
    ..abstract_text> ..
  17. VIRAL INFECTION INFLUENCE ON TRANSPLANTATION TOLERANCE
    DALE LESLIE GREINER; Fiscal Year: 2013
    ..This work will identify how infection during transplantation may affect the host immune system and ultimate fate of the graft. ..
  18. FISH-Flow platform for host-based tuberculosis diagnostics
    Maria Laura Gennaro; Fiscal Year: 2013
    ..The FISH-Flow platform in its manual and automated versions will be applicable to detection of receptor-mediated responses in many infectious and non-infectious pathologies. ..
  19. Immune Regulation of Virus Clearance and Tissue Injury at Sites of Infection
    Thomas J Braciale; Fiscal Year: 2013
    ..To determine the impact of viral infection on the production of Te-derived IL- 10. The proposed studies are designed to complement ongoing related studies in Projects 2, 3 and 4. ..
  20. Mucosal Immunity, Vaccines and Microbiota Interplay in Humans and Animal
    Marcelo B Sztein; Fiscal Year: 2013
    ..Given the shortcomings of available measures to successfully control this infection, and its bioterrorism potential, to develop a S. dysenteriae type 1 vaccine is of great importance. ..
  21. Martin Delaney Collaboratory to Eradicate HIV-1 Infection
    David M Margolis; Fiscal Year: 2013
    ..We are convinced that together we will catalyze advances that will ultimately lead to the eradication of HIV infection. ..