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A4303 - Resident CD4+ T Cells in the M. Tuberculosis-Induced BAL Cell Gene Expression Signature of Individuals with Latent TB Infection
Author Block: R. F. Silver1, J. R. Jarvela2, M. Moyer3, S. Weigel3, P. Leahy4, J. Dazard5; 1Pulmonary, Critical Care, and Sleep Medicine, Case Western Reserve University School of Medicine and the Louis Stokes Cleveland VA Medical Center, Cleveland, OH, United States, 2Pulmonary, Critical Care and Sleep Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States, 3Pulmonary, Critical Care, and Sleep Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, United States, 4Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, United States, 5Center for Proteomics and Bioinfomatics, Case Western Reserve University School of Medicine, Cleveland, OH, United States.
INTRODUCTION: Airway cells obtainable by bronchoalveolar lavage (BAL) provide the first line of defense against the airborne pathogen Mycobacterium tuberculosis (Mtb). Natural infection via the respiratory route may serve as a model for development of local immunity to Mtb. In particular, respiratory infection may optimally localize Mtb-specific CD4+ T cells to the airways where they can direct the initial recall response to the organism. We assessed the role of resident CD4+ T cells in Mtb-induced gene expression signature of individuals with latent tuberculosis infection (LTBI).
METHODS: Healthy non-smoking individuals aged 18-50 with LTBI as well as Mtb-naive controls underwent bronchoscopy with BAL. For LTBI subjects, magnetic separation was used to deplete CD4+ and CD8+ T-cell subsets. Both unsorted and T-cell depleted BAL samples were infected with Mtb strain H37Rv and evaluated for genome-wide gene expression using microarrays. Significant changes in gene expression were assessed using a Bayesian ANOVA model. Comparisons between unsorted and CD4+ T-cell depleted conditions, as well as a CD8+ T-cell depletion control, were entered into Ingenuity Knowledge Database (IPA) to identify pathways and gene networks specifically associated with CD4+ T-cell responses. Identification of significant pathways was done by imposing a stringent criterion of changes in enrichment p-value of > 2 logs, while controlling the false discovery rate. Confirmatory PCR was performed on the LTBI subjects and Mtb-naive controls.
RESULTS: In LTBI subjects, Mtb infection of unsorted BAL cells induced significant changes in expression of approximately 500 genes. By our criterion, the significance of 34 pathways was reduced following CD4 depletion. In contrast, only 8 pathways were affected by CD8 depletion, each of which were impacted by CD4 depletion as well. Of the 26 pathways uniquely affected by CD4 depletion, focus on those in which changes in expression were detected in at least 9 genes per pathway and on genes that were identified in at least 2 pathways resulted in a list of 39 candidate genes comprising the CD4+ T-cell mediated BAL gene cell expression signature induced by Mtb. Confirmatory PCR studies further clarified the specificity of these findings.
CONCLUSIONS: Our findings demonstrate the feasibility of identifying a Mtb-induced BAL cell gene expression signature in LTBI subjects that demonstrates the significance of resident CD4+ T cells in initial responses to re-exposure to the organism. These findings establish a standard for comparison with similar profiles developed via standard BCG vaccination and by novel TB vaccines currently in development.