The role of CD8 T cells during pulmonary infection with Mycobacterium tuberculosis

Abstract

With over 8 million deaths per year and incidence rates increasing in many countries, tuberculosis is a world-wide pandemic that is responsible for more human suffering, death, and lost productivity than any other infectious disease in the history of mankind. Although a vaccine for tuberculosis exists and is currently in widespread usage, experimental evidence indicates that the bacille Calmette-Guerin live attenuated vaccine provides limited protective efficacy that decreases with age. As the preponderance of cases typically occurs in underdeveloped nations with limited resources to combat the problem, a major multinational effort is currently underway to develop a safer, more effective vaccine. Identification of the immunological correlates of protection is therefore an essential prerequisite for vaccine development. Recent evidence indicates a role for CD8 T cells in protective immunity. We therefore sought to investigate the role that CD8 T cells play during pulmonary infection with Mycobacterium tuberculosis to elucidate the protective effector functions and ultimately determine if epitopes targeting CD8 T cells should be included in candidate tuberculosis vaccines. In order to understand the CD8 T cell response, we began these studies by characterizing the magnitude and the kinetics of CD8 T cell recruitment into the infected lung, in conjunction with phenotypic characterization of the CD8 T cell response to pulmonary infection with M. tuberculosis. Observations by our laboratory of the preferentially peripheral location of CD8 T cells around the murine granuloma led us to examine CD8 T cell expression of integrin adhesion molecules. Although we were ultimately unable to correlate poor migration with deficient integrin molecule expression, we were able to verify the presence of four principal integrin molecules on CD8 T cells and observe their upregulation and maintenance following infection. Further experiments were performed to phenotypically define the CD8 T cell population following aerosol infection with M. tuberculosis. We examined multiple surface markers of activation and costimulation, as well as markers associated with memory in mice infected with M. tuberculosis and in memory-immune mice that were infected and subsequently treated using a combination anti-tuberculosis drug regimen. In these experiments, we characterized not only the total CD8 T cell population in the lungs and spleens of mice, but also an antigen-specific CD8 T cell subpopulation using a novel MHC class I tetramer reagent containing a peptide epitope located within the M. tuberculosis-derived Mtb32 secreted protein. In subsequent experiments, we characterized the cytokine secretion profile and expression of cytolytic effector molecules by the responding CD8 T cell population following infection. These experiments allowed us to precisely observe the phenotypic changes that occurred following aerosol infection, as well as document the phenotypic changes associated with the emergence of CD8 memory T cells following chemotherapeutic-induced reduction of bacterial numbers. In chapter three, we utilized the MHC class I tetramer reagent to track Mtb32-specific CD8 T cells in the lungs of mice infected with M. tuberculosis. We were able to characterize the antigen-specific response to this protein over time in both infected mice and mice that were first immunized with the protective candidate Mtb72F vaccine. These experiments revealed an early, robust antigen-specific CD8 T cell response possessing interferon-gamma secreting ability in the lungs after aerosol infection, the size of which temporally correlated with immunologic control of bacterial replication. We were further able to demonstrate the induction of an antigen-specific memory response following immunization with a novel liposomal/protein vaccine adjuvant. Other experiments utilizing this tetramer reagent in conjunction with mice lacking CD4 T cells defined the requirement for CD4 T cell help in augmenting the CD8 T cell response. Chapter four outlines experiments examining the antigen-specific CD8 T cell response to the M tbl0.3/Mtbl0.4 vaccine candidate proteins. Although CD8 T cell-directed immunization with the minimal peptide epitope failed to confer protection in the lungs of mice, an exceptionally large and persistent antigen-specific CD 8 T cell response possessing cytolytic activity directed against this single antigenic peptide was observed. The significance of this large but nonprotective CD8 T cell response remains to be determined. In these studies, we were able to comprehensively characterize the pulmonary recruitment kinetics, surface phenotype and cytokine secretion profile of the CD8 T cells responding to pulmonary tuberculosis infection in a murine model. Importantly, we were able to track and characterize with precision the antigen-specific CD8 T cell response directed against the Mtb32 and Mtb10.3/Mtb10.4 M. tuberculosis-derived proteins over the course of infection. These experiments have further demonstrated that antigen-specific CD8 T cells can be targeted by multiple different immunization regimens, including liposomal, DNA, and protein in adjuvant combinations containing M. tuberculosis-derived antigens that possess CD8 T cell epitopes. In addition, we have shown that the cells elicited by immunization rapidly traffic to the lungs following aerosol infection and secrete interferon-gamma; a cytokine known to be protective during tuberculosis infection. We were able to monitor these cells over the timecourse of infection to determine the magnitude and duration of the CD8 T cell response. We have also shown that CD8 T cells elicited by vaccination can express phenotypic markers associated with a memory phenotype, which is the ultimate goal of a successful vaccine. These studies have contributed to the collective knowledge of the CD8 T cell response during pulmonary infection with M. tuberculosis in the hope that a better understanding of this response will aid in rational vaccine design and selection of candidate antigens for incorporation into future tuberculosis vaccines.