Innate Immunity and T Cell Priming in Human Tuberculosis

Patrick Haslett, M.D.

University of Miami

Funded in January, 2003: $ for 3 years


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Searching for an Immune Defect in Tuberculosis

The researchers will explore why only a small subset of people who are exposed to the tuberculosis bacterium develop the disease, while others who are exposed are able to contain the infection without treatment, even though they may not eliminate the bacterium. The study is prompted by a recent observation concerning leprosy, another bacterial disease in which only a small fraction of those exposed are clinically affected.

One-third of the world's population is infected with the bacterium that causes tuberculosis (TB). Yet 90 percent of these people have a natural immunity that is adequate to protect them from developing the disease. The researchers hypothesize that the remaining ten percent of infected people who develop TB have a defect in what are called "Toll-like" receptors. These receptors are part of the innate immune system's dendritic cells, sentries that recognize an invader and teach immune T cells to attack it. In people who develop TB, the researchers hypothesize, these Toll-like receptors malfunction and fail to recognize the bacterium as foreign and harmful. The investigators base this hypothesis on a recent finding that failure of Toll-like receptors to recognize the leprosy bacterium occurs in the small proportion of people who actually develop disfiguring leprosy after exposure to the leprosy germ. Leprosy and TB bacteria are similar; both are Mycobacteria. The Miami researchers now will see if the same immune defect that occurs in leprosy is at fault in TB.

To explore this possibility, the investigators will identify people who have active TB, but who nonetheless repeatedly have a negative skin test (which is a measure of the immune response to TB). The researchers will conduct a series of biochemical assays to determine whether these patients fail to mount the proper immune response to the TB bacterium, and instead have an immune "tolerance" to it. The assays also will enable the investigators to locate the Toll-like receptors that are malfunctioning. If no defect can be demonstrated, the researchers then will undertake tests on stored cultures of TB in the laboratory to see if they can identify factors of the bacterium that might enable them to elude the immune systems of those who develop the disease.


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Innate Immunity and T Cell Priming in Human Tuberculosis

One third of the world's population is infected with Mycobacterium tuberculosis (M.tb), the cause of tuberculosis (TB), yet only 10 percent of these people develop clinical disease during their lifetimes. The adaptive immune response that protects the great majority of infected individuals from TB is critically dependent upon M.tb-specific CD4+ T cells that secrete interferon- g(Th-1 cells). Activation and maturation of antigen-presenting dendritic cells (DC) is a crucial preliminary to initiating Th-1 immunity. The Toll-like family of pathogen receptors (TLR) provides a key mechanism for direct microbial maturation of DC. In the absence of maturation, DC can elicit antigen-specific T cell tolerance. The latter is manifested as an absence of Th-1 immunity, and may involve the concomitant development of antigen-specific regulatory CD4+ T cells that secrete IL-10.

 We hypothesize that the immune defect that permits the development of TB in some individuals is a consequence of inadequate TLR-mediated DC activation. This defect may reflect a host-determined abnormality in TLR responsiveness, or a pathogen-specific defect in the TLR ligand. In support of this notion, a TLR abnormality has recently been described in lepromatous leprosy, a mycobacterial infection where uncontrolled disease is associated with M. leprae-specific T cells that secrete IL-10 while failing to mount a Th-1 response.

In this project, we shall test the study hypothesis by characterizing DC and TLR functions in patients with differing levels of T cell immunity against M.tb. The primary comparison will be between patients who have been cured of TB with antibiotics and individuals who have naturally acquired immunity against M.tb. We shall also seek to identify a subset of cured TB patients who have apparent "deviant" IL-10-secreting M.tb-specific T cell responses in the absence of any detectable Th-1 response, since the clearest defects in TLR function may be identified in this group. We suggest that the latter subset of patients may represent an immunologic corollary to lepromatous leprosy.

 We shall stratify patients according to T cell responses, and subsequently analyze ex vivo DC responses to mycobacterial and generic TLR ligands. The identification of functional TLR abnormalities will lead to studies to characterize the underlying molecular defects. A complementary strategy will be to study the TLR-activating potential of M.tb isolates that have been stored from study subjects who were M.tb culture-positive at TB diagnosis. A deeper understanding of immune protection against TB disease, and its failure, will suggest new approaches to circumvent immune defects in susceptible individuals, as well as identify potentially important attributes of new vaccine candidates.


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Patrick Haslett, M.D.

Patrick Haslett, M.D., M.B., M.R.C.P., is an Infectious Disease Specialist, Assistant Professor in the Department of Microbiology and Immunology, and member of the new HIV Pathogenesis Research Group at the University of Miami. Dr. Haslett is also a Staff Physician in the Department of Medicine at the Miami Veterans Association Medical Center. He completed his medical education at the University of London and initial clinical training in the UK. Dr. Haslett is a Diplomate of the London School of Tropical Medicine and Hygiene. He spent several years as a clinician in rural Africa before coming to New York University for formal training in clinical Infectious Diseases, and subsequently in clinical investigation and cellular immunology at the Rockefeller University, where he was a Clinical Scholar.

His research laboratory conducts investigations into regulation of the immune response in patients with chronic infectious diseases, with an emphasis on diseases caused by mycobacteria and HIV. He discovered that the immune modulating drug thalidomide acts as a T cell costimulator. Recent work has focused on the immune regulating activities of a subpopulation of human T cells that bear the gamma-delta T cell receptor. These cells are activated and proliferate in mycobacterial infections, but are severely depleted in progressive HIV disease. Current projects include clinical investigations of the potential of thalidomide to enhance virus-specific immunity in patients with chronic HIV infection, and an exploration of the comodulatory relationships of dendritic cells and gamma delta T cells.


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Lay Results:
Leprosy is an ancient infectious scourge, caused by a microscopic organism called Mycobacterium leprae. This organism can live for years inside human cells, and has developed various, but poorly understood, mechanisms for escaping human immune defenses. In the absence of antibiotic treatment, a large proportion of leprosy patients suffer lifelong debilitating disease where the mycobacteria are able to reproduce to very high numbers. In these patients, defenses against other infections appear to be normal, so the immune system has a specific “blind spot” with regard to Mycobacterium leprae only. If we were able to understand how leprosy avoids immunity, we might be able to learn new ways to specifically shut down the immune response where it is unwanted, as in autoimmune diseases such as multiple sclerosis and diabetes.

We think that an important way in which these mycobacteria either avoid recognition by the immune system, or, if recognized, trick the system into making an ineffectual response, is by modifying the actions of a key set of immune cells called dendritic cells. Dendritic cells act as immune gatekeepers, strategically located in those parts of the body that interface with the environment. In a properly functioning immune response, upon contact with infectious organisms, these cells respond by moving into areas where other immune cells, called T cells, are concentrated, and generating various signals which “instruct” the T cells to seek, recognize, and destroy cells infected with the offending organism.

To test the idea that in leprosy patients, there is a defect in the function of dendritic cells in response to Mycobacterium leprae compared to other triggers of dendritic cell activity, we first developed a method for rapidly measuring dendritic cell function in the blood of patients, where dendritic cells are present in very low concentrations. Our technique involved the direct testing of dendritic cell function in a drop of blood taken from a patient, with a minimum of manipulation, which we think most closely reflects the way these cells might function in the body. Having developed this approach, we tested the blood of 11 leprosy patients and 11 controls, and observed no differences in the function of these cells. We did, however, note that the degree to which dendritic cells from both patients and controls were “excited” by Mycobacterium leprae was modest compared to other stimuli, including other types of mycobacteria. This led us to conclude that there was no obvious intrinsic defect in the dendritic cell response in leprosy patients, but that the mycobacterium itself might have ways of subverting the function of normal dendritic cells. We next tested another idea, that Mycobacterium leprae is able to subvert dendritic cell function by inserting some of its chemicals into the surface of dendritic cells in such a way that the T cell instructing properties of the dendritic cell are changed. We demonstrated that a chemical from the mycobacterium is indeed inserted into the dendritic cell surface, in such a location that it would be strategically positioned to affect an interacting T cell. Furthermore, we found that this chemical activates immune proteins that circulate in the blood that may further deviate the responses of T cells.

These findings represent a novel mechanism of immune escape by an infectious organism and suggest new approaches to developing therapies that could be used to influence immune function.

Scientific Results:
Mycobacterium leprae (M. leprae), the cause of leprosy, is an intracellular bacterium with a striking ability to elude effective Th1 immunity in patients who have otherwise normal immune function. To test whether this defect results from a specific failure of dendritic cell (DC) activation in leprosy patients, we developed a rapid flow cytometric method that enabled us to examine the function of both plasmacytoid and myeloid DCs in the peripheral blood of 11 leprosy patients and 11 demographically matched controls. We found no difference in the expression of the maturation markers, CD80, CD83, CD86 and CCR7, nor in the expression of the cytokines, tumor necrosis factor-α and interferon-α, following stimulation with lysates of M. leprae, M. tuberculosis, and generic ligands for TLR 2, 4, 7, 8 and 9. Thus, no intrinsic defect in DC function was observed in leprosy patients. However, we did note very modest responses of DCs to M. leprae lysates compared to M. tuberculosis lysates and other stimuli. Since M. leprae is known to contain multiple TLR2 ligands, we hypothesized that the organism is able to suppress the response of DCs to TLR signaling. In further experiments, we showed that a complex glycolipid called phenolic glycolipid-1 (PGL-1), which comprises up to 2% of the mass of M. leprae, is inserted into the lipid rafts of human dendritic cells infected with M. leprae. Since TLRs are co-located in lipid rafts, PGL-1 is strategically inserted to potentially interfere with the function of these receptors. We next demonstrated that M. leprae-infected, but not M. bovis-infected DCs are able to activate complement present in the serum of both leprosy patients, and to a lesser extent, healthy controls. This process was calcium and not magnesium-dependent, consistent with complement activation via either the classical or mannose-binding lectin pathway. This activation results in the colocalization of C3 and PGL-1 in the lipid rafts of the dendritic cell plasma membrane. This finding is consistent with previously published work demonstrating complement activation by PGL-1. The presence of C3 in lipid rafts of M. leprae-infected DCs suggests a mechanism by which DCs may subvert acquired immunity by engaging the complement receptor CD46 during T cell priming, a process that is known to stimulate potentially immunosuppressive IL-10 secreting regulatory T cells.

We conclude that M. leprae has the potential to subvert T cell responses by multiple mechanisms that include complement-dependent stimulation of regulatory T cells, as well as the physical disruption of TLR signaling by insertion of PGL-1 into lipid rafts.


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Abbo L., Vincek V., Dickinson G., Shrestha N., Doblecki S., and Haslett P.A.J.  Selective defect in plasmacyoid dendritic cell function in a patient with AIDS-associated atypical genital herpes simplex vegetans treated with imiquimod.  Clin Infect Dis. 2007 Feb 1;44(3):e25-7.

Ida J.A., Shrestha N., Desai S., Pahwa S., Hanekom W.A., and Haslett P.A.J.  A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation.  J Immunol Methods. 2006 Mar 20;310(1-2):86-99.

Shrestha N., Ida J.A., Lubinski A.S., Pallin M., Kaplan G., and Haslett P.A.J.  Regulation of acquired immunity by gamma delta T-cell/dendritic-cell interactions.  Ann N Y Acad Sci. 2005 Dec;1062:79-94.