Immunology Component

Role of the Host Immune System in Protection against Tuberculosis

M. tuberculosis bacilli are efficiently transmitted from person to person by aerosol. Only small numbers of bacilli are necessary to establish infection in the lung, because local innate immunity, mediated primarily by alveolar macrophages, fails to control growth of these slowly replicating bacilli.  In most healthy adults, acquired immunity mediated by T cells controls but does not eradicate M. tuberculosis infection.  One third of the world's population is thought to harbor persistent M. tuberculosis and ongoing immune surveillance by T cells is required to maintain control over them.  Acquired immunity likely also protects against re-infection, which is important in parts of the world with high levels of M. tuberculosis transmission.

The interaction of T cells and infected macrophages is central to protective immunity to M. tuberculosis.  CD4+ T cells have an essential role but are supported by other T cell subsets such as CD8+, gd TCR+ T cells (gd T cells), and CD1 restricted T cells.  Antigens for these T cells are being defined, and their function in the immune response elucidated.  Recent research has resulted in some insight into mechanisms used by macrophages to control M. tuberculosis.  How T cells help them perform this task, however, remains poorly understood.  TNF-alpha, IL-12 and IFN-gamma are central cytokines in regulation and effector phase of immune responses to M. tuberculosis. 

Macrophages are not only primary effector cells for control of M. tuberculosis but also essential for processing and presentation of antigens to T cells.  To survive (and thrive) in macrophages, M. tuberculosis has not only evolved mechanisms to live through the initial encounter with macrophage but also to block acquired immunity.  Modulation of phagosomes, neutralization of macrophage effector molecules, inciting the secretion of inhibitory cytokines, and interference with processing of antigens for T cells, all represent strategies for bacterial survival.  The relative importance of different T cell subsets and mechanisms employed by M. tuberculosis to interfere with macrophage and T cell function likely depends on the stage of infection.  During primary infection an acute acquired immune response develops in the lung as innate immune mechanisms fail to control dividing bacilli.  As replicating M. tuberculosis are controlled by activated T cells and macrophages, immune responses are down regulated, and the infection enters a chronic phase, in which memory T cells help macrophages in granulomas control persistent bacilli and provide surveillance against re-infection.  Failure of acquired immunity during the acute and/or chronic phases allows M. tuberculosis infection to become clinically apparent, most commonly with pulmonary manifestations but in some with extra-pulmonary or disseminated disease.  The balance of the interaction between T cells and infected macrophages determines the outcome of the host-pathogen interaction in M. tuberculosis infection.  The ability to resist microbicidal functions and to modulate antigen processing and presentation allows M. tuberculosis to survive inside macrophages for many years.  When host immunity fails, expectoration of reactivated bacilli allows the organism to seek a new host, perpetuating a cycle that allows M. tuberculosis to remain one of the most successful human pathogens. 

Research Challenges

The TBRU seeks to understand how the host immune system works to control the growth of M. tuberculosis and to identify immune components as markers of protective immunity during different stages of M. tuberculosis infection.

Work Statement [from RFP-NIH-NIAID-DMID-1999-2006]

As specified in the TBRU contract, the primary objective of the Immunology Component of the TBRU is to conduct a coordinated, multi-disciplinary investigation of host immunological response mechanisms to M. tuberculosis in order to identify surrogate markers for clinical use.

Sub-objectives Include:

• Establish and/or maintain appropriate animal, cell culture, or other model(s) to evaluate the immune response to M. tuberculosis associated with pulmonary disease progression/protection associated with M. tuberculosis, and its correlation to the human disease.
• Develop and/or utilize appropriate in vitro tests to assess the immunological status of individuals exposed to M. tuberculosis and correlate these results with clinical expression of the disease.
• Identify host immune factors associated with latent infection and reactivation.
• Identify host immune factors associated with disease protection.

Project Descriptions [1994-2000]

Overview

The primary focus of the Immunology Component activities is to develop and validate immunological assays for the evaluation of the human immune response to M. tuberculosis [MTB] infection.  Particular attention is focused on molecules and experimental approaches that could be used to assess response to anti-MTB therapy [surrogate markers] and to identify host immune responses characteristic of MTB infection/disease  [correlates of protective immunity].

Developmental Immunology

Developmental immunology studies were conducted at Case Western Reserve University (CWRU), Cleveland, Ohio under the tutelage of Drs Ellner, Toossi and Hirsch.  Assay systems/approaches and molecules most promising then were further evaluated in pilot immunology studies conducted at the international TBRU sites at Makerere University [MU], Kampala, Uganda and at the Universidade Federal do Espírito Santo (UFES), Vitoria, Brazil. The most informative markers then were used and further validated in the context of clinical trials [adjunctive treatment with heat-killed Mycobacterium vaccae immunotherapeutic agent and recombinant human interleukin (IL)-2] and the on-going TBRU household contact study {Kawempe Community Study] at the Kampala site.

Developmental studies included the establishment of a whole blood culture system to assess anti-MTB immunoreactivity in subjects exposed to MTB (who subsequently did or did not develop MTB infection) and in patients with active pulmonary TB, assessment of expression of molecules potentially involved in anti-MTB immunity at the cellular level (by quantitative RT-PCR using the Taqman methodology), and experiments evaluating the (enhanced) immunogenicity of purified and recombinant MTB antigens coupled to polystryrene beads.

Pilot Immunology Studies

Translational immunology studies in Uganda (involving both HIV-infected and -uninfected study subjects) focused on assessing dynamic changes in cytokine immunoreactivities during the course of anti-MTB therapy and thereafter (both at the protein and m-RNA level) and to pin-point mechanisms involved in depressed anti-MTB immunity during active TB. These studies identified the immunosuppressive cytokines TGF-b1 and IL-10, produced by mononuclear phagocytes, and apoptosis of MTB-reactive T cells as important contributing factors involved in suppression of T cell functions. Further, pleural TB was used as a model to study cytokine responses and apoptosis at sites of active MTB infection.

More recently translational immunology work also was initiated at the Universidade Federal do Espírito Santo (UFES), Vitoria, Brazil.  The initial study at UFES was designed to simultaneously assess immunological parameters in peripheral blood and spontaneously expectorated and induced sputum (using methodologies adapted from asthma research) as well as microbiological markers (in sputum specimens) during the course of anti-MTB therapy.  Therefore, results from this protocol will be most relevant to the goals of the TBRU contract.

Immunology Studies in the Context of Clinical Trials

As stated, immunological markers also were studied in the context of clinical trials. Parameters included as secondary endpoints in the IL-2 study indicate that administration of IL-2 as an adjunct to treatment for tuberculosis is systemically activetemic effects but had little effect on the outcome of treatment.  For example, the expression of IL-2 receptors was increased among peripheral blood T-cell subsets from study subjects receiving IL-2 when compared to those in the placebo arm.  Interestingly, production of IFN-g and IL-12 were higher and that of IL-10 was lower in MTB antigen-stimulated whole blood culture supernatants prepared from subjects receiving IL-2 as compared to similar materials prepared from patients in the placebo arm.

A late breaker abstract describing the main data from the trial was presented at the ATS meeting in Atlanta, Georgian on May 22, 2002 and at the IUATLD African Region Meeting in Durban, South Africa from June 11-14, 2002.   A manuscript describing the clinical trial results and some of the immunological data has been accepted for publication in the American Journal of Respiratory and Critical Care Medicine.  A separate manuscript describing all the immunological results is in preparation.

Collaborative Studies and Training Activities

In addition to studies primarily funded by TBRU, a number of collaborative immunology projects also were conducted in the US and at the TBRU site in Uganda with financial support through investigator initiated funding mechanisms (RO1 awards from NIAID to Drs. Ellner, Toossi and Hoft), Fogarty International (Masters Project of Dr. Mayanja-Kizza), The Wellcome Trust (Immunology of Pleural TB, Alison Elliott, PI), the Centers of Disease Control (US Household Contact Study), and the Damien Foundation (Guido Vanham, PI).

Current Activities

Treatment Shortening Study

Recruitment for the TBRU Treatment Shortening Study (DMID 01-009) started in April of 2002 at the TBRU Uganda site.  A detailed laboratory protocol for immunologic studies for subjects participating in this study has been developed by Dr. Hirsch and distributed to the relevant laboratory personnel in Uganda and Brazil. Immunologic parameters studied at both sites include MTB antigen-induced production of IFN-g and TNF-a in whole blood at multiple time points throughout the study. In addition, sputum cytokines will be measured in the first 100 patients enrolled in the treatment shortening study at the Brazil site. In addition to these "real-time" immunological assessments, viable peripheral blood mononuclear cells will be stored frozen for later use either testing new assays or validating additional immunological markers.

Household Contact Study

The next generation household contact study (Kawempe Community Study-KCS) in Uganda was launched in April 2002..  This study allows simultaneous assessment of immunologic and microbiologic parameters, both prospectively and retrospectively, in subjects at various stages of MTB infection/disease.  The particular focus will be to capture immunologic events in household members undergoing tuberculin skin test conversion and persons who remain skin test negative despite prolonged and close contact with a person with active tuberculosis.  Suggestions from the TBRU Workshop held in October 2001 were included in the immune function studies and an Immunology Working group for KCS was established with investigators from CWRU and University of Oregon.

TBRU Participation in CDC HHC Study

As was the case during the previous contract period, TBRU has continued its participation in the CDC HHC study in the US (through processing of whole blood samples sent to Dr Hirsch's laboratory to assess MTB culture-filtrate-induced production of IFN-g, TNF-a and IL-10).  Blood samples arrive in Cleveland from CDC supported TB clinics around the US and Canada and are set up as whole blood assays. Cytokine content is assessed by Elisa in whole blood culture supernatants collected following 24h and 5 days of culture.

Immunologic and Microbiologic Predictors of Response to Treatment

The immunology component has continued enrollment for the protocol simultaneously assessing Immunologic and Microbiologic Predictors of Response to anti-MTB Treatment in Brazil.  However, with follow-up of the first 40 patients with smear-positive pulmonary TB complete and measurements of cytokines undergoing final, the predictor study protocol has been revised and now focuses on recruiting AFB smear negative, AFB culture positive patients with pulmonary TB, undergoing bronchoscopy for diagnostic purposes.  It is hoped that analysis of paired sputum and BAL specimens from these patients will help in further validating the use of sputum cytokines as a means for assessing anti-MTB immune responses at sites of active MTB infection.

Pilot Immunology Studies in Uganda

Enrollment of study subjects for the Pilot Immunology Studies in Uganda has been re-initiated after approval in November 2002 of the amended study protocol by IRB boards in Cleveland and Kampala (Aids Research Council).  Revisions to the original protocol were made both to respond to questions raised by members of the Uganda IRB board during the most recent annual review of this protocol, and also to incorporate regulatory procedures governing future use of retained patient samples recently established by US sponsoring agencies. Studies currently ongoing focus on further delineating subsets of T-cells particularly prone to apoptosis and (using the Elisaspot technique) in establishing the role of cytotoxic CD8 T cells in anti-MTB immune responses.

Developmental Immunology

Developmental immunology studies in Dr Boom's laboratory in Cleveland presently focus on further defining the role of the 19 kDa Ag, and other molecules prepared from MTB, in regulating MHC I and II function, and in establishing the contribution of gd T-cells and of TLR-receptors in regulating anti-MTB immunity. Work in Dr Hirsch's laboratory evolves around further establishing mechanisms involved in apoptosis of MTB-reactive T-cells.  Further, efforts are being made to improve the whole blood assay to measure specific arms of the immune response.

Work at Imperial College (in Dr Douglas Young's laboratory in London) continues to focus on developing the luciferase assay for use with BCG lux constructs in whole blood or PBMC, as well as developing additional constructs under the control of differentially regulated promoters from MTB, that could respond to immune activation.

Organization and Operation

Immunologic studies are initiated primarily at CWRU under the direction of Dr. Henry Boom and Dr. Christina Hirsch.  Pilot studies are also accomplished at the sites of clinical trials with technology transfer to improve the services of the immunology laboratories at these sites. 

Both Dr. Boom and Dr. Hirsch work directly with the laboratory staff during periodic visits to the immunology laboratories in Uganda and Brazil.  They also participate in weekly conference calls to maintain contact with the local technical staff.

Technology Transfer

Overview

An important objective for the TBRU is the development and improvement of sustainable laboratory services at the laboratories located at the international clinical trials sites in Uganda and Brazil.  Efforts to develop the capacity for new immunologic testing in Uganda are directed primarily through the Immunology Laboratory the Joint Clinical Research Centre [JCRC] in Kampala.  Technical staff in Cleveland work with visiting experts to assist in the training needed to introduce new laboratory tests, procedures, and equipment to laboratories in Uganda.

Example of Technology Transfer

As a recent example, Mr. Keith Chervenak, the senior research assistant in Dr. Boom's laboratory, spent six weeks at the TBRU Immunology Laboratory at JCRC in Kampala, Uganda during March-April 2002, working with local technical staff to transfer assays developed at CWRU to the laboratory in Uganda. In particular, Mr. Chervenak trained the TBRU Uganda-based laboratory personnel in the use of the new tests, assisted the staff in determining how the assays could best be accomplished under existing field conditions and also was involved in the preparation of the required Standard Operating Procedures (SOP's).  Finally, he facilitated efforts to establish necessary coordination between the field-based TBRU Epidemiology team in Kawempe and the TBRU technical staff at the JCRC Immunology Laboratory.  The latter was achieved through education of the team of Home Visitors and on-site physicians with respect to the study-specific requirements of the immunology protocol.

Presently, Mr. Chervenak holds a monthly conference call with the Ugandan laboratory to discuss ongoing operations and to update staff on necessary protocol changes. Laboratory data are sent electronically to Cleveland for review by Mr. Chervenak before being added to the appropriate study database.

Laboratory Sites

JCRC Immunology Laboratory-Uganda

The immunology laboratory at the Joint Clinical Research Centre is a full service laboratory headed by Mr. Pierre Peters.  This laboratory works to provide immunologic laboratory services and participates in studies to evaluate the field and laboratory performance of immunologic tests needed to support TBRU clinical and basic research programs in Uganda.  The laboratory also assists the TBRU Specimen Repository in processing, storage and shipment of specimens collected during TBRU clinical trials to the US and other international sites, to facilitate testing of samples with assays not yet available in Uganda.

Nucleo Immunology Laboratory-Brazil

The TBRU has established a working relationship with the Universidade Federal do Espírito Santo [UFES], Vitoria, Brazil to conduct immunologic studies of host responses to infection by M. tuberculosis.  This site consists of a fully equipped laboratory as well as an isolation ward in the adjacent hospital dedicated for use in TBRU clinical studies.  The Laboratory Director is Dr. Reynaldo Dietze with Dr. Ribeiro Rodrigues responsible for immunologic studies.

Staff

The Director of the TBRU, Dr. Henry Boom leads the Immunology Component together with Dr. Christina Hirsch.  Dr. Hirsch provides the primary research link to the laboratories in Uganda and Brazil. 

Publications

Please click here for a list of TBRU publications