Differential T-Cell Recognition of Native and Recombinant Mycobacterium tuberculosis GroES

Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-gamma by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.     

T-Cell Recognition of Mycobacterium tuberculosis Culture Filtrate Fractions in Tuberculosis Patients and Their Household Contacts

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.     

In Vitro Stimulation of Lymphocytes in Leprosy Patients, Healthy Contacts of Leprosy Patients, and Subjects Not Exposed to Leprosy

In vitro lymphocyte stimulation was performed on peripheral blood lymphocytes from 48 leprosy patients, 15 healthy contacts of leprosy patients, and 16 normal controls who lived in a leprosy-free area and who had not been exposed to leprosy. Tuberculin PPD and an antigen fraction. MLW 1, prepared from M. leprae, were used as stimulants. The MLW 1 preparation contained one antibody-precipitable component when tested in crossed immunoelectrophoresis against a polyvalent anti-M. leprae immunoglobulin preparation, namely the ML 7 antigen. MLW 1 induced strong lymphocyte responses in patients with tuberculoid leprosy and healthy contacts of leprosy patients, but only a weak or no responses in lepromatous leprosy patients and non-exposed controls. A marked depression of the response to tuberculin PPD was observed in lepromatous leprosy patients. The specificity of the MLW 1 antigen is discussed, and a new estimator of specific lymphocyte stimulation, the delta cpm', is introduced.     

Mycobacterium leprae-Induced Alterations in Macrophage Fc Receptor Expression and Monocyte-Lymphocyte Interaction in Familial Contacts of Leprosy Patients

Macrophage Fc receptor expression and monocyte-lymphocyte interaction in the presence of Mycobacterium leprae were examined in familial contacts of leprosy patients. Defective Mφ functions similar to those of borderline and lepromatous patients could he observed in approximately 71% of consanguineous contacts and 43% of of spouses of index patients. Although the values in the latter group were markedly lower than those of the consanguineous contacts, they tended to be higher than those of normal individuals (20%). These in vitro Mφ functions were independent of age, sex, and age at onset of exposure and were only weakly associated with duration of exposure. The outcome of the monocyte-lymphocyte interaction lest paralleled to a large extent the in vivo Mitsuda lepromin response. Four contacts with defective Mφ functions also showed signs of leprosy. The value of these in vitro tests as markers of 'susceptibility' could therefore prove significant.     

T-Cell Recognition of Mycobacterial Proteins MPB70 and MPB64 in Cattle Immunized with Antigen and Infected with Mycobacterium bovis

Defined antigenic reagents and knowledge of T-cell responses are required for the design of improved diagnostic tests for bovine tuberculosis. The limited species distribution of Mycobacterium bovis antigens MPB70 and MPB64 has indicated their potential for inclusion in future tests. The strategy adopted in this study was to define bovine T-cell responses to these antigens at the epitope level, using cattle immunized with recombinant forms of the antigens, and to compare these responses with cattle which had been experimentally infected with M. bovis . Panels of synthetic peptides (20-mers with 10-residue overlaps) were used and five epitopes were identified and found to be powerful stimulators of T-cell responses in both types of animal (residues 81–100 and 174–190 for MPB70; and residues 1–20, 41–60 and 181–200 for MPB64). Further investigation in larger numbers of cattle ( n  = 14) of mixed breeds from tuberculosis-infected herds confirmed that each peptide produced response in several of the cattle, but no single peptide was recognized by all animals. However, the limited numbers of animals in this study suggest that peptide reagents may identify as many positive animals as the intact antigenic protein and could form components of a future diagnostic test. The use of cattle immunized with the proteins of interest has proved to be an interesting model for studying the nature of bovine T-cell responses to defined mycobacterial proteins.     

Mechanism of T-Cell Activation by Mycobacterial Antigens in Inflammatory Synovitis

Earlier studies demonstrated enhanced proliferative responses to an acetone precipitable Mycobacterium tuberculosis (AP-MT) antigenic complex by T lymphocytes from the synovial fluid, compared with the peripheral blood, of patients with inflammatory synovitis, including rheumatoid arthritis. In contrast, decreased proliferation and interleukin 2 (IL-2) production in response to mitogens by synovial fluid lymphocytes from patients with rheumatoid arthritis has been demonstrated. In order to determine if IL-2 was produced in response to AP-MT, the peripheral blood and synovial fluid of patients with inflammatory arthritis were analysed by measuring proliferation and IL-2 production in response to AP-MT and tetanus toxoid. A reduction of IL-2 production relative to proliferation was observed in some, but not all, synovial fluids of patients who responded to the AP-MT. Nevertheless, antibodies to IL-2 as well as interleukin 4 (IL-4), significantly inhibited proliferation of synovial fluid lymphocytes by AP-MT. There was no inhibition by antibodies to interleukin 6 (IL-6). We conclude that AP-MT induced proliferation by synovial fluid lymphocytes is mediated by both IL-2 and IL-4.     

A Comparative Study on the Effects of rIL-4, rIL-2, rIFN-γ, and rTNF-α on Specific T-Cell Non-Responsiveness to Mycobacterial Antigens in Lepromatous Leprosy Patients in Vitro

We have studied lepromatous leprosy (LL) as a human model disease for T-cell non-responsiveness to specific mycobacterial antigens and studied the effect of rIL-4, rIL-2, rIFN-gamma and rTNF-alpha thereon. T-cell non-responsiveness to Mycobacterium bovis bacillus Calmette-Guerin (BCG) or purified protein derivative of M. tuberculosis (PPD) antigens could be overcome in 5 out of 8 non-responder patients by rIL-2 and in 2 out of 8 by rIL-4. The ability of rIL-4 to overcome BCG/PPD non-responsiveness was strongly dose-dependent. When rIL-2 and rIL-4 were added simultaneously, they seemed to synergize in their effect. T-cell non-responsiveness to M. leprae could be overcome only in 2 out of 18 non-responders by rIL-2 but not by rIL-4 alone. The ability of rIL-2 to overcome T-cell non-responsiveness to M. leprae antigens became particularly marked when the recombinant 65-kDa heat shock antigen of M. leprae was used instead of whole bacilli. Exogenously added rIL-4, and to a lesser extent rIL-2, strongly enhanced existing T-cell responses to BCG or M. leprae in the majority (8 out of 11) of responders. These findings may have implications for the in vivo manipulation of the immune response by recombinant lymphokines and vaccines.     

T-Cell Responses to Fractionated Antigens of Mycobacterium w, a Candidate Anti-Leprosy Vaccine, in Leprosy Patients

Mycobacterium w, an atypical cultivable mycobacterium, is undergoing phase III clinical trials as a vaccine against leprosy in India. It has brought about lepromin conversion and histopathological upgradation in a significant number of patients studied so far. It is important to identify antigens of M. w that trigger T-cell responses in leprosy patients vaccinated with this organism. In the present study the peripheral T-cell repertoire of 12 M. w-vaccinated leprosy patients, 10 unimmunized leprosy patients, 8 tuberculoid and 5 healthy contacts was analysed with fractionated antigens of M. w. The lepromatous leprosy patients who are in general anergic to antigens of M. leprae did not respond to antigens of M. w. However, peripheral blood mononuclear cells obtained from leprosy patients who had been vaccinated with M. w responded to many antigens. These responses were frequently directed against low molecular weight entities of 14-45 kDa. T cells from tuberculoid leprosy patients and healthy contacts also responded predominantly to a number of low molecular weight antigens of M. w. The study also identified an immunodominant 28-31 kDa antigenic fraction carrying T- as well as B-cell activating determinants.     

Intradermal Recombinant Interleukin 2 Enhances Peripheral Blood T-Cell Responses to Mitogen and Antigens in Patients with Lepromatous Leprosy

Thirty-one patients with lepromatous leprosy received recombinant interleukin 2 (IL-2) intradermally in doses ranging from 10 to 30 micrograms. Before injection and at time intervals of 2-21 days thereafter, samples of peripheral blood mononuclear cells (PBMC) were obtained. Single or multiple injections (1-3) of IL-2 did not modify the total number of circulating lymphocytes or the number of T cells and the CD4/CD8 T-cell ratio. However, IL-2 had a pronounced influence on the [3H]thymidine incorporation in response to various stimuli 4-8 days after intradermal IL-2. Stimulation indices of three- to sevenfold above pre-IL-2 levels were observed with the polyclonal activator phytohaemagglutinin (PHA) and enhanced thymidine incorporation occurred in the presence of antigens to which the patients were already sensitized, such as purified protein derivative and BCG. IL-2 had no effect on the unresponsive state of lepromatous leprosy patient T cells to the antigens of Mycobacterium leprae.     

HLA Class-II-restricted Mycobacterium lepraereactive T-Cell Clones from Leprosy Patients Established with a Minimal Requirement for Autologous Mononuclear Cells

This report describes an effective method for the cloning of Mycobacterium leprae-reactive T lymphocytes with Epstein-Barr-virus transformed autologous B cells as antigen-presenting cells. The two advantages of this method are that it drastically reduces the number of autologous peripheral blood mononuclear cells (less than 10(7) cells) needed to obtain and propagate these T-cell clones (TLC), and that it enables us to expand individual TLC to large numbers of cells (greater than 10(8)). Thus the major obstacles for the cloning of T lymphocytes--especially important with regard to patients--are bypassed. Thus far, TLC from three leprosy patients have been established. These TLC are HLA class II restricted in their M. leprae-directed response. A marked enhancement in antigen responsiveness was observed after further expansion of several TLC, some of which turned from nonresponder into responder TLC. Four tested TLC display strikingly different antigen recognition patterns when tested against a number of other mycobacterial antigens; one TLC so far recognizes only M. leprae antigens.     

Lymphangiogenesis Is Induced by Mycobacterial Granulomas via Vascular Endothelial Growth Factor Receptor-3 and Supports Systemic T-Cell Responses against Mycobacterial Antigen

Granulomatous inflammation is characteristic of many autoimmune and infectious diseases. The lymphatic drainage of these inflammatory sites remains poorly understood, despite an expanding understanding of lymphatic role in inflammation and disease. Here, we show that the lymph vessel growth factor Vegf-c is up-regulated in Bacillus Calmette-Guerin– and Mycobacterium tuberculosis–induced granulomas, and that infection results in lymph vessel sprouting and increased lymphatic area in granulomatous tissue. The observed lymphangiogenesis during infection was reduced by inhibition of vascular endothelial growth factor receptor 3. By using a model of chronic granulomatous infection, we also show that lymphatic remodeling of tissue persists despite resolution of acute infection and a 10- to 100-fold reduction in the number of bacteria and tissue-infiltrating leukocytes. Inhibition of vascular endothelial growth factor receptor 3 decreased the growth of new vessels, but also reduced the proliferation of antigen-specific T cells. Together, our data show that granuloma–up-regulated factors increase granuloma access to secondary lymph organs by lymphangiogenesis, and that this process facilitates the generation of systemic T-cell responses to granuloma-contained antigens.The lymphatic system is made of a network of tissue-resident lymphatic endothelial vessels that drain extracellular fluid to the lymph nodes and back into blood circulation, a process that is critical in maintaining body fluid balance. Lymphatics also play a critical role in transporting dendritic cells (DCs) of the immune system, which may contain bacterial, viral, or fungal peptides, to T- and B-cell areas in the lymph nodes. Afferent lymph vessels express high levels of chemokines CCL19/21, which bind to CCR7 on activated DCs and induce their migration across lymphatic endothelial cells toward lymph nodes.^{1, 2, 3} Soluble antigen alone can also flow through the lymph to the lymph nodes, where it can be acquired by lymph node–resident DCs and presented to T and B cells.^{4, 5} Through these processes, adaptive immunity and clonal expansion of lymphocytes are initiated during infection.Although the role and requirement of lymphatics during steady-state conditions are well studied, the mechanisms and consequences of lymphangiogenesis during inflammation are far less so by comparison. Lymphangiogenesis is induced during neonatal development, as well as postdevelopment (inflammation, infection, and tumor growth) by vascular endothelial growth factor (VEGF)-C and VEGF-D binding to vessel-expressed VEGF receptor 3 (VEGFR3).^{6, 7, 8, 9} CD11b⁺ monocytes have been identified as an important initiators of lymphangiogenesis because they produce VEGF-C and VEGF-D after proinflammatory stimuli^{10, 11, 12} and can integrate into pre-existing lymph vessels and transdifferentiate into lymphatic endothelial-like cells.¹³ Recent evidence shows an unappreciated role for lymphatics and lymphangiogenesis beyond transportation of antigen-presenting cells and peptides to the lymph nodes. These functions include direct modulation of DC and T-cell activation or tolerance,^{14, 15, 16, 17} the presentation of antigens,^{18, 19} and egress of T cells from lymph nodes.^{20, 21} The growing appreciation of diversity in lymphatic function ensures the importance of understanding lymphangiogenesis during infection and inflammation.Granulomatous immune responses are associated with many infectious and autoimmune diseases. The granuloma itself is a macrophage-dominated collection of leukocytes that forms with defined spatial and organizational arrangement, and these sites are important in the protection and pathology during granulomatous diseases.^{22, 23, 24, 25} During infectious disease, granulomas contain the immune response-inducing antigens, and so engagement between the peripheral immune organs and these antigens is required. Lymphatic vessels are important because they are routes that soluble and DC-carried antigens use to reach the lymph nodes from granulomatous tissue. The relationship between the granulomas and lymphoid vessels, especially in the context of lymphangiogenesis, is not yet understood. Here, we used two different mycobacterial models of granulomatous inflammation to investigate this relationship. This first involves high-dose infection with the Bacillus Calmette-Guerin (BCG) strain of mycobacterium, which induces acute granulomatous inflammation in the liver 3 weeks after infection. Resolution of inflammation after 3 weeks results in reduced, but persistent, BCG-containing granulomas in the chronic stages of infection. Granulomatous inflammation of the liver is a characteristic pathology of diseases including histoplasmosis^{26, 27, 28} and schistosomiasis,^{29, 30, 31} and many tuberculosis patients also have tubercle granulomas in their livers.^{32, 33, 34} We also used a mouse model involving aerosol infection in the lung with Mycobacterium tuberculosis (MTB). This model is distinct from systemic BCG infection because acute granulomatous inflammation does not resolve, and mice eventually succumb to disease resulting from increasing granuloma and bacterial burden. Understanding the relationship between granulomatous inflammation and lymphangiogenesis will undoubtedly involve an understanding of the infectious context given that granulomas can occur in different organs and the fact that lymphatic form and function are adapted to the anatomy of the tissue.Here, using both models, we show that granulomatous inflammation induces lymphangiogenesis and that the biology of this process has a regulatory role in the proliferation of mycobacterial-specific T cells.     

T-Cell Stimulation with Purified Mycobacterial Antigens in Patients and Healthy Subjects infected with Mycobacterium leprae: Secreted Antigen 85 is Another Immunodominant Antigen

Peripheral blood leucocytes from 9 paucibacillary and 12 multibacillary leprosy patients, from 18 healthy controls and from 34 healthy leprosy contacts were stimulated with three mycobacterial heat shock proteins with respective molecular weights of 70,65 and 18 kDa and with the secreted 30–32 kDa protein, also called antigen 85. Antigen 85 was found to be the most powerful T-cell antigen (as measured by lymphoproliferation and IFN-γ secretion), eliciting a positive response in all (100%) paucibacillary patients and in all lepromin-positive controls and contacts. The three heat shock proteins (hsp) were less active T-cell stimuli. Reactivity to the 70 kDa hsp was found in only 44% of the paucibacillary patients, in 80% of the lepromin-positive controls and in 60% of the lepromin-positive leprosy contacts. The 65 kDa hsp stimulated T cells in 89% of the paucibacillary patients and in 80% of the lepromin-positive controls and contacts. Responsiveness to the 18 kDa hsp, finally, was clearly more frequent in tuberculoid leprosy patients (78%) than in lepromin-positive controls (40%) or lepromin-positive leprosy contacts (4%). T-cell reactivity of 8 lepromin-negative controls, of 9 lepromin-negative contacts and of 12 multibacillary leprosy patients was low to all the antigens tested. Although proliferative and IFN-γ responses were generally closely related, some subjects demonstrated a dissociation of these two immune parameters. Our data confirm previous findings on the powerful T-cell stimulatory properties of antigen 85 during M. leprae infection and suggest that this antigen is indeed a potentially protective T-cell immunogen.     

Consequences of Smallpox Vaccination in Leprosy Patients

This study illustrates the consequences of smallpox revaccination in 45 lepromatous, 28 tuberculoid, and 47 normal individuals. Results obtained with intradermal inoculations indicated that the patients with leprosy were associated with a relative anergy against the vaccinia virus, the anergy being minimal in the tuberculoid leprosy but marked in the cases with lepromatous leprosy. Major vaccinial reactions were observed more often in patients with lepromatous leprosy than in the controls or patients with tuberculoid leprosy. Furthermore in a patient with lepromatous leprosy, vaccinia necrosum also developed. The smallpox vaccination with live virus also appeared as a provocative factor for the precipitation of lepra reaction in the lepromatous leprosy cases. After 3 weeks of vaccination, the frequency of the specific humoral antibody response was the same in the tuberculoid patients and controls while it was higher in the cases with lepromatous leprosy. The prevaccination titer of total hemagglutination inhibition antibody was significantly higher in the lepromatous leprosy cases. However, the postvaccinial, humoral antibody response of the lepromatous patients was of the same magnitude as that observed in the normal individuals, and it was mainly due to a 2-mercaptoethanol-resistant antibody.     

Individuals from Different Populations Identify Multiple and Diverse T-Cell Determinants on Mycobacterial HSP70

The 70 kDa heat-shock protein (HSP) of Mycobacterium leprae stimulates both cellular and antibody responses in leprosy patients and subclinically infected individuals despite partial homology with host HSP70. Furthermore, mycobacterial HSP70 can act as a carrier protein in unprimed mice, suggesting the presence of widely shared T-cell determinants on this protein. In order to elucidate the frequency and genetic restriction of these T-cell epitopes, we have undertaken a systematic analysis of the proliferative responses to 20mer peptides encompassing the whole protein in different populations. Caucasian BCG vaccinees who responded to recombinant M. leprae HSP70 identilied multiple scattered T-cell determinants, four of which were recognized by 60% of subjects in association with a variety of HLA-DR haplotypes. When a group of Nepali leprosy and tuberculosis patients were tested, significant differences in the pattern of peptide recognition were observed. The dominant peptides recognized by Caucasian subjects were infrequently reactive and other peptides were stimulatory, again in association with a variety of HLA-DR phenotypes. The C-terminal 70 residues of the M. leprae HSP70 are specific to M. leprae and sera from lepromatous leprosy patients bind to this region. However, few T-cell determinants were identified in these residues, indicating that this region is unhelpful as a diagnostic tool for detecting M. leprae-specific T-cell responses. When compared with the equivalent regions of the human HSP70, the commonly recognized peptides showed significant differences in amino-acid sequence. When taken in conjunction with the failure of human HSP70 to stimulate M. leprae HSP70-reactive T-cell clones (E. Adams et al., unpublished observations), this finding indicates that the human T-cell response to this protein is largely directed at mycobacterial-specific determinants. The presence of multiple T-cell epilopes on M. leprae HSP70 with varied patterns of HLA-DR association suggests that the whole protein is required for stimulating effective T-cell responses in genetically diverse populations.     

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.