The role of macrophages in the lymphoproliferative response to Mycobacterium leprae in vitro.

Peripheral blood lymphocytes from patients suffering from lepromatous leprosy do not normally react in vitro to stimulation by Mycobacterium leprae antigens. In contrast, we found that T cells from non-responding patients in combination with macrophages from responding patients or healthy contacts did respond well to M. leprae. Conversely, T cells from responding patients or healthy contacts in combinations with macrophages from non-responding patients failed to respond. It seems, therefore, that the lack of response normally observed in in vitro tests using cells from lepromatous leprosy patients is due to a failure of their macrophages to present M. leprae antigens in an immunogenic form.     

Cytokine measurement in lymphocyte culture supernatant of inactive lepromatous leprosy patients

The aim of the present study was to determine the effects of stimulation of sonicated Mycobacterium leprae (MLS) extract and phorbol myristate acetate (PMA) on the pattern of cytokine production in peripheral blood mononuclear cells (PBMC) and to find out whether there is any difference between stimulation of MLS extract and PMA. Blood samples were collected and PBMC isolated from 43 inactive lepromatous leprosy patients. After culture for 24 hours, lymphocytes were stimulated with MLS extract and PMA. In the culture supernatant, IL-2, 4, 6, 8, TNF-alpha and TGF-beta levels were measured by using ELISA. M. leprae stimulated group IL-6, IL-8, TNF-alpha, TGF-beta levels were found significantly higher than PMA stimulated group (P < 0.05). However, there was no difference between the two groups for IL-4. Only IL-2 levels were higher in PMA stimulated group than M. leprae stimulated group. Sonicated M. leprae extract have a strong effect on cytokine levels in vitro. Our results suggest that antigens with varying specificities favour the production of distinct cytokine patterns following in vitro restimulation.     

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.     

Quantification of antigen-reactive cells among human T lymphocytes.

The number of antigen-reactive cells among human peripheral blood T lymphocytes was estimated by a limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of incorporation of tritiated thymidine [3H]dThd. We have studied the frequency of memory T cells for the bacterial antigens tuberculin PPD and tetanus toxoid in immune donors, as well as the frequency of alloantigen-reactive T cells. In 11 different donors, the observed frequencies of the antigen-reactive T cell ranged between 1:300 and 1:16 000 for PPD; for tetanus toxoid values, between 1:750 and 1:11 500 were obtained in 5 different donors. The frequency of alloantigen-reactive T cells was found to be higher: between 1:200 and 1:600 (n = 10). For 3 donors, the estimated frequencies proved to be reproducible over a period of several months. Finally, a correlation could be demonstrated between the frequency of PPD-reactive T cells and the [3H]dThd incorporation of 4 X 10(4) PPD-stimulated lymphocytes.     

In vitro proliferative response to M. leprae and PPD of isolated T cell subsets from leprosy patients

In vitro proliferative response to Mycobacterium leprae and PPD to T cell subsets, isolated by selective depletion procedure from peripheral blood using OKT4 or OKT8 monoclonal antibodies plus complement, was investigated in leprosy patients. Whole peripheral blood mononuclear cells (PBMC) developed a strong proliferative response to both M. leprae and PPD in most tuberculoid patients. This proliferation was confined to T cells, and concerned predominantly OKT4+ cells. Both antigens, however, induced a smaller, but significant proliferation oF OKT8+ cells. In lepromatous patients, proliferative response of whole PBMC incubated with M. leprae was in most cases unsignificant, at variance with PPD-induced proliferation, which was not significantly lower than that of PBMC from tuberculoid patients. In a majority of M. leprae non-responders, neither OKT4+ nor OKT8+ enriched PBMC developed a proliferative response to M. leprae. Unexpectedly in four M. leprae unreactive patients, control treatment of PBMC with complement alone restored a strong proliferative response to M. leprae. Taken together, these results suggest that in vitro unresponsiveness to M. leprae results at least in some patients, from an active suppressor mechanism but that the effector phase of such suppression does not directly involve OKT8+ T cells.     

The suppressive effect of M. leprae on the in vitro proliferative responses of lymphocytes from patients with leprosy.

Peripheral blood lymphocytes from sixty leprosy patients and eight healthy contacts known to be responsive to M. leprae, were stimulated in vitro with concanavalin A (Con A) or PPD alone or in combination with autoclaved, whole M. leprae. Time kinetics and the percentage of inhibition induced by M. leprae differed in the two disease groups and contacts. Antigen-generated suppression of Con A-stimulated lymphocyte transformation was observed on day 4 in seventeen of twenty-one (80%) tuberculoid patients and six of seventeen (35.3%) untreated lepromatous patients. Healthy contacts and 53% lepromatous individuals showed enhanced Con A responses in the presence of antigen. On prolongation of antigen presence to 6 days, a marginal effect was noted in the tuberculoid group. In contrast, all healthy individuals and some lepromatous patients showed increased inhibition of Con A responses. M. leprae antigens showed uniform inhibition of PPD-induced 3H-thymidine incorporation in leprosy patients and healthy contacts.     

Lymphocyte response of leprosy patients to human-derived and purified armadillo-derived Mycobacterium leprae, BCG and PPD.

The lymphocyte transformation test was applied to compare in vitro lymphocyte responses of tuberculoid (high resistant) and lepromatous (low resistant) leprosy patients to purified Mycobacterium leprae derived from experimentally infected armadillos and crude M. leprae derived from man, as well as to bacille Calmette-Guérin (BCG) and purified protein derivative (PPD). It was found that the purification procedure using enzymic digestion did not affect the immunogenicity of armadillo-derived M. leprae as compared with the crude human-derived preparation, although 2.5-5-fold higher doses of the purified organisms were required to elicitate equivalent lymphocyte responses. The result indicated the suitability of purified armadillo-derived M. leprae as the standard antigen for lymphocytes transformation tests in leprosy. The cross-reactivity studies show a close relationship between PPD and BCG, but not between M. leprae and PPD or BCG.     

A placebo controlled clinical trial of transfer factor in lepromatous leprosy.

The effects of repeated injections of transfer factor over a period of 20 weeks were investigated in fourteen bacteriologically positive patients at the lepromatous side of the leprosy spectrum. All patients showed negative (0 mm induration) skin tests to M. leprae antigens (i.e. leprolin and lepromin). Of these patients, seven were treated with transfer factor with a total of 9 units (1 unit being equivalent to 5 x 10(8) lymphocytes) and seven with a placebo. Maintenance treatment with clofazimine was continued. Transfer factor was prepared from the lymphocytes of donors who showed positive skin tests to M. leprae antigens (i.e. leprolin greater than or equal to 12 mm induration, average 15.5 mm or lepromin greater than or equal to 8 mm induration, average 13.6 mm), as well as a positive lymphocyte transformation in vitro to M. leprae (the average transformation being higher than the average transformation of lymphocytes of tuberculoid leprosy patients). No differences were found between the two groups as regards the clinical course of the disease, the histopathological and bacteriological evaluation of skin biopsies, changes in skin test reactivity to various antigens (i.e. lepromin, leprolin, PPD, Mumps, C. albicans, Tr. rubrum and Varidase), as well as the lymphocyte transformation in vitro to various mitogens (i.e. PHA, PWM, Con A) and antigens (i.e. M. leprae, leprolin, PPD, BCG, Mumps, C. albicans, Trichophyton and Varidase). No evidence was found to suggest that transfer factor is a valuable adjuvant in the treatment of lepromatous leprosy patients or that it increases cell-mediated immune reactivity towards M. leprae.     

Studies on the Defect in Cell-Mediated Immunity in Lepromatous Leprosy using HLA-D-Identical Siblings

Sixteen healthy siblings were identified as HLA-D-identical to 12 borderline lepromatous or polar lepromatous leprosy patients by the absence of a mixed lymphocyte reaction (MLR). The peripheral blood mononuclear cells (PBM) of the healthy siblings showed a lymphoproliferative response (delta cpm) to Mycobacterium leprae antigens which was about fivefold or more greater than that of the lepromatous patients. Lepromatous PBM, with or without mitomycin C treatment, were co-cultured with a constant number of normal PBM. In other experiments the two cell types were co-cultured in various proportions, with the total cell number kept constant. Neither approach revealed suppressor cells in lepromatous PBM capable of suppressing the lymphoproliferative response to M. leprae. On the contrary, we found that lepromatous PBM can respond to M. leprae antigens if the sensitized lymphocyte is provided by mitomycin-C treated normal PBM. Additionally, experiments in which isolated adherent cells and non-adherent cells of sibling pairs were recombined failed to reveal a defect in the M. leprae antigen-presenting function of lepromatous adherent cells. Since we found no evidence that sensitized cells are present in lepromatous PBM with their function unexpressed (due to a monocyte defect) or suppressed (due to suppressor cells), we conclude that lepromatous patients simply lack sufficient numbers of antigen-specific T lymphocytes to initiate a lymphoproliferative response to M. leprae antigens. The reason for their absence remains an important unanswered question.     

Natural suppressor cells in human leprosy: the role of HLA-D-identical peripheral lymphocytes and macrophages in the in vitro modulation of lymphoproliferative responses

Six families with HLA-D-identical siblings suffering from leprosy were studied. Lymphocytes and macrophages isolated from the peripheral blood were co-cultured with allogeneic, HLA-D-identical cells and stimulated with M. leprae antigens and concanavalin A. Tuberculoid patients had circulating lymphocytes which showed marked functional suppression of lymphoproliferative responses to antigen and mitogen. In contrast, lepromatous patients showed weak lymphocyte suppressor activity. Macrophages derived from responder individuals augmented, while those derived from lepromatous patients inhibited, M. leprae-induced proliferation of lymphocytes.     

Immunological cytokine correlates of protective immunity and pathogenesis in leprosy

The in vitro production of interferon (IFN)-gamma, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and IL-10 by blood mononuclear cells in response to whole Mycobacterium leprae and polyclonal stimulii of 23 individuals, representing a variety of conditions in relation to exposure/susceptibility to M. leprae, was assayed. In most cases, healthy household contacts of newly diagnosed multibacillary leprosy patients, designated exposed household contacts (EC), showed low-to-undetectable in vitro IFN-gamma production in addition to substantial TNF-alpha production in response to M. leprae. In contrast, peripheral blood mononuclear cells from previously exposed contacts (R) regarded as resistant-to-leprosy released low-to-moderate levels of IFN-gamma together with a mixed cytokine profile resembling a T helper (Th)0-type response. TNF-alpha/IL-10 ratios in response to M. leprae and Concanavalin A were significantly higher in EC than in R contacts suggesting a role for the TNF-alpha/IL-10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine profiles of leprosy patients were taken as reference points. Post-treatment lepromatous leprosy patients secreted relatively high levels of IL-10 in response to M. leprae, whereas one self-cured tuberculoid leprosy patient produced simultaneously high levels of IFN-gamma and TNF-alpha. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette-Guérin (BCG) vaccination were investigated. Vaccination induced amplification of IFN-gamma production with a concomitant decrease in TNF-alpha/IL-10 ratios that resembled the cytokine pattern observed in R contacts. IFN-gamma production was observed in response to both a cross-reactive antigen (Ag 85) and a M. leprae-specific protein (MMP-I), which attests to a BCG nonspecific stimulation of the immune system, thereby casting these antigens as likely candidates for inclusion in a subunit vaccine against leprosy. Finally, a model for protective x pathologic response to mycobacteria is presented.     

Cellular immune response to Mycobacterium leprae infection in human immunodeficiency virus-infected individuals.

The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous leprosy, and eight patients had borderline tuberculoid (BT) leprosy. Despite the low CD4+ T-cell count in the peripheral blood, no histologic or phenotypic change in the cellular infiltrate in either the lepromatous or tuberculoid lesions was observed when compared with HIV-1-negative patients. Lepromatous lesions contained heavily parasitized macrophages and few CD8+ T cells. Lesions from the patients with BT leprosy showed extensive CD4+ T-cell infiltration despite a significant reduction in CD4+ T-cell counts in the peripheral blood. No acid-fast bacilli were detected in the tuberculoid lesions. HIV-1 infection did not alter the lack of response in lepromatous leprosy to M. leprae antigens either in vitro or in vivo. In contrast, the skin test response to M. leprae antigens as well as the in vitro lymphoproliferative responses to mycobacterial antigens that are usually seen in patients with tuberculoid leprosy were abrogated in the BT HIV-1+ patients. However, production of gamma interferon in response to the same stimuli was preserved in most of the patients. Analysis of cytokine gene expression showed activation of additional cytokine genes in the unstimulated peripheral blood cells of patients with both leprosy and HIV-1 infections as compared with cells from patients with leprosy alone. These results suggest that granuloma formation in leprosy can be independent of the impaired CD4+ T-cell response of the HIV-1 infection. Furthermore, in HIV-1+ individuals with M. leprae infection, activation of cytokine genes is observed even when the circulating CD4+ T-cell count is significantly reduced.     

Presence of Mycobacterium leprae-Reactive Lymphocytes in Lymph Nodes of Lepromatous Leprosy Patients

A critical problem in leprosy is the relative deficiency of antigen-specific T cell-mediated immunity. We were successful in detecting a significant response to viable M. leprae in mononuclear cells isolated from the lymph nodes of lepromatous leprosy patients in contrast to the apparent M. leprae-specific energy seen in the peripheral blood. This observation suggests that antigen-reactive lymphocytes are generated in the lymph nodes of lepromatous patients but the inability to detect them in the circulation may be due either to a different processing and presentation of mycobacterial antigens within the peripheral blood and lymph node compartments or to a selective sequestration of lymphocytes within the lymph node.     

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.     

Evidence that the Mechanism of Immunological Tolerance (''Central Failure'') Is Operative in the Lack of Host Resistance in Lepromatous Leprosy

The cellular immune defect in lepromatous leprosy has been studied. The following observations have been made: 1) Three lepromatous patients who had been on anti-leprosy treatment for more than 10 years still failed to respond to M. leprae by lymphocyte transformation (mean 0.2%) while they responded strongly to BCG (mean 34.2%) and PPD (mean 56.1%). 2) Lepromatous serum failed to inhibit M. leprae -induced lymphocyte transformation and M. leprae -induced leukocyte migration inhibition. 3) Lepromatous lymphocytes revealed a reduced capacity to attach M. leprae to their surface. The only experimental condition compatible with the observed characteristics would seem to be a state of immunological tolerance to an antigen (or antigens) of M. leprae. The lasting nature of this non-responsive state suggests that it plays a primary role in the pathogenesis of lepromatous leprosy.     

Flow Cytometric Analysis of CD2 Modulation on Human Peripheral Blood T Lymphocytes by Dharmendra Preparation of Mycobacterium leprae

It has been reported previously that Mycobacterium leprae modulated CD2 on human peripheral blood T lymphocytes and that this modulation was accompanied by a marked reduction in the proliferative response of these cells to mitogens and antigens. In this study, we report that treatment of peripheral blood mononuclear cells from healthy individuals with Dharmedra preparation of M. leprae inhibited their ability to form rosettes with sheep red blood ceils. Flow cytometric analysis of Dharmendra lepromin-treated cells showed that, in addition to CD2. CD4 and CD8 were modulated while the surface expression of CD3 was not affected. The specificity of CD2 modulation was confirmed by similar effects of Dharmendra lepromin on thymocytes and lymph node cells from human CD2 transgenic mice. The modulatory effect of Dharmendra lepromin was not observed at lower temperatures. Dharmendra lepromin treatment of activated T cells resulted in reduced binding of monoclonal antibodies to IL-2R and D66 epitope of CD2. The modulatory effects were not observed with Dharmendra preparation of BCG or other preparations of M. leprae. Our results indicate that certain M. leprae. factor(s) specifically modulate(s) CD2, CD4, CD8 and IL-2R but not CD3 on T lymphocytes. The suppressive effect of Dharmendra lepromin on the T-cell proliferative response reported earlier may be explained by its modulatory effect on a number of T-cell surface molecules.     

Effect of Mycobacterium leprae on lymphocyte proliferation: suppression of mitogen and antigen responses of human peripheral blood mononuclear cells

Evidence is presented that Mycobacterium leprae suppresses the in vitro proliferative response of human peripheral blood mononuclear cells (PBM) to antigen and mitogen. Lymphoproliferation induced by PPD or alloantigen stimulation was inhibited by concentrations of M. leprae which were not cytotoxic for lymphoblasts. In contrast, the inhibition of mitogen-stimulated PBM was seen only at higher concentrations of M. leprae which proved to be cytotoxic for lymphoblasts. The inhibitory effect was found not to be dependent on a particular cell population present in leprosy patients, as PBM from normal were inhibited similarly. These findings may explain some of the immunological aberrations observed in lepromatous leprosy patients who harbour large numbers of M. leprae bacilli in their tissues.     

Limiting dilution analysis of human, tetanus-reactive helper T lymphocytes. A rapid method for the enumeration of helper T lymphocytes with specificity for soluble antigens.

The number of helper T lymphocytes (HTL) in human peripheral blood with specificity for the soluble protein, tetanus toxoid, was estimated by limiting dilution analysis (LDA). HTL were detected via antigen-induced interleukin-2 (IL-2) production, as measured by incorporation of tritiated thymidine by an IL-2-dependent indicator cell line, CTLL-20. Culture conditions optimizing assay sensitivity are described, and the ability to detect antigen-specific HTL using this LDA technique are demonstrated. Observed HTL frequencies in healthy human donors tested for tetanus-reactive helper T cells ranged from less than 1 HTL/268,749 peripheral blood mononuclear cells (PBMC) (undetectable) to 1 HTL/1486 PBMC. The LDA technique was also used to detect frequency shifts in human peripheral blood HTL following challenge with antigen. This assay offers distinct advantages over proliferative LDA techniques in that it is rapid (requiring only 2 days), and defines an antigen-reactive T cell subset with defined function (IL-2 secretion). Furthermore, the LDA technique can be adapted for the detection of other soluble protein antigens, such as PPD and Candida albicans. In general, this LDA technique provides a reliable, quantitative index of human HTL reactivity to any of a variety of soluble protein antigens, and has clinical as well as experimental applicability.     

T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae.

The 18-kilodalton (kDa) antigen of Mycobacterium leprae was expressed as a fusion protein with a 2-kDa leader peptide and used in proliferation assays with peripheral blood cells. Fifty percent of untreated tuberculoid leprosy patients and 93% of long-term leprosy contacts responded to the recombinant protein in lymphocyte transformation tests. Comparison of the stimulation indices in the two groups showed that the contacts responded more strongly than the tuberculoid leprosy patients. Seventy percent of Mycobacterium bovis BCG-vaccinated European donors responded, although with low stimulation indices. The isolation of 18-kDa antigen-responsive T-cell lines from a BCG-vaccinated British donor confirmed that the 18-kDa antigen contains at least one cross-reactive epitope. These results indicate that the 18-kDa protein is an important antigen in the immune response to leprosy.     

Mechanism of immunosuppression in leprosy: presence of suppressor factor(s) from macrophages of lepromatous patients.

Human peripheral blood mononuclear cell proliferation induced by Mycobacterium leprae could be inhibited by the suppressor factor in the lysate of the macrophages of lepromatous leprosy patients. Macrophages from normal subjects and tuberculoid patients did not show production of a suppressor factor. Inhibition occurred only when the factor was present in the initial stages of lymphocyte culture. The factor is heat stable and nondialyzable. Proliferation induced by some mycobacteria and concanavalin A could also be blocked by the factor. Interestingly, blastogenic response by a few other antigens and phytohemagglutinin could not be inhibited by the suppressor factor. Mononuclear cells pretreated with such lysate from lepromatous macrophages for 24 h could induce suppressive activity in the cells in vitro in an autologous system. Treatment of these cells with carbonyl iron after the induction phase, to remove phagocytic cells, did not abolish their suppressive activity. The lepromatous macrophage lysate also generated suppressive activity in a T-lymphocyte-enriched population of normal subjects. These studies are interpreted to indicate that immunosuppression in lepromatous patients is produced by both macrophages and T lymphocytes. The exact phase in which either of these cells acts as a suppressor may be different. Specific suppression by macrophages to M. leprae can be an early event, and nonspecific suppression by T lymphocytes may be a later event in the course of lepromatous leprosy.