Ability of lymphokine-activated killer cells to lyse mycobacteria-infected cells.

Lymphokine-activated killer (LAK) cells were generated by interleukin-2 activation of peripheral blood lymphocytes obtained from lepromatous leprosy (LL) patients and healthy individuals. The ability of LAK cells to lyse targets (macrophages and T-24, a bladder carcinoma cell line) infected with mycobacteria (Mycobacterium leprae and mycobacterial strain ICRC) was assessed in a 51 chromium-release assay. It was observed that LAK cells generated from LL patients and healthy individuals could preferentially lyse M. leprae or ICRC-pulsed macrophages and T-24 cells, compared to non-pulsed targets. The ability of LAK cells to kill intracellular mycobacteria was demonstrated in colony forming assays. These results indicate a promising role for LAK cells in immunotherapy of leprosy.

Immunoprecipitation of mycobacterial antigens with sera from patients with leprosy.

Pooled sera from leprosy patients across the clinical spectrum, tuberculosis patients and healthy individuals were tested for their reactivity with antigens of Mycobacterium leprae and a panel of cultivable mycobacteria by immunoprecipitation technique. Sera from lepromatous leprosy patients demonstrated exclusive reactivity with the 26-kDa protein of M. tuberculosis H37Ra, 28-kDa protein of M. kansasii, 45-kDa protein of M. smegmatis, and 158, 40 and 14 kDa proteins of M. phlei. Sera from patients with borderline tuberculoid leprosy, tuberculoid leprosy, tuberculosis and health individuals failed to identify these antigens. Our studies indicate that analysis and characterization of immunodominant antigenic epitopes present on proteins of cultivable mycobacteria, sharing cross-reactive epitopes with M. leprae may prove to be important in the serodiagnosis of multibacillary leprosy as well as for developing vaccines for immunotherapy of leprosy.

Immunoreactivity of mycobacterial strain ICRC and Mycobacterium leprae antigens with polyclonal and monoclonal antibodies.

ICRC, a cultivable mycobacterium, is undergoing clinical trials as an antileprosy vaccine in India. In the present study, we have investigated the crossreactivity between antigens of the mycobacterial strains of ICRC and Mycobacterium leprae using polyclonal and monoclonal antibodies in a radioimmunoprecipitation assay. It was observed that polyclonal anti-ICRC and anti-M. leprae antibodies showed predominant reactivity to a 21-kDa protein of the mycobacterial strain ICRC and the 21- and 14-kDa proteins of M. leprae. Crossreactivity between the antigens of the mycobacterial strains ICRC and M. leprae was established further by using M. leprae-specific monoclonal antibody WML06 (reacting with the 14-kDa protein of M. leprae), which identified the 21- and 14-kDa proteins of the mycobacterial strain ICRC. Thus, our studies demonstrate that the 14-kDa protein of M. leprae, which is known to harbor T- and B-cell epitopes, shares crossreactive antigenic determinants with the 21- and 14-kDa proteins of the mycobacterial strain ICRC. We believe that such proteins may provide important reagents for designing subunit vaccines and for determining skin-test reagents.

Lymphocyte proliferation, IFN-gamma production and limiting dilution analysis of T-cell responses to ICRC and Mycobacterium leprae antigens in leprosy patients.

Lymphocyte proliferative responses and interferon-gamma (IFN-gamma) production after stimulation with antigens of ICRC, Mycobacterium leprae, and purified protein derivative (PPD) were assessed in leprosy patients and healthy donors. The patients studied were newly diagnosed as having lepromatous leprosy (LL), multidrug therapy (MDT) responders (MDT-R LL), MDT nonresponders (MDT-NR LL), borderline lepromatous (BL), and borderline tuberculoid/tuberculoid (BT/TT) leprosy. The tuberculoid leprosy patients showed increased lymphocyte proliferation and IFN-gamma production in response to stimulation with ICRC, M. leprae, and PPD antigens compared to other groups of LL patients and healthy donors. Although lymphocytes from LL patients showed low responses to ICRC and M. leprae antigens, their responses to PPD were not grossly affected. MDT-R LL patients showed higher lymphocyte proliferative responses and IFN-gamma production after stimulation with ICRC and PPD but not with M. leprae antigens. Tuberculoid leprosy patients showed higher T-cell frequencies to ICRC and M. leprae antigens compared to MDT-R LL and MDT-NR LL patients. The increased lymphocyte proliferative responses to ICRC observed in the MDT-R LL patients was reflected in the increased T-cell frequency to ICRC compared to M. leprae.

T-cell responses of leprosy patients and healthy contacts toward separated protein antigens of Mycobacterium leprae.

Sonicated extracts of Mycobacterium leprae were separated by two-dimensional gel electrophoresis and electroeluted into 400 distinct soluble fractions. These fractions were probed with T lymphocytes from leprosy patients of different disease types, healthy contacts, and unexposed healthy individuals. Proliferative responses were visualized using three-dimensional stimulation profiles. T cells from many patients and contacts responded to a multitude of antigen fractions of different molecular masses and isoelectric points. T cells from unexposed individuals gave significant responses to lysates or whole organisms of M. leprae, but no or only marginal responses to separated antigen fractions. T cells of polar tuberculoid (TT) and the majority of polar lepromatous (LL) leprosy patients responded only to separated antigen fractions but not to lysates or whole organisms of M. leprae. The remaining LL patients were totally unresponsive and even failed to respond to separated M. leprae fractions. Thus, in some leprosy patients unresponsiveness to M. leprae seems to be caused by distinct components and can be broken by using separated antigen fractions; whereas in others, anergy remains. T cells of borderline tuberculoid (BT) patients, who were under chemotherapy, responded to separated antigen fractions as well as to lysates of M. leprae organisms. In contrast, BT patients who were untreated failed to react with any of the M. leprae preparations. Similarly, T cells of the majority of LL patients responding to separated fractions were under chemotherapy; whereas T cells from untreated LL patients gave no or only marginal responses to any of the M. leprae antigen preparations. These findings suggest some linkage between the degree of T-cell responsiveness and antileprosy drug treatment.

Major proteins of mycobacterial strain ICRC and Mycobacterium leprae, identified by antibodies in sera from leprosy patients and their contacts.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.

Anti-leprosy vaccines: current status and future prospects.

Different types of leprosy vaccines are currently used in field trials in India. The rationale behind their use, the parameters for determining their efficacy, their merits and demerits are discussed and the future prospects are highlighted.

Mitogen induced proliferation and cytokine production by lymphocytes from leprosy patients.

In this paper we have assessed the mitogen responses of leprosy patients and healthy donors in terms of proliferation and cytokine production (Interleukin 2 and Interferon gamma). The patients investigated included untreated and multidrug therapy non-responsive LL patients and MDT responsive LL and TT patients. The mitogen responses of untreated and multidrug therapy non responsive LL patients were not significantly different from those of the healthy donors. It was interesting to note that TT and multidrug therapy responsive LL patients showed higher responses to mitogens than healthy donors as assessed by all three parameters. The results suggest a correlation of increased mitogenic responses with improvement in clinical status in leprosy.

Natural killer-cell-mediated and antibody-dependent cellular cytotoxicity in leprosy.

We have assessed the natural killer (NK) cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) in the peripheral blood lymphocytes (PBL) from untreated lepromatous leprosy (LL) patients, LL patients on multidrug therapy (MDT) with favorable responses (MDT-R), LL patients clinically classified as nonresponders to MDT (MDT-NR), treated tuberculoid leprosy (TT) patients, and healthy donors. NK cytotoxicity was modulated by treating the PBL with recombinant interferon-alpha (IFN-alpha) and recombinant interleukin-2 (IL-2). The mean percent NK cytotoxicity of untreated LL patients (15 +/- 3), treated MDT-R patients (20 +/- 4), and treated MDT-NR patients (12 +/- 4) was significantly lower than that of TT patients (39 +/- 6) and healthy donors (37 +/- 5). Treatment of effectors with IL-2 or IFN-alpha enhanced NK cytotoxicity in 5 of 6 untreated LL patients, 6 of 6 treated MDT-R LL patients, 4 of 5 and 3 of 5 treated MDT-NR LL patients, respectively, and 5 of 8 and 3 of 8 treated TT patients, respectively. Although PBL from TT patients showed initial NK activity comparable to that of healthy donors, fewer TT patients showed modulation of NK activity by IL-2, and IFN-alpha to a lesser extent. The ADCC activity was lower in untreated LL patients compared to treated patients, while TT patients had normal ADCC activity. The results indicate that although LL patients show lowered spontaneous cytotoxicity, it can be modulated favorably by lymphokines.