Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens.

Antigens of armadillo-derived Mycobacterium leprae sonic extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane, and the unstained blot was converted into 20 fractions of antigen-bearing particles. These were tested in cellular proliferation assays, and reproducible results were obtained between batches of fractions. Peripheral blood mononuclear cells from healthy contacts of leprosy patients (presumed to have protective immunity) were tested with the fractions to investigate which antigens they recognized. A small group of tuberculoid leprosy patients were also tested. Both groups showed a wide range of responses. Almost every fraction stimulated proliferation with at least one donor, yet none was clearly immunodominant or inhibitory in either group. Thus, protective immunity did not appear to be associated with proliferation caused by any single fraction.     

Reduced suppressor cell response to Mycobacterium leprae in lepromatous leprosy.

We have previously shown that concanavalin A (ConA) induction of suppressor cell activity is impaired in patients with lepromatous leprosy (LL). In this study, we demonstrated that the proportion of cells bearing the Leu8 antigen (associated with suppressor-inducer cells) is low in LL patients and tends to normalize during the erythema nodosum leprosum (ENL) episode. Antigen-induced suppressor cell function was evaluated by a two-stage assay. In the first stage, peripheral blood mononuclear cells (PBMC) were cultured for 5 days either in the presence of gamma-irradiated Mycobacterium leprae or in tissue culture medium as a control. In the second stage, mitomycin C-treated suppressor or control cells were added to phytohemagglutinin (PHA)- or ConA-stimulated autologous PBMC. The results indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in patients with tuberculoid (TT) and intermediate clinical (BB, BL, BT) forms and Mycobacterium bovis BCG-immunized normal controls. In ENL patients, the percent suppression was between that of TT and normal individuals. M. leprae-induced suppression was more effective on ConA- than on PHA-triggered T-cell proliferation in all groups. In contrast, normal PBMC cultured for 5 days in RPMI 1640 medium (N-C) and cells from patients with leprosy (TT-C and LL-C) had effects of their own on PHA- or ConA-induced proliferation. LL-C depressed the response to ConA and enhanced PHA-induced proliferation of autologous cells. Conversely, TT-C reduced PHA-induced proliferation and increased the ConA response. Suppression of proliferation could not be overcome with exogenous interleukin-2 and was not related to the induction of the Tac antigen. The abilities of LL, TT, ENL, and normal cells to proliferate upon PHA or ConA stimulus were similar, indicating that the defect in the generation of in vitro suppression by M. leprae in LL patients occurred during the induction period (step 1 of assay).     

In vitro lymphocyte stimulation in leprosy; simultaneous stimulation with Mycobacterium leprae antigens and phytohaemagglutinin

Peripheral blood lymphocytes from 105 subjects with different forms of leprosy and healthy contacts of leprosy patients were stimulated in vitro with different preparations of mycobacterial antigens alone or in combination with a suboptimal dose of phytohaemagglutinin (PHA). In nearly all individuals sonicated leprosy bacilli and PHA together gave a lower ³H-thymidine incorporation than did the same dose of PHA alone. There was no difference in the degree of inhibition seen in the different patient groups or the healthy contacts. High doses of whole, washed Mycobacterium leprae, combined with PHA led to an increased thymidine incorporation in borderline tuberculoid leprosy patients who had experienced a reversal reaction, and in healthy contacts with more than 6 months of exposure, while most lepromatous patients and contacts with less than 6 months exposure did not show an augmentation of the PHA-induced thymidine incorporation. The inhibition exerted by sonicated M. leprae was dose-dependent, seen even with very low doses of antigen, and was not due to direct cytotoxicity. M. bovis, strain BCG, was weakly suppressive in combination with PHA, and sonicated M. duvalii had a very marked suppressive effect. There was no correlation between the suppressive effect of M. leprae antigens and the other mycobacteria neither was there any correlation with the responses to the mycobacterial antigens alone. Many lepromatous leprosy patients showed significant suppression of background incorporation with addition of M. leprae antigens. This paper discusses whether the apparent `non-responsiveness' in lepromatous leprosy could be due to active suppressor mechanisms operative in vivo.     

Suppression of delayed hypersensitivity skin reactions to tuberculin by M. leprae antigens in patients with lepromatous and tuberculoid leprosy.

Delayed hypersensitivity skin reactions to tuberculin when injected alone or in mixture with antigens of M. leprae were examined in leprosy patients and in healthy controls. The tuberculin reaction was significantly inhibited in more than one half of both LL and BT patients by the soluble extract of M. leprae (leprosin), the leprosin derived 12 kD protein or leprosin depleted of the 12 kD antigen. However, suppression was not found in healthy controls from a leprosy endemic region. These results suggest that multiple M. leprae-specific antigens have an immunoregulatory function. Since suppression was demonstrable not only in LL (leprosin-anergic), but also in BT (leprosin-responder) patients it is of interest that the 'mixed' skin test can discriminate the immune status of at least certain BT patients from that of the infected but self-healing healthy controls. Corollary lymphocyte cultures failed to show any suppression by leprosing of the lymphoproliferative responses to tuberculin.     

Evidence for the presence of M. leprae reactive T lymphocytes in patients with lepromatous leprosy.

Evidence for the presence of Mycobacterium leprae reactive T cells in many lepromatous leprosy (LL) patients was obtained using in vitro antigen-induced lymphoproliferative responses. (1) Co-cultures of T enriched cells from LL patients when combined with 2 h adherent cells (AC) from HLA-D compatible tuberculoid leprosy individuals showed significant levels of 3H-thymidine incorporation in the presence of soluble and integral M. leprae antigens. (2) More interestingly, autologous T cell + AC co-cultures also showed significant improvement in antigen-induced lymphoproliferation in nine of 16 lepromatous patients. Insignificant improvement was observed in similar co-cultures of tuberculoid leprosy patients. (3) Addition of exogenous, purified human interleukin-2 (IL-2) to antigen stimulated PBMC from some lepromatous patients showed the best improvement in terms of overall 3H-thymidine incorporation, indicating that lepromatous patients possess T cells which can differentiate to an IL-2 responsive state. Significantly, the level of proliferation varied within the group. A proportion of clinically similar lepromatous patients failed to show improvement by any of the above methods.     

Failure to induce delayed-type hypersensitivity to Mycobacterium leprae in long-term treated lepromatous leprosy patients.

Lepromatous leprosy (LL) patients whose bacillary load has decreased to almost undetectable levels by long-term chemotherapy failed to develop delayed-type hypersensitivity (DTH) to Mycobacterium leprae antigen following immunization with killed armadillo-derived M. leprae. When these LL patients were immunization with killed armadillo-derived M. leprae. When these LL patients were immunized with killed M. leprae in a mixture with live BCG, only DTH to purified protein derivative (PPD) was induced. These results are further evidence that immunological unresponsiveness to the leprosy antigen of patients with lepromatous leprosy is antigen-specific and non-reversible.     

Comparison of IgM, IgG and IgA responses to M.leprae specific antigens in leprosy

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.     

Lsr2 peptides of Mycobacterium leprae show hierarchical responses in lymphoproliferative assays, with selective recognition by patients with anergic lepromatous leprosy

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.     

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.     

Lymphocyte transformation test in leprosy: decreased lymphocyte reactivity to Mycobacterium leprae in lepromatous leprosy, with no evidence for a generalized impairment.

Untreated leprosy patients were examined with respect to lymphocyte transformation in vitro after stimulation with mycobacterial and other microbial antigens, allogeneic lymphocytes, or nonspecific mitogens. Methods were used to circumvent technical variability. The results were compared with those obtained in controls matched for age, sex, race, and environment. No evidence was found for a generalized impairment of lymphocyte transformation in vitro, whereas a specific defect towards Mycobacterium leprae was demonstrable in lepromatous leprosy patients. The response to M. leprae, investigated in untreated and treated leprosy patients, decreased along the leprosy spectrum. Moreover, the results of the one-way mixed lymphocyte cultures showed that lymphocytes from leprosy patients had a normal stimulator and responder capacity, when they were tested against a panel of allogeneic lymphocytes. The influence of serum factors was investigated in untreated leprosy patients in the mixed lymphocyte culture. On average, tuberculoid as well as lepromatous sera showed a low-level depressive effect, but some sera showed a stimulatory effect. Therefore, a depressive effect of serum factors cannot be considered to be a general feature of leprosy. The correlation between the Mitsuda type of lepromin skin test and the lymphocyte reactivity in vitro to M. leprae was studied, and a positive correlation was found.     

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.     

Evidence that the Mitsuda reaction to Mycobacterium leprae can be mediated by lymphocytes responsive to Mycobacterium tuberculosis.

A positive Mitsuda skin test for delayed type hypersensitivity to Mycobacterium leprae is associated with a high level of protection against lepromatous leprosy, while the value of tuberculin sensitivity in leprosy is less pronounced. Cutaneous lymphocytes, isolated from the Mitsuda reaction of a PPD-positive individual not previously exposed to M. leprae, were cloned with Dharmendra lepromin and analysed for antigen specificity. Thirteen lepromin-responsive cell lines were derived, with greater than 95% certainty that the number of true clones was at least five and the number of functionally monoclonal lines at least seven. All lepromin-responsive clones proliferated in response to PPD as well, implying that PPD-responsive cells can fulfill the helper T cell function required for the in vivo Mitsuda reaction.     

Leprosy patients with lepromatous disease recognize cross-reactive T cell epitopes in the Mycobacterium leprae 10-kD antigen

T cell responses play a critical role in determining protective responses to leprosy. Patients with self-limiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity. Since the T cell reactivity of lepromatous patients to purified protein derivative (PPD), a highly cross-reactive antigen, is similar to that of tuberculoid patients, we queried if lepromatous patients could recognize cross-reactive epitopes in Mycobacterium leprae antigens as well. T cell responses were analysed to a recombinant antigen 10-kD (a heat shock cognate protein) which is available from both M. tuberculosis (MT) and M. leprae (ML) and displays 90% identity in its amino acid sequence. Lymphoproliferative responses were assessed to ML and MT 10 kD in newly diagnosed leprosy patients (lepromatous, n = 23; tuberculoid, n = 65). Lepromatous patients showed similar, but low, lymphoproliferative responses to ML and MT 10 kD, while tuberculoid patients showed much higher responses to ML 10 kD. This suggests that the tuberculoid patients may be recognizing both species-specific and cross-reactive epitopes in ML 10 kD, while lepromatous patients may be recognizing only cross-reactive epitopes. This was further supported by linear regression analysis. Lepromatous patients showed a high concordance in T cell responses between ML and MT 10 kD (r = 0.658; P < 0.0006) not observed in tuberculoid patients (r = 0.203; P > 0.1). Identification of cross-reactive T cell epitopes in M. leprae which could induce protective responses should prove valuable in designing second generation peptide-based vaccines.     

HLA-DQ molecules and the control of Mycobacterium leprae-specific T cell nonresponsiveness in lepromatous leprosy patients.

The major histocompatibility complex (MHC) controls of the outcome of the immune response to T cell-dependent antigens by dictating whether T cell responsiveness will result (MHC-immune response [Ir]genes) or alternatively T cell nonresponsiveness will occur, possibly through the activation of suppressor cells (MHC-immune suppression [Is] genes). In mice, I-A molecules typically restrict antigen-specific helper T cells. In contrast, H-2 I-E molecules have been reported to control nonresponsiveness to a variety of antigens through antigen-specific suppressor cells. In analogy, HLA-DR molecules are the dominant restriction elements for helper T cells in man. This forces the question whether DQ molecules may be involved in controlling nonresponsiveness in man, e.g. through suppression. In one system, T cell nonresponsiveness to Schistosoma japonicum, evidence has been presented supporting this notion. We have now used a second system, Mycobacterium leprae-specific T cell nonresponsiveness, that is typically found in lepromatous leprosy patients. We find positive but limited evidence for a role for HLA-DQ molecules in controlling T cell nonresponsiveness to M. leprae of the 22 nonresponder patients tested, 4 showed a proliferative T cell response to M. leprae after the addition of DQ- but not DR-specific mAb to the cell cultures. In one of the four BCG nonresponders, anti-DQ mAb had a similar effect.     

Humoral immunity in leprosy: immunoglobulin G and M antibody responses to Mycobacterium leprae in relation to various disease patterns.

A solid-phase radioimmunoassay, applying whole Mycobacterium leprae as antigen and radiolabeled protein A from Staphylococcus aureus as antibody-detecting reagent, was used for the determination of specific immunoglobulin G (IgG) and IgM antibody responses in leprosy patients. High IgG anti-M. leprae antibody levels were found in lepromatous leprosy patients, whereas the antibody response in tuberculoid leprosy patients varied from negative, i.e., comparable with responses measured in normal individuals, to strongly positive. In tuberculoid leprosy patients, a significant increase in IgG anti-M. leprae antibody levels was observed in the more widespread forms of the disease, but positive antibody responses were especially predominant among patients with active lesions. Lepromatous leprosy patients generally demonstrated high levels of both IgG and IgM anti-M. leprae antibodies, but no relation was found between the antibody responses and bacillary load or other clinical parameters. A marked decrease in specific IgG and IgM antibody levels was observed in lepromatous leprosy patients during their first year of treatment. Differences in mechanisms regulating the humoral immune response in tuberculoid and lepromatous leprosy patients were indicated, and the application of antibody assessments in leprosy control programs is discussed.     

Accessory cell heterogeneity in lepromatous leprosy; dendritic cells and not monocytes support T cell responses.

Dendritic cell (DC)-enriched cell populations from anergic lepromatous leprosy (LL) patients were found to be several-hundred-fold more efficient than monocytes (MO) in promoting antigen-induced T cell responses in autologous accessory + T cell cultures. Whereas, the use of autologous monocytes over a wide concentration range failed to stimulate Mycobacterium leprae-induced T cell proliferation, DC at concentrations as low as 0.1% induced significant proliferation in 9/15 and interferon gamma production in 14/15 LL patients. Four of the LL patients who failed to show proliferation were, however, able to secrete interferon gamma in the same T cell + DC co-cultures. DC were able to present particulate leprae antigens to autologous T cells. This preference for DC as an accessory cell was not shown when the cross-reacting antigen PPD was used in parallel co-cultures. Though tuberculoid leprosy patients showed some improvement in T cell proliferation with DC as compared to MO constituted co-cultures, this was not statistically significant. These results suggest that there is a heterogeneity in accessory cell requirement across the leprosy spectrum and that many lepromatous patients possess M. leprae-reactive functional T cells.     

Low T lymphocyte responsiveness to Mycobacterium leprae antigens in association with HLA-DR3

The type of leprosy which develops after infection with Mycobacterium leprae is influenced by the presence or absence of HLA-DR3, as has been demonstrated in an ethnic group originating from Surinam. In the present study we investigated in this same ethnic group the role of HLA-DR, and of HLA-DR3 in particular, in monocyte-T cell interactions during leprosy specific proliferative responses in vitro. HLA-DR3 heterozygous T cells from tuberculoid leprosy patients were cultured with antigen and either HLA-DR3 positive or HLA-DR3 negative homozygous HLA-DR compatible allogeneic monocytes as antigen presenting cells (APCs). T cell proliferative responses with DR3 homozygous monocytes as APCs, were observed to be decreased as compared to T cell proliferative responses with DR3 negative homozygous monocytes as APCs. Furthermore, although the leprosy specific monocyte-T cell interactions were shown to be restricted for HLA-DR, in the anti-MLW-1 response HLA-DR3 appeared to function as a restricting element poorly or not at all. These observations may imply, that an in vitro correlate has been found for an (HLA associated) genetic control of leprosy in vivo.     

Serological responses of patients with lepromatous and tuberculoid leprosy to 30-, 31-, and 32-kilodalton antigens of Mycobacterium tuberculosis.

Sera from patients with lepromatous and tuberculoid leprosy were examined in immunoblot assays for antibodies to Mycobacterium tuberculosis culture filtrate antigens. Antibodies to 30- and 31-kilodalton proteins were present in 88 and 81%, respectively, of 16 patients with lepromatous disease and absent in 16 patients with tuberculoid disease. Antibodies to a 32-kilodalton protein were found in 12 and 38% of lepromatous and tuberculoid patients, respectively. These reactivities may be useful for distinguishing lepromatous and tuberculoid leprosy.     

Use of a whole blood assay to evaluate in vitro T cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects.

Development of an immunological tool to detect infection with Mycobacterium leprae would greatly benefit leprosy control programmes, as demonstrated by the contribution of the tuberculin test to tuberculosis control. In a new approach to develop a 'tuberculin-like' reagent for use in leprosy, two new fractions of M. leprae depleted of cross-reactive and immunomodulatory lipids- MLSA-LAM (cytosol-derived) and MLCwA (cell wall-derived)-have been produced in a form suitable for use as skin test reagents. T cell responses (interferon-gamma (IFN-gamma) and lymphoproliferation) to these two new fractions were evaluated in a leprosy-endemic area of Nepal using a simple in vitro whole blood test. The two fractions were shown to be highly potent T cell antigens in subjects exposed to M. leprae-paucibacillary leprosy patients and household contacts. Responses to the fractions decreased towards the lepromatous pole of leprosy. Endemic control subjects also showed high responses to the fractions, indicating high exposure to M. leprae, or cross-reactive mycobacterial antigens, in this Nepali population. The new fractions, depleted of lipids and lipoarabinomannan (LAM) gave enhanced responses compared with a standard M. leprae sonicate. The cell wall fraction appeared a more potent antigen than the cytosol fraction, which may be due to the predominance of the 65-kD GroEL antigen in the cell wall. The whole blood assay proved a robust field tool and a useful way of evaluating such reagents prior to clinical trials.     

The role of interleukin-2 (IL-2) in the specific unresponsiveness of lepromatous leprosy to Mycobacterium leprae: studies in vitro and in vivo

The role of IL-2 in the immunological deficiency of lepromatous leprosy patients towards Mycobacterium leprae have been studied further. After initial stimulation with M. leprae + IL-2, lepromatous lymphocytes could be restimulated with M. leprae alone. The specificity of the responses obtained varied. Some patients gave a stronger response to BCG as compared to M. leprae, while in others a stronger response to M. leprae as compared to BCG was obtained. Studies of the composition of lymphocytes in dermal infiltrates subsequent to injection of killed M. leprae revealed that in both tuberculoid and lepromatous patients, early accumulation of cell staining for both IL-2 receptor and IL-2 were seen. However, with time IL-2 receptor and IL-2 staining lymphocytes diminished in lepromatous infiltrates, while these were maintained in tuberculoid lesions.