The in vitro production of interferon (IFN)-g, interleukin (IL)-5, tumour necrosis factor (TNF)-a 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-g production in addition to substantial TNF-a 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-g together with a mixed cytokine pro®le resembling a T helper (Th)0-type response. TNF-a/IL-10 ratios in response to M. leprae and Concanavalin A were signi®cantly higher in EC than in R contacts suggesting a role for the TNF-a/IL-10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine pro®les 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-g and TNF-a. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette-Gue Ârin (BCG) vaccination were investigated. Vaccination induced ampli®cation of IFN-g production with a concomitant decrease in TNF-a/IL-10 ratios that resembled the cytokine pattern observed in R contacts. IFN-g production was observed in response to both a cross-reactive antigen (Ag 85) and a M. lepraespeci®c protein (MMP-I), which attests to a BCG nonspeci®c 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´pathologic response to mycobacteria is presented.

No abstract

Household contacts of multibacillary leprosy patients (HCMB) constitute the group of individuals at the highest risk of developing leprosy. Early diagnosis and treatment of their index cases combined with Bacille Calmette-Guerin (BCG) immunization remain important strategies adopted in Brazil to prevent HCMB from evolving into active disease. In the present study, we assessed the impact of these measures on the immune response to Mycobacterium leprae in HCMB. Peripheral blood mononuclear cells (PBMC) from HCMB (n = 16) were obtained at the beginning of leprosy index case treatment (T0). At this time point, contacts were vaccinated (n = 13) or not (n = 3) in accordance with their infancy history of BCG vaccination and PBMCs were recollected at least 6 months later (T1). As expected, a significant increase in memory CD4 and CD8 T cell frequencies responsive to M. leprae whole-cell sonicate was observed in most contacts. Of note, higher frequencies of CD4+ T cells that recognize M. leprae specific epitopes were also detected. Moreover, increased production of the inflammatory mediators IL1-β, IL-6, IL-17, TNF, IFN-γ, MIP1-β, and MCP-1 was found at T1. Interestingly, the increment in these parameters was observed even in those contacts that were not BCG vaccinated at T0. This result reinforces the hypothesis that the continuous exposure of HCMB to live M. leprae down regulates the specific cellular immune response against the pathogen. Moreover, our data suggest that BCG vaccination of HCMB induces activation of T cell clones, likely through “trained immunity”, that recognize M. leprae specific antigens not shared with BCG as an additional protective mechanism besides the expected boost in cell-mediated immunity by BCG homologues of M. leprae antigens.

In spite of hyporesponsivity to Mycobacterium leprae, borderline lepromatous (BL) patients show clinical and immunological instability, and undergo frequent acute inflammatory episodes such as type 1 reaction (T1R), which may cause nerve damages. This work focused on the participation of T cell subsets from blood and skin at T1R onset. We observed a significantly increased ex vivo frequency of both effector and memory CD4+ and CD8+ T cells in T1R group. Besides, ex vivo frequency of T cell homing receptor, the Cutaneous Leukocyte-associated Antigen (CLA) was significantly increased in T cells from T1R patients. M. leprae induced a higher frequency of CD4+ TEM and CD8+ TEF cells, as well as of CD8+/TEMRA (terminally differentiated effector T cells) subset, which expressed high CD69+. The presence of IFN-γ‒producing-CD4+ TEF and naïve and effector CD8+ T lymphocytes was significant in T1R. TBX21 expression was significantly higher in T1R, while BL showed increased GATA3 and FOXP3 expression. In T1R, TBX21 expression was strongly correlated with CD8+/IFN-γ‒ T cells frequency. The number of double positive CD8+/CLA+ and CD45RA+/CLA+ cells was significantly higher in skin lesions from T1R, in comparison with non-reactional BL group. The observed increase of ex vivo T cells at T1R onset suggests intravascular activation at the beginning of reactional episodes. The antigen-specific response in T1R group confirmed the higher number of CD8+/CLA+ and CD45RA+/CLA+ cells in T1R lesions suggests possible migration of these cells activated by M. leprae components inside the vascular compartment to skin and participation in T1R physiopathology.

BackgroundDespite the efficacy of multidrug therapy, surviving Mycobacterium leprae causes relapse in some leprosy patients, and these patients present signs and symptoms of disease after healing. This study focused on the cellular immune response in relapsed multibacillary patients but also included non-relapsed multibacillary cured individuals, newly diagnosed and untreated multibacillary patients, paucibacillary patients just before the beginning of treatment, and voluntary healthy individuals for comparative analysis.Methodology/Principal FindingsInhibition of CD86 expression in the blood-derived monocytes and dendritic cells of relapsed multibacillary patients, either ex vivo or after M. leprae antigen stimulation was observed by flow cytometry. In addition, no significant changes in Interferon-gamma (IFN-γ) expression were observed in 5-day culture supernatants of relapsed patients in response to M. leprae, neither before nor after treatment, as measured by ELISA. However, these patients demonstrated a significant increase in central memory CD4+ and CD8+ M. leprae-specific T cells, as assessed by multiparametric flow cytometry. The increase in frequency of central memory T cells in relapsed patients strongly correlated with the bacillary index and the number of skin lesions observed in these subjects. Moreover, cytokine multiplex analysis demonstrated significant antigen-specific production of Interlukin-1beta (IL-1b), IL-6, and Tumour Necrosis Factor (TNF) in the relapsed group with extremely low IL-10 production, which resulted in a high TNF/IL-10 ratio.Conclusions/SignificanceInhibition of CD86 expression may function to reduce effector T cell responses against the M. leprae antigen. Furthermore, the predominance of central memory T cells in association with the high TNF/IL-10 ratio and no observed IFN-γ production may be related to the pathogenesis of relapse in multibacillary leprosy. Therefore, our findings may be a direct result of the clinical presentation, including a number of skin lesions and bacterial load, of relapsed patients. To our knowledge, this is the first study correlating immune response parameters with the clinical presentation of relapsed multibacillary patients.