Expression of B7-1 costimulatory molecules in patients with multibacillary leprosy and reactional states.

BACKGROUND: The expression of B7 as a costimulatory molecule on the surface of antigen-presenting cells such as macrophages and on dendritic cells characterizes the efficiency of the cell-mediated immune response. AIMS: Our purpose was to evaluate B7-1 expression in peripheral blood mononuclear cells (PBMCs) immediately after cell isolation ('spontaneous' B7 expression), and in inflammatory cells from cutaneous lesions of patients with multibacillary leprosy (MB-L) without and during the reactional states of erythema nodosum leprosum (ENL) or reversal reaction (RR). METHODS: Peripheral blood samples and skin biopsies of eight patients without (MB-L) and with reactional episodes (ENL and RR) were studied using antibodies against B7-1, CD1b, DR and CD14 in flow-cytometry and immunohistochemistry experiments. RESULTS: The flow-cytometry studies (mean +/- SD% of fluorescent cells) revealed significant B7-1 expression on PBMCs isolated from patients with ENL (8.0 +/- 0.6%) and RR (15.0 +/- 1.4%) compared with that observed for patients with MB-L (0.4 +/- 0.2%). Similar results were observed for cutaneous lesions of these patients by immunohistochemical assays. One patient studied before and during ENL revealed weak B7 expression before the reactional episode (0.3% of cells) compared with the marked level of B7-expressing cells detected during ENL (8.5% fluorescent cells). Interestingly, an even higher B7 expression (15% of cells) was observed in patients with RR. CONCLUSIONS: Our results strongly suggest that B7 expression precedes reactional episodes in MB-L, which could be related to the acquisition of effective immunity to Mycobacterium leprae during reactional episodes in leprosy. We propose B7 expression as a marker of CMI response in reactional episodes in leprosy.

Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells potently activate autologous T cells via a B7 and interleukin-12-dependent mechanism.

Mycobacteria are potent adjuvants, can survive intracellularly and have been safely used for many years as vaccines against tuberculosis and leprosy. They are thus important potential vectors for recombinant vaccines. Many of their adjuvant properties are mediated following phagocytosis by dendritic cells (DC), which are in turn critical for priming naïve T cells. Although the maturation of DC in response to mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guérin (BCG), is well described the subsequent responses of autologous T cells to mycobacterium-infected DC remains uncharacterized. In our experiments DC infected with BCG expressed more co-stimulatory molecules than tumour-necrosis factor-alpha (TNF-alpha) -treated DC and stimulated more potent mixed leucocyte reactions. When autologous T cells were co-cultured with BCG-exposed DC they became highly activated, as determined by display of CD25, CD54 and CD71 on both CD4+ and CD8+ cells. In contrast, the response of T cells to TNF-alpha-matured DC was significantly less. Cytokine production from T cells cultured with BCG-exposed DC was enhanced with elevated secretion of interleukin-2 (IL-2), IL-10 and interferon-gamma (IFN-gamma) and was produced by both CD4+ and CD8+ lymphocytes as determined by intracellular staining. In particular, IFN-gamma secretion was increased from 50 pg/ml to 25 000 pg/ml and IL-10 secretion increased from 20 pg/ml to 300 pg/ml in BCG-exposed DC co-cultures. Blocking antibodies to B7.1 and B7.2 or IL-12 significantly reduced the secretion of IFN-gamma and reductions were also seen in the expression of CD25 and CD71 by CD4+ cells. These data demonstrate that mycobacterially infected DC are particularly potent activators of autologous T cells compared to TNF-alpha-exposed DC and that the resultant T cells are functionally superior.

Evaluation of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules and dendritic cells on the immune response in leprosy.

The cell activation depends on T cell antigen receptor binding to antigen plus MHC and costimulation. The binding of CD28, expressed on the T cell surface to B7 (B7-1 or CD80/B7-2 or CD86) present on the antigen--presenting cells (APCs), determines, in several T cell function models, if activation or anergy follows antigenic stimulation. In leprosy, the role of CD80 and CD86 as costimulatory signal in M. leprae-specific cellular immunity has not yet been defined. We investigated the role of B7-CD28 pathway of T cell activation in the in vitro response to M. leprae, following stimulation in the presence of monocytes or dendritic cells (DCs) as APCs. Monocytes were purified, by cold aggregation, from peripheral blood mononuclear leukocytes (PBMC), isolated from leprosy patients. In order to obtain DCs, the monocytes were cultured in the presence of IL-4 and GM-CSF. T cells were purified from PBMC by negative selection with mABs and C'. The phenotype of the cell populations was monitored by FACS. Lymphoproliferative assays were performed with T cells, in the presence of monocytes or DCs. The cells were stimulated by M. leprae in the presence of anti-CD80 antibody (Ab) and/or anti-CD86 antibody (Ab) (Innogenetics). In some experiments Il-10, Il-12 and anti-Il-12 Ab were also added to the culture. We observed a significantly more efficient APC function for DCs when compared to monocytes in T cell in vitro responses to M. leprae. Regardless of the clinical form of Leprosy, the M. leprae-specific immune response was markedly reduced in the presence of anti-CD86 Ab. Il-12 increase the immune response to M. leprae while IL-10 or anti-IL-12 Ab reduce this response when monocytes or DCs were used as APCs.

Addiction of anti-CD28 antibodies restores PBMC proliferation and IFN-gamma production in lepromatous leprosy patients.

During antigen recognition, T lymphocytes are primed by a physical interaction with antigen-presenting cells (APC). At least two signals are needed to activate T cells. One is provided by T cell receptor (TCR)/CD3 in the context of the mayor histocompatibility complex (MHC), and another signal is mediated by antigen-independent molecules, that is T cell membrane-bound CD28 and its specific ligand B7-1 (CD80) present in APC. Both signals trigger a series of metabolic events initiating right at the cell membrane and ending with activation and proliferation of T cells as well as specific cytokines synthesis. Our main goal was to determine whether deficiency in interferon-gamma (IFN-gamma) production shown by peripheral blood mononuclear cells (PBMC) from lepromatous leprosy (LL) patients, could be overcome by reconstituting in vitro the appropriate signals (by means of addition of anti-CD28 and anti-CD80 monoclonal antibodies). We also determined the stimulation index (SI) in the same PBMC. Our results demonstrated no significant differences in CD80 expression monocytes and B lymphocytes from LL patients when compared with healthy subjects. Nonetheless, CD28 expression significantly decreased in lymphocytes from LL patients (p < 0.01). Regarding IFN-gamma levels and SI, LL-PBMC failure before mitogenic stimuli could be reversed by further incubation with anti-CD28 antibody, but stimulation by specific antigen of Mycobacterium leprae was not changed. Addition of anti-CD80 antibody significantly increased IFN-gamma levels in phytohemagglutinin (PHA)-stimulated PBMC, although proliferation deficiency persisted. Cells stimulated with specific antigen did not modify either their proliferation or IFN-gamma levels.

B7-1, but not CD28, is crucial for the maintenance of the CD4+ T cell responses in human leprosy.

We used human leprosy as a model to compare patterns of costimulatory molecule expression in respect to the clinical/immunologic spectrum of disease. We found that B7-1, B7-2, and CD28 transcripts dominated in tuberculoid leprosy patients, who have potent T cell responses to Mycobacterium leprae. In contrast, CTLA-4 was more strongly expressed in lesions from lepromatous patients, who manifest specific T cell anergy to the leprosy bacterium. T cell clones from tuberculoid lesions were CD4+CD28+ or CD4+CD28-, and T cell clones from lepromatous lesions were predominantly CD8+CD28-. The M. leprae-specific recall response of CD4+ T cell clones from tuberculoid lesions was blocked by anti-B7-1 mAb, but not by anti-B7-2 mAb or CTLA-Ig. However, anti-CD28 and anti-CTLA-4 mAbs did not block activation of clones from tuberculoid lesions, suggesting that B7-1 may utilize another costimulatory pathway. Peripheral blood T cell responses in the lepromatous form were strongly regulated by CD28 during T cell activation, in contrast to the tuberculoid form. Thus, B7-1 costimulation could play a role in maintaining a strong immune response to the pathogen.

Potential role of B7-1 and CD28 molecules in immunosuppression in leprosy.

In order to understand the mechanism of unresponsiveness towards Mycobacterium leprae antigens in leprosy, we evaluated the role of M. leprae sonicate antigens in regulating the expression of the costimulatory molecules B7-1, CD28, intercellular adhesion molecule-1 (ICAM-1), LFA-1alpha, LFA-1beta and Mac-1 on the lymphocytes of both leprosy patients and healthy subjects. It was observed that the expression of B7-1 and CD28 was significantly decreased but the levels of ICAM-1 and LFA-1alpha were increased in patients with untreated borderline leprosy (BL)/lepromatous leprosy (LL) disease. No remarkable change was noticed in the case of borderline tuberculoid (BT) leprosy or treated BL/LL patients. Further, a striking finding was that lymphocytes from healthy subjects cultured with a particularly high dose of M. leprae sonicate antigens down-regulated the expression of B7-1 and CD28 molecules, but up-regulated the display of ICAM-1 and LFA-1alpha. Furthermore, proliferation induced by M. leprae sonicate was inhibited only by anti-B7-1 antibody. Mycobacterium leprae antigen-induced suppression of the proliferation of lymphocytes of healthy volunteers and LL patients was reversed by culturing the lymphocytes with purified protein derivative (PPD). It may be concluded from the findings in this study that down regulation of B7-1 and CD28 in BL/LL leprosy patients may be responsible for a defective T cell signalling by the B7-1/CD28 pathway caused by M. leprae antigens. This may lead to clonal inactivation of M. leprae-reactive T cells, consequently the bacilli grow without restriction in macrophages.