Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein.

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR alphabeta/gammadelta) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of alphabeta/gammadelta T cell receptor, the constitutive levels of TCR gammadelta were high in lepromatous patients. Thus, TCR bearing gammadelta appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-gamma but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNgamma.

Co-stimulatory signals increase the reactivity of gammadelta T cells towards mycobacterial antigens.

Although it has been shown that gammadelta T lymphocytes are able to react with different cell-associated or soluble antigens, the immune repertoire of these cells appears to be skewed to the recognition of mycobacterial antigens. We have studied the number and reactivity of gammadelta T cells towards several mycobacterial antigens in patients with tuberculosis and leprosy, as well as their healthy contacts and control individuals. We found an increased number of Vdelta2+ cells in healthy contacts (PPD+ and lepromin+) and tuberculoid leprosy patients. The gammadelta T cells from lepromatous leprosy showed a decreased response to all antigens tested, but some of these patients exhibited a significant response to the 30-kD glycoprotein of Mycobacterium tuberculosis. Interestingly, the reactivity of gammadelta T cells against mycobacterial antigens was significantly increased by costimulatory signals generated through CD7, LFA-1, CD50 and CD69 in all groups. However, signalling through CD69 did not enhance the responsiveness of gammadelta lymphocytes from lepromatous patients. On the other hand, the in vitro blockade of IL-10 with a specific antibody enhanced the cell proliferation of gammadelta lymphocytes from lepromatous leprosy patients, whereas exogenous IL-10 had an opposite effect in most individuals studied. These results suggest the potential role of different cell membrane receptors in the regulation of gammadelta T cell proliferation induced by mycobacteria, as well as the possible involvement of IL-10 in this phenomenon.

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.

Association of polymorphism at COL3A and CTLA4 loci on chromosome 2q31-33 with the clinical phenotype and in-vitro CMI status in healthy and leprosy subjects: a preliminary study.

Two genetic loci, viz. COL3A and CTLA4, located within the chromosome 2q31-33 region in the vicinity of the proposed syntenic site of the mouse "Bcg" locus were genotyped by the polymerase chain reaction in leprosy patients and healthy individuals. All the subjects studied were assessed as in-vitro responders/non-responders to mycobacterial antigens. Simple sequence length polymorphism analysis revealed five (236 to 312 bp) and eight (84 to 120 bp) allelomorphs for COL3A and CTLA4, respectively. Our preliminary analysis showed a significant association between the 250-bp COL3A allelomorph in the homozygous condition and the multibacillary form of leprosy (P < 0.05: relative risk = 5.5). Another allelic (312 bp) variant of COL3A was significantly correlated with non-responsiveness to M. leprae antigens in vitro (P < 0.01). The 104-bp allelomorph of CTLA4 was not observed in any of the 25 cases of leprosy. This absence was statistically significant (P < 0.05) when compared with normal healthy controls and depicted a high relative risk (RR = 25.83). An additional observation of the predominance of a unique 84-bp CTLA4/CTLA4-like allelomorph was observed in the Indian subjects studied.

The experimental granuloma. A hypothesis to explain the persistence of the lesion.

Granulomatous inflammation is the morphological substrate of a variety of important infectious diseases such as tuberculosis, leprosy, schistosomiasis and others. Nevertheless, although many aspects of this special type of inflammation are known, fundamental questions concerning granuloma formation, persistence, fate and significance for host-parasite relationships still remain to be elucidated. In this brief review, the basic and more relevant literature related to experimental investigations on granuloma physiopathology is presented. Based on recent investigations performed in our laboratory showing that MDF (Macrophage Deactivating Factor) secreted by epithelioid cells and characterized as the calcium-binding protein protein MRP-14 deactivates activated macrophages, a hypothesis to explain the persistence of granulomatous inflammation is put forward.

T lymphocyte subpopulations in leprosy patients and their relation with circulating immune complexes.

The possible relationship between circulating immune complexes (CIC) and peripheral T lymphocyte populations was studied in thirteen active multibacillary leprosy (10 lepromatous--LL--and 3 borderline lepromatous--BL--) and 19 matched controls. Theophylline-resistant T cells (The-R, a lymphocyte subpopulation displaying helper activity on B cells) and total T cells were assessed by means of the E rosette technique, with and without previous theophylline incubation, 1h 37 degrees C, respectively. CIC were quantified by 125I-C1q binding test. Although leprosy patients showed a statistical non significant light depression in total T cells the remarkable variability in circulating levels of The-R T cells enabled us to separate them into two well delineated groups (in relation to this variable p less than 0.001) with no difference in age, sex and bacteriologic state: a) leprosy patients with The-R T cells proportionally conserved (6LL and 2BL); b) leprosy patients with The-R T cells proportionally depressed (4LL and 1BL). Patients belonging to the latter group showed the highest statistically significant levels of CIC. Even though we do not discard an unknown factor being responsible for our findings, we believe that this inverse relationship between elevated CIC and depressed The-R circulating T cells might be representing a lower helper activity on antibody synthesis intending to reduce its excessive production.

CD1-positive epidermal Langerhans cells in regressed tuberculoid and lepromatous leprosy lesions.

A comparison was made of the numbers of epidermal Langerhans cells in active and regressed lesions of tuberculoid and lepromatous leprosy using the OKT6 and OKIa monoclonal antibodies. A reduction in the numbers of CD1+ epidermal Langerhans cells was noticed in the regressed lesions of both the tuberculoid and lepromatous leprosy lesions unlike the active lesions. The majority of infiltrates in both types of regressed lesions were HLA-DR+ and CD1-.

CD1-positive epidermal Langerhans cells in skin reactions to autologous peripheral-blood-derived mononuclear cells in leprosy patients.

A comparison was made on the characteristics of the infiltrates, the number and distribution of CD1-positive epidermal Langerhans cells (LC) at the sites of skin reaction induced by autologous peripheral-blood-derived mononuclear cells (PBMC) in leprosy patients. Clinically and histologically, the skin reaction was well expressed in tuberculoid patients as compared to lepromatous patients, erythema nodosum leprosum (ENL) patients and contacts. The quantum of lymphocytes in the infiltrates was maximal in the tuberculoid patients and it was minimal in lepromatous and ENL patients. The number and distribution of LC in the tuberculoid patients was significantly higher in the PBMC-inoculated sites as compared to control sites over 24 h. In contrast, no difference in the number and distribution of LC was noticed in the lepromatous and ENL patients. These observations indicate that the lymphocytes of tuberculoid patients in contrast to lepromatous leprosy patients are capable of sustenance in the local micro-environments of the skin and an effective interaction may be possible between LC and PBMC.

Characterization of a CD11c-reactive monoclonal antibody (HC1/1) obtained by immunizing with phorbol ester differentiated U937 cells.

A mouse monoclonal antibody (HC1/1) specific for a differentiation marker of human monocytes and granulocytes has been generated by using as immunogen the monocytic cell line U937 differentiated with phorbol esters. This differentiation antigen has been characterized as the p150/95 member of the CD11 family of glycoproteins by cellular distribution studies, immunoprecipitation and competition experiments with MAb 3.9 specific for the CD11c antigen. Immuno-alkaline phosphatase staining of normal tissue sections with the HC1/1 MAb demonstrated that the CD11c antigen is a useful macrophage marker. Furthermore, the MAb HC1/1 stains specifically populations of macrophages on skin and lymph node sections from different pathologies such as Sarcoidosis, Granuloma annulare, Lepromatous leprosy and Toxoplasmosis.