Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

Multifunctional CD4(+) T cells correlate with active Mycobacterium tuberculosis infection.

Th1 CD4(+) T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN-γ/IL-2/TNF-α triple expressors, IFN-γ/IL-2, IFN-γ/TNF-α or TNF-α/IL-2 double expressors or IFN-γ, IL-2 or TNF-α single expressors) of CD4(+) T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85-90%TB patients, were only present in 10-15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12- to 15-fold) proportions of IL-2/IFN-γ double and IFN-γ single expressors as compared with the other CD4(+) T-cell subsets. Proportions of the other double or single CD4(+) T-cell expressors did not differ between TB and LTBI subjects. These distinct IFN-γ, IL-2 and TNF-α profiles of M. tuberculosis-specific CD4(+) T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB-infected patients after completion of anti-mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4(+) T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti-mycobacterial therapy.

Mycobacterial ESAT-6 and katG are recognized by sarcoidosis CD4+ T cells when presented by the American sarcoidosis susceptibility allele, DRB1*1101.

INTRODUCTION: Genetic associations of American sarcoidosis susceptibility implicate MHC class II allele, DRB1*1101. We previously reported immune recognition of Mycobacterium peptides from peripheral cells of 26 sarcoidosis subjects, 24 PPD- healthy volunteers, and eight with latent tuberculosis infection. MATERIALS AND METHODS: In order to further link these genetic and immunologic pillars of sarcoidosis pathogenesis, we performed flow cytometry on these same subjects to identify the cells responsible for immune responses to ESAT-6 and katG peptides, followed by HLA typing to determine allelic associations with recognition. DISCUSSION AND CONCLUSION: Sarcoidosis CD4+ T cells were primarily responsible for the systemic responses. Recognition was inhibited by monoclonal antibody against HLA-DR and HLA-DQ, but not HLA-DP. Immune recognition of ESAT-6 peptide NNALQNLARTISEAG was associated with possession of DRB1*1101. ESAT-6 and katG presented by antigen-presenting cells expressing DRB1*1101-induced Th-1 responses from sarcoidosis T cells, thus providing a mechanistic insight for the association of HLA DRB1*1101 with sarcoidosis, and sarcoidosis T cell interaction with microbial antigens.

Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli.

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Raf-mitogen-activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. leprae activated Erk1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKCepsilon-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, G1/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival.

A study of autologous melanocyte transfer in treatment of stable vitiligo.

BACKGROUND: Replenishing melanocytes selectively in vitiliginous macules by autologous melanocytes is a promising treatment. With expertise in culturing melanocytes, it has now become possible to treat larger recipient areas with smaller skin samples. AIM: To study the extent of repigmentation after autologous melanocyte transplantation in patients with stable vitiligo. METHODS: The melanocytes were harvested as an autologous melanocyte rich cell suspension from a donor split thickness graft. Melanocyte culture was performed in selected cases where the melanocyte cell count was insufficient to meet the requirement of the recipient area. These cells were then transplanted to the recipient area that had been superficially dermabraded. RESULTS: An excellent response was seen in 52.17% cases with the autologous melanocyte rich cell suspension (AMRCS) technique and in 50% with the melanocyte culture (MC) technique. CONCLUSION: Autologous melanocyte transplantation can be an effective form of surgical treatment in stable but recalcitrant lesions of vitiligo.

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.

In vitro tumor necrosis factor production by mononuclear cells from lepromatous leprosy patients and from patients with erythema nodosum leprosum.

The production of tumor necrosis factor (TNF) by Mycobacterium leprae-stimulated phagocyte cells, isolated from lepromatous leprosy patients (LL) and normal individuals, was evaluated, using the highly TNF-sensitive mouse fibrosarcoma cell line WEHI164cl13. Mononuclear cells, isolated from all individuals studied, showed a low level of spontaneous TNF production, except for patients undergoing erythema nodosum leprosum (ENL), in which we found significantly higher levels of TNF. Addition of M. leprae to the phagocyte cell culture enhanced TNF production in all groups studied, except in the group with untreated leprosy patients. Strongest M. leprae-induced TNF release was found in mononuclear cell cultures derived from ENL patients. Patients in the postreactional state showed significantly higher TNF levels than healthy controls. These findings support the idea that TNF plays a key role in the complex symptomatology of ENL.

Mycobacterial-induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients.

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr-labeled monocytes pulsed with bacillus Calmette Guerin (BCG) or Mycobacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen-pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho-proliferation. Killing was significantly higher against antigen-pulsed vs. nonpulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K-562 target cell. Cross-reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae- and BCG-activated cytotoxic cells were found in both the CD4-CD8+ and CD4+CD8- populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.

T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae.

The 18-kilodalton (kDa) antigen of Mycobacterium leprae was expressed as a fusion protein with a 2-kDa leader peptide and used in proliferation assays with peripheral blood cells. Fifty percent of untreated tuberculoid leprosy patients and 93% of long-term leprosy contacts responded to the recombinant protein in lymphocyte transformation tests. Comparison of the stimulation indices in the two groups showed that the contacts responded more strongly than the tuberculoid leprosy patients. Seventy percent of Mycobacterium bovis BCG-vaccinated European donors responded, although with low stimulation indices. The isolation of 18-kDa antigen-responsive T-cell lines from a BCG-vaccinated British donor confirmed that the 18-kDa antigen contains at least one cross-reactive epitope. These results indicate that the 18-kDa protein is an important antigen in the immune response to leprosy.

Accessory cell function of cells isolated from Mycobacterium leprae-induced granulomas.

The large cells from Mycobacterium leprae-induced granulomas in guinea pig lymph nodes were separated by Percoll discontinuous density gradient centrifugation and on a fluorescence-activated cell sorter (FACS) using cross-reacting monoclonal antibody to human MHC Class II antigens. Large Percoll-separated cells (83% Class II antigen positive and 52% macrophage-specific antigen positive) and FACS-separated cells are able to act as antigen-presenting cells for T-cell proliferation to PPD. In previous studies, macrophage antigen-positive cells consistently failed to act as accessory cells. This indicates that there is a population of accessory cells which are macrophage antigen negative and MHC Class II antigen positive present in these M. leprae-induced granulomas.

Linfócitos em hanseníase: I. Linfócitos B do sangue periférico de portadores do Mal de Hansen / Lymphocytes in hanseniasis: I-B lymphocytes in the bloodstream of patients with Haansen's disease

Os autores estudaram os valores dos linfócitos do tipo B em doentes portadores do mal de hansen. Examinaram o sangue de 125 pessoas nao portadoras de nenhuma doença aparente . Concomitantemente examinaram o sangue de 413 doentes, distribuídos nas formas: Virchowiana, tuberculóide e indeterminada. Os resultados mostraram nåo haver diferença entre controles e portadores na forma Virchowiana, para linfócitos do tipo B, porém diminuiçåo em relaçåo as outras duas formas: tuberculóide e indeterminada. Os autores também nåo encontraram diferenças estatísticamente significantes quando foram relacionados os resultados obtidos portadores das três formas estudadas. Verificaram ainda que o tempo de tratamento nåo modifica o número dos linfócitos do sangue periférico

Linfócitos em hanseníase: II. Linfócitos T do sangue periférico de portaddores do mal de Hansen / Lymphocytes in hanseniasis: II-Lymphocytes in the bloodstream of patients with Hansen's disease

Os autores estudaram a aplicaçåo do teste de rosetas na determinaçåo do número dos linfócitos do tipo T, da concorrente circulatória de portadores do Mal de Hansen, com vistas ao emprego no diagnóstico da hanseníase. Determinaram os parâmetros necessários à execuçåo do teste. Examinaram o sangue de 125 pessoas tidas como sadias, tomadas como controle e examinaram concomitantemente o sangue de 413 outras pessoas portadoras de hanseníase, nas formas: virchowina, tuberculóide e indeterminada. Os resultados mostraram nåo haver diferenças estatisticamente significantes entre o grupo de doentes portadores da forma virchowiana e controles mas encontraram diferenças estatisticamente significantes entre os resultados apresentados pelos controles e portadores das formas tuberculóide e indeterminada, aumentandos nessas

In vitro studies on dermal granulomas of human leprosy--cellular characteristics.

Single-cell suspensions from the granulomas of leprosy cases were prepared for an in vitro study of the properties of the infiltrating cells. Biopsies from 44 untreated patients with tuberculoid and lepromatous leprosy were analyzed. The granulomas were found to contain lymphocytes and "large cells" (epithelioid cells and macrophages). The number of lymphocytes was significantly higher in the suspensions from the tuberculoid granulomas in comparison to the suspensions from the lepromatous granulomas. A high percentage of lymphocytes from the tuberculoid granulomas formed rosettes with sheep erythrocytes, and also showed the presence of esterase as dots in the cytoplasm. However, the lymphocytes did not form rosettes with EAC. Most of the "large cells" from both types of granulomas were esterase positive, exhibited peroxidase activity, and did not carry receptors for C3. A high percentage of "large cells" in the tuberculoid granulomas was nonadherent to a plastic surface, while the lepromatous granulomas contained a high proportion of adherent "large cells."

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

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.

Reduction of a subset of T cells bearing Fc receptors for IgG in lepromatous leprosy.

Enumeration of a subpopulation of T cells with receptors for the Fc portion of IgG (T gamma) in the peripheral blood of 14 normal subjects and 43 patients with leprosy was undertaken. Tuberculoid leprosy patients showed normal levels of T gamma cells. In contrast, bacillary positive patients with lepromatous leprosy revealed a significant reduction of circulating T gamma cells (p less than 0.001).