Bacillus Calmette-Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally.

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Dermal dendrocytes FXIIIa+ are essential antigen-presenting cells in indeterminate leprosy.

Indeterminate leprosy (IL) is the early phase of Hansen disease and reword (APCs). Langerhans cells and dermal dendrocytes FXIIIa positive (DDFXIIIa) are the major APCs in the skin and can be identified by the expression of CD1a and FXIIIa, respectively, by immunohistochemical techniques. Plasmacytoid dendritic cells (PDCs) are another type of dermal dendrocytes with a questionable antigen-presenting function and can be highlighted by anti-CD123 expression. To our knowledge, there are no studies evaluating DDFXIIIa and PDC in IL. The purpose was to investigate the involvement of these cells in the pathogenesis of IL. The authors performed a retrospective study on 18 cases of IL (10 confirmed and 8 suspected) to investigate expression of FXIIIa, CD1a, and CD123. The results were compared with normal skin (for CD1a and FXIIIa only). A higher amount of FXIIIa-positive cells (P , 0.05) in confirmed and suspected IL cases was noted when comparing with normal skin. However, CD1a showed no quantitative differences in the epidermis of IL lesions when comparing with normal skin and CD123 expression was negligible. Based on these findings, the authors postulate that Langerhans cells and PDCs do not have a major role in IL and that DDFXIIIa may be the main APCs in IL. Further study is required to establish this.

Newer targeted therapies in psoriasis.

Psoriasis is a common, chronic, inflammatory skin disease that can have a significant impact on the quality of life of those who are afflicted due to chronicity of the disease and frequent remissions and relapses. Many available systemic therapies, however, are unsuitable for chronic administration due to the risk of cumulative toxicity. Recent advances in the understanding of the pathophysiology of psoriasis have led to the development of new, genetically engineered, targeted therapies for this disease. These include approaches targeting antigen presentation and co-stimulation, T-cell activation and leukocyte adhesion, action on pro-inflammatory mediators, and modulating the cytokine balance. Although only preliminary data are available so far and there is limited data supporting their use, these trials contribute to a further understanding of the disease and will eventually lead to new therapeutic options for psoriasis.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis.

BACKGROUND: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. RESULTS: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 µg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). CONCLUSIONS: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

The role of toll-like receptors in host defenses and their relevance to dermatologic diseases.

The family of toll-like receptors (TLRs) plays a central role in the cutaneous immune defense system. To date, different TLRs have been found on several major cell populations of the skin, such as keratinocytes, fibroblasts, antigen-presenting cells, and melanocytes. Activation of TLRs leads, via different intracellular signaling pathways, to the production of pro-inflammatory stimuli, and is considered a danger signal that should transform the skin in to the functional state of defense. However, TLRs have also been implicated in tissue homeostasis and renewal. Within the group of TLRs, two types have been identified: surface-expressed TLRs, which are predominantly active against bacterial cell wall compounds; and intracellular receptors, which preferentially recognize virus-associated pattern molecules. In addition, surface-expressed receptors trigger phagocytotic and maturation signals, while the intracellular TLRs lead to the induction of antiviral genes. Our review aims to outline the importance of TLRs in the pathogenesis of numerous skin diseases and the potential of TLR agonists as a treatment option for various skin diseases.

[Genetic involvement of bacterial sensor molecules in Japanese leprosy].

Occurrence of new patients of leprosy, caused by Mycobacterium leprae infection, is now almost absent in Japan but is still uncontrolled in developing countries. As one factor affecting the disease development, genetic predisposition of a host has been considered to be associated. Actually, various gene mutations have been reported to be associated at two stages of the disease progression, not only establishment of the disease but also determination of the phenotype, such as lepromatous (L)-type, tuberculoid (T)-type and reversal reaction. On the basis of recent progress of the research on innate immunity, here we analyzed single nucleotide polymorphisms (SNPs) of the genes of major bacterial sensor molecules expressed in antigen-presenting cells, TLR2, DC-SIGN, NOD1 and NOD2, in Japanese leprosy patients. As a result, frequency of polymorphisms in DC-SIGN -336 showed significant difference between the leprosy patients and the healthy controls, reflecting its role in establishment of the disease. Especially, among those with a particular TLR2 -16934 genotype, frequency of the polymorphisms in DC-SIGN -336 showed significant difference between the patients and the controls, suggesting any cooperation of these SNPs.

Identification of an Immunomodulating Agent from Mycobacterium leprae.

A search for an immunomodulating agent from mycobacteria was carried out using Mycobacterium leprae. The antigenicity of each fraction of the bacterial membrane, which contains the most antigenic components of M. leprae, was assessed by using sera from paucibacillary leprosy. N-terminal sequencing of the serum-reactive protein and functional assessment of the membrane fractions using monocyte-derived dendritic cells (DCs) identified major membrane protein II (MMP-II) as one of the efficient T-cell-activating candidates. Purified MMP-II stimulated DCs from healthy individuals to produce interleukin-12 p70 and up-regulated the surface expression of major histocompatibility complex class I and II, CD86, and CD83 molecules. Also, there was an increase in the percentage of CD83(+) cells in the DC population. Furthermore, MMP-II-pulsed DCs expressed their derivatives on their surfaces. Using Toll-like receptor 2 (TLR-2)-dependent receptor constructs, we found that TLR-2 signaling was involved in DC maturation induced by MMP-II. Taken together, MMP-II can be recognized as an immunomodulating protein in terms of activation of antigen-presenting cells and innate immunity.

Upregulation of T-cell-stimulating activity of mycobacteria-infected macrophages.

Macrophages are one of the most abundant host cells to come in contact with mycobacteria. However, the infected macrophages less efficiently stimulate autologous T cells in vitro. We investigated the effect of the induction of phenotypic change of macrophages on the host cell activities by using Mycobacterium leprae as a pathogen. The treatment of macrophages with interferon-gamma (IFN-gamma), GM-CSF and interleukin-4 deprived macrophages of CD14 antigen expression but instead provided them with CD1a, CD83 and enhanced CD86 antigen expression. These phenotypic features resembled those of monocyte-derived dendritic cells (DC). These macrophage-derived DC-like cells (MACDC) stimulated autologous CD4+ and CD8+ T cells when infected with M. leprae. Further enhancement of the antigen-presenting function and CD1a expression of macrophages was observed when treated with IFN-gamma. The M. leprae-infected and -treated macrophages expressed bacterial cell membrane-derived antigens on the surface and were efficiently cytolysed by the cell membrane antigen-specific CD8+ cytotoxic T lymphocytes (CTL). These results suggest that the induction of phenotypic changes in macrophages can lead to the upregulation of host defence activity against M. leprae.

[Immunopathology of leprosy: the complexity of the mechanisms of host immune response to Mycobacterium leprae]. / Imunopatologia da hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae.

Leprosy, whose etiologic agent Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic forms, varying from a vigorous immune response mediated by cells to M. leprae, with Th1 standard in the tuberculoid polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of Th2 response and exacerbation of humoral response. It is probable that different polymorphic genes determine susceptibility to M. leprae. Additional studies are necessary to clarify the complex interactions between cytokines and the role of the phenotypic diversity of cells network that contribute to the host defense. The comprehension of such mechanisms will provide new insights for the identification of agonists and/or antagonists for pro- or anti-inflammatory effects, and also will indicate possible situations for its appropriate use in immunologic and/or immunotherapeutic interventions.

Imunopatologia da hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae / Immunopathology of leprosy: the complexity of the mechanisms of host immune response to Mycobacterium leprae

Leprosy, whose etiologic agent Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic forms, varying from a vigorous immune response mediated by cells to M. leprae, with Th1 standard in the tuberculoid polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of Th2 response and exacerbation of humoral response. It is probable that different polymorphic genes determine susceptibility to M. leprae. Additional studies are necessary to clarify the complex interactions between cytokines and the role of the phenotypic diversity of cells network that contribute to the host defense. The comprehension of such mechanisms will provide new insights for the identification of agonists and/or antagonists for pro- or anti-inflammatory effects, and also will indicate possible situations for its appropriate use in immunologic and/or immunotherapeutic interventions.

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.

Cross-reactive epitopes and HLA-restriction elements in human T cell recognition of the Mycobacterium leprae 18-kD heat shock protein.

We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M. leprae immunization and that the peptide 38-50 serves as an immunodominant epitope recognized by CD4+ T cell clones. By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M. leprae HSP18 and peptide 38-50 were recognized by memory T cells 8 years after immunization with M. leprae. The finding that M. bovis BCG-induced T cell lines responded to M. leprae HSP18, but not to the peptide 38-50, suggested the existence of additional T cell epitopes of a cross-reactive nature. Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38-148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1-38 and 41-55. While peptide 38-50-reactive T cell clones showed limited cross-reactivity by responding to M. leprae, M. avium and M. scrofulaceum, the T cell lines specific to the epitopes 1-38 and 41-55 were broadly cross-reactive, as demonstrated by their response to M. leprae, M. tuberculosis complex, M. avium and other mycobacteria. MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1-38 and 38-50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41-55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells. The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M. leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M. tuberculosis complex or environmental mycobacteria.

The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Interleukin-15 in mycobacterial infection of antigen-presenting cells.

Interleukin-15 (IL-15) shares many biological functions with IL-2 but also exhibits unique effects. Some of these represent the potent chemoattractant activity and expansion of distinct T-cell subsets, particularly memory T cells. IL-15 may therefore modulate the quality and quantity of cellular immune responses directed against intracellular pathogens. Immunohistochemical examination of skin lesions obtained from patients with the lepromatous or the tuberculoid form of Hansen's disease revealed intralesional IL-15 protein in both forms of the disease. In addition to Mycobacterium leprae, a number of different mycobacterial species are capable of effectively inducing IL-15 secretion in infected macrophages. In this work, increased IL-15 secretion was observed in IL-4/granulocyte-macrophage colony-stimulating factor (GM-CSF)-activated antigen-presenting cells (APC) compared with unstimulated macrophages. Immunocytological detection of intracellular IL-15 revealed that infection with different mycobacterial species resulted in different staining patterns of anti-IL-15 immunoreactive material in APC. In contrast to IL-2 or IL-7, IL-15 enhanced the cytolytic potential of immune effector cells in vitro and favoured the expansion of CD1b-restricted immune cells recognizing mycobacterial-associated antigens presented by autologous APC. IL-15 produced by infected cells in situ may represent one of the key cytokines involved in granuloma formation and may aid the augmentation of cellular immune responses directed against mycobacterial-infected cells.

Isolation of recombinant phage clones expressing mycobacterial T cell antigens by screening a recombinant DNA library with human CD4+ Th1 clones.

A lambda gt11 recombinant DNA library of Mycobacterium leprae was screened to isolate recombinant phage clones expressing mycobacterial antigens important for T cell reactivity. The library was plated on a lawn of Escherichia coli Y1090 and recombinant antigens were expressed from isolated phage clones in 96-well plates. Pools of recombinant antigens from 12 wells were tested in T cell proliferation assays with MHC class II restricted human CD4+ Th1 clones secreting interferon-gamma and cytotoxic for antigen pulsed antigen presenting cells. By screening 1750 pools of recombinant antigens with a mixture of eight Th1 clones, we identified two recombinant phage clones that expressed recombinant mycobacterial antigens stimulatory for T cells. MHC restriction analysis and reactivity to a battery of mycobacterial antigens suggested that the two responding Th1 clones recognized mycobacterial antigens/epitopes with different MHC class II (HLA-DR) restriction requirements. Our results suggest that the methodology described in this paper is suited to isolate recombinant phage clones expressing mycobacterial recombinant antigens stimulatory for T cells of protective phenotype. Such antigens may be useful in designing new vaccines and diagnostic reagents against mycobacterial diseases.

Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset.

The efficacy of thalidomide (alpha-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor alpha. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2-mediated T cell proliferation and interferon gamma production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.

Differential ability of T cell subsets to undergo activation-induced cell death.

Human T cell clones were analyzed for their susceptibility to activation-induced cell death (AICD) in response to CD3/T cell receptor ligation. AICD was observed only in Th1 clones and was Fas-mediated, whereas Th2 clones resisted AICD. Analysis of a panel of Th0 clones, characterized by their ability to secrete both Th1 and Th2 cytokines, revealed that this subset included both AICD-sensitive (type A) and -resistant (type B) clones. Resistance to AICD by Th2 and Th0-type B clones was not due to lack of expression of either Fas receptor or its ligand. Paradoxically, the AICD-resistant clones were susceptible to apoptosis when Fas receptor was directly ligated by anti-Fas antibodies. However, prior activation of the resistant clones by monoclonal antibodies to CD3/TCR complex induced resistance against Fas-mediated apoptosis. Thus, the Fas-FasL pathway is critical for the induction of AICD in T cells, and moreover this pathway can be negatively regulated in the AICD-resistant clones by signals that are generated from ligation of the CD3/TCR complex.

[Regulation of the immune response: the TH1/TH2 concept]. / Regulation der Immunantwort: das TH1-/TH2-Konzept.

The immune system has different possible ways of reacting to an antigen. The choice of an appropriate immune response is determined by the manner of antigen presentation, the amount of antigen, the localization of antigen uptake, the type of antigen presenting cell, the genetic predisposition of the individual and the presence of certain cytokines released by antigen presenting or other inflammatory cells. An immune response which is not not appropriate can lead to clinical symptoms or insufficient clearance of an infectious agent. This is well-illustrated in the example of lepra lepromatosa (insufficient, since humoral immune response to an intracellular agent) or lepra tuberculosa (complete clearance of Mycobacterium leprae). A decisive step for the type of immune response is the stimulation of different T-cell subpopulations. CD4 or CD8 T-cells can be further subdivided by a distinct cytokine production. So-called TH1 cells predominantly produce cytokines, which stimulate a cellular immune response (IFN gamma, IL-12, IL-2). In contrast, TH2 cells predominantly produce IL-4 and IL-5. These cytokines boost an IgE-mediated allergic reaction and inflammation. Although the TH1/ TH2 distinction is frequently not absolute, as overlaps can frequently be observed, this classification is useful for better understanding of immune reactions in various diseases. Moreover, since TH1- and TH2-related cytokines act antagonistically, therapeutic strategies are under development which strengthen e.g. a TH2 immune response in TH1 dominated diseases and vice versa.

CD1-restricted T cell recognition of microbial lipoglycan antigens.

It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.