Attenuated Listeria monocytogenes carrier strains can deliver an HIV-1 gp120 T helper epitope to MHC class II-restricted human CD4+ T cells

Listeria monocytogenes is a facultative intracellular pathogen which, following uptake by macrophages, escapes from the phagosome and replicates in the cytoplasm. This property has been exploited using recombinant L. monocytogenes as a carrier for the intracytoplasmic expression of antigens when MHC class I-restricted cytotoxic T lymphocyte responses are required. Much less is known of the ability of these bacteria to trigger MHC class II-restricted responses. Here, we demonstrate that after ingestion of L. monocytogenes expressing a T helper epitope from the gp120 envelope glycoprotein of HIV, human adherent macrophages and dendritic cells can process and present the epitope to a specific CD4+ T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR Vbeta 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well-established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.     

Regulation of class II MHC expression

The class II genes of the major histocompatibility complex (MHC) encode the alpha/beta heterodimeric glycoproteins that play a critical role in the induction of immune responses through presentation of processed antigen to CD4+ T lymphocytes. The constitutive expression of class II MHC antigens is restricted primarily to B cells, dendritic cells, thymic epithelium, and macrophages, although a wide variety of other cell types can be induced to express class II antigens after exposure to cytokines. The appropriate constitutive and inducible te constitutive and inducible expression of class II MHC antigens is essential for normal immune function; thus it is not surprising that aberrant expression on cell types normally class II MHC negative has been correlated with various autoimmune disorders, and lack of expression results in a severe combined immunodeficiency disorder called bare lymphocyte syndrome (BLS). In this review, we discuss the agents that both induce and inhibit class II MHC expression, the function of class II MHC antigens with an emphasis on the ability of these proteins to act as signal transducing molecules, and the molecular regulation of class II MHC expression.     

CD40-CD154 interaction and IFN-gamma are required for IL-12 but not prostaglandin E2 secretion by microglia during antigen presentation to Th1 cells

IL-12 and PGE2 promote and inhibit, respectively, the development of Th1 responses. Production of these mediators by APC residing in the central nervous system (CNS) may be involved in the local regulation of the T cell phenotype during infectious and autoimmune CNS diseases. In the present study we have examined IL-12 and PGE2 secretion by cultured microglia and astrocytes from the mouse brain upon Ag-dependent interaction with I-Ad-restricted, OVA323-339 specific TCR transgenic Th1 and Th2 cell lines. We show that microglia, which restimulate efficiently both Th1 and Th2 cells, secrete IL-12 upon Ag-dependent interaction with Th1, but not with Th2 cells. Th1-driven IL-12 production depends on TCR ligation by MHC class II/peptide complexes, CD40 engagement on microglia, and IFN-gamma secretion by activated Th1 cells. Th1 and, to a lesser extent, Th2 cells also stimulate the production of PGE2 by microglia. T cell-mediated induction of PGE2 requires MHC class II/peptide/TCR interactions but does not depend on CD40 engagement or on the presence of IFN-gamma. Astrocytes, which preferentially activate Th2 cells, fail to produce IL-12 and secrete negligible amounts of PGE2 upon interaction with either Th1 or Th2 cells. These results suggest that during CNS infection or immunopathology, IL-12 produced by microglia upon Ag-specific interaction with Th1 cells may further skew the immune response to Th1, whereas the T cell-dependent production of PGE2 by microglia may represent a negative feedback mechanism, limiting the propagation of Th1 responses.     

Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA

Genetic vaccination depends at least in part on the adjuvant properties of plasmids, properties that have been ascribed to unmethylated CpG dinucleotides in bacterial DNA. Because dendritic cells (DC) participate in the T cell priming that occurs during genetic vaccination, we reasoned that CpG-containing DNA might activate DC. Thus, we assessed the effects of CpG oligodeoxynucleotides (CpG ODN) on Langerhans cell (LC)-like murine fetal skin-derived DC (FSDDC) in vitro and on LC in vivo. Treatment with CpG ODN as well as LPS induced FSDDC maturation, manifested by decreased E-cadherin-mediated adhesion, up-regulation of MHC class II and costimulator molecule expression, and acquisition of enhanced accessory cell activity. In contrast to LPS, CpG ODN stimulated FSDDC to produce large amounts of IL-12 but only small amounts of IL-6 and TNF-alpha. Injection of CpG ODN into murine dermis also led to enhanced expression of MHC class II and CD86 Ag by LC in overlying epidermis and intracytoplasmic IL-12 accumulation in a subpopulation of activated LC. We conclude that immunostimulatory CpG ODN stimulate DC in vitro and in vivo. Bacterial DNA-based vaccines may preferentially elicit Th1-predominant immune responses because they activate and mobilize DC and induce them to produce large amounts of IL-12.     

Functional connectivity in depressive, obsessive-compulsive, and schizophrenic disorders: an explorative correlational analysis of regional cerebral metabolism

Cellular elements of the vascular wall, such as endothelium (En) and smooth muscle cells/pericytes (SM/P) possess important immunologic properties. We have previously reported that murine brain microvessel En cells and SM/P express Major Histocompatibility (MHC) class II molecules and activate syngeneic CD4+ T cells in a class II dependent way. Herein we compare MHC class II expression on brain microvessel En to aorta large vessel En cells in order to explore the mechanisms of immune responses in brain tissue versus other peripheral tissues. Interestingly, we demonstrate that En cells from brain microvessel and large aortic vessel express the I-A but not the I-E subunit of MHC class II molecules. The expression of I-A class II molecules can be upregulated on brain microvessel and aortic En cells by interferon-gamma (IFN-gamma). Similarly, the expression of I-A, but not I-E, MHC class II molecules on brain microvessel endothelial cells was upregulated in the presence of activated T cells. Interleukin-10 (IL-10) was found to inhibit IFN-gamma-mediated upregulation of I-A class II molecule expression on aortic but not on microvessel En cells. Our data may indicate that some differences in organ-specific immune responses, are defined by local parameters, such as MHC distribution and regulation.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.     

Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency

BACKGROUND: Major-histocompatibility-complex (MHC) class II deficiency is an autosomal recessive primary immunodeficiency disease in which MHC class II molecules are absent. It is a genetically heterogeneous disease of gene regulation resulting from defects in several transactivating genes that regulate the expression of MHC class II genes. The mutations responsible for MHC class II deficiency are classified according to complementation group (a group in which the phenotype remains uncorrected in pairwise fusions of cells). There are three known complementation groups (A, B, and C). METHODS: To elucidate the genetic defect in patients with MHC class II deficiency that was not classified genetically, we performed direct complementation assays with the three genes known to regulate the expression of MHC class II genes, CIITA, RFX5, and RFXAP, and the relevant mutations were identified in each patient. RESULTS: Mutations in the RFXAP gene were found in three patients from unrelated families, and the resulting defect was classified as belonging to a novel complementation group (D). Transfection with the wild-type RFXAP gene restored the expression of MHC class II molecules in the patients' cells. CONCLUSIONS: Mutations in a novel MHC class II transactivating factor, RFXAP, can cause MHC class II deficiency. These mutations abolish the expression of MHC class II genes and lead to the same clinical picture of immunodeficiency as in patients with mutations in the other two MHC class II regulatory genes.     

Activation of human dendritic cells following infection with Mycobacterium tuberculosis

Dendritic cells (DC) play an essential role in the initiation of primary T cell responses to foreign Ag. It is likely that these potent APC are critical in the initiation of immune responses to pathogens, such as bacteria or parasites. However, little is known about the interaction of these important APC with pathogens. To address this issue, the interaction of the bacterium Mycobacterium tuberculosis with human DC was studied. DC generated from human peripheral blood by short term culture in medium containing recombinant human cytokines granulocyte-macrophage-CSF and IL-4 were capable of phagocytosing M. tuberculosis. Infection of DC with live M. tuberculosis bacilli resulted in increased APC surface expression of the costimulatory molecules CD54, CD40, and B7.1, as well as MHC class I molecules. In addition, infected DC secreted elevated levels of inflammatory cytokines, including TNF-alpha, IL-1, and IL-12. M. tuberculosis-infected human monocytes also secreted inflammatory cytokines, but exhibited no enhancement of costimulatory or MHC class I molecule expression. These data indicate that infection with M. tuberculosis results in the direct activation and maturation of these DC. In vivo, such activation may facilitate migration to the lymph nodes, and enhance presentation of Ag to T cells, thereby facilitating the induction of the immune response against this pathogen.     

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)-Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2(b), H-2(d), H-2(k), and H-2(q) was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5-8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.     

Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II-restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen-presenting cell types. This study compares the ability of different antigen-presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II-restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen-presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.     

Anti-TNF antibody modulates cytokine and MHC expression in cardiac allografts

Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine evoked in response to alloantigen stimulation and may be involved in lymphocyte activation, adhesion molecule expression, and regulation of MHC class II antigens. Anti-TNF treatment prolongs cardiac allograft survival. We investigated the role of anti-TNF in the regulation of MHC class II antigens and cytokine mRNA expression of TNF, interferon-gamma (IFN), IL-2, IL-4, and IL-10 in cardiac allografts to elucidate its immunological mechanism. These in vivo studies were conducted using a rat MHC mismatch Brown-Norway to Lewis (BN to LEW) heterotopic cardiac transplant model. In control untreated rats, allografts were rejected at 6.8 +/- 0.6 days. Allograft survival was significantly prolonged to 12.7 +/- 1.4 days with anti-TNF treatment. MHC class II expression, analyzed by indirect immunofluorescence cytometry, demonstrated that MHC class II-positive cells increased by 25% in spleens of untreated allografted rats compared to naive rats, while anti-TNF-treated allografted rats had a similar percentage of MHC II cells as naives. Further, naive, untransplanted rats and both anti-TNF and untreated, transplanted rats had heart and spleens harvested on Day 5 post-transplant. Cytokine mRNA expression was determined by semiquantitative RT-PCR. In heart and spleen cells from naives, TNF mRNA expression was undetectable or very weak. However, in rejecting allografts and spleen cells from untreated recipients, TNF expression was remarkably increased, while anti-TNF attenuated this TNF expression in both heart graft and spleen cells. Furthermore, IL-2, IL-10, and IFN expression were absent in naive hearts. However, in untreated allografts IL-2, IL-10, and IFN were strongly expressed, which was markedly decreased after anti-TNF treatment. Finally, IL-4 expression was found equally in naive hearts, untreated allografts, and anti-TNF-treated allografts. These results suggest that anti-TNF antibody treatment may not only neutralize TNF activity but also play a role in altering cytokine mRNA expression and MHC class II expression.     

Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods

In this study we have shown that an antibody to CD18 identified a population of cells in the rabbit retina that resembled the perivascular macrophage found in other regions of the central nervous system. In the normal retina these cells possessed a ramified morphology and presented in an ordered array on the vitreal surface in association with the epiretinal vessels. Approximately 50% of the perivascular macrophages constitutively expressed MHC class II. In response to interleukin-1 beta (IL-1 beta)-induced inflammation, these cells became activated, as evidenced by a change from a ramified to an ameboid morphology and increased expression of MHC class II, and migrated away from the vessels. These changes were first detected around 3 h post-intraocular challenge coincident with the onset of inflammation. At the peak of the inflammatory response (approximately 24 h post-challenge), many activated perivascular macrophages were no longer associated with the vessels and formed long "cord" of MHC class II+ cells associated with underlying deposits of fibrin. In eyes challenged with heat-inactivated IL-1, no change in the morphology or distribution of the perivascular macrophage was noted. At 3 weeks post-challenge with IL-1, the number and distribution of the perivascular macrophages were restored to baseline values, although with a reduced cell size. Since these changes closely resemble those that occur in non-lymphoid dendritic cells in the skin, heart, and/or kidney following activation with cytokines or bacterial products, the results suggest that the perivascular macrophage represents the dendritic cell of the retina and may thus play an important role in immune surveillance in the eye and maintenance of the blood-retina barrier.     

Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro

Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12-deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide-major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II-associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.     

Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the "default" T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2-3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-gamma by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing- associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor alpha or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.     

Natural killer activating receptors trigger interferon gamma secretion from T cells and natural killer cells

Proliferation of human CD4+ alphabeta T cells expressing a natural killer cell activating receptor (NKAR) has been shown to be enhanced, particularly in response to low doses of antigen, if the target cells present appropriate human class I major histocompatibility complex (MHC) molecules. Here, we show that NKAR also enhance proliferation and killing of target cells by subsets of CD8+ alphabeta and CD8+ gammadelta T cells, as well as by NK cells. Strikingly, interferon gamma secretion from all of these types of lymphocytes was markedly increased by interaction of the NKAR with their MHC class I ligands, independently of enhancement of proliferation. Thus, the recognition of class I MHC molecules by NKAR on both T cells and NK cells may provide a regulatory mechanism that affects immune responses through the secretion of interferon gamma and possibly other cytokines. It represents a signal for cytokine secretion alternative and/or augmentative to that through the T cell receptor.