SIGN-R1+MHC II+ cells of the splenic marginal zone--a novel type of resident dendritic cells

In the spleen, the MZ forms an interface between red and white pulp. Its major function is to trap blood-borne antigens and to reorient them to APCs and lymphocytes. SIGN-R1(+) cells are of the MZ inherent cell population, which for a long time, have been considered as macrophages. We now show that one subpopulation of SIGN-R1(+) cells that express MHC II molecules should be considered as a resident DC. Histological analysis indicated that SIGN-R1(+) cells have dendritic-like protrusions extending into T and B cell areas. Flow cytometry analysis revealed an expression profile of adhesion, costimulatory, and MHC molecules similar to cDCs but distinct from macrophages. Most importantly, SIGN-R1(+)MHC(+) cells were able to present antigen to na?ve CD4 T cells, as well as to cross-present soluble, particulate antigens secreted by Listeria monocytogenes to CD8 T cells in vitro and in vivo. Our experiments identified SIGN-R1(+)MHC II(+) cells as professional APCs and indicate their nature as splenic resident DCs.     

Differential expression of alternative H2-M isoforms in B cells, dendritic cells and macrophages by proinflammatory cytokines

Major histocompatibility (MHC) class II heterodimers bind peptides generated by degradation of endocytosed antigens and display them on the surface of antigen presenting cells (APCs) for recognition by CD4+ T cells. Efficient loading of MHC class II molecules with peptides is catalyzed by the MHC class II-like molecule H2-M. The coordinate regulation of MHC class II and H2-M expression is a prerequisite for efficient MHC class II/peptide assembly in APCs determining both the generation of the T cell repertoire in the thymus and cellular immune responses in the periphery. Here we show that expression of H2-M and MHC class II genes is coordinately and cell type-specific regulated in splenic B cells, splenic dendritic cells (DCs) and peritoneal macrophages (Mphi) in response to proinflammatory and immunoregulatory cytokines, including GM-CSF, IFN-gamma, TGF-beta2, IL-4, IL-10 and viral IL-10. In addition, ratio-RT-PCR expression analysis of the duplicated H2-Mbeta-chain loci demonstrates for the first time that Mbl and Mb2 genes are differentially expressed in individual APC types. Mb2 is preferentially expressed in IL-4, GM-CSF, IL-10, vIL-10 and IFN-gamma stimulated splenic B cells, whereas splenic DCs express both Mb genes at almost equal levels. In contrast, peritoneal Mphi express predominantly Mb2 but stimulation with IFN-gamma induces a switch towards Mb1 expression. These data suggest a common mechanism that regulates coordinate expression of H2-M and MHC class II genes in professional APCs. Differential expression of Mb1 and Mb2, and by consequence alternative H2-M isoforms (Malphabeta1 or Malphabeta2), may influence the nature of the peptide repertoire presented by different APC types.     

Maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs). Microglia also express CD11c during this phase of the infection. CD11c(+) macrophages in the infected CNS exhibit variable properties of immature antigen-presenting cells (APCs), with modestly increased CD40 and MHC expression, and equivalent potent antigen uptake when compared with CD11c(-) macrophages. Furthermore, CDllc(+) and F4/80(+) macrophages and microglia are localized to areas of demyelination, in some instances directly associated with damaged axons. These results suggest that chronic CNS infection results in the appearance of CD11c-expressing macrophages from the blood that exhibit properties of immature APCs, are closely associated with areas of demyelination, and may act as final effectors of myelin destruction.     

Legionella pneumophila infection up-regulates dendritic cell Toll-like receptor 2 (TLR2)/TLR4 expression and key maturation markers

Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.     

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation,T-cell priming,and peptide association with MHC class II

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-γ, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC.     

Deciphering new mechanisms on T‐cell costimulation by human mast cells

It is well established that full activation of T cells to recognize a specific antigen requires additional signals. These secondary signals are generated by the interaction of costimulatory molecules expressed on APCs. Classical APCs include DCs, macrophages, Langerhans cells, and B cells. However, in recent years, several haematopoietic and nonhaematopoietic cells have been described to express MHC class II antigens and, in appropriate conditions, costimulatory molecules. In this issue, Suurmond et al. [Eur. J. Immunol. 2016. 46: 1132–1141] show, for the first time, that human mast cells not only express costimulatory molecules of the TNF‐receptor and CD28 families, but can also costimulate T cells through a yet‐to‐be‐defined CD28‐independent interaction.     

Activation, cytokine production, and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen causing localized gastroenteritis in humans. Macrophages (Mphis) and dendritic cells (DCs) play an important role in innate immunity against Salmonella. In this report, we have compared the consequences of infection of human Mphis and DCs with wild-type S. typhimurium and an isogenic PgtE-defective strain. PgtE is an outer membrane protein hypothesized to have a role in intracellular survival of Salmonella. We observed that DCs undergo full maturation in response to Salmonella infection, as indicated by up-regulation of cell-surface marker proteins CD80, CD83, CD86, and human leukocyte antigen class II. CC chemokine ligand 5 (CCL5), CXC chemokine ligand 10, tumor necrosis factor alpha, interleukin (IL)-12, and IL-18 gene expression and protein production were readily induced by Salmonella-infected Mphis and DCs. CCL20 was preferentially produced by Mphis, whereas DCs secreted higher levels of CCL19 as compared with Mphis. DCs and Mphis infected with S. typhimurium also produced high levels of interferon-gamma (IFN-gamma). Cytokine neutralization and stimulation experiments suggest that the production was partly regulated by Salmonella-induced type I IFNs, IL-12, and IL-18. DC cytokine production induced by Salmonella was much higher as compared with the responses induced by Salmonella lipopolysaccharide or flagellin. Mphis and DCs were capable of internalizing and harboring Salmonella for several days. S. enterica PgtE provided no survival advantage for the bacteria in human Mphis or DCs. Our results demonstrate that although Mphis and DCs share similar functions, they may have different roles during Salmonella infection as a result of differential production of certain chemokines and cytokines.     

Migration of Salmonella typhimurium --harboring bone marrow--derived dendritic cells towards the chemokines CCL19 and CCL21

Macrophages are considered as main cellular target encountered by the facultative intracellular bacterium Salmonella typhimurium. However, in orally infected mice these pathogens are first internalized by dendritic cells (DCs) that are located in the subepithelial dome of Peyer's patches. Moreover, DCs can penetrate the intestinal epithelium to sample bacteria. Here, we examined the interaction of Salmonella with bone marrow-derived DCs (BM-DCs). In order to study the role of DCs as vehicles for the dissemination of Salmonella, an in vitro model was established. In this model, Salmonella -activated BM-DCs enhanced surface expression of MHC class II and co-stimulatory molecules. We found that, upon maturation, BM-DCs upregulated chemokine receptor 7 (CCR7) mRNA and surface molecule expression. Salmonella -exposed DCs as well as mature DCs, but not immature DCs, were recruited towards the CC chemokines CCL19 and CCL21, two ligands of CCR7. The maturation process of DCs did neither require bacterial internalization nor viability. About one third of the migrated BM-DCs harbored intracellular bacteria, whereas the remaining two third did not contain bacteria. Salmonella, but not an apathogenic E. coli laboratory strain was capable to survive within BM-DCs. Taken together, our data implicate that DCs are first activated and subsequently utilized as carriers by Salmonella.     

Marked suppression of T cells by a benzothiophene derivative in patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis

In a search for new anti-autoimmune agents that selectively suppress activation of autoreactive T cells, one such agent, 5-methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide (CI-959-A), was found to be effective. This compound, which is known to suppress tumor necrosis factor alpha (TNF-alpha)-induced CD54 expression, inhibited the primary proliferative response of the T cell to antigen (Ag)-presenting cells (APCs) including allogenic dendritic cells (DCs), autologous Epstein-Barr virus-infected B cells, and human T lymphotropic virus type I (HTLV-I)-infected T cells. Autoreactive T cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) spontaneously proliferate in vitro, and their activation is reported to be associated with CD54 expression. The spontaneous proliferation of T cells from patients with HAM/TSP was entirely blocked by CI-959-A. However, in this study, the T-cell proliferation in 15 patients with HAM/TSP was found to depend more extensively on major histocompatibility complex (MHC) class II and CD86 than on CD54 Ags. Since most important APCs for the development of HAM/TSP are DCs and HTLV-I-infected T cells, the effect of CI-959-A on DC generation and on the expression of surface molecules on activated T cells is examined. CI-959-A suppressed recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)- and recombinant interleukin-4-dependent differentiation of DCs from monocytes and inhibited the expression of CD54 and, more extensively, MHC class II and CD86 Ags. CI-959-A showed little toxicity toward lymphoma or HTLV-I-infected T-cell lines or toward monocytes and cultured DCs. These results suggest that CI-959-A might be a potent anti-HAM/TSP agent.     

Mycobacterium tuberculosis diverts alpha interferon-induced monocyte differentiation from dendritic cells into immunoprivileged macrophage-like host cells

Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-alpha) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMphi) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-alpha, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMphi, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.     

Lack of effect of methimazole on dendritic cell (DC) function and DC-induced Graves' hyperthyroidism in mice

In addition to the biochemical inhibition of thyroid hormone synthesis, antithyroid drugs including methimazole (MMI) may have immunosuppressive effect through inhibition of major histocompatibility complex (MHC) class I and II expressions on non-professional (thyrocytes) and professional (macrophages and B cells) antigen presenting cells (APCs). Dendritic cells (DCs) are another professional APCs and very likely play the most important role in the primary immune response. Therefore, we focused in this study on evaluating the effect of MMI on DC function in mice. Bone marrow cells cultured with granulocyte macrophage colony stimulating factor and interleukin (IL)-4 expressed high levels of CD11c and moderate levels of MHC class II, both of which are widely used markers for DCs. In vitro incubation of this DC-containing cell population with 10(- 6)-10(- 4) M MMI for 2 days did not change basal- and maturation signal (adenoviral infection and lipopolysaccharide)-induced levels of the cell surface marker expressions such as MHC class I and II, CD86, CD40 and DEC205, and of proinflammatory cytokine IL-6 release. Further we found that treatment of the DC-containing cell population with MMI did not influence the incidence of Graves' hyperthyroidism and anti-thyrotropin receptor (TSHR) antibody titers in a mouse Graves' model we have recently established with DCs infected with adenovirus expressing the TSHR A subunit. Although we cannot completely exclude immunosuppressive effect of MMI on other immune cells, our data indicate that DCs do not appear to be the primary target for the immunosuppressive effect of MMI.     

Phenotype of Resting and Activated Monocyte-Derived Dendritic Cells Grown from Peripheral Blood of Patients with Ankylosing Spondylitis

Decreased levels of class II major histocompatibility complex (MHC) expression and impaired formation of immunological synapse by dendritic cells (DCs) of HLA-B27 transgenic rats have been recently demonstrated. The resulting dysfunction of DCs may be implicated in the pathogenesis of the HLA-B27-related disease in transgenic animals. The phenotype of DCs in patients with ankylosing spondylitis (AS) has not been evaluated. Monocyte-derived DCs (MDDCs) were grown from patients with active AS and age-matched healthy volunteers. Surface expression of HLA-DR, co-stimulation molecules CD80, CD86 and CD40, as well as CD83 was assessed by flow cytometry and compared between the groups under 3 conditions: in resting state, after stimulation by lipopolysaccharide (LPS) and after stimulation by LPS in the presence of etanercept, a soluble receptor of tumor necrosis factor α. Lower baseline expression of class II MHC molecules (HLA-DR) was observed by MDDCs grown from AS patients, as compared to healthy subjects. Post-stimulated levels of HLA-DR were comparable in both groups, suggesting greater up-regulation of class II MHC molecules by MDDCs from AS in response to LPS. No difference between groups in the levels of expression of co-stimulation molecules and CD83 was observed. Lower basic expression of class II MHC by the MDDCs grown from patients with AS may be associated with impaired regulation of their activity. Functional studies on DCs from patients with AS are needed to evaluate the integrity of their antigen-presenting function.     

Legionella reveal dendritic cell functions that facilitate selection of antigens for MHC class II presentation

To understand how adaptive immune responses are generated against bacteria that avoid being delivered to lysosomes, interactions between professional antigen-presenting cells (APCs) and the intracellular pathogen Legionella pneumophila were examined. In contrast to murine bone marrow-derived macrophages (BMMs), we show that dendritic cells (DCs) restrict the growth of intracellular Legionella. Similar to what has been reported in BMMs, phagosomes containing Legionella matured into endoplasmic reticulum (ER)-derived organelles after DC internalization. Biogenesis of an ER-derived vacuole did not effectively sequester Legionella antigens from presentation on MHC class II molecules (MHC II). It was determined that proteins synthesized after Legionella had established residence in an ER-derived vacuole were presented by infected APCs. These data indicate that the ability of DCs to restrict intracellular growth of Legionella could be an important property that facilitates priming of protective T cell-mediated immune responses to vacuolar pathogens.     

Lack of effect of methimazole on dendritic cell (DC) function and DC-induced Graves' hyperthyroidism in mice

In addition to the biochemical inhibition of thyroid hormone synthesis, antithyroid drugs including methimazole (MMI) may have immunosuppressive effect through inhibition of major histocompatibility complex (MHC) class I and II expressions on non-professional (thyrocytes) and professional (macrophages and B cells) antigen presenting cells (APCs). Dendritic cells (DCs) are another professional APCs and very likely play the most important role in the primary immune response. Therefore, we focused in this study on evaluating the effect of MMI on DC function in mice. Bone marrow cells cultured with granulocyte macrophage colony stimulating factor and interleukin (IL)-4 expressed high levels of CD11c and moderate levels of MHC class II, both of which are widely used markers for DCs. In vitro incubation of this DC-containing cell population with 10^{? 6}–10^{? 4} M MMI for 2 days did not change basal- and maturation signal (adenoviral infection and lipopolysaccharide)-induced levels of the cell surface marker expressions such as MHC class I and II, CD86, CD40 and DEC205, and of proinflammatory cytokine IL-6 release. Further we found that treatment of the DC-containing cell population with MMI did not influence the incidence of Graves' hyperthyroidism and anti-thyrotropin receptor (TSHR) antibody titers in a mouse Graves' model we have recently established with DCs infected with adenovirus expressing the TSHR A subunit. Although we cannot completely exclude immunosuppressive effect of MMI on other immune cells, our data indicate that DCs do not appear to be the primary target for the immunosuppressive effect of MMI.     

Activation of macrophages and expansion of specific T lymphocytes in the lungs of mice intratracheally inoculated with Cryptococcus neoformans

A quantitative and qualitative change in inflammatory cells in the lungs of mice after intratracheal inoculation of heat-killed Cryptococcus neoformans was examined by direct analysis of the pulmonary intraparenchymal leucocytes. Macrophages and T and B lymphocytes increased, peaked at day 7, and then gradually decreased to the basal level, Macrophages were activated 7 days after the inoculation, as indicated by the enhanced expression of MHC class II, intercellular adhesion molecule-1 (ICAM-I)and Fc receptor (FcR), which have been known as their activation markers. T cells were also activated, as indicated by the induction of IL-2 receptor (IL-2R) and the enhanced expression of leucocyte function-associated molecule-1 (LFA-1) and ICAM-1, a pair of adhesion molecules which have also been regarded as T cell activation markers. CD4⁺ T cells preferentially accumulated in lungs, and proliferated in vitro by stimulation with heat-killed whole yeast cells, suggesting that at least some of the infiltrated T cells expand locally in response to the organisms. These results demonstrate that the activation of macrophages and T cells reactive to C. neoformans is induced in lungs after intratracheal inoculation of heat-killed organisms, and suggest that these cells interact to eliminate organisms more efficiently from the host.     

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKCdelta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKCdelta and conclude that this event is pivotal for optimal CD4 T cell activation.     

Regulation of MHC II and CD1 antigen presentation: from ubiquity to security

MHC class II and CD1-mediated antigen presentation on various APCs [B cells, monocytes, and dendritic cells (DC)] are subject to at least three distinct levels of regulation. The first one concerns the expression and structure of the antigen-presenting molecules; the second is based on the extracellular environment and signals of danger detected. However, a third level of regulation, which has been largely overlooked, is determined by lateral associations between antigen-presenting molecules and other proteins, their localization in specialized microdomains within the plasma membrane, and their trafficking pathways. This review focuses on features common to MHC II and CD1 molecules in their ability to activate specific T lymphocytes with the objective of addressing one basic question: What are the mechanisms regulating antigen presentation by MHC II and CD1 molecules within the same cell? Recent studies in immature DC, where MHC II and CD1 are coexpressed, suggest that the invariant chain (Ii) regulates antigen presentation by either protein. Ii could therefore favor MHC II or CD1 antigen presentation and thereby discriminate between antigens.     

Unraveling features of the natural MHC class II peptidome of skin-migrated dendritic cells

Dendritic cells (DCs) migrating from peripheral tissues at steady state are considered the most efficient antigen-presenting cells (APCs) involved in the induction of peripheral T-cell tolerance via self-antigen presentation on MHC class II molecules. However, difficulties in obtaining sufficient numbers of such DCs have precluded previous analyses of their natural MHC class II peptidome in laboratory animals or humans. Here, we overcome this difficulty by collecting the large quantities of sheep DCs that migrate from the skin via the afferent lymphatics at steady state to the draining lymph node. We compared the repertoire of MHC class II-bound peptides from afferent lymph DCs with autologous APCs derived from peripheral blood. A large fraction of the MHC class II peptidome from skin DCs was derived from membrane-recycling proteins (59%) and from proteins of the antigen presentation machinery (50%), whereas these types of peptides constituted a more limited fraction in blood APCs (21 and 11%, respectively). One sheep cytokeratin peptide was identified in the skin DC peptidome indicating active processing of epithelium-derived antigens. Conversely, peptides derived from cytosolic and soluble antigens of the extracellular milieu were more represented in blood APCs than skin DCs. The biased peptidome of skin-migrated DCs indicates that these cells express a peptide repertoire for the generation of self-reactive and/or regulatory T cells mainly directed toward DC molecules from internal and external membranes and to a lesser extent toward antigens of the extracellular milieu, including some tissue-specific peptides.     

Early activation markers of human peripheral dendritic cells

Two major populations of dendritic cells (DCs), myeloid and plasmacytoid, can be isolated from human peripheral blood, and are distinguished by differential expression of the cell surface markers CD11c and CD123. These two populations of DCs also are different in their expression of Toll-like receptor (TLRs), which are involved in their activation. To investigate the early events during activation of peripheral DCs, the cells were stimulated in vitro with ligands for TLR-4 (as in lipopolysaccharides [LPS]) or TLR-9 (CpG-containing oligonucleotide [CpG]). The earliest change in protein expression detected after stimulating peripheral DCs with lipopolysaccharide (LPS) or CpG was increased production of the chemokine interleukin (IL)-8. Enhanced production of IL-8 occurred already within 2 hours of stimulation in both myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs), and preceded expression of the well established activation marker CD40. Although both populations of DCs secreted IL-8 upon activation, the levels of IL-8 produced was several times higher within the M-DCs compared with the P-DCs population. Before activation, both subsets of DCs expressed the IL-8 receptor type B (CD128b); but after stimulation the IL-8 receptor was down-regulated in both populations of DCs. Increased expression of MHC class II molecules is generally regarded as an early activation marker of DCs. However, only the P-DCs showed a significant up-regulation of MHC class II after stimulation. The M-DC population up-regulated MHC class II without any prior activation; thus care should be taken using increased expression of MHC class II molecules as an early activation marker of peripheral M-DCs after activation in vitro. In conclusion, we propose that during activation of human DCs the production of IL-8 and loss of CD128b are the earliest signs of activation preceding both MHC class II, CD40, CD80, and CD86 expression.