Heterogeneous populations from in vitro cultures of antigen presenting cells in pigs.

Dendritic cells (DCs) are the most potent antigen presenting cells (APCs). Because of the difficulty in obtaining these cells directly from tissues, different sources of DCs are frequently used for in vitro experimentation and many of their biological and functional characteristics were studied using these systems. Until recently, it was assumed that specific culture conditions polarized the differentiation of either DCs or macrophages (Macs); however, it was shown that some DC culture systems in other species generate heterogeneous cell populations that can be identified according to their CD11c and MHC class II (MHC-II) expression. Following this approach, porcine DCs were directly isolated from peripheral blood or differentiated in vitro by culturing bone marrow (BM) progenitor cells or blood monocytes treated with growth factors. Mostly homogeneous monocyte-derived DCs (MoDCs) were obtained with similar phenotype and phagocytic characteristics to that of blood DCs. On the contrary, BM-derived DC (BMDC) cultures generated two distinct heterogeneous populations identified as MHC-II+ and MHC-II++ cells. BMDCs MHC-II+ had similar phenotypic and phagocytic characteristics to those of MoDCs and blood DCs. However, BMDCs MHC-II++ population expressed a higher amount of surface markers and transcribed genes associated with Macs-lineage exhibiting a higher phagocytic capacity than all the other cells. Noteworthy, every cell system expressed different genetic signatures. These results will help interpreting and re-interpreting data obtained using in vitro systems.

Identification of genes and pathways in human antigen-presenting cell subsets in response to polio vaccine by bioinformatical analysis.

BACKGROUND: Polio eradication has been achieved in the world except for three countries due to the widespread use of the inactivated poliovirus vaccine (IPV) and the live-attenuated oral poliovirus vaccine. Following polio eradication, the IPV would be the only polio vaccine available. However, the mechanisms of the interactions between IPV and human antigen-presenting cells (APCs) remain largely unclear. METHODS: To investigate the involvement of the IPV in human monocytes, we downloaded the gene chip GSE44721 from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the GEO2R analysis tool. Functional and pathway enrichment analyses were performed for DEGs using the Metascape database. DEG-associated protein-protein-interactions (PPIs) were established by the Search Tool for the Retrieval of Interacting Genes website and visualized by Cytoscape. RESULTS: There were 240 DEGs (51 upregulated and 189 downregulated genes) identified from the GSE44721 data set, and they were significantly enriched in several biological processes, including antigen processing and presentation of lipid antigen via MHC class Ib, adaptive immune response, and response to interferon-gamma. One hundred thirty-six nodes were screened from the DEG PPI network. There were six significant hub proteins (WDR36, MRTO4, RPF2, PPAN, CD40, and BMS1) that regulated the IPV in human monocytes. CONCLUSIONS: In summary, using bioinformatical analysis, we have information for the immunization activated by the IPV in monocytes. Moreover, hormones and cytokines regulate the activation of APCs.

Cellular immunological changes in patients with LADA are a mixture of those seen in patients with type 1 and type 2 diabetes.

There is currently scarce knowledge of the immunological profile of patients with latent autoimmune diabetes mellitus in the adult (LADA) when compared with healthy controls (HC) and patients with classical type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective of this study was to investigate the cellular immunological profile of LADA patients and compare to HC and patients with T1D and T2D. All patients and age-matched HC were recruited from Uppsala County. Peripheral blood mononuclear cells were isolated from freshly collected blood to determine the proportions of immune cells by flow cytometry. Plasma concentrations of the cytokine interleukin (IL)-35 were measured by enzyme-linked immunosorbent assay (ELISA). The proportion of CD11c+ CD123- antigen-presenting cells (APCs) was lower, while the proportions of CD11c+ CD123+ APCs and IL-35+ tolerogenic APCs were higher in LADA patients than in T1D patients. The proportion of CD3- CD56high CD16+ natural killer (NK) cells was higher in LADA patients than in both HC and T2D patients. The frequency of IL-35+ regulatory T cells and plasma IL-35 concentrations in LADA patients were similar to those in T1D and T2D patients, but lower than in HC. The proportion of regulatory B cells in LADA patients was higher than in healthy controls, T1D and T2D patients, and the frequency of IL-35+ regulatory B cells was higher than in T1D patients. LADA presents a mixed cellular immunological pattern with features overlapping with both T1D and T2D.

MITAP-compliant characterization of human regulatory macrophages.

This article provides a transparent description of Mreg_UKR cell products, including manufacture and quality-control processes, using the structure and vocabulary of the 'Minimum Information about Tolerogenic Antigen-presenting Cells' reporting guidelines. This information is intended as a resource for those in the field, as well as a stimulus to develop a new wave of immunoregulatory and tissue-reparative monocyte-derived cell therapies.

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.

Extramedullary hematopoiesis leading to the production of a novel antigen-presenting cell type in murine spleen.

The concept of extramedullary hematopoiesis for production of organ-specific antigen presenting cells has importance in immunity in terms of the compartmentalisation of the immune response in different tissue sites. A new and distinct dendritic-like antigen presenting cell subtype is described which is dependent on the spleen microenvironment for development. Cells arise by a unique developmental pathway distinct from other dendritic cells (DC). In particular, a self-renewing progenitor of these cells has been identified in spleen upstream of the earliest DC progenitor currently identified in bone marrow. This progenitor depends on the splenic microenvironment for maintenance and proliferation, adding further support for spleen as a site for hematopoiesis.

Quantitative immunophenotypic analysis of antigen-presenting cells involved in ectromelia virus antigen presentation in BALB/c and C57BL/6 mice.

During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c(+) , CD11b(+) , and CD19(+) cells. CD11c(+) and CD11b(+) cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19(+) cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b(+) DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox.

Distinct APCs explain the cytokine bias of α-galactosylceramide variants in vivo.

α-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 cytokines. Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1(fl/fl) mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by α-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-γ, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Th1 or Th2 cellular networks by changing APC targeting in vivo.

Antigen-presenting cell marker expression and phagocytotic activity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) cells are the main cellular constituents of the periodontium, maintain the integrity of the connective tissue, and impact pathology in periodontitis. The aim of this study was to analyze whether PDL cells recognize foreign particles and participate in the immune response to periodontal pathogens. METHODS: Expression of surface proteins characteristic of antigen-presenting cells (APCs) (major histocompatibility complex [MHC] class II, CD40, CD80, CD86) was analyzed in PDL cells after challenge with the cytokines interleukin (IL)-1ß, IL-17A, and interferon-gamma (IFN-γ) or with heat-killed Aggregatibacter actinomycetemcomitans using real-time PCR and flow cytometry. Confocal laser scanning microscopy, transmitted light microscopy, flow cytometry, and time-lapse microscopy were applied to analyze their phagocytotic capacity of collagen (carboxylate-modified microspheres), non-periodontal (Escherichia coli) and periodontal (Aggregatibacter actinomycetemcomitans) pathogens. Furthermore, it was examined whether cytokine activation of PDL cells affects the phagocytosis of collagen or bacteria. RESULTS: PDL cells upregulated MHC class II after cytokine stimulation on transcriptional level, whereas co-stimulatory molecules characteristic of professional APCs were not induced. Analyses on protein level revealed that MHC class II was not constitutively expressed in all PDL cell lines used. PDL cells phagocytosed both collagen and bacteria via acidic vesicles, suggesting the formation of phagosomes. Phagocytosis could be partially inhibited by inhibitors of phagocytosis, i.e., dynasore and wortmannin. Pre-incubation with cytokines did not further enhance the phagocytosis rate of collagen or bacteria. CONCLUSIONS: These results suggest that PDL cells do not only represent bystanders in periodontal infections, but display non-professional APC characteristics, suggesting possible participation in immune reactions of the oral cavity.

Nuocytes: expanding the innate cell repertoire in type-2 immunity.

Activation and differentiation of the Th1 cell population lead to their production of the classical type-1 cytokines IFN-γ, IL-2, and TNF-ß, thus promoting type-1 immunity. This is thought to occur via the ligation of TLRs by bacterial and viral products, which in turn, drive production of the essential Th1 cell differentiation factor, IL-12, by dendritic cells (DCs). Concurrent studies have been able to identify the effector cytokines produced by Th2 cells (IL-4, IL-5, IL-9, and IL-13) as being essential for parasitic immunity and also as essential factors in allergic asthma. However, the factors that are critical for initiation of the type-2 response remained obscure. Recently however, two critical observations have led to a more detailed understanding of the innate type-2 response. First, two novel, type-2-inducing cytokines-IL-25 and IL-33-were identified as being necessary for the up-regulation of the type-2 effector cytokines, mirroring the role of IL-12 in the type-1 response. Second, studies focused on target cell populations of IL-25 and IL-33 have identified novel, innate cell populations, which potentially bridge the gap between presentation of the type-2-inducing cytokine and the later adaptive Th2 cell response. In this review, we will discuss these new type-2 innate cell populations, in particular, the recently discovered nuocyte population, which are required for type-2 responses against helminthic parasites.

Antigen-presenting cell (APC) subsets in ovarian cancer.

The major human antigen-presenting cells (APCs) include monocytes/macrophages, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and B cells. These APC subsets have been observed in ovarian tumor environments. Their phenotypes and functionalities are subjected to alteration by multiple factors in the tumor environment. In this review, we summarize the nature, cellular interactions, and prognostic significance of the main APC populations in ovarian cancer, and discuss the relevance of manipulating APC subsets for patient treatment.

Primary porcine CD11R1+ antigen-presenting cells isolated from small intestinal mucosa mature but lose their T cell stimulatory function in response to cholera toxin treatment.

Antigen-presenting cells (APCs) in the small intestinal mucosa perform dual functions of maintaining tissue homeostasis and of protecting against intestinal pathogens as key inducers of both innate and adaptive immune responses. Intestinal APCs are thus important regulators of intestinal immunity and also potential target cells for mucosal adjuvants such as cholera toxin (Ctx), which was used successfully in several oral vaccination studies in pigs. The aims of the present study were (1) to isolate porcine small intestinal APCs and evaluate the feasibility of using these cells for functional in vitro studies and (2) to determine the response of intestinal APCs to Ctx. Microscopic and flow cytometric analyses using antibodies to CD1, CD11R1, CD16, and SIRPalpha (SWC3) revealed the presence of multiple subsets of MHC-II(++) APCs in porcine small intestinal mucosa. The alpha-integrin subunit CD11R1 was most frequently expressed and therefore chosen as a selection marker. CD11R1(+) cells were enriched from total lamina propria cells to >90% purity by immunomagnetic separation. Within the CD11R1 cells, we identified two populations with distinct forward and side scatter characteristics: (1) APCs identified by their high expression of MHC-II and consisting of SIRPalpha(+) and SIRPalpha(-) subsets, and (2) contaminating eosinophils. In culture, intestinal APCs spontaneously matured, as shown by significant (>5-fold) increase in CD80/CD86 expression. The SIRPalpha(+) APCs quickly disappeared from the cultures, likely due to increased apoptotic cell death. However, the observed spontaneous changes in the isolated cell population did not mask the effects of stimulation with Ctx, which resulted in a 2.5-fold increase in the expression of maturation markers CD80/CD86, but significant loss of T cell stimulatory function, corroborating previous results obtained with MoDC.

Cathepsin G is differentially expressed in primary human antigen-presenting cells.

Cathepsins are required for the processing of antigens in order to make them suitable for loading on major histocompatibility complex (MHC) class II molecules, for subsequent presentation to CD4(+) T cells. It was shown that antigen processing in monocyte-derived dendritic cells (DC), a commonly used DC model, is different from that of primary human DC. Here, we report that the two subsets of human myeloid DC (mDC) and plasmacytoid DC (pDC) differ in their cathepsin distribution. The serine protease cathepsin G (CatG) was detected in mDC1, mDC2, pDC, cortical thymic epithelial cells (cTEC) and high levels of CatG were determined in pDC. To address the role of CatG in the processing and presentation of a Multiple Sclerosis-associated autoantigen myelin basic protein (MBP), we used a non-CatG expressing fibroblast cell line and fibroblasts, which were preloaded with purified CatG. We find that preloading fibroblasts with CatG results in a decrease of MBP84-98-specific T cell proliferation, when compared to control cells. Our data suggest a different processing signature in primary human antigen-presenting cells and CatG may be of functional importance.

Human peripheral blood dendritic cells and monocyte subsets display similar chemokine receptor expression profiles with differential migratory responses.

Human antigen presenting cells (APC) found in peripheral blood are considered to be precursors that have been released from the bone marrow and are in transit to the peripheral tissues. These APC populations include myeloid dendritic cells (mDC), plasmacytoid DC (pDC) and monocytes (Mo). To assign specialized functional roles and stages of development for APCs, CD33 expressing APC subsets were examined for their capacity to respond to chemokines. Three major CD33(+) subsets including CD33(bright)CD14(bright) Mo, CD33(bright)CD14(-) CD11c(+) mDC and CD33(dim)CD14(-) pDC were present. Dendritic cells subsets and Mo expressed low levels of CC and CXC receptors, but distinctive chemokine receptor expression profiles were not observed. The percentage of cells expressing a particular chemokine receptor varied from donor to donor and over time in the same donor. Myeloid DC and Mo but not pDC migrated toward CXCL12 in a concentration dependent manner. Monocytes and pDC, but not myeloid DC, were attracted by high concentrations of CXCL10. All CD33(+) subsets migrated in a concentration dependent manner toward CCL19, but responded less robustly to CCL21. CCL20 was not chemoattractant for any population. Despite the finding that APC did not exhibit unique surface chemokine receptor expression patterns, they exhibited differential migration to CXCL12, CXCL10 and CCL21 but not to CCL20 or CCL19.

Cellular expression requirements for inhibition of type 1 diabetes by a dominantly protective major histocompatibility complex haplotype.

The H2(g7) (K(d), A(g7), E(null), and D(b)) major histocompatibility complex (MHC) is the primary genetic contributor to type 1 diabetes in NOD mice. NOD stocks congenically expressing other MHC haplotypes such as H2(nb1) (K(b), A(nb1), E(k), and D(b)) in a heterozygous state are type 1 diabetes resistant. Hematopoietically derived antigen-presenting cells (APCs) expressing H2(nb1) MHC molecules delete or inactivate autoreactive diabetogenic T-cells. Thus, provided a relatively benign preconditioning protocol is ultimately developed, hematopoietic chimerization by APCs expressing dominantly protective MHC molecules could conceivably provide a means for type 1 diabetes prevention in humans. Before hematopoietic chimerization can be considered for type 1 diabetes prevention, it must be determined what subtype(s) of APCs (B-cells, macrophages, and/or dendritic cells) expressing protective MHC molecules most efficiently inhibit disease, as well as the engraftment level they must achieve to accomplish this. These issues were addressed through analyses of NOD background bone marrow chimeras in which H2(nb1) molecules were selectively expressed on variable proportions of different APC subtypes. While a modest B-cell effect was observed, the strongest type 1 diabetes protection resulted from at least 50% of dendritic cells and macrophages expressing H2(nb1) molecules. At this engraftment level, H2(nb1)-expressing dendritic cells and macrophages mediated virtually complete deletion of a highly pathogenic CD8 T-cell population.

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry.

AIM: To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. METHODOLOGY: Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. RESULTS: Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). CONCLUSIONS: Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.

Ins and outs of dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells which are strategically positioned at the boundaries between the inner and the outside world, in this way bridging innate and adaptive immunity. DC can initiate T cell responses against microbial pathogens and tumors due to their capacity to stimulate naïve T cells. The development of DC occurs in distinct stages. DC precursors develop in the bone marrow and home to a large variety of tissues. Immature DC capture antigen (Ag) and, following proinflammatory signals, migrate to the lymphoid organs where, after maturation, they present captured Ag to naïve T cells, thereby inducing differentiation of naïve T cells into effector T cells. An important cognate event in the development of cell-mediated immunity is the interaction between CD40 and CD40 ligand. Ligation of CD40 on DC by its ligand results in maturation of the DC. In addition to CD40 ligand (expressed by activated Th cells), inflammatory cytokines, bacterial components or Ag-Ab immune complexes can induce maturation of DC. Maturation of DC is crucial for the priming of efficient T cell responses and is characterized by a decreased Ag processing capacity, an increased cell surface expression of MHC and costimulatory molecules, and rearrangement of cytoskeleton, adhesion molecules, and cytokine receptors. Mature DC migrate from peripheral tissues to secondary lymphoid organs, where T cell priming occurs. DC are not only critical in initiating T cell immunity, they also play a role in the induction of T cell tolerance and the regulation of the type of T cell response that is induced. Here we give an overview of the dendritic cell system.

Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses.

Optimal Ag targeting and activation of APCs, especially dendritic cells (DCs), are important in vaccine development. In this study, we report the effects of different Toll-like receptor (TLR)-binding compounds to enhance immune responses induced by human APCs, including CD123(+) plasmacytoid DCs (PDCs), CD11c(+) myeloid DCs (MDCs), monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to imidazoquinolines (imiquimod and R-848) and to CpG oligodeoxynucleotides stimulation, resulting in enhancement in expression of costimulatory molecules and induction of IFN-alpha and IL-12p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7, responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high production of IL-12 p70 without producing detectable IFN-alpha. Optimally TLR ligand-stimulated PDCs or MDCs exposed to CMV or HIV-1 Ags enhanced autologous CMV- and HIV-1-specific memory T cell responses as measured by effector cytokine production compared with TLR ligand-activated monocytes and B cells or unstimulated PDCs and MDCs. Together, these data show that targeting specific DC subsets using TLR ligands can enhance their ability to activate virus-specific T cells, providing information for the rational design of TLR ligands as adjuvants for vaccines or immune modulating therapy.

The adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo.

The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response. BALB/c mice were injected intravenously or subcutaneously with OM-174. The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype. The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS).