MHC-II Antigen Expression Research Articles

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells

IntroductionWhile amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in humans and horses and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications.MethodsIn our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion.ResultsAECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic marker SSEA-4, but not SSEA-3, as demonstrated by immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity.ConclusionsIn conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic cell-based therapies in order to treat tissue lesions, especially with loss of substance.

Long-Term Cultured Human Term Placenta-Derived Mesenchymal Stem Cells of Maternal Origin Displays Plasticity

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics.

Human PD-L1-overexpressing porcine vascular endothelial cells induce functionally suppressive human CD4+CD25hiFoxp3+ Treg cells

In xenotransplantation models, direct activation of hCD4(+) T cells by porcine VECs leads to a robust proliferation of T cells. To investigate the underlying mechanisms, human antiporcine MLEC culture was used to investigate cross-species cell interactions, proliferation of hCD4(+) T cells, and induction of human cytokines. We report that xenoantigen presentation by PIEC expands hCD4(+) Foxp3(+) Tregs and hCD4(+) Foxp3(-) Teffs, and this process is dependent on porcine MHC-II antigen expression. Stable transfection of hPD-L1 into PIEC inhibits Teff proliferation, but Treg proliferation is not affected. Surprisingly, IL-10 production by hCD4(+) T cells is augmented significantly by PIEC(hPD-L1). Notably, hPD-L1-induced Tregs have higher suppressive potency and mediate suppressive function partially through IL-10 and CD73. This study opens the possibility of using hPD-L1-overexpressing porcine VECs as a novel therapeutic to allow tolerance of xenotransplants and also supports the possibility of using hPD-L1 transgenic pigs as xenotransplant donors.

Expression of IFNα-inducible genes and modulation of HLA-DR and thyroid stimulating hormone receptors in Graves’ disease

Interferon alpha (IFNα) plays an important role in the pathogenesis of different autoimmune diseases. IFNα is widely used for the treatment of chronic viral infections, particularly chronic hepatitis C virus infection; however, several case reports have emerged describing autoimmune conditions, such as Graves’ disease (GD), that have developed in the patients receiving IFNα. The mechanism by which IFNα is involved in GD remains poorly understood. We investigated the expression of IFNα and IFNα-inducible genes (IFIGs) in GD and found that IFIGs were overexpressed in 60% of 54 clinical diagnostic GD patients. These elevated IFIGs correlated with serological levels of autoantibody to thyroid stimulating hormone receptor (TSHR). Recombinant human IFNα stimulated primary cultured thyrocytes resulted in not only high level expression of IFIGs, but also, more importantly, expression of MHC-II antigens (HLA-DR3 and HLA-DR5) and TSHR in GD subjects. Furthermore, thyroid gland tissues from GD patients over express HLA-DR, TSHR and IFNα receptors at both messenger RNA and protein levels. Taken together, these data indicated that in GD patients, IFNα can function on thyroid tissue to induce a number of genes, particularly MHC class II molecules which may enhance autoantigen presentation of TSHR on thyrocytes.

In vivo immunostimulatory effects of CpG ODN in newborn piglets

The in vivo immunoadjuvant effects of CpG oligodeoxynucleotides (CpG ODN) have been studied extensively in mice and relatively fewer studies have been done in other species. But so far, the innate immunostimulatory effects of CpG ODN have been demonstrated just in mouse, monkey, sheep and chicken in some reports. The purpose of this study is to determine the potential effects of CpG ODN in newborn piglets. The proportion of CD4 +, CD8 + T lymphocytes subpopulations and the major histocompability complex (MHC-II) antigen expression of peripheral blood mononuclear cells (PBMCs) and IFN-γ in serum were tested at various time-points. The results suggested that, the CD4 +/CD8 + ratio decreased over time in piglets inoculated with phosphate buffer saline (PBS) alone, however, it was stable in CpG ODN-inoculated piglets; the use of CpG ODN can prevent effectively the reduction of the proportion of CD4 + T lymphocytes. The MHC-II antigen expression and IFN-γ level of CpG ODN-injected piglets were significantly higher than those of PBS-injected piglets. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than in PBS. The innate immunostimulatory activity of CpG ODN appeared to be in dose-dependent manner. These in vivo data demonstrate for the first time that CpG ODN can stimulate innate immune system in newborn piglets.

Differential expression of MHC class II molecules in highly metastatic breast cancer cells is mediated by the regulation of the CIITA transcription: Implication of CIITA in tumor and metastasis development

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNγ or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNγ induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNγ-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.

Human Embryonic Stem Cells: Potential Tool for Achieving Immunotolerance?

The derivation of human embryonic stem cells (hESCs), whose in vitro differentiation might be directed toward different cell types, has raised the hope for cell replacement therapies. Despite the emerging reports to differentiate hESCs into specific lineages and then to distinct mature cell subsets, there are still several issues that need to be resolved before transplantation of these cells can be realized. In this context, immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation. However, recent data support the concept that hESCs and/or their differentiated derivatives possess immune-privileged properties, suggesting that cells derived from hESC may provide a potential tool for induction of immunetolerance. Currently, our understanding of the tolerogenic potential of hESCs is limited to assessment by in vitro assays or xenogenic transplantation approaches in vivo. Human ESCs express low levels of major histocompatability complex (MHC)-I antigens and lack expression of MHC-II antigens and costimulatory molecules, and are not recognized by natural killer cells and inhibit T-cell induced-stimulation by third-party antigen-presenting cells. Upon injection into immunocompetent mice, hESCs are unable to induce an immune response as demonstrated by their inability to induce an inflammatory response. Based on these initial observations, further studies in hESCs immunobiology are warranted and may reveal unique mechanisms that account for the immunological properties of hESCs. Here, we explore the prospect of using hESCs and their derivatives for immunomodulation and tolerance induction.

Down-regulation of CXCR1 and CXCR2 expression on human neutrophils upon activation of whole blood by S. aureus is mediated by TNF-alpha.

It was suggested that bacterial products can inhibit the expression of leucocyte chemokine receptors during sepsis and affect leucocyte functions in septic syndrome. Superantigens and toxins produced by Staphylococcus aureus are capable of activating leucocytes via binding to MHC-II antigens on monocytes and T-cell receptor molecules on T lymphocytes. It was recently shown that staphylococcal enterotoxins directly down-regulate the expression of CC chemokine receptors on monocytes through binding to MHC class II molecules. We studied the effects of killed S. aureus on the expression of interleukin-8 receptors, CXCR1 and CXCR2, on polymorphonuclear leucocytes (PMN), which are known to lack the expression of MHC-II antigens. It was shown that S. aureus down-regulated the cell-surface expression of CXCR1 and CXCR2 on PMN in the whole blood and total blood leucocyte fraction containing PMN and monocytes, but did not modulate IL-8 receptor expression in purified PMN suspension. Antibody to TNF-alpha abrogated down-regulation of IL-8 receptors induced by S. aureus. In contrast, LPS reduced CXCR1 and CXCR2 expression in purified PMN and whole blood in a TNF-alpha-independent manner. We further showed that TNF-alpha-induced decrease of CXCR1 and CXCR2 expression was associated with lower IL-8 binding and lower CXCR1 and CXCR2 mRNA levels, and was abrogated by protease inhibitors. We suggest that during septicemia, S. aureus may inhibit neutrophil responsiveness to IL-8 and other CXC chemokines via TNF-alpha- mediated down-regulation of CXCR1 and CXCR2.

Spontaneous Borna disease in sheep and horses: immunophenotyping of inflammatory cells and detection of MHC-I and MHC-II antigen expression in Borna encephalitis lesions

Borna disease (BD) has been recognized as a virally induced T-cell dependent immunopathological disorder of the central nervous system (CNS), as shown by experimental infection of rats with Borna disease virus (BDV). In contrast to the rat model, little is known about the pathogenesis of spontaneous BD in sheep and horses. The present study describes the brain lesions of 12 ovine and 11 equine cases of naturally occurring BD. A set of monoclonal and polyclonal antibodies was used in order to determine the cells operative in encephalitic lesions and to detect expression of MHC-I and MHC-II products in the brains of affected animals. In all cases investigated, a reaction pattern similar to that reported for the acute phase of BD in experimentally infected rats was noted. In brief, the majority of inflammatory cells in perivascular infiltrates (PVI) as well as parenchymal and meningeal infiltrates were CD3+. CD4+ cells outnumbered CD8+ cells in PVI as well as in the parenchyma. Macrophages (defined by lysozyme immunoreactivity) were seen less often and B-cells or plasma cells (cells positive for λ or κ light chains) were demonstrated at lower numbers. TCR-1+ cells were found on very rare occasions in PVI of some sheep. MHC-I and MHC-II products were constantly expressed on inflammatory cells but inconsistently on astrocytes and neurons. Neuronal degeneration was not a major feature.

Increased proliferative activity due to necroses induced by pre-operative embolization in benign meningiomas.

The proliferative behaviour of 35 benign intracranial meningiomas was investigated which were embolized for devascularization 3 to 268 hours prior to surgical exstirpation. The nuclear proliferation antigen Ki-67 was visualized by means of the monoclonal antibody MIB1 on formalin fixed and paraffin embedded tissue. Tumor cells and inflammatory reactions were recognized by means of conventional staining procedures and by immunohistochemical detection of HAM56, LCA, HLA-DR, CD15-epitope and vimentin. Extravasation and proliferation of granulocytes, macrophages, lymphocytes and the degree of expression of MHCII antigens was estimated according to a 7-point ordinal scale. Confirming preliminary observations of others the proliferation index within the perinecrotic tumor rim (PIperinec) exceeded that of intact tissue highly significantly. PIperinec peaked at the third to fourth day after embolization and kept this level until the seventh day. The time course of PIperinec was paralleled by that of macrophages, whereas--expectedly--granulocytes occured earlier and lymphocytes and HLA-DR-positivity somewhat later. The timely relationships suggested that the perinecrotic increase in tumor cell proliferation was mainly due to macrophage-born mitogens. Perinecrotic proliferative activity in embolized meningiomas does not reflect genuine tumor proliferation and should not be used for assessment of the presence or degree of malignancy in a given tumor.

Cloning, expression and characterization of biologically active feline tumour necrosis factor-α

We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (fTNF-α). Messenger RNA was extracted from feline peritoneal macrophage cultures and used to synthesize cDNA for polymerase chain reaction (PCR) amplification. The PCR products were cloned into the plasmid vector pCRII and sequenced, showing 99.3% homology with a published fTNF-α gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-α and glutathione S-transferase (GST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-α antibody in Western blotting, but not with a polyclonal anti-murine TNF-α serum. Recombinant fTNF-α (rfTNF-α) and rfTNF-α-GST had a CD 50 of 15 ng ml −1 and 230 ng ml −1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-α-GST intravenously manifested the typical biological effects of TNF-α, including fever, depression, and piloerection. The rfTNF-α-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-α receptor and MHC-I antigen expression.

Comparative ultrastructure and immunolabeling of MHC-II antigens of alveolar macrophages obtained from patients with paracoccidioidomycosis and other lung diseases

Samples of alveolar macrophages (AM) obtained by bronchoalveolar lavage from patients with either paracoccidioidomycosis, silicosis, sarcoidosis, or allergic alveolitis were investigated by electron microscopy and immunocytochemistry to compare cellular ultrastructure and expression of MHC-II antigens in the AM cell surface. All samples of AM obtained from patients with these pathologies showed heterogeneous structural features. Although, this morphological diversity is also present in AM of healthy donors, our observations seem to indicate that in the diseases studied this morphofunctional diversity is associated with additional ultrastructural characteristics inherent to each disease. In paracoccidioidomycosis the proportion of vacuolated macrophages is significantly lower than in other diseases; this might indicate that in paracoccidioidomycosis the proportion of activated AM is smaller. We observed significant differences in the expression of MHC-II antigens. Silicosis, sarcoidosis, and allergic alveolitis do not differ significantly in the quantity of immunolabeled AM or in the distribution of the label. The percentage of AM from paracoccidioidomycosis that exhibit the MHC-II molecule is very low with poor immunolabeling. In this disease the low expression of the MHC-II molecule could be related to a decrease of the antigen presenting function by AM.

The generation of nitric oxide participates in γIFN-induced MHC class II antigen expression by cultured astrocytoma cells

The effects of interferon gamma (γIFN) on the MHCII antigen expression by human cultured astrocytoma cells were investigated. The co-incubation of γIFN with T67 astrocytoma cells produced a dose-dependent increase of MHCII antigen expression as evaluated by flow cytometric (FACS) analysis and confocal laser microscopy analysis. The number of NHCII molecules expressed by γIFN-pretreated astrocytoma cells was reduced by co-incubation with N ω-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of the nitric oxide (NO)-synthesizing enzyme. In addition, methylene blue, which inhibits the biological activity of NO acting at the guanylate cyclase level, strongly antagonized the MHCII antigen expression on astrocytoma cells induced by γIFN. Furthermore, γIFN increased the activity of the inducible isoform of NO-synthase as well as the concentration of nitrite, one of the breakdown products of NO and the antiplatelet activity of astrocytoma cells. In conclusion, the present data show that γIFN increases the synthesis and release of NO by cultured astrocytoma cells and this could co-participate in the MHCII antigen expression by this cell type. Therefore, the generation of NO by cultured astrocytoma cells may represent an important step in the development of the immunocompetent activity of astrocytes.

Effect of selection of swine for high and low immune responsiveness on monocyte superoxide anion production and Class II MHC antigen expression

Monocyte function was investigated in second (G 2) and third (G 3) generation pigs selected for high and low antibody and cell-mediated immune responsiveness. In groups of pigs from the high- and low-immune response lines, monocyte release of superoxide anion (O 2 −) was assayed in response to phorbol 12-myristate-13-acetate and expression of the Class II MHC (MHC-II) antigens SLA-DR and SLA-DQ, determined using flow cytometry. Analysis of variance using a linear model demonstrated no significant intergroup differences in O 2 − production by lymphokine-activated monocytes from G 2 pigs. In G 3 pigs, there were no significant intergroup differences in the percentage of MHC-II + cells or in the density of expression of either SLA-DR or SLA-DQ. In individual pigs, monocyte SLA-DR and SLA-DQ expression was similar in terms of the percentage of MHC-II + cells and in the magnitude of MHC-II expression. Litter contributed significantly to variation in monocyte O 2 − production in G 2 pigs ( P ≤ 0.005) and SLA-DQ ( P ≤ 0.01) expression. Although the lines differed significantly in correlates of antibody and cell-mediated immune response, there was no apparent effect of selection for high and low immune responsiveness in swine on monocyte O 2 − production and MHC-II expression.