Differential cytokine expression in direct and indirect co-culture of islets and mesenchymal stromal cells.

BACKGROUND: Transplantation of allogenic Langerhans islets (ISL) has been employed as an alternative to pancreas transplantation to provide endogenous supply of insulin and treat hypoglycemia unawareness in type 1 diabetes. Nevertheless, the process of islets isolation exposes the islets to hypoxia and other aggressive conditions that results in the recover of less than half of the islets present in the pancreas. Several studies demonstrated that co-culturing islets with mesenchymal stromal cells (MSC) before implantation enhances islets survival and function and this effect is mediated by cytokines. However, it remains unclear if the profile of cytokines secreted by MSC in co-culture with islets changes upon the type of co-culture: direct and indirect. MATERIALS AND METHODS: In 3 series of experiments with human islets of 3 different donors, we compared the levels of a panel of cytokines measured in the supernatant of ISL cultured alone, Wharton Jelly MSC (WJMSC) cultured alone, direct co-culture of ISL-WJMSC and indirect co-culture using a permeable transwell membrane to separate ISL and WJMSC. RESULTS: Comparing the profile of cytokines secreted by islets alone with islets in direct co- culture with WJMSC, we found higher expression of IL1b, IL17, IFγ, IL4, IL10, IL13, Granulocyte-macrophage colony-stimulating factor (GMCSF) and Leptin, in the supernatant of the co-cultures. In contrast, when comparing islets cultured alone with islets in indirect co-culture with MSC, we found no significant differences in the levels of cytokines we analyzed. CONCLUSION: Direct contact between human WJMSC and pancreatic islets is required for elevated expression of a range of immune cytokines, including both those considered inflammatory, and anti-inflammatory.

Transitional B lymphocytes are associated with protection from kidney allograft rejection: a prospective study.

Recent cross-sectional studies suggest an important role for transitional B lymphocytes (CD19 + CD24hiCD38hi) in promoting transplant tolerance, and protecting from late antibody-mediated rejection (ABMR). However, prospective studies are lacking. This study enrolled 73 de novo transplant recipients, and collected serial clinical, immunological and biochemical information over 48 ± 6 months. Cell phenotyping was conducted immediately prior to transplantation, and then on five occasions during the first year posttransplantation. When modeled as a time-dependent covariate, transitional B cell frequencies (but not total B cells or "regulatory" T cells) were associated with protection from acute rejection (any Banff grade; HR: 0.60; 95% CI: 0.37-0.95; p = 0.03). No association between transitional B cell proportions and either de novo donor-specific or nondonor-specific antibody (dnDSA; dnNDSA) formation was evident, although preserved transitional B cell proportions were associated with reduced rejection rates in those patients developing dnDSA. Three episodes of ABMR occurred, all in the context of nonadherence, and all associated with in vitro anti-HLA T cell responses in an ELISPOT assay (p = 0.008 versus antibody-positive patients not experiencing ABMR). This prospective study supports the potential relevance of transitional ("regulatory") B cells as a biomarker and therapeutic intervention in transplantation, and highlights relationships between humoral immunity, cellular immunity and nonadherence.

A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are of interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. Umbilical cord blood (UCB) is reported to contain MSC, and therefore may be a useful source of these cells for clinical applications. METHODS: We evaluated protocols for isolating MSC from UCB and characterized the surface phenotype, differentiation potential and immunoregulatory properties of the cells obtained. RESULTS: Ten of 25 UCB units processed yielded MSC-like colonies, with depletion of lineage+ cells providing a higher efficiency. Only two of the cultures could be expanded satisfactorily; the remainder failed to proliferate. One culture generated transformed lines that were grossly aneuploid, had up-regulated TERT transcripts and had lost CD90 expression and the capacity to differentiate. The two propagated UCB-MSC lines were similar to those from bone marrow but were not identical to each other, with differences seen in expression of surface markers and cytoskeletal proteins. Both underwent osteogenesis, but at different rates and to different degrees, while neither generated adipocytes. When added as a third party to a mixed lymphocyte culture, both suppressed proliferation. DISCUSSION: MSC-like cells can be isolated from UCB, but at low efficiencies, and they exhibit a variety of morphologies, growth rates and differentiation potentials and can transform in culture.

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies.

BACKGROUND AND OBJECTIVES: Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro, as a means for predicting their therapeutic potential after myocardial infarction. MATERIALS AND METHODS: Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs. RESULTS: Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (approximately 0.07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine. CONCLUSION: Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.

Mesenchymal stem cells of cord blood origin are effective at preventing but not treating graft-versus-host disease.

The immunosuppressive properties of mesenchymal stem cells (MSC) make them particularly attractive to manipulate graft-versus-host disease (GVHD). So far, the experience of using MSC to treat GVHD is limited to a few cases, controversial results come from preclinical models and several issues remain to be clarified. The present studies were designed to address these questions in a xenogenic model testing the ability of umbilical cord blood-derived MSC (UCB-MSC) to prevent and/or treat GVHD. Sublethally irradiatiated non-obese diabetic/severe combined immunodeficiency NOD/SCID mice transplanted with human peripheral blood mononuclear cells (huPBMC) showed extensive human T-cell proliferation in the peripheral blood, lymphoid and non-lymphoid tissues, which evolved in extensive GVHD (wasting, ruffled hair and hunched back). The mice treated with a single dose of UCB-MSC did not behave differently form the controls. However, when UCB-MSC were given at weekly intervals, there was a marked decrease in human T-cell proliferation and none of the mice developed GVHD. No therapeutic effect was obtained if UCB-MSC were administered at onset of GVHD. This work supports the clinical use of MSC in stem cell transplantation as a prophylaxis rather than treatment of GVHD.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

Diagnosis of defects in the type 1 cytokine pathway.

Patients with inherited defects in the interleukin-12 (IL-12)-dependent, 'high-output' interferon-gamma (IFN-gamma) pathway exhibit selective susceptibility to poorly pathogenic mycobacterial and salmonella infections. This review summarises the extended clinical spectrum seen in this group of patients and indicates a strategy for the identification of putative defects in the type 1 cytokine pathway.

Protocols for high efficiency, stage-specific retroviral transduction of murine fetal thymocytes and thymic epithelial cells.

Viral vectors have the potential to provide a fast and economic alternative to transgenic methods for manipulating gene expression in studies of immune system development and function. Although protocols exist for the infection of hematopoietic precursors and peripheral T cells in vitro, critical stages of T cell differentiation are strictly dependent upon a three-dimensional thymic architecture and their analysis poses unique technical challenges. Whole fetal thymic lobes have been used as targets for retroviral and adenoviral infection, both in situ and in vitro, but this approach does not allow for discrimination between lymphoid and stromal components. Isolated thymocytes have been infected by co-culture with viral producer cells, but under these conditions they rapidly lose their developmental potential. To overcome these problems we have combined a number of efficient techniques for retroviral production, concentration, and infection that allow us to rapidly achieve significant transduction rates of purified populations of double-negative (DN) and double-positive (DP) thymocytes, single-positive (SP) T lymphocytes, as well as fetal thymic MHC II(+) epithelial cells without the need for co-culture with viral producer cells. Reaggregate thymic organ culture (RTOC) techniques were used to assess the development and function of transduced cells in defined cellular environments. As a demonstration of the utility of these methods, CD80 (B7.1) was transduced into thymic epithelial cells and shown to allow them to mediate negative selection of DP thymocytes, and to act as antigen-presenting cells (APC) to mature T cells. The ability to genetically manipulate primary cells of a specified type and differentiation stage provides a powerful complement to RTOC techniques for the study of T cell development.

Recall antigen presentation by gamma-interferon-activated microglia results in T cell activation and propagation of the immune response.

The interaction between microglia and T cells is important in the development of central nervous system inflammation. This may result in full T cell activation, a partial state of activation, anergy or apoptosis of the 'responding' T cell. Here, we demonstrate that neonatal rodent microglia not only fail to initiate a mixed lymphocyte reaction (MLR), but suppress background T cell proliferation. Even after activation with gamma-IFN or following phagocytosis, microglia remain unable to support a MLR. By contrast, gamma-IFN-activated microglia are able to activate memory T cells in a recall assay resulting in cytokine (gamma-IFN) release and modest T cell proliferation. Although the stimulation index is small, functional relevance is demonstrated. Supernatants from the recall assay stimulate gamma-IFN-dependent activation of a STAT (signal transducer and activator of transcription) factor within resting microglia. This demonstrates that memory T cells not only receive sufficient stimulation from the gamma-IFN-activated microglia to proliferate and produce cytokines, but that there is also a reciprocal stimulation of resting microglia. Importantly, this provides evidence that activated microglia have the potential to propagate immune responses in the central nervous system, but are unlikely to initiate a primary response.

An HLA-B regulatory element binds a factor immunologically related to the upstream stimulation factor.

HLA-A and -B are expressed by most cell types, and their levels can be increased by treatment with interferons (IFNs). The relative basal levels of HLA-A and -B expression can vary, and HLA-B loci are induced much more strongly by IFNs. Constitutive activity is dependent on an upstream enhancer (ENH) which contains a rel (KBF, NF kappa B) binding motif, and induction is mediated by an interferon response element (IRE) which binds members of the IRF family. Reported here is the identification of a regulatory element, 'R', which overlaps the IRE of HLA-B loci, but which is absent from the equivalent region of HLA-A or H2 class I genes. The core of the element, CACGAG, is bound by a nuclear factor which is recognized by an antiserum raised against the upstream stimulation factor (USF), a member of the helix-loop-helix/leucine zipper family. The use of reporter gene constructs shows that mutation of the R element results in increased induction by IFN alpha in some cell lines, which appears to be due to competitive binding of USF with IRF proteins.

Synergistic induction of HLA class I expression by RelA and CIITA.

The major histocompatibility complex (MHC) class I genes are induced synergistically by interferons (IFN) and tumor necrosis factor (TNF), a response thought to involve the cooperative action of Rel/NF-kB and interferon regulatory factor (IRF) transcription factors. The IFN-gamma-inducible class II transcriptional activator (CIITA) has recently been shown to transactivate MHC class I as well as class II genes, and this investigation shows that CIITA synergizes strongly with RelA to stimulate HLA class I expression. The functional interaction of CIITA and RelA requires both promoter elements and the upstream Rel binding site and is not seen with a class II reporter. The promoter elements necessary for CIITA action are also required for induction by IFN-alpha. HLA-A and HLA-B loci respond differentially to IFNs, and we identify locus-specific differences in critical promoter elements in addition to known polymorphisms in the Rel and IRF binding sites. The HLA-A promoter is transactivated relatively poorly by CIITA and does not interact detectably with CREB proteins implicated in CIITA recruitment, but the synergism with RelA can compensate for this weakness. The present findings illustrate that multiple transcription factors cooperate to regulate class I expression and that their relative importance differs according to the locus and cell type examined. (Blood. 2000;95:3804-3808)

Differential expression and interferon response of HLA class I genes in thymocyte lines and response variants.

cDNA clones which seem to represent the alleles of HLA-A and -B expressed by the thymoma MOLT-4 have been isolated and used as locus-specific probes to measure the corresponding mRNA levels in MOLT-4 and other human thymocyte lines, and the effect of interferon (IFN)-alpha and -gamma on these levels. It is shown in MOLT-4, and in its derived line YHHH, that HLA-B mRNA levels are undetectable before treatment but respond to IFN-alpha and -gamma more markedly than those of HLA-A. This differential induction is best shown with YHHH, which is hypersensitive to IFN-alpha, where the HLA-B mRNA levels increase to a level threefold those of HLA-A. Other thymocyte lines tested also showed preferential induction by IFN-alpha of HLA-B, although the basal levels of HLA-A and -B tended to be similar. The effect of the altered ratio of HLA-A to -B mRNA on surface expression of the antigens and the correlation between basal level expression and inducibility are discussed.

Autocrine activation by interferon-gamma of STAT factors following T cell activation.

The activation of T cells requires engagement of the T cell receptor/CD3 complex and co-stimulatory molecules, and results in the triggering of several signaling pathways which lead rapidly to the nuclear translocation of several transcription factors, such as nuclear factor (NF)-kappa B and NF-AT. A result of this activation process is the induction of a number of genes, including those encoding cytokines such as interleukin-2, tumor necrosis factor-alpha, and interferon (IFN)-gamma which have important immunoregulatory effects. We report here that a DNA-binding factor containing STAT1 also becomes activated in human peripheral blood T lymphocytes or Jurkat cells, although not until 1-2 h after stimulation. Activation is delayed a further 1-2 hr when mononuclear cell cultures are stimulated by an antigen which requires processing. Appearance of the STAT1 factor is significantly reduced in the presence of cyclosporin A, and blocked by cycloheximide, indicating that its activation is dependent upon a protein(s) synthesized in response to initial signaling events. Neutralizing antiserum against IFN-gamma, but not other cytokines tested, blocked activation of the factor almost completely, and IFN-gamma was found in the culture supernatants of stimulated cells at levels at which recombinant IFN-gamma could activate the factor in naive cells. Therefore, a STAT1 transcription factor is activated by IFN-gamma synthesized and released upon stimulation of T lymphocyte populations. While Jurkat cells both secrete and respond to IFN-gamma in an autocrine loop, it seems likely that the responding cells may differ from those synthesizing this cytokine in the mononuclear cell cultures in the light of the recent report that Th1 cells lack the IFN-gamma receptor chain necessary for activation of STAT1 (Pernis, A., Gupta, S., Gollob, K.J., Garfein, E., Coffman, R.L., Schindler, C., and Rothman, P., Science 1995. 269:245).

IFN-alpha super-induction of HLA class I expression by a variant thymoma cell line involves nuclear translocation of Rel complexes.

Variant thymoma lines have been described which exhibit a substantially increased level of HLA class I induction by IFN-alpha, but not by IFN-gamma, and an unchanged response of other IFN-alpha-stimulated genes (Burrone et al., EMBO J. 1985. 4: 2855-2860). We report that their amplified response correlates with the nuclear translocation of Rel transcription factors upon prolonged treatment with IFN-alpha. The variant cells contain an IkappaBalpha subset with a significantly shortened half-life, and a constitutively active form of IkappaBalpha efficiently blocks HLA class I induction. Therefore, in addition to STAT-mediated induction, prolonged exposure to IFN-alpha can affect transcription involving Rel factors, which are implicated in the regulation of numerous immune response and viral genes.

Identification of early neuronal subpopulations in avian neural crest cell cultures.

Gangliosides were employed as early differentiation markers to investigate how phenotypic diversity is generated in the vertebrate neutral crest cell population. Chromatographic analysis of metabolically labeled glycolipids from neural crest derivatives revealed that glia cell precursors synthesize a characteristic subset of the ganglioside types produced by dorsal root ganglion neurons. The ganglioside synthesis pattern of neural crest cultures is similar to that of glial precursors, but gangliosides characteristic of neurons are also detectable. To determine whether neuronal gangliosides are expressed by every cell in neural crest cultures, or by discrete cell subpopulations, crest cultures were stained immunocytochemically with monoclonal antibody A2B5 which recognizes a neuron-specific ganglioside in the GQ fraction. A2B5 was found to bind to about 1% of migratory stage neural crest cells isolated from neural tube explants after 1 day in culture. These A2B5+ cells were postmitotic and exhibited a uni- or bipolar neuronal morphology. A second, larger (10-20%) population of A2B5+ cells appears after culturing cells from 1-day crest cell clusters an additional 1-2 days. These cells initially have the typical small, stellate morphology of crest cells but later extend one or more processes. [3H]Thymidine incorporation and cell counting studies show that the precursors to these cells had divided at least once in culture before becoming postmitotic and expressing A2B5 immunoreactivity. The second A2B5+ population does not appear in secondary cultures of crest cell clusters isolated from 2-day-old explants of neural tubes. Another monoclonal antibody, R24, which recognizes ganglioside GD3, binds to subpopulations of both neurons and nonneurons in sensory ganglion cultures. In neural crest cultures R24 binds to a large subpopulation of cells, but not to A2B5+ ones. The significance of this immunostaining pattern is not yet understood. The early appearance of subpopulations, and the presence of heterogeneity in neural crest cultured under a variety of conditions suggest that intrinsic cellular mechanisms might generate subpopulations within the neural crest upon which environmental factors act.

Positive cooperativity in a (dissected) lectin-membrane glycoprotein binding event.

A transmembrane glycoprotein, glycophorin, was assembled into large liposomes that may be handled like intact cells. Under appropriate conditions, lectin binding to these simple model cells accurately mimics the positive cooperative behavior commonly reported for binding of various lectins to real cells. Hence we suggest that the simple observation of cooperativity in such a cell-surface recognition event does not necessarily imply an involvement of complex cellular machinery; rather, it may simply reflect interaction of a multivalent ligand with conformationally deformable headgroups.

Transcriptional regulation of HLA-A and -B: differential binding of members of the Rel and IRF families of transcription factors.

HLA-A and -B transplantation antigens can be expressed differentially at the basal level and in response to interferons (IFNs). To determine which DNA control elements and nuclear factors are responsible for these differences, HLA-A and -B upstream regulatory regions were used in expression and mobility-shift analyses. The HLA-A enhancer was found to contain two Rel (KBF/NF-kappa B) binding motifs, while the HLA-B enhancer has only one and is transactivated less well by overexpression of the NF-kappa B p65 subunit. On the other hand, the HLA-B IFN response element mediates a much stronger induction by IFNs and has a higher affinity for IRF-1 and -2, which are transcription factors implicated in the regulation of major histocompatibility complex class I genes. These results suggest a molecular basis for the way in which HLA-A and -B loci have adapted to be differentially expressed and to respond to different sets of cytokine signals.

Interaction of succinate--ubiquinone reductase (complex II) with (arylazido)phospholipids.

The interaction of purified succinate dehydrogenase and succinate--ubiquinone reductase (complex II) with lipids was explored by using two (arylazido)phospholipids, one with the reactive nitrene in the head-group region of the bilayer [1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (PLII)] and one with the nitrene on the methyl terminus of one of the fatty acid chains [1-myristoyl-2-[12-[(2-azido-4-nitrophenyl)amino]lauroyl]-sn-glycero-3-[14C]phosphocholine (PLI)]. Protein was reacted with vesicles of egg lecithin containing radioactive (arylazido)-phospholipids and the covalent cross-linking of lipid and protein induced by irradiation under UV light. Purified succinate dehydrogenase was found to bind to lipid vesicles through both subunits as both were labeled by PLII. The smaller subunit was inserted into the interior of the bilayer and labeled by PLI. Complex II was found to interact with lipid vesicles, with the smaller subunit of succinate dehydrogenase, CII-3, and CII-4 all inserted into the interior of the bilayer. The large subunit of succinate dehydrogenase was found to be held above the bilayer in complex II and not labeled by either probe. Results are used to derive a picture of the arrangement of subunits in complex II and to evaluate the utility of (arylazido)-phospholipids in membrane studies.