Rat Mesenchymal Stromal Cells Inhibit T Cell Proliferation but Not Cytokine Production Through Inducible Nitric Oxide Synthase

Mesenchymal stromal cells (MSC) have important immunomodulatory properties, they inhibit T lymphocyte allo-activation and have been used to treat graft-versus-host disease. How MSC exert their immunosuppressive functions is not completely understood but species specific mechanisms have been implicated. In this study we have investigated the mechanisms for rat MSC mediated inhibition of T lymphocyte proliferation and secretion of inflammatory cytokines in response to allogeneic and mitogenic stimuli in vitro. MSC inhibited the proliferation of T cells in allogeneic mixed lymphocyte reactions and in response to mitogen with similar efficacy. The anti-proliferative effect was mediated by the induced expression of nitric oxide (NO) synthase and production of NO by MSC. This pathway was required and sufficient to fully suppress lymphocyte proliferation and depended on proximity of MSC and target cells. Expression of inducible NO synthase by MSC was induced through synergistic stimulation with tumor necrosis factor α and interferon γ secreted by activated lymphocytes. Conversely, MSC had a pronounced inhibitory effect on the secretion of these cytokines by T cells which did not depend on NO synthase activity or cell contact, but was partially reversed by addition of the cyclooxygenase (COX) inhibitor indomethacin. In conclusion, rat MSC use different mechanisms to inhibit proliferative and inflammatory responses of activated T cells. While proliferation is suppressed by production of NO, cytokine secretion appears to be impaired at least in part by COX-dependent production of prostaglandin E2

Prediction, diagnosis and treatment of experimental graft-versus-host disease : an investigation of genomic, molecular and cellular factors in graft-versus-host disease and mesenchymal stromal cell therapy in a rat model of allogeneic stem cell transplantation

Patients who suffer from leukemia or lymphoma can be cured by the transplantation of bone marrow stem cells from a volunteer donor (allogeneic hematopoietic cell transplantation). Recipients frequently acquire an immune disorder known as graft-versus-host disease (GvHD) which represents the main hindrance of this therapeutic procedure at present. Graft-borne donor T cells are activated by disparate antigens in the host leading to a cascade of adverse immune reactions and damage of host tissues that give rise to clinical GvHD. The objectives of this thesis were twofold: firstly, to develop diagnostic tools and identify reliable biomarkers of GvHD; secondly, to improve GvHD treatment by mesenchymal stromal cell (MSC) therapy. In a collaborative effort, we helped develop the rat skin explant, an ex-vivo assay of GvHD, as well as a DNA microarray for the study of genetic factors that regulate GvHD. Analysis of gene expression in rat skin explants revealed MHC and innate immune receptor genes that were associated with graft-versus-host reactions and are thus of potential use as diagnostic biomarkers of GvHD. In a model of MHC-mismatched bone marrow transplantation that caused acute GvHD in rats, we isolated natural killer cell and T cell subsets including regulatory T cells that were differentially distributed in disease. Using this animal model, we also tested whether MSC injections could prevent GvHD. We concluded that repeated interventions failed to alleviate disease or improve survival in transplanted rats. In contrast, MSC potently suppressed T cell proliferation and cytokine secretion in vitro. Proliferation was inhibited through the enzymatic synthesis of nitric oxide. In summary, this work resulted in the characterization of genetic and cellular markers of GvHD in the skin ex vivo and after transplantation in vivo as well as the isolation of nitric oxide synthesis as a key pathway used by rat MSC to inhibit allogeneic T cell responses in vitro

Mycoplasma Contamination Revisited: Mesenchymal Stromal Cells Harboring Mycoplasma hyorhinis Potently Inhibit Lymphocyte Proliferation In Vitro

Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination

Expression Profiling of Major Histocompatibility and Natural Killer Complex Genes Reveals Candidates for Controlling Risk of Graft versus Host Disease

Background: The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. Methodology/Principal Findings: To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. Conclusions/Significance: We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients

Vurdering av tester for PD-L1 ved urotelialt karsinom: en forenklet metodevurdering

Hovedbudskap: Programmed cell death ligand 1 (PD-L1) på tumorceller kan binde til Programmed cell death protein 1 på immunceller og hindre disse i å bekjempe og nøytralisere kreftsvulster. Det finnes flere legemidler som blokkerer bindingen og er tilgjengelige i behandling av ulike krefttyper. I vurdering av denne formen for immunterapi kan tilstedeværelse av PD-L1 på kreftceller si noe om forventet effekt i forkant av en behandling (prediksjon). Flere tester og testmetoder har blitt utviklet for å måle PD-L1 på vevssnitt fra kreftbiopsier. Bestillerforum for nye metoder har gitt Folkehelseinstituttet i oppdrag å utarbeide en metodevurdering om testegenskaper til kommersielt tilgjengelige PD-L1 tester til bruk ved urotelialt karsinom. Vi har besvart oppdraget ved å utarbeide en forenklet metodevurdering der vi oppsummerer systematiske oversikter som undersøker samsvar mellom ulike PD-L1 tester og testmetoder uavhengig av krefttype. Vi inkluderte to systematiske oversikter som vurderte samsvar mellom ulike PD-L1 tester og testmetoder. • Ingen av de inkluderte systematiske oversiktene var begrenset til PD-L1 testing av urotelialt karsinom. • Studier gjort på PD-L1 testing av ikke-småcellet lungekreft viser stor grad av samsvar mellom testresultater fra 22C3, 28-8 og SP263. SP142 viste noe lavere samsvar sammenlignet med de andre testene. • Det var stor grad av samsvar mellom hvordan PD-L1 uttrykk ble vurdert av patologer og i ulike laboratorier. Denne metodevurderingen oppsummerer grad av samsvar mellom kommersielt tilgjengelig PD-L1 tester. Metodevurderingen gir ingen konklusjoner om effekt av ulike legemidler i behandlingen av urotelialt karsinom hos voksne

Organoids in the clinic: A systematic review of outcomes

Research on organoids has undergone significant advances during the last decade. However, outcomes from the use of organoids in clinical trials have not yet been documented. Therefore, there is an urgent need to assess the reporting of clinically relevant outcomes from organoid research in the scientific literature. This article presents a systematic review and appraisal of the published literature in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, together with a synopsis of recent relevant reviews. Surprisingly, no randomized controlled trials have reported clinical outcomes with any types of organoids. We found very few ongoing and registered studies that may provide clinically relevant results within this decade. Our screening and interpretation of the literature, including review articles, indicate a focus on technical and preclinical aspects of organoid research. This is the first systematic review of clinical trials involving organoids. Few clinical studies are planned or already underway, and, so far, no high-quality evidence relating to the clinical outcomes of organoid research has been published. The many promises of organoid research still need to be translated from bench to bed.</p