The hUCMSCs Exerts Excellent Anti-inflammation Effect which is Different from TCZ IL-6 Antagonist by Inducing High IL-6 Secretion

BACKGROUND The biologic and cellular IL-6-related therapies have been used to treat the autoimmune diseases (AID), which prompting us to furtherly explore the IL-6 role in human umbilical cord mesenchymal stem cells (hUCMSCs) therapy.MATERIALS & METHODS Peripheral blood mononuclear cells (PBMCs) were responder co-cultured with hUCMSCs or exogenous IL-6. PBMC suppression assay was used to analyze the anti-inflammatory effects by using the MTT assay. The IL-6 concentration in supernatant was measured using ELISA. The correlation between anti-inflammation effect of hUCMSCs and IL-6 levels, and the relevant roles of IL-6, IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was furtherly evaluated.RESULTS Initially, hUCMSCs did not exert any inhibitory effect on PBMCs, however, a potent inhibitory effect on PBMCs was observed and the IL-6 concentration reached about 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 could inhibit PBMCs inflammations but less than that of hUCMSCs. The hUCMSCs exerts excellent anti-inflammation effect by inducing higher IL-6 level which is different from TCZ IL-6 antagonistCONCLUSIONS High concentration IL-6 cytokine secretion plays an important role in the anti-inflammation effect of hUCMSCs cell therapy.

Combination of Cord Blood T Regulatory Cells with Ruxolitinib Decreases Side Effects and Improves Survival in Xenogenic Graft Vs. Host Disease

Background: Recent approval of ruxolitinib (rux) for steroid refractory graft versus host disease (GvHD) has revolutionized the field and provided tremendous choice for the patients. However, the side effects including thrombocytopenia leads to dug discontinuation and intolerance. We have previously shown that adoptive therapy with cord blood (CB) derived T regulatory (Treg) cells can prevent and treat GvHD. We hypothesized that the addition of CB Treg therapy to rux based therapy can augment overall efficacy. Methods: CellTrace Violet suppression assay was performed to evaluate the suppressor function of CB Treg cells in the presence or absence of rux. Xenogenic GvHD mouse model was utilized where the NSG mice underwent sublethal irradiation on day -1 followed by injection of 1x107 donor peripheral blood (PB) mononuclear cells (MNCs) on day 0. Oral rux at 1 mg daily was fed continuously to the mice in the presence or absence of 1x107 CB Treg cells, tagged with CellTrace Violet dye, administered on days +4, +7, +11, +18. Mice were followed every other day for weight, GvHD score and survival. Serial blood draws were performed to analyze for cell compartment and cytokine assays. Results: We examine whether the addition of rux impacts the suppressor function of CB Treg cells. Varying concentration of rux including 0.01, 0.05, 0.1 and 0.5 µM were added at 24, 48 and 96 hours of the cell suppression assay. The addition of rux 0.05 µM at 48 hours of the cell suppression culture led to a restoration of poor cell function (Figure A). Lowest GVHD score was reported in the rux+CB Treg combination arms at day +14 when compared to rux alone or CB Treg alone arm which (Figure B) translated into a superior survival in the rux +CB Treg arm (Figure C). Furthermore, an increase in the hemoglobin level (Figure D) and the platelet count (Figure E) was demonstrated in the rux+CB Treg arm. Addition of rux led to longer persistence of the injected CB Treg cells on day 14 (data not shown) which correlated with the increase in the plasma level of the pro-Treg cell signaling markers including IL-7 (Figure F) and IL-15 (Figure G). A decrease in the IL-4 production supported the increased Treg cell function (Figure H). A synergistic suppression of inflammatory cytokines including IL-17 (Figure I) and IL1A (Figure J) was evident in the rux+CB Treg arm. Conclusion: The combination of CB Tregs with ruxolitinib leads to improved overall survival, decreased inflammatory cytokines and improved hematologic parameters. Such combination should be explored in a clinical setting Figure Disclosures Sadeghi: Cellenkos Inc.: Current Employment. Parmar:Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

779 Immune checkpoint blockade impacts the suppressive phenotype and function of regulatory T cells in an endogenous mouse lymphoma model

BackgroundAntibodies against programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have become established part of anti-cancer therapy. However, the mechanisms contributing to the therapeutic success have not been entirely uncovered by now. Here we focus on the impact of PD-1/CTLA-4-blocking antibodies on regulatory T cells (Tregs), which are known to be involved in tumor immune evasion in many cancer types.MethodsTo evaluate how Tregs are affected by anti-PD-1/CTLA-4 therapy, we used a MYC-transgenic mouse model of spontaneously arising B-cell lymphoma, which can be effectively treated by immune checkpoint inhibition. Data were acquired by flow cytometry.ResultsAs earlier shown, Tregs were involved in immune escape of MYC tumors. The Treg to effector T cell (Teff) ratio was elevated within the CD4-positive cell compartment. Tumor-infiltrating Tregs were predominantly thymic Tregs, which recognized overexpressed tumor-derived self-peptides in an MHC class II-restricted manner and showed upregulated expression of activation markers, Foxp3, CD25 and IL-10. To examine whether these phenotypic alterations correlated with the immunosuppressive capability of Tregs, an in vitro suppression assay was established. In this setting, MYC Tregs turned out to suppress proliferation and IFN-γ release of Teff cells more effectively than wildtype Tregs. The suppression observed in vitro was mediated by cell contacts and IL-10. Further suppressive mechanisms are likely to play a role, such as competition for MHC-II ligands and a consumption of IL-2. To investigate if immune checkpoint blockade interferes with these Treg-dependent immunosuppressive pathways, MYC mice were treated with a combination of anti-PD-1 and anti-CTLA-4 antibodies. Tregs from treated MYC mice showed decreased expression of CD69, CD137, Foxp3, CD25 and IL-10 as compared to Tregs from untreated MYC mice. This correlated with a lower suppressive capacity of Tregs from treated animals in the in vitro suppression assay.ConclusionsTaken together, the data show that immune checkpoint blockade impairs the development of the suppressive phenotype of intratumoral Tregs. Thus, apart from the initially described Teff reactivation, other mechanisms are also relevant for unfolding the therapeutic effect of immune checkpoint inhibitors.Ethics ApprovalAll animal experiments were approved by Regierung von Oberbayern, approval number 55.2-1-54.

Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

Two gut microbes are necessary and sufficient for normal cognition in Drosophila melanogaster

It is widely accepted that the gut microbiome can affect various aspects of brain function, including anxiety, depression, learning, and memory. However, we know little about how individual microbial species contribute to communication along the gut-brain axis. Vertebrate microbiomes are comprised of hundreds of species, making it difficult to systematically target individual microbes and their interactions. Here, we use Drosophila melanogaster as a simple model organism to tease apart individual and combined effects of gut microbes on cognition. We used an aversive phototactic suppression assay to show that two dominant gut commensals in our lab stock, Lactobacillus and Acetobacter, are necessary and sufficient for normal learning and short-term memory relative to flies with a conventional microbiome. We also demonstrate that microbes did not affect their hosts’ ability to detect the aversive learning stimulus (quinine), suggesting that our results were due to decreased cognition and not sensory deficits. We thus establish Drosophila as a model for elucidating mechanisms of gut-brain communication at the level of individual bacterial species.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs. We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities. Next, we employed a well-described murine model of allogeneic BMT (BL/6-->Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro. Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector‐R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS‐2, and GPA/RBP‐1) and one mutated R gene—Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.