Inhibition Of Mixed Lymphocyte Reaction Research Articles

Thrombin receptor activating peptide-6 decreases acute graft-versus-host disease through activating GPR15.

G-protein coupled receptor 15 (GPR15) is expressed on T-cells. We previously reported knockout of GPR15 increased acute graft-versus-host disease (GvHD) in mice. In this study, we identified thrombin receptor activating peptide-6 (TRAP-6, peptide sequence: SFLLRN) as an activator of GPR15. GRP15 and β-arrestin2 were needed for TRAP-6-mediated inhibition of mixed lymphocyte reactions. TRAP-6 decreased acute GvHD in allotransplant models in mice, an effect dependent on GPR15-expression in donor T-cells. RNA-seq and protein analyses indicated TRAP-6 increased binding of β-arrestin2/TAB1 and inhibited phosphorylation of TAK1 and NF-κB-P65. GPR15 is expressed differently on CD4+ T-cells and CD8+ T-cells. TRAP-6 inhibited phosphorylation of NF-κB-P65 in CD4+ T-cells but increased granzyme B expression in CD8+ T-cells. TRAP-6 decreased acute GvHD without inhibiting graft-versus-tumor (GvT) efficacy against A20 lymphoma cells. SALLRN, a mutant of TRAP-6, preserved the anti-acute GvHD effect but avoided the adverse effects of TRAP-6. TRAP-6 and SALLRN also decreased allogeneic and xenogeneic reactions induced by human blood mononuclear cells. In conclusion, TRAP-6 activated GPR15 on T-cells and decreased acute GvHD in mice without impairing GvT efficacy.

The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum

BackgroundBone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture.MethodsA comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.ResultsPrimary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.ConclusionsTraditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells.

Kindlin-2 is a multidomain intracellular protein that can be recruited to β-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies.

High Concentration of Immunoglobulin Allows Long-Lasting Suppression of Allo-Immune Reaction By Directly Inhibiting T-Cell Activation

Intravenous immunoglobulin (IVIg) has been used after allogeneic stem cell transplants (HSCT) mainly to prevent infections. In addition, some studies have also demonstrated that IVIg reduced GVHD and GVHD-related mortality, but others failed to show these effects. To answer whether IVIg could reduce GVHD and/or has preventive effects, as a first step, we compared the mixed lymphocyte reactions (MLRs) between a variety of responder and stimulator combinations (Balb/c and C57BL/6) with/without immunoglobulin (Ig). We showed that the MLR was strongly inhibited when high concentration of Ig was added to the cocluture (Stimulator (S): bone marrow-derived dendritic cells (BM-DCs) obtained from C57BL/6 using GM-CSF, Responder (R): splenocytes obtained from Balb/c). Interestingly, dose dependent inhibition was not observed only when high concentration of Ig could suppresses allo-reaction. The next concern was whether Ig could suppresse MLR via modulation of DCs or T-cell directly, or both. To test this, splenocytes were stimulated with phytohemagglutinin (PHA) in the presence of various Ig concentrations. We observed that Ig suppressed PHA-induced T-cell proliferation in a dose dependent manner. To eliminate the possibility that Ig might have direct cytotoxicity against lymphocytes and consequently allo-immune reaction seemed to be inhibited, we performed dead cell analysis by flowcytometry using 7-AAD and annexin V on T-cells in the various concentrations of Ig. Even in high concentration of Ig, the frequency of apoptotic or dead cells were observed as same levels as in low concentration or absence of Ig, suggesting that Ig inhibited T-lymphocyte activation directly. Next concern was when Ig allowed tolerogenic function to T-cells and/or DCs. That is, only pretreatment of Ig before MLR was enough to suppress allo-reaction or persistent high concentration of Ig exposure was needed during MLR. To test this, splenocytes and BM-DCs were cultured with Ig for 24 hours before MLRs, and then washed twice and we compared the MLRs. Ig pretreatment to either splenocytes or BM-DCs or both did not show any suppression of MLRs as shown in the figure below, indicating that persistent high levels of Ig during interaction were needed to induce maximum inhibiting effects. In conclusion, we have clearly demonstrated that Ig has a strong capacity to suppress allo-immune reaction. Repetitive high dose IVIg treatment would be a promising approach to reduce or prevent sever acute GVHD, only if serum Ig concentration could be kept in high level. In addition, to apply this in clinical settings, disadvantage of high dose IVIg should be further investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expansion of Functionally Active Alloantigen-Specific Regulatory T Cells Using Activated B Cells.

Background: Achieving transplant tolerance to eliminate chronic immunosuppression is the holy grail of transplantation. Polyclonally expanded regulatory T cells (Tregs) prolong graft survival in animal models without immunosuppression, but antigen-specific Tregs may have more potency and selectivity. B cells can be activated to serve as antigen presenting cells to stimulate Tregs in culture, and multimeric CD40L (Ultra-CD40L) may improve B cell expansion compared to monomeric CD40L. The purpose of the present study was to expand and activate B cells with Ultra-CD40L and use these to expand antigen-specific Tregs and test their functional capabilities. Methods & Results: Peripheral blood mononuclear cells (PBMC) were isolated from healthy laboratory volunteers and cultured under B cell-promoting conditions with Ultra-CD40L, IL-4, and cyclosporine A for 21 days (n=5). The B cells expanded 100-1,000 fold by day 14-21 and displayed increased expression of CD80, CD86, and HLA-DR, the hallmark of mature antigen presenting cells. Next, CD4+CD127-CD25+ Tregs were isolated from PBMCs of a different volunteer and stimulated with irradiated allogeneic B cells under Treg-promoting conditions with exogenous IL-2 and sirolimus (SRL) for 28 days. These cultured Tregs expanded 13-fold when SRL was removed after 21 days (n=5) and up to 28-fold when SRL was removed after 14 days (n=2). Cultured Tregs maintained the CD4+CD127-CD25+Foxp3+ phenotype and demonstrated dose-dependent inhibition of mixed lymphocyte reactions (MLR) of autologous responders in an antigen-specific manner (p<0.01). These Tregs also displayed antigen-specific infectious tolerance in MLR by generating and amplifying new Tregs in CFSE-labeled autologous responder cells stimulated with the specific allostimulator used in expanding the Tregs (n=4). Conclusions: B cells display robust expansion using Ultra-CD40L and can stimulate production of alloantigen-specific Tregs ex vivo. These Tregs retain antigen specificity and functionally behave like Tregs in vitro by inhibiting MLR proliferation and infectiously generating new Tregs. Upon further development such Tregs might be effectively utilized clinically to achieve transplant tolerance. DISCLOSURE:Kornbluth, R.: Other, Multimeric Biotherapeutics, President and Chief Scientific Officer.

Both Host-Type and Donor-Type Mast Cells Protect Against Acute Graft-Versus-Host Disease

Originally mast cells have been known as effector cells in the IgE-mediated allergic responses. In addition, recent studies demonstrated that mast cells exerted pro-inflammatory or anti-inflammatory effects in complicated immune response depending on the situation. In allogeneic hematopoietic stem cell transplantation (HSCT), exact role of mast cells in graft-versus-host disease (GVHD) remains unclear. First we investigated whether host mast cells protect acute GVHD or not in a murine HSCT model using mast cell deficient mice. When lethally irradiated wild type (WT) B6 or mast cell deficient mice were transplanted with bone marrow cells and spleen cells from WT Balb/c mice, mast cell deficient B6 host mice showed significantly lower survival rate than WT B6 mice (p<0.01) due to acute GVHD. Moreover when irradiated mast cell deficient host mice (B6 derived) were given BM cells and splenocytes from mast cell deficient Balb/c mice, these mice died much faster compared to the mice receiving WT BM plus splenocytes (p<0.01) as shown below. Next, we demonstrated that bone marrow derived cultured mast cells (BMCMCs) inhibited mixed lymphocyte reaction (MLR) in a dose-dependent manner, when added to the coculture (Stimulator: dendritic cells (DCs) from B6, Responder: spleen cells of Balb/c, BMCMCs from B6). In addition when mast cells generated from the same strain of responder cells (Stimulator: DCs from B6, Responder: spleen cells from Balb/c, BMCMCs from Balb/c) were added to the MLR mixture, MLR was also suppressed even in this condition. Taken together, we have clearly demonstrated that both host-type and donor-type mast cells suppressed alloreaction in vivo and in vitro. However when we used anti-CD3 and anti-CD28 monoclonal antibodies to stimulate T-cells instead of DCs, BMCMCs could not suppress the lymphoproliferation any more. It suggested that mast cells could suppress MLR via the regulation of DCs directly, not T-cells. Next we have shown that BMCMCs dominantly regulated the alloreaction in a cell contact-dependent manner by using transwell system, though mast cells contain many kinds of substances to regulate immune reactions such as IL-10. DCs highly expressed costimulatory molecules such as CD80, CD86, CD40, I-A, however expression level of these molecules was not changed during coculture of BMCMCs and DCs. Finally, we compared the MLR suppression rate between naïve and activated BMCMCs using IgE and specific antigens, however no difference was observed between them. In conclusion, both host and donor-type mast cells have a protective role against acute GVHD.▪ Disclosures:No relevant conflicts of interest to declare.

Genetically engineered fixed K562 cells: potent “off-the-shelf” antigen-presenting cells for generating virus-specific T cells

Background aimsThe human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. MethodsIn order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. ResultsIn preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8+ T cells in HLA-A∗0201+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. ConclusionsFixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Serum-free culture of umbilical cord mesenchymal stem cells

This study was purposed to observe the culture of umbilical cord mesenchymal stem cells (UC-MSC) with serum-free medium, and compared it with the medium containing 10% fetal bovine serum (FBS). The normal umbilical cords were acquired during cesarean section, and then were cultured with MesenCult-XF serum-free medium or medium containing 10% fetal bovine serum (FBS). The morphology, immunophenotype, cell cycle, proliferation and differentiation potential of mesenchymal stem cells and the inhibition of mixed lymphocyte reaction were observed through different medium culture method. The results showed that the MSC cultured with serum-free MesenCult(-)XF medium could transfer and multiply for average of 6.57 ± 0.7 times, and the serum medium-cultured MSC could transfer and multiply for average of 4.59 ± 0.45 times (P < 0.05). Two kinds of medium cultured MSC all expressed CD44, CD90, CD73, CD105 antigen, but did not expressed CD31, CD45, HLA-DR and CD34 antigen, and their expression levels were not significantly different. The serum-free medium-cultured MSC (65 ± 5.2%) were all at Go/G1 phase, and the serum-contained medium-cultured MSC (62+3.1%) were at Go/G1 phase(P > 0.05); the 2 kinds of media-cultured MSC all could differentiate into fat and ossification; when serum-free medium cultured umbilical cord MSC were inoculated at the the density of 10(3), 5×10(3), 10(4), and 2×10(4) cells/well, then co-cultured with the reactant and stimulating cells, the CPM were (6.43 ± 0.47)×10(4), (4.30 ± 0.38)×10(4), (1.97 ± 0.13)×10(4) and (0.24 ± 0.03)×10(4), respectively, and the serum-containing medium-cultured MSC were incubated with different density of mixed lymphocyte, displaying CPM that were (7.85 ± 0.07)×10(4), (5.64 ± 0.12)×10(4), (3.09 ± 0.18)×10(4) and (1.73 ± 0.05)×10(4). It is concluded that the serum-free medium has been confirmed to culture MSC, which have potential of transfer and differentiation with count for clinical application, and can avoid foreign protein sensitization.

IVIg-induced anti-inflammatory monocytes (CD14 low, HLA-DR high) with a low CD80 and high PDL1 expression inhibits T cell activation in allogeneic mixed lymphocytes reactions (P2194)

Abstract Intravenous immunoglobulin (IVIg) is a therapeutic preparation of human IgG isolated from plasma donations and has been proposed as a therapy to improve the rate of graft survival in patients with a high risk for antibody-mediated-rejection. Blocking of anti-HLA antibodies by anti-idiotypic IgG present in IVIg was proposed to explain the rapid effect of IVIg. However, a long-term reduction of anti-HLA alloreactivity in IVIg-treated patients suggests that IVIg modulates the functions of immune cells. In the present study, we showed using the allogeneic mixed lymphocyte reaction (MLR) as an in vitro model of allograft rejection and GvHD that IVIg strongly inhibits IL-2 secretion (T cell activation) and modulates the level of other pro- and anti-inflammatory cytokines secretion. To determine the mechanisms underlying the inhibition of T cell activation in MLR, we studied the effect of IVIg on the phenotype of the cells involved in MLR. Our results revealed that MLR inhibition by IVIg correlates with the induction of anti-inflammatory monocytes (CD14 low, HLA-DR high) with a low CD80 and high PDL1 expression. To evaluate the importance of PDL1 on the MLR inhibition, anti-PDL1 was added during the MLR. Blocking of PDL1 restored the MLR, as evaluated by IL-2 secretion. We thus propose that PDL1 plays a central role in the inhibition of MLR. Our results help to better understand how IVIg induces long-term peripheral tolerance and improves graft survival in transplanted patients.

IVIg suppresses allogeneic mixed lymphocyte reactions by induction of anti-inflammatory monocytes (CD14LOW, HLA-DRHIGH) with CD80LOW and PDL1HIGH expression.

Event Abstract Back to Event IVIg suppresses allogeneic mixed lymphocyte reactions by induction of anti-inflammatory monocytes (CD14LOW, HLA-DRHIGH) with CD80LOW and PDL1HIGH expression. Lauriane PADET1, 2* and Renée BAZIN1, 2 1 Héma-Québec, Research and Development, Canada 2 Laval University, Department of Biochemistry, Microbiology and Bioinformatics, Canada Intravenous immunoglobulin (IVIg) is a therapeutic preparation of human IgG isolated from thousands of plasma donations and has been proposed as a therapy to improve the rate of graft survival in patients with a high risk for antibody-mediated-rejection. Blocking of anti-HLA antibodies by anti-idiotypic IgG present in IVIg was proposed to explain the rapid anti-inflammatory effect of IVIg. However, peripheral tolerance beyond the half-life of IVIg was observed in IVIg-treated patients, suggesting that IVIg modulates the functions of immune cells. In the present study we showed, using the allogeneic mixed lymphocyte reaction (MLR) as an in vitro model of allograft rejection and GvHD, that IVIg strongly inhibits IL-2 secretion (T cell activation) and modulates the level of other pro- and anti-inflammatory cytokines secretion (IL-6, INF-γ, TNF-α, IL-1RA). To determine the mechanisms underlying the inhibition of T cell activation in MLR, we studied the effect of IVIg on the phenotype of the cells involved in MLR (mainly T cells and monocytes). Our results revealed that MLR inhibition by IVIg correlates with the induction of anti-inflammatory monocytes (CD14LOW, HLA-DRHIGH) with a low CD80 and high PDL1 expression. To evaluate the importance of PDL1 on the MLR inhibition, anti-PDL1 was added during the MLR. Blocking of PDL1 restored the MLR, as evaluated by IL-2 secretion by activated T cells. We thus propose that PDL1 plays a central role in the inhibition of MLR. Our results help to better understand how IVIg induces long-term peripheral tolerance and improves graft survival in transplanted patients. Keywords: Mixed Lymphocyte Reaction, IVIgG, anti-inflammatory monocytes, PDL1, CD80 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: PADET L and BAZIN R (2013). IVIg suppresses allogeneic mixed lymphocyte reactions by induction of anti-inflammatory monocytes (CD14LOW, HLA-DRHIGH) with CD80LOW and PDL1HIGH expression.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00315 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Ms. Lauriane PADET, Héma-Québec, Research and Development, Québec, Canada, lauriane.padet@hema-quebec.qc.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lauriane PADET Renée BAZIN Google Lauriane PADET Renée BAZIN Google Scholar Lauriane PADET Renée BAZIN PubMed Lauriane PADET Renée BAZIN Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation, potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here, we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF, containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically, UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally, XF and SF-cultured UCMSC have comparable adipogenic, osteogenic, and endothelial differentiation potential. Interestingly, the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression, transwell experiments revealed that in neither UCMSC culture a direct cell–cell contact is necessary for inhibiting T cell proliferation, and that the major effector molecule is prostaglandin E 2 (PGE 2). Taken together, GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine.

Galectin-9 Ameriorates Acute GVHD by Inducing T-Cell Apoptosis and Increasing Regulatory T Cell.

Abstract 1338Poster Board I-360Galectins are a family of soluble animal lectins that differ in their affinity for b-galactosides. Fifteen members of the human galectin family have been described to date. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently found to serve as a ligand for T cell immunoglobulin and mucin domain-3 (Tim-3), which is a Th1-specific type 1 membrane protein. Gal-9 modulates immune reactions, such as induction of apoptosis in Th1 cells. We herein investigated the effects of Gal-9 treatment on acute graft-versus-host disease (aGVHD) in a murine model. First, we assessed whether recombinant human (rh) Gal-9 can inhibit the mixed lymphocyte reaction (MLR) by culturing irradiated (30 Gy) splenic cells from 7- to 8-week-old female BDF1 mice with splenic cells from 7- to 8-week-old female C57BL/6J mice in the presence of various concentrations of Gal-9 for 10 days. rhGal-9 inhibited MLR in a dose-dependent manner. We then studied whether this effect was mediated by rhGal-9-induced apoptosis by culturing splenic cells from BDF1 mice with plate-bound anti-CD3 monoclonal antibody and Gal-9. Flow cytometric analysis revealed that rhGal-9 increased the number of Annexin-V+ cells in a dose-dependent manner (Figure. 1A). Thus, rhGal-9 inhibited MLR by inducing splenic cell apoptosis. This suppressive effect of Gal-9 on MLR was inhibited by lactose but not by sucrose (Figure. 1B). Taken together, Gal-9 induces T cell apoptosis through Ca2+ influx induced by binding to b-galactoside, resulting in the suppression of MLR. We then assessed whether rhGal-9 treatment altered aGVHD. Recipient B6D2F1 mice received a myeloablative dose (9 Gy) of total body irradiation from an X-ray irradiator. Six to eight hours later, each recipient mouse was injected i.v. with 4 × 106 TCD-BM cells and 5 × 106 mononuclear splenocytes. aGVHD clinical scores were evaluated once or twice a week. After aGVHD developed, recipient mice were treated with rhGal-9 (30 mg/mouse) or vehicle by i.p. injection for 14 consecutive days. The administration of rhGal-9 significantly attenuated aGVHD as compared to vehicle-treated mice (Figure. 2). Histological analyses also confirmed that aGVHD was declined by rhGal-9 treatment. Additionally, we investigated the effects of Gal-9 treatment on different T ell subsets. To analyze the effect of Gal-9 on donor lymphocytes, splenic mononuclear cells from GFP Tg mice were used for the induction of aGVHD. The gating parameter was first set to isolate the lymphocyte population of peripheral blood leukocytes, and then set for GFP+ cells. Gal-9 treatment decreased the frequency of CD4+/Tim-3+ cells and CD8+/intracellular IFN-g+ cells, whereas CD4+/CD25+ and CD25+/Foxp3+ Treg cells were increased by rhGal-9 treatment. These results suggest that Gal-9 regulates aGVHD by increasing regulatory T cells. In conclusion, Gal-9 ameliorates aGVHD by inducing T-cell apoptosis and also by increasing regulatory T cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tolerance Induction by Immature Dendritic Cells Is Mediated by Distinct MHC Dependent and Independent Mechanisms: A Novel Role for Perforin, Granzyme A and Toll Like Receptor 7.

Abstract 65The mechanism underlining tolerance induction by immature myeloid dendritic cells (IMDC) is poorly understood. To address this issue, we generated mouse IMDC from hematopoietic stem cells (HSC) via differentiation to early myeloid cells (10 days with a cytokine cocktail containing: TPO, Flt-3L, SCF, IL-3 and IL-6) and further differentiation to IMDC (10% GM-CSF for an additional 10 days). The phenotype of the cells obtained at the end of this procedure was CD11c+, CD11b+, B220−, CD80+, MHC class II + Low and CD86−.Addition of the IMDC to long term ( 3 days) mixed lymphocyte reaction (MLR) in which TCR transgenic (Tg) CD4+ or CD8+ alloreactive T cells are reacted against their cognate MHC antigens, led to a marked inhibition of proliferation (reaction ratio 1 IMDC: 2 T cells). This suppression was found to be mediated In short term MLR ( 5 hours) through killing of the effector cells.Killing of CD4+ alloreactive T cells was found to be MHC independent; namely, IMDC expressing MHC not recognized by the TCR transgene exhibited similar inhibition as that exhibited by IMDC expressing the cognate MHC (55±3% versus 59±4% killing upon addition of cognate MHC or non-cognate MHC IMDC, respectively).As previously shown, this non-specific killing is likely mediated by the NO system, as it could be completely abrogated by pre-treatment with N (G)-nitro-L- arginine methyl ester (L-NAME ) (5±4% and 4±3% killing upon addition of pre- treated IMDC bearing cognate MHC or non-cognate MHC, respectively).In contrast, killing of CD8+ alloreactive T cells by IMDC was only slightly reversed by L-NAME (74±3% killing with non-treated IMDC bearing cognate MHC, compared to 68±4% after pre treatment with L-NAME), and the remaining killing activity was found to be MHC dependent. Thus, in contrast to cognate IMDC, very low killing of alloreactive TCR transgenic CD8 T cells was exhibited by non-cognate IMDC in the presence of L-NAME (5±2% killing). When tested in a mouse model of T cell mediated bone marrow (BM) allograft rejection, only IMDC of donor, but not of third party origin, could prolong graft survival upon infusion of 3×106 IMDC (median survival of 24 versus 10 days respectively, p<0.05) suggesting that only the MHC dependent tolerance mechanism is operative in this graft rejection model.Surprisingly, the MHC dependent killing exhibited by IMDC in short term MLR was completely lost when using IMDC generated from perforin KO mice or when IMDC were pre-treated with Bapta-AM or Concanamycin A (6±4%, 5±3% and 3±3% killing, respectively). Thus, killing of the responding alloreactive CD8+ T cells by their cognate IMDC involves a perforin/granzyme mediated mechanism. Indeed, Electron Microscopy (EM) analysis revealed that IMDC were loaded with granules containing perforin and Granzyme A, mostly localized in proximity to the cell membrane. While these granules could barely be detected in early myeloid cells, formation of granules was markedly enhanced upon differentiating into IMDC.Further EM analysis revealed that recognition of the IMDC by alloreactive CD8+ T cells induced granules polarization towards the area of contact. Live video imaging (using perforin and granzyme A immunostaining) further supported the suggestion that this granule polarization leads to specific killing of the responding CD8+ T cell. Furthermore, pre-treatment of IMDC with a blocking peptide for Toll like receptor 7 (TLR7) but not for TLR1, TLR5, TLR6, TLR9, TLR10, or TLR11, completely abolished this granule degranulation, and the eventual killing of alloreactive CD8+ T cells (blocking TLR7 reduced the MLR inhibition from 68±4% to 8±3%, p<0.01). Thus, MHC dependent killing of alloreactive CD8+ T cells by IMDC is likely triggered by TCR recognition of the cognate MHC on the IMDC which, in turn, activate Toll receptor signaling leading to degranulation and granule mediated killing.In conclusion, IMDC can induce immune tolerance through two distinct mechanisms. The first, mediated by the NO system, is MHC independent and is limited to ablation of CD4+ T cell responses. The second, mainly targeting CD8+ T cells, is MHC dependent and is mediated by cytotoxic granules in the IMDC which polarize towards the contact area upon recognition by the T cells. This MHC- specific mechanism is clearly dependent on perforin and is specifically triggered by TLR7 ligation. The latter mechanism was predominant when testing IMDC in a mouse model for T cell mediated BM allograft rejection. Disclosures:No relevant conflicts of interest to declare.

Immunogenicity of Allogeneic Adipose-Derived Stem Cells in a Rat Spinal Fusion Model

Adipose-derived stem cells (ASCs) express a nonimmunogenic profile as shown by in vitro studies that demonstrate a lack of T cell proliferation to allogeneic ASCs as well as ASC-mediated suppression of mixed lymphocyte reactions. To determine whether these observations would translate in vivo, immune monitoring studies were carried out in conjunction with a rat spinal fusion study. ASCs derived from Fischer or ACI strain rats were loaded onto scaffolds and implanted in Fischer recipients that had undergone the following treatments: (1) No treatment; (2) Scaffold only; (3) Syngeneic ASCs+Scaffold; or (4) Allogeneic ASCs+Scaffold. Half of each group was sacrificed at 4 weeks postimplantation, and the remaining animals were sacrificed at 8 weeks. As determined in a separate study, allogeneic and syngeneic ASCs were equally efficacious in accelerating spinal fusion compared to No treatment and Scaffold only control groups. To determine whether donor ASCs induced an immune response in recipient rats, lymph nodes were harvested for T cell proliferation studies and serum was collected to assess antibody responses. Although T cell priming was not detected to donor alloantigens in recipients at either time point, significant antibody responses were detected to ACI ASCs in animals implanted with syngeneic or allogeneic ASCs. Antibodies were of the IgG isotype, noncytotoxic in the presence of complement, and reactive to fetal bovine serum. These results support the use of allogeneic ASCs for spinal fusion.

Sequential mechanisms of cyclophosphamide-induced skin allograft tolerance including the intrathymic clonal deletion followed by late breakdown of the clonal deletion

The cellular basis of the transplantation tolerance in a model system of BALB/c (Mls-1b) mice rendered cyclophosphamide (CP)-induced tolerant to DBA/2 (Mls-1a) skin allograft was investigated by assessing V beta 6+ T cells. From our results, three major mechanisms that are essential to the CP-induced skin allograft tolerance were sequentially elucidated. The first mechanism was destruction of donor-Ag-stimulated T cells in the periphery by CP treatment. The second mechanism was intrathymic clonal deletion of donor-reactive T cells, such as V beta 6+ T cells, correlating strongly with intrathymic mixed chimerism. The clonal deletion, however, was not always essential for the maintenance of the skin allografts, because DBA/2 skin survived even after the clonal deletion terminated and V beta 6+ T cells reappeared in the periphery of the recipient BALB/c mice. The third mechanism was generation of tolerogen-specific suppressor T cells, especially in the late stage of the tolerance. In contrast, the clonal anergy that is evidenced by the specific suppression of mixed lymphocyte reaction in the recipient BALB/c mice after injecting with DBA/2 spleen cells alone was not considered as a significant mechanism in prolonging skin allograft survival because such anergic mice showed accelerated rejection of the skin allografts. These results may suggest practical hierarchy of the mechanisms of CP-induced allograft tolerance.

Immunological tolerance induced by liver grafting in the rat: splenic macrophages and T cells mediate distinct phases of immunosuppressive activity

In the rat combination DA into PVG, liver grafts are not rejected but induce donor-specific transplantation tolerance. We have examined the immunosuppressive properties of spleen cells from PVG recipients of DA liver grafts at various times post-grafting. The results indicate the development of two phases of cell-mediated suppressor activity, which appear to be mediated by separate spleen cell populations. Mitomycin-C-treated spleen cells taken from animals between 5 and 28 days post-grafting were able to suppress rat mixed lymphocyte reactions (MLRs). These 'early' suppressor cells were glass adherent and absent from populations purified by passage through nylon wool or G10 Sephadex columns. Suppression of MLR by purified glass adherent cells was not specific for either stimulator or responder haplotypes and was blocked by indomethacin. Nylon wool purified T cells were not suppressive at this time. Spleen cell suppressor activity declined to background levels after 35 days post-grafting. However, spleen cells from long-term surviving liver graft recipients (20 weeks or more) were again able to suppress MLR; the 'late' suppressor cells were nylon wool non-adherent and suppression was specific for the donor (DA) MHC type. We conclude that liver grafting in this combination generates early and late phases of suppression among spleen cells, that the early phase is produced by macrophages and mediated by prostaglandins and that the late phase is dependent on allospecific suppressor T cells.

IFN-λ contributes to dendritic cell-mediated expansion of regulatory T cells during HCV infection. (44.38)

Abstract IFN-λ inhibits HCV replication and triggers IFN-dependent genes. We hypothesized that IFN-λ is immunomodulatory and evaluated its effects on myeloid dendritic cells (DC). DCs generated with IFN-λ (IFN-λ-DC) expressed normal levels of DC markers, but showed reduced allostimulatory capacity in mixed lymphocyte reaction (MLR), similar to DCs of patients with chronic HCV infection (HCV-DC). No additive effects of IFN-λ subtypes were observed. The inhibition of MLR was due to preferential expansion of CD4+CD25+ T cells in the presence of HCV-DCs or IFN-λ-DC, but not normal DCs. Purified CD4+CD25+ T cells from MLR were Foxp3+, CD45RA+, produced IL-10 and TGFb, and inhibited CD4+CD25− T cell proliferation. HCV-DCs produced increased levels of IFN-λ compared to controls. In vitro generation of DCs in the presence of LPS increased expression of IFN-λ. Interestingly, we found increased plasma levels of LPS in HCV patients compared to controls suggesting a possible in vivo mechanism for IFN-λ induction. Further, in vitro DCs generation with LPS resulted in expansion of Tregs and inhibition of MLR, thus mimicking the effects of HCV-DCs and IFN-λ-DCs. We also identified an increased frequency of Tregs in peripheral blood of HCV patients (12±2%) compared to controls (5±1%) (p&amp;lt;0.05). In conclusion, increased frequency of Tregs in HCV infection is associated with increased IFN-λ production by DCs that promote Treg generation.

Control of Allograft Rejection by Applying a Novel Nuclear Factor-κB Inhibitor, Dehydroxymethylepoxyquinomicin

Nuclear factor (NF)-kappaB plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-kappaB and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. DHMEQ significantly suppressed alphaCD3 + alphaCD28 monoclonal antibody-triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-gamma production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-kappaB but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-gamma producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. DHMEQ inhibits nuclear translocation of NF-kappaB but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-kappaB and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.

Combination Effect of Mycophenolate Mofetil with Mizoribine on Cell Proliferation Assays and in a Mouse Heart Transplantation Model

Mycophenolate mofetil (MMF) is a highly potent immunosuppressant that suppresses the proliferation of T and B cells by the uncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH). Consequently, under MMF immunosuppression, good graft survival has been achieved in clinical organ transplantation, although MMF shows adverse gastrointestinal effects. Mizoribine (MZ) also inhibits IMPDH in a competitive manner and is used clinically for organ transplantation in Japan as an immunosuppressant with fewer adverse gastrointestinal effects. Therefore, in this study we investigated the synergistic effects on in vitro and in vivo assays of mice of a combination of MMF with MZ immunosuppression. Mixed lymphocyte reaction (MLR) assay and a mouse heart transplantation model were used to evaluate the immunosuppressive effect of MMF with MZ. The median-effect principle and a combination index (CI) were employed to determine synergism, an additive effect, or antagonism. Combination of MMF with MZ resulted in mild synergistic effects in the inhibition of MLR (CI = 0.854-1.143). In the mouse heart transplantation model, C57BL/6 recipients who received a BALB/c heart under the combination of MMF and MZ at 40 + 40 or 80 + 80 mg/kg/day showed a strong synergistic prolongation of graft survival with 19.7 +/- 18.9 (P = 0.0012, CI = 0.438) and 78.4 +/- 36.9 days (P = 0.0002, CI = 0.317), respectively, compared with recipients treated with MMF or MZ monotherapy. CONCLUSIONS.: The combination of MMF and MZ showed mild synergistic effects in the inhibition of MLR and strong synergistic effects in a mouse heart transplantation model.