Proliferation In Mixed Lymphocyte Reaction Research Articles

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD

AbstractDespite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

Drug repurposing of ilepcimide that ameliorates experimental autoimmune encephalomyelitis via restricting inflammatory response and oxidative stress

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS) that remains incurable. Herein, we demonstrated that ilepcimide (Antiepilepsirine), an antiepileptic drug used for decades, protects mice from experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Our studies found that ilepcimide treatment effectively ameliorates demyelination, blood-brain barrier leakage and infiltration of CD4+ and CD8+ T cells in EAE mice. On the one hand, ilepcimide can inhibit dihydroorotate dehydrogenase (DHODH), an important therapeutic target for MS. Computer molecular docking, thermal shift and fluorescence quenching assay demonstrated the directly interaction between ilepcimide and DHODH. Accordingly, ilepcimide observably repressed T cell proliferation in mixed lymphocyte reaction (MLR) assay and concanavalin A (Con-A) model in a DHODH-dependent manner. On the other hand, ilepcimide exhibited neuroprotective effect possibly through activating NRF2 antioxidant pathway in mouse neural crest-derived Neuro2a cells. Collectively, our findings have revealed the therapeutic potential of ilepcimide in EAE mouse model via restricting inflammatory response and oxidative stress, offering a potential opportunity for repurposing existing drug ilepcimide for MS therapy.

Investigating immune tolerance: characterization of immunoregulatory DN T cells

Abstract Autoimmunity results from defects in immune tolerance pathways that impair the capacity of immune cells to distinguish between self and non-self. One of these immune tolerance processes involves immunoregulatory TCRαβ+CD4−CD8− double negative T cells (DN T). Indeed, DN T cells protect from autoimmune pathologies and graft rejection in mice. Similarly, high DN T cell numbers correlate with a low incidence of chronic graft-versus-host disease in humans, an autoimmune-like pathology. While the thymic differentiation process of DN T cells was recently described, the function of these unconventional T cells is still poorly documented. To characterize DN T cells, we performed bulk RNA sequencing on CD4, CD8, γδ and DN T cells isolated from mouse spleen. Comparison of the transcriptome profiles revealed that DN T cells express a unique signature. Moreover, following in vitro stimulation, we find that DN T cells also present a distinct cytokine secretion pattern. Interestingly, we observed a lower induction of CD69, LAG-3 and PD-1 on DN T cells in comparison to CD4 and CD8 T cells, whereas LCK phosphorylation is unabated. DN T cell proliferation in mixed lymphocyte reactions is also lower than that of CD4 and CD8 T cells. Overall, our transcriptomic and cytokine analyses clearly identify DN T cells as a unique T cell subset. Moreover, while DN T cells do not express an exhaustion profile, they are more refractory to in vitro T cell stimulation than conventional T cells. A better characterization of DN T cell function will help conceive novel immunotherapies to treat autoimmune diseases. Supported by the Canadian Institutes of Health Research - PJT 159603. S.P. was supported by a postdoctoral CIHR fellowship

Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells.

A subset of T regulatory cells (Tregs), identified by TIRC7 (T cell immune response cDNA 7) expression is designated as Immune Regulatory 1 Cells (IR1 cells). TIRC7 is an immune checkpoint inhibitor, co-localized with the T- cell receptor, HLA-DR and CTLA-4 during T-cell activation, which delivers regulatory signals via binding to its ligand, HLA-DR α2 domain. IR1 cells express FOXP3, and multiple other markers associated with immune suppression. They constitute as much as 10% of Tregs. IR1 cells strongly inhibit proliferation in mixed lymphocyte reactions, where they express high levels of IL-10. Ex vivo expansion of Tregs over 2 weeks in the presence of an agonist TIRC7 antibody disproportionately expands the IR1 Treg subset, while maintaining high expression of suppressive markers including CD39, IL-10, LAP and GARP. Ex vivo expanded IR1 cells are a potent, homogeneous, stable set of suppressor Tregs with the potential to modulate immune dysregulation. The characteristics of IR1 cells suggest a therapeutic advantage over polyclonal Tregs for therapeutic interventions. Early restoration of immune homeostasis using IR1 cells has the potential to fundamentally alter the natural history of conditions characterized by abnormalities in the T regulatory cell compartment.

Wharton's Jelly Mesenchymal Stromal Cells from Human Umbilical Cord: a Close-up on Immunomodulatory Molecules Featured In Situ and In Vitro.

Therapeutic options for end-stage organ failure are often limited to whole organ transplantation. The tolerance or rejection of the transplanted organ is driven by both early non-specific innate and specific adaptive responses. The use of mesenchymal stromal cells (MSCs) is considered a promising tool in regenerative medicine. Human umbilical cord (HUC) is an easily available source of MSCs, without relevant ethical issues. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs), showed consistent immunomodulatory features that may be useful to promote immune tolerance in the host after transplantation. Few data are available on the phenotype of WJ-MSCs in situ. We investigated the expression of immune-related molecules, such as HLAs, IDO, CD276/B7-H3, and others, both in situ (HUC) and in in vitro-cultured WJ-MSCs. Morphological and biochemical techniques were used to define the expression of such molecules. In addition, we focused on the possible role of CD276/B7-H3 on T cells proliferation inhibition. We assessed CD276/B7-H3 expression by WJ-MSCs both in situ and alongside cell culture. WJ-MSCs were able to suppress T cell proliferation in mixed lymphocyte reaction (MLR). Moreover, we describe for the first time a specific role for CD276/B7-H3, since the immunomodulatory ability of WJ-MSCs was abolished upon anti-CD276/B7-H3 antibody addition to the MLR. These results further detail the immune regulation properties and tolerance induction exerted by human WJ-MSCs, in particular pointing to CD276/B7-H3 as one of the main involved factors. These data further suggest WJ-MSCs as potent tools to modulate local immune response in "support-type" regenerative medicine approaches.

Exopolysaccharide from a commensal bacterium improves outcome of graft versus host disease by decreasing donor T cell activation

Abstract We previously showed that exopolysaccharide (EPS) produced by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease, induced by the enteric pathogen Citrobacter rodentium. Here, we show that EPS induces clinical improvement in another T cell-mediated disease, graft versus host disease (GVHD). GVHD is a common complication of allogenic bone marrow transplantation and is caused by the activation of alloreactive donor T cells. To monitor the inflammatory response of allogeneic bone marrow cells in recipient mice, we used donor cells from transgenic mice that express a luciferase-based biosensor that is cleaved by caspase-1, a protein activated during inflammation. We found that donor inflammatory responses and T cell activation was decreased in EPS-treated mice. Our previous data indicate that EPS does not have a direct effect on T cells. To identify the cell type affected by EPS, we tested the activity of EPS on bone marrow-derived dendritic cells (BMDCs) and found that these cells have upregulated expression of the inhibitory molecule, PD-L1, and the cells inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the upregulation of PD-L1 expression on splenic dendritic cells. We conclude that EPS ameliorates clinical manifestations of GVHD by decreasing the activation of donor T cells, and we suggest that EPS affects T cells by inducing inhibitory dendritic cells.

Harnessing MSC Osteoprogenitor Subpopulations to Augment Hematopoietic Transplantations

Adjuvant MSC infusions could improve transplantation outcomes via suppression of graft-vs-host reactions and enhancement of HSPC engraftment. However, bone marrow (BM) MSCs are a heterogeneous cell population, particularly when culture expanded to obtain therapeutically relevant cell numbers. A systematic study of expanded subpopulations is hindered by the lack of unique surface markers. In lieu of immunophenotype, we hypothesized and verified that biophysical properties of MSCs can be a useful strategy for isolating unique subpopulations. Here, we will describe a clinically relevant biophysical strategy for deriving different MSC subpopulations and discuss their potential utility in the context of hematopoietic transplantation. Cell diameter, membrane stiffness and nuclear membrane fluctuations are collectively predictive of at least two MSC subpopulations (multipotent and osteoprogenitors). Both MSC subpopulations are distinct in their transcriptome (different signatures of VEGF, IL8, ANG1, etc) and differentiation potential (osteogenic and adipogenic) but no surface marker reliably distinguishes them apart. In functional assays, osteoprogenitor vs multipotent MSCs show stronger capacities for CD34+CD38−HSPC expansion (Day 10: 42.7 ± 6.21 vs 29.1 ± 8.27, n = 5, respectively), but no significant differences (n = 3 biological repeats) in their ability to suppress CD8+T-cell proliferation in mixed lymphocyte reactions. When used as a rescue therapy for lethal irradiation, osteoprogenitors improved survival rates by 30-40% compared to multipotent MSCs. Part of the recovery process was prompted by MSC secretome mediated stimulation of BM tissue repair, including increased cell proliferation, reduced inflammation and the restoration of niche environments. These paracrine effects may be beneficial in fostering favorable post-myeloablative BM environments for donor HSPCs to home, engraft and survive. In murine transplantation models, we observed a higher rate of early engraftment following adjuvant therapy with osteoprogenitors. Week 4 CD45 chimerism in peripheral blood was 2- to 3- fold higher (∼11% vs ∼4%) in the osteoprogenitor treated group compared to the other MSC groups. These results demonstrate heterogeneity in expanded MSCs populations that would lead to inconsistencies in their overall properties. Newer biophysical technologies have the potential for isolating useful subpopulations defined by key quality attributes for specific forms of MSC-therapy; these capabilities may advance MSC-based therapy for regenerative medicine. Here, we demonstrate the augmentation of HSPC transplants in murine models using a biophysically isolated osteogenic MSC subpopulation (osteoprogenitors) which led to the fastest engraftment rates; this may be due to their superior abilities for promoting BM tissue regeneration as well as hematopoietic expansion.

Miniaturized High-Throughput Multiparameter Flow Cytometry Assays Measuring In Vitro Human Dendritic Cell Maturation and T-Cell Activation in Mixed Lymphocyte Reactions

Enhancing antitumor activities of the human immune system is a clinically proven approach with the advent of monoclonal antibodies recognizing programmed cell death protein-1 (PD1) receptors on immune cell surfaces. Historically, using flow cytometry as a means to assess next-generation agent activities was underused, largely due to limits on cell number and assay sensitivity. Here, we leveraged an IntelliCyt high-throughput flow cytometry platform to monitor human dendritic cell maturation and lymphocyte proliferation in mixed lymphocyte reactions. Specifically, we established flow cytometry–based immunophenotyping and screening methodologies capable of measuring T-cell activation as a result of cell-associated antigens presented on dendritic cell surfaces, as indicated by cell proliferation, cytokine secretion, and surface marker expression. Together, the overall novelty of this 384-well platform is its capability to measure multiple functional readouts in one well and consistently evaluate large numbers of compounds in a single study, as well as its ability to show increased assay sensitivity requiring considerably fewer primary cells and less reagents compared to more traditional 96-well flow cytometry methods.

Abstract 5629: Preclinical characterization of Sym023 a human anti-TIM3 antibody with a novel mechanism of action

Abstract Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. The next wave of immunomodulatory targets that are being explored for cancer therapy include T cell immunoglobulin and mucin domain protein 3 (TIM3). TIM3 is constitutively expressed on cells of myeloid origin whereas the TIM3 expression is induced on T-cells upon activation. The exact function of TIM3 on the different immune cells is not clear and may be context dependent suggesting that TIM3 is not a classical immune check-point. Sym023 is a human anti-TIM3 antibody, which binds human TIM3 and cross-reacts with cynomolgus monkey TIM3. Sym023 blocks binding of phosphatidyl serine but not galectin 9 and stimulates T-cell proliferation in mixed lymphocyte reactions and tumor growth inhibition in vivo. Here, we present data demonstrating that ligation of TIM3 by Sym023 increase cytokine production and T cell proliferation in vitro through a novel mechanism of action. Citation Format: Trine Lindsted, Monika Gad, Michael V. Grandal, Camilla Frölich, Vikram K. Bhatia, Torben Gjetting, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Koefoed, Mikkel W. Pedersen. Preclinical characterization of Sym023 a human anti-TIM3 antibody with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5629.

Reduction of autoantibody in SLE by probiotic exopolysaccharide-induced inhibitory dendritic cells

Abstract Exopolysaccharide (EPS) secreted by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease induced by the enteric pathogen Citrobacter rodentium. We tested the activity of EPS on dendritic cells (DCs), and found that in vitro, EPS not only induced the expression of inhibitory molecules, PDL1 and PDL2, but also that these DCs inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the generation of DCs expressing PD-L1 and PD-L2. To test if the EPS-induced DCs are inhibitory in vivo, we used a model that mimics Systemic Lupus Erythematosus (SLE) and tested if the level of autoantibody was decreased. We injected Bm12 cells into EPS-treated C57BL/6 mice to induce SLE-like symptoms, and found that the titer of autoantibodies was significantly decreased in the EPS-injected mice compared to control mice. We conclude that EPS can generate inhibitory DCs both in vitro and in vivo, and we suggest that these DCs have the potential to protect from SLE and other autoimmune diseases.

Immunomodulatory properties of titanium dioxide nanostructural materials.

OBJECTIVES:Although titanium dioxide (TiO2) nanostructural materials have been widely used in Biology and Medicine, very little is known about immunomodulation mechanism of these materials. Objectives of this study are to investigate in vitro immunomodulatory effects of TiO2. Immunosuppressant may lower immune responses and are helpful for the treatment of graft versus host diseases and autoimmune disorders.MATERIALS AND METHODS:In this study, we used H2Ti3O7 titanium dioxide nanotubes (TNT) nanotubes along with commercial TiO2 nanoparticles (TNP) and TiO2 fine particles (TFP). We investigated the in vitro immunomodulatory effects of TNP, TNT, and TFP using mixed lymphocyte reaction (MLR). Suppression was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cytokine profile was measured by enzyme-linked immunosorbent assay (ELISA).RESULTS AND CONCLUSIONS:The results from this study illustrated that the TiO2 nanostructural materials strongly suppressed splenocytes proliferation in MLR. For TNP and TNT, at 50 μg/ml suppression of 20%–25% and 30%–35%, respectively, and for TFP at 100 μg/ml suppression was 25%–30% was observed. Suppression of splenocytes proliferation in the presence of TNP, TNT, and TFP demonstrated that these nanostructural materials probably block T-cell-mediated responses in vitro. Our ELISA results confirmed that significantly lower levels of Th1 type cytokines (interleukin-2, interferon-γ) in the 48 h MLR culture supernatants. Our data suggest that TiO2 nanostructural materials suppress splenocytes proliferation by suppressing Th1 cytokines.

Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells.

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.

Slowly-adhered primary bone marrow mesenchymal stem cells may exhibit more potent immuno-regulatory activity

This study was to investigate the relationship between the adherent ability of freshly isolated MSCs with their inhibitory effect on lymphocyte activation. Human bone marrow mononucleated cells were maintained in culture for 48 hours, the attached and the non-attached cells were then cultured separately and the adherent cells were collected and passaged. Cellular surface markers were analyzed with flow cytometry. Alkaline phosphatase activity and the intracellular lipid droplets were measured by histological staining, the in vitro osteogenesis and adipogenesis were identified. One-way mixed lymphocyte reaction was used to evaluate the suppressive activity of the adherent cells on lymphocyte proliferation, the prostaglandin E2 level in supernatant of cultured cells was detected by ELISA. Some cells attached to the plastic after the bone marrow mononucleated cells were allowed to adhere for 48 hours. The slowly-attached cells were fibroblast-like in morphology, homogenously positive for CD44 and CD73 and negative for CD31 and CD45. They could be coaxed into osteoblasts and adipoblasts under the standard inductive conditions. These cells were able to inhibit lymphocyte proliferation in mixed lymphocyte reaction and their effect was more potent than those from the adherent cells appeared within 48 hours. The concentration of prostaglandin E-2 in the supernatants of the slowly-adhered cells was significantly higher than that in the MSCs cultured with the traditional method (90.8 ± 10.37 ng/ml vs 70.2 ± 8.98 ng/ml) (P < 0.01). The MSCs exist in the marrow mononucleated cells after adherent culture for 48 hours, and the MSCs may exhibit more potently inhibitory activity on lymphocyte activation.

Immunostimulatory effects of Leishmania infantum HSP70 recombinant protein on dendritic cells in vitro and in vivo.

Activation of dendritic cells (DCs) has an important role in immunity against Leishmania. We investigated the effect of Leishmania infantum (L. infantum) heat shock protein 70 recombinant protein (rHSP70) as a vaccine on DC maturation and function. BALB/c mouse splenic DCs were isolated and treated with different concentrations of rHSP70. Maturation markers, cytokine production and capability of DCs to proliferate allogeneic T cells were evaluated. Furthermore, this recombinant protein was injected into BALB/c mice, and expression of CD86, CD40 and MHC class II molecules by their splenic DCs were evaluated. rHSP70 significantly increases the production of IL-12p70 by DCs. It had no effect on allogeneic T-cell proliferation in mixed lymphocyte reaction. It increased IFN-γ and decreased IL-4 cytokine level in mixed lymphocyte reaction supernatant. The in vitro study showed that rHSP70 had no significant effect neither on the percentage of CD40(+), CD86(+) and MHC class II(+) DCs nor on the mean fluorescent intensity. However, in vivo results showed that rHSP70 increases the percentage of CD86-, CD40- and MHC class II-expressing cells as well as mean fluorescent intensity of CD40 and MHC class II. This study demonstrated the capability of L. infantum-derived rHSP70 in maturating BALB/c mice splenic DCs and in vivo polarization of immunity to a Th1 response.

Increased expression of herpesvirus entry mediator in 1,25-dihydroxyvitamin D3-treated mouse bone marrow-derived dendritic cells promotes the generation of CD4+CD25+Foxp3+ regulatory T cells

Dendritic cells (DCs) can initiate immune responses or induce immune tolerance. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a secosteroid hormone that can induce tolerogenic dendritic cells favoring the induction of regulatory T cells (Treg). The present study revealed a tolerogenic effect of 1,25(OH)2D3 on the phenotype and function of ovalbumin (OVA)‑activated mouse bone marrow‑derived DCs. Three inhibitory molecules associated with tolerogenic DCs, programmed death‑ligand-1 (PD‑L1), PD‑L2 and herpesvirus entry mediator (HVEM) and the expression of co‑stimulatory molecules (CD80, CD86 and CD40) on 1,25(OH)2D3‑treated DCs were examined. The levels of interleukin (IL)‑2, IL‑6 and IL‑10 secreted by 1,25(OH)2D3‑treated DCs were analyzed. The capability of 1,25(OH)2D3‑treated DCs to induce CD4+CD25+Foxp3+ Treg and the stimulation of allogeneic CD4+ T‑cell proliferation in mixed lymphocyte reaction (MLR) was studied. 1,25(OH)2D3‑treated DCs induced up to 21.0‑fold upregulation of the HVEM expression and 4.1‑fold enhancement of the HVEM expression upon activation with OVA [OVA‑D3/immature (im)DCs]. PD‑L1 was not affected by 1,25(OH)2D3‑treated DCs and downregulated PD‑L2 expression on 1,25(OH)2D3‑treated DCs and OVA‑D3/imDC. The expression of co‑stimulatory molecules (CD80, CD86 and CD40) was downregulated in 1,25(OH)2D3‑treated DCs and OVA‑D3/imDC. Furthermore, 1,25(OH)2D3‑treated DCs secreted much higher levels of IL‑10, however lower levels of IL‑2 and IL‑6 compared with the activated control DCs. Together with this pattern of cytokines, 1,25(OH)2D3‑treated DCs exhibited low allogeneic CD4+ T‑cell stimulatory activity and a higher number of CD4+CD25+Foxp3+ cells in the MLR cultures but not in the activated control DCs. These findings indicate that 1,25(OH)2D3 possesses the immunosuppressive properties by upregulating the expression of the inhibitory molecules, HVEM, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases.

Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy

Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc-mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, β-galactosidase (β-Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co-culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4(+) cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc-MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0-G1 phase, without significant differences between SSc and HC. SSc-MSCs showed an increased positive β-Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, β-Gal staining increased significantly in SSc-MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc- and HC-MSCs. Interleukin (IL)-6 and transforming growth factor (TGF)-β-related transcripts and their protein levels were significantly higher in SSc-MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL-6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.

Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

Adiponectin Mediated MHC Class II Mismatched Cardiac Graft Rejection in Mice Is IL-4 Dependent

BackgroundAdiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined.Methodology/Principal FindingsWild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6) and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4+ and CD8+ T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2.ConclusionsOur results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.

Donor-Derived Mesenchymal Stem Cells Facilitate Minimization of Calcineurin Inhibitors in Kidney Transplant Patients and the Immune Profile Monitoring: A Clinical Pilot Study

Background: The deleterious side effects of calcineurin inhibitors (CNIs) such as nephrotoxicity, cardiovascular events, infection and malignancy, have impaired patients long-term survival. New immunotherapy regimens with minimization or elimination of CNIs are required to improve transplant outcome. Mesenchymal stem cells (MSCs) represent a unique cell population with potent immunosuppressive function, and can prolong allograft survival in experimental organ transplant models. In this present study, MSCs combined with sparing tacrolimus (TAC) were administered to de novo living related kidney transplant (KTx) recipients to prevent acute rejection, and post-transplant immune profile was monitored. Patients and methods: Twelve patients were enrolled in this prospective study. Six patients received donor-derived MSCs combined with minimized TAC, and the other six without MSCs were assigned as control. MSCs were infused into renal allograft via renal artery prior to vessel anastomosis (5×106 cells), and were subsequently given intravenously one month later (2×106 cells/kg). Safety of MSCs infusion, renal function and complications were observed. Donor-specific mixed lymphocyte reaction (MLR) was performed prior to KTx (day 0) and three (mon 3) or six months (mon 6) post-transplant. Chimerism was examined. Immune cells including CD4+ and CD8+ T cell, CD4+CD25+CD127lowFoxp3+ regulatory T cell (Tregs), NK cell, and CD19+CD27+ memory B cell were monitored by flow cytometry. Intracellular cytokines including IFN-γ, TNF-α, IL-4 and IL-10 were examined. Results: Tacrolimus dose in MSCs group was significantly reduced compared to control group (0.047 ± 0.005 vs 0.073 ± 0.013 mg/kg). One acute rejection occurred in control group, and was reversed by antithymocyte globulin and methylprednisolone therapy. None acute rejection was observed in MSCs group. All patients survived with stable renal function at mon 6. There was no significant different in MLR proliferation analysis at mon 3 and mon 6 between two groups. The proportion and absolute count of peripheral blood lymphocyte was not significantly different between two groups. Total T cells, CD4+ T cells, CD8+ T cells, and CD4+/CD8+ ratio were significantly different neither. Tregs proportion in MSCs group increased at mon 3 and returned at mon 6, compared to Tregs at day 0. However, Tregs significantly decreased at mon 3 in control group. B cells proportion in MSCs group increased from 9.5% (day 0) to 16.7% at mon 3, and returned at mon 6. While in control group, B cells constantly decreased within six months (3.8% vs 7.9%). The proportion of memory B cells increased by 27.9% at mon 3 in MSCs group, in contrast to 23% of decrease in control group. NK cells proportion in control group continually increased post KTx, while it showed transient decrease at mon 3 in MSCs group. No chimerism was detected in both groups. The proportion of IL-10 producing cells showed constant increase tendency within six months after KTx in MSCs group, while it transiently decreased at mon 3 in control group. Conclusions: Donor-derived MSCs may guarantee efficient hypo-responsiveness to allograft while minimizing calcineurin inhibitors in living related kidney transplantation. The long-term therapeutic effect and its mechanism of action need to be further investigated.

Mesenchymal stem cells administered after liver transplantation prevent acute graft-versus-host disease in rats

Acute graft-versus-host disease is a serious and life-threatening complication of liver transplantation (LT) that occurs in 1% to 2% of liver allograft recipients. It is associated with a high mortality rate, and effective therapies are lacking. In our established rat model, a relative decrease in regulatory T cells (Tregs) was previously shown to be associated with acute graft-versus-host disease after liver transplantation (LT-aGVHD). Mesenchymal stem cells (MSCs) have been used to treat graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, and they have been shown to induce Tregs, which have immunomodulatory effects. In this study, when a treatment with donor- or recipient-derived MSCs was administered from day 8 to day 14 after the typical symptoms of LT-aGVHD started, the recipients were not cured, and their survival time was not prolonged. However, when MSCs of different origins were administered from day 0 to day 6 after LT, the recipients survived significantly longer than the control group, and the surviving MSC-treated rats did not show typical LT-aGVHD symptoms. In vivo tracings of carboxyfluorescein diacetate succinimidyl ester-stained MSCs did not show significant accumulations in the target organs after administration. Flow cytometry analysis showed that the Treg ratios in peripheral blood were more higher for the MSC-treated groups versus the control group. More immunohistochemically stained forkhead box P3-positive cells were also found in the intestines of the MSC-treated groups versus the control group. Further investigations of the function of MSCs showed that they could increase the Treg ratio in a mixed lymphocyte reaction (MLR) and lead to a greater reduction in MLR proliferation in vitro. In conclusion, the post-LT administration of MSCs of either donor or recipient origin could prevent the onset of LT-aGVHD in our rat model.