Induction Of Specific Tolerance Research Articles

Mechanistic and Biomarker Studies to Demonstrate Immune Tolerance in Multiple Sclerosis.

The induction of specific immunological tolerance represents an important therapeutic goal for multiple sclerosis and other autoimmune diseases. Sound knowledge of the target antigens, the underlying pathomechanisms of the disease and the presumed mechanisms of action of the respective tolerance-inducing approach are essential for successful translation. Furthermore, suitable tools and assays to evaluate the induction of immune tolerance are key aspects for the development of such treatments. However, investigation of the mechanisms of action underlying tolerance induction poses several challenges. The optimization of sensitive, robust methods which allow the assessment of low frequency autoreactive T cells and the long-term reduction or change of their responses, the detection of regulatory cell populations and their immune mediators, as well as the validation of specific biomarkers indicating reduction of inflammation and damage, are needed to develop tolerance-inducing approaches successfully to patients. This short review focuses on how to demonstrate mechanistic proof-of-concept in antigen-specific tolerance-inducing therapies in MS.

Interferon-γ/Interleukin-27 Axis Induces Programmed Death Ligand 1 Expression in Monocyte-Derived Dendritic Cells and Restores Immune Tolerance in Central Nervous System Autoimmunity.

Antigen (Ag)-specific tolerance induction by intravenous (i. v.) injection of high-dose auto-Ags has been explored for therapy of autoimmune diseases, including multiple sclerosis (MS). It is thought that the advantage of such Ag-specific therapy over non-specific immunomodulatory treatments would be selective suppression of a pathogenic immune response without impairing systemic immunity, thus avoiding adverse effects of immunosuppression. Auto-Ag i.v. tolerance induction has been extensively studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and limited clinical trials demonstrated that it is safe and beneficial to a subset of MS patients. Nonetheless, the mechanisms of i.v. tolerance induction are incompletely understood, hampering the development of better approaches and their clinical application. Here, we describe a pathway whereby auto-Ag i.v. injected into mice with ongoing clinical EAE induces interferon-gamma (IFN-γ) secretion by auto-Ag-specific CD4+ T cells, triggering interleukin (IL)-27 production by conventional dendritic cells type 1 (cDC1). IL-27 then, via signal transducer and activator of transcription 3 activation, induces programmed death ligand 1 (PD-L1) expression by monocyte-derived dendritic cells (moDCs) in the central nervous system of mice with EAE. PD-L1 interaction with programmed cell death protein 1 on pathogenic CD4+ T cells leads to their apoptosis/anergy, resulting in disease amelioration. These findings identify a key role of the IFN-γ/IL-27/PD-L1 axis, involving T cells/cDC1/moDCs in the induction of i.v. tolerance.

Development of ImmTOR Tolerogenic Nanoparticles for the Mitigation of Anti-drug Antibodies.

The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and hypersensitivity reactions for many biologics. The focus of this review is the development of ImmTOR, a platform technology designed to prevent the formation of ADAs that can be applied broadly across a wide variety of biologics by inducing immunological tolerance with ImmTOR nanoparticles encapsulating rapamycin. The induction of tolerance is antigen-specific and dependent on the incorporation of rapamycin in nanoparticles and the presence of the antigen at the time of administration of ImmTOR. Evidence for the induction of specific immune tolerance vs. general immune suppression is supported by the findings that: (1) ImmTOR induces regulatory T cells specific to the co-administered antigen; (2) tolerance can be transferred by adoptive transfer of splenocytes from treated animals to naïve recipients; (3) the tolerance is durable to subsequent challenge with antigen alone; and (4) animals tolerized to a specific antigen are capable of responding to an unrelated antigen. ImmTOR nanoparticles can be added to new or existing biologics without the need to modify or reformulate the biologic drug. The ability of ImmTOR to mitigate the formation of ADAs has been demonstrated for coagulation factor VIII in a mouse model of hemophilia A, an anti-TNFα monoclonal antibody in a mouse model of inflammatory arthritis, pegylated uricase in hyperuricemic mice and in non-human primates, acid alpha-glucosidase in a mouse model of Pompe disease, recombinant immunotoxin in a mouse model of mesothelioma, and adeno-associated vectors in a model of repeat dosing of gene therapy vectors in mice and in non-human primates. Human proof-of concept for the mitigation of ADAs has been demonstrated with SEL-212, a combination product consisting of ImmTOR + pegadricase, a highly immunogenic enzyme therapy for the treatment of gout. ImmTOR represents a promising approach to preventing the formation of ADAs to a broad range of biologic drugs.

Virus specific tolerance enhanced efficacy of cancer immuno-virotherapy

BackgroundActivation of the immune system to fight cancer is a major goal in immunology and oncology. Although cancer treatment using oncolytic viruses shows promising results, virus mediated oncolysis induces a weak anti-tumor immune response. Upon application of viruses, immune responses against the virus play a significant role in limiting tumor virotherapy. Although suppression of host immunity increases the efficacy of virotherapy against the tumor, but inhibits anti-tumor immune responses. Induction of viral specific tolerance before viral replication may cause the virus to efficiently replicate in tumor cells without affecting the immune responses against tumor antigens. Investigation of the combined strategy of virotherapy and immunotherapy using irradiated tumor cells along with IL-2 and interferon-alpha in virus specific tolerant mice was the goal of this study. Materials and methodsFor tolerance induction, the newborn mice were injected with vesicular stomatitis virus (VSV) subcutaneously. After injection of TC-1 tumor cells to adult tolerant mice and formation of a tumor, irradiated TC-1 cells along with IL-2 and Interferon-alpha expression plasmid were injected twice in mice and followed by virotherapy. Size of tumors and CTL activity against the virus and tumor cells were measured. ResultThe results showed increased efficacy of virotherapy in combination with immune-stimulators and tumor cells injection in tolerant mice compared to normal mice. ConclusionSpecific tolerance against the oncolytic virus enhances the efficacy of virotherapy both in monotherapy and in combination with immunotherapy.

Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response.

The induction of specific and sustainable tolerance is a challenging issue in organ transplantation. The discovery of the immunosuppressive properties of apoptotic cells in animal models has paved the way for their use in human transplantation. In this work, we aimed to define a stable, reproducible, and clinically compatible production procedure of human apoptotic cells (Apo-cells). Using a clinically approved extracorporeal photopheresis technique, we have produced and characterized phenotypically and functionally human apoptotic cells. These Apo-cells have immunosuppressive properties proved in vitro and in vivo in NOD/SCID/γC mice by their capacity to modulate an allogeneic response following both a direct and an indirect antigen presentation. These results brought the rationale for the use of Apo-cells in tolerance induction protocol for organ transplantation.

Mechanisms of Immune Tolerance in Liver Transplantation-Crosstalk Between Alloreactive T Cells and Liver Cells With Therapeutic Prospects.

Liver transplantation (LTx) is currently the most powerful treatment for end-stage liver disease. Although liver allograft is more tolerogenic compared to other solid organs, the majority of LTx recipients still require long-term immune suppression (IS) to control the undesired alloimmune responses, which can lead to severe side effects. Thus, understanding the mechanism of liver transplant tolerance and crosstalk between immune cells, especially alloreactive T cells and liver cells, can shed light on more specific tolerance induction strategies for future clinical translation. In this review, we focus on alloreactive T cell mediated immune responses and their crosstalk with liver sinusoidal endothelial cells (LSECs), hepatocytes, hepatic stellate cells (HSCs), and cholangiocytes in transplant setting. Liver cells mainly serve as antigen presenting cells (APCs) to T cells, but with low expression of co-stimulatory molecules. Crosstalk between them largely depends on the different expression of adhesion molecules and chemokine receptors. Inflammatory cytokines secreted by immune cells further elaborate this crosstalk and regulate the fate of naïve T cells differentiation within the liver graft. On the other hand, regulatory T cells (Tregs) play an essential role in inducing and keeping immune tolerance in LTx. Tregs based adoptive cell therapy provides an excellent therapeutic option for clinical transplant tolerance induction. However, many questions regarding cell therapy still need to be solved. Here we also address the current clinical trials of adoptive Tregs therapy and other tolerance induction strategies in LTx, together with future challenges for clinical translation from bench to bedside.

IL-10 signaling in dendritic cells is required for tolerance induction in a murine model of allergic airway inflammation.

Allergen specific tolerance induction efficiently ameliorates subsequent allergen induced inflammatory responses. The underlying regulatory mechanisms have been attributed mainly to interleukin (IL)-10 produced by diverse hematopoietic cells, while targets of IL-10 in allergen specific tolerance induction have not yet been well defined. Here, we investigate potential cellular targets of IL-10 in allergen specific tolerance induction using mice with a cell type specific inactivation of the IL-10 receptor gene. Allergic airway inflammation was effectively prevented by tolerance induction in mice with IL-10 receptor (IL-10R) deficiency in T or B cells. Similarly, IL-10R on monocytes/macrophages and/or neutrophils was not required for tolerance induction. In contrast, tolerance induction was impaired in mice that lack IL-10R on dendritic cells: those mice developed an allergic response characterized by a pronounced neutrophilic lung infiltration, which was not ameliorated by tolerogenic treatment. In conclusion, our results show that allergen specific tolerance can be effectively induced without a direct impact of IL-10 on cells of the adaptive immune system, and highlight dendritic cells, but not macrophages nor neutrophils, as the main target of IL-10 during tolerance induction.

Design and molecular dynamic simulation of a new double-epitope tolerogenic protein as a potential vaccine for multiple sclerosis disease.

One of the debilitating diseases affecting the central nervous system is multiple sclerosis (MS). As there is no definitive treatment for MS, researchers have mainly consented with optimization of strategies which slows down the progression of the disease such as specific auto-antigens tolerance induction. In this regard, the aim of this study was design of a new double-epitope protective vaccine based on interleukin (IL)-16-neuroantigens fusion proteins. First, we selected highly antigenic epitopes of myelin basic protein (MBP) (aa 84-104) and myelin oligodendrocyte glycoprotein (MOG) (aa 99-107) from available literature and our bioinformatics analysis. The correct cleavage of our constructs and major histocompatibility complex class II binding affinities of cleaved epitopes were checked and evaluated using Pepcleave and IEDB servers, respectively. Then, different combination of MOG and MBP epitopes with or without fusion to C-terminal active part of IL-16 were designed as constructs. Afterward, Modeller and Gromacs softwares used for the investigation of the MBP, and MOG epitopes antigenicity in these constructs. The results of molecular dynamics simulations showed that IL-16 in MOG + linker + MBP + IL-16 construct does not interfere with final epitopes antigenicity of MOG + linker + MBP construct. To sum up, the construct with IL-16 is suggested as a new double-epitope tolerogenic vaccine for prevention and amelioration of MS in human.

Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance.

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.

Mucosal Gamma/Delta T Cells Increased by i.n. Administration of Gliadin to Four-Week-Old NOD Mice Prevent Diabetes in the NOD-SCID Model of Adoptive Cotransfer of Diabetes

Beta-cell autoantigens, e.g., insulin, proinsulin, or GAD65, have been investigated both in animal models and human trials for induction of beta-cell specific tolerance to prevent the development of type 1 diabetes (T1D). On the other hand, environmental factors that significantly contribute to the recent outbreak of T1D were less studied, probably due to their complexity. The dietary wheat protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevented T1D in animal models. We have previously reported that i.n. administration of gliadin prevented diabetes in NOD mice. Herewith we investigated mucosal gamma/delta T cells that were induced by i.n. administration of gliadin for their regulatory capacity to prevent diabetes in the NOD-SCID model of diabetes co-transfer. I.n. administration of gliadin to 4-week-old NOD mice increased proportion of gamma/delta T cells in pancreatic and mesenteric lymph nodes. Adoptive co-transfer of 2x106 gamma/delta T cells with 1x107 diabetogenic splenocytes led to statistically significant (p<0.01) diabetes prevention. The gamma/delta T cells were characterized for their memory-naïve and mucosal homing properties, or NK-like features by assessing expression of CD8alpha, CD45RB, CD62L, CCR7, CD103 and NKG2D by flowcytometry. In conclusion, similar to i.n. administration of insulin, i.n. vaccination with gliadin, an environmental antigen with possible ethiological influence in T1D, led to increased proportion of mucosal gamma/delta T cells. These gamma/delta T cells possess regulatory capacity to prevent diabetes transfer in the NOD-SCID model and may represent a novel and safer strategy for prevention or an early intervention in T1D. Disclosure D. Funda: None. J. Golias: None. M. Haupt-Jorgensen: None. K. Buschard: None.

Effect of immune tolerance induced by immature dendritic cells and CTLA4-Ig on systemic lupus erythematosus: An in vivo study.

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease in which tissue damage is caused by autoantibodies. The induction of specific immune tolerance, including the utilization of immune regulatory cells, may enhance the therapeutic effects of organ transplantation in patients with SLE. Furthermore, inhibiting immune responses has been reported to be an effective treatment for SLE. However, few studies have explored the association between an increased immune tolerance and a decreased immune response in SLE treatment. Dendritic cells (DCs), which are highly efficient antigen-presenting cells, are able to induce specific tolerance, while cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) inhibits the immune response. In the present study, interleukin (IL)-10-treated DCs and CTLA4-Ig were administered to mice with SLE alone or in combination and the therapeutic effects were investigated. IL-10 was added into the culture medium of bone marrow-derived DCs to prevent them from differentiating into mature cells. Low levels of major histocompatibility complex II, cluster of differentiation (CD)40, CD80 and CD86 were detected, which indicated that the immature state of DCs was maintained. IL-10-treated DCs were subsequently injected into the caudal vein of B6.MRL-Faslpr/J lupus mice, which are an established animal model of SLE. To amplify the tolerance effect, mice were simultaneously injected with CTLA4-Ig. Compared with the IL-10-treated DC and CTLA4-Ig groups, combined treatment with IL-10-treated DCs and CTLA4-Ig strongly induced immune tolerance in mice with SLE, as indicated by the significantly reduced levels of urine protein, anti-nuclear antibody, double-stranded DNA and IL-17A. A significant decrease in the proportion of T helper cells and an increase in the proportion of CD4+ forkhead box protein P3+ Treg cells was also observed, further confirming the induction of immune tolerance. These results suggest that combined treatment with IL-10-DCs and CTLA4-Ig may be a promising novel therapeutic strategy for the treatment of SLE.

Translating Mechanism of Regulatory Action of Tolerogenic Dendritic Cells to Monitoring Endpoints in Clinical Trials.

Tolerogenic dendritic cells (tolDCs) have reached patients with autoimmune and inflammatory disease, at least in clinical trials. The safety of tolDCs as intervention therapy has been established, but the capacity to modulate autoimmune response in vivo remains to be demonstrated. Studies have revealed a diversity of regulatory mechanisms that tolDCs may employ in vivo. These mechanisms differ between various types of modulated tolDC. The most often foreseen action of tolDCs is through regulatory polarization of naïve T cells or activation of existing regulatory T cells, which should ultimately diminish autoimmune inflammation. Yet, selection of a target autoantigen remains critical to expedite tissue specific tolerance induction, while measuring immune modulation incited by tolDCs in vivo provides a great challenge. We will discuss the regulatory action of different types of tolDCs and the possible methods to monitor immunological efficacy endpoints for the next generation clinical trials.

Cyclophosphamide-induced tolerance in kidney transplantation avoids long-term immunosuppressive therapy.

There has recently been remarkable progress in immunosuppressive agents, such as tacrolimus and cyclosporine. Therefore, the rate of organ establishment has improved in transplantation. However, immunosuppressive agents generally suppress the function of Tcells. Thus, opportunistic infections, such as cytomegalovirus infection, are still a major problem in kidney transplantation. Induction of specific tolerance to avoid immunosuppressive drug therapy after kidney transplantation is considered as the ultimate goal of transplantation. Various factors induce tolerance that involves establishment of hematopoietic chimerism and various cell subsets. In particular, we have carried out various studies regarding the cyclophosphamide-induced tolerance system. Tolerance is induced after establishment of hematopoietic chimerism after donor bone marrow transplantation. At the clinical stage, kidney transplantation before administration of cyclophosphamide after transfusion of bone marrow to create hematopoietic chimera is considered to be one of the most successful protocols. Furthermore, recent studies have shown the involvement of multiple populations of immune cells in preserving immunological tolerance and promoting long-term renal grafts. The present review focuses on how cyclophosphamide and other immune factors induce tolerance in kidney transplantation.

Combined Bone Marrow and Kidney Transplantation for the Induction of Specific Tolerance.

The induction of specific tolerance, in order to avoid the detrimental effects of lifelong systemic immunosuppressive therapy after organ transplantation, has been considered the “Holy Grail” of transplantation. Experimentally, tolerance has been achieved through clonal deletion, through costimulatory blockade, through the induction or infusion of regulatory T-cells, and through the establishment of hematopoietic chimerism following donor bone marrow transplantation. The focus of this review is how tolerance has been achieved following combined bone marrow and kidney transplantation. Preclinical models of combined bone marrow and kidney transplantation have shown that tolerance can be achieved through either transient or sustained hematopoietic chimerism. Combined transplants for patients with multiple myeloma have shown that organ tolerance and prolonged disease remissions can be accomplished with such an approach. Similarly, multiple clinical strategies for achieving tolerance in patients without an underlying malignancy have been described, in the context of either transient or durable mixed chimerism or sustained full donor hematopoiesis. To expand the chimerism approach to deceased donor transplants, a delayed tolerance approach, which will involve organ transplantation with conventional immunosuppression followed months later by bone marrow transplantation, has been successful in a primate model. As combined bone marrow and organ transplantation become safer and increasingly successful, the achievement of specific tolerance may become more widely applicable.

Systemic Administration of Proteoglycan Protects BALB/c Retired Breeder Mice from Experimental Arthritis.

This study was undertaken to evaluate the prophylactic potential of proteoglycan (PG) administration in experimental arthritis. Female BALB/c retired breeder mice received two (2xPG50 and 2xPG100 groups) or three (3xPG50 group) intraperitoneal doses of bovine PG (50 μg or 100 μg) every three days. A week later the animals were submitted to arthritis induction by immunization with three i.p. doses of bovine PG associated with dimethyldioctadecylammonium bromide adjuvant at intervals of 21 days. Disease severity was daily assessed after the third dose by score evaluation. The 3xPG50 group showed significant reduction in prevalence and clinical scores. This protective effect was associated with lower production of IFN-γ and IL-17 and increased production of IL-5 and IL-10 by spleen cells restimulated in vitro with PG. Even though previous PG administration restrained dendritic cells maturation this procedure did not alter the frequency of regulatory Foxp3+ T cells. Lower TNF-α and IL-6 levels and higher expression of ROR-γ and GATA-3 were detected in the paws of protected animals. A delayed-type hypersensitivity reaction confirmed specific tolerance induction. Taken together, these results indicate that previous PG inoculation determines a specific tolerogenic effect that is able to decrease severity of subsequently induced arthritis.

Current novel approaches in systemic therapy of atopic dermatitis: specific inhibition of cutaneous Th2 polarized inflammation and itch.

This present review gives an overview on different approaches on systemic treatment of atopic dermatitis with a focus on molecules that are presently investigated as defined target structures in clinical studies with patients with atopic dermatitis. Available systemic drugs for the treatment of atopic dermatitis are limited. There is a high need for the development of new treatment regimen. Current studies address novel biologicals or small molecules for the treatment of atopic dermatitis. In particular, interventions in the T-helper 2 cell (Th2) allergic inflammation are promising. Dupilumab, a humanized monoclonal antibody to the interteukin-4R is the first antibody (i.e. 'biological') with published efficacy shown in controlled prospective studies in atopic dermatitis. A number of other target molecules is currently addressed in clinical studies with blocking antibodies against target molecules of Th2 polarized inflammation in atopic dermatitis (e.g. interleukins 13, 22, 31, thymic stromal lymphopoetin). Inhibiting phospodiesterase-4, chemoattractant receptor-homologous molecule 2, the histamine-4 receptor, or the neurokine-1 receptor may lead to the approval of novel 'small drugs' for a specific treatment of atopic dermatitis. Antifungals may be helpful in the variant of head and neck dermatitis associated with sensitizations against Malassezia species. Induction of specific immune tolerance (e.g. with allergen-specific immunotherapy) should be further studied in terms of the management of atopic dermatitis because available clinical studies resulted in inconsistent results on the skin condition. Current studies with new substances for the systemic treatment of atopic dermatitis have tremendous implications for the future management of atopic dermatitis.

Personalized Immunomodulatory Therapy for Atopic Dermatitis: An Allergist's View.

The current standard medical therapy for atopic dermatitis (AD) mainly focuses on symptomatic relief by controlling skin inflammation with topical corticosteroids and/or topical calcineurin inhibitors. However, the clinical efficacy of pharmacological therapy is often disappointing to both patients and physicians. The terminology of AD contains a historical meaning of eczematous dermatitis caused by hypersensitivity reaction to environmental inhalant or food allergen. Complex interrelationships among genetic abnormalities, environmental triggers, skin barrier defects, and immune dysfunction resulting in a vicious domino-circle seem to be involved in the development and maintenance of AD. In the viewpoint of AD as an allergic disease, complete avoidance of clinically relevant allergen or induction of specific immune tolerance through administrations of allergen (allergen immunotherapy) can provide clinical remission by breaking the vicious domino-circle maintaining a chronic disease state. In recent clinical studies, monoclonal antibodies including the anti-interleukin-4 receptor antibody and anti-B cell antibody induced significant clinical improvements in patients with AD. The detailed characteristics of immune dysfunction are heterogeneous among patients with AD. Therefore, a personalized combination of immunomodulatory therapies to reduce hypersensitivity (allergen immunotherapy) and correct immune dysfunction (monoclonal antibody therapy) could be a reasonable therapeutic approach for patients with AD. Future immunomodulatory therapies for AD should be developed to achieve long-term treatment-free clinical remission by induction of immune tolerance.

Immunosuppression By Mesenchymal Stromal Cells Derived from Human Induced Pluripotent Stem Cells : Evaluation in an aGVHD Model

▪BackgroundOne major problem of allogeneic hematopoietic stem cell transplantation (HSCT) is acute Graft versus Host Disease (aGVHD). aGVHD has been managed until now with HLA matching and a constant evolving repertoire of immunosuppressive drugs. One alternative would be to generate in the host a permanent tolerance state toward the graft. Tolerant inducing cell therapy has been proposed with adult mesenchymal stromal (MSCs) cells. Ex vivo isolated somatic MSCs have been implicated in immunoregulatory functions on cells from both the innate and adaptive immune system. They were proposed for cell therapies for the treatment of aGVHD,Nevertheless, their use is restricted because of the few number that can be recovered from adult tissues, their limited in vitro expansion, and the absence of a full phenotypic characterization. Therefore other sources of well-defined and unlimited number of MSCs are needed, and MSCs derived in vitro from human Induced pluripotent stem cell (huIPS) would be a valuable tool for therapeutic approaches.AimsBecause of our expertise in pluripotent stem cell differentiation, we generated huiPS-MSCs that present a strong immunosuppressive activity on allogeneic T cell responses. Our objectives are: 1/ To evaluate and characterize in vitro this immunosuppression. 2/ To validate in vivo these results using a xenoGVHD model.MethodsTo characterize the huIPS-MSCs in vitro, FACS phenotyping and multipotency were tested. Their immunogenicity in vitro was monitored in co-cultures with allogenic peripheral blood mononuclear cells (PBMC). The in vivo immunosuppressive activity of huiPS-MSCs was evaluated using a xenoGVHD model in immunodeficient NSG (NOD/SCID/IL2rγKO) mice in which human PBMC were injected intra-peritonally. We established 3 groups : 1) huIPS-MSCs (control) 2) PBMC 3) PBMC + IPS-MSCs. We repeated huIPS-MSCs injection weekly with median number of injection n=3 (range 2-3). The activation state of human allogeneic T lymphocytes recovered from mice between 5 to 8 weeks after initial injection was evaluated and indicated the level of the xenoGVHD process and the efficiency of huiPS-MSCs to prevent it.Resultsa) In vitro characterization of huIPS-MSCsAs expected, the huiPS-MSCs were positive for CD73, CD90, CD105, HLA-I Ags and negative for CD45, CD34, HLA-II Ags and they were capable of differentiation into the classical mesenchymal-derived cells (osteoblast, chondrocytes and adipocytes). To test their immunosuppressive properties, we analyzed their action on the proliferation of human T lymphocytes stimulated in an allogeneic manner (Fig 1). The stimulation of PBMC in mixed lymphocyte reaction resulted in CD4 and CD8 T cell proliferation (28 ± 7% and 47 ± 8%, respectively), which was significantly reduced in co-culture with huiPS-MSCs (4 ± 2% and 10 ± 2, respectively, n=3 p<0,05). We were able to demonstrate using blocking antibodies that part of the inhibition exerted by the iPS-MSCs is due to a) B7H1, a membrane receptor for the B7 family, known for its inhibitory action on the activation of T lymphocytes b) and B7H3 (from the same family) whose role remains controversial.b) In vivo characterization of huIPS-MSCsAfter sacrifice of mice, human circulating cells, those present in the peritoneal cavity and in the spleen were analysed by FACS. Mostly T lymphocytes were detected, and their number was significantly reduced in mice treated with huIPS-MSCs p<0,05 (Fig 2).Intracytoplasmic labelling of recovered T cells showed that untreated mice displayed high percentages of human differentiated T cells producing IFN # and TNF # (typical of a inflammatory Th1 cytokine polarization profile), while little or none produced low inflammatory (IL-4) or anti-inflammatory (IL-10) cytokines. In contrast, in mice treated with the huiPS-MSCs, the proportion of T cells of the Th1 type was substantially reduced, while that of T cells producing IL-4 and / or IL-10 was slightly increased (Fig 3). In parallel, T cells expressing FoxP3 appeared.ConclusionWe were able to generate immune-modulatory huiPS-MSCs that can be used to reduce activation of T cells in a xeno-aGVHD model through a switch from a Th1 inflammatory differentiation pathway to a T cell regulatory pathway. Our results may favor the development of new tools and strategies based on the use of pluripotent stem cells and their derivatives to prevent aGVHD but also for the induction of specific tolerance. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In utero hematopoietic cell transplantation: induction of donor specific immune tolerance and postnatal transplants.

In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative non-immunosuppressive transplant approach that allows for donor cell engraftment across immunologic barriers. Successful engraftment is associated with donor-specific tolerance. IUHCT has the potential to treat a large number of congenital hematologic, immunologic, and genetic diseases either by achieving high enough engraftment levels following a single IUHCT or by inducing donor specific tolerance to allow for non-toxic same-donor postnatal transplants. This review evaluates donor specific tolerance induction achieved by IUHCT. Specifically it addresses the need to achieve threshold levels of donor cell engraftment following IUHCT to consistently obtain immunologic tolerance. The mechanisms of tolerance induction including partial deletion of donor reactive host T cells by direct and indirect antigen presentation and the role of regulatory T cells in maintaining tolerance are reviewed. Finally, this review highlights the promising clinical potential of in utero tolerance induction to provide a platform on which postnatal cellular and organ transplants can be performed without myeloablative or immunosuppressive conditioning.

Genetically engineered Lactococcus lactis protect against house dust mite allergy in a BALB/c mouse model.

BackgroundMucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.MethodsThree strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.ResultsDer p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.ConclusionOral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.