Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

A diametric role for OX40 in the response of effector/memory CD4+ T cells and regulatory T cells to alloantigen.

OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40 ligand (OX40L) pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. In this article, we show that the alloantigen-mediated activation of naive and memory CD4(+) T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 had no effect on the activation and proliferation of T cells; rather, effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death among proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting, rather than diminishing, regulatory T cell survival. These data show that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-Ags.

Renal ischemia-reperfusion injury amplifies the humoral immune response.

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

Mesenchymal stromal cells in transplantation rejection and tolerance.

Mesenchymal stromal cells (MSCs) have recently emerged as promising candidates for cell-based immunotherapy in solid organ transplantation (SOT). In addition to immune modulation, MSCs possess proreparative properties and preclinical studies indicate that MSCs have the capacity to prolong graft survival and in some cases induce tolerance. Currently, the application of MSCs in SOT is being evaluated in phase I/II clinical trials. Whereas the mechanisms of action used by MSC immunomodulation have been somewhat elucidated in vitro, the data from preclinical transplant models have been unclear. Furthermore, the optimal timing, dose, and route of administration remain to be elucidated. Importantly, MSCs have the ability to sense their environment, which may influence their function. In this article, we discuss the impact of the local microenvironment on MSCs and the mechanisms of MSC immunomodulation in the setting of SOT.

Pathways analysis of differential gene expression induced by engrafting doses of total body irradiation for allogeneic bone marrow transplantation in mice.

A major challenge in allogeneic bone marrow (BM) transplantation is overcoming engraftment resistance to avoid the clinical problem of graft rejection. Identifying gene pathways that regulate BM engraftment may reveal molecular targets for overcoming engraftment barriers. Previously, we developed a mouse model of BM transplantation that utilizes recipient conditioning with non-myeloablative total body irradiation (TBI). We defined TBI doses that lead to graft rejection, that conversely are permissive for engraftment, and mouse strain variation with regards to the permissive TBI dose. We now report gene expression analysis, using Agilent Mouse 8x60K microarrays, in spleens of mice conditioned with varied TBI doses for correlation to the expected engraftment phenotype. The spleens of mice given engrafting doses of TBI, compared with non-engrafting TBI doses, demonstrated substantially broader gene expression changes, significant at the multiple testing-corrected P <0.05 level and with fold change ≥2. Functional analysis revealed significant enrichment for a down-regulated canonical pathway involving B-cell development. Genes enriched in this pathway suggest that suppressing donor antigen processing and presentation may be pivotal effects conferred by TBI to enable engraftment. Regardless of TBI dose and recipient mouse strain, pervasive genomic changes related to inflammation was observed and reflected by significant enrichment for canonical pathways and association with upstream regulators. These gene expression changes suggest that macrophage and complement pathways may be targeted to overcome engraftment barriers. These exploratory results highlight gene pathways that may be important in mediating BM engraftment resistance.

Chimerism-based experimental models for tolerance induction in vascularized composite allografts: Cleveland clinic research experience.

The preclinical experimental models of vascularized composite allografts (VCAs) have been rapidly developed for the assessment of immunomodulatory protocols for clinical application. Recently, researchers have focused on immunomodulatory protocols which overcome the immunologic barrier between the allogeneic donor and recipient and may lead to tolerance induction. In order to test the feasibility of chimerism induction, experimental VCAs have been performed in different models including rodents, large animals, and nonhuman primates. These models differ in the complexity of transplanted tissue and in their responses to immunomodulatory protocols. In most applications, VCA contains multiple-tissue components; however, each individual component of CTA possesses unique immunologic characteristics that ultimately contribute to the chimerism induction and successful outcome of the VCA. Heterogenic character and complexity of tissue components in different VCA models determine the quality and robustness of donor-specific chimerism. As introduced in experimental studies, variable immunomodulatory options have been studied to achieve tolerance to VCA in rodents and large animal models allowing for widespread application in clinic. In this paper, based on our own experience, we have analyzed the current knowledge of tolerance-inducing strategies via chimerism induction in VCA experimental models in the context of immunomodulatory protocols and VCA complexity and their relevance and applicability to clinical practice.

T cell depletion in paediatric stem cell transplantation.

Haematopoietic stem cell transplantation (HSCT) can be a curative procedure for a growing number of paediatric diseases, but as the indications for HSCT grow, so does the need to find suitable stem cell donors. When the preferred option of a genoidentical sibling donor is not available alternative donors, including unrelated adult or umbilical cord blood donors, or haploidentical related donors may be considered. Outcome following alternative donor HSCT has improved over the past 20 years but graft-versus-host disease (GvHD) remains a significant obstacle. T cell depletion (TCD) for non-genoidentical grafts aims to reduce the morbidity and mortality associated with GvHD, but this intervention has not led directly to improved survival due to delayed immune reconstitution and increased infections, graft rejection and increased rates of disease relapse. Limited data from the paediatric population, however, suggest some encouraging results for children undergoing haploidentical HSCT: a move from positive selection of CD34(+) haematopoietic stem cells towards negative depletion of specific cell subsets in order to retain useful accessory cells within the graft appears to enhance immune reconstitution and improve disease-free survival. Here we review recent paediatric outcome data for T cell-depleted HSCT, explore the role of serotherapy in conditioning regimens and look at future possibilities to improve outcome, including novel allodepletion techniques, suicide gene therapy and pathogen-specific immunotherapy.

Monoclonal anti-interleukin-6 receptor antibody attenuates donor-specific antibody responses in a mouse model of allosensitization.

Interleukin 6 is an immune regulatory cytokine that impacts the development and maturation of T-cell, B-cell, and antibody producing plasma cells. A monoclonal antibody to the IL-6R (Tocilizumab®) was recently approved by the FDA for treatment of rheumatoid arthritis. Although anti-IL-6R anitbodies can reduce autoantibody levels in human disease, the use of anti-IL-6R for alloantibody suppression has not been examined. Here, we report on our experience with a mousenized rat-anti-mouse IL-6R (mMR16-1) for attenuating donor-specific antibody (DSA) responses. C57BL/6 mice were sensitized with skin allografts from a HLA.A2 transgenic mouse, and treated with intraperitoneal injections of mMR16-1 or control antibody. DSA responses were monitored weekly for 5weeks by measurement of serum anti-HLA.A2 antibodies in a flow cytometric antibody binding assay. Results show that mMR16-1 significantly reduced DSA IgM, IgG2a and IgG1 responses, respectively, while normalizing serum amyloid A (SAA), an acute phase reactant induced by IL-6 (p<0.01 vs. control). mMR16-1 injections increased mononuclear cell apoptosis in the spleens, as detected by annexin V staining and TUNEL. In conclusion, anti-IL6R attenuates de novo DSA responses and suppresses inflammatory markers (SAA). The data indicate that antibody therapy targeting the IL-6/IL-6R pathway may serve as a strategy to suppress DSA generation.

Standardizing skin biopsy sampling to assess rejection in vascularized composite allotransplantation.

Over 70 hands and 20 faces have been transplanted during the past 13 yr, which have shown good to excellent functional and esthetic outcomes. However, (skin) rejection episodes complicate the post-operative courses of hand and face transplant recipients and are still a major obstacle to overcome after reconstructive allotransplantation. This article summarizes all relevant information on the skin component and rejection of a vascularized composite allograft. As more and more centers plan to implement a vascularized composite allotransplantation (VCA) program, we further develop guidelines and recommendations on collection and processing of skin biopsies from hand and face allograft recipients. This will help to standardize post-operative monitoring, avoid pitfalls for those new in the field and facilitate comparison of data on VCA between centers.

Site-specific immunosuppression in vascularized composite allotransplantation: prospects and potential.

Skin is the most immunogenic component of a vascularized composite allograft (VCA) and is the primary trigger and target of rejection. The skin is directly accessible for visual monitoring of acute rejection (AR) and for directed biopsy, timely therapeutic intervention, and management of AR. Logically, antirejection drugs, biologics, or other agents delivered locally to the VCA may reduce the need for systemic immunosuppression with its adverse effects. Topical FK 506 (tacrolimus) and steroids have been used in clinical VCA as an adjunct to systemic therapy with unclear beneficial effects. However, there are no commercially available topical formulations for other widely used systemic immunosuppressive drugs such as mycophenolic acid, sirolimus, and everolimus. Investigating the site-specific therapeutic effects and efficacy of systemically active agents may enable optimizing the dosing, frequency, and duration of overall immunosuppression in VCA with minimization or elimination of long-term drug-related toxicity.

Allogeneic hematopoietic stem cell transplantation may restore gluten tolerance in patients with celiac disease.

We report on 2 patients affected by both celiac disease (CD) and ß-thalassemia major who underwent successful myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) for the latter condition. After HSCT, the introduction of a gluten-containing diet did not cause the reappearance of clinical, serological, and histological markers of CD in up to 5 years of follow-up. After transplantation, in both patients, dendritic cells and regulatory FoxP3T cells showed a recovery of normal values and no proliferative T-cell response upon gliadin stimulation was found. These data suggest that allogeneic HSCT may lead to induction of gluten tolerance in patients with CD.

Delta-like ligand 4 identifies a previously uncharacterized population of inflammatory dendritic cells that plays important roles in eliciting allogeneic T cell responses in mice.

Graft-versus-host disease (GVHD) reflects an exaggerated inflammatory allogeneic T cell response in hosts receiving allogeneic hematopoietic stem cell transplantation (HSCT). Inhibition of pan-Notch receptor signaling in donor T cells causes reduction of GVHD. However, which Notch ligand(s) in what APCs is important for priming graft-versus-host reaction remains unknown. We demonstrate that δ-like ligand-4 (Dll4) and Dll4-positive (Dll4(high)) inflammatory dendritic cells (i-DCs) play important roles in eliciting allogeneic T cell responses. Host-type Dll4(high) i-DCs occurred in the spleen and intestine of HSCT mice during GVHD induction phase. These Dll4(high) i-DCs were CD11c(+)B220(+)PDCA-1(+), resembling plasmacytoid dentritic cells (pDCs) of naive mice. However, as compared with unstimulated pDCs, Dll4(high) i-DCs expressed higher levels of costimulatory molecules, Notch ligands Jagged1 and Jagged2, and CD11b, and produced more Ifnb and Il23 but less Il12. In contrast, Dll4-negative (Dll4(low)) i-DCs were CD11c(+)B220(-)PDCA-1(-), and had low levels of Jagged1. In vitro assays showed that Dll4(high) i-DCs induced significantly more IFN-γ- and IL-17-producing effector T cells (3- and 10-fold, respectively) than Dll4(low) i-DCs. This effect could be blocked by anti-Dll4 Ab. In vivo administration of Dll4 Ab reduced donor-alloreactive effector T cells producing IFN-γ and IL-17 in GVHD target organs, leading to reduction of GVHD and improved survival of mice after allogeneic HSCT. Our findings indicate that Dll4(high) i-DCs represent a previously uncharacterized i-DC population distinctive from steady state DCs and Dll4(low) i-DCs. Furthermore, Dll4 and Dll4(high) i-DCs may be beneficial targets for modulating allogeneic T cell responses, and could facilitate the discovery of human counterparts of mouse Dll4(high) i-DCs.

Prolonged, low-dose anti-thymocyte globulin, combined with CTLA4-Ig, promotes engraftment in a stringent transplant model.

BACKGROUND: Despite significant nephrotoxicity, calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression in solid organ transplantation. We, along with others, have reported tolerogenic properties of anti-thymocyte globulin (ATG, Thymoglobulin®), evinced by its ability both to spare Tregs from depletion in vivo and, when administered at low, non-depleting doses, to expand Tregs ex vivo. Clinical trials investigating B7/CD28 blockade (LEA29Y, Belatacept) in kidney transplant recipients have proven that the replacement of toxic CNI use is feasible in selected populations. METHODS: Rabbit polyclonal anti-murine thymocyte globulin (mATG) was administered as induction and/or prolonged, low-dose therapy, in combination with CTLA4-Ig, in a stringent, fully MHC-mismatched murine skin transplant model to assess graft survival and mechanisms of action. RESULTS: Prolonged, low-dose mATG, combined with CTLA4-Ig, effectively promotes engraftment in a stringent transplant model. Our data demonstrate that mATG achieves graft acceptance primarily by promoting Tregs, while CTLA4-Ig enhances mATG function by limiting activation of the effector T cell pool in the early stages of treatment, and by inhibiting production of anti-rabbit antibodies in the maintenance phase, thereby promoting regulation of alloreactivity. CONCLUSION: These data provide the rationale for development of novel, CNI-free clinical protocols in human transplant recipients.

Implication of indolamine 2,3 dioxygenase in the tolerance toward fetuses, tumors, and allografts.

Mammalian IDO is a heme-containing enzyme whose main activity in mammals is to degrade the essential amino acid tryp into l-kynurenine. Although the link between its enzymatic activity and the immune response is not straightforward, several lines of evidence suggest that this enzyme is involved in fighting infections and paradoxically, also in the establishment of the immune tolerance associated with fetus implantation and with the development of oncogenic processes. IDO is associated with the successful development of the fetus. It participates early in pregnancy to the efficient invasion of the uterine mucosa by the nascent trophoblast and remains active throughout the whole process, as illustrated by the decrease in systemic tryp from the second trimester of gestation and the return to normal values after delivery. The short-term activation of IDO in response to invading pathogens and emerging tumors participates in the elimination of these threats, whereas the sustained activation of IDO often results in a state of immune tolerance that may favor chronic infections and the uncontrolled proliferation of malignant cells. However, despite these potential deleterious effects of IDO, the enzyme is instrumental in maintaining the peripheral tolerance that is required to avoid autoimmune diseases. Below, we review the implication of IDO activation upon the physiological development of the fetus and the pathological development of tumors and discuss whether such an enzyme could be used as a therapeutic tool to decrease the rate of allograft rejections via its potent immunomodulatory properties.

Blockade of p-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo.

Donor-specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody-mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody-mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc-independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody-mediated rejection, in which B6.RAG1(-/-) recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P-selectin, which supports monocyte binding. In the experimental model, donor-specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL-1-Ig abolished antibody-induced monocyte infiltration in the allograft, but had little effect on antibody-induced endothelial injury. Our data suggest that antagonism of P-selectin may ameliorate accumulation of macrophages in the allograft during antibody-mediated rejection.

Systemic immunoregulatory and proteogenomic effects of tacrolimus to sirolimus conversion in liver transplant recipients.

UNLABELLED: Immunosuppression (IS) withdrawal from calcineurin inhibitors is only possible in ≈ 20% of liver transplant recipients. However, mammalian target of rapamycin inhibitors (e.g., sirolimus; SRL) appear to be more immunoregulatory and might promote a tolerant state for withdrawal. Our aim was to determine whether systemic (i.e., blood, marrow, and allograft) signatures of immunoregulation are promoted by conversion from tacrolimus (TAC) to SRL. We therefore performed the following serial assays before and after SRL conversion in liver transplant recipients to test for enhanced markers of immunoregulation: (1) flow-cytometry immunophenotyping of peripheral blood mononuclear cells (PBMCs) and bone marrow aspirates for regulatory T cells (Tregs) (e.g., CD4(+) CD25(+++) FOXP3(+) ) and regulatory dendritic cells (DCregs) (immunoglobulin-like transcript 3(+) /4(+) ); (2) liver biopsy immunohistochemical staining (e.g., FOXP3:CD3 and CD4:CD8 ratios) and immunophenotyping of biopsy-derived Tregs after growth in culture; (3) effects of pre- versus postconversion sera on Treg generation in mixed lymphocyte reactions; (4) peripheral blood nonspecific CD4 responses; and (5) peripheral blood gene transcripts and proteomic profiles. We successfully converted 20 nonimmune, nonviremic recipients (age, 57.2 ± 8.0; 3.5 ± 2.1 years post-liver transplantation) from TAC to SRL for renal dysfunction. Our results demonstrated significant increases in Tregs in PBMCs and marrow and DCregs in PBMCs (P < 0.01) after conversion. In biopsy staining, FOXP3:CD3 and CD4:CD8 ratios were significantly higher after conversion and a number of biopsy cultures developed new or higher FOXP3(+) cell growth. Nonspecific CD4 responses did not change. Both pre- and postconversion sera inhibited mixed lymphocyte reactions, although only TAC sera suppressed Treg generation. Finally, 289 novel genes and 22 proteins, several important in immunoregulatory pathways, were expressed after conversion. CONCLUSIONS: TAC to SRL conversion increases systemic Tregs, DCregs, and immunoregulatory proteogenomic signatures in liver transplant recipients and may therefore facilitate IS minimization or withdrawal.

Inhibiting CXCL12 blocks fibrocyte migration and differentiation and attenuates bronchiolitis obliterans in a murine heterotopic tracheal transplant model.

OBJECTIVES: Fibrocytes are integral in the development of fibroproliferative disease after lung transplantation. Undifferentiated fibrocytes (CD45+anti-collagen 1+CXCR4+) preferentially traffic by way of the CXCR4/CXCL12 axis and differentiate into smooth muscle actin-producing (CD45+CXCR4+α-smooth muscle actin+) cells. We postulated that an antibody directed against CXCL12 would attenuate fibrocyte migration and fibro-obliteration of heterotopic tracheal transplant allografts. METHODS: A total alloantigenic mismatch murine heterotopic tracheal transplant model of obliterative bronchiolitis was used. The mice were treated with either goat-anti-human CXCL12 F(ab')(2) or goat IgG F(ab')(2). Buffy coat, bone marrow, and trachea allografts were collected and analyzed using flow cytometry. Tracheal luminal obliteration was assessed using hematoxylin-eosin and Direct Red 80 collagen stain. RESULTS: Compared with the controls, the anti-CXCL12-treated mice showed a significant decrease in tracheal allograft fibrocyte populations at 7 and 21 days after transplantation. Bone marrow and buffy coat aspirates showed the same trend at 7 days. In the anti-CXCL12-treated mice, there was a 35% decrease in luminal obliteration at 21 days (65% vs 100% obliterated; interquartile range, 38% vs 10%; P = .010) and decreased luminal collagen deposition at 21 and 28 days after transplantation (P = .042 and P = .012, respectively). CONCLUSIONS: Understanding the role of fibrocytes in airway fibrosis after lung transplantation could lead to a paradigm shift in treatment strategy. Anti-CXCL12 antibody afforded protection against infiltrating fibrocytes and reduced the deterioration of the tracheal allografts. Thus, the CXCR4/CXCL12 axis is a novel target for the treatment of fibro-obliteration after lung transplantation, and the quantification of fibrocyte populations could provide clinicians with a biomarker of fibrosis, allowing individualized drug therapy.

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis.

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.