Induction Of Allograft Tolerance Research Articles

A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products

Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg-cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a Phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4LVFOXP3. To facilitate the preclinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR/Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10-12 weeks post-transplant and T-cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4LVFOXP3 into the FOXP3KO hu-mice restored in vivo regulatory functions including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This humanized-mouse disease model can be reproducibly established and constitutes an ideal model to assess preclinical efficacy of human Treg cell investigational products.

Induced pluripotent stem cell–derived hematopoietic stem and progenitor cells induce mixed chimerism and donor-specific allograft tolerance

In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage− phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Research Highlights.

Research Highlights.

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.

Highly purified and functionally stable in vitro expanded allospecific Tr1 cells expressing immunosuppressive graft-homing receptors as new candidates for cell therapy in solid organ transplantation.

The development of new strategies based on the use of Tr1 cells has taken relevance to induce long-term tolerance, especially in the context of allogeneic stem cell transplantation. Although Tr1 cells are currently identified by the co-expression of CD49b and LAG-3 and high production of interleukin 10 (IL-10), recent studies have shown the need for a more exhaustive characterization, including co-inhibitory and chemokines receptors expression, to ensure bona fide Tr1 cells to be used as cell therapy in solid organ transplantation. Moreover, the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, therefore stability of Tr1 cells needs to be further investigated. Here, we establish a new protocol that allows long-term in vitro expansion of highly purified expanded allospecific Tr1 (Exp-allo Tr1). Our expanded Tr1 cell population becomes highly enriched in IL-10 producers (> 90%) and maintains high expression of CD49b and LAG-3, as well as the co-inhibitory receptors PD-1, CTLA-4, TIM-3, TIGIT and CD39. Most importantly, high dimensional analysis of Exp-allo Tr1 demonstrated a specific expression profile that distinguishes them from activated conventional T cells (T conv), showing overexpression of IL-10, CD39, CTLA-4 and LAG-3. On the other hand, Exp-allo Tr1 expressed a chemokine receptor profile relevant for allograft homing and tolerance induction including CCR2, CCR4, CCR5 and CXCR3, but lower levels of CCR7. Interestingly, Exp-allo Tr1 efficiently suppressed allospecific but not third-party T cell responses even after being expanded in the presence of proinflammatory cytokines for two extra weeks, supporting their functional stability. In summary, we demonstrate for the first time that highly purified allospecific Tr1 (Allo Tr1) cells can be efficiently expanded maintaining a stable phenotype and suppressive function with homing potential to the allograft, so they may be considered as promising therapeutic tools for solid organ transplantation.

Treg Therapy for the Induction of Immune Tolerance in Transplantation-Not Lost in Translation?

The clinical success of solid organ transplantation is still limited by the insufficiency of immunosuppressive regimens to control chronic rejection and late graft loss. Moreover, serious side effects caused by chronic immunosuppressive treatment increase morbidity and mortality in transplant patients. Regulatory T cells (Tregs) have proven to be efficient in the induction of allograft tolerance and prolongation of graft survival in numerous preclinical models, and treatment has now moved to the clinics. The results of the first Treg-based clinical trials seem promising, proving the feasibility and safety of Treg therapy in clinical organ transplantation. However, many questions regarding Treg phenotype, optimum dosage, antigen-specificity, adjunct immunosuppressants and efficacy remain open. This review summarizes the results of the first Treg-based clinical trials for tolerance induction in solid organ transplantation and recapitulates what we have learnt so far and which questions need to be resolved before Treg therapy can become part of daily clinical practice. In addition, we discuss new strategies being developed for induction of donor-specific tolerance in solid organ transplantation with the clinical aims of prolonged graft survival and minimization of immunosuppression.

Distinct Immune Homeostasis Remodeling Patterns after HLA-Matched and Haploidentical Transplantation

The homeostatic immune system in a healthy state represents a dynamic balance between immunogenicity and immune tolerance. Allogenic hematopoietic stem cell transplantation (allo-HSCT) is not only a curative therapy for hematological malignancies, but also has been used to remodel the immune system to a homeostatic state, such as inducing allograft tolerance in solid organ transplantation and treating autoimmune diseases. Therefore, the homeostatic immune system after allo-HSCT represents donor-derived hematopoietic cells that reconstitute the immune system in recipients and establish tolerance to recipients. However, little is known about the responsible genes or relevant cell types that remodel immune homeostasis after allo-HSCT. Moreover, whether haploidentical stem cell transplantation (haplo-SCT) shares a similar pattern of immune homeostasis remodeling with HLA-matched sibling donor transplantation (MSDT) is also unclear. In this study, we used a comprehensive single-cell RNA-seq (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) approach coupled with bulk transcriptome (RNA-seq) and epigenome (ATAC-seq) analysis to dissect the landscape of the rebalanced homeostatic immune system in recipients after allo-HSCT (Fig. 1A). We identified 35 subpopulations in the 5 lineages based on the expression of cell type-associated genes, including the hematopoietic progenitor compartment with highly proliferative ability, T/NK lineage cells, B lineage cells, myeloid lineage cells and platelets (Fig. 1B). All cell subsets were present in both donors and recipients, which indicated that the immune system was successfully reconstituted in the recipients after allo-HSCT (Fig. 1B). Moreover, we found HSPC compartments adequately reconstitute in those recipients who have rebalanced to homeostasis after allo-HSCT. Although all immune cell subpopulations from donors were reconstituted in the paired recipients, there were different patterns of immune subpopulation distributions in recipients after allo-HSCT. In MSDT, donor-derived immune cell reconstitution in recipients as similar to that in their paired donors, while in haplo-SCT, there were low levels of CD4+ and CD8+ naïve T cells, Tcm cells, CD4+ Tem cells and CD4 Treg cells and high levels of CD4+ Teff cells, CD8+ Teff cells, CD8+ Tem cells and CD8+ Trp cells in recipients compared with their paired donors (Fig. 1C). We observed that memory-like CD8 T cells with immunoregulatory functions (CD8 regulatory precursors, CD8 Trp) were expanded in haplo-SCT recipients. CD8 Trp showed similar transcriptional profiling compared with CD4 Treg indicated that increased CD8 Trp subpopulations might act as regulatory cells and contribute to maintaining the tolerance of the donor-derived immune system in recipients of haplo-SCT. To investigate the dynamic changes in TCR clonotype after allo-HSCT, we integrated the analyzed single-cell transcriptomes and TCR sequences. We found that the dominant clonotype of CD8 Teff cells in MSDT recipients is the clonotype consistent with that in their paired donors, while the dominant clonotype of CD8 Teff cells in haplo-SCT recipients is the recipient's unique clonotype and educated by recipient thymic with high ZNF683 expression (Fig. 1D-E). Combined analysis of bulk CD8 T cell RNA-seq and ATAC-seq, we found that transcription factor ZNF683 was specifically activated in CD8+ T cells from haplo-SCT recipients compared with their paired donors (Fig. 1F). In vitro experiments indicated that a high expression level of ZNF683 in human primary T cells promoted inactivation of CD8+ T cells, maintained CD8+ T-cell replicative capacity and inhibited CD8+ T-cell apoptosis, which might suppress CD8 T cell senescence and corelated with CD8 T cell persistence. Moreover, ZNF683 could be downregulated when CD8+ T cells were activated, which suggests that ZNF683 might be a checkpoint of long-lived quiescent CD8+ T cells. In summary, our study provides a rich dataset of cellular states, gene programs, and T-cell repertoires in recipients whose donor-derived immune system rebalanced toward homeostasis after allo-HSCT. Understanding the molecular mechanisms underlying different patterns of immune homeostasis remodeling in different transplantation models will help us provide more precise medical care for patients who receive allo-HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical trials for renal allograft tolerance induction through combined hematopoietic stem cell transplantation: A narrative review.

During the last four decades, development of effective immunosuppressants has significantly improved short-term results of organ transplantation. However, long-term results have not been satisfactory due to chronic rejection or complications caused by immunosuppressive drugs. Therefore, induction of immunological tolerance of the transplanted organ is considered essential to improve the long-term results. Despite numerous tolerance strategies that have been successful in murine models, inducing hematopoietic chimerism through donor hematopoietic stem cell transplantation is the only method that reproducibly induces kidney allograft tolerance in nonhuman primates or humans. Combining kidney and hematopoietic stem cell transplantation to achieve allograft tolerance has now been attempted with different chimerism strategies. This review summarizes the status of current clinical trials on the induction of allograft tolerance. We also summarize recent studies to extend the chimerism approach to deceased donor transplant recipients.

Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote liver allograft tolerance in humans

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregs invivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. We conducted a clinical trial in stable liver transplant recipients 2-6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.gov NCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. The study is registered at ClinicalTrials.gov (NCT02949492). The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

231.2: Selective Bcl-2 Inhibition Promotes Hematopoietic Chimerism and Allograft Tolerance Without Myelosuppressive Complications

Introduction: Hematopoietic stem cell transplantation (HSCT) has been an only approach that reproducibly achieved renal allograft tolerance in humans. However, severe myelosuppression and toxicities after myeloablative conditioning have hampered wider clinical application. To achieve hematopoietic stem cell (HSC) engraftment, it is essential to make space in the bone marrow (BM) niches by depleting host HSCs. This has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. Therefore, a novel approach more selectively capable of depleting HSCs is needed to widen the clinical application of HSCT. Based on murine studies that showed successful induction of mixed chimerism and allograft tolerance without irradiation by Bcl-2 inhibition, we evaluated an FDA approved Bcl-2 inhibitor (Venetoclax, Vntx) in our mixed chimerism inducing conditioning regimen. Methods: Cynomolgus monkeys received kidney and bone marrow transplantation after conditioned with various nonmyeloablative regimens which included total body irradiation (TBI), thymic irradiation (TI) and anti-thymocyte globulin (ATG), followed by a short post-transplant course of costimulatory blockade (CoB) and Cyclosporine. The study groups were consisted of 3Gy TBI, no Vntx (Group A), 1.5Gy TBI, no Vntx (Group B), TBI 1.5Gy with Vntx(10 mg/kg x 11 on days -4 to 6) (Group C), TBI 1.5Gy with Vntx but Belatacept as CoB (Group D), no TBI with Vntx (Group E), no TI with Vntx (Group F), no CoB with Vntx (Group G). Results: Most recipients treated with 3Gy TBI (Group A) developed mixed chimerism and long-term immunosuppression (I.S.)-free renal allograft survival (Table 1), but suffered from transient but severe pancytopenia. With reduced TBI (1.5Gy), all recipients failed to develop chimerism, and 4/5 developed acute rejection. By adding Vntx, despite the dose of TBI being reduced to half (1.5Gy), all six recipients (Group C) developed significantly superior chimerism (Figure 1) than regimens without Bcl-2 inhibition (Groups A and B), and 5/6 achieved long-term I.S.-free allograft without pancytopenia (Figure 2). With Belatacept in place of anti-CD154 mAb, 2/3 achieved chimerism and long-term I.S. free allograft survival. The current studies also revealed that a minimal non-toxic dose of TBI, TI and CoB remain to be essential for long-term I.S.-free renal allograft survival. Without TBI (Group E) or CoB (Group G), the recipients failed to develop chimerism and rejected allografts despite administration of Vntx. Without TI (Group F), all recipients developed mixed chimerism but failed to achieve tolerance. Conclusion: Bcl-2 inhibition with Vntx significantly promoted mixed chimerism and renal allograft tolerance without myelosuppressive complications. A minimal non-toxic dose of TBI, TI, and CoB were still required for chimerism and allograft tolerance induction even with Bcl-2 inhibition.

Pharmacogenomics of mycophenolic acid in kidney transplantation: Contribution of immune response-related genes

Mycophenolic acid (MPA) inhibits IMPDH, involved in the guanosine nucleotides synthesis, and prevents DNA replication in immune cells. The repression of cell and humoral immunity by MPA induces allograft tolerance preventing acute rejection in solid organ transplantation. MPA is an effective and safe drug, but genetic and non-genetic factors have been implicated in the interindividual variability of drug response. Several studies have shown the impact of variants of pharmacokinetics or pharmacodynamics-related genes on MPA response in kidney transplantation. This review explored further the influence of genes involved in the immune response on clinical outcomes of kidney recipients on short- or long-term MPA treatment. Variants in genes related to T cell activation (CD28, CTL4, ICOS, PDPC1), pro-inflammatory cytokines (IL2, IL6, IL12A, IL12B, TNF, IFNG), immunomodulatory cytokines (IL4, IL10, TGFB1), and innate immune response (CD14, TLR2, TLR4) were shown to be associated with increased risk of acute rejection, graft function or survival, chronic graft nephropathy, viral infections or MPA-induced myelotoxicity. Some of the significant pharmacogenetic associations were confirmed by meta-analyses of kidney transplantation. These findings are suggestive that variants in immune response-related genes contribute to the variability of MPA response, and have potential application as biomarkers of acute rejection in kidney transplantation.

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Dendritic Cell-Mediated Regulation of Liver Ischemia-Reperfusion Injury and Liver Transplant Rejection.

Liver allograft recipients are more likely to develop transplantation tolerance than those that receive other types of organ graft. Experimental studies suggest that immune cells and other non-parenchymal cells in the unique liver microenvironment play critical roles in promoting liver tolerogenicity. Of these, liver interstitial dendritic cells (DCs) are heterogeneous, innate immune cells that appear to play pivotal roles in the instigation, integration and regulation of inflammatory responses after liver transplantation. Interstitial liver DCs (recruited in situ or derived from circulating precursors) have been implicated in regulation of both ischemia/reperfusion injury (IRI) and anti-donor immunity. Thus, livers transplanted from mice constitutively lacking DCs into syngeneic, wild-type recipients, display increased tissue injury, indicating a protective role of liver-resident donor DCs against transplant IRI. Also, donor DC depletion before transplant prevents mouse spontaneous liver allograft tolerance across major histocompatibility complex (MHC) barriers. On the other hand, mouse liver graft-infiltrating host DCs that acquire donor MHC antigen via “cross-dressing”, regulate anti-donor T cell reactivity in association with exhaustion of graft-infiltrating T cells and promote allograft tolerance. In an early phase clinical trial, infusion of donor-derived regulatory DCs (DCreg) before living donor liver transplantation can induce alterations in host T cell populations that may be conducive to attenuation of anti-donor immune reactivity. We discuss the role of DCs in regulation of warm and liver transplant IRI and the induction of liver allograft tolerance. We also address design of cell therapies using DCreg to reduce the immunosuppressive drug burden and promote clinical liver allograft tolerance.

The mTOR Deficiency in Monocytic Myeloid-Derived Suppressor Cells Protects Mouse Cardiac Allografts by Inducing Allograft Tolerance.

BackgroundMyeloid-derived suppressor cells (MDSCs) can prevent allograft rejection and induce immune tolerance in transplantation models. Previous studies have demonstrated that inhibition of mTOR signaling can enhance the MDSC protective effect in heart transplantation (HTx) by promoting MDSC expansion. In addition, mTOR inhibition is related to autophagy. The present study investigated the protective mechanism of mTOR-deficient monocytic MDSCs (M-MDSCs) in mouse HTx.MethodsMyeloid-specific mTOR conditional knockout mice were generated to obtain mTOR−/− M-MDSCs. The proliferation and immunosuppressive function of mTOR−/− M-MDSCs were determined by flow cytometry and T cell proliferation assays. The mTOR−/− M-MDSC intracellular autophagy levels were determined using western blotting and electron microscopy. RNAseq analysis was performed for wild-type (WT) and mTOR−/− M-MDSCs. Allogeneic HTx mouse model was established and treated with WT or mTOR−/− M-MDSCs. Enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry assays were performed to determine WT and mTOR−/− M-MDSC-induced immune tolerance.ResultsThe mTOR deficiency promoted M-MDSC differentiation and enhanced intracellular autophagy levels in vivo and in vitro. mTOR deficiency also enhanced the immunosuppressive function of M-MDSCs. In addition, infusing with WT and mTOR−/− M-MDSCs prolonged cardiac allograft survival and established immune tolerance in recipient mice by inhibiting T cell activation and inducing regulatory T cells.ConclusionmTOR deficiency enhances the immunosuppressive function of M-MDSCs and prolongs mouse cardiac allograft survival.

Tolerance, immunosuppression, and immune modulation: impacts on lung allograft survival.

Immune responses following lung transplantation continue to result in high rates of allograft failure and rejection, and current immunosuppression does not address the unique immunologic properties of the lung. Here, we review recent studies on lung allograft tolerance and alloimmunity and discuss implications for immunosuppression. Processes governing tolerance and alloimmunity in lung allografts differ from other solid organs. Recent studies have suggested that allorecognition is regulated at the level of the lung graft. Furthermore, certain cell populations essential for lung allograft tolerance may facilitate rejection in other organs. Induction of lung allograft tolerance is associated with the formation of tertiary lymphoid organs, which are enriched in regulatory T cells and play an important role in preventing rejection. Recent discoveries regarding alloactivation and the regulation of tolerance following lung transplantation have introduced exciting potential avenues for the development of lung-specific immunosuppression.

Using Selective Bcl2 Inhibition to Induce Cardiac Allograft Tolerance

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. B cell lymphoma 2 (Bcl2) inhibition has allowed for durable mixed chimerism with reduced conditioning in the murine skin transplant model. We investigated whether the Bcl2 inhibitor ABT199 could enhance mixed chimerism and induce cardiac allograft tolerance in the absence of kidney cotransplantation. <h3>Methods</h3> Fifteen cynomolgus recipients underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplantation. Group 1 recipients received heart alone (n=5), Group 2 recipients received heart and kidney cotransplants (n=7), and in Group 3, heart recipients received ABT199 in lieu of kidney cotransplants (n=3).Group 1 and 2 received 3Gy total body irradiation (TBI) while Group 3 received 1.5Gy TBI. All recipients underwent a 28-day course of immunosuppression with a calcineurin inhibitor and anti-CD154 mAb after which all immunosuppression was stopped. <h3>Results</h3> All five Group 1 recipients demonstrated cellular rejection (ISHLT 3R) by 96 days post bone marrow transplant (pBMTx). In contrast, no Group 2 recipients showed early rejection and 6/7 survived >400 days pBMTx. The first two Group 3 recipients developed GvHD and succumbed by days 59 and 68 pBMTx without evidence of rejection. However, after reducing the size of the donor BMT, the third recipient is now 76 days pBMTx with excellent allograft contractility and no evidence of GVHD. Mean peak lymphoid chimerism was 10% on day 26 pBMTx for Group 1, 26% on day 33 pBMTx for Group 2, and 57% on day 54 pBMTx for Group 3. (Figure 1) <h3>Conclusion</h3> After further refinement of the conditioning regimen, ABT199 may allow for the induction of heart allograft tolerance without the need for kidney co-transplantation and with a reduction in TBI thereby improving safety.

Detection and Monitoring of Regulatory Immune Cells Following Their Adoptive Transfer in Organ Transplantation.

Application of cell-based immunotherapy in organ transplantation to minimize the burden of immunosuppressive medication and promote allograft tolerance has expanded significantly over the past decade. Adoptively transferred regulatory immune cells prolong allograft survival and transplant tolerance in pre-clinical models. Many cell products are currently under investigation in early phase human clinical trials designed to assess feasibility and safety. Despite rapid advances in manufacturing practices, defining the appropriate protocol that will optimize in vivo conditions for tolerance induction remains a major challenge and depends heavily on understanding the fate, biodistribution, functional stability and longevity of the cell product after administration. This review focuses on in vivo detection and monitoring of various regulatory immune cell types administered for allograft tolerance induction in both pre-clinical animal models and early human clinical trials. We discuss the current status of various non-invasive methods for tracking regulatory cell products in the context of organ transplantation and implications for enhanced understanding of the therapeutic potential of cell-based therapy in the broad context of control of immune-mediated inflammatory disorders.

Galectin-9 is required for endometrial regenerative cells to induce long-term cardiac allograft survival in mice

BackgroundEndometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cells, were identified as an attractive candidate for immunoregulation and induction of cardiac allograft tolerance. However, the underlying mechanisms of ERCs in immune regulation still remain largely unclear. The present study is designed to determine whether the expression of Galectin-9 (Gal-9), a soluble tandem-repeat member of the galectin family, is crucial for ERC-based immunomodulation.MethodsIn this study, we measured Gal-9 expression on ERCs and then co-cultured Gal-9-ERCs, ERCs, and ERCs+lactose (Gal-9 blocker) with activated C57BL/6-derived splenocytes. Furthermore, we performed mouse heart transplantation between BALB/c (H-2d) donor and C57BL/6 (H-2b) recipient. ERCs were administrated 24 h after the surgery, either alone or in combination with rapamycin.ResultsOur data demonstrate that ERCs express Gal-9, and this expression is increased by IFN-γ stimulation in a dose-dependent manner. Moreover, both in vitro and in vivo results show that Gal-9-ERC-mediated therapy significantly suppressed Th1 and Th17 cell response, inhibited CD8+ T cell proliferation, abrogated B cell activation, decreased donor-specific antibody production, and enhanced the Treg population. The therapeutic effect of ERCs was further verified by their roles in prolonging cardiac allograft survival and alleviating graft pathological changes.ConclusionsTaken together, these data indicate that Gal-9 is required for ERC-mediated immunomodulation and prevention of allograft rejection.

Unraveling the Crucial Roles of FoxP3+ Regulatory T Cells in Vascularized Composite Allograft Tolerance Induction and Maintenance.

The role of regulatory T cells (Treg) in tolerance induction of vascularized composite allotransplantation (VCA) remains unclear. This study was designed to examine characteristics of Treg after VCA and their capacity to rescue allografts from rejection. Osteomyocutaneous allografts were transplanted from Balb/c to C57BL/6 mice. All mice received costimulatory blockade and a short course of rapamycin. To elucidate the role of Treg for tolerance induction, Treg depletion was performed at postoperative day (POD) 0, 30, or 90. To assess capacity of Treg to rescue allografts from rejection, an injection of 2 × 106 Treg isolated from tolerant mice was applied. Eighty percent of VCA recipient mice using costimulatory blockade and rapamycin regimen developed tolerance. The tolerant recipients had a higher ratio of circulating Treg to effector T cells and elevated interleukin-10 at POD 30. A significantly higher rejection rate was observed when Treg were depleted at POD 30. But Treg depletion at POD 90 had no effect on tolerance. Treg from tolerant recipients showed stronger suppressive potential and the ability to rescue allografts from rejection. Furthermore, transplanted Treg-containing skin grafts from tolerant mice delayed rejection elicited by adoptively transferred effector T cells to Rag2-/- mice. Circulating Treg are crucial for inducing VCA tolerance in the early posttransplant phase, and allograft-residing Treg may maintain tolerance. Treg may, therefore, serve as a potential cellular therapeutic to improve VCA outcomes.

Bacterial products in donor airways prevent the induction of lung transplant tolerance.

Although postoperative bacterial infections can trigger rejection of pulmonary allografts, the impact of bacterial colonization of donor grafts on alloimmune responses to transplanted lungs remains unknown. Here, we tested the hypothesis that bacterial products present within donor grafts at the time of implantation promote lung allograft rejection. Administration of the toll-like receptor 2 (TLR2) agonist Pam3 Cys4 to Balb/c wild-type grafts triggered acute cellular rejection after transplantation into B6 wild-type recipients that received perioperative costimulatory blockade. Pam3 Cys4 -triggered rejection was associated with an expansion of CD8+ T lymphocytes and CD11c+ CD11bhi MHC (major histocompatibility complex) class II+ antigen-presenting cells within the transplanted lungs. Rejection was prevented when lungs were transplanted into TLR2-deficient recipients but not when MyD88-deficient donors were used. Adoptive transfer of B6 wild-type monocytes, but not T cells, following transplantation into B6 TLR2-deficient recipients restored the ability of Pam3 Cys4 to trigger acute cellular rejection. Thus, we have demonstrated that activation of TLR2 by a bacterial lipopeptide within the donor airways prevents the induction of lung allograft tolerance through a process mediated by recipient-derived monocytes. Our work suggests that donor lungs harboring bacteria may precipitate an inflammatory response that can facilitate allograft rejection.