Blocking CCL8-CCR8-Mediated Early Allograft Inflammation Improves Kidney Transplant Function.

BACKGROUND: In kidney transplantation, early allograft inflammation impairs long-term allograft function. However, precise mediators of early kidney allograft inflammation are unclear, making it challenging to design therapeutic interventions. METHODS: We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS: Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period, they were also induced to express a high level of Ccl8, which, in turn, promoted recipient monocyte graft infiltration, their differentiation to resident macrophages, and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed, including CD4, CD8, and γδ T cells. Consequently, blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS: Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation.

Acute murine cytomegalovirus disrupts established transplantation tolerance and causes recipient allo-sensitization.

We have previously shown that acute cytomegalovirus (CMV) infection disrupts the induction of transplantation tolerance. However, what impact acute CMV infection would have on the maintenance of established tolerance and on subsequent recipient allo-sensitization is a clinically important unanswered question. Here we used an allogeneic murine islet transplantation tolerance model to examine the impact of acute CMV infection on: (a) disruption of established transplantation tolerance during tolerance maintenance; and (b) the possibility of recipient allo-sensitization by CMV-mediated disruption of stable tolerance. We demonstrated that acute CMV infection abrogated transplantation tolerance during the maintenance stage in 50%-60% recipients. We further demonstrated that acute CMV infection-mediated tolerance disruption led to recipient allo-sensitization by reverting the tolerant state of allo-specific T cells and promoting their differentiation to allo-specific memory cells. Consequently, a second same-donor islet allograft was rejected in an accelerated fashion by these recipients. Our study therefore supports close monitoring for allo-sensitization in previously tolerant transplant recipients in whom tolerance maintenance is disrupted by an episode of acute CMV infection.

Murine cytomegalovirus dissemination but not reactivation in donor-positive/recipient-negative allogeneic kidney transplantation can be effectively prevented by transplant immune tolerance.

Cytomegalovirus (CMV) reactivation from latently infected donor organs post-transplantation and its dissemination cause significant comorbidities in transplant recipients. Transplant-induced inflammation combined with chronic immunosuppression has been thought to provoke CMV reactivation and dissemination, although sequential events in this process have not been studied. Here, we investigated this process in a high-risk donor CMV-positive to recipient CMV-negative allogeneic murine kidney transplantation model. Recipients were either treated with indefinite immunosuppression or tolerized in a donor-specific manner. Untreated recipients served as controls. Kidney allografts from both immunosuppressed and tolerized recipients showed minimal alloimmunity-mediated graft inflammation and normal function for up to day 60 post-transplantation. However, despite the absence of such inflammation in the immunosuppressed and tolerized groups, CMV reactivation in the donor positive kidney allograft was readily observed. Interestingly, subsequent CMV replication and dissemination to distant organs only occurred in immunosuppressed recipients in which CMV-specific CD8 T cells were functionally impaired; whereas in tolerized recipients, host anti-viral immunity was well-preserved and CMV dissemination was effectively prevented. Thus, our studies uncoupled CMV reactivation from its dissemination, and underscore the potential role of robust transplantation tolerance in preventing CMV diseases following allogeneic kidney transplantation.

Donor apoptotic cell-based therapy for effective inhibition of donor-specific memory T and B cells to promote long-term allograft survival in allosensitized recipients.

Allosensitization constitutes a major barrier in transplantation. Preexisting donor-reactive memory T and B cells and preformed donor-specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long-term immunosuppression-free allograft survival. We demonstrate that donor-specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti-CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen-presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti-CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long-term islet allograft survival in such recipients.

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft-infiltrating Th17 cells and activated B cells.

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig-to-humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45+ cells comprising activated human B cells, CD4+ CD44+ IL-17+ Th17 cells, and CD68+ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long-term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI-SP), and peri-transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI-SP treatment in controlling human immune cells in promoting long-term islet xenograft survival.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Apoptotic cell-based therapies for promoting transplantation tolerance.

PURPOSE OF REVIEW: This article is aimed to provide readers with an updated review on the applicability, efficacy, and challenges of employing donor apoptotic cell-based therapies to promote transplantation tolerance in various experimental and clinical settings. RECENT FINDINGS: Recently, donor apoptotic cell-based therapies have been employed in various models of cell (including pancreatic islets and bone marrow hematopoietic stem cells) and solid organ (heart and kidney) transplantation to promote donor-specific tolerance. Published data, thus far, have revealed a high potential of this approach in inducing robust transplantation tolerance. Recent clinical trials have also underscored the safety and potential efficacy of this approach in alleviating graft-versus-host disease (GVHD) in bone marrow transplantation (BMT). Host factors including prior allo-sensitization and opportunistic infections pose major obstacles in establishing transplantation tolerance employing this strategy. However, emerging data provide strategies for overcoming such obstacles in these clinically relevant settings. SUMMARY: Donor apoptotic cell therapy is an emerging strategy in promoting transplantation tolerance, with recent data emphasizing its efficacy and applicability for transplantation tolerance in the clinic.

Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNß, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNß, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNß antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNß, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.

A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model, in which HSCs are depleted from the normal liver. METHODS: We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 µl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 µg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment. RESULTS: CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64-72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice. CONCLUSIONS: HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.

Selective expansion of allogeneic regulatory T cells by hepatic stellate cells: role of endotoxin and implications for allograft tolerance.

Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines and expressing Ag-presenting and T cell coregulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally occurring immunosuppressive CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4(+) T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs upregulated Fas (CD95) expression on conventional CD4(+) T cells and induced their apoptosis in a Fas/Fas ligand-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell-cell contact and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80, and CD86 and stimulated the production of IL-1α, IL-1ß, IL-6, IL-10 and TNF-α by HSCs. Interestingly, production of IL-1α, IL-1ß, IL-6, and TNF-α was strongly inhibited, but that of IL-10 enhanced in LPS-pretreated HSC/Treg cocultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxin-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs and reducing inflammation and alloimmunity.

Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO.

Hepatic stellate cells (HSCs) are critical for hepatic wound repair and tissue remodeling. They also produce cytokines and chemokines that may contribute to the maintenance of hepatic immune homeostasis and the inherent tolerogenicity of the liver. The functional relationship between HSCs and the professional migratory APCs in the liver, that is, dendritic cells (DCs), has not been evaluated. In this article, we report that murine liver DCs colocalize with HSCs in vivo under normal, steady-state conditions, and cluster with HSCs in vitro. In vitro, HSCs secrete high levels of DC chemoattractants, such as MΙP-1α and MCP-1, as well as cytokines that modulate DC activation, including TNF-α, IL-6, and IL-1ß. Culture of HSCs with conventional liver myeloid (m) DCs resulted in increased IL-6 and IL-10 secretion compared with that of either cell population alone. Coculture also resulted in enhanced expression of costimulatory (CD80, CD86) and coinhibitory (B7-H1) molecules on mDCs. HSC-induced mDC maturation required cell-cell contact and could be blocked, in part, by neutralizing MΙP-1α or MCP-1. HSC-induced mDC maturation was dependent on activation of STAT3 in mDCs and, in part, on HSC-secreted IL-6. Despite upregulation of costimulatory molecules, mDCs conditioned by HSCs demonstrated impaired ability to induce allogeneic T cell proliferation, which was independent of B7-H1, but dependent upon HSC-induced STAT3 activation and subsequent upregulation of IDO. In conclusion, by promoting IDO expression, HSCs may act as potent regulators of liver mDCs and function to maintain hepatic homeostasis and tolerogenicity.

Single cell transcriptomics of mouse kidney transplants reveals a myeloid cell pathway for transplant rejection.

Myeloid cells are increasingly recognized as major players in transplant rejection. Here, we used a murine kidney transplantation model and single cell transcriptomics to dissect the contribution of myeloid cell subsets and their potential signaling pathways to kidney transplant rejection. Using a variety of bioinformatic techniques, including machine learning, we demonstrate that kidney allograft-infiltrating myeloid cells followed a trajectory of differentiation from monocytes to proinflammatory macrophages, and they exhibited distinct interactions with kidney allograft parenchymal cells. While this process correlated with a unique pattern of myeloid cell transcripts, a top gene identified was Axl, a member of the receptor tyrosine kinase family Tyro3/Axl/Mertk (TAM). Using kidney transplant recipients with Axl gene deficiency, we further demonstrate that Axl augmented intragraft differentiation of proinflammatory macrophages, likely via its effect on the transcription factor Cebpb. This, in turn, promoted intragraft recruitment, differentiation, and proliferation of donor-specific T cells, and it enhanced early allograft inflammation evidenced by histology. We conclude that myeloid cell Axl expression identified by single cell transcriptomics of kidney allografts in our study plays a major role in promoting intragraft myeloid cell and T cell differentiation, and it presents a potentially novel therapeutic target for controlling kidney allograft rejection and improving kidney allograft survival.

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite.

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.

Emerging approaches and technologies in transplantation: the potential game changers.

Newly emerging technologies are rapidly changing conventional approaches to organ transplantation. In the modern era, the key challenges to transplantation include (1) how to best individualize and possibly eliminate the need for life-long immunosuppression and (2) how to expand the donor pool suitable for human transplantation. This article aims to provide readers with an updated review of three new technologies that address these challenges. First, single-cell RNA sequencing technology is rapidly evolving and has recently been employed in settings related to transplantation. The new sequencing data indicate an unprecedented cellular heterogeneity within organ transplants, as well as exciting new molecular signatures involved in alloimmune responses. Second, sophisticated nanotechnology platforms provide a means of therapeutically delivering immune modulating reagents to promote transplant tolerance. Tolerogenic nanoparticles with regulatory molecules and donor antigens are capable of targeting host immune responses with tremendous precision, which, in some cases, results in donor-specific tolerance. Third, CRISPR/Cas9 gene editing technology has the potential to precisely remove immunogenic molecules while inserting desirable regulatory molecules. This technology is particularly useful in generating genetically modified pigs for xenotransplantation to solve the issue of the shortage of human organs. Collectively, these new technologies are positioning the transplant community for major breakthroughs that will significantly advance transplant medicine.

Murine CMV induces type 1 IFN that impairs differentiation of MDSCs critical for transplantation tolerance.

Clinical tolerance without immunosuppression has now been achieved for organ transplantation, and its scope will likely continue to expand. In this context, a previously understudied and now increasingly relevant area is how microbial infections might affect the efficacy of tolerance. A highly prevalent and clinically relevant posttransplant pathogen is cytomegalovirus (CMV). Its impact on transplantation tolerance and graft outcomes is not well defined. Employing a mouse model of CMV (MCMV) infection and allogeneic pancreatic islet transplantation in which donor-specific tolerance was induced by infusing donor splenocytes rendered apoptotic by treatment with ethylenecarbodiimide, we investigated the effect of CMV infection on transplantation tolerance induction. We found that acute MCMV infection abrogated tolerance induction and that this abrogation correlated with an alteration in the differentiation and function of myeloid-derived suppressor cells (MDSCs). These effects on MDSCs were mediated in part through MCMV induced type 1 interferon (IFN) production. During MCMV infection, the highly immunosuppressive Gr1HI-granulocytic MDSCs were markedly reduced in numbers, and the accumulating Ly6CHI-monocytic cells lost their MDSC-like function but instead acquired an immunostimulatory phenotype to cross-present alloantigens and prime alloreactive CD8 T cells. Consequently, the islet allograft exhibited an altered effector to regulatory T-cell ratio that correlated with the ultimate graft demise. Blocking type 1 IFN signaling during MCMV infection rescued MDSC populations and partially restored transplantation tolerance. Our mechanistic studies now provide a solid foundation for seeking effective therapies for promoting transplantation tolerance in settings of CMV infection.

Harnessing Apoptotic Cells for Transplantation Tolerance: Current Status and Future Perspectives.

PURPOSE OF REVIEW: The use of donor apoptotic cells is an emerging therapy for inducing transplantation tolerance. In this review, we will discuss current understanding of mechanisms of this approach, as well as crucial aspects necessary for successful translation of this approach to clinical transplantation. RECENT FINDINGS: Transplantation tolerance by donor apoptotic cells is mediated by their homeostatic interaction with recipient phagocytes, and subsequent expansion of suppressor cell populations as well as inhibition of effector T cells via deletion and anergy. To ensure their tolerogenicity, it is critical to procure non-stressed donor cells, and to induce and arrest their apoptosis at the appropriate stage prior to their administration. Equally important is the monitoring of dynamics of recipient immunological status, and its influences on tolerance efficacy and longevity. Emerging concepts and technologies may significantly streamline tolerogen manufacture and delivery of this approach, and smooth its transition to clinical application. SUMMARY: Hijacking homeostatic clearance of donor apoptotic cells is a promising strategy for transplantation tolerance. Timing is now mature for concerted efforts for transitioning this strategy to clinical transplantation.

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice.

BACKGROUND: Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge. METHODS: The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine antidonor xenogeneic immune responses during early and late rejection and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival. RESULTS: We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220 B cells and a robust antipig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-γ production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide-treated splenocytes significantly inhibited antidonor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in approximately 65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 and 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust antidonor IFN-γ, but not IL-17, response. CONCLUSIONS: These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of antixenogeneic immune responses.