Wild-type Grafts Research Articles

Blockade of VIP-Signaling Enhanced Anti-Leukemic Activity in Murine Allogeneic BMT without Significantly Increased GvHD.

Abstract 3001 Background and Objective:Vasoactive intestinal peptide (VIP) has potent immune-suppressive activity and can generate tolerogenic dendritic cells (DC) in vitro that block graft versus host disease (GvHD) in mouse model of BMT. We have previously published that absence of VIP signaling dramatically decreases PD-1 expression on activated CD8 T-cells and increases cellular antiviral immunity (JI 2011, 187:1057). To determine whether blockade of VIP-signaling enhances the graft-versus-leukemia (GvL) activity of donor T-cells in an allogeneic BMT model, we treated tumor bearing B6B′10BR allogeneic transplant recipients with a short course of daily s.c. injections of a small molecule VIP antagonist - VIPhyb or used VIP-knockout (VIP-KO) mice as BM donors. Methods:Recipient mice were inoculated with luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) by i.v. injection one day after lethal total body irradiation, then transplanted with the combination of 5 × 106 T cell-depleted BM (TCD-BM) plus splenocytes from either VIP-KO mice or wild-type (WT) littermates two days after irradiation. One group transplanted with WT BM and splenocytes received daily injections of 10 μg VIPhyb for one week; another group received saline injections. Survival, GvHD clinical sores (body weight, activity, posture, fur texture and skin), and bioluminescence imaging (BLI) were collected daily, twice a week, and weekly, respectively. Results:Transplantation of low dose (0.5 × 106) splenocytes from VIP-KO donors or low dose WT splenocytes in conjunction with VIPhyb-treatment dramatically improved tumor-free survival in the B6B′10BR allogeneic BMT model compared with PBS-treated recipients of WT grafts (Figure 1). The best overall survival (70%) and lowest number of mice with detectable tumor (10%) were seen in the VIPhyb-treated group. VIPhyb-treated mice did not have increased GvHD as assessed by clinical scoring. Recipients of VIP-KO grafts had 40% survival with no detectable tumors by BLI, and were without significant GvHD by clinical scoring. In contrast, the recipients transplanted with TCD-BM alone (without added splenocytes) and recipients that received 0.5 × 106 splenocytes and TCD-BM from WT donors and treated with PBS had increased tumor growth detected by BLI following BMT, and all of the mice died by 2 months post-BMT.Moreover, in non-tumor bearing mice transplanted with an intermediate dose (1 × 106) of splenocytes, survival was not different among recipients engrafted with VIP-KO BM and T-cells (84 ± 6 %), WT BM and T-cells treated with VIPhyb (89 ± 8 %) and WT BM and T-cells treated with PBS (94 ± 5 %). A similar enhancement of the GvL effect and a corresponding survival advantage for VIP-signaling blockade was seen in tumor-bearing transplant recipients of TCD-BM plus 1 × 106 splenocytes, with significantly better survival among recipients of VIP-KO donor cells (50%), recipients of WT cells treated with VIPhyb (60%) compared with recipients of WT cells treated with PBS (20%; p=0.04). Furthermore, in non-tumor bearing mice that received a higher dose (3 × 106) of splenocytes, recipients of VIP-KO BM and recipients of WT BM treated with VIPhyb had no significant increase in GvHD compared with recipients of WT BM treated with PBS (66 ± 9 %, 71 ± 8 % and 71 ± 8 % survival at 80 days, respectively). The mechanism by which administration of a VIP antagonist enhanced anti-tumor immunity includes the effect of blocking VIP-signaling induction of cAMP, leading to fewer Treg and fewer tolerogenic DC. Of note, blocking VIP-signaling led to significant decreases in expression of PD-1 and PD-L1 on CD8+ T-cells and DCs, respectively. Conclusion:Treatment with a small molecule antagonist of VIP-signaling, VIPhyb, dramatically increased anti-leukemic activity of donor T-cells without significantly increased GvHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Protective Effects of Transgenic Human Endothelial Protein C Receptor Expression in Murine Models of Transplantation

Thrombosis and inflammation are major obstacles to successful pig-to-human solid organ xenotransplantation. A potential solution is genetic modification of the donor pig to overexpress molecules such as the endothelial protein C receptor (EPCR), which has anticoagulant, anti-inflammatory and cytoprotective signaling properties. Transgenic mice expressing human EPCR (hEPCR) were generated and characterized to test this approach. hEPCR was expressed widely and its compatibility with the mouse protein C pathway was evident from the anticoagulant phenotype of the transgenic mice, which exhibited a prolonged tail bleeding time and resistance to collagen-induced thrombosis. hEPCR mice were protected in a model of warm renal ischemia reperfusion injury compared to wild type (WT) littermates (mean serum creatinine 39.0 ± 2.3 μmol/L vs. 78.5 ± 10.0 μmol/L, p < 0.05; mean injury score 31 ± 7% vs. 56 ± 5%, p < 0.05). Heterotopic cardiac xenografts from hEPCR mice showed a small but significant prolongation of survival in C6-deficient PVG rat recipients compared to WT grafts (median graft survival 6 vs. 5 days, p < 0.05), with less hemorrhage and edema in rejected transgenic grafts. These data indicate that it is possible to overexpress EPCR at a sufficient level to provide protection against transplant-related thrombotic and inflammatory injury, without detrimental effects in the donor animal.

ICAM-1 Upregulation in Ethanol-Induced Fatty Murine Livers Promotes Injury and Sinusoidal Leukocyte Adherence after Transplantation

Background. Transplantation of ethanol-induced steatotic livers causes increased graft injury. We hypothesized that upregulation of hepatic ICAM-1 after ethanol produces increased leukocyte adherence, resulting in increased generation of reactive oxygen species (ROS) and injury after liver transplantation (LT). Methods. C57BL/6 wildtype (WT) and ICAM-1 knockout (KO) mice were gavaged with ethanol (6 g/kg) or water. LT was then performed into WT recipients. Necrosis and apoptosis, 4-hydroxynonenal (4-HNE) immunostaining, and sinusoidal leukocyte movement by intravital microscopy were assessed. Results. Ethanol gavage of WT mice increased hepatic triglycerides 10-fold compared to water treatment (P < 0.05). ICAM-1 also increased, but ALT was normal. At 8 h after LT of WT grafts, ALT increased 2-fold more with ethanol than water treatment (P < 0.05). Compared to ethanol-treated WT grafts, ALT from ethanol-treated KO grafts was 78% less (P < 0.05). Apoptosis also decreased by 75% (P < 0.05), and 4-HNE staining after LT was also decreased in ethanol-treated KO grafts compared to WT. Intravital microscopy demonstrated a 2-fold decrease in leukocyte adhesion in KO grafts compared to WT grafts. Conclusions. Increased ICAM-1 expression in ethanol-treated fatty livers predisposes to leukocyte adherence after LT, which leads to a disturbed microcirculation, oxidative stress and graft injury.

The mRNA of a Knotted1-like transcription factor of potato is phloem mobile

Potato Homeobox1 (POTH1) is a Knotted1-like transcription factor from the Three Amino Acid Loop Extension (TALE) superfamily that is involved in numerous aspects of development in potato (Solanum tuberosum L). POTH1 interacts with its protein partner, StBEL5, to facilitate binding to specific target genes to modulate hormone levels, mediate leaf architecture, and enhance tuber formation. In this study, promoter analyses show that the upstream sequence of POTH1 drives β-glucuronidase activity in response to light and in association with phloem cells in both petioles and stems. Because POTH1 transcripts have previously been detected in phloem cells, long-distance movement of its mRNA was tested. Using RT-PCR and transgenic potato lines over-expressing POTH1, in vitro micrografts demonstrated unilateral movement of POTH1 RNA in a rootward direction. Movement across a graft union into leaves from newly arising axillary shoots and roots of wild type stocks was verified using soil-grown tobacco heterografts. Leaves from the wild type stock containing the mobile POTH1 RNA exhibited a reduction in leaf size relative to leaves from wild type grafts. Both untranslated regions of POTH1 when fused to an expression marker β-glucuronidase, repressed its translation in tobacco protoplasts. RNA/protein binding assays demonstrated that the UTRs of POTH1 bind to two RNA-binding proteins, a polypyrimidine tract-binding protein and an alba-domain type. Conserved glycerol-responsive elements (GRE), specific to alba-domain interaction, are duplicated in both the 5' and 3' untranslated regions of POTH1. These results suggest that POTH1 functions as a mobile signal in regulating development.

Osteopontin deficiency delays inflammatory infiltration and the onset of muscle regeneration in a model of muscle injury

SUMMARYOsteopontin is secreted by skeletal muscle myoblasts and stimulates their proliferation. Expression of osteopontin in skeletal muscle is upregulated in pathological conditions including Duchenne muscular dystrophy, and recent evidence suggests that osteopontin might influence the course of this disease. The current study was undertaken to determine whether osteopontin regulates skeletal muscle regeneration. A whole muscle autografting model of regeneration in osteopontin-null and wild-type mice was used. Osteopontin expression was found to be strongly upregulated in wild-type grafts during the initial degeneration and subsequent early regeneration phases that are observed in this model. Grafted muscle from osteopontin-null mice degenerated more slowly than that of wild-type mice, as determined by histological assessment, fibre diameter and fibre number. The delayed degeneration in osteopontin-null grafts was associated with a delay in neutrophil and macrophage infiltration. Centrally nucleated (regenerating) muscle fibres also appeared more slowly in osteopontin-null grafts than in wild-type grafts. These results demonstrate that osteopontin plays a non-redundant role in muscle remodelling following injury.

Blocking Vasoactive Intestinal Peptide Signaling Enhances Anti-Viral Immunity without Increased Graft Versus Host Disease in Murine Allogeneic Bone Marrow Transplantation

Abstract 1002 Background:Vasoactive intestinal peptide (VIP) is a neuropeptide hormone and type 2 cytokine that inhibits Th1 immunity and induces the generation of regulatory T-cells. We have recently reported that non-transplanted mice “knocked-out” for VIP and syngeneic transplant recipients of VIP-knockout (KO) BM had dramatically improved survival, viral clearance, and increased numbers of specific antiviral CD8+ T-cells following murine cytomegalovirus (mCMV) infection (JI 2011. 187:1057–65). In this study, we used a small molecule VIP antagonist as well as VIP-KO mice to further investigate effects and mechanisms of VIP-signaling on antiviral immune responses in wild type (WT) non-transplanted mice and following allogeneic BMT. Methods:B10BR (CD45.2, H-2Kk) and CB6/J F1 (CD45.2, H-2Kb/d) mice were transplanted with 3 × 103 FACS-sorted hematopoietic stem cells (HSC), 5 × 104 dendritic cells (DC), and 0.3, 1, or 3 × 106 splenic T-cells either from VIP-KO (CD45.2, H-2Kb) or WT donors after myeloablative conditioning (11Gy). WT mice and BMT recipients transplanted with WT grafts were treated with daily subcutaneous injection of VIP antagonist (10 μg/100μL per mouse) or PBS for 7 days (from one day prior to infection to 6 days post-infection). BALB/C mice, B6 VIP-KO and WT littermates, as well as CB6/J F1 BMT recipients, were infected with graded doses (LD10, LD50 and LD90) of mCMV by intraperitoneal injection. Survival, viral load, antigen specific T-cells, and clinical scores of graft versus host disease (GvHD) were assessed at distinct time-points post-BMT or after mCMV infection. The expression of co-stimulatory or co-inhibitory markers (CD25, CD62L, CD69, PD-1, FoxP3, PD-L1, CD80, CD86, and MHC-II) and intracellular expression of cytokines (IL-10, IFN-γ, TNF-α, and IL-12) on T-cells and DC from the mice were measured by flow cytometry. Results:Improved survival was seen in mCMV-infected allogeneic B6→CB6/J F1 transplant recipients of VIP-KO grafts (100%) compared with recipients of WT grafts treated with PBS (40%). Allogeneic recipients of VIP-KO grafts and allogeneic recipients of WT grafts treated with VIP antagonist had increased viral clearance and enhanced in vivo killing of viral-peptide-pulsed targets compared with PBS-treated recipients of WT grafts. No difference in the incidence or severity of acute GvHD was seen in allogeneic BMT recipients of graded doses of VIP-KO versus WT splenic T-cells (0.3, 1, and 3 × 106) in murine MHC mis-matched BMT models. Allogeneic transplant recipients of VIP-KO grafts and WT grafts treated with VIP antagonist, infected with low dose mCMV, had lower levels of PD-L1 and PD-1 expression on DC and T-cells, respectively, and higher levels of CD80, CD86 and MHC-II expression on conventional DC (cDC) and plasmacytoid DC (pDC) compared with recipients of WT allografts treated with PBS. Recipients of VIP-KO grafts and recipients treated with VIP antagonist had higher-levels of IL-12+ cDC, activated CD25+/CD69+ CD4 and CD8 T-cells, and more mCMV-M45-peptide MHC-I tetramer+ CD8+ T-cells compared with recipients of WT grafts treated with PBS. Absence of VIP-signaling led to enhanced intracellular expression of IFN-γ and less IL-10 expression in T-cells from mCMV-infected recipients of VIP-KO B6→CB6/J F1 allogeneic transplants, and mCMV-infected, VIP antagonist-treated recipients of WT allogeneic transplants. In the absence of mCMV infection, the numbers of regulatory T cells (Treg) were similar among VIP-KO mice, WT mice treated with VIP antagonist, and PBS-treated WT controls. In contrast, mCMV-infected VIP-KO mice had significantly fewer Treg compared with mCMV- infected WT mice, non-infected WT mice and non-infected VIP-KO mice. Conclusion:Genetic or pharmacological blockade of VIP-signaling enhanced both innate and adaptive antiviral immune responses in allogeneic BMT recipients without significantly elevating GvHD. Selective targeting of VIP-signaling represents a novel therapeutic approach to enhance antiviral immunity in the setting of immunodeficiency and allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Tissue-intrinsic dysfunction of circadian clock confers transplant arteriosclerosis

The suprachiasmatic nucleus of the brain is the circadian center, relaying rhythmic environmental and behavioral information to peripheral tissues to control circadian physiology. As such, central clock dysfunction can alter systemic homeostasis to consequently impair peripheral physiology in a manner that is secondary to circadian malfunction. To determine the impact of circadian clock function in organ transplantation and dissect the influence of intrinsic tissue clocks versus extrinsic clocks, we implemented a blood vessel grafting approach to surgically assemble a chimeric mouse that was part wild-type (WT) and part circadian clock mutant. Arterial isografts from donor WT mice that had been anastamosed to common carotid arteries of recipient WT mice (WT:WT) exhibited no pathology in this syngeneic transplant strategy. Similarly, when WT grafts were anastamosed to mice with disrupted circadian clocks, the structural features of the WT grafts immersed in the milieu of circadian malfunction were normal and absent of lesions, comparable to WT:WT grafts. In contrast, aortic grafts from Bmal1 knockout (KO) or Period-2,3 double-KO mice transplanted into littermate control WT mice developed robust arteriosclerotic disease. These lesions observed in donor grafts of Bmal1-KO were associated with up-regulation in T-cell receptors, macrophages, and infiltrating cells in the vascular grafts, but were independent of hemodynamics and B and T cell-mediated immunity. These data demonstrate the significance of intrinsic tissue clocks as an autonomous influence in experimental models of arteriosclerotic disease, which may have implications with regard to the influence of circadian clock function in organ transplantation.

IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinflammatory Cytokine Release and Pancreatic Islet Graft Dysfunction

Islets from patients with type 2 diabetes exhibit β cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-α, IL-1α, IL-1β, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to β cell dysfunction in type 2 diabetes and islet transplantation.

Prolonged Xenograft Survival Induced by Inducible Costimulator-Ig Is Associated With Increased Forkhead Box P3+ Cells

Blockade of the inducible costimulator (ICOS) pathway has been shown to prolong allograft survival; however, its utility in xenotransplantation is unknown. We hypothesize that local expression of ICOS-Ig by the xenograft will suppress the T-cell response resulting in significant prolonged graft survival. Pig iliac artery endothelial cells (PIEC) secreting ICOS-Ig were generated and examined for the following: (1) inhibition of allogeneic and xenogeneic proliferation of primed T cells in vitro and (2) prolongation of xenograft survival in vivo. Grafts were examined for Tregs by flow cytometry and cytokine levels determined by quantitative reverse-transcriptase polymerase chain reaction. Soluble ICOS-Ig markedly decreased allogeneic and xenogeneic primed T-cell proliferation in a dose-dependent manner. PIEC-ICOS-Ig grafts were significantly prolonged compared with wild-type grafts (median survival, 34 and 12 days, respectively) with 20% of PIEC-ICOS-Ig grafts surviving more than 170 days. Histological examination showed a perigraft cellular accumulation of Forkhead box P3 (Foxp3(+)) cells in the PIEC-ICOS-Ig grafts, these were also shown to be CD3(+)CD4(+)CD25(+). Survival of wild-type PIEC grafts in a recipient simultaneously transplanted with PIEC-ICOS-Ig were also prolonged, with a similar accumulation of Foxp3(+) cells at the periphery of the graft demonstrating ICOS-Ig induces systemic graft prolongation. However, this prolongation was specific for the priming xenograft. Intragraft cytokine analysis showed an increase in interleukin-10 levels, suggesting a potential role in induction/function of CD4(+)CD25(+)Foxp3(+) cells. This study demonstrates prolonged xenograft survival by local expression of ICOS-Ig, we propose that the accumulation of CD4(+)CD25(+)Foxp3(+) cells at the periphery of the graft and secretion of interleukin-10 is responsible for this novel observation.

Tissue‐intrinsic circadian clock dysfunction confers transplant rejection

To determine the role of intrinsic tissue clocks versus extrinsic clocks in peripheral tissue disease, we implemented an organ transplant approach (blood vessel grafting) to surgically assemble a chimeric mouse that was in part wild-type and in part circadian clock mutant. Aortic isografts from donor wild-type mice that had been anastamosed to common carotid arteries of recipient WT mice (WT:WT) were pulsating with blood flow and oscillating with a circadian rhythm after four weeks. Surprisingly, when wild-type grafts were anastamosed to mice with disrupted circadian clocks, the structural adaptation was comparable to WT:WT grafts. In contrast, aortic grafts from Bmal1-KO or PerCdKO mice transplanted into litter-mate control WT developed arteriosclerotic lesions, suggesting that circadian clock gene dysfunction intrinsic to peripheral tissues is necessary to confer pathological responses. These data demonstrate the significance of tissue circadian clocks as an autonomous influence in peripheral tissue disease and additionally reveal the potential influence of circadian rhythm in the therapeutic success of organ transplantation.

Cardiac myocyte-specific overexpression of human GTP cyclohydrolase I protects against acute cardiac allograft rejection.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for tetrahydrobiopterin (BH(4)) synthesis. Decreases in GTPCH activity and expression have been shown in late stages of acute cardiac rejection, suggesting a deficit in BH(4). We hypothesized that increasing intracellular levels of BH(4) by cardiac myocyte-targeted overexpression of GTPCH would diminish acute cardiac allograft rejection. Transgenic mice overexpressing GTPCH in the heart were generated and crossed on C57BL6 background. Wild-type and transgenic mouse donor hearts were transplanted into BALB/c recipient mice. Left ventricular (LV) function, histological rejection, BH(4) levels, and inflammatory cytokine gene expression (mRNA) were examined. Expression of human GTPCH was documented by PCR, Western analysis, and function by a significant (P < 0.001) increase in cardiac BH(4) levels. GTPCH transgene decreased histological rejection (46%; P < 0.003) and cardiac myocyte injury (eosin autofluorescence; 56%; P < 0.0001) independent of changes in inflammatory cytokine expression or nitric oxide content. GTPCH transgene decreased IL-2 (88%; P < 0.002), IL-1R2 (42%; P < 0.0001), and programmed cell death-1 (67%; P < 0.0001) expression, whereas it increased fms-like tyrosine kinase 3 (156%; P < 0.0001) and stromal-derived factor-1 (2; 190%; P < 0.0001) expression. There was no difference in ejection fraction or fractional shortening; however, LV mass was significantly increased (P < 0.05) only in wild-type grafts. The decreases in LV mass, cardiac injury, and histological rejection support a protective role of cardiac GTPCH overexpression and increased BH(4) synthesis in cardiac allografts. The mechanism of the decreased rejection appears related to decreased T cell proliferation and modulation of immune function by higher expression of genes involved in hematopoietic/stromal cell development and recruitment.

Recipient Toll-like receptors contribute to chronic graft dysfunction by both MyD88- and TRIF-dependent signaling

Toll-like receptors (TLRs) recognize specific molecular patterns derived from microbial components (exogenous ligands) or stressed cells (endogenous ligands). Stimulation of these receptors leads to a pronounced inflammatory response in a variety of acute animal models. Chronic allograft dysfunction (CAD) was regarded as a candidate disease to test whether TLRs influence chronic fibrosing inflammation. Potential endogenous renal TLR ligands, specifically for TLR2 and TLR4, have now been detected by a significant upregulation of glucose regulated protein (GRP)-94, fibrinogen, heat shock protein (HSP)-60, HSP-70, biglycan (Bgn) and high-mobility group box chromosomal protein 1 (HMGB1) in the acute and chronic transplant setting. In a genetic approach to define the contribution of TLR2 and TLR4, and their adaptor proteins MyD88 and TRIF [Toll/interleukin (IL)-1 receptor domain-containing adaptor-protein inducing interferon beta], to CAD, kidney transplantation of TLR wild-type grafts to recipients who were deficient in TLR2, TLR4, TLR2/4, MyD88 and TRIF was performed. TLR and adaptor protein deficiencies significantly improved the excretory function of chronic kidney grafts by between 65% and 290%, and histopathologic signs of chronic allograft damage were significantly ameliorated. T cells, dendritic cells (DCs) and foremost macrophages were reduced in grafts by up to 4.5-fold. The intragraft concentrations of IL-6, IL-10, monocyte chemotactic protein-1 (MCP-1) and IL-12p70 were significantly lower. TLR-, MyD88- and TRIF-deficient recipients showed a significant reduction in fibrosis. alpha-smooth muscle actin (alpha-SMA)-positive cells were decreased by up to ninefold, and collagen I and III were reduced by up to twofold. These findings highlight the functional relevance of TLRs and their two major signaling pathways in graft-infiltrating mononuclear cells in the pathophysiology of CAD. A TLR signaling blockade may be a therapeutic option for the prevention of CAD.

Critical Role of Interferon Regulatory Factor-1 in Murine Liver Transplant Ischemia Reperfusion Injury

Interferon regulatory factor-1 (IRF-1) is a transcription factor that regulates gene expression during immunity. We hypothesized that IRF-1 plays a pivotal role in liver transplant (LTx) ischemia/reperfusion (I/R) injury. Mouse orthotopic LTx was conducted after 24 hours cold storage in University of Wisconsin (UW) solution in wildtype (WT) C57BL/6 and IRF-1 knockout (KO) mice. IRF-1 deficiency in liver grafts, but not in recipients, resulted in significant reduction of hepatocyte apoptosis and liver injury, as well as improved survival. IRF-1 mRNA up-regulation was typically seen in graft hepatocytes in WT-->WT LTx. Deficiency of IRF-1 signaling in graft resulted in significantly reduced messenger RNA (mRNA) levels for death ligands and death receptors in hepatocytes, as well as decreased caspase-8 activities, indicating that IRF-1 mediates death ligand-induced hepatocyte death. Further, a smaller but significant IRF-1 mRNA up-regulation was seen in WT graft nonparenchymal cells (NPC) and associated with interferon gamma (IFN-gamma) mRNA up-regulation exclusively in NPC. IFN-gamma mRNA was significantly reduced in IRF-1 KO graft. Thus, IRF-1 in graft hepatocytes and NPC has distinct effects in hepatic I/R injury. However, LTx with chimeric liver grafts showed that grafts lacking hepatocellular IRF-1 had better protection compared with those lacking IRF-1 in NPC. The study identifies a critical role for IRF-1 in liver transplant I/R injury.

Role of CCR7 in Facilitating Direct Allosensitization and Regulatory T-Cell Function in High-Risk Corneal Transplantation

Chemokine receptor 7 (CCR7) is a key homing molecule for immune cell trafficking, including corneal antigen-presenting cell (APC) migration from the inflamed cornea to draining lymph nodes (LNs). Here, the authors investigated the effect of CCR7-facilitated donor APC trafficking on allosensitization, regulatory T-cell (Treg) function, and graft survival in corneal transplantation. CCR7(-/-) or wild-type (WT) allogeneic corneal grafts were transplanted onto the neovascularized high-risk recipient beds. Two weeks later, the frequency of directly alloprimed host T cells was measured by the IFN-gamma ELISPOT assay. Treg function was tested by a coculture suppression assay and an IFN-gamma ELISPOT assay. Kaplan-Meier analysis was performed to evaluate graft survival. The recipients of CCR7(-/-) grafts had fewer migrated donor APCs and lower frequency of IFN-gamma-producing T cells in the draining LNs. However, there was no statistically significant difference in transplant survival between recipients of CCR7(-/-) and those of WT grafts. Tregs from the CCR7(-/-) graft recipient group showed reduced regulatory potential for the suppression of proliferation of naive T cells and direct alloprimed T cells and expressed lower Foxp3 levels. In vitro studies confirmed that mature CCR7(+) major histocompatibility complex class II(+) CD86(+) graft-derived dendritic cells were critical for Treg function. Not only is CCR7-mediated donor-derived APC trafficking to the draining LNs important in the initiation of host T-cell priming, it is crucial for Treg-mediated tolerance.

Interleukin 23 Secretion by Donor Antigen Presenting Cells Is Critical for Organ-Specific Pathology in Graft Versus Host Disease

The initiation of graft-versus-host disease (GVHD) is dependent upon donor T cell recognition of host alloantigens presented within the context of host antigen presenting cells (APCs). Eventually host APCs are replaced by donor APCs, however the role of the latter in the propagation of GVHD remains largely unknown. Recently, interleukin 23 (IL-23) has been identified as a cytokine that is secreted exclusively by APCs, and has been shown to play a pivotal role in several inflammatory disorders. The purpose of this study therefore was to address whether secretion of IL-23 by donor APCs played a role in the pathophysiology of GVHD. Transplantation of bone marrow and spleen cells from IL-23 deficient C57BL/6 (B6) (H-2b) donors into lethally irradiated MHC-incompatible Balb/c (H-2d) or FVB (H-2q) mice resulted in significantly less weight loss and proinflammatory cytokine production, as well as improved immune reconstitution, when compared to animals transplanted with marrow grafts from normal B6 mice. Histological examination of GVHD target tissues revealed that, in the absence of donor APC-derived IL-23 secretion, there was a selective and profound reduction in pathological damage in the colon, without any corresponding reduction in either the lung or liver. This was accompanied by a marked decrease in lipopolysaccharide (LPS) and proinflammatory cytokine production (IFN-γ, G-CSF, TNF-α, IL-1β and IL-6) in the colon microenvironment. To further investigate the preferential protection observed in the colon, we examined IL-23 mRNA levels in the spleen, liver and colon to determine whether there was differential gene expression during GVHD. In comparison to normal nontransplanted Balb/c mice, there was a selective increase in IL-23 (3-fold) mRNA levels in the colon of GVHD animals, but there was no difference between normal and GVHD mice in the spleen or liver. Examination of IL-23 receptor mRNA levels in these same tissues revealed a marked increase only in the colon of GVHD animals. To define the donor APC populations in the colon responsible for secretion of IL-23, CD11c+ CD11b+ dendritic and CD11b+ CD11c− myeloid/monocytic cells were examined for expression of IL-23 mRNA. Levels were significantly increased (600-fold) in CD11c+ CD11b+ cells derived from the colon as opposed to the spleen of GVHD mice where IL-23 expression was undetectable. Similarly, gene expression of IL-23 was 20-fold higher in colon versus splenic-derived CD11b+ CD11c− cells. Expression of the IL-23R was most dramatically increased (~1000-fold) in purified colon-derived CD4+ T cells whereas CD4+ T cells isolated from the spleen or liver of these same mice had negligible expression, suggesting that APC-derived IL-23 acted primarily through donor-derived CD4+ T cells. Since IL-23 induces secretion of IL-17 and IFN-γ by memory CD4+ T cells, we performed studies to determine if pathological damage was mediate by either of these cytokines. Transplantation with marrow grafts from IL-17-deficient donors had no protective effect on either overall or colon-specific pathology, providing evidence that the proinflammatory effects of IL-23 were independent of IL-17. Conversely, transplantation with IFN-γ−/− marrow grafts resulted in a significant reduction in colonic pathology compared to recipients of wild type grafts and was associated with a marked reduction in LPS and IL-23 levels as well as proinflammatory cytokine production demonstrating that the inflammatory effects of IL-23 in the colon were mediated, in large measure, through donor-derived IFN-γ. Finally, we observed that TLR-4 gene expression was selectively augmented in the colon when compared to the spleen and liver. The importance of the LPS/TLR4 signaling pathway was further confirmed by studies demonstrating that mice transplanted with TLR4- deficient marrow grafts had significantly reduced IL-23 and LPS levels as well as pathological damage in the colon. In summary, these data demonstrate that IL-23 has a unique and selective role in the induction of colonic inflammation during acute GVHD and serves as a critical mediator linking conditioning regimen-induced mucosal injury and LPS translocation to subsequent proinflammatory cytokine production and GVHD-associated pathological damage. These findings define a novel organ-specific role for IL-23 in the pathophysiology of GVHD and identify IL-23 as a therapeutic target for the prevention of this disease.

Soluble Lymphotoxin Plays a Critical Role in Acute Graft-Versus-Host Disease

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies.We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P<0.0002). Having observed a potentially TNF-independent but TNFR dependent pathway in the generation of GVHD we next examined mRNA expression by real-time PCR. This demonstrated that the majority of LTα mRNA was within donor CD4 T cells and that expression was unbalanced (> 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P<0.01) in both systems while LTβ−/− grafts induced GVHD that was equivalent to, or more severe than in recipients of WT grafts. This data confirms soluble LTα3 but not membrane bound LTα1β2 as an additional pathogenic molecule in GVHD. Using a luciferase-expressing, host-type leukemia (P815) in the B6 → B6D2F1 system, relapse occurred at an identical rate in recipients of WT and LTα−/− grafts confirming that GVL is maintained in the absence of donor derived soluble LTα3. Thus the combined neutralization of both LTα3 and TNF represents an important and logical therapeutic intervention in transplant medicine.

Donor Deficiency of Decay-Accelerating Factor Accelerates Murine T Cell-Mediated Cardiac Allograft Rejection

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation, DAF has been thought to primarily regulate antibody-mediated allograft injury, which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses, we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response, in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression, by controlling local complement activation on interacting T cell APC partners, regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation.

COX-2 from the injury milieu is critical for the initiation of periosteal progenitor cell mediated bone healing

Although a critical role of COX-2 in bone repair has been established, the mechanism involved remains unclear. During early inflammatory phase of bone healing, COX-2 is produced by the surrounding inflammatory cells as well as bone/cartilage progenitors. Based on the temporal and spatial expression of COX-2 during the early phase of fracture healing, we hypothesize that COX-2 from both sources is critical for progenitor cell activation, proliferation and differentiation. To directly test this we utilized a murine femoral grafting model, in which live segmental grafts from the same strains were transplanted and donor versus host cell involvement in healing was assessed. Specifically, fresh femur cortical bone grafts of 4 mm in length from COX-2 −/− (KO) mice were transplanted into wild type (WT) mice with the same sized segmental defect in femurs. Similarly, grafts from WT were transplanted into the defects in KO mice. As controls, transplantations between wild types, and transplantations between KO were also performed. Histologic analyses showed that WT-to-WT transplantation resulted in normal endochondral bone healing as evidenced by markedly induction of neovascularization and periosteal bone formation on donor graft. In contrast, transplantation of KO graft into KO host led to 96% reduction of bone formation and near elimination of donor cell-initiated periosteal bone formation. Similarly, transplantation of WT graft into a KO host resulted in 87% reduction of bone formation ( n = 8, p > 0.05), indicating that KO host impaired WT donor progenitor cell expansion and differentiation. When a KO graft was transplanted into WT host, KO donor periosteal cell-initiated endochondral bone formation was restored. Histomorphometric analyses demonstrated 10-fold increase in bone formation and 3-fold increase in cartilage formation compared to KO-to-KO transplantation ( n = 8, p < 0.05), suggesting that COX-2 deficient donor cells were capable to differentiate and form bone when placed in a WT host. Taken together, our data strongly suggest that COX-2 is critical for initiation of periosteal cortical bone healing. The early induction of COX-2 constitutes a crucial host-healing environment for activation and differentiation of donor periosteal progenitors. Elimination of COX-2 at the early stage of healing could lead to detrimental effects on periosteal progenitor cell-initiated cortical bone repair.

Importance of Donor- and Recipient-Derived Selectins in Cardiac Allograft Rejection

The selectins expressed on activated endothelial cells (E- and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP-/-). In BALB/c recipients, survival of fully allomismatched hearts from ELP-/- C57BL/6 donors was almost double that of wild-type grafts. In ELP-/- cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP-/- grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP-/- recipients promptly rejected wild-type hearts. Alternative adhesive molecules such as alpha4beta7 integrin may compensate for the lack of selectins and may mediate rejection in ELP-/- recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2(bm12) mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP-/- grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation.

PDL1 Is Required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection

The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-gamma-producing alloreactive T cells and expansion of effector CD8(+) T cells in the periphery, and a decline in the percentage of Foxp3(+) graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.