Heterogeneity in allospecific T cell function in transplant-tolerant hosts determines susceptibility to rejection following infection.

Even when successfully induced, immunological tolerance to solid organs remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC class I-derived peptide become hypofunctional if the allograft is accepted for more than 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC class II, an alloantigen whose expression declines after transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities after tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.

Implantable bioelectronic systems for early detection of kidney transplant rejection.

Early-stage organ transplant rejection can be difficult to detect. Percutaneous biopsies occur infrequently and are risky, and measuring biomarker levels in blood can lead to false-negative and -positive outcomes. We developed an implantable bioelectronic system capable of continuous, real-time, long-term monitoring of the local temperature and thermal conductivity of a kidney for detecting inflammatory processes associated with graft rejection, as demonstrated in rat models. The system detects ultradian rhythms, disruption of the circadian cycle, and/or a rise in kidney temperature. These provide warning signs of acute kidney transplant rejection that precede changes in blood serum creatinine/urea nitrogen by 2 to 3 weeks and approximately 3 days for cases of discontinued and absent administration of immunosuppressive therapy, respectively.

Single-cell sequencing of a novel model of neonatal bile duct ligation in mice identifies macrophage heterogeneity in obstructive cholestasis.

Macrophages (MΦ) play a role in neonatal etiologies of obstructive cholestasis, however, the role for precise MΦ subsets remains poorly defined. We developed a neonatal murine model of bile duct ligation (BDL) to characterize etiology-specific differences in neonatal cholestatic MΦ polarization. Neonatal BDL surgery was performed on female BALB/c mice at 10 days of life (DOL) with sham laparotomy as controls. Comparison was made to the Rhesus Rotavirus (RRV)-induced murine model of biliary atresia (BA). Evaluation of changes at day 7 after surgery (BDL and sham groups) and murine BA (DOL14) included laboratory data, histology (H&E, anti-CD45 and anti-CK19 staining), flow cytometry of MΦ subsets by MHCII and Ly6c expression, and single cell RNA-sequencing (scRNA-seq) analysis. Neonatal BDL achieved a 90% survival rate; mice had elevated bile acids, bilirubin, and alanine aminotransferase (ALT) versus controls (p < 0.05 for all). Histology demonstrated hepatocellular injury, CD45+ portal infiltrate, and CK19+ bile duct proliferation in neonatal BDL. Comparison to murine BA showed increased ALT in neonatal BDL despite no difference in histology Ishak score. Neonatal BDL had significantly lower MHCII-Ly6c+ MΦ versus murine BA, however, scRNA-seq identified greater etiology-specific MΦ heterogeneity with increased endocytosis in neonatal BDL MΦ versus cellular killing in murine BA MΦ. We generated an innovative murine model of neonatal obstructive cholestasis with low mortality. This model enabled comparison to murine BA to define etiology-specific cholestatic MΦ function. Further comparisons to human data may enable development of immune modulatory therapies to improve patient outcomes.

Antibody-suppressor CXCR5+ CD8+ T cellular therapy ameliorates antibody-mediated rejection following kidney transplant in CCR5 KO mice.

CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2b ) were transplanted with A/J kidneys (H-2a ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5+ CD8+ T cells and CD8-mediated cytotoxicity to alloprimed IgG+ B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5+ CD8+ T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG+ B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5+ CD8+ T cells (but not alloprimed CXCR5- CD8+ or third-party primed CXCR5+ CD8+ T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5+ CD8+ T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function.

Delayed graft function due to transplant ischemia/reperfusion injury adversely affects up to 50% of deceased-donor kidney transplant recipients. However, key factors contributing to the severity of ischemia/reperfusion injury remain unclear. Here, using a clinically relevant mouse model of delayed graft function, we demonstrated that donor genetic background and kidney-intrinsic MyD88/Trif-dependent innate immunity were key determinants of delayed graft function. Functional deterioration of kidney grafts directly corresponded with the duration of cold ischemia time. The graft dysfunction became irreversible after cold ischemia time exceeded six hours. When cold ischemia time reached four hours, kidney grafts displayed histological features reflective of delayed graft function seen in clinical kidney transplantation. Notably, kidneys of B6 mice exhibited significantly more severe histological and functional impairment than kidneys of C3H or BALB/c mice, regardless of recipient strains or alloreactivities. Furthermore, allografts of B6 mice also showed an upregulation of IL-6, neutrophil gelatinase-associated lipocalin, and endoplasmic reticulum stress genes, as well as an increased influx of host neutrophils and memory CD8 T-cells. In contrast, donor MyD88/Trif deficiency inhibited neutrophil influx and decreased the expression of IL-6 and endoplasmic reticulum stress genes, along with improved graft function and prolonged allograft survival. Thus, kidney-intrinsic factors involving genetic characteristics and innate immunity serve as critical determinants of the severity of delayed graft function. This preclinical murine model allows for further investigations of the mechanisms underlying delayed graft function.

MCMV Dissemination from Latently-Infected Allografts Following Transplantation into Pre-Tolerized Recipients.

Transplantation tolerance is achieved when recipients are unresponsive to donor alloantigen yet mobilize against third-party antigens, including virus. After transplantation, cytomegalovirus (CMV) reactivation in latently-infected transplants reduces allograft viability. To determine if pre-tolerized recipients are resistant to viral dissemination in this setting, we transfused chemically-fixed donor splenocytes (1-ethyl-3- (3'-dimethyl-aminopropyl)-carbo-diimide (ECDI)-treated splenocytes (ECDIsp)) to induce donor antigen tolerance without immunosuppression. In parallel, we implanted donor islet cells to validate operational tolerance. These pre-tolerized recipients were implanted with murine CMV (MCMV) latently-infected donor kidneys (a validated model of CMV latency) to monitor graft inflammation and viral dissemination. Our results indicate that tolerance to donor islets was sustained in recipients after implantation of donor kidneys. In addition, kidney allografts implanted after ECDIsp and islet implantation exhibited low levels of fibrosis and tubulitis. In contrast, kidney cellular and innate immune infiltrates trended higher in the CMV group and exhibited increased markers of CD8+ T cell activation. Tolerance induction was unable to prevent increases in MCMV-specific CD8+ T cells or dissemination of viral IE-1 DNA. Our data suggest that latently-infected allografts are inherently more susceptible to inflammation that is associated with viral dissemination in pre-tolerized recipients. Thus, CMV latently-infected allografts require enhanced strategies to protect allograft integrity and viral spread.

A novel murine model of reversible bile duct obstruction demonstrates rapid improvement of cholestatic liver injury.

There are limited murine models of cholestatic liver diseases characterized by chronic biliary obstruction and resumption of bile flow. While murine bile duct ligation (BDL) is a well-established model of obstructive cholestasis, current models of BDL reversal (BDLR) alter biliary anatomy. We aimed to develop a more physiologic model of BDLR to evaluate the time course and mechanism for resolution of hepatic injury after biliary obstruction. In the present study, we restored bile flow into the duodenum without disruption of the gall bladder after murine BDL using biocompatible PE-50 tubing. After establishing the technique, overall survival for BDLR at 7 or 14 days after BDL was 88%. Sham laparotomy was performed in control mice. Laboratory data, liver histology, and hepatic gene expression were compared among BDL, BDLR, and controls. Laboratory evidence of cholestatic liver injury was observed at day 7 after BDL and rapid improvement occurred within 48 hr of BDLR. After BDLR there was also enhanced gene expression for the bile acid transporter Abcb11, however, bile duct proliferation persisted. Assessment of the immune response showed increased gene and protein expression for the general immune cell marker Cd45 in BDLR versus BDL mice suggesting a reparative immune response after BDLR. In summary, we have established a novel murine model of BDLR that allows for the investigation into bile acid and immune pathways responsible for hepatic repair following obstructive cholestasis. Future studies with our model may identify targets for new therapies to improve outcome in pediatric and adult cholestatic liver disease.

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.

Rats provide a superior model of human stress erythropoiesis.

Mouse models are widely used to study human erythropoiesis in vivo. One important caveat using mouse models is that mice often develop significant extramedullary erythropoiesis with anemia, which could mask important phenotypes. To overcome this drawback in mice, here we established in vitro and in vivo rat models for the studies of stress erythropoiesis. Using flow cytometry-based assays, we can monitor terminal erythropoiesis in rats during fetal and adult erythropoiesis under steady state and stress conditions. We used this system to test rat erythropoiesis under phenylhydrazine (PHZ)-induced hemolytic stress. In contrast to mice, rats did not have an increased proportion of early-stage erythroid precursors during terminal differentiation in the spleen or bone marrow. This could be explained by the abundant bone marrow spaces in rats that allow sufficient erythroid proliferation under stress. Consistently, the extent of splenomegaly in rats after PHZ treatment was significantly lower than that in mice. The level of BMP4, which was significantly increased in mouse spleen after PHZ treatment, remained unchanged in rat spleen. We further demonstrated that the bone marrow c-Kit positive progenitor population underwent a phenotype shift and became more CD71 positive and erythroid skewed with the expression of maturing erythroid markers under stress in rats and humans. In contrast, the phenotype shift to an erythroid-skewed progenitor population in mice occurred mainly in the spleen. Our study establishes rat in vitro and in vivo erythropoiesis models that are more appropriate and superior for the study of human stress erythropoiesis than mouse models.

Vascular scaffolds with enhanced antioxidant activity inhibit graft calcification.

There is a need for off-the-shelf, small-diameter vascular grafts that are safe and exhibit high long-term patency. Decellularized tissues can potentially be used as vascular grafts; however, thrombogenic and unpredictable remodeling properties such as intimal hyperplasia and calcification are concerns that hinder their clinical use. The objective of this study was to investigate the long-term function and remodeling of extracellular matrix (ECM)-based vascular grafts composited with antioxidant poly(1, 8-octamethylene-citrate-co-cysteine) (POCC) with or without immobilized heparin. Rat aortas were decellularized to create the following vascular grafts: 1) ECM hybridized with POCC (Poly-ECM), 2) Poly-ECM subsequently functionalized with heparin (Poly-ECM-Hep), and 3) non-modified vascular ECM. Grafts were evaluated as interposition grafts in the abdominal aorta of adult rats at three months. All grafts displayed antioxidant activity, were patent, and exhibited minimal intramural cell infiltration with varying degrees of calcification. Areas of calcification co-localized with osteochondrogenic differentiation of vascular smooth muscle cells, lipid peroxidation, oxidized DNA damage, and cell apoptosis, suggesting an important role for oxidative stress in the calcification of grafts. The extent of calcification within grafts was inversely proportional to their antioxidant activity: Poly-ECM-Hep > ECM > Poly-ECM. The incorporation of antioxidants into vascular grafts may be a viable strategy to inhibit degenerative changes.

Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction.

Primary graft dysfunction is the predominant driver of mortality and graft loss after lung transplantation. Recruitment of neutrophils as a result of ischemia-reperfusion injury is thought to cause primary graft dysfunction; however, the mechanisms that regulate neutrophil influx into the injured lung are incompletely understood. We found that donor-derived intravascular nonclassical monocytes (NCMs) are retained in human and murine donor lungs used in transplantation and can be visualized at sites of endothelial injury after reperfusion. When NCMs in the donor lungs were depleted, either pharmacologically or genetically, neutrophil influx and lung graft injury were attenuated in both allogeneic and syngeneic models. Similar protection was observed when the patrolling function of donor NCMs was impaired by deletion of the fractalkine receptor CX3CR1. Unbiased transcriptomic profiling revealed up-regulation of MyD88 pathway genes and a key neutrophil chemoattractant, CXCL2, in donor-derived NCMs after reperfusion. Reconstitution of NCM-depleted donor lungs with wild-type but not MyD88-deficient NCMs rescued neutrophil migration. Donor NCMs, through MyD88 signaling, were responsible for CXCL2 production in the allograft and neutralization of CXCL2 attenuated neutrophil influx. These findings suggest that therapies to deplete or inhibit NCMs in donor lung might ameliorate primary graft dysfunction with minimal toxicity to the recipient.

CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.

Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal.

Mechanocompatible Polymer-Extracellular-Matrix Composites for Vascular Tissue Engineering.

Small-diameter vascular grafts developed from vascular extracellular matrix (ECM) can potentially be used for bypass surgeries and other vascular reconstruction and repair procedures. The addition of heparin to the ECM improves graft hemocompatibility but often involves chemical cross-linking, which increases ECM mechanical stiffness compared to native arteries. Herein, the importance of maintaining ECM mechanocompatibility is demonstrated, and a mechanocompatible strategy to immobilize heparin onto the ECM via a biodegradable elastomer is described. Specifically, poly(1,8-octamethylene citrate)-co-cysteine is hybridized to the ECM, forming a polymer-ECM composite that allows for heparin immobilization via maleimide-thiol "click" chemistry. Heparinized composites reduce platelet adhesion by >60% in vitro, without altering the elastic modulus of the ECM. In a rat abdominal aortic interposition model, intimal hyperplasia in heparinized mechanocompatible grafts is 65% lower when compared to ECM-only control grafts at four weeks. In contrast, grafts that are heparinized with carbodiimide chemistry exhibit increased intimal hyperplasia (4.2-fold) and increased macrophage infiltration (3.5-fold) compared to ECM-only control grafts. All grafts show similar, partial endothelial cell coverage and little to no ECM remodeling. Overall, a mechanocompatible strategy to improve ECM thromboresistance is described and the importance of ECM mechanical properties for proper in vivo graft performance is highlighted.

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

Ethylenecarbodiimide-fixed donor splenocyte infusions differentially target direct and indirect pathways of allorecognition for induction of transplant tolerance.

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c(+) dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c(+) dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4(+)CD25(+)Foxp3(+) T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Effect of dietary supplementation with copper sulfate or tribasic copper chloride on the growth performance, liver copper concentrations of broilers fed in floor pens, and stabilities of vitamin E and phytase in feeds.

An experiment was conducted using a total of 840, 1-day-old, Arbor Acres commercial male broilers to compare copper (Cu) sulfate and tribasic Cu chloride (TBCC, Cu(2)(OH)(3)Cl) as sources of supplemental Cu for broilers fed in floor pens. Chicks were randomly allotted to one of seven treatments for six replicate pens of 20 birds each, and were fed a basal corn-soybean meal diet (10.20 mg/kg Cu) supplemented with 0, 100, 150, or 200 mg/kg Cu from either Cu sulfate or TBCC for 21 days. Chicks fed 200 mg/kg Cu as TBCC had a higher (P < 0.05) average daily gain (ADG) than those consuming other diets. Liver Cu contents of broilers fed diets supplemented with TBCC were numerically lower (P > 0.05) than those of broilers fed diets supplemented with Cu sulfate. The vitamin E contents and the phytase activities in the feed fortified with TBCC were higher (P < 0.01) and numerically higher (P > 0.05) compared with those in the feeds fortified with Cu sulfate stored at room temperature, respectively. The vitamin E contents in liver and plasma of broilers fed diets supplemented with TBCC were higher (P < 0.05) than those of birds fed diets supplemented with Cu sulfate. This result indicates that TBCC is more effective than Cu sulfate in improving the growth of broilers fed in floor pens, and it is chemically less active than Cu sulfate in promoting the undesirable oxidation of vitamin E in feeds.