Daniela Cugini

Pretransplant Infusion of Mesenchymal Stem Cells Prolongs the Survival of a Semiallogeneic Heart Transplant through the Generation of Regulatory T Cells

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day −7) or double (portal vein, day −7 and tail vein, day −1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4+CD25+Foxp3+ Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.

Sirolimus versus cyclosporine therapy increases circulating regulatory T cells, but does not protect renal transplant patients given alemtuzumab induction from chronic allograft injury.

Background. In kidney transplant recipients with alemtuzumab induction maintained on mycophenolate mofetil (MMF) immunosuppression, sirolimus (SRL) promotes significant expansion of circulating CD4+CD25high regulatory T cells (Treg). This might translate into more effective protection against chronic graft injury compared to cyclosporin A (CsA), which, in the same clinical setting, does not affect Treg. Methods. To assess this hypothesis, in the extension of a single-center, prospective, randomized, open, blind endpoint study aimed to assess the effect of low-dose SRL or CsA on circulating Treg, we compared the outcomes of renal transplant recipients on SRL (n=11) or CsA (n=10) by per-protocol biopsies and serial measurements of glomerular filtration rate (GFR), renal plasma flow (RPF), and 24-hour proteinuria over 30 months posttransplant. Results. Despite 4-fold higher CD4+CD25high Treg counts (22.1±12.2% vs. 5.7±4.2% of CD3+CD4+ T cells), SRL-treated patients, compared to CsA-treated patients, had a significantly higher tubular C4d staining score (1.1±0.6 vs. 0.2±0.3, P<0.01), with nonsignificant trends to higher chronic allograft damage index score (5.6±2.4 vs. 3.7±3.3), faster GFR (−2.92±0.33 vs. −0.28±0.44 ml/min/1.73m2 per year), and RPF (−10.80±5.45 vs. −1.86±3.09 ml/min/1.73 m2 per year) decline, and more clinical proteinuria (n=6 vs. 4). There was no significant correlation between Treg counts and any considered outcome variable in the study group as a whole and within each cohort. Conclusions. These data suggest that, despite enhanced Treg expression, low-dose SRL combined to alemtuzumab induction and MMF-based steroid-free maintenance therapy, does not appreciably protect renal transplant recipients from chronic allograft injury and dysfunction.

The Toll-IL-1R Member Tir8/SIGIRR Negatively Regulates Adaptive Immunity against Kidney Grafts

Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8−/− grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8−/− than in Tir8+/+ grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8−/− dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-α than Tir8+/+ cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8−/− kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.

Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generating regulatory T cells.

Background. It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells. Methods. Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer. Results. Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti–third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of naïve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti–IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a naïve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into naïve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a naïve MLR against donor antigens. Conclusions. Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.

Erythropoietin, but not the correction of anemia alone, protects from chronic kidney allograft injury

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.

DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties

Background. We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4+ T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. Methods. Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4+ T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4+ T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. Results. CD4+ T cells pre-exposed to dnIKK2-DCs were CD4+CD25−/dim and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:105 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). Conclusions. DnIKK2-Treg is a unique population of CD4+CD25− T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.

Both darbepoetin alfa and carbamylated erythropoietin prevent kidney graft dysfunction due to ischemia/reperfusion in rats.

Background. Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction. Increases in cold and warm ischemia times lead to a higher risk of early posttransplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term graft loss in kidney transplant patients. Methods. Darbepoetin alfa (DA) and carbamylated nonerythropoietic derivative of erythropoietin (CEPO) protective effects were evaluated in a model of I/R injury after kidney transplantation in both syngeneic and allogeneic combinations. The effects of wortmannin (phosphorylated Akt [p-Akt] inhibitor) administration were also investigated. Serum creatinine was evaluated at 16, 24, 48 hr and at 4 and 7 days posttransplant. Animals were killed 24 hr or 7 days after transplant and kidneys were processed for histological analysis, immunohistochemistry assessment of erythropoietin receptor (EPOR) and &bgr;-common chain receptor expression, granulocyte infiltration, nitrotyrosine staining, p-Akt expression, peritubular capillary (PTC) density, apoptosis, antioxidant, and antiapoptotic gene expression. Results. DA and CEPO significantly reduced serum creatinine, tubular injury, tubular nitrotyrosine staining, and prevented I/R-induced tubular apoptosis, but only when given both to the donor and to the recipient. DA and CEPO cytoprotection was associated with prevention of I/R-induced drop of p-Akt expression in tubuli, and almost complete preservation of capillary density in the tubulointerstitium of the graft. CEPO was more effective than DA in reducing tubular oxidative stress and preserving PTCs. Conclusion. DA and CEPO when given both to the donor and to the recipient, prevented renal graft dysfunction, tubular oxidative stress, and apoptosis after I/R injury in kidney transplantation. Their cytoprotection was mediated by tubular p-Akt activation and PTC density preservation.

Embryonic Stem Cells, Derived Either after In Vitro Fertilization or Nuclear Transfer, Prolong Survival of Semiallogeneic Heart Transplants

Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE2 as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC– and NT-ESC–tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

Propionyl-L-carnitine prevents early graft dysfunction in allogeneic rat kidney transplantation

Ischemia-reperfusion injury is an important cause of graft failure. Because carnitine regulates substrate flux and energy balance across membranes which may be deranged in ischemia we determined whether its use was effective in preventing kidney injury in an allogeneic transplant model. Brown Norway rats received a Lewis rat kidney transplant and were then treated with cyclosporine A to avoid rejection. The grafts were stored in Belzer solution supplemented with propionyl-L-carnitine during the cold ischemia period. Compared to rats receiving untreated kidneys but with equal cold ischemia times, the post-transplant serum creatinine values of the carnitine-treated transplants were significantly lower. Histological evaluation 16 h after transplant showed that propionyl-L-carnitine significantly inhibited tubular necrosis and neutrophil infiltration of the allografts and improved the 3 month graft survival. Treated transplants also had decreased lipid peroxidation, inducible nitric oxide synthase expression and protein nitration compared to the untreated grafts. Post-transplant serum creatinine levels were significantly reduced and graft survival was slightly prolonged in rats not receiving cyclosporine A treatment and transplanted with a kidney treated with propionyl-L-carnitine. The efficacy of propionyl-L-carnitine to modulate ischemia-reperfusion injury during transplantation suggests that its use in human transplantation is worth testing.

Moderate salt restriction with or without paricalcitol in type 2 diabetes and losartan-resistant macroalbuminuria (PROCEED): a randomised, double-blind, placebo-controlled, crossover trial

BACKGROUND Macroalbuminuria predicts renal and cardiovascular events in patients with type 2 diabetes. We aimed to assess the albuminuria-lowering effects of salt restriction, paricalcitol therapy, or both, in this population. METHODS In this randomised, double-blind, placebo-controlled, crossover trial, we recruited adult patients with type 2 diabetes from six diabetology outpatient clinics in northern Italy, with 24 h albuminuria of more than 300 mg despite 100 mg per day losartan therapy, blood pressure of less than 140/90 mm Hg, serum creatinine concentration of less than 2 mg/dL, stable renal function on stable renin-angiotensin system inhibitor therapy with a fixed dose of losartan, parathyroid hormone concentration of 20 pg/mL to <110 pg/mL, serum calcium concentration of less than 9·5 mg/dL, and serum phosphate concentration of less than 5 mg/dL, who had been more than 80% compliant with placebo treatment during a 1 month placebo run-in. We allocated patients 1:1 with computer-generated randomisation to an open-label 3 month high-sodium (>200 mEq [4·8 g] per day) or low-sodium (<100 mEq [2·4 g] per day) diet and, within each diet group, to a 1 month double-blind treatment period of oral paricalcitol (2 μg per day) or placebo, followed by 1 month of placebo washout and then a further 1 month double-blind treatment period of paricalcitol or placebo in which patients crossed over to the opposite treatment period. The primary outcome was 24 h albuminuria (median of three consecutive measurements). Analyses were modified intention-to-treat (including all randomly allocated patients who took at least one dose of study drug and had an efficacy measurement after the first treatment period). Patients and investigators were masked to paricalcitol and placebo assignment. Those assessing outcomes were masked to both study drug and diet assignment. This study is registered with ClinicalTrials.gov, number NCT01393808, and the European Union Clinical Trials Register, number 2011-001713-14. FINDINGS Between Dec 13, 2011, and Feb 17, 2015, we randomly allocated 57 (50%) patients to a low-sodium diet (28 [49%] to paricalcitol then placebo and 29 [51%] to placebo then paricalcitol) and 58 (50%) to a high-sodium diet (29 [50%] to paricalcitol then placebo and 29 [50%] to placebo then paricalcitol). In the low-sodium group (30 mEq of daily sodium intake reduction, equivalent to approximately 1·7-1·8 g per day), 24 h albuminuria was reduced by 36·6% (95% CI 28·5-44·9) from 724 mg (441-1233) at baseline to 481 mg (289-837) at month 3 (p<0·0001), but no significant change occurred in the high-sodium group (from 730 mg [416-1227] to 801 mg [441-1365]; 2·9% [-16·8 to 16·4] increase; p=0·50). Changes between diet groups differed by 32·4% (17·2-48·8; p<0·0001) and correlated with changes in natriuresis (r=0·43; p<0·0001). On the high-sodium diet, paricalcitol reduced the salt-induced albuminuria increase by 17·8% (3·9-32·3) over the month of treatment compared with placebo (p=0·02), whereas on the low-sodium diet, paricalcitol did not have a significant effect versus placebo (increase of 4·1% [-9·3 to 21·6]; p=0·59). During placebo treatment, albuminuria decreased with the low-sodium diet (p=0·0002) and did not significantly change with the high-sodium diet, but changes were significantly different between diet groups (p=0·0004). Treatment was well tolerated and no patients withdrew from the study because of treatment-related effects. 67 adverse events occurred in 52 (45%) patients during paricalcitol treatment and 44 events occurred in 36 (31%) patients during placebo treatment. During paricalcitol therapy, 14 cases of hypercalciuria, six cases of hypercalcaemia, and five cases of hyperphosphataemia were reported in one patient each, all of which were possibly treatment related. One case of hypercalciuria was reported in one patient during the placebo treatment period. One stroke and one coronary event occurred during paricalcitol therapy. No patients died during the study. INTERPRETATION In patients with macroalbuminuria and type 2 diabetes, moderate salt restriction enhances the antialbuminuric effect of losartan, an effect that could be nephroprotective and cardioprotective in the long term. The finding that paricalcitol prevents a sodium-induced increase in albuminuria provides support for trials to test the long-term risk-benefit profile of paricalcitol add-on therapy in patients with type 2 diabetes and macroalbuminuria refractory to dietary salt restriction, including patients refractory to even moderate salt restriction. FUNDING AbbVie.