Fibrosis Progression According to Epithelial‐Mesenchymal Transition Profile: A Randomized Trial of Everolimus Versus CsA

Markers of epithelial‐mesenchymal transition (EMT) may identify patients at high risk of graft fibrogenesis who could benefit from early calcineurin inhibitor (CNI) withdrawal. In a randomized, open‐label, 12‐month trial, de novo kidney transplant patients received cyclosporine, enteric‐coated mycophenolate sodium (EC‐MPS) and steroids to month 3. Patients were stratified as EMT+ or EMT? based on month 3 biopsy, then randomized to start everolimus with half‐dose EC‐MPS (720 mg/day) and cyclosporine withdrawal (CNI‐free) or continue cyclosporine with standard EC‐MPS (CNI). The primary endpoint was progression of graft fibrosis (interstitial fibrosis/tubular atrophy [IF/TA] grade increase ≥1 between months 3–12) in EMT+ patients. 194 patients were randomized (96 CNI‐free, 98 CNI); 153 (69 CNI‐free, 84 CNI) were included in histological analyses. Fibrosis progression occurred in 46.2% (12/26) CNI‐free EMT+ patients versus 51.6% (16/31) CNI EMT+ patients (p = 0.68). Biopsy‐proven acute rejection (BPAR, including subclinical events) occurred in 25.0% and 5.1% of CNI‐free and CNI patients, respectively (p < 0.001). In conclusion, early CNI withdrawal with everolimus initiation does not prevent interstitial fibrosis. Using this CNI‐free protocol, in which everolimus exposure was relatively low and administered with half‐dose EC‐MPS, CNI‐free patients were overwhelmingly under‐immunosuppressed and experienced an increased risk of BPAR.

     

Effect of Empagliflozin on Tacrolimus‐Induced Pancreas Islet Dysfunction and Renal Injury

An inhibitor of sodium glucose co‐transporter type 2 (SGLT‐2) is recommended in type 2 diabetes mellitus (DM) but its use is still undetermined in tacrolimus (TAC)‐induced DM. We evaluated the effect of empagliflozin (Em) on TAC‐induced pancreatic islet dysfunction and renal injury in an experimental model of TAC‐induced DM and in vitro. TAC induced a twofold increase in SGLT‐2 expression, while Em decreased SGLT‐2 expression and further increased urinary glucose excretion compared to the TAC group. Em reduced hyperglycemia and increased plasma insulin level, pancreatic islet size, and glucose‐stimulated insulin secretion compared to the TAC group. In kidney, Em alleviated TAC‐induced renal dysfunction and decreased albumin excretion and histological injury compared with the TAC group. Increased oxidative stress and apoptotic cell death by TAC was remarkably decreased with Em in serum and pancreatic and renal tissues. In in vitro study, TAC decreased cell viability and increased reactive oxygen species (ROS) production in both insulin‐secreting beta‐cell derived (INS‐1) and human kidney‐2 (HK‐2) cell lines. Addition of Em increased cell viability and decreased ROS production in HK‐2 but not in INS‐1 cell lines. This suggests that Em is effective in controlling TAC‐induced hyperglycemia and has direct protective effect on TAC‐induced renal injury.     

Age‐Dependent Metabolic and Immunosuppressive Effects of Tacrolimus

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age‐specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon‐γ cytokine production and promote interleukin‐10 production in old CD4⁺ T cells. In addition, TAC administration decreased interleukin‐2 secretion in old CD4⁺ T cells more effectively while inhibiting the proliferation of CD4⁺ T cells in old mice. Both TAC‐treated murine and human CD4⁺ T cells demonstrated an age‐specific suppression of intracellular calcineurin levels and Ca²⁺ influx, two critical pathways in T cell activation. Of note, depletion of CD8⁺ T cells did not alter allograft survival outcome in old TAC‐treated mice, suggesting that TAC age‐specific effects were mainly CD4⁺ T cell mediated. Collectively, our study demonstrates age‐specific immunosuppressive capacities of TAC that are CD4⁺ T cell mediated. The suppression of calcineurin levels and Ca²⁺ influx in both old murine and human T cells emphasizes the clinical relevance of age‐specific effects when using TAC.     

A molecular biopsy test based on arteriolar under‐hyalinosis reflects increased probability of rejection related to under‐immunosuppression

Calcineurin inhibitor immunosuppressive drugs induce changes such as arteriolar hyalinosis (ah) in kidney transplants, raising the possibility that molecular changes in biopsies related to histologic ah can provide information about drug exposure. We hypothesized that molecular changes associated with less‐than‐expected hyalinosis might highlight a subpopulation of patients with under‐immunosuppression/nonadherence at intermediate times of biopsy posttransplant (TxBx). Using gene expression data from 562 indication biopsies, we developed a molecular classifier for predicting the expected ah lesions (M_ah) at a particular TxBx. M_ah‐scores increased linearly with log(TxBx), but some biopsies had lower scores than expected for TxBx. The deviation of individual M_ah‐scores below the predicted regression line of M_ah‐scores vs TxBx is defined as “low hyalinosis index.” Low hyalinosis indices were frequent in biopsies between 3 months and 3 years posttransplant, particularly among biopsies lacking histologic hyalinosis (ah0), and were associated with T cell–mediated rejection and a subset of recent‐onset antibody‐mediated rejection without glomerular double contours. In patients with medical records available for review, low hyalinosis indices were frequently associated with physician‐recorded concerns about nonadherence (suspected or proven). We conclude that the M_ah classifier and hyalinosis index identify indication biopsies with rejection for which the possibility of patient nonadherence should be considered.     

Posttransplant reduction in preexisting donor‐specific antibody levels after belatacept‐ versus cyclosporine‐based immunosuppression: Post hoc analyses of BENEFIT and BENEFIT‐EXT

BENEFIT and BENEFIT‐EXT were phase III studies of cytotoxic T‐cell crossmatch–negative kidney transplant recipients randomized to belatacept more intense (MI)‐based, belatacept less intense (LI)‐based, or cyclosporine‐based immunosuppression. Following study completion, presence/absence of HLA‐specific antibodies was determined centrally via solid‐phase flow cytometry screening. Stored sera from anti‐HLA–positive patients were further tested with a single‐antigen bead assay to determine antibody specificities, presence/absence of donor‐specific antibodies (DSAs), and mean fluorescent intensity (MFI) of any DSAs present. The effect of belatacept‐based and cyclosporine‐based immunosuppression on MFI was explored post hoc in patients with preexisting DSAs enrolled to BENEFIT and BENEFIT‐EXT. In BENEFIT, preexisting DSAs were detected in 4.6%, 4.9%, and 6.3% of belatacept MI‐treated, belatacept LI‐treated, and cyclosporine‐treated patients, respectively. The corresponding values in BENEFIT‐EXT were 6.0%, 5.7%, and 9.2%. In both studies, most preexisting DSAs were of class I specificity. Over the first 24 months posttransplant, a greater proportion of preexisting DSAs in belatacept‐treated versus cyclosporine‐treated patients exhibited decreases or no change in MFI. MFI decline was more apparent with belatacept MI‐based versus belatacept LI‐based immunosuppression in both studies and more pronounced in BENEFIT‐EXT versus BENEFIT. Although derived post hoc, these data suggest that belatacept‐based immunosuppression decreases preexisting DSAs more effectively than cyclosporine‐based immunosuppression.     

Prospective Analyses of Circulating B Cell Subsets in ABO‐Compatible and ABO‐Incompatible Kidney Transplant Recipients

Immunosuppressive strategies applied in renal transplantation traditionally focus on T cell inhibition. B cells were mainly examined in the context of antibody‐mediated rejection, whereas the impact of antibody‐independent B cell functions has only recently entered the field of transplantation. Similar to T cells, distinct B cell subsets can enhance or inhibit immune responses. In this study, we prospectively analyzed the evolution of B cell subsets in the peripheral blood of ABO‐compatible (n = 27) and ABO‐incompatible (n = 10) renal transplant recipients. Activated B cells were transiently decreased and plasmablasts were permanently decreased in patients without signs of rejection throughout the first year. In patients with histologically confirmed renal allograft rejection, activated B cells and plasmablasts were significantly elevated on day 365. Rituximab treatment in ABO‐incompatible patients resulted in long‐lasting B cell depletion and in a naïve phenotype of repopulating B cells 1 year following transplantation. Acute allograft rejection was correlated with an increase of activated B cells and plasmablasts and with a significant reduction of regulatory B cell subsets. Our study demonstrates the remarkable effects of standard immunosuppression on circulating B cell subsets. Furthermore, the B cell compartment was significantly altered in rejecting patients. A specific targeting of deleterious B cell subsets could be of clinical benefit in renal transplantation.     

Spontaneous Resolution of Acute Rejection and Tolerance Induction With IL‐2 Fusion Protein in Vascularized Composite Allotransplantation

Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife‐threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance‐inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL‐2/Fc (a long‐lasting human IL‐2 fusion protein), in combination with antilymphocyte serum (ALS) and short‐term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind‐limb transplant model. We demonstrate that hIL‐2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long‐term allograft survival and donor‐antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL‐2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon‐γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.     

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin‐15

Proinflammatory, cytotoxic CD4⁺CD28^null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4⁺CD28^null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28⁺ T cells. CD4⁺CD28^null T cells contained alloreactive (CD137⁺) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4⁺CD28^null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a⁺CD4⁺CD28^null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4⁺CD28^null T cells did not show significant suppression. Thus, CD4⁺CD28^null T cells represent a population with absent alloreactivity unless IL‐15 is present.     

Prospective randomized study of conversion from tacrolimus to cyclosporine A to improve glucose metabolism in patients with posttransplant diabetes mellitus after renal transplantation

Tacrolimus (TAC) increases the risk of posttransplant diabetes (PTDM) compared with cyclosporine A (CYC). The present 12‐month, multicenter, investigator‐driven, prospective, randomized study was designed to assess whether conversion from tacrolimus to CYC can reverse PTDM after renal transplantation. Predominantly white patients with PTDM according to the 2005 American Diabetes Association criteria were randomized to either replacement of TAC with CYC or continuation of their TAC‐based regimen after stratification for type of glucose‐lowering therapy, steroid therapy, and hepatitis C status. At 12 months, 14 of 41 patients with complete data in the CYC arm (34%; 95%CI 19%‐49%) were free of diabetes, whereas this was the case in only 4 of 39 patients (10%; 95%CI 3%‐20%) in the TAC arm (P = .01). At 12 months, 39% of patients in the CYC arm were off glucose‐lowering medication vs 13% of patients in the TAC arm (P = .01). The CYC group decreased glycated hemoglobin level during the 12‐month follow‐up, resulting in significantly lower levels compared with the TAC group (6.0 ± 0.9% vs 7.1 ± 1.7% at 12 months; P = .002). In conclusion, replacement of TAC with CYC significantly improves glucose metabolism and has the potential to reverse diabetes during the first year after conversion. (EU Clinical Trials Register No. 2006‐001765‐42)     

A Randomized Controlled Trial Comparing the Efficacy of Cyp3a5 Genotype‐Based With Body‐Weight‐Based Tacrolimus Dosing After Living Donor Kidney Transplantation

Patients expressing the cytochrome P450 (CYP) 3A5 gene require a higher tacrolimus dose to achieve therapeutic exposure compared with nonexpressers. This randomized‐controlled study investigated whether adaptation of the tacrolimus starting dose according to CYP3A5 genotype increases the proportion of kidney transplant recipients being within the target tacrolimus predose concentration range (10–15 ng/mL) at first steady‐state. Two hundred forty living‐donor, renal transplant recipients were assigned to either receive a standard, body‐weight‐based or a CYP3A5 genotype‐based tacrolimus starting dose. At day 3, no difference in the proportion of patients having a tacrolimus exposure within the target range was observed between the standard‐dose and genotype‐based groups: 37.4% versus 35.6%, respectively; p = 0.79. The proportion of patients with a subtherapeutic (i.e. <10 ng/mL) or a supratherapeutic (i.e. >15 ng/mL) Tac predose concentration in the two groups was also not significantly different. The incidence of acute rejection was comparable between both groups (p = 0.82). Pharmacogenetic adaptation of the tacrolimus starting dose does not increase the number of patients having therapeutic tacrolimus exposure early after transplantation and does not lead to improved clinical outcome in a low immunological risk population.     

Kaempferol Promotes Transplant Tolerance by Sustaining CD4+FoxP3+ Regulatory T Cells in the Presence of Calcineurin Inhibitor

Calcineurin inhibitor cyclosporine is widely used as an immunosuppressant in clinic. However, mounting evidence has shown that cyclosporine hinders tolerance induction by dampening Tregs. Therefore, it is of paramount importance to overcome this pitfall. Kaempferol was reported to inhibit DC function. Here, we found that kaempferol delayed islet allograft rejection. Combination of kaempferol and low‐dose, but not high‐dose, of cyclosporine induced allograft tolerance in majority of recipient mice. Although kaempferol plus either dose of cyclosporine largely abrogated proliferation of graft‐infiltrating T cells and their CTL activity, both proliferation and CTL activity in mice treated with kaempferol plus low‐dose, but not high‐dose, cyclosporine reemerged rapidly upon treatment withdrawal. Kaempferol increased CD4+FoxP3+ Tregs both in transplanted mice and in vitro, likely by suppressing DC maturation and their IL‐6 expression. Reduction in Tregs by low dose of cyclosporine was reversed by kaempferol. Kaempferol‐induced Tregs exhibited both allospecific and non‐allospecific suppression. Administering IL‐6 abrogated allograft tolerance induced by kaempferol and cyclosporine via diminishing CD4+FoxP3+ Tregs. Thus, for the first time, we demonstrated that kaempferol promotes transplant tolerance in the presence of low dose of cyclosporine, which allows for sufficient Treg generation while minimizing side effects, resulting in much‐needed synergy between kaempferol and cyclosporine.     

Immunosuppressive effect of the gut microbiome altered by high‐dose tacrolimus in mice

The alterations induced in gut microbiota by tacrolimus may affect immune function and organ transplantation. Mice were treated with high‐dose tacrolimus for 14 days. The fecal microbiota were analyzed by pyrosequencing the 16S rRNA genes, and the effect on metabolism was predicted using the sequence data. The subgroups of T cells in the serum, gut‐associated lymphoid tissue, and draining lymph nodes were determined by flow cytometry. Tacrolimus treatment significantly altered the relative abundance of Allobaculum, Bacteroides, and Lactobacillus and CD4⁺CD25^hiFoxP3⁺ regulatory T cells in the colonic mucosa and the circulation. These were significantly increased after either tacrolimus treatment or treatment by fecal microbiota transfer from tacrolimus‐treated donors. Further, treatment with low‐dose tacrolimus plus fecal microbiota transfer from high‐dose tacrolimus–altered mice increased skin allograft survival rate in a skin transplantation model. Thus, high‐dose tacrolimus alters the compositions and taxa of the gut microbiota. Administration of these conditioned gut microbiota plus low‐dose tacrolimus resulted in regulation of colonic and systemic immune responses and an increased allograft survival rate. This study demonstrated a new strategy for controlling allograft rejection by combining an immunosuppressive agent with gut microbiome transplantation.     

Early Conversion to Prednisolone/Everolimus as an Alternative Weaning Regimen Associates With Beneficial Renal Transplant Histology and Function: The Randomized‐Controlled MECANO Trial

In renal transplantation, use of calcineurin inhibitors (CNIs) is associated with nephrotoxicity and immunosuppression with malignancies and infections. This trial aimed to minimize CNI exposure and total immunosuppression while maintaining efficacy. We performed a randomized controlled, open‐label multicenter trial with early cyclosporine A (CsA) elimination. Patients started with basiliximab, prednisolone (P), mycophenolate sodium (MPS), and CsA. At 6 months, immunosuppression was tapered to P/CsA, P/MPS, or P/everolimus (EVL). Primary outcomes were renal fibrosis and inflammation. Secondary outcomes were estimated glomerular filtration rate (eGFR) and incidence of rejection at 24 months. The P/MPS arm was prematurely halted. The trial continued with P/CsA (N = 89) and P/EVL (N = 96). Interstitial fibrosis and inflammation were significantly decreased and the eGFR was significantly higher in the P/EVL arm. Cumulative rejection rates were 13% (P/EVL) and 19% (P/CsA), (p = 0.08). A post hoc analysis of HLA and donor‐specific antibodies at 1 year after transplantation revealed no differences. An individualized immunosuppressive strategy of early CNI elimination to dual therapy with everolimus was associated with decreased allograft fibrosis, preserved allograft function, and good efficacy, but also with more serious adverse events and discontinuation. This can be a valuable alternative regimen in patients suffering from CNI toxicity.     

Long‐Term Clinical Impact of Adaptation of Initial Tacrolimus Dosing to CYP3A5 Genotype

Pretransplantation adaptation of the daily dose of tacrolimus to CYP3A5 genotype is associated with improved achievement of target trough concentration (C₀), but whether this improvement affects clinical outcomes is unknown. In the present study, we have evaluated the long‐term clinical impact of the adaptation of initial tacrolimus dosing according to CYP3A5 genotype: The transplantation outcomes of the 236 kidney transplant recipients included in the Tactique study were retrospectively investigated over a period of more than 5 years. In the Tactique study, patients were randomly assigned to receive tacrolimus at either a fixed dosage or a dosage determined by their genotype, and the primary efficacy end point was the proportion of patients for whom tacrolimus C₀ was within target range (10–15 ng/mL) at day 10. Our results indicate that the incidence of biopsy‐proven acute rejection and graft survival were similar between the control and the adapted tacrolimus dose groups, as well as between the patients who achieve the tacrolimus C₀ target ranges earlier. Patients' death, cancer, cardiovascular events, and infections were also similar, and renal function did not change. We conclude that optimization of initial tacrolimus dose using pharmacogenetic testing does not improve clinical outcomes.     

Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor,Elafin, to Maintain Graft Perfusion During Acute Rejection

The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b‐9. Elafin was also found to promote angiogenesis through activation of the extracellular signal‐regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.