IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor.

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rß during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rß(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity.

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

Adequate tacrolimus exposure modulates the impact of HLA class II molecular mismatch: a validation study in an American cohort.

Clinicians have few tools to predict the risk of alloimmune injury that would guide immunosuppression management in renal transplant patients. We evaluated human leukocyte antigen (HLA)-DR/DQ molecular mismatch to predict de novo donor-specific antibodies (DSAs) during the first year of transplant and explored how differences in tacrolimus exposure may modulate this risk. HLA-DR and -DQ eplet mismatches were determined between 444 donor-recipient pairs in Denver, Colorado between 2007 and 2013. Previously defined mismatch thresholds stratified recipients into low- (N = 119), intermediate- (N = 153), and high- (N = 172) risk categories. The area under the curve for DSA at 1 year was 0.84 and 0.82 for HLA-DR and HLA-DQ eplet mismatches, respectively. Compared to low-risk patients, there was a graded increase in risk of DR/DQ DSA in intermediate (HR 15.39, 95% CI 2.01-118.09, p = .009) and high-risk (HR 23.81, 95% CI 3.17-178.66, p = 0.002) categories. Intermediate- and high-risk patients with a mean tacrolimus <6 ng/ml versus >8 ng/ml had increased risk of DR/DQ DSA at 1 year (HR 2.34, 95% CI 1.05-5.22, p = .04). HLA molecular mismatch represents a reproducible, objective, and clinically relevant tool to stratify patients by alloimmune risk and may help guide personalized immunosuppression management.

A noninferiority design for a delayed calcineurin inhibitor substitution trial in kidney transplantation.

Improving long-term kidney transplant outcomes requires novel treatment strategies, including delayed calcineurin inhibitor (CNI) substitution, tested using informative trial designs. An alternative approach to the usual superiority-based trial is a noninferiority trial design that tests whether an investigational agent is not unacceptably worse than standard of care. An informative noninferiority design, with biopsy-proven acute rejection (BPAR) as the endpoint, requires determination of a prespecified, evidence-based noninferiority margin for BPAR. No such information is available for delayed CNI substitution in kidney transplantation. Herein we analyzed data from recent kidney transplant trials of CNI withdrawal and "real world" CNI- based standard of care, containing subjects with well-documented evidence of immune quiescence at 6 months posttransplant-ideal candidates for delayed CNI substitution. Our analysis indicates an evidence-based noninferiority margin of 13.8% for the United States Food and Drug Administration's composite definition of BPAR between 6 and 24 months posttransplant. Sample size estimation determined that ~225 randomized subjects would be required to evaluate noninferiority for this primary clinical efficacy endpoint, and superiority for a renal function safety endpoint. Our findings provide the basis for future delayed CNI substitution noninferiority trials, thereby increasing the likelihood they will provide clinically implementable results and achieve regulatory approval.

Mus musculus papillomavirus 1 is a key driver of skin cancer development upon immunosuppression.

Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.

SARS Cov-2 infection in a renal-transplanted patient: A case report.

The clinical manifestation of COVID-19 can vary from an asymptomatic course to ARDS requiring invasive mechanical ventilation and extracorporeal membrane oxygenation. A kidney transplanted patient infected with SARS CoV-2 infection showed a mild disease despite immune suppression. It is possible that Immunosuppression can "be protective" as the cytokine storm is an important factor in the disease story. Despite the good outcome reported in the present case report, is remains of vital importance the solid organ transplant patients use precautions in order to avoid the infection.

A comprehensive review of the impact of tacrolimus intrapatient variability on clinical outcomes in kidney transplantation.

Tacrolimus (Tac) is widely used to prevent rejection and graft loss in solid organ transplantation. A limiting characteristic of Tac is the high intra and interpatient variability associated with its use. Routine therapeutic drug monitoring (TDM) is necessary to facilitate Tac management and to avoid undesirable clinical outcomes. However, whole blood trough concentrations commonly utilized in TDM are not strong predictors of the detrimental clinical outcomes of interest. Recently, researchers have focused on Tac intrapatient variability (Tac IPV) as a novel marker to better assess patient risk. Higher Tac IPV has been associated with a number of mechanisms leading to shortened graft survival. Medication nonadherence (MNA) is considered to be the primary determinant of high Tac IPV and perhaps the most modifiable risk factor. An understanding of the methodology behind Tac IPV is imperative to its recognition as an important prognostic measure and integration into clinical practice. Therapeutic interventions targeting MNA and reducing Tac IPV are crucial to improving long-term graft survival.

Distinct peripheral blood molecular signature emerges with successful tacrolimus withdrawal in kidney transplant recipients.

Tacrolimus (Tac) is an effective anti-rejection agent in kidney transplantation, but its off-target effects make withdrawal desirable. Although studies indicate that Tac can be safely withdrawn in a subset of kidney transplant recipients, immune mechanisms that underlie successful vs unsuccessful Tac removal are unknown. We performed microarray analyses of peripheral blood mononuclear cells (PBMC) RNA from subjects enrolled in the Clinical Trials in Organ Transplantation-09 study in which we randomized stable kidney transplant recipients to Tac withdrawal or maintenance of standard immunosuppression beginning 6 months after transplant. Eight of 14 subjects attempted but failed withdrawal, while six developed stable graft function for ≥2 years on mycophenolate mofetil plus prednisone. Whereas failed withdrawal upregulated immune activation genes, successful Tac withdrawal was associated with a downregulatory and proapoptotic gene program enriched within T cells. Functional analyses suggested stronger donor-reactive immunity in subjects who failed withdrawal without evidence of regulatory T cell dysfunction. Together, our data from a small, but unique, patient cohort support the conclusion that successful Tac withdrawal is not simply due to absence of donor-reactive immunity but rather is associated with an active immunological process.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

Clinical course of COVID-19 in a liver transplant recipient on hemodialysis and response to tocilizumab therapy: A case report.

The novel coronavirus disease 2019 (COVID-19) is a highly infectious and rapidly spreading disease. There are limited published data on the epidemiology and outcomes of COVID-19 infection among organ transplant recipients. After initial flulike symptoms, progression to an inflammatory phase may occur, characterized by cytokine release rapidly leading to respiratory and multiorgan failure. We report the clinical course and management of a liver transplant recipient on hemodialysis, who presented with COVID-19 pneumonia, and despite completing a 5-day course of hydroxychloroquine, later developed marked inflammatory manifestations with rapid improvement after administration of off-label, single-dose tocilizumab. We also highlight the role of lung ultrasonography in early diagnosis of the inflammatory phase of COVID-19. Future investigation of the effects of immunomodulators among transplant recipients with COVID-19 infection will be important.

The tacrolimus-induced glucose homeostasis imbalance in terms of the liver: From bench to bedside.

Tacrolimus (TAC), the mainstay of maintenance immunosuppressive agents, plays a crucial role in new-onset diabetes after transplant (NODAT). Previous studies investigating the diabetogenic effects of TAC have focused on the ß cells of islets. In this study, we found that TAC contributed to NODAT through directly affecting hepatic metabolic homeostasis. In mice, TAC-induced hypoglycemia rather than hyperglycemia during starvation via suppressing gluconeogenetic genes, suggesting the limitation of fasting blood glucose in the diagnosis of NODAT. In addition, TAC caused hepatic insulin resistance and triglyceride accumulation through insulin receptor substrate (IRS)2/AKT and sterol regulatory element binding protein (SREBP1) signaling, respectively. Furthermore, we found a pivotal role of CREB-regulated transcription coactivator 2 (CRTC2) in TAC-induced metabolic disorders. The restoration of hepatic CRTC2 alleviated the metabolic disorders through its downstream molecules (eg, PCK1, IRS2, and SREBP1). Consistent with the findings from bench, low CRTC2 expression in graft hepatocytes was an independent risk factor for NODAT (odds ratio = 2.692, P = .023, n = 135). Integrating grafts' CRTC2 score into the clinical model could significantly increase the predictive capacity (areas under the receiver operating characteristic curve: 0.71 vs 0.79, P = .048). Taken together, in addition to its impact on pancreatic cells, TAC induces "hematogenous diabetes" via CRTC2 signaling. Liver-targeted management may be of help to prevent or heal TAC-associated diabetes.

Cyclosporine and COVID-19: Risk or favorable?

The coronavirus disease 2019 (COVID-19) pandemic is declared a global health emergency. COVID-19 is triggered by a novel coronavirus: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Baseline characteristics of admitted patients with COVID-19 show that adiposity, diabetes, and hypertension are risk factors for developing severe disease, but so far immunosuppressed patients who are listed as high-risk patients have not been more susceptible to severe COVID-19 than the rest of the population. Multiple clinical trials are currently being conducted, which may identify more drugs that can lower mortality, morbidity, and burden on the society. Several independent studies have convincingly shown that cyclosporine inhibit replication of several different coronaviruses in vitro. The cyclosporine-analog alisporivir has recently been shown to inhibit SARS-CoV-2 in vitro. These findings are intriguing, although there is no clinical evidence for a protective effect to reduce the likelihood of severe COVID-19 or to treat the immune storm or acute respiratory distress syndrome (ARDS) that often causes severe morbidity. Here, we review the putative link between COVID-19 and cyclosporine, while we await more robust clinical data.

A case of SARS-CoV-2 pneumonia with successful antiviral therapy in a 77-year-old man with a heart transplant.

The SARS-CoV-2 infection can be seen as a single disease, but it also affects patients with relevant comorbidities who may have an increased risk of a severe course of infection. In this report, we present a 77-year-old patient with a heart transplant receiving relevant immunosuppressive therapy who tested positive for SARS-CoV-2 after several days of dyspnea, dry cough, and light general symptoms. Computed tomography confirmed interstitial pneumonia. The patient received antiviral therapy with hydroxychloroquine and showed no further deterioration of the clinical state. After 12 days of hospitalization, the patient was released; he was SARS-CoV-2 negative and completely asymptomatic.

Butyric acid normalizes hyperglycemia caused by the tacrolimus-induced gut microbiota.

Approximately 33.6% of nondiabetic solid organ transplant recipients who received tacrolimus developed hyperglycemia. Whether the tacrolimus-induced gut microbiota is involved in the regulation of hyperglycemia has not been reported. Hyperglycemia was observed in a tacrolimus-treated mouse model, with reduction in taxonomic abundance of butyrate-producing bacteria and decreased butyric acid concentration in the cecum. This tacrolimus-induced glucose metabolic disorder was caused by the gut microbiota, as confirmed by a broad-spectrum antibiotic model. Furthermore, oral supplementation with butyrate, whether for remedy or prevention, significantly increased the butyric acid content in the cecum and arrested hyperglycemia through the regulation of glucose-regulating hormones, including glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and insulin, in serum. The butyrate-G-protein-coupled receptor 43-GLP-1 pathway in the intestinal crypts may be involved in the pathogenesis of normalization of hyperglycemia caused by the tacrolimus. Therefore, tacrolimus affects glucose metabolism through the butyrate-associated GLP-1 pathway in the gut, and oral supplementation with butyrate provides new insights for the prevention and treatment of tacrolimus-induced hyperglycemia in transplant recipients.

Tacrolimus troughs and genetic determinants of metabolism in kidney transplant recipients: A comparison of four ancestry groups.

Tacrolimus trough and dose requirements vary dramatically between individuals of European and African American ancestry. These differences are less well described in other populations. We conducted an observational, prospective, multicenter study from which 2595 kidney transplant recipients of European, African, Native American, and Asian ancestry were studied for tacrolimus trough, doses, and genetic determinants of metabolism. We studied the well-known variants and conducted a CYP3A4/5 gene-wide analysis to identify new variants. Daily doses, and dose-normalized troughs were significantly different between the four groups (P < .001). CYP3A5*3 (rs776746) was associated with higher dose-normalized tacrolimus troughs in all groups but occurred at different allele frequencies and had differing effect sizes. The CYP3A5*6 (rs10264272) and *7 (rs413003343) variants were only present in African Americans. CYP3A4*22 (rs35599367) was not found in any of the Asian ancestry samples. We identified seven suggestive variants in the CYP3A4/5 genes associated with dose-normalized troughs in Native Americans (P = 1.1 × 10-5 -8.8 × 10-6 ) and one suggestive variant in Asian Americans (P = 5.6 × 10-6 ). Tacrolimus daily doses and dose-normalized troughs vary significantly among different ancestry groups. We identified potential new variants important in Asians and Native Americans. Studies with larger populations should be conducted to assess the importance of the identified suggestive variants.

Late intra-patient tacrolimus trough level variability as a major problem in kidney transplantation: A Collaborative Transplant Study Report.

Intra-patient variability (IPV) of tacrolimus trough level has been associated with poor outcome after kidney transplantation. These findings were derived from single-center analyses and restricted mainly to measurements early after transplantation. We analyzed in a multicenter effort whether high IPV of tacrolimus levels at posttransplant years 1, 2, and 3 was associated with impaired clinical outcome. More than 6600 patients who received a deceased donor kidney transplant during 2000-2014 and had a functioning graft for >3 years were studied. Graft survival was significantly impaired with increasing IPV (P < 0.001). As compared to patients with a low IPV of <30%, the risk of graft loss during years 4-6 increased 32% in patients with an IPV of 30% to 44% and 66% in patients with an IPV of ≥45% (P = 0.002 and P < 0.001). About one-third of patients showed an IPV of ≥30% with substantially impaired outcome. Even in patients with good outcome during the first 3 posttransplant years, a high IPV was associated with inferior graft survival. Our data indicate that a fluctuating tacrolimus trough level at years 1, 2, and 3 posttransplant is a major problem in kidney transplantation.

Assessment of tacrolimus intrapatient variability in stable adherent transplant recipients: Establishing baseline values.

The purpose of this study was to determine the intrapatient (within the same patient) variability of tacrolimus in adherent patients. Daily tacrolimus trough levels were obtained at home using dried blood spot technology in kidney and liver transplant recipients. Patients were randomized to receive 3 formulations of tacrolimus, each for two 1-week periods. Adherence was monitored by patient diary, pill counts, and use of the Medication Event Monitoring System (MEMS). Variability was quantified as the coefficient of variation (CV). Comparison of CV between groups was by independent t test or one-way ANOVA as appropriate. The population was found to be adherent with a rate of 99.9% with a mean interval between the evening and morning dose of tacrolimus of 11.86 hours. The median CV for the entire population was 15.2% (range 4.8%-110%). There were no differences in CV by allograft type or tacrolimus formulation. The multivariate analysis did not identify any demographic characteristics associated with a CV > 30%. In a highly adherent population, tacrolimus did not display high intrapatient variability. Given the association between IPV and poor allograft outcomes, future studies are needed to quantitate the influence of adherence and establish target IPV goals.

Evidence of a clinically significant drug-drug interaction between cannabidiol and tacrolimus.

Cannabidiol (CBD), a major purified nonpsychoactive component of cannabis with anticonvulsant properties, was approved by the U.S. Food and Drug Administration (FDA) in June 2018 as an adjuvant treatment for refractory epilepsy (Epidiolex; GW Pharmaceuticals). CBD is metabolized by cytochrome P450 (CYP)3A4 and CYP2C19 with a growing body of evidence suggesting it is also a potent inhibitor of these pathways. We report for the first time a significant drug-drug interaction between the purified CBD product and tacrolimus. A participant in a CBD clinical trial for epilepsy who was also receiving tacrolimus showed an approximately 3-fold increase in dose-normalized tacrolimus concentrations while receiving 2000-2900 mg/day of CBD. Our report delineates an important concern for the transplant community with the increasing legalization of cannabis and advent of an FDA-approved CBD product. Larger studies are needed to better understand the impact of this drug-drug interaction in solid organ transplant recipients.

Absence of recipient C3aR1 signaling limits expansion and differentiation of alloreactive CD8+ T cell immunity and prolongs murine cardiac allograft survival.

Activation, differentiation, and expansion of alloreactive CD8+ T cells, the dominant effectors that mediate murine heart allograft rejection, requires allorecognition, costimulation, and cytokine-initiated signals. While previous work showed that alloreactive CD4+ T cell immunity entails immune cell-produced and locally activated complement, whether and how C3a receptor 1 (C3aR1) signaling impacts transplant outcomes and the mechanisms linking C3aR1 to alloreactive CD8+ T cell activation/expansion remain unclear. Herein we show that recipient C3aR1 deficiency or pharmacological C3aR1 blockade synergizes with tacrolimus to significantly prolong allograft survival versus tacrolimus-treated controls (median survival time 21 vs. 14 days, P < .05). Recipient C3aR1-deficiency reduced the frequencies of posttransplant, donor-reactive CD8+ T cells twofold. Reciprocal adoptive transfers of naive WT or C3ar1-/- CD8+ T cells into syngeneic WT or C3ar1-/- allograft recipients showed that T cell-expressed C3aR1 induces CD8+ T proliferation, mTOR activation and transcription factor T-bet expression. Host C3aR1 indirectly facilitates alloreactive CD8+ T cell proliferation/expansion by amplifying antigen presenting cell costimulatory molecule expression and innate cytokine production. In addition to expanding mechanistic insight, our findings identify C3aR1 as a testable therapeutic target for future studies aimed at improving human transplant outcomes.

Inhibition of the mTOR pathway: A new mechanism of ß cell toxicity induced by tacrolimus.

The mechanisms of tacrolimus-induced ß cell toxicity are unknown. Tacrolimus (TAC) and rapamycin (Rapa) both bind to FK506-binding protein 12 (FKBP12). Also, both molecular structures are similar. Because of this similarity, we hypothesized that TAC can also inhibit the mTOR signalling, constituting a possible mechanism of ß cell toxicity. Thus, we studied the effect of TAC and Rapa over the mTOR pathway, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin secretion and content in INS-1 ß cells treated with or without glucose and palmitate and in islets from lean or obese rats. TAC and Rapa inhibited the mTOR pathway as reflected by lower levels of phospho-mTOR, phospo-p70S6K, and phospo-S6. The effect of Rapa was larger than TAC. Both drugs reduced the levels of MafA, insulin secretion, and content although these effects were larger with TAC. The changes on MafA and insulin metabolism were observed in cells on glucose and palmitate, in obese animals, and were absent in cells on maintenance medium or in lean animals. In silico docking and immunoprecipitation experiments confirmed that TAC can form a stable noncovalent interaction with FKBP12-mTOR. Thus, the mTOR inhibition may be a mechanism contributing to the diabetogenic effect of TAC.