Interleukin-6 blockade with tocilizumab increases Tregs and reduces T effector cytokines in renal graft inflammation: A randomized controlled trial

Interleukin-6 (IL-6) is a proinflammatory cytokine and key regulator of Treg: T effector cell (Teff) balance. We hypothesized that IL-6 blockade with tocilizumab, a monoclonal antibody to IL-6R, would increase Tregs, dampen Teff function, and control graft inflammation. We conducted a randomized controlled clinical trial (2014–2018) of clinically stable kidney transplant recipients on calcineurin inhibitor, mycophenolate mofetil, and prednisone, with subclinical graft inflammation noted on surveillance biopsies during the first year posttransplant. Subjects received tocilizumab (8 mg/kg IV every 4 weeks; 6 doses; n = 16) or no treatment (controls; n = 14) on top of usual maintenance immunosuppression. Kidney biopsies pre- and post-treatment were analyzed using Banff criteria. Blood was analyzed for serum cytokines, Treg frequencies, and T cell effector molecule expression (IFN-γ, IL-17, granzyme B) post-stimulation ex vivo. Tocilizumab-treated subjects were more likely to show improved Banff ti-score (62.5% vs. 21.4%, p = .03), increased Treg frequency (7.1% ± 5.55% vs. 3.6% ± 1.7%, p = .0168), and a blunted Teff cytokine response compared to controls. Changes in Banff i- and t-scores were not significantly different. The treatment was relatively well tolerated with no patient deaths or graft loss. Blockade of IL-6 is a novel and promising treatment option to regulate the T cell alloimmune response in kidney transplant recipients. NCT02108600.     

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney–Bone Marrow Transplantation to Induce Transplantation Tolerance

We examined tolerance mechanisms in patients receiving HLA‐mismatched combined kidney–bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high‐throughput sequencing of T cell receptor‐β hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non‐Tregs revealed marked early enrichment of Tregs (CD3⁺CD4⁺CD25^highCD127^lowFoxp3⁺) in blood that resulted from peripheral proliferation (Ki67⁺), possibly new thymic emigration (CD31⁺), and, in one tolerant subject, conversion from non‐Tregs. Among recovering conventional T cells, central memory CD4⁺ and CD8⁺ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor‐reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia‐driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.     

Relationship between antithymocyte globulin,T cell phenotypes,and clinical outcomes in pediatric kidney transplantation

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4⁺ naïve and increased CD4⁺ effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients.     

Short‐term adverse effects of early subclinical allograft inflammation in kidney transplant recipients with a rapid steroid withdrawal protocol

The impact of subclinical inflammation (SCI) noted on early kidney allograft biopsies remains unclear. This study evaluated the outcome of SCI noted on 3‐month biopsy. A total of 273/363 (75%) kidney transplant recipients with a functioning kidney underwent allograft biopsies 3‐months posttransplant. Among those with stable allograft function at 3 months, 200 biopsies that did not meet the Banff criteria for acute rejection were identified. These were Group I: No Inflammation (NI, n = 71) and Group II: Subclinical Inflammation (SCI, n = 129). We evaluated differences in kidney function at 24‐months and allograft histology score at 12‐month biopsy. SCI patients had a higher serum creatinine (1.6 ± 0.7 vs 1.38 ± 0.45; P = .02) at 24‐months posttransplant, and at last follow‐up at a mean of 42.5 months (1.69 ± 0.9 vs 1.46 ± 0.5 mg/dL; P = .027). The allograft chronicity score (ci + ct + cg + cv) at 12‐months posttransplant was higher in the SCI group (2.4 ± 1.35 vs 1.9 ± 1.2; P = .02). The incidence of subsequent rejections within the first year in SCI and NI groups was 24% vs 10%, respectively (P = .015). De novo donor‐specific antibody within 12 months was more prevalent in the SCI group (12/129 vs 1/71, P = .03). SCI is likely not a benign finding and may have long‐term implications for kidney allograft function.     

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4⁺CD25⁺CD127^?Foxp3⁺ (Treg) in clinically relevant large animal models. We infused ex vivo–expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin–lymphodepleted, MHC‐mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti–interleukin‐6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon‐γ production by host CD8⁺ T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed “Treg‐friendly” agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.     

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.     

Tolerance of Vascularized Islet‐Kidney Transplants in Rhesus Monkeys

We previously reported that transplantation (Tx) of prevascularized donor islets as composite islet‐kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cell (HSC) Tx in an attempt to induce tolerance in nonhuman primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IK Tx. HSC Tx was performed after mobilization and leukapheresis of the donors and conditioning of the recipients with total body irradiation, T cell depletion, and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IK Tx was performed either 20–22 (n = 3) or 208 (n = 1) days after HSC Tx. All animals accepted IKs without rejection. All recipients required >20 U/day insulin before IK Tx to maintain <200 mg/dL, whereas after IK Tx, three animals required minimal doses of insulin (1–3 U/day) and one animal was insulin free. These results constitute a proof‐of‐principle that this IK tolerance strategy may provide a cure for both end‐stage renal disease and diabetes without the need for immunosuppression.     

Development and clinical validity of a novel blood‐based molecular biomarker for subclinical acute rejection following kidney transplant

Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead to chronic rejection and graft loss. We conducted a multicenter study to develop a blood‐based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%‐88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%‐61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy‐proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor‐specific antibodies. We also found that <50% showed histologic improvement of subAR on follow‐up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood‐based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.     

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation

Short‐term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen‐related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end‐stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3⁺CD4⁺CD25⁺CD127^lo surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg‐specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.     

Complement Polymorphisms in Kidney Transplantation: Critical in Graft Rejection?

The complement system, as part of the innate immune system, plays an important role in renal transplantation. Complement is involved in the protection against foreign organisms and clearance of apoptotic cells but can also cause injury to the renal allograft, for instance, via antibody binding or in ischemia–reperfusion injury. Numerous polymorphisms in complement factors have been identified thus far; some of them result in different functionalities or alter complement levels. In this review, we provide an overview of the literature on the role of complement polymorphisms in renal transplantation. Furthermore, we discuss functional complement polymorphisms that have not yet been investigated in kidney transplantation. By investigating multiple polymorphisms both in donor and recipient at the same time, a complotype can be constructed. Because the combination of multiple polymorphisms is likely to have a greater impact than a single one, this could provide valuable prognostic information.     

Nonchimeric HLA‐Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)‐identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34⁺ stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection‐free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow‐up including 5‐year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time‐dependent increases of circulating CD4⁺CD25⁺⁺⁺CD127^?FOXP3⁺ Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA‐identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

     

Complement Alternative Pathway Deficiency in Recipients Protects Kidney Allograft From Ischemia/Reperfusion Injury and Alloreactive T Cell Response

Despite the introduction of novel and more targeted immunosuppressive drugs, the long‐term survival of kidney transplants has not improved satisfactorily. Early antigen‐independent intragraft inflammation plays a critical role in the initiation of the alloimmune response and impacts long‐term graft function. Complement activation is a key player both in ischemia/reperfusion injury (IRI) as well as in adaptive antigraft immune response after kidney transplantation. Since the alternative pathway (AP) amplifies complement activation regardless of the initiation pathways and renal IR injured cells undergo uncontrolled complement activation, we speculated whether selective blockade of AP could be a strategy for prolonging kidney graft survival. Here we showed that Balb/c kidneys transplanted in factor b deficient C57 mice underwent reduced IRI and diminished T cell–mediated rejection. In in vitro studies, we found that fb deficiency in T cells and dendritic cells conferred intrinsic impaired alloreactive/allostimulatory functions, respectively, both in direct and indirect pathways of alloantigen presentation. By administering anti‐fB antibody to C57 wt recipients in the early post Balb/c kidney transplant phases, we documented that inhibition of AP during both ischemia/reperfusion and early adaptive immune response is necessary for prolonging graft survival. These findings may have implication for the use of AP inhibitors in clinical kidney transplantation.     

Easier Control of Late‐Onset Cytomegalovirus Disease Following Universal Prophylaxis Through an Early Antiviral Immune Response in Donor‐Positive,Recipient‐Negative Kidney Transplants

Universal prophylaxis for cytomegalovirus (CMV) prevention is viable but, compared with a preemptive strategy, leads to higher incidence of late‐onset disease (LOD) associated with poor patient and graft survival. The purpose of this study was to compare LOD with early onset disease (EOD), with a focus on the highest risk kidney transplant recipients (KTRs): CMV seronegative recipients transplanted from seropositive donors (D+R?). Since CMV control depends on both antiviral treatment and specific immune response, we also compared Vδ2‐negative (Vδ2^neg) γδ T cell expansion involved in CMV infection resolution. EOD was defined as occurring <3 mo and LOD as occurring >3 mo after transplantation. Depending on the period, universal prophylaxis or preemptive treatment was used. Overall, 168 D+R? KTRs were included between 2003 and 2011. LOD was associated with a lower peak DNAemia (p = 0.04), fewer recurrences (odds ratio 0.16; 95% confidence interval 0.05–0.55; p = 0.01) and shorter anti‐CMV curative treatment (40 vs. 60 days, p < 0.0001). As a corollary, we found that Vδ2^neg γδ T cell expansion was faster in LOD than in EOD (31 vs. 168 days after the beginning of CMV disease, p < 0.0001). In D+R? KTRs, universal prophylaxis is associated with more LOD, which had better infection management and a faster immune response. These results support the use of universal prophylaxis over a preemptive strategy and reappraise outcomes of LOD.     

Feasibility,long-term safety,and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1–10 × 10⁶ Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.     

The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell–mediated rejection,antibody‐mediated rejection,and prospects for integrative endpoints for next‐generation clinical trials

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i‐IFTA) and its relationship to T cell–mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody‐mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i‐IFTA is associated with reduced graft survival. Furthermore, these groups presented that i‐IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i‐IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor‐specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next‐generation clinical trials.