Regulatory B Cell‐Dependent Islet Transplant Tolerance Is Also Natural Killer Cell Dependent

Immunologic tolerance to solid organ and islet cell grafts has been achieved in various rodent models by using antibodies directed at CD45RB and Tim‐1. We have shown that this form of tolerance depends on regulatory B cells (Bregs). To elucidate further the mechanism by which Bregs induce tolerance, we investigated the requirement of natural killer (NK) and NKT cells in this model. To do so, hyperglycemic B6, μMT, Beige, or CD1d^?/? mice received BALB/c islet grafts and treatment with the tolerance‐inducing regimen consisting of anti‐CD45RB and anti‐TIM1. B6 mice depleted of both NK and NKT cells by anti‐NK1.1 antibody and mice deficient in NK activity (Beige) did not develop tolerance after dual‐antibody treatment. In contrast, transplant tolerance induction was successful in CD1d^?/? recipients (deficient in NKT cells), indicating that NK, but not NKT, cells are essential in B cell–dependent tolerance. In addition, reconstitution of Beige host with NK cells restored the ability to induce transplant tolerance with dual‐antibody treatment. Transfer of tolerance by B cells from tolerant mice was also dependent on host Nk1.1⁺ cells. In conclusion, these results show that regulatory function of B cells is dependent on NK cells in this model of transplantation tolerance.     

Identifying Subphenotypes of Antibody‐Mediated Rejection in Kidney Transplants

The key lesions in antibody‐mediated kidney transplant rejection (ABMR) are microcirculation inflammation (peritubular capillaritis and/or glomerulitis lesions, abbreviated “pg”) and glomerular double contours (cg lesions). We used these features to explore subphenotypes in 164 indication biopsies with ABMR‐related diagnoses: 137 ABMR (109 pure and 28 mixed with T cell–mediated rejection [TCMR]) and 27 transplant glomerulopathy (TG), identified from prospective multicenter studies. The lesions indicated three ABMR subphenotypes: pgABMR, cgABMR, and pgcgABMR. Principal component analysis confirmed these subphenotypes and showed that TG can be reclassified as pgcgABMR (n = 17) or cgABMR (n = 10). ABMR‐related biopsies included 45 pgABMR, 90 pgcgABMR, and 25 cgABMR, with four unclassifiable. Dominating all time intervals was the subphenotype pgcgABMR. The pgABMR subphenotype presented earliest (median <2 years), frequently mixed with TCMR, and was most associated with nonadherence. The cgABMR subphenotype presented late (median 9 years). Subphenotypes differed in their molecular changes, with pgABMR having the most histologic–molecular discrepancies (i.e. potential errors). Donor‐specific antibody (DSA) was not identified in 29% of pgcgABMR and 46% of cgABMR, but failure rates and molecular findings were similar to cases where DSA was known to be positive. Thus, ABMR presents distinct subphenotypes, early pg‐dominant, late cg‐dominant, and combined pgcg phenotype, differing in time, molecular features, accompanying TCMR, HLA antibody, and probability of nonadherence.     

Immediate and Catastrophic Antibody‐Mediated Rejection in a Lung Transplant Recipient With Anti–Angiotensin II Receptor Type 1 and Anti–Endothelin‐1 Receptor Type A Antibodies

Preexisting donor‐specific anti‐HLA antibodies (DSAs) have been associated with reduced survival of lung allografts. However, antibodies with specificities other than HLA may have a detrimental role on the lung transplant outcome. A young man with cystic fibrosis underwent lung transplantation with organs from a suitable deceased donor. At the time of transplantation, there were no anti‐HLA DSAs. During surgery, the patient developed a severe and intractable pulmonary hypertension associated with right ventriular dysfunction, which required arteriovenous extracorporeal membrane oxygenation. After a brief period of clinical improvement, a rapid deterioration in hemodynamics led to the patient's death on postoperative day 5. Postmortem studies showed that lung specimens taken at the end of surgery were compatible with antibody‐mediated rejection (AMR), while terminal samples evidenced diffuse capillaritis, blood extravasation, edema, and microthrombi, with foci of acute cellular rejection (A3). Immunological investigations demonstrated the presence of preexisting antibodies against the endothelin‐1 receptor type A (ET_AR) and the angiotensin II receptor type 1 (AT₁R), two of the most potent vasoconstrictors reported to date, whose levels slightly rose after transplantation. These data suggest that preexisting anti‐ET_AR and anti‐AT₁R antibodies may have contributed to the onset of AMR and to the catastrophic clinical course of this patient.     

Assessment of Tocilizumab (Anti–Interleukin‐6 Receptor Monoclonal) as a Potential Treatment for Chronic Antibody‐Mediated Rejection and Transplant Glomerulopathy in HLA‐Sensitized Renal Allograft Recipients

Extending the functional integrity of renal allografts is the primary goal of transplant medicine. The development of donor‐specific antibodies (DSAs) posttransplantation leads to chronic active antibody‐mediated rejection (cAMR) and transplant glomerulopathy (TG), resulting in the majority of graft losses that occur in the United States. This reduces the quality and length of life for patients and increases cost. There are no approved treatments for cAMR. Evidence suggests the proinflammatory cytokine interleukin 6 (IL‐6) may play an important role in DSA generation and cAMR. We identified 36 renal transplant patients with cAMR plus DSAs and TG who failed standard of care treatment with IVIg plus rituximab with or without plasma exchange. Patients were offered rescue therapy with the anti–IL‐6 receptor monoclonal tocilizumab with monthly infusions and monitored for DSAs and long‐term outcomes. Tocilizumab‐treated patients demonstrated graft survival and patient survival rates of 80% and 91% at 6 years, respectively. Significant reductions in DSAs and stabilization of renal function were seen at 2 years. No significant adverse events or severe adverse events were seen. Tocilizumab provides good long‐term outcomes for patients with cAMR and TG, especially compared with historical published treatments. Inhibition of the IL‐6–IL‐6 receptor pathway may represent a novel approach to stabilize allograft function and extend patient lives.     

A Probabilistic Approach to Histologic Diagnosis of Antibody‐Mediated Rejection in Kidney Transplant Biopsies

Histologic diagnosis of antibody‐mediated rejection (ABMR) in kidney transplant biopsies uses lesion score cutoffs such as 0 versus >0 rather than actual scores and requires donor‐specific antibody (DSA); however, cutoffs lose information, and DSA is not always reliable. Using microarray‐derived molecular ABMR scores as a histology‐independent estimate of ABMR in 703 biopsies, we reassessed criteria for ABMR to determine relative importance of various lesions, the utility of equations using actual scores rather than cutoffs, and the potential for diagnosing ABMR when DSA is unknown or negative. We confirmed that the important features for ABMR diagnosis were peritubular capillaritis (ptc), glomerulitis (g), glomerular double contours, DSA and C4d staining, but we questioned some features: arterial fibrosis, vasculitis, acute tubular injury, and sum of ptc+g scores. Regression equations using lesion scores predicted molecular ABMR more accurately than score cutoffs (area under the curve 0.85–0.86 vs. 0.75). DSA positivity improved accuracy, but regression equations predicted ABMR with moderate accuracy when DSA was unknown. Some biopsies without detectable DSA had high probability of ABMR by regression, although most had HLA antibody. We concluded that regression equations using lesion scores plus DSA maximized diagnostic accuracy and can estimate probable ABMR when DSA is unknown or undetectable.     

The natural killer cell activating receptor,NKG2D,is critical to antibody-dependent chronic rejection in heart transplantation

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor–recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag^−/− recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.     

Deletion of the activating NK cell receptor NKG2D accelerates rejection of cardiac allografts

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co‐stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC‐disparate BALB/c‐derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1^?/?). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46⁺ NK cells in Klrk1^?/? recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4⁺, but a lesser infiltration of CD8⁺ T cell frequencies. Contrary to published observations, co‐stimulation blockade with CTLA4‐Ig resulted in a significant acceleration of cardiac rejection by Klrk1^?/? recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1^?/? recipients were characterized by significantly higher levels of interferon‐γ mRNA, and both CD4⁺ and CD8⁺ T cells displayed a greater capacity for degranulation and interferon‐γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.     

Cytotoxicity of Natural Killer Cells Activated Through NKG2D Contributes to the Development of Bronchiolitis Obliterans in a Murine Heterotopic Tracheal Transplant Model

Bronchiolitis obliterans after lung transplantation is a major cause of postoperative mortality in which T cell–mediated immunity is known to play an important role. However, the exact contribution of natural killer (NK) cells, which have functions similar to CD8⁺ T cells, has not been defined. Here, we assessed the role of NK cells in murine bronchiolitis obliterans through heterotopic tracheal transplantations and found a greater percentage of NK cells in allografts than in isografts. Depletion of NK cells using an anti‐NK1.1 antibody attenuated bronchiolitis obliterans in transplant recipients compared with controls. In terms of NK cell effector functions, an improvement in bronchiolitis obliterans was observed in perforin‐KO recipient mice compared to wild type (WT). Furthermore, we found upregulation of NKG2D‐ligand in allografts and demonstrated the significance of this using grafts expressing Rae‐1, a murine NKG2D‐ligand, which induced severe bronchiolitis obliterans in WT and Rag‐1 KO recipients. This effect was ameliorated by injection of anti‐NKG2D blocking antibody. Together, these results suggest that cytotoxicity resulting from activation of NK cells through NKG2D leads to the development of murine bronchiolitis obliterans.     

Plasma‐Derived C1 Esterase Inhibitor for Acute Antibody‐Mediated Rejection Following Kidney Transplantation: Results of a Randomized Double‐Blind Placebo‐Controlled Pilot Study

Antibody‐mediated rejection (AMR) is typically treated with plasmapheresis (PP) and intravenous immunoglobulin (standard of care; SOC); however, there is an unmet need for more effective therapy. We report a phase 2b, multicenter double‐blind randomized placebo‐controlled pilot study to evaluate the use of human plasma‐derived C1 esterase inhibitor (C1 INH) as add‐on therapy to SOC for AMR. Eighteen patients received 20 000 units of C1 INH or placebo (C1 INH n = 9, placebo n = 9) in divided doses every other day for 2 weeks. No discontinuations, graft losses, deaths, or study drug‐related serious adverse events occurred. While the study's primary end point, a difference between groups in day 20 pathology or graft survival, was not achieved, the C1 INH group demonstrated a trend toward sustained improvement in renal function. Six‐month biopsies performed in 14 subjects (C1 INH = 7, placebo = 7) showed no transplant glomerulopathy (TG) (PTC+cg≥1b) in the C1 INH group, whereas 3 of 7 placebo subjects had TG. Endogenous C1 INH measured before and after PP demonstrated decreased functional C1 INH serum concentration by 43.3% (p < 0.05) for both cohorts (C1 INH and placebo) associated with PP, although exogenous C1 INH–treated patients achieved supraphysiological levels throughout. This new finding suggests that C1 INH replacement may be useful in the treatment of AMR.     

Using Molecular Phenotyping to Guide Improvements in the Histologic Diagnosis of T Cell–Mediated Rejection

Recognition that some lesions typical of T cell–mediated rejection (TCMR) also occur in antibody‐mediated rejection requires revision of the histologic TCMR definition. To guide this process, we assessed the relative importance of various lesions and the performance of new histology diagnostic algorithms, using molecular TCMR scores as histology‐independent estimates of true TCMR. In 703 indication biopsies, random forest analysis and logistic regression indicated that interstitial infiltrate (i‐lesions) and tubulitis (t‐lesions) were the key histologic predictors of molecular TCMR, with arteritis (v‐lesions) having less importance. Histology predicted molecular TCMR more accurately when diagnoses were assigned by strictly applying the Banff rules to the lesion scores and redefining isolated v‐lesion TCMR. This improved prediction from area under the curve (AUC) 0.70 with existing rules to AUC 0.80. Further improvements were achieved by introducing more categories to reflect inflammation (AUC 0.84), by summing the lesion scores (AUC 0.85) and by logistic regression (AUC 0.90). We concluded that histologic assessment of TCMR can be improved by placing more emphasis on i‐ and t‐lesions and incorporating new algorithms for diagnosis. Nevertheless, some discrepancies between histologic and molecular diagnoses persist, partially due to the inherent nonspecificity of i‐ and t‐lesions, and molecular methods will be required to help resolve these cases.     

Molecular Assessment of Microcirculation Injury in Formalin‐Fixed Human Cardiac Allograft Biopsies With Antibody‐Mediated Rejection

Precise diagnosis of antibody‐mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin‐fixed, paraffin‐embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor‐specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti‐HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy‐based molecular assessment of antibody‐mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.     

Back signaling of HLA class I molecules and T/NK cell receptor ligands in epithelial cells reflects the rejection‐specific microenvironment in renal allograft biopsies

The role of endothelial cells in the pathophysiology of antibody‐mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal‐transducing capacity to mediate back signaling after encounter with either T/NK cells or donor‐specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL‐6, CXCL1,8,10, CCL2, and sICAM‐1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.     

Proteasome Inhibitor Carfilzomib‐Based Therapy for Antibody‐Mediated Rejection of the Pulmonary Allograft: Use and Short‐Term Findings

We present this observational study of lung transplant recipients (LTR) treated with carfilzomib (CFZ)‐based therapy for antibody‐mediated rejection (AMR) of the lung. Patients were considered responders to CFZ if complement‐1q (C1q)‐fixing ability of their immunodominant (ID) donor‐specific anti‐human leukocyte antibody (DSA) was suppressed after treatment. Treatment consisted of CFZ plus plasma exchange and immunoglobulins. Fourteen LTRs underwent CFZ for 20 ID DSA AMR. Ten (71.4%) of LTRs responded to CFZ. DSA IgG mean fluorescence intensity (MFI) fell from 7664 (IQR 3230–11 874) to 1878 (653–7791) after therapy (p = 0.001) and to 1400 (850–8287) 2 weeks later (p = 0.001). DSA C1q MFI fell from 3596 (IQR 714–14 405) to <30 after therapy (p = 0.01) and <30 2 weeks later (p = 0.02). Forced expiratory volume in 1s ( FEV₁) fell from mean 2.11 L pre‐AMR to 1.92 L at AMR (p = 0.04). FEV₁ was unchanged after CFZ (1.91 L) and subsequently rose to a maximum of 2.13 L (p = 0.01). Mean forced expiratory flow during mid forced vital capacity (25–75) (FEF_25–75) fell from mean 2.5 L pre‐AMR to 1.95 L at AMR (p = 0.01). FEF_25–75 rose after CFZ to 2.54 L and reached a maximum of 2.91 L (p = 0.01). Responders had less chronic lung allograft dysfunction or progression versus nonresponders (25% vs. 83%, p = 0.04). No deaths occurred within 120 days and 7 patients died post CFZ therapy of allograft failure. Larger prospective interventional studies are needed to further describe the benefit of CFZ‐based therapy for pulmonary AMR.     

Antigen‐Specificity of T Cell Infiltrates in Biopsies With T Cell–Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing

This study interrogates the antigen‐specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). Peripheral blood mononuclear cells (PBMC) from five healthy HLA‐A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing of the T cell–receptor complementary DNA was performed on peptide‐stimulated PBMC and 23 biopsies with T cell–mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus‐reactive clones. Biopsies with TCMR also contained BKPyV‐specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV‐reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V‐family and J‐gene utilization patterns. Dendrograms also revealed that V‐gene, J‐gene, and D‐gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus‐reactive clones. The T cell component of tissue injury in viral nephropathy appears to be mediated primarily by an “innocent bystander” mechanism in which the principal element is secondary T cell influx triggered by both antiviral and anti‐HLA immunity.     

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody‐Mediated Rejection Diagnostics: Strengths,Unmet Needs,and Future Directions

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody‐mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide‐ranging multidisciplinary discussion that was generated, and considerations for future endeavors.     

Exercise increases skin graft resistance to rejection

Regular exercise reduces risk of various chronic diseases and can prevent the development and recurrence of cancer, making it a promising nonpharmacological modulator of disease. Yet the effect of regular exercise on solid organ transplant outcome remains uncertain. Using a model of voluntary wheel‐running exercise and skin transplantation in mice, we hypothesized that exercise strengthens the alloimmune response, leading to an increased rate of rejection. Instead, we found that regular exercise in mice resulted in prolonged graft survival, with mean allograft survival time increasing by almost 50%. We observed this graft survival extension in exercised mice despite evidence of a slightly enhanced alloimmune response, comprised of increased proliferation of alloreactive CD4⁺ T cells, as well as increased interferon‐γ production by these cells. Exercise was not associated with significant changes in numbers of conventional CD4⁺ or CD8⁺ T cells, NK cells, or Foxp3⁺ regulatory T cells. In conclusion, our study suggests that exercise increases skin graft resistance to a similar or slightly higher level of alloimmunity and supports regular exercise as an important beneficial pursuit for transplant recipients.