Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.     

Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7-10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T-dependent, failing to develop in T-cell-deficient hosts. Allografts into immunoglobulin-deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody-independent. However at day 21 kidneys rejecting in immunoglobulin-deficient hosts showed decreased edema, arteritis, venulitis, and necrosis. Thus the three groups of lesions are: T-cell-mediated interstitial infiltration, MHC induction, and venulitis, which develops rapidly then stabilizes; slower but progressive T-cell-mediated tubulitis and arteritis; and late antibody-mediated endothelial injury, which contributes to late edema, arteritis, and venulitis.     

Transplant Glomerulopathy, Late Antibody-Mediated Rejection and the ABCD Tetrad in Kidney Allograft Biopsies for Cause

To define the relative frequency of phenotypes of transplant glomerulopathy, we retrospectively reviewed the findings in 1036 biopsies for clinical indications from 1320 renal transplant patients followed in our clinics between 1997 and 2005. Transplant glomerulopathy, defined by double contours of glomerular basement membranes (D), was diagnosed in 53 biopsies (5.1%) from 41 patients (3.1%) at a median of 5.5 years post-transplant (range 3.8-381 months). In cases with D, we studied the frequency of circulating anti-HLA alloantibody (A), peritubular capillary basement membrane multilayering (B) and peritubular capillary C4d deposition (C). B was present in 48 (91%) of D biopsies. C4d staining by indirect immunofluorescence was detected in 18 of 50 D biopsies studied (36%). By Flow PRA Screening or ELISA, A was detected in 33 (70%) in 47 D cases with available sera, of which 28/33 or 85% were donor-specific. Class II (13/33) or class I and II (17/33) were more common than class I (3/33) antibodies. Thus 73% of transplant glomerulopathy has evidence of alloantibody-mediated injury (A and/or C), with ABCD and ABD being the common phenotypes in biopsies for cause. The remaining 27%, mostly BD, may be a different disease or a stage in which A and C are undetectable.     

Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients

Granzyme B (GZMB) and perforin 1 gene (PRF1) are key effector molecules of cytotoxic T lymphocytes, in causing acute and chronic solid organ transplant rejection. In this study, we analyzed the impact of GZMB and PRF1 polymorphism on kidney allograft outcomes. In all, 527 de novo kidney Hispanic allograft recipients were genotyped for PRF1 (rs10999426, rs35947132) and GZMB (rs8192917, rs7144366). PRF1 (rs10999426, rs35947132) G alleles and GG genotypes were negatively associated with allograft rejection, demonstrating protection against allograft rejection (OR = 0.61, p = 0.005 for rs1099946; OR = 0.4, p = 0.01 for rs 35947132). On the other hand, the GA heterozygosity of PRF1 was found marginally associated with the rejection group (OR = 1.53, p = 0.05 for rs10999426; OR = 2.24, p = 0.07 for rs35947132). There was a significant increase in allograft survival in time period studied for the PRF1 (rs10999426) GG genotype, while the GA heterozygosity was associated with graft failure. We found no association for polymorphic markers in GZMB gene with allograft rejection. Survival was significantly improved for patients who were homozygous TT for the GZMB (rs8192917) (TT vs. CC/TT, p = 0.041). The result suggests that PRF1 and GZMB gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.     

IFN-γ Prevents Early Perforin-Granzyme-Mediated Destruction of Kidney Allografts by Inducing Donor Class I Products in the Kidney

Interferon-γ (Ifng) protects organ allografts: mouse kidney allografts lacking Ifng receptors rapidly fail with massive ischemic necrosis around days 5 to 7, reflecting microcirculation failure. We hypothesized that Ifng protects the graft by preventing perforin-granzyme-mediated cytotoxic damage to the microcirculation by inducing class Ia and/or Ib products. We transplanted kidney allografts lacking Ifng receptors into various knockout hosts. The necrosis/congestion phenotype did not require host B cells or IL-4 and IL-13 receptors, but required the T-cell alloresponse: it did not occur if the hosts were syngeneic or T-cell deficient. However, host perforin-granzyme mechanisms were required: no necrosis developed if hosts lacked either perforin or granzymes A and B. The ability of Ifng to protect the allograft required donor class I products: allografts lacking class I products due to Tap1 or β2 microglobulin deficiency developed a similar necrosis-congestion phenotype at day 7 despite Ifng receptors being present. Thus when host cytotoxic T cells infiltrate organ allografts, Ifng prevents their perforin-granzyme mechanism from compromising the microcirculation by a mechanism requiring donor class Ia or Ib products. We propose that donor class Ia or Ib products are needed to trigger inhibitory receptors on effector T cells.     

Patterns of De Novo Allo B Cells and Antibody Formation in Chronic Cardiac Allograft Rejection After Alemtuzumab Treatment

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody‐mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti‐human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody‐mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody‐mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long‐term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor‐specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.     

Donor Specificity but Not Broadness of Sensitization Is Associated With Antibody‐Mediated Rejection and Graft Loss in Renal Allograft Recipients

Panel‐reactive antibodies are widely regarded as an important immunological risk factor for rejection and graft loss. The broadness of sensitization against HLA is most appropriately measured by the “calculated population‐reactive antibodies” (cPRA) value. In this study, we investigated whether cPRA represent an immunological risk in times of sensitive and accurate determination of pretransplantation donor‐specific HLA antibodies (DSA). Five hundred twenty‐seven consecutive transplantations were divided into four groups: cPRA 0% (n = 250), cPRA 1–50% (n = 129), cPRA 51–100% (n = 43), and DSA (n = 105). Patients without DSA were considered as normal risk and received standard immunosuppression without T cell–depleting induction. Patients with DSA received an enhanced induction therapy and maintenance immunosuppression. Surveillance biopsies were performed at 3 and 6 months. Median follow‐up was 5.7 years. Among the three cPRA groups, there were no differences regarding the 1‐year incidence of ABMR (p = 0.16) and TCMR (p = 0.75). The 5‐year allograft survival rates were similar and around 87% (p = 0.28). The estimated glomerular filtration rate at last follow‐up was 50–53 mL/min (p = 0.45). On multivariable Cox proportional hazard analysis, the strongest independent predictor for ABMR and (death‐censored) graft survival was pretransplantation DSA. cPRA were not predictive for ABMR, TCMR, or (death‐censored) graft survival. We conclude that with current DSA assignment, the broadness of sensitization measured by cPRA does not imply an immunological risk.     

Cluster Analysis of Lesions in Nonselected Kidney Transplant Biopsies: Microcirculation Changes,Tubulointerstitial Inflammation and Scarring

Banff classification empirically established scoring of histologic lesions, but the relationships of lesions to each other and to underlying biologic processes remain unclear. We hypothesized that class discovery tools would reveal new relationships between individual lesions, and relate lesions to C4d staining, anti‐HLA donor‐specific antibody (DSA) and time posttransplant. We studied 234 nonselected renal allograft biopsies for clinical indications from 173 patients. Silhouette plotting and principal component analysis revealed three groups of lesions: microcirculation changes, including inflammation (glomerulitis, capillaritis) and deterioration (double contours, mesangial expansion); scarring/hyalinosis; and tubulointerstitial inflammation. DSA and C4d grouped with microcirculation inflammation, whereas time posttransplant grouped with scarring/hyalinosis lesions. Intimal arteritis clustered with DSA, C4d and microcirculation inflammation, but also showed correlations with tubulitis. Fibrous intimal thickening in arteries clustered with scarring/hyalinosis. Capillary basement membrane multilayering showed intermediary relationships between microcirculation deterioration and time‐dependent scarring. Correlation analysis and hierarchical clustering confirmed the lesion relationships. Thus, we propose that the pathologic lesions in biopsies are not independent but are members of groups that represent distinct pathogenic forces: microcirculation changes, reflecting the stress of DSA; scarring, hyalinosis and arterial fibrosis, reflecting the cumulative burden of injury over time; and tubulointerstitial inflammation. Interpretation of lesions should reflect these associations.     

A Novel,Blocking, Fc‐Silent Anti‐CD40 Monoclonal Antibody Prolongs Nonhuman Primate Renal Allograft Survival in the Absence of B Cell Depletion

CD40–CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti‐CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti‐CD40 antibody can still promote graft survival. The parent anti‐CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC‐mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc‐silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98–100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well‐tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc‐silent anti‐CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.     

HLA‐A, ‐B, ‐C, ‐DR,and ‐DQ Matching in Pancreas Transplantation: Effect on Graft Rejection and Survival

To enhance selection of appropriate deceased donors for pancreas transplants, we sought to determine whether HLA matching improved posttransplantation outcomes. In this single‐center study of 1219 pancreas transplants, we correlated posttransplantation outcomes with HLA‐A, ‐B, ‐C, ‐DR, and ‐DQ matches and mismatches. Rejection was linearly correlated with the number of mismatches. The individual number of HLA mismatches reached significance at four or more with a 2.3‐ to 2.9‐fold increase in rejection. The effect was most predominant with HLA‐B (1.8‐fold with one mismatch and 2.0‐fold with two mismatches) and ‐DR (1.9‐fold with two mismatches) loci, whereas HLA‐A, ‐C, and ‐DQ matches or mismatches did not independently predict acute rejection. The affect was strongest in solitary pancreas transplants, with little impact for simultaneous pancreas and kidney (SPK). In contrast, HLA matching did not affect graft or patient survival rates but was associated with a reduced risk of opportunistic infection. Avoidance of acute rejection saved an estimated $32 000 for solitary pancreas recipients and $52 000 for SPK recipients in hospital costs. Our data do not support the use of HLA matching for predicting pancreas graft survival but do support its significance for the reduction of acute rejection, particularly for solitary pancreas recipients.     

Reduction of Extended‐Release Tacrolimus Dose in Low‐Immunological‐Risk Kidney Transplant Recipients Increases Risk of Rejection and Appearance of Donor‐Specific Antibodies: A Randomized Study

The aim of this study (ClinicalTrials.gov, NCT01744470) was to determine the efficacy and safety of two different doses of extended‐release tacrolimus (TacER) in kidney transplant recipients (KTRs) between 4 and 12 mo after transplantation. Stable steroid‐free KTRs were randomized (1:1) after 4 mo: Group A had a 50% reduction in TacER dose with a targeted TacER trough level (C₀) >3 μg/L; group B had no change in TacER dose (TacER C₀ 7–12 μg/L). The primary outcome was estimated GFR at 1 year. Of 300 patients, the intent‐to‐treat analysis included 186 patients (group A, n = 87; group B, n = 99). TacER C₀ was lower in group A than in group B at 6 mo (4.1 ± 2.7 vs. 6.7 ± 3.9 μg/L, p < 0.0001) and 12 mo (5.6 ± 2.0 vs. 7.4 ± 2.1 μg/L, p < 0.0001). Estimated GFR was similar in both groups at 12 mo (group A, 56.0 ± 17.5 mL/min per 1.73 m²; group B, 56.0 ± 22.1 mL/min per 1.73 m²). More rejection episodes occurred in group A than group B (11 vs. 3; p = 0.016). At 1 year, subclinical inflammation occurred more frequently in group A than group B (inflammation score [i] >0: 21.4% vs. 8.8%, p = 0.047; tubulitis score [t] >0: 19.6% vs. 8.7%, p = 0.076; i + t: 1.14 ± 1.21 vs. 0.72 ± 1.01, p = 0.038). Anti‐HLA donor‐specific antibodies appeared only in group A (6 vs. 0 patients, p = 0.008). TacER C₀ should be maintained >7 μg/L during the first year after transplantation in low‐immunological‐risk, steroid‐free KTRs receiving a moderate dose of mycophenolic acid.