An objective definition for clinical suspicion of T‐cell‐mediated rejection after liver transplantation

A uniform definition of clinical suspicion of T‐cell‐mediated rejection (TCMR) in liver transplantation (LT) is needed to homogenize clinical decisions, especially within randomized trials. This multicenter study included a total of 470 primary LT recipients. The derivation cohort consisted of 142 patients who had clinically driven liver biopsies at any time after LT. The external validation cohort included 328 patients who underwent protocol biopsies at day 7‐10 after LT. The rates of moderate‐severe histological TCMR were 33.8% in the derivation cohort and 43.6% in the validation cohort. Independent predictors (ie, risk factors) of moderate‐severe TCMR in the derivation cohort were as follows: serum bilirubin >4 mg/dL (OR=5.83; P<.001), rising bilirubin within the 4 days prior to liver biopsy (OR=4.57; P=.003), and blood eosinophils count >0.1×10⁹/L (OR=3.81; P=.004). In the validation cohort, the number of risk factors was an independent predictor of moderate‐severe TCMR (OR=1.74; P=.001), after controlling for hepatitis C status. The number of risk factors paralleled the rates of moderate‐severe TCMR in the derivation and validation cohorts (P<.001 in both comparisons). In conclusion, increased serum bilirubin, rising bilirubin and eosinophilia are validated risk factors for moderate‐severe histological TCMR and could be used as objective criteria to select candidates for liver biopsy.     

IL‐6 production by monocytes is associated with graft function decline in patients with borderline changes suspicious for acute T‐cell‐mediated rejection: a pilot study

Although borderline changes (BL ) suspicious for acute T‐cell‐mediated rejection represent a diagnostic category, its clinical relevance is questioned leading to heterogeneous therapeutic management. We hypothesized that measuring IL ‐6 secretion by peripheral blood mononuclear cells identifies patients with ongoing graft damage. We examined the association between secreted IL ‐6 and the change in estimated glomerular filtration rate at 6 months after the biopsy (ΔeGFR ). We then conducted phenotypic and functional studies on patient and mouse innate immune cells in the blood and the kidney. In a training set, ΔeGFR was strongly associated with IL ‐6 levels, showing a clinically meaningful decline of 4.6 ± 1.5 ml /min per increase in log10 IL ‐6 (P = 0.001). These results were consistent after adjustment and were reproduced in a validation cohort. Phenotyping of peripheral blood cells revealed that the main source of IL ‐6 was CD 14⁺CD 16^?CCR 2⁺HLA‐DR ⁺CD 86⁺CD 11c⁺ inflammatory monocytes. There was a significant correlation between IL ‐6 secretion and interstitial dendritic cell density in the biopsy. Finally, characterization of mouse kidney dendritic cells revealed that they share features with macrophages and function as effector cells secreting IL ‐6. In conclusion, measuring IL ‐6 secreted by peripheral blood cells can be useful in the management of patients with BL in the absence of a concurrent inflammatory condition.     

Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation

Oh JY, Kim MK, Lee HJ, Ko JH, Kim Y, Park CS, Kang HJ, Park CG, Kim SJ, Lee JH, Wee WR. Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation. Xenotransplantation 2010; 17: 140–146. © 2010 John Wiley & Sons A/S. Abstract: Background: We have demonstrated earlier that porcine corneal xenografts underwent an acute cell‐mediated rejection in mice despite the absence of T cells. In the present study, we investigated the effect of complement depletion by cobra venom factor (CVF) on the corneal xenograft rejection in a pig‐to‐mouse model. Methods: Porcine corneas were orthotopically transplanted into C57BL/6 (B6) and severe combined immunodeficiency (SCID) mice. For complement depletion, 25 μg of CVF (1 g/kg bodyweight) was injected intraperitoneally on the day before and 1, 3, 5, and 7 days after transplantation. Graft survival was clinically assessed by slit lamp biomicroscopy and the median survival time (MST) was calculated. The grafts were histologically evaluated serially after transplantation using antibodies against CD4, CD8, NK1.1, and F4/80. Results: The CVF treatment significantly prolonged the porcine corneal xenograft survival in both B6 (MST 9.4 vs. 15.5 days; P = 0.0011) and SCID mice (MST 16.4 vs. 20.5 days; P = 0.0474). Histologically, whereas macrophages and CD4⁺ T cells were progressively infiltrated into porcine corneal grafts in CVF‐untreated B6 mice, the infiltration by both cells was markedly delayed and decreased in the xenografts in CVF‐treated B6 mice. Likewise, macrophage infiltration, which was prominent in rejected porcine xenografts in SCID mice, was also reduced in CVF‐treated SCID mice. Conclusions: Our results suggest that complement depletion by CVF delayed, although did not prevent, an acute cell‐mediated rejection in a pig‐to‐mouse corneal xenotransplantation.     

Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response

Sun L, Yi S, O’Connell PJ. Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response. Xenotransplantation 2010; 17: 121–130. © 2010 John Wiley & Sons A/S. Abstract: Backgrounds: Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Foxp3 expressing human naturally occurring CD4+CD25+ regulatory T cells (nTregs) have been shown to suppress pathological and physiological immune responses, including the CD4+ T‐cell‐mediated anti‐pig xenogeneic response in vitro. Although Foxp3 is required for nTreg development and their function, the precise role of Foxp3 in regulating Treg suppressive function in xenoimmune response remains to be identified. Methods: In vitro expanded human nTregs were transfected with fluorescein isothiocyanate ‐conjugated Foxp3 small interfering RNA (siRNA) by Lipofectamine 2000. Transfected nTregs were sorted by fluorescence‐activated cell sorting, and then analyzed for Foxp3 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25? T cells were stimulated with xenogeneic pig peripheral blood mononuclear cell in the presence or absence of nTregs in a coculture or transwell system for evaluation of nTreg suppressive activity. The production of effector cytokines by xenoreactive CD4+CD25? T cells as well as suppressive cytokine by nTregs in their cocultures was examined by ELISA. Results: The siRNA‐mediated Foxp3 knockdown resulted in impaired nTreg anergic state, downregulated expression of nTreg function associated molecules, and reduced production of suppressive cytokines by nTregs, which together leading to impaired nTreg‐mediated suppression of CD4+CD25? T‐cell proliferation and their effector cytokine production in response to xenogeneic stimulation. Conclusions: This study demonstrates that Foxp3 expression is required for human nTregs to maintain their suppressive function in the xenoimmune response.     

The clinical and pathological significance of borderline T cell–mediated rejection

The pathological diagnosis of borderline rejection (BL‐R) denotes possible T cell–mediated rejection (TCMR), but its clinical significance is uncertain. This single‐center, cross‐sectional cohort study compared the functional and histological outcomes of consecutive BL‐R diagnoses (n = 146) against normal controls (n = 826) and acute TCMR (n = 55) from 551 renal transplant recipients. BL‐R was associated with the following: contemporaneous renal dysfunction, acute tubular necrosis, and chronic tubular atrophy (P < .001); progressive tubular injury with fibrosis by longitudinal sequential histology (45.3% at 1 year); increased subsequent acute rejection (39.4%), allograft failure (P < .001), and patient mortality (P = .007). BL‐R detected by biopsy indicated for impaired function was followed by suboptimal functional recovery (46.3%), persistent inflammation (27.2%), and acute rejection episodes (50.0%) despite antirejection treatment in 83.3%. By 1 year after BL‐R, the incidence of new‐onset microvascular inflammation (9.3%), C4d staining (22.3%), transplant glomerulopathy (13.3%), and de novo donor‐specific antibodies (31.5%) exceeded normal controls (P < .05‐.001). BL‐R inflammation in protocol biopsy persisted in 28.0% and progressed to acute rejection in 32.6%; however, it resolved in 61.6% of the untreated cases. In summary, BL‐R is a heterogeneous diagnostic grouping, ranging from mild inconsequential inflammation to clinically significant TCMR, which is capable of immune‐mediated tubular injury resulting in inferior functional, immunological, and histological consequences.     

Response to treatment and long‐term outcomes in kidney transplant recipients with acute T cell–mediated rejection

The recent recognition of complex and chronic phenotypes of T cell–mediated rejection (TCMR) has fostered the need to better evaluate the response of acute TCMR—a condition previously considered to lack relevant consequences for allograft survival—to the standard of care. In a prospective cohort of kidney recipients (n = 256) with biopsy‐proven acute TCMR receiving corticosteroids, we investigated clinical, histological, and immunological phenotypes at the time of acute TCMR diagnosis and 3 months posttreatment. Independent posttreatment determinants of allograft loss included the glomerular filtration rate (GFR) (HR = 0.94; 95% CI = 0.92‐0.96; P < .001), proteinuria (HR = 1.40; 95% CI = 1.10‐1.79; P = .007), time since transplantation (HR = 1.02; 95% CI = 1.00‐1.03; P = .016), peritubular capillaritis (HR = 2.27; 95% CI = 1.13‐4.55; P = .022), interstitial inflammation in sclerotic cortical parenchyma (i‐IF/TA) (HR = 1.87; 95% CI = 1.08‐3.25; P = .025), and donor‐specific anti‐HLA antibodies (DSAs) (HR = 2.67; 95% CI = 1.46‐4.88; P = .001). Prognostic value was improved using a composite evaluation of response to treatment versus clinical parameters only (cNRI = 0.68; 95% CI = 0.41‐0.95; P < .001). A classification tree for allograft loss identified five patterns of response to treatment based on the posttreatment GFR, i‐IF/TA, and anti‐HLA DSAs (cross‐validated accuracy = 0.80). Compared with responders (n = 155, 60.5%), nonresponders (n = 101, 39.5%) had a higher incidence of de novo DSAs, antibody‐mediated rejection, and allograft loss at 10 years (P < .001 for all comparisons). Thus, clinical, histological, and immunological assessment of response to treatment of acute TCMR revealed different profiles of the response to treatment with distinct outcomes.     

Outside‐in HLA class I signaling regulates ICAM‐1 clustering and endothelial cell‐monocyte interactions via mTOR in transplant antibody‐mediated rejection

Antibody‐mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long‐term survival of solid organ transplants. AMR is caused by donor‐specific antibodies to HLA, which contribute to TAV by initiating outside‐in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab–mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab–activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM‐1) clustering, and monocyte firm adhesion to HLA I Ab–activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1^?/? recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM‐1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell‐monocyte interactions during AMR.     

Memory T cell–mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells

Donor‐reactive memory T cells generated via heterologous immunity represent a potent barrier to long‐term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor‐specific effector/memory CD8⁺ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8⁺ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB‐expressing CD8⁺ donor‐reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b^?/? CD8⁺ T cells resulted in an accumulation of donor‐specific CD8⁺ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8⁺ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor‐specific CD8⁺ memory T cells precipitated costimulation blockade‐resistant rejection. These data therefore identify a novel cell‐intrinsic inhibitory pathway that functions to limit the risk of memory T cell–mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.     

Evidence for the alloimmune basis and prognostic significance of Borderline T cell–mediated rejection

Prognostic biomarkers of T cell–mediated rejection (TCMR) have not been adequately studied in the modern era. We evaluated 803 renal transplant recipients and correlated HLA‐DR/DQ molecular mismatch alloimmune risk categories (low, intermediate, high) with the severity, frequency, and persistence of TCMR. Allograft survival was reduced in recipients with Banff Borderline (hazard ratio [HR] 2.4, P = .003) and Banff ≥ IA TCMR (HR 4.3, P < .0001) including a subset who never developed de novo donor‐specific antibodies (P = .002). HLA‐DR/DQ molecular mismatch alloimmune risk categories were multivariate correlates of Banff Borderline and Banff ≥ IA TCMR and correlated with the severity and frequency of rejection episodes. Recipient age, HLA‐DR/DQ molecular mismatch category, and cyclosporin vs tacrolimus immunosuppression were independent correlates of Banff Borderline and Banff ≥ IA TCMR. In the subset treated with tacrolimus (720/803) recipient age, HLA‐DR/DQ molecular mismatch category, and tacrolimus coefficient of variation were independent correlates of TCMR. The correlation of HLA‐DR/DQ molecular mismatch category with TCMR, including Borderline, provides evidence for their alloimmune basis. HLA‐DR/DQ molecular mismatch may represent a precise prognostic biomarker that can be applied to tailor immunosuppression or design clinical trials based on individual patient risk.     

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.     

Antibody‐mediated rejection despite inhibition of terminal complement

Terminal complement blockade has been shown to decrease the incidence of early acute antibody‐mediated rejection (eAMR) in the first month after positive cross‐match kidney transplant recipients, yet some patients still develop eAMR. The current study investigated possible mechanisms of eAMR despite eculizumab treatment. Of the 26 patients treated with eculizumab, two developed clinical eAMR and another patient developed histologic signs of eAMR without graft dysfunction (‘subclinical eAMR’). Twenty‐three did not have histologic injury on early surveillance biopsies. All 26 patients had therapeutic levels of eculizumab and showed complete blockade of complement in hemolytic assays. High levels of donor‐specific alloantibody (DSA) including total IgG, IgG3, and C1q+ DSA were present in patients with and without eAMR, and none correlated well with eAMR. In contrast, IgM DSA was present in only four patients after transplantation: the two patients with clinical eAMR, one patient with subclinical AMR, and one patient without eAMR (P = 0.006 correlation with eAMR). Both clinical eAMR episodes were easily treated with plasma exchange which removed IgM more completely and rapidly than IgG, resulting in normalization of function and histology. These data suggest a possible role of antidonor IgM DSA in the pathogenesis of eAMR in patients treated with terminal complement blockade (ClinicalTrials.gov Identifier: NCT00670774).     

T cell–mediated rejection is a major determinant of inflammation in scarred areas in kidney allografts

Inflammation in fibrosis areas (i‐IF/TA) of kidney allografts is associated with allograft loss; however, its diagnostic significance remains to be determined. We investigated the clinicohistologic phenotype and determinants of i‐IF/TA in a prospective cohort of 1539 kidney recipients undergoing evaluation of i‐IF/TA and tubulitis in atrophic tubules (t‐IF/TA) on protocol allograft biopsies performed at 1 year posttransplantation. We considered donor, recipient, and transplant characteristics, immunosuppression, and histological diagnoses in 2260 indication biopsies performed within the first year posttransplantation. Nine hundred forty‐six (61.5%) patients presented interstitial fibrosis/tubular atrophy (IF/TA Banff grade > 0) at 1 year posttransplant, among whom 394 (41.6%) showed i‐IF/TA. i‐IF/TA correlated with concurrent t‐IF/TA (P < .001), interstitial inflammation (P < .001), tubulitis (P < .001), total inflammation (P < .001), peritubular capillaritis (P < .001), interstitial fibrosis (P < .001), and tubular atrophy (P = .02). The independent determinants of i‐IF/TA were previous T cell–mediated rejection (TCMR) (P < .001), BK virus nephropathy (P = .007), steroid therapy (P = .039), calcineurin inhibitor therapy (P = .011), inosine‐5′‐monophosphate dehydrogenase inhibitor therapy (P = .011), HLA‐B mismatches (P = .012), and HLA‐DR mismatches (P = .044). TCMR patients with i‐IF/TA on posttreatment biopsy (N = 83/136, 61.0%) exhibited accelerated progression of IF/TA over time (P = .01) and decreased 8‐year allograft survival (70.8% vs 83.5%, P = .038) compared to those without posttreatment i‐IF/TA. Our results support that i‐IF/TA may represent a manifestation of chronic active TCMR.     

Prevention and treatment of alloantibody‐mediated kidney transplant rejection

Summary Antibody‐mediated rejection (AMR), which is commonly caused by preformed and/or de novo HLA alloantibodies, has evolved as a leading cause of early and late kidney allograft injury. In recent years, effective treatment strategies have been established to counteract the deleterious effects of humoral alloreactivity. One major therapeutic challenge is the barrier of a positive pretransplant lymphocytotoxic crossmatch. Several apheresis‐ and/or IVIG‐based protocols have been shown to enable successful crossmatch conversion, including a strategy of peritransplant immunoadsorption for rapid crossmatch conversion immediately before deceased donor transplantation. While such protocols may increase transplant rates and allow for acceptable graft survival, at least in the short‐term, it has become evident that, despite intense treatment, many patients still experience clinical or subclinical AMR. This reinforces the need for innovative strategies, such as complementary allocation programs to improve transplant outcomes. For acute AMR, various studies have suggested efficiency of plasmapheresis‐ or immunoadsorption‐based protocols. There is, however, no established treatment for chronic AMR and the development of strategies to reverse or at least halt chronic active rejection remains a big challenge. Major improvements can be expected from studies evaluating innovative therapeutic concepts, such as proteasome inhibition or complement blocking agents.     

The possible critical role of T‐cell help in DSA‐mediated graft loss

In this review, we discuss a possible central role of T‐cell help in severe forms of graft damage mediated by donor‐specific HLA antibodies (DSA). Some kidney transplant recipients with pretransplant DSA show a high graft failure rate, whereas in other patients DSA do not harm the transplanted kidney and in most cases, disappear shortly after transplantation. Analyzing 80 desensitized highly immunized kidney transplant recipients and another multicenter cohort of 385 patients with pretransplant HLA antibodies, we reported recently that an ongoing T‐cell help from an activated immune system, as measured by an increased level of soluble CD30 in serum, might be necessary for the DSA to exert a deleterious effect. Patients positive for both pretransplant DSA and sCD30 appear to require special measures, such as the elimination of DSA from the circulation, potent immunosuppression, good HLA‐matching, and intense post‐transplant monitoring, whereas exclusion of DSA‐positive patients from transplantation in the absence of high sCD30 may not be justified in all cases, even if the pretransplant DSA are strong and complement‐activating.