Claudin-2 upregulation enhances intestinal permeability, immune activation, dysbiosis, and mortality in sepsis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients.

Banff 2022 Vascularized Composite Allotransplantation Meeting Report: Diagnostic criteria for vascular changes.

As more data become available, the Banff 2007 working classification of skin-containing vascularized composite allograft (VCA) pathology is expected to evolve and develop. This report represents the Banff VCA Working Group's consensus on the first revision of the 2007 scoring system. Prior to the 2022 Banff-CanXadian Society of Transplantation Joint Meeting, 83 clinicians and/or researchers were invited to a virtual meeting to discuss whether the 2007 Banff VCA system called for a revision. Unanimously, it was determined that the vascular changes were to be included in the first revision. Subsequently, 2 international online surveys, each followed by virtual discussions, were launched. The goals were (1) to identify which changes define severe rejection, (2) to grade their importance in the evaluation of severe rejection, and (3) to identify emerging criteria to diagnose rejection. A final hybrid (in-person and virtual) discussion at the Banff/Canadian Society of Transplantation Joint Meeting finalized the terminology, the definition, a scoring system, and a reporting system of the vascular changes. This proposal represents an international consensus on this topic and establishes the first revision of the Banff 2007 working classification of skin-containing vascularized composite allograft pathology.

Banff 2022 pancreas transplantation multidisciplinary report: Refinement of guidelines for T cell-mediated rejection, antibody-mediated rejection and islet pathology. Assessment of duodenal cuff biopsies and noninvasive diagnostic methods.

The Banff pancreas working schema for diagnosis and grading of rejection is widely used for treatment guidance and risk stratification in centers that perform pancreas allograft biopsies. Since the last update, various studies have provided additional insight regarding the application of the schema and enhanced our understanding of additional clinicopathologic entities. This update aims to clarify terminology and lesion description for T cell-mediated and antibody-mediated allograft rejections, in both active and chronic forms. In addition, morphologic and immunohistochemical tools are described to help distinguish rejection from nonrejection pathologies. For the first time, a clinicopathologic approach to islet pathology in the early and late posttransplant periods is discussed. This update also includes a discussion and recommendations on the utilization of endoscopic duodenal donor cuff biopsies as surrogates for pancreas biopsies in various clinical settings. Finally, an analysis and recommendations on the use of donor-derived cell-free DNA for monitoring pancreas graft recipients are provided. This multidisciplinary effort assesses the current role of pancreas allograft biopsies and offers practical guidelines that can be helpful to pancreas transplant practitioners as well as experienced pathologists and pathologists in training.

The Banff 2022 Kidney Meeting Work Plan: Data-driven refinement of the Banff Classification for renal allografts.

The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell-mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Banff Digital Pathology Working Group: Image Bank, Artificial Intelligence Algorithm, and Challenge Trial Developments.

The Banff Digital Pathology Working Group (DPWG) was established with the goal to establish a digital pathology repository; develop, validate, and share models for image analysis; and foster collaborations using regular videoconferencing. During the calls, a variety of artificial intelligence (AI)-based support systems for transplantation pathology were presented. Potential collaborations in a competition/trial on AI applied to kidney transplant specimens, including the DIAGGRAFT challenge (staining of biopsies at multiple institutions, pathologists' visual assessment, and development and validation of new and pre-existing Banff scoring algorithms), were also discussed. To determine the next steps, a survey was conducted, primarily focusing on the feasibility of establishing a digital pathology repository and identifying potential hosts. Sixteen of the 35 respondents (46%) had access to a server hosting a digital pathology repository, with 2 respondents that could serve as a potential host at no cost to the DPWG. The 16 digital pathology repositories collected specimens from various organs, with the largest constituent being kidney (n = 12,870 specimens). A DPWG pilot digital pathology repository was established, and there are plans for a competition/trial with the DIAGGRAFT project. Utilizing existing resources and previously established models, the Banff DPWG is establishing new resources for the Banff community.

A novel role of BK potassium channel activity in preventing the development of kidney fibrosis.

Kidney fibrosis is a common characteristic of chronic kidney disease and while the large conductance voltage and calcium-activated potassium channel (BK) is widely expressed in kidneys, its role in kidney fibrosis is unknown. To evaluate this, we found that BK protein expression was decreased in the fibrotic kidneys. Accompanying this was increased fibrotic marker protein expression of fibronectin, vimentin and α-smooth muscle actin and increased mRNA expressions of fibronectin, α-smooth muscle actin, collagen III and collagen I. These changes occurred in the unilateral ureteral obstruction and folic acid models of fibrosis and were more pronounced in BK knockout than in wild-type mice. Activation of BK activity by chemical NS1619 or BMS191011 channel openers attenuated kidney fibrosis in these two models while protecting kidney function in wild-type mice. BK deficiency up-regulated transforming growth factor-ß (TGF-ß)/transcription factor Smad2/3 signaling in the fibrotic kidney, whereas activation of BK activity inhibited this signaling pathway both in vivo and in vitro. BK channel activation increased the degradation of TGF-ß receptors induced by TGF-ß1 in vivo and in vitro. Furthermore, in cell lines HK-2, NRK49, and NRK-52E, BK channel activation by NS1619 led to increased caveolae formation and facilitated localization of TGF-ß receptors in the microdomains of lipid rafts. Thus, our data demonstrated that BK activation has an anti-fibrotic effect on kidney fibrosis by inhibiting the TGF-ß signaling pathway through accelerating TGF-ß receptor degradation via the caveolae route. Hence, our study provides innovative insight into BK as a potential therapeutic target for the treatment of kidney fibrosis.

Addition of interleukin-6 receptor blockade to carfilzomib-based desensitization in a highly sensitized nonhuman primate model.

Sensitized patients, those who had prior exposure to foreign human leukocyte antigens, are transplanted at lower rates due to challenges in finding suitable organs. Desensitization strategies have permitted highly sensitized patients to undergo kidney transplantation, albeit with higher rates of rejection. This study assesses targeting plasma cell and interleukin (IL)-6 receptor for desensitization in a sensitized nonhuman primate kidney transplantation model. All animals were sensitized using two sequential skin transplants from maximally major histocompatibility complex-mismatched donors. Carfilzomib (CFZ)/tocilizumab (TCZ) desensitization (N = 6) successfully decreased donor-specific antibody (DSA) titers and prevented the expansion of B cells compared to CFZ monotherapy (N = 3). Dual desensitization further delayed, but did not prevent humoral rebound, as evidenced by a delayed increase in post-kidney transplant DSA titers. Accordingly, CFZ/TCZ desensitization conferred a significant survival advantage over CFZ monotherapy. A trend toward increased T follicular helper cells was also observed in the dual therapy group along the same timeline as an increase in DSA and subsequent graft loss. Cytomegalovirus reactivation also occurred in the CFZ/TCZ group but was prevented with ganciclovir prophylaxis. In accordance with prior studies of CFZ-based dual desensitization strategies, the addition of IL-6 receptor blockade resulted in desensitization with further suppression of posttransplant humoral response compared to CFZ monotherapy.

Assessment of a computerized quantitative quality control tool for whole slide images of kidney biopsies.

Inconsistencies in the preparation of histology slides and whole-slide images (WSIs) may lead to challenges with subsequent image analysis and machine learning approaches for interrogating the WSI. These variabilities are especially pronounced in multicenter cohorts, where batch effects (i.e. systematic technical artifacts unrelated to biological variability) may introduce biases to machine learning algorithms. To date, manual quality control (QC) has been the de facto standard for dataset curation, but remains highly subjective and is too laborious in light of the increasing scale of tissue slide digitization efforts. This study aimed to evaluate a computer-aided QC pipeline for facilitating a reproducible QC process of WSI datasets. An open source tool, HistoQC, was employed to identify image artifacts and compute quantitative metrics describing visual attributes of WSIs to the Nephrotic Syndrome Study Network (NEPTUNE) digital pathology repository. A comparison in inter-reader concordance between HistoQC aided and unaided curation was performed to quantify improvements in curation reproducibility. HistoQC metrics were additionally employed to quantify the presence of batch effects within NEPTUNE WSIs. Of the 1814 WSIs (458 H&E, 470 PAS, 438 silver, 448 trichrome) from n = 512 cases considered in this study, approximately 9% (163) were identified as unsuitable for subsequent computational analysis. The concordance in the identification of these WSIs among computational pathologists rose from moderate (Gwet's AC1 range 0.43 to 0.59 across stains) to excellent (Gwet's AC1 range 0.79 to 0.93 across stains) agreement when aided by HistoQC. Furthermore, statistically significant batch effects (p < 0.001) in the NEPTUNE WSI dataset were discovered. Taken together, our findings strongly suggest that quantitative QC is a necessary step in the curation of digital pathology cohorts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model.

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

Association Between Molecular Subtypes of Colorectal Tumors and Patient Survival, Based on Pooled Analysis of 7 International Studies.

BACKGROUND AND AIMS: The heterogeneity among colorectal tumors is probably due to differences in developmental pathways and might associate with patient survival times. We studied the relationship among markers of different subtypes of colorectal tumors and patient survival. METHODS: We pooled data from 7 observational studies, comprising 5010 patients with colorectal cancer. All the studies collected information on microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and mutations in KRAS and BRAF in tumors. Tumors with complete marker data were classified as type 1 (MSI-high, CIMP-positive, with pathogenic mutations in BRAF but not KRAS), type 2 (not MSI-high, CIMP-positive, with pathogenic mutations in BRAF but not KRAS), type 3 (not MSI-high or CIMP, with pathogenic mutations in KRAS but not BRAF), type 4 (not MSI-high or CIMP, no pathogenic mutations in BRAF or KRAS), or type 5 (MSI-high, no CIMP, no pathogenic mutations in BRAF or KRAS). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for associations of these subtypes and tumor markers with disease-specific survival (DSS) and overall survival times, adjusting for age, sex, stage at diagnosis, and study population. RESULTS: Patients with type 2 colorectal tumors had significantly shorter time of DSS than patients with type 4 tumors (HRDSS 1.66; 95% CI 1.33-2.07), regardless of sex, age, or stage at diagnosis. Patients without MSI-high tumors had significantly shorter time of DSS compared with patients with MSI-high tumors (HRDSS 0.42; 95% CI 0.27-0.64), regardless of other tumor markers or stage, or patient sex or age. CONCLUSIONS: In a pooled analysis of data from 7 observational studies of patients with colorectal cancer, we found that tumor subtypes, defined by combinations of 4 common tumor markers, were associated with differences in survival time. Colorectal tumor subtypes might therefore be used in determining patients' prognoses.

Decreased Hepatocyte Autophagy Leads to Synergistic IL-1ß and TNF Mouse Liver Injury and Inflammation.

BACKGROUND AND AIMS: The proinflammatory cytokine IL-1ß has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL-1ß promotes liver injury in these diseases is unclear, as no IL-1ß receptor-linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy's function in hepatotoxicity from proinflammatory cytokines. APPROACH AND RESULTS: AML12 cells with decreased autophagy from a lentiviral autophagy-related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL-1ß, which was markedly amplified by TNF co-treatment. IL-1ß/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin-1s to prevent death. IL-1ß/TNF depleted autophagy-deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL-1ß/TNF-induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy-deficient cells. Mice with a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were similarly sensitized to cathepsin-dependent hepatocellular injury and death from IL-1ß/TNF in combination, but neither IL-1ß nor TNF alone. Knockout mice had increased hepatic inflammation, and IL-1ß/TNF-treated, autophagy-deficient AML12 cells secreted exosomes with proinflammatory damage-associated molecular patterns. CONCLUSIONS: The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL-1ß/TNF-induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage-associated molecular patterns.

Artificial intelligence and algorithmic computational pathology: an introduction with renal allograft examples.

Whole slide imaging, which is an important technique in the field of digital pathology, has recently been the subject of increased interest and avenues for utilisation, and with more widespread whole slide image (WSI) utilisation, there will also be increased interest in and implementation of image analysis (IA) techniques. IA includes artificial intelligence (AI) and targeted or hypothesis-driven algorithms. In the overall pathology field, the number of citations related to these topics has increased in recent years. Renal pathology is one anatomical pathology subspecialty that has utilised WSIs and IA algorithms; it can be argued that renal transplant pathology could be particularly suited for whole slide imaging and IA, as renal transplant pathology is frequently classified by use of the semiquantitative Banff classification of renal allograft pathology. Hypothesis-driven/targeted algorithms have been used in the past for the assessment of a variety of features in the kidney (e.g. interstitial fibrosis, tubular atrophy, inflammation); in recent years, the amount of research has particularly increased in the area of AI/machine learning for the identification of glomeruli, for histological segmentation, and for other applications. Deep learning is the form of machine learning that is most often used for such AI approaches to the 'big data' of pathology WSIs, and deep learning methods such as artificial neural networks (ANNs)/convolutional neural networks (CNNs) are utilised. Unsupervised and supervised AI algorithms can be employed to accomplish image or semantic classification. In this review, AI and other IA algorithms applied to WSIs are discussed, and examples from renal pathology are covered, with an emphasis on renal transplant pathology.

Western diet-induced increase in colonic bile acids compromises epithelial barrier in nonalcoholic steatohepatitis.

There is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)-induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r-/- . WD-fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD-fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD-fed mice, increased paracellular permeability, while the BA-binding resin sevelamer hydrochloride protected against CDCA-induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD-fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD-induced impairment of the intestinal epithelial barrier in NASH.

Deep-learning-based accurate hepatic steatosis quantification for histological assessment of liver biopsies.

Hepatic steatosis droplet quantification with histology biopsies has high clinical significance for risk stratification and management of patients with fatty liver diseases and in the decision to use donor livers for transplantation. However, pathology reviewing processes, when conducted manually, are subject to a high inter- and intra-reader variability, due to the overwhelmingly large number and significantly varying appearance of steatosis instances. This process is challenging as there is a large number of overlapped steatosis droplets with either missing or weak boundaries. In this study, we propose a deep-learning-based region-boundary integrated network for precise steatosis quantification with whole slide liver histopathology images. The proposed model consists of two sequential steps: a region extraction and a boundary prediction module for foreground regions and steatosis boundary prediction, followed by an integrated prediction map generation. Missing steatosis boundaries are next recovered from the predicted map and assembled from adjacent image patches to generate results for the whole slide histopathology image. The resulting steatosis measures both at the pixel level and steatosis object-level present strong correlation with pathologist annotations, radiology readouts and clinical data. In addition, the segregated steatosis object count is shown as a promising alternative measure to the traditional metrics at the pixel level. These results suggest a high potential of artificial intelligence-assisted technology to enhance liver disease decision support using whole slide images.

Banff Digital Pathology Working Group: Going digital in transplant pathology.

The Banff Digital Pathology Working Group (DPWG) was formed in the time leading up to and during the joint American Society for Histocompatibility and Immunogenetics/Banff Meeting, September 23-27, 2019, held in Pittsburgh, Pennsylvania. At the meeting, the 14th Banff Conference, presentations directly and peripherally related to the topic of "digital pathology" were presented; and discussions before, during, and after the meeting have resulted in a list of issues to address for the DPWG. Included are practice standardization, integrative approaches for study classification, scoring of histologic parameters (eg, interstitial fibrosis and tubular atrophy and inflammation), algorithm classification, and precision diagnosis (eg, molecular pathways and therapeutics). Since the meeting, a survey with international participation of mostly pathologists (81%) was conducted, showing that whole slide imaging is available at the majority of centers (71%) but that artificial intelligence (AI)/machine learning was only used in ≈12% of centers, with a wide variety of programs/algorithms employed. Digitalization is not just an end in itself. It also is a necessary precondition for AI and other approaches. Discussions at the meeting and the survey highlight the unmet need for a Banff DPWG and point the way toward future contributions that can be made.

Avoidance of CNI and steroids using belatacept-Results of the Clinical Trials in Organ Transplantation 16 trial.

Immunosuppression devoid of corticosteroids has been investigated to avoid long-term comorbidities. Likewise, alternatives to calcineurin inhibitors have been investigated as a strategy to improve long-term kidney function following transplanion. Costimulatory blockade strategies that include corticosteroids have recently shown promise, despite their higher rates of early acute rejection. We designed a randomized clinical trial utilizing depletional induction therapy to mitigate early rejection risk while limiting calcineurin inhibitors and corticosteroids. This trial, Clinical Trials in Organ Transplantation 16 (CTOT-16), sought to evaluate novel belatacept-based strategies employing tacrolimus and corticosteroid avoidance. Sixty-nine kidney transplant recipients were randomized from 4 US transplant centers comparing a control group of with rabbit antithymocyte globulin (rATG) induction, rapid steroid taper, and maintenance mycophenolate and tacrolimus, to 2 arms using maintenance belatacept. There were no graft losses but there were 2 deaths in the control group. However, the trial was halted early because of rejection in the belatacept treatment groups. Serious adverse events were similar across groups. Although rejection was not uniform in the belatacept maintenance therapy groups, the frequency of rejection limits the practical implementation of this strategy to avoid both calcineurin inhibitors and corticosteroids at this time.

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection.

The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell-mediated rejection (TCMR), borderline, and antibody-mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor-specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.

Pretransplant Desensitization with Costimulation Blockade and Proteasome Inhibitor Reduces DSA and Delays Antibody-Mediated Rejection in Highly Sensitized Nonhuman Primate Kidney Transplant Recipients.

BACKGROUND: Patients with broad HLA sensitization have poor access to donor organs, high mortality while waiting for kidney transplant, and inferior graft survival. Although desensitization strategies permit transplantation via lowering of donor-specific antibodies, the B cell-response axis from germinal center activation to plasma cell differentiation remains intact. METHODS: To investigate targeting the germinal center response and plasma cells as a desensitization strategy, we sensitized maximally MHC-mismatched rhesus pairs with two sequential skin transplants. We administered a proteasome inhibitor (carfilzomib) and costimulation blockade agent (belatacept) to six animals weekly for 1 month; four controls received no treatment. We analyzed blood, lymph node, bone marrow cells, and serum before desensitization, after desensitization, and after kidney transplantation. RESULTS: The group receiving carfilzomib and belatacept exhibited significantly reduced levels of donor-specific antibodies (P=0.05) and bone marrow plasma cells (P=0.02) compared with controls, with a trend toward reduced lymph node T follicular helper cells (P=0.06). Compared with controls, carfilzomib- and belatacept-treated animals had significantly prolonged graft survival (P=0.02), and renal biopsy at 1 month showed significantly reduced antibody-mediated rejection scores (P=0.02). However, four of five animals with long-term graft survival showed gradual rebound of donor-specific antibodies and antibody-mediated rejection. CONCLUSIONS: Desensitization using proteasome inhibition and costimulation blockade reduces bone marrow plasma cells, disorganizes germinal center responses, reduces donor-specific antibody levels, and prolongs allograft survival in highly sensitized nonhuman primates. Most animals experienced antibody-mediated rejection with humoral-response rebound, suggesting desensitization must be maintained after transplantation using ongoing suppression of the B cell response.

Daratumumab in Sensitized Kidney Transplantation: Potentials and Limitations of Experimental and Clinical Use.

BACKGROUND: Donor-specific antibodies are associated with increased risk of antibody-mediated rejection and decreased allograft survival. Therefore, reducing the risk of these antibodies remains a clinical need in transplantation. Plasma cells are a logical target of therapy given their critical role in antibody production. METHODS: To target plasma cells, we treated sensitized rhesus macaques with daratumumab (anti-CD38 mAb). Before transplant, we sensitized eight macaques with two sequential skin grafts from MHC-mismatched donors; four of them were also desensitized with daratumumab and plerixafor (anti-CXCR4). We also treated two patients with daratumumab in the context of transplant. RESULTS: The animals treated with daratumumab had significantly reduced donor-specific antibody levels compared with untreated controls (57.9% versus 13% reduction; P<0.05) and prolonged renal graft survival (28.0 days versus 5.2 days; P<0.01). However, the reduction in donor-specific antibodies was not maintained because all recipients demonstrated rapid rebound of antibodies, with profound T cell-mediated rejection. In the two clinical patients, a combined heart and kidney transplant recipient with refractory antibody-mediated rejection and a highly sensitized heart transplant candidate, we also observed a significant decrease in class 1 and 2 donor-specific antibodies that led to clinical improvement of antibody-mediated rejection and to heart graft access. CONCLUSIONS: Targeting CD38 with daratumumab significantly reduced anti-HLA antibodies and anti-HLA donor-specific antibodies in a nonhuman primate model and in two transplant clinical cases before and after transplant. This supports investigation of daratumumab as a potential therapeutic strategy; however, further research is needed regarding its use for both antibody-mediated rejection and desensitization.