Thalachallour Mohanakumar

2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody‐mediated liver allograft rejection at the 11th (Paris, France, June 5–10, 2011), 12th (Comandatuba, Brazil, August 19–23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5–10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody‐mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Donor-Derived Exosomes With Lung Self-Antigens in Human Lung Allograft Rejection.

The immunological role of exosomes in allograft rejection remains unknown. We sought to determine whether exosomes are induced during lung allograft rejection and to define the antigenic compositions of HLA, lung‐associated self‐antigens (SAgs) and microRNAs (miRNAs). Exosomes were isolated from sera and bronchoalveolar lavage fluid from 30 lung transplant recipients (LTxRs) who were stable or who had acute rejection (AR) or bronchiolitis obliterans syndrome (BOS). Exosomes were defined by flow cytometry for CD63 and western blotting for annexin V SAgs, collagen V (Col‐V) and Kα1 tubulin were examined by electron microscopy; miRNAs were profiled by a miRNA array. Donor HLA and SAgs were detected on exosomes from LTxRs with AR and BOS but not from stable LTxRs. Exosomes expressing Col‐V were isolated from sera from LTxRs 3 mo before AR and 6 mo before BOS diagnosis, suggesting that exosomes with SAgs may be a noninvasive rejection biomarker. Exosomes isolated from LTxRs with AR or BOS also contained immunoregulatory miRNAs. We concluded that exosomes expressing donor HLA, SAgs and immunoregulatory miRNAs are present in the circulation and local site after human lung transplantation and play an important role in the immune pathogenesis of acute allograft rejection and BOS.

Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch

Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection. The method involves the initial development of a panel of probes in which each probe will specifically target a unique sequence of a human leukocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR (ddPCR) used as a quantitative assay to accurately track the trace amount of DcfDNA in an ample excess of recipient DNA background. The average false positive rate noted was about 1 per 800,000 molecules. Serially 2-fold diluted cfDNA, representing donor fractions of cfDNA, were spiked into a constant level of cfDNA representing the recipient cfDNA. The fraction of spiked cfDNA was measured and quantitative linearity was observed across seven serially diluted cfDNA samples. We were able to measure the minor portion of cfDNA as low as 0.2% of total cfDNA. We subsequently applied the method to a pilot set of 18 LTx recipients grouped into biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or stable groups. Serial plasma samples were used to identify the percentage of DcfDNA over total cfDNA. The level of DcfDNA was significantly elevated in patients diagnosed with acute rejection (10.30±2.80, n=18), compared to that from stable (1.71±0.50, n=24) or from BOS patients (2.52±0.62, n=20). In conclusion, we present results validating the application of digital PCR to quantify DcfDNA assay in primary clinical specimens, which demonstrate that DcfDNA can be used as an early non-invasive biomarker for acute lung allograft rejection.

Immune Responses to Tissue-Restricted Nonmajor Histocompatibility Complex Antigens in Allograft Rejection

Chronic diseases that result in end-stage organ damage cause inflammation, which can reveal sequestered self-antigens (SAgs) in that organ and trigger autoimmunity. The thymus gland deletes self-reactive T-cells against ubiquitously expressed SAgs, while regulatory mechanisms in the periphery control immune responses to tissue-restricted SAgs. It is now established that T-cells reactive to SAgs present in certain organs (e.g., lungs, pancreas, and intestine) are incompletely eliminated, and the dysregulation of peripheral immuneregulation can generate immune responses to SAgs. Therefore, chronic diseases can activate self-reactive lymphocytes, inducing tissue-restricted autoimmunity. During organ transplantation, donor lymphocytes are tested against recipient serum (i.e., cross-matching) to detect antibodies (Abs) against donor human leukocyte antigens, which has been shown to reduce Ab-mediated hyperacute rejection. However, primary allograft dysfunction and rejection still occur frequently. Because donor lymphocytes do not express tissue-restricted SAgs, preexisting Abs against SAgs are undetectable during conventional cross-matching. Preexisting and de novo immune responses to tissue-restricted SAgs (i.e., autoimmunity) play a major role in rejection. In this review, we discuss the evidence that supports autoimmunity as a contributor to rejection. Testing for preexisting and de novo immune responses to tissue-restricted SAgs and treatment based on immune responses after organ transplantation may improve short- and long-term outcomes after transplantation.

Exosomes expressing the self-antigens myosin and vimentin play an important role in syngeneic cardiac transplant rejection induced by antibodies to cardiac myosin

Long‐term success of heart transplantation is hindered by humoral and cell‐mediated immune responses. We studied preexisting antibodies to cardiac self‐antigens, myosin and vimentin, and exosomes induced by antibodies to self‐antigens in eliciting immune responses to cardiac grafts. After syngeneic heterotopic murine heart transplantation, rabbit anti‐myosin or normal rabbit immunoglobulin was administered at day 0 or 7. Sera were collected after heartbeat cessation, cellular infiltration was analyzed, and exosomes were isolated from sera. Histopathologic examination of the controls transplanted hearts demonstrated normal architecture, and their sera demonstrated neither antibodies to self‐antigens nor exosomes expressing self‐antigens. Administration of antibodies to cardiac myosin immediately posttransplantation (day 0) but not on day 7 triggered graft failure on day 7, and histopathologic examination revealed marked cellular infiltration with neutrophils and lymphocytes. Histopathologic examination of rejected hearts also demonstrated myocyte damage as sera had increased antibodies to myosin and vimentin and development of exosomes expressing self‐antigens. Administration of exosomes isolated from failed grafts containing self‐antigens induced graft dysfunction; exosomes isolated from stable mice did not induce graft failure. Antibodies to self‐antigens can induce exosomes containing self‐antigens, initiating an immune response and causing graft failure after cardiac transplantation.

Autologous and Allogenous Antibodies in Lung and Islet Cell Transplantation

The field of organ transplantation has undoubtedly made great strides in recent years. Despite the advances in donor–recipient histocompatibility testing, improvement in transplantation procedures, and development of aggressive immunosuppressive regimens, graft-directed immune responses still pose a major problem to the long-term success of organ transplantation. Elicitation of immune responses detected as antibodies to mismatched donor antigens (alloantibodies) and tissue-restricted self-antigens (autoantibodies) are two major risk factors for the development of graft rejection that ultimately lead to graft failure. In this review, we describe current understanding on genesis and pathogenesis of antibodies in two important clinical scenarios: lung transplantation and transplantation of islet of Langerhans. It is evident that when compared to any other clinical solid organ or cellular transplant, lung and islet transplants are more susceptible to rejection by combination of allo- and autoimmune responses.

Circulating Exosomes with Distinct Properties during Chronic Lung Allograft Rejection

Circulating exosomes containing donor HLA and lung-associated self-antigens (SAg) are thought to play an important role in allograft rejection after human lung transplantation. We characterized exosomes isolated from serum of 10 lung transplant recipients (LTxR) diagnosed with bronchiolitis obliterans syndrome (BOS) and compared them with exosomes isolated from serum of 10 stable LTxR. Lung-associated SAg (K-α-1-tubulin [Kα1T] and collagen V [Col-V]), MHC class II molecules, costimulatory molecules CD40, CD80, and CD86, and transcription factors class II MHC trans-activator, NF-κB, hypoxia-inducible factor 1-α, IL-1R–associated kinase 1, MyD88, and 20S proteasome were detected in exosomes from BOS, but not stable LTxR. In contrast, adhesion molecules were present in both groups. C57BL/6 mice immunized with exosomes from BOS but not stable LTxR demonstrated Ab to SAg (Col-V, 33.5 ± 15.7 versus 10.4 ± 6.4, p = 0.021; Kα1T, 925 ± 403 versus 317 ± 285, p = 0.044) and HLA (mean fluorescence intensity: BOS, 8450; stable, 632; p < 0.05). Furthermore, splenic lymphocytes demonstrated increased frequency of lung SAg-specific IL-17 (Col-V, 128 ± 46 versus 31 ± 21, p = 0.013; Kα1T, 194 ± 47 versus 67 ± 43, p = 0.014) and IFN-γ (Col-V, 165 ± 79 versus 38 ± 40, p = 0.042; Kα1T, 232 ± 64 versus 118 ± 39, p = 0.012). Reduced levels of IL-10–producing cells were seen in BOS exosome immunized mice compared with mice immunized with stable exosomes (Col-V, 59 ± 23 versus 211 ± 85, p = 0.016; Kα1T, 78 ± 49 versus 295 ± 104, p = 0.017). Owing to the unique immune-stimulating properties of exosomes induced during rejection, we propose that they play an important role in eliciting both alloantigen- and SAg-specific immunity, leading to chronic rejection after lung transplantation.

Zbtb7a induction in alveolar macrophages is implicated in anti-HLA–mediated lung allograft rejection

Zbtb7a in alveolar macrophages influences early inflammation and autoimmunity in antibody-mediated rejection of transplanted lungs. Curbing culprits of chronic rejection De novo donor-specific antibodies generated after organ transplantation can lead to chronic rejection, and Nayak et al. sought to understand the mechanisms leading to production of these antibodies in lung transplantation. Using mouse models and data from human patients, they identified expression of the transcription factor Zbtb7a in alveolar macrophages as a crucial mediator. Patients eventually diagnosed with chronic rejection had higher expression of this transcription factor early on. Preventing macrophages from expressing Zbtb7a ameliorated models of obliterative airway disease and prevented chronic rejection of lung transplants in mice. Interrupting macrophage presentation of donor antigens may be a solution to prevent generation of these destructive antibodies and the ensuing chronic rejection. Chronic rejection significantly limits long-term success of solid organ transplantation. De novo donor-specific antibodies (DSAs) to mismatched donor human leukocyte antigen after human lung transplantation predispose lung grafts to chronic rejection. We sought to delineate mediators and mechanisms of DSA pathogenesis and to define early inflammatory events that trigger chronic rejection in lung transplant recipients and obliterative airway disease, a correlate of human chronic rejection, in mouse. Induction of transcription factor zinc finger and BTB domain containing protein 7a (Zbtb7a) was an early response critical in the DSA-induced chronic rejection. A cohort of human lung transplant recipients who developed DSA and chronic rejection demonstrated greater Zbtb7a expression long before clinical diagnosis of chronic rejection compared to nonrejecting lung transplant recipients with stable pulmonary function. Expression of DSA-induced Zbtb7a was restricted to alveolar macrophages (AMs), and selective disruption of Zbtb7a in AMs resulted in less bronchiolar occlusion, low immune responses to lung-restricted self-antigens, and high protection from chronic rejection in mice. Additionally, in an allogeneic cell transfer protocol, antigen presentation by AMs was Zbtb7a-dependent where AMs deficient in Zbtb7a failed to induce antibody and T cell responses. Collectively, we demonstrate that AMs play an essential role in antibody-induced pathogenesis of chronic rejection by regulating early inflammation and lung-restricted humoral and cellular autoimmunity.

Lung Retransplantation for Chronic Rejection: A Single-Center Experience

BACKGROUNDnChronic lung allograft dysfunction (CLAD) is nonreversible and remains the biggest obstacle to long-term survival after lung transplantation (LTx). Retransplantation is the sole definitive therapeutic option for CLAD. We analyzed our single-center experience with retransplantation as a treatment option for CLAD.nnnMETHODSnFrom March 1, 2010, to May 31, 2016, 419 consecutive patients underwent LTx at our institution; 29 of these procedures were retransplantations for CLAD. We analyzed demographic characteristics, lung allocation score, operation type, length of stay, and perioperative outcomes. Actuarial survival was estimated using Kaplan-Meier survival curves.nnnRESULTSnIn total, 29 of 419 patients (6.9%) underwent retransplantation for CLAD. Median time from primary LTx to retransplantation was 1,163 days (range: 304 to 3,971 days). Patients undergoing retransplantation were younger and had higher lung allocation scores than primary transplantation patients. Most LTx procedures were bilateral (93% of retransplantations, 95% of primary LTx). Rates of cardiopulmonary bypass, extracorporeal membrane oxygenation support for severe primary graft dysfunction, and re-exploration for bleeding were higher in retransplantation patients (pxa0= 0.010, pxa0= 0.019, and pxa0= 0.029, respectively). One- and 5-year survival rates in the retransplantation group were similar to those of the primary LTx group (89.2% and 64.3% versus 89.7% and 58.2%, respectively; pxa0= 0.79).nnnCONCLUSIONSnLung retransplantation is a viable treatment option for CLAD after LTx. In this study, retransplantation patients were younger, had higher lung allocation scores, and were more likely to require cardiopulmonary bypass and postoperative extracorporeal membrane oxygenation support than primary LTx patients. Postoperative length of stay and short- and mid-term survival were comparable with those of primary LTx patients.

Human leukocyte antigens antibodies after lung transplantation: Primary results of the HALT study

Donor‐specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) are associated with worse outcomes after lung transplantation. To determine the incidence and characteristics of DSA early after lung transplantation, we conducted a prospective multicenter observational study that used standardized treatment and testing protocols. Among 119 transplant recipients, 43 (36%) developed DSA: 6 (14%) developed DSA only to class I HLA, 23 (53%) developed DSA only to class II HLA, and 14 (33%) developed DSA to both class I and class II HLA. The median DSA mean fluorescence intensity (MFI) was 3197. We identified a significant association between the Lung Allocation Score and the development of DSA (HR = 1.02, 95% CI: 1.001‐1.03, P = .047) and a significant association between DSA with an MFI ≥ 3000 and acute cellular rejection (ACR) grade ≥ A2 (HR = 2.11, 95% CI: 1.04‐4.27, P = .039). However, we did not detect an association between DSA and survival. We conclude that DSA occur frequently early after lung transplantation, and most target class II HLA. DSA with an MFI ≥ 3000 have a significant association with ACR. Extended follow‐up is necessary to determine the impact of DSA on other important outcomes.