Clinical relevance of human leukocyte antigen antibodies in liver, heart, lung and intestine transplantation.

PURPOSE OF REVIEW: Solid phase assays identify human leukocyte antigen (HLA) antibodies with a great sensitivity. Whether to accept or decline an organ if the virtual crossmatch is positive, when to monitor and whether to treat de-novo donor-specific antibody (DSA) posttransplant remain challenging issues for the transplant clinician. RECENT FINDINGS: Technologies that can differentiate which antibodies pose the greatest risk for antibody-mediated rejection (AMR) are evolving. Complement fixing luminex assays have been used to predict high-risk antibodies, but using these assays alone will miss some preformed antibodies. How these technologies fit into the laboratory's testing algorithm will likely need to be individualized. Posttransplant de-novo DSAs are associated with inferior outcomes. In hearts, similar to renal transplantation, acute rejection is a risk factor for developing de-novo DSA. Further data are needed to determine whether other risk factors are similar to those reported for renal transplants. Antibodies to self-antigens are increasingly recognized posttransplant and how the alloimmune response contributes to altered autoregulation is a current research focus. SUMMARY: Identification of DSA enables the clinician to make informed decisions regarding whether or not to accept an organ and if augmented immunosuppression is indicated. Monitoring for DSA posttransplant identifies recipients at a greater risk for AMR and can guide management. However, the best approach to dealing with de-novo DSA remains unclear.

Tacrolimus versus cyclosporin as primary immunosuppression for lung transplant recipients.

BACKGROUND: Lung transplantation is a well-accepted treatment for people with most end-stage lung diseases. Although both tacrolimus and cyclosporin are used as primary immunosuppressive agents in lung transplant recipients, it is unclear which of these drugs is better in reducing rejection and death without causing adverse effects. OBJECTIVES: To assess the benefits and harms of tacrolimus versus cyclosporin for primary immunosuppression in lung transplant recipients. SEARCH METHODS: We searched the Cochrane Renal Group's Specialised Register to 10 April 2013 through contact with the Trials Search Co-ordinator using search terms relevant to this review. We also searched Science Citation Index Expanded and the Transplant Library to 20 April 2013. SELECTION CRITERIA: We included all randomised controlled trials (RCT) that compared any dose and duration of administration of tacrolimus versus cyclosporin as primary immunosuppressive treatment in lung transplant recipients. Our selection criteria required that all included patients received the same additional immunosuppressive therapy within each study. DATA COLLECTION AND ANALYSIS: Three authors extracted data. For dichotomous data we used risk ratio (RR) and used mean difference (MD) for continuous data, each with 95% confidence intervals (CI). Methodological components of the included studies were used to assess risk of systematic errors (bias). Trial sequential analysis was used to assess risk of random errors (play of chance). MAIN RESULTS: We included three studies that enrolled a total of 413 adult patients that compared tacrolimus with microemulsion or oral solution cyclosporin. All studies were found to be at high risk of bias. Tacrolimus seemed to be significantly superior to cyclosporin regarding the incidence of bronchiolitis obliterans syndrome (RR 0.46, 95% CI 0.29 to 0.74), lymphocytic bronchitis score (MD -0.60, 95% CI -1.04 to -0.16), treatment withdrawal (RR 0.27, 95% CI 0.16 to 0.46), and arterial hypertension (RR 0.67, 95% CI 0.50 to 0.89). However, the finding for arterial hypertension was not confirmed when analysed using a random-effects model (RR 0.54, 95% CI 0.17 to 1.73). Furthermore, trial sequential analysis found that none of the meta-analyses reached the required information sizes and cumulative Z-curves did not cross trial sequential monitoring boundaries. Diabetes mellitus occurred more frequently among people in the tacrolimus group compared with the cyclosporin group when the fixed-effect model was applied (RR 4.24, 95% CI 1.58 to 11.40), but no difference was found when the random-effects model was used for analysis (RR 4.43, 95% CI 0.75 to 26.05). Again, trial sequential analysis found that the required information threshold was not reached and cumulative Z-curve did not cross the trial sequential monitoring boundary. No significant difference between treatment groups was observed regarding mortality (RR 1.06, 95% CI 0.75 to 1.49), incidence of acute rejection (RR 0.89, 95% CI 0.77 to 1.03), numbers of infections/100 patient-days (MD -0.15, 95% CI -0.30 to 0.00), cancer (RR 0.21, 95% CI 0.04 to 1.16), kidney dysfunction (RR 1.41, 95% CI 0.93 to 2.14), kidney failure (RR 1.57, 95% CI 0.28 to 8.94), neurotoxicity (RR 7.06, 95% CI 0.37 to 135.19), and hyperlipidaemia (RR 0.60, 95% CI 0.30 to 1.20). Trial sequential analysis showed the required information thresholds were not reached for any of these outcome measures. AUTHORS' CONCLUSIONS: Tacrolimus may be superior to cyclosporin regarding bronchiolitis obliterans syndrome, lymphocytic bronchitis, treatment withdrawal, and arterial hypertension, but may be inferior regarding development of diabetes. No difference in mortality and acute rejection was observed between patients treated with tacrolimus and cyclosporin. There were few studies comparing tacrolimus and cyclosporin after lung transplantation, and the numbers of patients and events in the included studies were limited. Furthermore, the included studies were deemed to be at high risk of bias. Hence, more RCTs are needed to assess the results of the present review. Such studies ought to be conducted with low risks of systematic errors (bias) and of random errors (play of chance).

CMV driven CD8(+) T-cell activation is associated with acute rejection in lung transplantation.

Lung transplantation is the definitive treatment for terminal respiratory disease, but the associated mortality rate is high. Acute rejection of the transplanted lung is a key determinant of adverse prognosis. Furthermore, an epidemiological relationship has been established between the occurrence of acute lung rejection and cytomegalovirus infection. However, the reasons for this association remain unclear. Here, we performed a longitudinal characterization of CMV-specific T-cell responses and immune activation status in the peripheral blood and bronchoalveolar lavage fluid of forty-four lung transplant patients. Acute rejection was associated with high levels of cellular activation in the periphery, reflecting strong CMV-specific CD8(+) T-cell activity post-transplant. Peripheral and lung CMV-specific CD8(+) T-cell responses were very similar, and related to the presence of CMV in the transplanted organ. These findings support that activated CMV-specific CD8(+) T-cells in the lung may play a role in promoting acute rejection.

Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation.

We hypothesized alterations in gene expression could identify important pathways involved in transplant lung injury. Broncho alveolar lavage fluid (BALF) was sampled from donors prior to procurement and in recipients within an hour of reperfusion as part of the NIAID Clinical Trials in Organ Transplantation Study. Twenty-three patients with Grade 3 primary graft dysfunction (PGD) were frequency matched with controls based on donor age and recipient diagnosis. RNA was analyzed using the Human Gene 1.0 ST array. Normalized mRNA expression was transformed and differences between donor and postreperfusion values were ranked then tested using Gene Set Enrichment Analysis. Three-hundred sixty-two gene sets were upregulated, with eight meeting significance (familywise-error rate, FWER p-value <0.05), including the NOD-like receptor inflammasome (NLR; p < 0.001), toll-like receptors (TLR; p < 0.001), IL-1 receptor (p = 0.001), myeloid differentiation primary response gene 88 (p = 0.001), NFkB activation by nontypeable Haemophilus influenzae (p = 0.001), TLR4 (p = 0.008) and TLR 9 (p = 0.018). The top five ranked individual transcripts from these pathways based on rank metric score are predominantly present in the NLR and TLR pathways, including IL1ß (1.162), NLRP3 (1.135), IL1α (0.952), IL6 (0.931) and CCL4 (0.842). Gene set enrichment analyses implicate inflammasome-mediated and innate immune signaling pathways as key mediators of the development of PGD in lung transplant patients.

Human leucocyte antigen-defined microchimerism early post-transplant does not predict for stable lung allograft function.

Microchimerism is the presence of foreign cells in an individual below 1% of total cells, which can occur in the setting of solid organ transplantation. This study quantitated donor-derived cellular subsets longitudinally in human leucocyte antigen (HLA)-mismatched lung transplant recipients (LTR) during the first post-operative year and evaluated the pattern of peripheral microchimerism with clinical outcomes. Peripheral blood mononuclear cells (PBMC) isolated from non-HLA-B44 LTR who received HLA-B44 allografts were sorted flow cytometrically into three cellular subsets. Real-time quantitative polymerase chain reaction (q-PCR) demonstrated that donor-derived HLA-B44 microchimerism is a common phenomenon, observed in 61% of patients. The level of donor-derived cells varied across time and between LTR with frequencies of 38% in the B cells/monocytes subset, 56% in the T/NK cells subset and 11% in the dendritic cells (DC) subset. Observations highlighted that microchimerism was not necessarily associated with favourable clinical outcomes in the first year post-lung transplantation.

Cytokine complex-expanded natural killer cells improve allogeneic lung transplant function via depletion of donor dendritic cells.

RATIONALE: Natural killer (NK) cells are innate lymphocytes that target virus-infected and tumor cells. Much less is known about their ability to limit adaptive immune responses. OBJECTIVES: Thus, we investigated to what extent NK cells can influence mouse lung allograft rejection. METHODS: For this purpose, we employed an orthotopic lung transplantation model in mice. MEASUREMENTS AND MAIN RESULTS: We demonstrate here that NK cells infiltrate mouse lung allografts before T cells and thereby diminished allograft inflammation, and that NK-cell deficiency enhanced allograft rejection. In contrast, expansion of recipient NK cells through IL-15/IL-15Rα complex treatment resulted in decreased T-cell infiltration and alloreactive T-cell priming as well as improved function of the allogeneic lung transplant. Only perforin-competent, but not perforin-deficient, NK cells were able to transfer these beneficial effects into transplanted NK cell-deficient IL-15Rα(-/-) mice. These NK cells killed allogeneic dendritic cells (DCs) in vitro and significantly decreased the number of allogeneic DCs in transplanted lungs in vivo. Furthermore, DC-depleted lung allografts presented decreased signs of rejection. CONCLUSIONS: These results suggest that NK cells favor allograft acceptance by depleting donor-derived DCs, which otherwise would prime alloreactive T-cell responses. Thus, conditioning regimens that augment NK-cell reactivity should be clinically explored to prepare lung allograft recipients.

Detection of Epstein-Barr virus-specific memory CD4+ T cells using a peptide-based cultured enzyme-linked immunospot assay.

Approaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-γ secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.

Association of human cytomegalovirus DNAaemia and specific granzyme B responses in lung transplant recipients.

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNAaemia could be associated with HCMV disease and reduced allograft survival. In the present study we analysed whether or not HCMV-specific granzyme B (Grz-B) responses indicating CD8(+) T cell cytotoxicity exert an impact on HCMV DNAaemia and relate to specific interferon (IFN)-γ secretion. HCMV-specific Grz-B responses were quantitated by enzyme-linked immunosorbent assay (ELISA) in 70 samples from 39 HCMV seropositive LTRs who were prospectively investigated for HCMV DNA plasma levels and IFN-γ kinetics using a standardized CD8(+) T cell assay (QuantiFERON®-CMV assay). In all LTRs who were protected from HCMV DNAaemia by early and persistent IFN-γ responses, Grz-B responses were also detected. In LTRs who developed episodes of HCMV DNAaemia, the Grz-B responses which were detected prior to viral DNA detection differed significantly in patients who experienced episodes with high (exceeding 1000 copies/ml) and low plasma DNA levels (P = 0·0290, Fisher's exact test). Furthermore, the extent of Grz-B release prior to viral DNAaemia correlated statistically with the detected levels of IFN-γ (P < 0·0001, Spearman's rank test). Of note, simultaneous detection of Grz-B and IFN-γ secretion was associated significantly with protection from high HCMV DNA plasma levels during the subsequent follow-up (P = 0·0057, Fisher's exact test), and this association was stronger than for IFN-γ detection alone. We conclude that, in addition to IFN-γ responses, Grz-B secretion by CD8(+) T cells is essential to control HCMV replication and a simultaneous measurement of IFN-γ and Grz-B could contribute to the immune monitoring of LTRs.

Early administration of FTY720 prevents chronic airway as well as vascular destruction in experimental rat lung transplantation.

BACKGROUND: Chronic rejection (CR) in terms of bronchiolitis obliterans (BO) and vascular sclerosis (VS) still represents the major obstacle for pulmonary graft survival in the medium and long term course after lung transplantation (LTX). Aside from nonspecific stimuli, early acute rejection (AR) seems to be causative especially in cases of a late diagnosis or inadequate treatment. This study investigated the effects of FTY720, a new immunosuppressant that promotes lymphocyte sequestration into lymph nodes and Peyer's patches, on the development of CR after experimental LTX. METHODS: A total of 50 rats underwent allogenic (F344-to-WKY) and syngenic (WKY-to-WKY) left LTX. Group 1 animals had no treatment. Group 2 animals were administered FTY720 (3 mg/kg body weight per day) at the maximum time of AR (day 14) and continued up to day 100 after LTX. Group 3 animals were treated with the same dosage of FTY720 from day 0 to 100. The grades of AR and CR were classified according to the criteria of the International Society for Heart and Lung Transplantation. RESULTS: Within 14 days after allogenic LTX, all nontreated rats developed early AR followed by severe CR with VS and BO. Similar data were observed for FTY720 treatment of existing AR (group 2). Only early administration of FTY720 (at the time of LTX) significantly reduced the proportion of animals with severe acute vascular rejection (P < .001). However, all of these allografts showed high-grade acute airway inflammation. After long-term application, the chronic inflammatory response was absent; none of the allografts developed BO and VS. CONCLUSION: Only application of FTY720 immediately after LTX prevented lymphocyte recirculation and lung injury.

Potential of targeting TGF-ß for organ transplant patients.

TGF-ß was originally considered as an immunoregulatory cytokine, but accumulating data demonstrate that it also plays a critical role in development of effector immunity. Since TGF- ß has a potent ability to alter immune responses, modulation of the TGF-ß pathway for treatment of transplantation patients could be effective if carried out in a target selective manner. This review will focus on the role of TGF-ß in T cell differentiation and discuss the prospect of TGF-ß as the therapeutic target of lung transplantation acceptance.

Lessons and insights from ABO-incompatible lung transplantation.

With ABO blood group incompatibility (ABOi) between donor and recipient becoming a part of mainstream living-donor renal transplantation, the applicability of ABOi to other areas of transplantation is being reconsidered. Here we present a case of inadvertent ABOi lung retransplantation managed successfully with initial plasmapheresis, antithymocyte globulin and intravenous immunoglobulin; and subsequently with oral cyclophosphamide and sirolimus in addition to tacrolimus and prednisolone. Interestingly, in the setting of solid levels of tacrolimus and sirolimus, the patient developed a fatal thrombotic microangiopathy of uncertain origin subsequent to the cessation of cyclophosphamide at 9 years posttransplant. It is apparent that ABOi lung transplantation can result in surprisingly successful long-term outcomes. Low pretransplant antibody titers likely aid this and, in pediatric neonatal or infant cases, this may not be uncommon. We must proceed cautiously as there are significant risks. Understanding the monitoring, prevention and treatment of lung transplant antibody-mediated rejection, while avoiding the long-term complications of overimmunosuppression, will be the keys to the success of future cases.

Cross-reactive anti-viral T cells increase prior to an episode of viral reactivation post human lung transplantation.

Human Cytomegalovirus (CMV) reactivation continues to influence lung transplant outcomes. Cross-reactivity of anti-viral memory T cells against donor human leukocyte antigens (HLA) may be a contributing factor. We identified cross-reactive HLA-A*02:01-restricted CMV-specific cytotoxic T lymphocytes (CTL) co-recognizing the NLVPMVATV (NLV) epitope and HLA-B27. NLV-specific CD8+ T cells were expanded for 13 days from 14 HLA-A*02:01/CMV seropositive healthy donors and 11 lung transplant recipients (LTR) then assessed for the production of IFN-γ and CD107a expression in response to 19 cell lines expressing either single HLA-A or -B class I molecules. In one healthy individual, we observed functional and proliferative cross-reactivity in response to B*27:05 alloantigen, representing approximately 5% of the NLV-specific CTL population. Similar patterns were also observed in one LTR receiving a B27 allograft, revealing that the cross-reactive NLV-specific CTL gradually increased (days 13-193 post-transplant) before a CMV reactivation event (day 270) and reduced to basal levels following viral clearance (day 909). Lung function remained stable with no acute rejection episodes being reported up to 3 years post-transplant. Individualized immunological monitoring of cross-reactive anti-viral T cells will provide further insights into their effects on the allograft and an opportunity to predict sub-clinical CMV reactivation events and immunopathological complications.

Small animal models of experimental obliterative bronchiolitis.

Despite significant progress in the treatment of chronic lung allograft rejection, obliterative bronchiolitis (OB) remains the major limitation to long-term survival after lung transplantation. The use of animal models is critical to an understanding of the pathological mechanisms behind OB, and to develop therapeutic strategies for OB. For almost 20 years, the technique of heterotopic tracheal transplantation was the leading experimental model in OB research. To address the need for a more physiologic experimental setup, a variety of small animal models have been proposed during the past two decades, such as the orthotopic tracheal transplantation model or the intrapulmonary trachea implantation model. The recent introduction of the orthotopic lung transplantation model in the mouse fulfilled the criteria for a physiologic lung transplantation setup, and also presents the advantage of being genetically modifiable. Here we review the evolution of OB models and their applications, from their beginning to the rapidly emerging physiologic models of chronic lung allograft rejection.

Pathologic findings in lung allografts with anti-HLA antibodies.

BACKGROUND: Despite data indicating a positive correlation between donor-specific anti-HLA antibodies (DSAs) and early development of bronchiolitis obliterans syndrome (BOS) in lung allografts, the role of an antibody-mediated process in acute and chronic lung allograft rejection has not been elucidated. In this study we evaluated pathologic features of transplant lung biopsies in patients with and without DSAs. METHODS: Forty-one lung transplant biopsies from 41 patients at our institution were included in our study. The biopsy H&E slides were reviewed in a blinded fashion, and scored for presence of microvascular inflammation, acute rejection, bronchiolar inflammation and acute lung injury, as well as diffuse alveolar damage (DAD). Microvascular inflammation was graded by the presence of capillary neutrophils on a scale of 0 to 4(+). For immunohistochemical analysis, the pattern and intensity of staining for C4d and C3d deposition were evaluated in airways and alveolar capillaries. RESULTS: Histopathology suspicious for antibody-mediated rejection (AMR)-defined as≥2(+) neutrophilic infiltration and/or DAD-were more common in DSA-positive cases than controls (11 of 16 vs 6 of 25, p<0.01). Evidence of allograft dysfunction was significantly more common among patients with both DSA and suspicious histopathology compared with controls (5 of 10 vs 3 of 25, p = 0.03). The combination of DSAs and histopathology suspicious for AMR was associated with both BOS (p = 0.002) and mortality (p = 0.03). Immunohistochemistry for C3d and C4d showed no correlation with each other, DSAs or histopathology. CONCLUSIONS: Grade 2(+) neutrophilic infiltration is the histopathologic finding most closely related to DSAs with graft dysfunction and development of BOS in lung transplant recipients and may be a marker for AMR.

Implications for human leukocyte antigen antibodies after lung transplantation: a 10-year experience in 441 patients.

BACKGROUND: Long-term survival after lung transplant is limited by the development of chronic and progressive airflow obstruction, a condition known as bronchiolitis obliterans syndrome (BOS). While prior studies strongly implicate cellular rejection as a strong risk factor for BOS, less is known about the clinical significance of human leukocyte antigen (HLA) antibodies and donor HLA-specific antibodies in long-term outcomes. METHODS: A single-center cohort of 441 lung transplant recipients, spanning a 10-year period, was prospectively screened for HLA antibodies after transplant using flow cytometry-based methods. The prevalence of and predictors for HLA antibodies were determined. The impact of HLA antibodies on survival after transplant and the development of BOS were determined using Cox models. RESULTS: Of the 441 recipients, 139 (32%) had detectable antibodies to HLA. Of these 139, 54 (39%) developed antibodies specific to donor HLA. The detection of posttransplant HLA antibodies was associated with BOS (HR, 1.54; P=.04) and death (HR, 1.53; P=.02) in multivariable models. The detection of donor-specific HLA antibodies was associated with death (HR, 2.42; P<.0001). The detection of posttransplant HLA antibodies was associated with pretransplant HLA-antibody detection, platelet transfusions, and the development of BOS and cytomegalovirus pneumonitis. CONCLUSIONS: Approximately one-third of lung transplant recipients have detectable HLA antibodies, which are associated with a worse prognosis regarding graft function and patient survival.

Experimental models of lung transplantation.

Lung transplantation is a life saving treatment for end stage pulmonary diseases. The development and refinement of this therapy required the utilization of various animal models, without which this procedure would not have become a clinical reality. Canine models were critical in the initial breakthroughs in surgical technique and immunosuppressive regimens, which directly led to the first successful human lung transplantation. Orthotopic lung transplant models in the rat provided a platform for more detailed investigation of immune responses to pulmonary grafts. Investigation of chronic rejection of lungs has significantly been advanced through the use of mouse tracheal transplant experiments. And finally, the advent of orthotopic, vascularized lung transplantation in the mouse opened the door to the use of genetic and molecular tools that are necessary for the rigorous mechanistic study of alloimmune and non-alloimmune factors contributing to lung graft failure. Taken together, animal models will continue to be a cornerstone in the advancement of clinical success in lung transplantation.

Therapeutic apheresis in transplantation medicine, experience with cardiac and lung transplantation in Jena.

Immunological problems like preformed donor specific antibodies (DSA) or high degree of immunization complicate the transplantation (TX) and can limit the therapeutic success. Essential pillars of antihumoral therapy are the extracorporeal procedures like therapeutic plasma exchange (TPE) and high plasma volume immunoadsorption (IA). Both extracorporeal treatments have the ability to remove pre-existing or newly formed antibodies quickly and effectively. In the last 5 years a total of 27 patients, 12 heart transplant (HTX) patients, 3 patients after combined lung and heart transplantation (Lu/HTX) and 12 lung transplant (LuTX) patients were treated for prevention (6 patients) or in case of rejection (20 patients) with TPE, cascade filtration (CF) or IA. 6 HTX recipients and 5 LuTX recipients were tested positive for HLA-ab either donor specific or de novo HLA-ab. Altogether 7 patients were tested positive for non-HLA-ab. 6 patients were treated prior to TX with 1-3 TPEs. After TX we treated 24 patients initially with TPE and performed on average 4 TPEs (1-11) for antibody elimination. 22 of 27 treated patients survived with stable graft function. The extracorporeal procedures we performed are qualified for rescue therapy of antibody mediated rejection (AMR).