HLA‐DRB1 Typing by Micro‐Bead Array Assay Identifies the Origin of Early Lymphoproliferative Disorder in a Heart Transplant Recipient

We report the case of a 68‐year‐old woman who underwent heart transplantation for hypertrophic cardiomyopathy. Two months after the transplant she developed mild fever and dyspnea with a marked drop in left ventricle ejection fraction of 31%. Coronary angiography was negative for cardiac allograft vasculopathy. Endomyocardial biopsy revealed ischemic damage with no evidence of acute cellular rejection, antibody‐mediated rejection or viral myocarditis. A neoplastic process was suspected even though full‐body computerized tomography was negative for malignancy. The patient died 4 months after transplantation. The autopsy showed acute antero‐septal myocardial infarction due to a nodular epicardial EBV‐related posttransplant lymphoproliferative disorder (PTLD) infiltrating the left anterior descending coronary artery with occlusive neoplastic thrombosis. We highlight two major aspects of this case: (1) the unusual occurrence of early PTLD involving the cardiac allograft and causing a fatal outcome, (2) the application of an immunological technique for HLA‐DRB1 typing to posttransplant paraffin‐embedded autopsy material to identify the recipient origin of this early malignancy, thus excluding a possible donor‐transmitted neoplasm.     

Human pregnancy and generation of anti‐angiotensin receptor and anti‐perlecan antibodies

Non‐HLA antibodies against the angiotensin II type 1 receptor (AT₁R) and the C‐terminal fragment of perlecan (i.e., LG3) are associated with the development of renal allograft rejection. It is currently unknown how humans develop anti‐AT₁R or anti‐LG3 antibodies. The aim of this study was to investigate whether pregnancy—as a model of sensitization to polymorphic proteins—induces anti‐AT₁R and/or anti‐LG3 antibodies. We included 104 samples from women obtained after physiologic full‐term pregnancy and 80 samples from healthy nonsensitized controls (40 women and 40 men). Both anti‐AT₁R and anti‐LG3 antibody levels were lower in pregnancy samples than in controls (both P < 0.05). By multivariate analysis, male gender was an independent predictor for high anti‐AT₁R antibody levels (OR 3.66, P = 0.04) and pregnancy was predictive for low anti‐LG3 antibody levels (OR 6.53, P = 0.0001). There was no correlation of anti‐AT₁R with anti‐LG3 antibody levels, either in the pregnancy or in the control samples (r² ≤ 0.03, P ≥ 0.26). In conclusion, physiologic full‐term pregnancy does not induce anti‐AT₁R or anti‐LG3 antibodies and may even lower their levels. Therefore, anti‐AT₁R and anti‐LG3 antibodies are likely not caused by allosensitization. The lack of correlation of anti‐AT₁R with anti‐LG3 antibodies suggests different mechanisms of generation, which remain to be elucidated.     

Costimulatory blockade with mTor inhibition abrogates effector T‐cell responses allowing regulatory T‐cell survival in renal transplantation

The advent of novel immunosuppressive strategies in renal transplantation, with immunomodulatory properties, might facilitate long‐term allograft survival. T‐cell depletion, costimulation‐blockade and mTor inhibition have been shown to favour anti‐donor hyporesponsiveness. Recently, the combination of rATG, belatacept (Bela) and sirolimus (SRL) has been used in kidney transplantation, showing very low incidence of acute rejection and excellent 12‐month graft and patient survival. Herein, we have analysed the 1‐year evolution of memory/effector and regulatory T cells and assessed the donor‐specific T‐cell alloimmune response in a group of these patients and compared with others treated with a calcineurin‐inhibitor(CNI)‐based (rATG/tacrolimus/MMF), and two other Bela‐based regimens (rATG/Bela/MMF and basiliximab/Bela/MMF/steroids). During the first year after transplantation, patients receiving rATG/Bela/SRL had significantly higher percentage of Tregs upon the memory T‐cell compartment and showed a potent anti‐donor suppressive activity. In an in vitro naive and memory/effector T‐cell co‐culture, the combination of costimulation‐blockade and SRL could abrogate both antigen‐specific T‐cell responses as efficiently as using a CNI drug. The combination of T‐cell depletion, costimulation‐blockade and mTor inhibition seems to be able to allow Treg survival and inhibit donor‐specific alloreactive effector immune responses after kidney transplantation in humans.     

No effect of HLA‐C mismatch after allogeneic hematopoietic stem cell transplantation with unrelated donors and T‐cell depletion in patients with hematological malignancies

HLA‐C mismatch in unrelated donor's hematopoietic stem cell transplantation (HSCT) has been associated with poor patient outcome. However, the impact of HLA‐C mismatch in the context of HSCT combined with in vivo T‐cell depletion remains unclear. We therefore performed a single‐center, retrospective analysis of the clinical outcome on patients with hematological malignancies treated with allo‐HSCT, who underwent T‐cell depletion. The majority of the patients (n=276) received a HLA‐A, HLA‐B, HLA‐DRB1‐matched graft that were either also HLA‐C matched (n=260), or patients with the permissive HLA‐C*03:03/03:04 mismatch (n=16), while the remaining patients (n=95) received a HLA‐C‐mismatched graft (excluding HLA‐C*03:03/03:04 mismatches). We did not observe any significant differences between the HLA‐C‐matched patients (including the permissive HLA‐C*03:03/03:04 mismatch) and the HLA‐C‐mismatched patients regarding cumulative proportion surviving, graft failure, relapse‐free survival, relapse, or acute graft‐versus‐host disease. Our data suggest that in the context of high dose T lymphocyte‐depleting agents, HLA‐C matching is not essential for patients with hematological malignancies.     

HLA‐DRB1 alleles are associated with the clinical course of disease and steroid dependence in Mexican patients with ulcerative colitis

Aim The aim of this study was to study the association between the HLA‐DRB1 alleles and the clinical course of ulcerative colitis (UC). Method Seventy‐five Mexican patients with UC were studied. High resolution HLA typing was performed using Polymerase Chain Reaction‐Sequence Specific Oligonucleotide PCR‐SSO reverse dot blot and Polymerase Chain Reaction‐single specific primer PCR‐SSP. Molecular typing techniques were applied to define HLA‐DRB1 alleles. Results Seventy‐five patients (36 female patients, 39 male patients) were studied. Significant associations were found between some HLA‐DRB1 alleles and the clinical course of disease: initial active and then inactive and the HLA‐DRB1*14 allele (P = 0.03; OR = 4.63; 95% CI: 1.08–21.23); and HLA‐DRB1*08 allele (P = 0.04; OR = 4.34; 95% CI: 1.9–33.3). On the other hand, the HLA‐DRB1*07 (P = 0.001; OR = 9.76 95% CI: 1.55–65.56) was significantly associated with steroid dependence in UC patients. Conclusions This study suggests that HLA‐DRB1 alleles were associated with the clinical course of disease and steroid dependence in UC patients.     

Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells

Ezzelarab C, Ayares D, Cooper DKC, Ezzelarab MB. Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells. Xenotransplantation 2012; 19: 311–316. © 2012 John Wiley & Sons A/S. Abstract: Background: Thrombin formation is a key feature in the activation of coagulation in pig xenograft recipients. As thrombin is known to activate endothelial and immune cells, we explored whether thrombin activation of pig endothelial cells (EC) was associated with an increased human T‐cell response. Methods: α1,3‐galactosyltransferase gene‐knockout (GTKO) pig aortic EC (pAEC) were activated by porcine interferon‐gamma (pIFNγ), human (h)IFN‐γ, or thrombin. Swine leukocyte antigen (SLA) class I and class II expression were measured. Human peripheral blood mononuclear cells (PBMC) and CD4⁺ T‐cell proliferation in response to activated pAEC, the effect of thrombin on pig CD80/CD86 mRNA, and the effect of thrombin inhibition by hirudin were evaluated. Results: After pAEC activation, SLA I expression did not change, and only pIFNγ upregulated SLA II expression. PBMC proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. CD4⁺ T‐cell proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. Thrombin inhibition by hirudin reduced thrombin‐induced upregulation of pAEC CD86 mRNA, and significantly reduced human PBMC proliferation to pAEC in comparison with thrombin alone (P < 0.05). Conclusions: Thrombin upregulates CD86 mRNA on pAEC, which is associated with increased human T‐cell proliferation against pAEC. Hirudin reduces CD86 mRNA in thrombin‐activated pAEC and is associated with downregulation of the human T‐cell proliferative response. The transplantation of organs from GTKO pigs transgenic for human thrombomodulin, and/or endothelial protein C receptor, in addition to therapeutic regulation of thrombin activation may reduce the cellular response to a pig xenograft and thus reduce the need for intensive immunosuppressive therapy.     

Dynamics of anti‐human leukocyte antigen antibodies after renal transplantation and their impact on graft outcome

The purpose of this study was to sequentially monitor anti‐HLA antibodies and correlate the results with antibody‐mediated rejection (AMR), graft survival (GS), and graft function (GF). We collected sera from 111 kidney transplant recipients on transplant days 0, 7, 14, 30, 60, 90, 180, and 360 and analyzed PRA levels by ELISA. DSAs were analyzed by single‐antigen beads in rejecting kidneys. At pre‐transplant, 79.3% of the patients were non‐sensitized (PRA = 0%) and 20.7% were sensitized (PRA > 1%). After transplant, patients were grouped by PRA profile: no anti‐HLA antibodies pre‐ or post‐transplant (group HLApre?/post?; n = 80); de novo anti‐HLA antibodies post‐transplant (group HLApre?/post+; n = 8); sensitized pre‐transplant/increased PRA post‐transplant (group HLApre+/post↑; n = 9); and sensitized pre‐transplant/decreased PRA post‐transplant (group HLApre+/post↓; n = 14). De novo anti‐HLA antibodies were detected at 7–180 d. In sensitized patients, PRA levels changed within the first 30 d post‐transplant. Incidence of AMR was higher in HLApre?/post+ and HLApre+/post↑ than in HLApre?/post?, and HLApre+/post↓ (p < 0.001) groups. One‐yr death‐censored GS was 36% in group HLApre+/post↑, compared with 98%, 88% and 100% in groups HLApre?/post?, HLApre?/post+, and HLApre+/post↓, respectively (p < 0.001). Excluding first‐year graft losses, GF and GS were similar among the groups. In conclusion, post‐transplant antibody monitoring can identify recipients at higher risk of AMR.     

De novo donor‐specific anti‐HLA antibodies after kidney transplantation are associated with impaired graft outcome independently of their C1q‐binding ability

Many aspects of post‐transplant monitoring of donor‐specific (DSA) and non‐donor‐specific (nDSA) anti‐HLA antibodies on renal allograft survival are still unclear. Differentiating them by their ability to bind C1q may offer a better risk assessment. We retrospectively investigated the clinical relevance of de novo C1q‐binding anti‐HLA antibodies on graft outcome in 611 renal transplant recipients. Acute rejection (AR), renal function, and graft survival were assessed within a mean follow‐up of 6.66 years. Post‐transplant 6.5% patients developed de novo DSA and 11.5% de novo nDSA. DSA (60.0%; P < 0.0001) but not nDSA (34.1%, P = 0.4788) increased rate of AR as compared with controls (27.4%). C1q‐binding anti‐HLA antibodies did not alter rate of AR in both groups. Renal function was only significantly diminished in patients with DSAC1q⁺. However, DSA significantly impaired 5‐year graft survival (65.2%; P < 0.0001) in comparison with nDSA (86.7%; P = 0.0054) and controls (90.7%). While graft survival did not differ between DSAC1q⁻ and DSAC1q⁺ recipients, 5‐year allograft survival was reduced in nDSAC1q⁺ (80.9%) versus nDSAC1q⁻ (90.7%, P = 0.0251). De novo DSA independently of their ability to bind C1q are associated with diminished graft survival.     

Human CMV‐specific T‐cell responses in kidney transplantation; toward changing current risk‐stratification paradigm

Despite the great efficacy of current antiviral preventive strategies, hCMV infection is still a major complication after renal transplantation, significantly challenging patient and graft survival. This issue seems to be explained because of the rather poor immunologic monitoring of the antiviral immune response. An important body of evidence has shown that monitoring the hCMV‐specific T‐cell response, at different time points of the transplant setting, seems to add crucial information for predicting the risk of viral infection, thus potentially helping individualization of therapeutic decision‐making in clinical transplantation. While several immune‐cellular assays have shown its capability for accurately monitoring hCMV‐specific T‐cell responses, only few such as the IFN‐γ ELISPOT and the ELISA based technology assays might be reliable for its application in the clinic. Nonetheless, an important effort has to be made among the transplant community to standardize and validate such immune assays. Noteworthy, large‐scale prospective randomized trials are highly warranted to ultimately introduce them in current clinical practice as a part of the highly desired personalized medicine.     

Significance of qualitative and quantitative evaluations of anti‐HLA antibodies in kidney transplantation

In this study, we retrospectively investigated the relationship between the presence/titers of donor‐specific (DSA)/nondonor‐specific antibody (NDSA) and the rate of graft rejection after transplantation. The subjects comprised 34 recipients who tested positive by FlowPRA^® Screening. The recipients were divided into two groups; 22 recipients with DSA and 12 recipients with NDSA, as detected using FlowPRA^® Single Antigen I and II beads. The antibodies were also quantitatively examined using the molecules of equivalent soluble fluorochrome (MESF) method. Nine of the 22 recipients with DSA (9/22, 40%) developed antibody‐mediated rejection (AMR), while none of the 12 recipients with NDSA (0/12, 0%) developed AMR (P < 0.01). In a quantitative analysis of the MESF data, patients with DSA with MESF values of over 3000 frequently showed AMR (8/11, 73%). In contrast, one of the patients with DSA with MESF values of <3000 showed AMR (1/11, 9%). One of the 12 patients (1/12, 8%) with NDSA showed cellular rejection (T‐cell‐mediated rejection), regardless of the MESF values. In patients with DSA, an MESF value of 3000 may be a useful cutoff value for identifying patients at a high risk for AMR.     

Clinical relevance of the de novo production of anti‐HLA antibodies following intestinal and multivisceral transplantation

Despite a negative pretransplant cross‐match, intestinal transplant recipients can mount humoral immune responses soon after transplantation. Moreover, the development of donor‐specific anti‐HLA antibodies (DSAs) is associated with severe graft injury. Between June 2000 and August 2011, 30 patients (median age 37.6 ± 9.8 years) received isolated intestinal transplantations (ITX, n = 18) or multivisceral transplantations (MVTXs, n = 12) at our center. We screened for human leukocyte antigen (HLA) antibodies pre‐ and post‐transplant. If patients produced DSAs, treatment with plasmapheresis and intravenous immunoglobulin (IVIG) was initiated. In the event of DSA persistence and/or treatment‐refractory rejection, rituximab and/or bortezomib were added. Ten patients developed DSAs and simultaneously showed significant signs of rejection. These patients received plasmapheresis and IVIG. Eight patients additionally received rituximab, and two patients were treated with bortezomib. DSA values decreased upon antirejection therapy in 8 of the 10 patients. The development of DSAs following ITX is often associated with acute rejection. We observed that the number of mismatched antigens and epitopes correlates with the probability of developing de novo DSAs. Early diagnosis and therapy, including B‐cell depletion and plasma cell inhibition, are crucial to preventing further graft injury.     

Pretransplant human leukocyte antigen antibodies detected by single‐antigen bead assay are a risk factor for long‐term kidney graft loss even in the absence of donor‐specific antibodies

Clinical relevance of ELISA‐ and single‐antigen bead assay (SAB)‐detected pretransplant HLA antibodies (SAB‐HLA‐Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death‐censored graft loss was found in patients with pretransplant SAB‐HLA‐Ab [HR: 4.46; 95% confidence interval (CI): 1.47–13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB‐HLA‐Ab but without SAB‐detected donor‐specific Ab (SAB‐DSA) (HR: 4.91; 95% CI of 1.43–16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA‐DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long‐term graft loss in patients with pretransplant SAB‐HLA, even in the absence of DSA. SAB‐HLA‐Ab‐positive patients, being negative in ELISA or CDC assay, might profit from a well‐HLA‐DR‐matched graft and intensified immunosuppression.     

Impact of cyclosporine‐A concentration in T‐cell replete haploidentical allogeneic stem cell transplantation

This paper aims to study whether cyclosporine‐A (CSA) levels have an impact on the clinical outcome of patients with T‐cell replete haploidentical allogeneic hematopoietic stem cell transplantation (allo‐HSCT). We analyzed 140 consecutive patients who had been given T‐cell replete haploidentical allo‐HSCT in our institute to assess the effect of CSA concentration in the early stages of allo‐HSCT on clinical outcomes, such as hematopoietic recovery, acute graft vs host disease (aGVHD), infection, disease‐free survival (DFS), and overall survival (OS). The median concentrations of CSA in the blood in the 1st, 2nd, 3rd, and 4th week after allo‐HSCT were 218, 235, 263, and 270 ng/mL, respectively. Additionally, 46%, 40%, 27%, and 18% of the patients had CSA blood levels below 200 ng/mL during those weeks. In total, 39 patients developed aGVHD (grade II‐IV), for a cumulative incidence of 27.8%, at a median of 32 days. Patients having a low CSA concentration (below 200 ng/mL) in the 3rd week had a higher cumulative incidence of grade II‐IV aGVHD (P = .02). In addition, multivariate logistic regression analysis showed that low CSA concentration (below 200 ng/mL) in the 3rd week was an independent risk factor of grade II‐IV aGVHD (P = .02; odds ratio = 2.66; 95% CI, 1.15‐6.17). However, CSA levels during the first 4 weeks did not have a significant impact on the patients’ hematopoietic recovery, infection, DFS, and OS. Our data indicated that adequate management of CSA levels during the peri‐engraftment period might improve clinical outcomes for those with T‐cell replete haploidentical allo‐HSCT.