Absence Of anti-HLA Antibodies Research Articles

Basiliximab vs. No Induction Therapy in Kidney Transplant Recipients with a Low Immunological Risk Profile Receiving Tacrolimus/Mycophenolate/Steroids Maintenance Immunosuppression.

Background: Induction therapy with basiliximab is recommended in kidney transplant (KT) recipients with a low immunological risk (LIR) profile. Whether basiliximab is associated with a decreased risk of acute rejection (AR) and graft loss is controversial. Methods: In our institution, LIR patients (absence of anti-HLA antibodies before KT) are inducted with basiliximab in case of living-donor KT, while deceased-donor KT recipients receive no induction. Maintenance immunosuppression is similar, including a combination of tacrolimus (Tac), mycophenolate (MPA) and steroids. In this single-center retrospective study, we included all adult LIR patients who underwent KT between 1 January 2015 and 31 December 2022. Results: Of the 471 patients included, 354 received no induction and 117 received basiliximab. The median (IQR) number of HLA A-B-DR mismatches was 3 (2-3) and 2 (2-4) in the no induction group and the basiliximab group, respectively. The cumulative incidences in the no induction group vs. the basiliximab group of acute rejection and graft loss over 5 years post-KT were similar at 8.9% vs. 7.8% (p = 0.8) and 8.5% vs. 4.2% (p = 0.063), respectively. In multivariable Cox regression analysis, delayed graft function emerged as an independent risk factor for acute rejection (hazard ratio [HR] 2.75, 95% confidence interval [CI] 1.23-6.13, p = 0.014) and graft loss (HR 9.32, CI 4.10-21.1, p < 0.001). Conclusions: Basiliximab did not provide any advantage in terms of rate of acute rejection and graft survival within 5 years post KT compared with a strategy without induction therapy in patients with a low immunological risk profile receiving triple maintenance immunosuppression Tac/MPA/steroids.

#4856 HISTOLOGICAL FINDINGS OF ACUTE HUMORAL REJECTION IN PATIENTS WITH STANDARD IMMUNOLOGICAL RISK: ANTIBODY-MEDIATED DAMAGE OR MICROVASCULAR INJURY?

Abstract Background and Aims The development of acute humoral rejection (AHR) is increased in patients with high immunological risk. Taking into account the highly sensitivity from the techniques used to determine anti-HLA antibodies, the histological findings of AHR in the immediate post-transplantation period in patients without preformed anti-HLA antibodies (standard immunological risk) is unexpected. We propose the implication of non-immunological factors as the origin of these findings. Our aim was to evaluate the histological findings of AHR in the immediate post-transplantation period in patients with standard immunological risk and to analyze factors related to its appearance. Method A multicenter retrospective study was carried out including renal transplants performed between January-2014 and december-2022, excluding patients with pre-transplant HLA sensitization and failures due to thrombosis. A descriptive analysis of transplant recipients with standard immunological risk who developed histological findings of AHR was made (AHRh) .In addition, we performed a comparative analysis of this group with the patients who developed AHR with pre-transplant HLA sensitization (AHRs). Results Of the 351 patients, 18 (5.1%) presented AHRh, all were first transplants without pre-transplant anti-HLA antibodies. Fourteen donors were brain death and four due to controlled circulatory arrest. Banff AHR was grade I in 1 patient, grade II in 13 patients, and III in 3 cases. 50% showed thrombotic microangiopathy (TMA). Seven (38.9%) failed after a mean follow-up of 28.7±26.1 months. The failure etiologies were chronic rejection (3) and primary dysfunction (4). No patient presented anti-HLA antibodies during the follow-up and 3 have received a second transplant without complications. When comparing the AHRh group with the AHRs (n = 14), we observed a greater age of the donor in AHRh (median 71,5 vs 56.5, p = 0.02) a higher percentage of donor due to controlled circulatory arrest (22% vs 0%, p = 0.075) and with history of Hypertension (66.7% vs 42.9%, p = 0.09). Recipient age was higher in AHRh (median 64.5 vs 51, p = 0.025), with a higher proportion of retransplant in AHRs (42.9% vs 0, p = 0.002). In AHRh, DGF (100% vs 57.1%, p = 0.002) and thrombotic microangiopathy (50% vs 14.3%, p = 0.03) were more frequent. Renal function was worse during the follow-up in AHRh (p&amp;lt;0.05). No differences have been observed in graft and patient survival. Conclusion The histological finding of RAH in patients with standard immunological risk appears as an aggressive process with negative consequences on graft survival. The absence of anti-HLA antibodies in subsequent follow-up suggests the involvement of non-immunological factors in its developing. Processes related to the maintenance and/or extraction of organs could favor the appearance of microvascular damage, with histological findings suggestive of AHR especially in older grafts and with comorbidity. Studying these cases is essential to identify risk groups and establish appropriate therapeutic strategies.

Association of HLA Mismatches and Histology Suggestive of Antibody-Mediated Injury in the Absence of Donor-Specific Anti-HLA Antibodies.

The histology of antibody-mediated rejection after kidney transplantation is observed frequently in the absence of detectable donor-specific anti-HLA antibodies. Although there is an active interest in the role of non-HLA antibodies in this phenotype, it remains unknown whether HLA mismatches play an antibody-independent role in this phenotype of microcirculation inflammation. To study this, we used the tools HLAMatchmaker, three-dimensional electrostatic mismatch score, HLA solvent accessible amino acid mismatches, and mismatched donor HLA-derived T cell epitope targets to determine the degree of HLA molecular mismatches in 893 kidney transplant recipients with available biopsy follow-up. Multivariable Cox proportional hazards models were applied to quantify the cause-specific hazard ratios of the different types of HLA mismatch scores for developing antibody-mediated rejection or histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies. In all survival analyses, the patients were censored at the time of the last biopsy. In total, 121 (14%) patients developed histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies, of which 44 (36%) patients had concomitant T cell-mediated rejection. In multivariable Cox analysis, all different calculations of the degree of HLA mismatch associated with developing histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies. This association was dependent neither on the presence of missing self (potentially related to natural killer cell activation) nor on the formation of de novo HLA antibodies. Also, glomerulitis and complement C4d deposition in peritubular capillaries associated with the degree of HLA mismatch in the absence of anti-HLA antibodies. The histology of antibody-mediated rejection and its defining lesions are also observed in patients without circulating anti-HLA antibodies and relate to the degree of HLA mismatch.

N-glycosylated IgG in patients with kidney transplants increases calcium/calmodulin kinase IV in podocytes and causes injury.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.

Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens.

Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.

Anti MICA Antibody (major histocompatibility complex class I related Chain A) Whether to Treat or Avoid?

Aims To study the role of MICA antibody in living renal transplantation in the absence of anti HLA antibodies. Methods A retrospective study of patients undergone living renal transplantation between 2000-2014 was performed. Recipients were classified in two groups, pre transplantation Anti MICA antibody positive group (n=17) and antibody negative comparison group (n=17). Patients with anti HLA antibodies were excluded and only isolated MICA positive patients were included in the study group. All recipients undergone tolerance protocol and post transplant T-regulatory cells, and were comparable in recipient age, donor age, donor relation, HLA and immunosuppression including induction. Results Patients with pre transplant MICA antibody positivity were associated with increased acute rejection as compared to comparison group (47% vs 11.7 %, p value= 0.02 ). Renal function in MICA positive group and comparison group were comparable over a mean follow up of 6.5 years (mean s.creatinine 1.58 vs 1.53 mg/dl ). Rate of chronic rejection was same in both groups (5.8% ). No patient or graft loss in either group over mean follow up of 6.5 years. Conclusion Isolated Anti MICA antibody positivity is uncommon event. Pre transplant anti MICA antibody positivity is associated with increased acute rejection rates. However chronic rejection rates and renal function are comparable in both groups. Thus our study emphasizes that MICA antibody positive patients may require more aggressive immunosuppression, role of desensitization has to be defined.

Xenoreactive antibodies and latent fibrin formation in VAD and cardiac transplant recipients can confound the detection and measurement of anti-AT1R antibodies.

Autoantibodies to the angiotensin II type 1 receptor (AT1R) are thought to be important in antibody-mediated rejection (AMR), especially in the absence of anti-HLA antibodies. We used a variety of methods to examine the specificity of a commercially available kit designed to quantitate anti-AT1R antibodies. We found that fibrin formation in serum samples from patients awaiting cardiac transplantation with ventricular assist devices (VADs) can produce falsely elevated anti-AT1R values. In addition, absorption studies with a variety of cell lines with or without expression of human AT1R, and those that express xenoantigens, suggest that many of the antibodies detected in the AT1R test system are heterophilic and have reactivity to xenoantigens. Furthermore, we provide data that show that reactivity to the sialic acid Neu5Gc is a common finding among samples that are highest in anti-AT1R levels. We conclude that a common laboratory method for quantitation of anti-AT1R antibodies is nonspecific and overestimates the frequency of true positives. A reevaluation of the role that anti-AT1R antibodies play in allograft function and patient outcomes is warranted.

Acute Humoral Rejection in the Absence of Anti-HLA Antibodies, a French Nation-Wide Study.

Antibody mediated rejection (ABMR) is usually associated with donor-specific anti-HLA antibodies (DSA). Evidence of ABMR in patients with no DSA strongly suggests the implication of non-HLA antibodies. We implemented a retrospective nation-wide study to improve undertanding of non-HLA ABMR. Inclusion criteria were: first or re-transplantion, deceased or living-donor; acute allograft dysfunction during the first three months; allograft biopsy showing a total score of glomerulitis and peritubular capillaritis≥3 according to the Banff classification; absence of identified anti-HLA DSAs using Luminex®. Demographical and clinical parameters were collected for 36 cases. Recipient age was 53±19 yrs. Donor age was 53±12 yrs. Cold ischemia time was 12±8 hrs. All patients received induction therapy (94% basiliximab, 6% thymoglobuline), a CNI (69% tacrolimus, 31% cyclosporine), MPA and steroids. Rejection was diagnosed at day 12±9 in the absence of graft function recovery in 30% of patients. The remaining 25 patients defined a poor serum creatinine nadir of 289±169 μmol/L at day 15±22. Acute graft dysfunction (393±230 μmol/L) led to the diagnosis of acute rejection at day 25±34. No anti-HLA DSA were observed. Mean g+ptc score was 3,8 [3-6]. In addition, intimal arteritis and interstitial inflammation (i ≥1) were observed in 50% and 69% of cases, respectively. Additional lesions included haemorragic suffusion and thrombotic microangiopathy in 26% and 9% of cases, respectively. Therapeutic strategies included steroids pulses (86%), thymoglobulines (37%), rituximab (31%), IVIG (49%) and plasmapheresis (49%). Primary non function was observed in 2 cases. At last follow up (3±1 yrs) serum creatinine was 178±92 μmol/l. 6% of patients were back to dialysis. This study provides a close clinico-pathological description of non-HLA ABMR, and will be followed by additional histological and mechannistic analyses.

Research Highlights

Research Highlights

HLA-Universal Platelet Transfusions Prevent Platelet Refractoriness in a Mouse Model

Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34(+) cells, using a short hairpin RNA (shRNA) to target β2-microglobulin (β2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34(+) cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2Rγc(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 × 10(6) HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.

Impact of anti-HLA antibodies on allogeneic hematopoietic stem cell transplantation outcomes after reduced-intensity conditioning regimens

Anti-human leukocyte antigen (HLA) antibodies are associated with several complications in solid organ transplantations, but their impact after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not yet well defined. To evaluate the relevance of anti-HLA antibodies, we have retrospectively analyzed 107 peripheral blood allo-HSCTs after reduced-intensity conditioning regimen between 2005 and 2010. Acute myeloid leukemia and multiple myeloma were the most frequent malignancies in the cohort. The detection of anti-HLA antibodies was systematically performed in all patients before transplantation. Anti-HLA antibodies were present in 24 patients (22%). There was no significant impact of anti-HLA antibodies on engraftment, incidence of relapse, and incidence of acute graft-vs-host disease. The presence of anti-HLA antibodies was associated with significantly worse overall survival (p = 0.006) and event-free survival (p = 0.024) after stratification on sex. The 3-year probability of overall survival was 34% without anti-HLA antibodies and 16% in their presence. Patients with anti-HLA antibodies had a higher transplant-related mortality associated with life-threatening vascular complications. Our study supports that anti-HLA antibodies should be tested and considered as an important impacting factor for transplantation outcomes after reduced-intensity conditioning allo-HSCT. We recommend its consideration before allo-HSCT in the donor-recipient selection parameters.

Anti-glutathione S-transferase T1 antibody-mediated rejection in C4d-positive renal allograft recipients

Chronic humoral rejection is a progressive form of graft injury, with defined diagnostic criteria, the crucial one being the evidence of circulating anti-donor antibodies. These antibodies are mainly directed against human leucocyte antigens (HLA), but other targets have also been described. We previously reported that antibodies against the Glutathione S-transferase T1 (GSTT1) enzyme appear in recipients without the GSTT1 gene who receive a graft from a GSTT1-positive donor. The primary aim of this study was to analyse the role of GSTT1 in cases of antibody-mediated rejection (AMR) in the absence of anti-HLA antibodies. A second objective was to describe the distribution of the GSTT1 enzyme in the human kidney. Four renal biopsies from four renal transplanted patients with declined renal function and circulating anti-donor GSTT1 antibodies were studied for C4d deposits in sections of paraffin-embedded tissue samples. Anti-donor-specific HLA and MICA antibody detection was done with the Luminex platform and anti-GSTT1 antibodies were tested by indirect immunofluorescence on rat tissues and ELISA assay. DNA of the patients was extracted for GSTT1 genotyping. Four patients with the GSTT1 donor/recipient mismatch developed anti-GSTT1 antibodies 32, 42, 48 and 60 months after the transplant. One patient also had donor-specific anti-HLA antibodies. Their biopsies showed pathologic lesions compatible with chronic antibody-mediated rejection (CAMR), along with positive C4d deposition in peritubular capillaries in three of them, being no valuable in the other case. This is the first study reporting an association between the appearance of chronic antibody-mediated renal allograft rejection and the occurrence of de novo production of anti-GSTT1 antibodies, in the absence of anti-HLA donor-specific antibodies. This fact suggests a potential role of the GSTT1 system in anti-graft immune response.

Anti-HLA class I antibodies and pulmonary homograft function after the Ross procedure

Background. The Ross procedure provides excellent long-term results in the majority of patients. However, degeneration of the pulmonary homograft in some patients remains an unresolved problem that may be related to immunologic factors. Therefore, we studied the prevalence of antihuman leukocyte antigen (HLA) class I antibodies and echocardiographic results of homograft function at rest.Methods. Forty-seven patients (37 men, 10 women; 47 ± 15 years) were seen for echocardiography 1.1 to 63.9 months (median, 27 months) postoperatively. The presence of anti-HLA antibodies was tested against a panel of lymphocytes of 50 donors.Results. Twenty-seven (57%) of the patients produced anti-HLA class I antibodies. No difference in the maximal or mean transhomograft pressure gradient, or in the frequency of homograft regurgitation according to the presence or absence of anti-HLA antibodies was found. However, the right ventricle was slightly but significantly larger in antibody-positive patients (26.3 ± 4.2 versus 30.7 ± 3.5 mm; p = 0.001).Conclusions. In the first years after the Ross procedure, we could not detect significant evidence of an association between anti-HLA class I antibodies and echocardiographic results of homograft function at rest in adults.

Activation of human airway epithelial cells by non-HLA antibodies developed after lung transplantation: a potential etiological factor for bronchiolitis obliterans syndrome.

The main cause of morbidity and mortality after lung transplantation (LT) is bronchiolitis obliterans syndrome (BOS). Anti-HLA antibodies development after LT has been shown to play an important role in BOS pathogenesis. However, the nature of non-HLA antibodies developed after LT and their role in BOS pathogenesis have not been determined. Sera from 16 BOS+ patients and 11 BOS- patients were collected at 12, 24, 36, and 48 months after LT. Anti-HLA class I and class II antibodies were absorbed with pooled human platelets and pooled human lymphoblastoid cell lines, respectively. Then, the presence of non-HLA antibodies against several cell lines from different origin was determined by flow cytometric analysis. Antibody-positive samples were tested for induction of proliferation and growth factor production in two selected airway epithelial cell (AEC) lines. Five of 16 BOS+ patients (31.2%) and 0 of 11 BOS- patients (0%) developed anti-AEC antibodies after LT (P=0.05). No reactivity against endothelial cells, lymphocytes, monocytes, or granulocytes was detected. Further analysis of two selected sera demonstrated the development of reactivity against a 60-kDa antigen expressed by 60% of AEC lines and only 12% of cell lines from other tissues. Antibody binding to this antigen induced intracellular Ca++ influx, tyrosine phosphorylation, proliferation, and up-regulation of transforming growth factor-beta and heparin-binding epidermal growth factor mRNA transcription in AECs. These results indicate that anti-AEC antibodies may play a role in the immunopathogenesis of BOS in the absence of anti-HLA antibodies.

Antibodies eluted from acutely rejected renal allografts bind to and activate human endothelial cells

This study was designed to investigate how antiendothelial antibodies (EAbs) are involved in acute irreversible renal graft rejection. Eluates from 25 renal allografts, lost by irreversible rejection ( n = 22) and by renal vein thrombosis (controls n = 3), were tested against a panel of cultured human umbilical vein endothelial cells (HUVEC). All patients were under immunosuppression at the time of nephrectomy. EAbs binding and membrane expression of adhesion molecules ELAM-1 and VCAM-1 were analyzed by flow cytometry (FACS) and by semiquantitative RT-PCR for mRNAs coding for those molecules. The absence of anti-HLA antibodies against the donor was ascertained at transplant, and before and after nephrectomy by the negativity of specific crossmatches performed using the most sensitive techniques. EAbs eluted from eight rejected kidneys bound to HUVEC. They did not induce any cytotoxicity, but their incubation with HUVEC (4 h at 37°C; 2.5 mg/ml) led to upregulation of mRNAs coding for VCAM-1 (35- to 60-fold increases) and ICAM-1 (8- to 12-fold increases) as compared with control EAbs. Membrane expression of adhesion molecules was also strikingly increased, with 80% of the cells expressing VCAM-1 and 65% expressing ELAM-1 upon incubation. EAbs were detected in eight out of nine (88.8%) eluates from kidneys lost from acute vascular rejection, but in none of the 13 (0.0%) kidneys lost from other types of rejection ( p < 0.0001). We conclude that EAbs, capable of activating human endothelial cells, can be recovered from acutely rejected kidneys and may play a direct role in the pathogenesis of acute rejection.