RESUMO
ABO-incompatible (ABOi) kidney transplantation has become an established strategy to increase the number of available living donors. At our center, the conditioning protocol for ABOi patients is based on anti-A/B antibody removal and depletion of B cells with the anti-CD20 mAb rituximab (Mabthera®). It is known that even low amounts of remaining rituximab in serum of patients results in false positive B cell cross match results, masking detection of potentially harmful donor human leukocyte antigen (HLA) specific antibodies. Treatment of donor cells with high concentrations (>1â¯mg/mL) of pronase is currently standard procedure for elimination of rituximab (RIT) interference. It is, however, troublesome that recent reports indicate that pronase treatment per se can induce incorrect flow cytometry cross match (FCXM) results. The aim of this study was to evaluate an alternative pronase-free FCXM for crossmatching of patients treated with rituximab. FCXM with an anti-RIT monoclonal antibody (mAb) pre-blocking step were evaluated on normal human sera (NHS) and patient sera supplemented with RIT. NHS supplemented with RIT or patient sera, without donor specific antibodies (DSA), resulted in high B cell median channel shift (>200 IgG) above background. This shift was eliminated by a serum pre-blocking step with 2-fold excess of anti-RIT (clone MB2A4). Blocking with anti-RIT did not influence the T cells crossmatch results. We present data supporting proof-of-concept that blocking with anti-RIT antibody prior to XM can enable reliable detection of anti-HLA class I and II donor specific antibodies without use of pronase treated donor cells.
Assuntos
Linfócitos B/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Citometria de Fluxo/métodos , Rejeição de Enxerto/prevenção & controle , Teste de Histocompatibilidade/métodos , Transplante de Rim , Rituximab/uso terapêutico , Sistema ABO de Grupos Sanguíneos/genética , Reações Falso-Positivas , Antígenos HLA/imunologia , Humanos , Isoanticorpos/metabolismo , Depleção Linfocítica , Pronase/metabolismo , Condicionamento Pré-TransplanteRESUMO
The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.
Assuntos
Anticorpos/imunologia , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Antígenos HLA/imunologia , Teste de Histocompatibilidade , Anticorpos/sangue , Proteínas do Sistema Complemento/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Teste de Histocompatibilidade/métodos , Humanos , Separação Imunomagnética/métodos , Leucócitos Mononucleares/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Allogeneic mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but little is known about their immunogenicity. In this study, we monitored the therapeutic and immunogenic effects of decidual stromal cells (DSCs) from term placentas when used as a therapy for generalized severe junctional epidermolysis bullosa (JEB) (previously termed Herlitz JEB), a lethal condition caused by the lack of functional laminin-332. An 11-month-old JEB patient was treated with five infusions of allogeneic DSCs within a 3-month period. Amniotic membranes (AMs) were applied to severe wounds. After the treatment, wounds started to heal in the middle of the blisters, but the improvements were transient. After two infusions of DSCs, the JEB patient had developed multispecific anti-HLA class-I antibodies. No antibodies to laminin-332 were detected, but the patient had high levels of anti-bovine serum albumin antibodies, which could bind to DSCs. Peripheral blood mononuclear cells (PBMCs) from the patient had a higher proliferative response to DSCs than to third-party PBMCs, which contrasts with the pattern observed in healthy donors. Human DSCs and MSCs induced similar xenoreactivity in mice. Two of 16 allogeneic stem cell-transplanted patients, treated with DSCs for graft-versus-host disease or hemorrhagic cystitis, showed a positive flow cytometric crossmatch test. One patient had anti-HLA antibodies before DSC infusion, whereas the other had no anti-HLA antibodies at any time. AM and DSC infusions may have improved the healing process in the JEB patient, but DSCs appeared to induce anti-HLA antibodies. The risk of alloimmunization by DSCs seems to be low in immunocompromised patients.
Assuntos
Epidermólise Bolhosa/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Fenômenos Imunogenéticos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Adolescente , Adulto , Idoso , Animais , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Idiotípicos/imunologia , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/imunologia , Criança , Decídua/imunologia , Decídua/transplante , Epidermólise Bolhosa/patologia , Epidermólise Bolhosa/terapia , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA/imunologia , Xenoenxertos/imunologia , Humanos , Lactente , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Masculino , Células-Tronco Mesenquimais/imunologia , Camundongos , Pessoa de Meia-Idade , Soroalbumina Bovina/imunologia , CalininaRESUMO
Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody-mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement-fixing HLA antibodies is commonly performed via the complement-dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read-out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry-based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead-enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells.
Assuntos
Anticorpos/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Linfócitos B/imunologia , Tipagem e Reações Cruzadas Sanguíneas , Humanos , Microesferas , Linfócitos T/imunologia , Doadores de TecidosRESUMO
The presence of human leukocyte antigen (HLA) and non-HLA antibodies (Abs) in kidney transplant recipients is associated with graft rejections. This study reports the results of an endothelial precursor cell crossmatch (EPCXM) test for detection of non-HLA Abs and its correlation to lymphocyte crossmatch (LXM) test results, the degree and type of sensitization, and transplantation (Tx) outcome in patients evaluated for living donor (LD) kidney transplantation (KTx). Patients were tested before any pre-transplantation (pre-Tx) treatment and at Tx. Pre-Tx treatments included B cell depletion and Ab removal. Patient records were reviewed for assessment of renal graft function, results of biopsies, and identification of complications affecting the graft. Pre-Tx sera from 32% of the LD patients had IgG and/or IgM-binding donor EPCs. Twenty-five percent of the patients were EPCXM IgM+. Of the patients with negative LXM tests, 25% had EPC Abs mainly of IgM class not reactive with HLA. There was no difference in rejection frequency or serum creatinine levels between the EPCXM+ and EPCXM- groups. The pre-Tx EPCXM+ group had significantly more patients with delayed graft function. Prospective studies with appropriate control groups are needed to establish whether pre-treatments aiming at removing anti-endothelial cell antibodies, as detected by the EPCXM pre-Tx, have a beneficial effect on short-term and long-term graft survival.
Assuntos
Função Retardada do Enxerto/diagnóstico , Células Endoteliais/metabolismo , Rejeição de Enxerto/diagnóstico , Transplante de Rim , Complicações Pós-Operatórias/diagnóstico , Células-Tronco/metabolismo , Adolescente , Adulto , Criança , Função Retardada do Enxerto/epidemiologia , Função Retardada do Enxerto/etiologia , Células Endoteliais/imunologia , Feminino , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/etiologia , Teste de Histocompatibilidade/estatística & dados numéricos , Humanos , Isoanticorpos/sangue , Doadores Vivos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Valor Preditivo dos Testes , Prognóstico , Células-Tronco/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
Natural killer (NK) cells are implicated in T cell-mediated autoimmune diseases. Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated animal model of the Guillain-Barré syndrome in human. The role of NK cells in the initiation and development of EAN remains unclear. In the present study, we demonstrate that anti-NKR-P1A monoclonal antibody (mAb) treatment in vivo did not affect the initiation and development of clinical EAN in Lewis rats induced by immunization with peripheral nerve myelin P0 protein peptide 180-199 and Freund's complete adjuvant, as well as the proportion of NKR-P1A+ cells (including NK cells and NKT cells) in the spleen. Furthermore, inflammatory cell infiltrations and demyelination in the peripheral nervous system (PNS) and in vitro P0 peptide 180-199-specific splenocyte proliferation were not different in anti-NKR-P1A mAb-treated rats compared to the control antibody-treated rats. The cytotoxic activity of NKR-P1A+ cells, determined by NK cell-sensitive K562 cells as target cells, decreased markedly in anti-NKR-P1A mAb-treated rats, suggesting that decrease of the cytotoxic activities of NKR-P1A+ cells is not sufficient to alter clinical EAN, although NKR-P1A+ cells may participate in the pathogenesis of T cell-mediated autoimmune diseases, such as EAN, by the mechanisms that involve the release of cytokines.
Assuntos
Antígenos de Superfície/imunologia , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/fisiopatologia , Animais , Anticorpos Monoclonais/farmacologia , Citotoxicidade Imunológica/imunologia , Doenças Desmielinizantes/imunologia , Citometria de Fluxo , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/fisiopatologia , Masculino , Proteína P0 da Mielina/farmacologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Fragmentos de Peptídeos/farmacologia , Nervos Periféricos/imunologia , Nervos Periféricos/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Baço/citologia , Baço/imunologiaRESUMO
The hepatitis B virus (HBV) core antigen (HBcAg) has a unique ability to bind a high frequency of naive human and murine B cells. The role of HBcAg-binding naive B cells in the immunogenicity of HBcAg is not clear. The HBcAg-binding properties of naive B cells were characterized using HBcAg particles with mutated spike region (residues 76-85) sequences. Deletion of residues 76-85 (HBcDelta76-85) destroyed naive B cell binding, whereas deletion of residues 79-85 did not. HBcAg particles with an Ile instead of the natural Ala at position 80 did not bind naive B cells, whereas reversion of Ile80-->Ala restored B cell binding. Destroying the B cell-binding ability of HBcAg had a marginal effect on the overall B cell immunogenicity of the different particles, suggesting that they were equally efficient in priming T helper cells. Therefore, the importance of HBcAg-binding B cells is studied with relation to the priming of HBcAg-specific cytotoxic T cells (CTLs). The role of HBcAg-binding B cells in the priming of HBcAg-specific CTLs was evaluated by immunization with endogenous HBcAg (DNA immunization) and exogenous recombinant HBcAg particles. Endogenous HBcAg primed HBcAg-specific CTLs in wild-type and B cell-deficient mice, whereas exogenous HBcAg primed HBcAg-specific CTLs only in wild-type mice. Importantly, HBcDelta76-85 did not prime CTLs despite the presence of B cells. Thus, the ability of exogenous HBcAg particles to prime specific CTLs is B cell dependent, suggesting a possible role for HBcAg-binding B cells in HBV infections.