RESUMO
Optimization of individual immunosuppression, which reduces the risks of both graft loss and patients' death, is considered the best approach to improve long-term outcomes of renal transplantation. Torque Teno Virus (TTV) DNAemia has emerged as a potential biomarker reflecting the depth of therapeutic immunosuppression during the initial year post-transplantation. However, its efficacy in long-term monitoring remains uncertain. In a cohort study involving 34 stable kidney transplant recipients and 124 healthy volunteers, we established lower and upper TTV DNAemia thresholds (3.75-5.1 log10 cp/mL) correlating with T-cell activatability, antibody response against flu vaccine, and risk for subsequent serious infections or cancer over 50 months. Validation in an independent cohort of 92 recipients confirmed that maintaining TTV DNAemia within this range in >50% of follow-up time points was associated with reduced risks of complications due to inadequate immunosuppression, including de novo DSA, biopsy-proven antibody-mediated rejection, graft loss, infections, or cancer. Multivariate analysis highlighted "in-target" TTV DNAemia as the sole independent variable significantly linked to decreased risk for long-term complications due to inadequate immunosuppression (odds ratio [OR]: 0.27 [0.09-0.77]; p = 0.019). Our data suggest that the longitudinal monitoring of TTV DNAemia in kidney transplant recipients could help preventing the long-term complications due to inadequate immunosuppression.
Assuntos
Infecções por Vírus de DNA , DNA Viral , Terapia de Imunossupressão , Transplante de Rim , Torque teno virus , Transplantados , Humanos , Torque teno virus/genética , Transplante de Rim/efeitos adversos , Masculino , Feminino , Pessoa de Meia-Idade , DNA Viral/sangue , Adulto , Infecções por Vírus de DNA/virologia , Infecções por Vírus de DNA/sangue , Infecções por Vírus de DNA/imunologia , Terapia de Imunossupressão/efeitos adversos , Estudos Longitudinais , Idoso , Rejeição de Enxerto , Imunossupressores/uso terapêutico , Imunossupressores/efeitos adversos , Estudos de Coortes , ViremiaRESUMO
Imlifidase recently received early access authorization for highly sensitized adult kidney transplant candidates with a positive crossmatch against an ABO-compatible deceased donor. These French consensus guidelines have been generated by an expert working group, in order to homogenize patient selection, associated treatments and follow-up. This initiative is part of an international effort to analyze properly the benefits and tolerance of this new costly treatment in real-life. Eligible patients must meet the following screening criteria: cPRA ≥ 98%, ≤ 65-year of age, ≥ 3 years on the waiting list, and a low risk of biopsy-related complications. The final decision to use Imlifidase will be based on the two following criteria. First, the results of a virtual crossmatch on recent serum, which shall show a MFI for the immunodominant donor-specific antibodies (DSA) > 6,000 but the value of which does not exceed 5,000 after 1:10 dilution. Second, the post-Imlifidase complement-dependent cytotoxicity crossmatch must be negative. Patients treated with Imlifidase will receive an immunosuppressive regimen based on steroids, rATG, high dose IVIg, rituximab, tacrolimus and mycophenolic acid. Frequent post-transplant testing for DSA and systematic surveillance kidney biopsies are highly recommended to monitor post-transplant DSA rebound and subclinical rejection.
Assuntos
Transplante de Rim , Adulto , Humanos , Pré-Escolar , Transplante de Rim/métodos , Teste de Histocompatibilidade/métodos , Antígenos HLA , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Anticorpos , IsoanticorposRESUMO
The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.
Assuntos
Formação de Anticorpos , Isoanticorpos , Animais , Rejeição de Enxerto , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Humanos , Isoantígenos , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos , Receptores de Antígenos de Linfócitos BRESUMO
BACKGROUND: The standard-of-care protocol, based on plasma exchanges, high-dose intravenous immunoglobulin and optimization of maintenance immunosuppression, can slow down the evolution of antibody-mediated rejection (AMR), but with high interindividual variability. Identification of a reliable predictive tool of the response to AMR treatment is a mandatory step for personalization of the follow-up strategy and to guide second-line therapies. METHODS: Interrogation of the electronic databases of 2 French university hospitals (Lyon and Strasbourg) retrospectively identified 81 renal transplant recipients diagnosed with AMR without chronic lesions (cg score ≤1) at diagnosis and for whom a follow-up biopsy had been performed 3-6 months after initiation of therapy. RESULTS: The evolution of humoral lesions on follow-up biopsy (disappearance versus persistence versus progression) correlated with the risk for allograft loss (logrank test, P = .001). Patients with disappearance of humoral lesions had â¼80% graft survival at 10 years. The hazard ratio for graft loss in multivariate analysis was 3.91 (P = .04) and 5.15 (P = .02) for patients with persistence and progression of lesions, respectively. The non-invasive parameters classically used to follow the intensity of humoral alloimmune response (evolution of immunodominant DSA mean fluorescence intensity) and the decline of renal graft function (estimated glomerular filtration rate decrease and persistent proteinuria) showed little clinical value to predict the histological response to AMR therapy. CONCLUSION: We conclude that invasive monitoring of the evolution of humoral lesions by the mean of follow-up biopsy performed 3-6 months after the initiation of therapy is an interesting tool to predict long-term outcome after AMR treatment.
Assuntos
Rejeição de Enxerto , Transplante de Rim , Humanos , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Estudos Retrospectivos , Seguimentos , Transplante de Rim/efeitos adversos , Sobrevivência de Enxerto , Biópsia , Anticorpos , IsoanticorposRESUMO
Transplant recipients, who receive therapeutic immunosuppression to prevent graft rejection, are characterized by high coronavirus disease 2019 (COVID-19)-related mortality and defective response to vaccines. We observed that previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but not the standard two-dose regimen of vaccination, provided protection against symptomatic COVID-19 in kidney transplant recipients. We therefore compared the cellular and humoral immune responses of these two groups of patients. Neutralizing anti-receptor-binding domain (RBD) immunoglobulin G (IgG) antibodies were identified as the primary correlate of protection for transplant recipients. Analysis of virus-specific B and T cell responses suggested that the generation of neutralizing anti-RBD IgG may have depended on cognate T-B cell interactions that took place in germinal center, potentially acting as a limiting checkpoint. High-dose mycophenolate mofetil, an immunosuppressive drug, was associated with fewer antigen-specific B and T follicular helper (TFH) cells after vaccination; this was not observed in patients recently infected with SARS-CoV-2. Last, we observed that, in two independent prospective cohorts, administration of a third dose of SARS-CoV-2 mRNA vaccine restored neutralizing titers of anti-RBD IgG in about 40% of individuals who had not previously responded to two doses of vaccine. Together, these findings suggest that a third dose of SARS-CoV-2 mRNA vaccine improves the RBD-specific responses of transplant patients treated with immunosuppressive drugs.
Assuntos
COVID-19 , Transplante de Rim , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Estudos Prospectivos , SARS-CoV-2 , Transplantados , Vacinas Sintéticas , Vacinas de mRNARESUMO
Detection of mismatched human leukocyte antigens by adaptive immune cells is considered as the main cause of transplant rejection, leading to either T-cell mediated rejection or antibody-mediated rejection. This canonical view guided the successful development of immunosuppressive therapies and shaped the diagnostic Banff classification for kidney transplant rejection that is used in clinics worldwide. However, several observations have recently emerged that question this dichotomization between T-cell mediated rejection and antibody-mediated rejection, related to heterogeneity in the serology, histology, and prognosis of the rejection phenotypes. In parallel, novel insights were obtained concerning the dynamics of donor-specific anti-human leukocyte antigen antibodies, the immunogenicity of donor-recipient non-human leukocyte antigen mismatches, and the autoreactivity against self-antigens. Moreover, the potential of innate allorecognition was uncovered, as exemplified by natural killer cell-mediated microvascular inflammation through missing self, and by the emerging evidence on monocyte-driven allorecognition. In this review, we highlight the gaps in the current classification of rejection, provide an overview of the expanding insights into the mechanisms of allorecognition, and critically appraise how these could improve our understanding and clinical approach to kidney transplant rejection. We argue that consideration of the complex interplay of various allorecognition mechanisms can foster a more integrated view of kidney transplant rejection and can lead to improved risk stratification, targeted therapies, and better outcome after kidney transplantation.
Assuntos
Transplante de Rim , Anticorpos , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/prevenção & controle , Antígenos HLA , Humanos , Terapia de Imunossupressão , Transplante de Rim/efeitos adversos , Complicações Pós-Operatórias , Doadores de TecidosRESUMO
BACKGROUND: Circulating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I-mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA-negative MVI. However, missing self's clinical importance as a nonhumoral trigger of allograft rejection remains unclear. METHODS: In a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene. RESULTS: We identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA-associated MVI. CONCLUSION: Missing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA-negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.
Assuntos
Rejeição de Enxerto/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Células Matadoras Naturais/imunologia , Receptores KIR/genética , Vasculite/genética , Adulto , Idoso , Anticorpos/sangue , Feminino , Genótipo , Sobrevivência de Enxerto , Antígeno HLA-A11/genética , Antígeno HLA-A11/imunologia , Antígeno HLA-A3/genética , Antígeno HLA-A3/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Teste de Histocompatibilidade , Humanos , Transplante de Rim , Masculino , Microvasos , Pessoa de Meia-Idade , Receptores KIR2DL2/genética , Receptores KIR2DL3/genética , Doadores de Tecidos , Transplantados , Vasculite/complicaçõesRESUMO
The current transplant immunology dogma defends that allograft rejection is initiated by recipient's adaptive immune system. In this prevalent model, innate immune cells in general, and natural killer (NK) cells in particular, are merely considered as downstream effectors which participate in the destruction of the graft only upon recruitment by adaptive effectors: alloreactive T cells or donor-specific antibodies (DSA). Challenging this vision, recent data demonstrated that recipients' NK cells are capable of a form of allorecognition because they can sense the absence of self HLA class I molecules on the surface of graft endothelial cells. Missing-self triggers mTORC1-dependent activation of NK cells, which in turn promote the development of graft microvascular inflammation and detrimentally impact graft survival. The fact that some patients develop chronic vascular rejection in absence of DSA or genetically-predicted missing self suggests that other molecular mechanisms could underly NK cell allorecognition. This review provides an overview of these proven and putative molecular mechanisms and discusses future research directions in this emerging field in organ transplant immunology.
Assuntos
Células Endoteliais , Rejeição de Enxerto , Alelos , Aloenxertos , Rejeição de Enxerto/genética , Humanos , Células Matadoras NaturaisRESUMO
The analysis of immune cell signaling is critical for the understanding of the biology and pathology of the immune system, and thus a mandatory step for the development of efficient biomarkers and targeted therapies. Phosflow, which has progressively replaced the traditional western blot approach, relies on flow cytometry to analyze various signaling pathways at a single-cell level. This technique however suffers a lack of sensitivity largely due to the low signal/noise ratio that characterizes cell signaling analysis. In this study, we describe a new technique, which combines the use of biofunctionalized nanospheres (i.e., synthetic particulate antigens, SPAg) to stimulate the immune cells in suspension and imaging flow cytometry to identify homogenously-stimulated cells and quantify the activity of the chosen signaling pathway in selected subcellular regions of interest. Using BCR signaling as model, we demonstrate that SIBERIAN (SPAg-assIsted suB-cEllulaR sIgnaling ANalysis) allows assessing immune cell signaling with unprecedented sensitivity and specificity.
Assuntos
Nanosferas , Citometria de Fluxo , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR. METHODS AND RESULTS: Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (n=73, 54%) had a higher risk of transplant failure (P=0.002). Among the remaining patients with complement-independent chronic AMR (n=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (P=0.02). In multivariable analysis, only proteinuria (HR: 7.24; P=0.01) and the presence of missing self (HR: 3.57; P=0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage. CONCLUSIONS: The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.
Assuntos
Aloenxertos/patologia , Ativação do Complemento/fisiologia , Rejeição de Enxerto/etiologia , Antígenos de Histocompatibilidade Classe I/sangue , Transplante de Rim/efeitos adversos , Células Matadoras Naturais/fisiologia , Adulto , Aloenxertos/imunologia , Técnicas de Cultura de Células , Complemento C3d/metabolismo , Células Endoteliais/fisiologia , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Humanos , Células Matadoras Naturais/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemAssuntos
Rejeição de Enxerto/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Transplantes/irrigação sanguínea , Transplantes/imunologia , Animais , Doença Crônica , Rejeição de Enxerto/patologia , Rejeição de Enxerto/terapia , Humanos , Ativação Linfocitária/fisiologia , Transplante de Órgãos/efeitos adversos , Tolerância a Antígenos Próprios/fisiologiaRESUMO
BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.
Assuntos
Rejeição de Enxerto/etiologia , Antígenos HLA/imunologia , Isoanticorpos/sangue , Transplante de Rim/efeitos adversos , Transcrição Gênica , Adulto , Idoso , Feminino , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos , Transplante HomólogoRESUMO
Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of ß2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/métodos , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Receptores KIR/imunologia , Animais , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/patologia , Humanos , Células K562 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Doadores de Tecidos , Transplante Homólogo , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia , Microglobulina beta-2/metabolismoRESUMO
Clinical outcome in antibody-mediated rejection (AMR) shows high inter-individual heterogeneity. Sialylation status of the Fc fragment of IgGs is variable, which could modulate their ability to bind to C1q and/or Fc receptors. In this translational study, we evaluated whether DSA sialylation influence AMR outcomes. Among 938 kidney transplant recipients for whom a graft biopsy was performed between 2004 and 2012 at Lyon University Hospitals, 69 fulfilled the diagnosis criteria for AMR and were enrolled. Sera banked at the time of the biopsy were screened for the presence of DSA by Luminex. The sialylation status of total IgG and DSA was quantified using Sambucus nigra agglutinin-based chromatography. All patients had similar levels of sialylation of serum IgGs (~2%). In contrast, the proportion of sialylated DSA were highly variable (median = 9%; range = 0-100%), allowing to distribute the patients in two groups: high DSA sialylation (n = 44; 64%) and low DSA sialylation (n = 25; 36%). The two groups differed neither on the intensity of rejection lesions (C4d, ptc, and g; p > 0.05) nor on graft survival rates (Log rank test, p = 0.99). in vitro models confirmed the lack of impact of Fc sialylation on the ability of a monoclonal antibody to trigger classical complement cascade and activate NK cells. We conclude that DSA sialylation status is highly variable but has not impact on DSA pathogenicity and AMR outcome.
Assuntos
Rejeição de Enxerto/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Isoanticorpos/imunologia , Ácido N-Acetilneuramínico/imunologia , Doadores de Tecidos , Adulto , Ativação do Complemento , Feminino , Humanos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.
Assuntos
Anticorpos/imunologia , Rejeição de Enxerto/diagnóstico , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Células Matadoras Naturais/imunologia , Adulto , Idoso , Aloenxertos/citologia , Aloenxertos/imunologia , Aloenxertos/patologia , Biomarcadores/análise , Biópsia , Estudos de Casos e Controles , Biologia Computacional , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Rim/citologia , Rim/imunologia , Rim/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Prognóstico , Resultado do Tratamento , Adulto JovemRESUMO
Antibody-mediated rejection is currently the leading cause of transplant failure. Prevailing dogma predicts that B cells differentiate into anti-donor-specific antibody (DSA)-producing plasma cells only with the help of CD4+ T cells. Yet, previous studies have shown that dependence on helper T cells decreases when high amounts of protein antigen are recruited to the spleen, two conditions potentially met by organ transplantation. This could explain why a significant proportion of transplant recipients develop DSA despite therapeutic immunosuppression. Using murine models, we confirmed that heart transplantation, but not skin grafting, is associated with accumulation of a high quantity of alloantigens in recipients' spleen. Nevertheless, neither naive nor memory DSA responses could be observed after transplantation of an allogeneic heart into recipients genetically deficient for CD4+ T cells. These findings suggest that DSA generation rather result from insufficient blockade of the helper function of CD4+ T cells by therapeutic immunosuppression. To test this second theory, different subsets of circulating T cells: CD8+, CD4+, and T follicular helper [CD4+CXCDR5+, T follicular helper cells (Tfh)], were analyzed in 9 healthy controls and 22 renal recipients. In line with our hypothesis, we observed that triple maintenance immunosuppression (CNI + MMF + steroids) efficiently blocked activation-induced upregulation of CD25 on CD8+, but not on CD4+ T cells. Although the level of expression of CD40L and ICOS was lower on activated Tfh of immunosuppressed patients, the percentage of CD40L-expressing Tfh was the same than control patients, as was Tfh production of IL21. Induction therapy with antithymocyte globulin (ATG) resulted in prolonged depletion of Tfh and reduction of CD4+ T cells number with depleting monoclonal antibody in murine model resulted in exponential decrease in DSA titers. Furthermore, induction with ATG also had long-term beneficial influence on Tfh function after immune reconstitution. We conclude that CD4+ T cell help is mandatory for naive and memory DSA responses, making Tfh cells attractive targets for improving the prevention of DSA generation and to prolong allograft survival. Waiting for innovative treatments to be translated into the clinical field ATG induction seems to currently offer the best clinical prospect to achieve this goal.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim , Transplante de Pele , Subpopulações de Linfócitos T/imunologia , Animais , Formação de Anticorpos , Soro Antilinfocitário/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Antígeno HLA-A2/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Memória Imunológica , Terapia de Imunossupressão , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
The generation of antibodies against protein antigens (such as donor-specific HLA molecules) requires that T follicular helper cells (Tfh) provide help to B cells. Immunosuppressive (IS) armamentarium prevents T cell activation, yet a significant proportion of renal transplant patients develop donor-specific antibodies (DSA), which suggests that IS drugs do not efficiently block T follicular helper cells. To test this hypothesis, the number of circulating Tfh, their polarization profile, and ability to up-regulate (i) the co-stimulatory molecules CD40L and ICOS, and (ii) the activation marker CD25, following in vitro stimulation in presence of IS drugs, were compared between 36 renal transplant patients (6-72 months post transplantation) and nine healthy controls. IS drugs reduced the number of Tfh1 and 2 but had little impact on Tfh17, which was the dominant subset in transplant patients. Although, IS drugs decreased activation-induced expression of co-stimulatory molecules by Tfh, the impact was highly variable between individuals. Furthermore, 20% of transplant patients displayed normal expression of CD25 on Tfh following in vitro stimulation (i.e., "residual activatability"). To test whether residual activatability of Tfh correlates with antibody response against thymo-dependent antigens we took advantage of the 2015 influenza vaccination campaign, which provided a normalized setting for antigenic stimulation. In line with our hypothesis, responders to influenza vaccine exhibited significantly higher percentage of CD25-expressing Tfh17 after in vitro stimulation. A results that was confirmed retrospectively in nine transplanted patients at the time of first DSA detection. We concluded that "residual activatability" of Tfh17 might be used as a non-invasive biomarker to identify transplant patients at higher risk to develop DSA under immunosuppression. If validated in larger studies, this assay might help optimizing the prevention of DSA through personalized adaptation of immunosuppressive regimen.
Assuntos
Antígenos/imunologia , Imunidade Humoral , Terapia de Imunossupressão , Ativação Linfocitária/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Adulto , Idoso , Formação de Anticorpos/efeitos dos fármacos , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Curva ROCRESUMO
Humoral rejection is the most common cause of solid organ transplant failure. Here, we evaluated a cohort of 49 patients who were successfully grafted with allogenic islets and determined that the appearance of donor-specific anti-HLA antibodies (DSAs) did not accelerate the rate of islet graft attrition, suggesting resistance to humoral rejection. Murine DSAs bound to allogeneic targets expressed by islet cells and induced their destruction in vitro; however, passive transfer of the same DSAs did not affect islet graft survival in murine models. Live imaging revealed that DSAs were sequestrated in the circulation of the recipients and failed to reach the endocrine cells of grafted islets. We used murine heart transplantation models to confirm that endothelial cells were the only accessible targets for DSAs, which induced the development of typical microvascular lesions in allogeneic transplants. In contrast, the vasculature of DSA-exposed allogeneic islet grafts was devoid of lesions because sprouting of recipient capillaries reestablished blood flow in grafted islets. Thus, we conclude that endothelial chimerism combined with vascular sequestration of DSAs protects islet grafts from humoral rejection. The reduced immunoglobulin concentrations in the interstitial tissue, confirmed in patients, may have important implications for biotherapies such as vaccines and monoclonal antibodies.
Assuntos
Endotélio Vascular/metabolismo , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas , Isoanticorpos/metabolismo , Quimeras de Transplante/metabolismo , Aloenxertos , Animais , Endotélio Vascular/patologia , Feminino , Rejeição de Enxerto/patologia , Humanos , Masculino , CamundongosRESUMO
NK cell education is the process through which chronic engagement of inhibitory NK cell receptors by self MHC-I molecules preserves cellular responsiveness. The molecular mechanisms responsible for NK cell education remain unclear. Here, we show that mouse NK cell education is associated with a higher basal activity of the mTOR/Akt pathway, commensurate to the number of educating receptors. This higher activity was dependent on the SHP-1 phosphatase and essential for the improved responsiveness of reactive NK cells. Upon stimulation, the mTOR/Akt pathway amplified signaling through activating NK cell receptors by enhancing calcium flux and LFA-1 integrin activation. Pharmacological inhibition of mTOR resulted in a proportional decrease in NK cell reactivity. Reciprocally, acute cytokine stimulation restored reactivity of hyporesponsive NK cells through mTOR activation. These results demonstrate that mTOR acts as a molecular rheostat of NK cell reactivity controlled by educating receptors and uncover how cytokine stimulation overcomes NK cell education.
Assuntos
Células Matadoras Naturais/imunologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Antibody-mediated rejection is associated with heterogeneous kidney allograft outcomes. Accurate evaluation of risk for graft loss at time of diagnosis is necessary to offer personalized treatment. In contrast with serological and molecular assessment, morpho-histological evaluation of antibody-mediated rejection lesions has not significantly evolved. This relies on Banff classifications designed to be of diagnostic discriminatory power rather than prognostic and face quantitative and qualitative limitations. Here we developed a method of Computer-assisted Analysis of Graft Inflammation (CAGI) to improve the classification of allograft inflammation. Digitization of immunostained biopsy sections, image processing and algorithm-driven analysis allowed quantification of macrophages, T cells, B cells, and granulocytes per unit surface of interstitium, capillaries or glomeruli. CAGI was performed on biopsy specimens of 52 patients with extensively phenotyped antibody-mediated rejection. Macrophage numbers in capillaries and interstitium, but not Banff scores or the amount of other immune cell subsets, correlated with donor-specific antibody (DSA) mean fluorescence intensity and DSA-C3d status. The quantity of macrophages in the interstitium and DSA-C3d status were the only independent predictors for significant allograft loss at the time of antibody-mediated rejection diagnosis (hazard ratio 3.71 and 2.34, respectively). A significant strategy integrating the DSA-C3d assay and the quantification of interstitial macrophages allowed identification of three groups with distinct renal prognosis: DSA-C3d-, DSA-C3d+/macrophages-low and DSAC3d+/macrophages-high. Thus, CAGI brings a missing piece to the antibody-mediated rejection puzzle by identifying morpho-histological processes that bridge in vitro parameters of DSA pathogenicity and graft loss. Hence, this approach could be useful in future integrated strategies of risk evaluation.