RESUMEN
High HLA sensitization (HS) limits access to compatible transplantation. New CD38-targeting agents have shown to reduce anti-HLA antibodies, although with important inter-patient variability thus, pre-treatment identification of responder and non-responder patients is needed for treatment decision-making. We analyzed 26 HS patients from two desensitization trials using anti-CD38 mAb. Hierarchical clustering identified three serological responder groups: high, low, and non-responders. Spectral flow-cytometry and functional HLA-specific memory B-cell (mBc) assessment was first conducted on PBMC and bone marrow samples from 16 patients treated with isatuximab (NCT04294459). Isatuximab effectively depleted bone marrow plasma cells, peripheral CD38-expressing plasmablasts, plasma cells, transitional B cells, and class-switch mBc, ultimately reducing frequencies of HLA-specific IgG-producing mBc. Multidimensional spectral flow cytometry with PLS-DA analysis revealed that pre-treatment abundance of specific circulating mBcs phenotypes, especially CD38neg class-switch mBc, accurately distinguished between high serological responders and low or non-responders (AUC 0.958, 0.860-1.000, p=0.009), who also displayed significantly lower frequencies of HLA-specific IgG-producing mBc (p<0.0001). This phenotypical mBc signature predicting response to therapy was validated in an external HS patient cohort (n=10) receiving daratumumab (NCT04204980). This study identifies critical circulating mBc subset phenotypes that distinguish HS patients with successful serological response to CD38-targeting desensitization therapies, potentially guiding treatment decision-making.
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HLA-DRB1*07:143N and HLA-DRB1*07:144 differ from DRB1*07:01:01:01 by single mismatches in exons 3 and 2 respectively.
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Cadenas HLA-DRB1 , Humanos , Cadenas HLA-DRB1/genética , Secuencia de Bases , Alineación de Secuencia , Alelos , Exones/genéticaRESUMEN
The novel HLA-C*05:278N allele has a premature stop codon in exon 4.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Exones/genética , Codón sin Sentido , Análisis de Secuencia de ADNRESUMEN
The novel HLA-DPB1*1455:01N allele differs from DPB1*05:01:01:01 by one amino acid deletion in exon 3.
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Donantes de Tejidos , Humanos , Secuencia de Bases , Alelos , Cadenas beta de HLA-DP/genéticaRESUMEN
Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer ). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.
Asunto(s)
Trasplante de Riñón , Humanos , Alelos , Prueba de Histocompatibilidad , Antígenos de Histocompatibilidad Clase I , Donantes de Tejidos , Antígenos HLA , Isoanticuerpos , Rechazo de InjertoRESUMEN
The novel HLA-C*08:258 and -C*12:374 alleles differ from HLA-C*08:02:01:01 and -C*12:03:01:01 by one nucleotide substitution.
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Antígenos HLA-C , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Antígenos HLA-C/genética , Alelos , Prueba de Histocompatibilidad/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Genes MHC Clase I , Análisis de Secuencia de ADN/métodosRESUMEN
The new HLA-DRB1*15:213 allele results from one nucleotide substitution in exon 3 of HLA-DRB1*15:02:01.
Asunto(s)
Trasplante de Corazón , Humanos , Cadenas HLA-DRB1/genética , Alelos , Secuencia de Bases , ExonesRESUMEN
The novel allele B*08:304 differs from B*08:01:01:01 by one nucleotide substitution in exon 2.
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Antígenos HLA-B , Nucleótidos , Humanos , Secuencia de Bases , Alelos , Exones/genética , Antígenos HLA-B/genética , Análisis de Secuencia de ADNRESUMEN
The novel HLA-C*03:618N allele has one change in exon 1 leading to a premature stop codon.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Exones/genética , Codón sin Sentido , Análisis de Secuencia de ADNRESUMEN
The novel HLA-A*01:420N allele has two changes in exon 4 leading to premature stop codon.
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Codón sin Sentido , Antígenos HLA-A , Humanos , Secuencia de Bases , Alelos , Exones/genética , Antígenos HLA-A/genética , Análisis de Secuencia de ADNRESUMEN
The novel allele HLA-C*17:64Q differs from HLA-C*17:01:01:02 by insertion of a Lysine in exon 2.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Análisis de Secuencia de ADN/métodos , Exones/genéticaRESUMEN
Heart transplantation in cardiac amyloidosis (CA) patients is possible and generally considered for transplantation if other organs are not affected. In this study, we aimed to describe and assess outcome in patients following heart transplantations at our CA referral center. Methods: We assessed all CA patients that had heart transplantations at our center between 2005 and 2018. Patients with New York Heart Association status 3 out of 4, with poor short-term prognosis due to heart failure, despite treatment, and without multiple myeloma, systemic disease, severe neuropathic/digestive comorbidities, cancer, or worsening infections were eligible for transplantation. Hearts were transplanted by bicaval technique. Standard induction and immunosuppressive therapies were used. Survival outcome of CA patients after transplantation was compared with recipients with nonamyloid pathologies in France. Results: Between 2005 and 2018, 23 CA patients had heart transplants: 17 (74%) had light chain (light chain amyloidosis [AL]) and 6 (26%) had hereditary transthyretin (hereditary transthyretin amyloidosis [ATTRv]) CA. Also, 13 (57%) were male, and the mean age at diagnosis was 56.5 y (range, 47.7-62.8). Among AL patients, 13 had heart-only and 5 had heart-kidney transplantations. Among ATTRv patients, 1 had heart-only and 5 had heart-liver transplantations. The 1-y survival rate after transplantation was 78%, 70% with AL, and 100% with ATTRv. At 2 y, 74% were alive: 65% with AL and 100% with ATTRv. Conclusion: After heart transplantation, French CA and nonamyloid patients have similar survival outcomes. Among CA patients, ATTRv patients have better prognosis than those with AL, possibly due to the combined heart-liver transplantation. Selected CA patients should be considered for heart transplantations.
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Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.
Asunto(s)
Antígenos HLA , Isoanticuerpos , Alelos , Proteínas del Sistema Complemento , Ácido Edético , Colorantes Fluorescentes , Prueba de Histocompatibilidad/métodos , Humanos , FicoeritrinaRESUMEN
Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody-antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to "shared" antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.
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Biología Computacional/métodos , Antígenos HLA-A/inmunología , Antígenos HLA-B/inmunología , Antígenos HLA-C/inmunología , Trasplante de Órganos , Adulto , Anciano , Estudios de Cohortes , Reacciones Cruzadas , Epítopos , Humanos , Isoanticuerpos/sangre , Aprendizaje Automático , Persona de Mediana Edad , Análisis de Componente Principal , Sistema de Registros , Inmunología del TrasplanteRESUMEN
Background: Sensitized patients, i.e. recipients with preformed donor-specific HLA antibodies (pfDSA), are at high-risk of developing antibody-mediated rejections (AMR) and dying after heart transplantation (HTx). Perioperative desensitization procedures are associated with better outcomes but can cause sensitization, which may influence their efficacy. Methods: In sensitized patients (pfDSA>1000 mean immunofluorescence (MFI) units), we assessed the effect of perioperative desensitization by comparing treated patients to a historical control cohort. Multivariable survival analyses were performed on the time to main outcome, a composite of death and biopsy-proven AMR with 5-year follow-up. Results: The study included 68 patients: 31 control and 37 treated patients. There was no difference in preoperative variables between the two groups, including cumulative pfDSA [4026 (1788;8725) vs 4560 (3162;13392) MFI units, p=0.28]. The cause of sensitization was pregnancy in 24/68, 35.3%, transfusion in 61/68, 89.7%, and previous HTx in 4/68, 5.9% patients. Multivariable analysis yielded significant protective association between desensitization and events (adjusted (adj.) hazard ratio (HR)=0.44 (95% confidence interval (95CI)=0.25-0.79), p=0.006) and deleterious association between cumulative pfDSA and events [per 1000-MFI increase, adj.HR=1.028 (1.002-1.053), p=0.031]. There was a sex-difference in the efficacy of desensitization: in men (n=35), the benefit was significant [unadj.HR=0.33 (95CI=0.14-0.78); p=0.01], but not in women (n=33) [unadj.HR=0.52 (0.23-1.17), p=0.11]. In terms of the number of patients treated, in men, 2.1 of patients that were treated prevented 1 event, while in women, 3.1 required treatment to prevent 1 event. Conclusion: Perioperative desensitization was associated with fewer AMR and deaths after HTx, and efficacy was more pronounced in men than women.
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Desensibilización Inmunológica , Trasplante de Corazón , Atención Perioperativa , Adulto , Biomarcadores , Biopsia , Desensibilización Inmunológica/métodos , Femenino , Estudios de Seguimiento , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Rechazo de Injerto/terapia , Antígenos HLA/inmunología , Trasplante de Corazón/efectos adversos , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Atención Perioperativa/métodos , Embarazo , Pronóstico , Estudios Retrospectivos , Factores Sexuales , Resultado del TratamientoRESUMEN
The 2007 Banff working classification of skin-containing Tissue Allograft Pathology addressed only acute T cell-mediated rejection in skin. We report the longitudinal long-term histological follow-up of six face transplant recipients, focusing on chronic and mucosal rejection. We identified three patterns suggestive of chronic rejection (lichen planus-like, vitiligo-like and scleroderma-like). Four patients presented lichen planus-like and vitiligo-like chronic rejection at 52 ± 17 months posttransplant with severe concomitant acute T cell-mediated rejection. After lichen planus-like rejection, two patients developed scleroderma-like alterations. Graft vasculopathy with C4d deposits and de novo DSA led to subsequent graft loss in one patient. Chronic active rejection was frequent and similar patterns were noted in mucosae. Concordance between 124 paired skin and mucosal biopsies acute rejection grades was low (κ = 0.2, p = .005) but most grade 0/I mucosal rejections were associated with grade 0/I skin rejections. We defined discordant (grade≥II mucosal rejection and grade 0/I skin rejection) (n = 55 [70%]) and concordant (grade≥II rejection in both biopsies) groups. Mucosal biopsies of the discordant group displayed lower intra-epithelial GranzymeB/FoxP3 ratios suggesting a less aggressive phenotype (p = .08). The grading system for acute rejection in mucosa may require phenotyping. Whether discordant infiltrates reflect a latent allo-immune reaction leading to chronic rejection remains an open question.
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Trasplante Facial , Trasplante de Riñón , Biopsia , Estudios de Seguimiento , Rechazo de Injerto/etiología , Humanos , Membrana MucosaRESUMEN
Allele specific antibody response against the polymorphic system of HLA is the allogeneic response marker determining the immunological risk for graft acceptance before and after organ transplantation and therefore routinely studied during the patient's workup. Experimentally, bead bound antigen- antibody reactions are detected using a special multicolor flow cytometer (Luminex). Routinely for each sample, antibody responses against 96 different HLA antigen groups are measured simultaneously and a 96-dimensional immune response vector is created. Under a common experimental protocol, using unsupervised clustering algorithms, we analyzed these immune intensity vectors of anti HLA class II responses from a dataset of 1,748 patients before or after renal transplantation residing in a single country. Each patient contributes only one serum sample in the analysis. A population view of linear correlations of hierarchically ordered fluorescence intensities reveals patterns in human immune responses with striking similarities with the previously described CREGs but also brings new information on the antigenic properties of class II HLA molecules. The same analysis affirms that "public" anti-DP antigenic responses are not correlated to anti DR and anti DQ responses which tend to cluster together. Principal Component Analysis (PCA) projections also demonstrate ordering patterns clearly differentiating anti DP responses from anti DR and DQ on several orthogonal planes. We conclude that a computer vision of human alloresponse by use of several dimensionality reduction algorithms rediscovers proven patterns of immune reactivity without any a priori assumption and might prove helpful for a more accurate definition of public immunogenic antigenic structures of HLA molecules. Furthermore, the use of Eigen decomposition on the Immune Response generates new hypotheses that may guide the design of more effective patient monitoring tests.
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Citometría de Flujo , Antígenos HLA/inmunología , Prueba de Histocompatibilidad , Histocompatibilidad , Isoanticuerpos/sangre , Isoantígenos/inmunología , Trasplante de Riñón , Aprendizaje Automático , Reconocimiento de Normas Patrones Automatizadas , Adulto , Análisis por Conglomerados , Femenino , Rechazo de Injerto/sangre , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Humanos , Inmunosupresores/uso terapéutico , Trasplante de Riñón/efectos adversos , Masculino , Persona de Mediana Edad , Análisis de Componente Principal , Resultado del TratamientoRESUMEN
After heart transplant, adding everolimus (EVL) to standard immunosuppressive regimen mostly relies on converting calcineurin inhibitors (CNIs) into EVL. The aim of this study was to describe the effects of combining low-dose EVL and CNIs in maintenance immunosuppression regimen (quadritherapy) and compare it with standard tritherapy associating standard-dose CNIs, mycophenolate mofetil, and corticosteroids. In the 3-year registry cohort of heart transplanted patients, those who received quadritherapy were compared with those who received tritherapy. EVL was added after 3 months posttransplant. Three analyses were performed to control for confounders: propensity score matching, multivariable survival, and inverse probability score weighting analyses. Among 213 patients who were included (75 with quadritherapy), propensity score matching selected 64 unique pairs of patients with similar characteristics. In the matched cohort (n = 128), quadritherapy was associated with fewer deaths (3 [4.7%] vs 17 [21.9%], P = .007) and biopsy-proven acute rejections (15 [23.4%] vs 31 [48.4%], P = .002). These results were confirmed in the overall cohort (n = 213), after multivariable and inverse probability score weighting analyses. Renal function and donor-specific HLA-antibodies remained similar in both groups. Low-dose combination quadritherapy was associated with fewer deaths and rejections, compared with standard immunosuppression tritherapy.
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Trasplante de Corazón , Inmunosupresores , Estudios de Cohortes , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Humanos , Inmunosupresores/uso terapéutico , Ácido Micofenólico/uso terapéutico , Puntaje de PropensiónRESUMEN
BACKGROUND: Eukaryote initiation factor 2 subunit ß (eIF2ß) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2ß contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2ß was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2ßΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS: eIF2ßΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2ß translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2ßΔ3K did not make this translocation. DISCUSSION: eIF2ß is involved in the protein synthesis process and should act in nuclear processes as well. eIF2ßΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2ßΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2ß polylysine stretch domains are promising targets for this.
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Proliferación Celular/genética , Factor 2B Eucariótico de Iniciación/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Polilisina/genética , Biosíntesis de Proteínas/genética , Eliminación de Secuencia/genética , Apoptosis/genética , Sitios de Unión , Núcleo Celular/metabolismo , Supervivencia Celular/genética , Citoplasma/metabolismo , Factor 2B Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Terapia Molecular Dirigida/métodos , Mutagénesis Sitio-Dirigida , Neoplasias/terapia , Unión Proteica , Transporte de Proteínas , ARN Mensajero/metabolismoRESUMEN
Current strategies to improve graft outcome following kidney transplantation consider information at the human leukocyte antigen (HLA) loci. Cell surface antigens, in addition to HLA, may serve as the stimuli as well as the targets for the anti-allograft immune response and influence long-term graft outcomes. We therefore performed exome sequencing of DNA from kidney graft recipients and their living donors and estimated all possible cell surface antigens mismatches for a given donor/recipient pair by computing the number of amino acid mismatches in trans-membrane proteins. We designated this tally as the allogenomics mismatch score (AMS). We examined the association between the AMS and post-transplant estimated glomerular filtration rate (eGFR) using mixed models, considering transplants from three independent cohorts (a total of 53 donor-recipient pairs, 106 exomes, and 239 eGFR measurements). We found that the AMS has a significant effect on eGFR (mixed model, effect size across the entire range of the score: -19.4 [-37.7, -1.1], P = 0.0042, χ2 = 8.1919, d.f. = 1) that is independent of the HLA-A, B, DR matching, donor age, and time post-transplantation. The AMS effect is consistent across the three independent cohorts studied and similar to the strong effect size of donor age. Taken together, these results show that the AMS, a novel tool to quantify amino acid mismatches in trans-membrane proteins in individual donor/recipient pair, is a strong, robust predictor of long-term graft function in kidney transplant recipients.