RESUMO
We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR and TCR) sequences from the blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires were associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation, and counterregulation by the co-receptors BTLA, Tim-3, PD-1, TIGIT, and CD73. Tfh, Th17-like, and nonconventional (but not classical antiviral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.
Assuntos
Infecções por Coronavirus , Citocinas , Sequenciamento de Nucleotídeos em Larga Escala , Pandemias , Pneumonia Viral , Betacoronavirus , COVID-19 , Humanos , Receptores de Antígenos de Linfócitos B/genética , SARS-CoV-2 , Índice de Gravidade de DoençaRESUMO
The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.
Assuntos
Aprendizado de Máquina , Receptores de Antígenos de Linfócitos B , Humanos , Receptores de Antígenos de Linfócitos B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Biologia Computacional/métodos , Linfoma de Células B/genética , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/classificação , AlgoritmosRESUMO
Post-acute sequelae of COVID-19 (PASC) are long-term consequences of SARS-CoV-2 infection that can substantially impair the quality of life. Underlying mechanisms ranging from persistent viruses to innate and adaptive immune dysregulation have been discussed. Here, we profiled the plasma of 181 individuals from the cohort study for digital health research in Germany (DigiHero), including individuals after mild to moderate COVID-19 with or without PASC and uninfected controls. We focused on soluble factors related to monocyte/macrophage biology and on circulating SARS-CoV-2 spike (S1) protein as a potential biomarker for persistent viral reservoirs. At a median time of 8 months after infection, we found pronounced dysregulation in almost all tested soluble factors, including both pro-inflammatory and pro-fibrotic cytokines. These immunological perturbations were remarkably independent of ongoing PASC symptoms per se, but further correlation and regression analyses suggested PASC-specific patterns involving CCL2/MCP-1 and IL-8 that either correlated with sCD162, sCD206/MMR, IFN-α2, IL-17A and IL-33, or IL-18 and IL-23. None of the analyzed factors correlated with the detectability or levels of circulating S1, indicating that this represents an independent subset of patients with PASC. These data confirm prior evidence of immune dysregulation and persistence of viral protein in PASC and illustrate its biological heterogeneity that still awaits correlation with clinically defined PASC subtypes.
Assuntos
Síndrome de COVID-19 Pós-Aguda , Glicoproteína da Espícula de Coronavírus , Humanos , Biomarcadores , Estudos de Coortes , COVID-19/complicações , Progressão da Doença , Síndrome de COVID-19 Pós-Aguda/diagnóstico , Síndrome de COVID-19 Pós-Aguda/metabolismo , Qualidade de Vida , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/sangue , Glicoproteína da Espícula de Coronavírus/química , Macrófagos/metabolismoRESUMO
Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Superantígenos/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Motivos de Aminoácidos , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , COVID-19 , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Enterotoxinas/química , Epitopos de Linfócito T , Humanos , Molécula 1 de Adesão Intercelular/química , Modelos Moleculares , Mutação , Neurotoxinas/química , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Superantígenos/química , Superantígenos/genética , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/patologiaRESUMO
Activating BRAF mutations occurs in 50-60% of malignant melanomas. Although initially treatable, the development of resistance to BRAF-targeted therapies (BRAFi) is a major challenge and limits their efficacy. We have previously shown that the BRAFV600E signaling pathway mediates the expression of EZH2, an epigenetic regulator related to melanoma progression and worse overall survival. Therefore, we wondered whether inhibition of EZH2 would be a way to overcome resistance to vemurafenib. We found that the addition of an EZH2 inhibitor to vemurafenib improved the response of melanoma cells resistant to BRAFi with regard to decreased viability, cell-cycle arrest and increased apoptosis. By next-generation sequencing, we revealed that the combined inhibition of BRAF and EZH2 dramatically suppresses pathways of mitosis and cell cycle. This effect was linked to the downregulation of Polo-kinase 1 (PLK1), a key regulator of cell cycle and proliferation. Subsequently, when we inhibited PLK1, we found decreased cell viability of melanoma cells resistant to BRAFi. When we inhibited both BRAF and PLK1, we achieved an improved response of BRAFi-resistant melanoma cells, which was comparable to the combined inhibition of BRAF and EZH2. These results thus reveal that targeting EZH2 or its downstream targets, such as PLK1, in combination with BRAF inhibitors are potential novel therapeutic options in melanomas with BRAF mutations.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Neoplasias Cutâneas , Humanos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Indóis/farmacologia , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Quinase 1 Polo-LikeRESUMO
BACKGROUND AND AIMS: Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T-cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH. APPROACH AND RESULTS: T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next-generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV-J genes. More importantly, we found strong AIH-specific TRBV-J skewing not attributable to the HLA-DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies. CONCLUSIONS: Patients with AIH show profound and persisting T-cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T-cell clusters shared between patients may mediate liver damage and warrant further study.
Assuntos
Hepacivirus , Hepatite C/imunologia , Hepatite Autoimune/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cirrose Hepática Biliar/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Cadeias HLA-DRB1/genética , Hepatite C/sangue , Hepatite Autoimune/sangue , Hepatite Autoimune/terapia , Humanos , Terapia de Imunossupressão/métodos , Cirrose Hepática Biliar/sangue , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/genética , Recidiva , Adulto JovemRESUMO
The B-cell architecture of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is complex since it is composed of malignant lymphocyte-predominant cells along with a rich B-cell bystander environment. To gain insight into molecular determinants of disease transformation, we studied B-cell evolutionary trajectories in lymphoma tissue from diagnosis to relapse or transformation to non- Hodgkin lymphoma by next-generation sequencing of immunoglobulin heavy chains. Patients with NLPHL that later transformed were older and showed IgD negativity, absence of the characteristic IGHV3/IGHD3/IGHJ6 lymphocyte-predominant rearrangement and high repertoire clonality. We constructed phylogenetic trees within the compartment of the malignant clone to investigate clonal evolution. In all relapsing cases, the lymphocyte-predominant rearrangement was identical at diagnosis and relapse. NLPHL cases with transformation showed more complex trajectories with strong intraclonal diversification. The dominant founder clone in transformations showed clonal evolution if derived from the same cell of origin, or arose from a different cell of origin. Together, our data point to a significant role of antigenic drive in the transformation of NLHPL and identify high B-cell repertoire clonality with dominant intraclonal lymphocyte-predominant cell diversification as a hallmark of transformation. Sequencing of initial paraffin-embedded tissue may therefore be applied diagnostically to identify NLPHL cases with high risk of transformation.
Assuntos
Doença de Hodgkin , Diagnóstico Diferencial , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/genética , Humanos , Linfócitos , Recidiva Local de Neoplasia , FilogeniaRESUMO
Coeliac disease (CD) is a clinically heterogeneous autoimmune disease with variable presentation and progression triggered by gluten intake. Molecular or genetic factors contribute to disease heterogeneity, but the reasons for different outcomes are poorly understood. Transcriptome studies of tissue biopsies from CD patients are scarce. Here, we present a high-resolution analysis of the transcriptomes extracted from duodenal biopsies of 24 children and adolescents with active CD and 21 individuals without CD but with intestinal afflictions as controls. The transcriptomes of CD patients divide into three groups-a mixed group presenting the control cases, and CD-low and CD-high groups referring to lower and higher levels of CD severity. Persistence of symptoms was weakly associated with subgroup, but the highest marsh stages were present in subgroup CD-high, together with the highest cell cycle rates as an indicator of virtually complete villous atrophy. Considerable variation in inflammation-level between subgroups was further deciphered into immune cell types using cell type de-convolution. Self-organizing maps portrayal was applied to provide high-resolution landscapes of the CD-transcriptome. We find asymmetric patterns of miRNA and long non-coding RNA and discuss the effect of epigenetic regulation. Expression of genes involved in interferon gamma signaling represent suitable markers to distinguish CD from non-CD cases. Multiple pathways overlay in CD biopsies in different ways, giving rise to heterogeneous transcriptional patterns, which potentially provide information about etiology and the course of the disease.
Assuntos
Doença Celíaca/genética , Adolescente , Estudos de Casos e Controles , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Criança , Pré-Escolar , Epigênese Genética , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Marcadores Genéticos , Humanos , Lactente , Interferon gama/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Aprendizado de Máquina , Masculino , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Índice de Gravidade de Doença , TranscriptomaRESUMO
Cerebral gliomas of World Health Organization (WHO) grade II and III represent a major challenge in terms of histological classification and clinical management. Here, we asked whether large-scale genomic and transcriptomic profiling improves the definition of prognostically distinct entities. We performed microarray-based genome- and transcriptome-wide analyses of primary tumor samples from a prospective German Glioma Network cohort of 137 patients with cerebral gliomas, including 61 WHO grade II and 76 WHO grade III tumors. Integrative bioinformatic analyses were employed to define molecular subgroups, which were then related to histology, molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q co-deletion and telomerase reverse transcriptase (TERT) promoter mutations, and patient outcome. Genomic profiling identified five distinct glioma groups, including three IDH1/2 mutant and two IDH1/2 wild-type groups. Expression profiling revealed evidence for eight transcriptionally different groups (five IDH1/2 mutant, three IDH1/2 wild type), which were only partially linked to the genomic groups. Correlation of DNA-based molecular stratification with clinical outcome allowed to define three major prognostic groups with characteristic genomic aberrations. The best prognosis was found in patients with IDH1/2 mutant and 1p/19q co-deleted tumors. Patients with IDH1/2 wild-type gliomas and glioblastoma-like genomic alterations, including gain on chromosome arm 7q (+7q), loss on chromosome arm 10q (-10q), TERT promoter mutation and oncogene amplification, displayed the worst outcome. Intermediate survival was seen in patients with IDH1/2 mutant, but 1p/19q intact, mostly astrocytic gliomas, and in patients with IDH1/2 wild-type gliomas lacking the +7q/-10q genotype and TERT promoter mutation. This molecular subgrouping stratified patients into prognostically distinct groups better than histological classification. Addition of gene expression data to this genomic classifier did not further improve prognostic stratification. In summary, DNA-based molecular profiling of WHO grade II and III gliomas distinguishes biologically distinct tumor groups and provides prognostically relevant information beyond histological classification as well as IDH1/2 mutation and 1p/19q co-deletion status.
Assuntos
Neoplasias Encefálicas/genética , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Glioma/genética , Isocitrato Desidrogenase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glioma/classificação , Glioma/patologia , Glioma/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores/métodos , Prognóstico , Regiões Promotoras Genéticas , Deleção de Sequência , Organização Mundial da Saúde , Adulto JovemRESUMO
The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. To elucidate the molecular aberrations in glioblastomas of long-term survivors, we performed genome- and/or transcriptome-wide molecular profiling of glioblastoma samples from 94 patients, including 28 long-term survivors with >36 months overall survival (OS), 20 short-term survivors with <12 months OS and 46 patients with intermediate OS. Integrative bioinformatic analyses were used to characterize molecular aberrations in the distinct survival groups considering established molecular markers such as isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations, and O(6) -methylguanine DNA methyltransferase (MGMT) promoter methylation. Patients with long-term survival were younger and more often had IDH1/2-mutant and MGMT-methylated tumors. Gene expression profiling revealed over-representation of a distinct (proneural-like) expression signature in long-term survivors that was linked to IDH1/2 mutation. However, IDH1/2-wildtype glioblastomas from long-term survivors did not show distinct gene expression profiles and included proneural, classical and mesenchymal glioblastoma subtypes. Genomic imbalances also differed between IDH1/2-mutant and IDH1/2-wildtype tumors, but not between survival groups of IDH1/2-wildtype patients. Thus, our data support an important role for MGMT promoter methylation and IDH1/2 mutation in glioblastoma long-term survival and corroborate the association of IDH1/2 mutation with distinct genomic and transcriptional profiles. Importantly, however, IDH1/2-wildtype glioblastomas in our cohort of long-term survivors lacked distinctive DNA copy number changes and gene expression signatures, indicating that other factors might have been responsible for long survival in this particular subgroup of patients.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Transcriptoma , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Genoma Humano , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Humanos , Isocitrato Desidrogenase/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Estudos Prospectivos , Sobreviventes , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Chronic HCV infection leads to a complex interplay with adaptive immune cells that may result in B cell dyscrasias like cryoglobulinemia or lymphoma. While direct-acting antiviral therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear. METHODS: We sequenced B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection and patients with HCV with a sustained virological response (SVR) after direct-acting antiviral therapy. This data set was mined for highly neutralizing HCV antibodies and compared to a diffuse large B cell lymphoma data set. The TKO model was used to test the signaling strength of selected B-BCRs in vitro. Single-cell RNA sequencing of chronic HCV and HCV SVR samples was performed to analyze the transcriptome of B cells with HCV-neutralizing antigen receptors. RESULTS: We identified a B cell fingerprint with high richness and somatic hypermutation in patients with chronic HCV and SVR. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 networks. In addition, we observed that IGHV1-69 CDR1 and FR3 mutations characterizing highly neutralizing HCV antibodies corresponded to recurrent point mutations found in clonotypic BCRs of high-grade lymphomas. These BCRs did not show autonomous signaling but a lower activation threshold in an in vitro cell model for the assessment of BCR signaling strength. Single-cell RNA sequencing revealed that B cells carrying these point mutations showed a persisting oncogenic transcriptome signature with dysregulation in signaling nodes such as CARD11, MALT1, RelB, MAPK, and NFAT. CONCLUSIONS: We provide evidence that lymphoma-like cells derive from the anti-HCV immune response. In many patients, these cells persist for years after SVR and can be interpreted as a mechanistic basis for HCV-related B cell dyscrasias and increased lymphoma risk even beyond viral elimination.
Assuntos
Linfócitos B , Hepacivirus , Hepatite C Crônica , Receptores de Antígenos de Linfócitos B , Transcriptoma , Humanos , Hepatite C Crônica/imunologia , Hepatite C Crônica/genética , Hepatite C Crônica/complicações , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos B/imunologia , Hepacivirus/imunologia , Hepacivirus/genética , Resposta Viral Sustentada , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/virologia , Anticorpos Neutralizantes/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Masculino , Antivirais/uso terapêutico , Mutação , Feminino , Pessoa de Meia-IdadeRESUMO
The rapid development of safe and effective vaccines helped to prevent severe disease courses after SARS-CoV-2 infection and to mitigate the progression of the COVID-19 pandemic. While there is evidence that vaccination may reduce the risk of developing post-COVID-19 conditions (PCC), this effect may depend on the viral variant. Therapeutic effects of post-infection vaccination have been discussed but the data for individuals with PCC remains inconclusive. In addition, extremely rare side effects after SARS-CoV-2 vaccination may resemble the heterogeneous PCC phenotype. Here, we analyze the plasma levels of 25 cytokines and SARS-CoV-2 directed antibodies in 540 individuals with or without PCC relative to one or two mRNA-based COVID-19 vaccinations as well as in 20 uninfected individuals one month after their initial mRNA-based COVID-19 vaccination. While none of the SARS-CoV-2 naïve individuals reported any persisting sequelae or exhibited PCC-like dysregulation of plasma cytokines, we detected lower levels of IL-1ß and IL-18 in patients with ongoing PCC who received one or two vaccinations at a median of six months after infection as compared to unvaccinated PCC patients. This reduction correlated with less frequent reporting of persisting gastrointestinal symptoms. These data suggest that post-infection vaccination in patients with PCC might be beneficial in a subgroup of individuals displaying gastrointestinal symptoms.
RESUMO
Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 34 patients with HA20, we show that heterozygous TNFAIP3 loss skews immune repertoires toward lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas. This skewing was mediated by a feed-forward tumor necrosis factor (TNF)/A20/nuclear factor κB (NF-κB) loop that shaped pre-lymphoma transcriptome signatures in clonally expanded B (CD81, BACH2, and NEAT1) or T (GATA3, TOX, and PDCD1) cells. The skewing was reversed by anti-TNF treatment but could also progress to overt lymphoma. Analysis of conditional TNFAIP3 knock-out mice reproduced the wiring of the TNF/A20/NF-κB signaling axis with permissive antigen receptors and suggested a distinct regulation in B and T cells. Together, patients with the genetic disorder HA20 provide an exceptional window into A20/TNF/NF-κB-mediated control of immune homeostasis and early steps of lymphomagenesis that remain clinically unrecognized.
Assuntos
Haploinsuficiência , Homeostase , NF-kappa B , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Humanos , Camundongos , NF-kappa B/metabolismo , Camundongos Knockout , Feminino , Masculino , Transdução de Sinais , Pessoa de Meia-Idade , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Adulto , Fator de Necrose Tumoral alfa/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfoma/genética , Linfoma/imunologia , Linfoma/patologiaRESUMO
BACKGROUND: Variant syndromes of autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) share diagnostic features of both entities, but their immunological underpinnings remain largely unexplored. METHODS: We performed blood profiling of 23 soluble immune markers and immunogenetics in a cohort of 88 patients with autoimmune liver diseases (29 typical AIH, 31 typical PBC and 28 with clinically PBC/AIH variant syndromes). The association with demographical, serological and clinical features was analyzed. RESULTS: While T and B cell receptor repertoires were highly skewed in variant syndromes compared to healthy controls, these biases were not sufficiently discriminated within the spectrum of autoimmune liver diseases. High circulating checkpoint molecules sCD25, sLAG-3, sCD86 and sTim-3 discriminated AIH from PBC on top of classical parameters such as transaminases and immunoglobulin levels. In addition, a second cluster of correlated soluble immune factors encompassing essentially TNF, IFNγ, IL12p70, sCTLA-4, sPD-1 and sPD-L1 appeared characteristic of AIH. Cases with complete biochemical responses to treatment generally showed a lower level of dysregulation. Unsupervised hierarchical clustering of classical and variant syndromes identified two pathological immunotypes consisting predominantly of either AIH or PBC cases. Variant syndromes did not form a separate group, but clustered together with either classical AIH or PBC. Clinically, patient with AIH-like variant syndromes were less likely to be able discontinue immunosuppressive treatment. CONCLUSIONS: Our analyses suggest that variants of immune mediated liver diseases may represent an immunological spectrum from PBC to AIH-like disease reflected by their pattern of soluble immune checkpoint molecules rather than separate entities.
Assuntos
Hepatite Autoimune , Cirrose Hepática Biliar , Hepatopatias , Humanos , Hepatite Autoimune/diagnóstico , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/tratamento farmacológico , Imunossupressores/uso terapêutico , BiomarcadoresRESUMO
Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N = 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished in vitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid in vitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Humanos , Medula Óssea , Antígeno CD146/metabolismo , Células da Medula Óssea , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Mesenquimais/metabolismo , Proliferação de CélulasRESUMO
T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.
RESUMO
Cancer/testis antigens (CTA) are a heterogeneous group of antigens that are expressed preferentially in tumor cells and testis. Based on this definition the human membrane-associated phospholipase A1 beta (lipase family member I, LIPI) has been identified as CTA. The high homology of LIPI and the membrane-associated phospholipase A1 alpha (lipase family member H, LIPH) suggests that both genes are derived from a common ancestor by gene duplication. In contrast to human LIPI, human LIPH is expressed in several tissues. LIPI sequences have only been identified in mammals. Here, we describe the identification of LIPI in non-mammalian vertebrates. Based on the conserved genomic organization of LIPI and LIPH we identified sequences for both lipases in birds and fishes. In all vertebrates the LIPI locus is neighbored by a member of the RNA binding motif (RBM) family, RBM11. By sequencing of reverse transcriptase-polymerase chain reaction products we determined the sequences of LIPI and LIPH messenger RNA from broilers. We found that the sequence homology between LIPI and LIPH is much higher in non-mammalian species than in mammals. In addition, we found broad expression of LIPI in broilers, resembling the expression profile of LIPH. Our data suggest that LIPI is a CTA only in mammalian species and that the unique sequence features of the mammalian LIPI/RBM11 locus have evolved together with the CTA-like expression pattern of LIPI.
Assuntos
Antígenos de Neoplasias/genética , Galinhas/genética , Isoenzimas/genética , Fosfolipases A1/genética , Animais , Sequência de Bases , Primers do DNA/genética , Evolução Molecular , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da EspécieRESUMO
Hypomethylating agents (HMA) like azacitidine are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic stem cell transplantation. Biomarker-driven identification of HMA-responsive patients may facilitate the choice of treatment, especially in the challenging subgroup above 60 years of age. Since HMA possesses immunomodulatory functions that constitute part of their anti-tumor effect, we set out to analyze the bone marrow (BM) immune environment by next-generation sequencing of T cell receptor beta (TRB) repertoires in 51 AML patients treated within the RAS-AZIC trial. Patients with elevated pretreatment T cell diversity (11 out of 41 patients) and those with a boost of TRB richness on day 15 after azacitidine treatment (12 out of 46 patients) had longer event-free and overall survival. Both pretreatment and dynamic BM T cell metrics proved to be better predictors of outcome than other established risk factors. The favorable broadening of the BM T cell space appeared to be driven by antigen since these patients showed significant skewing of TRBV gene usage. Our data suggest that one course of AZA can cause reconstitution to a more physiological T cell BM niche and that the T cell space plays an underestimated prognostic role in AML.Trial registration: DRKS identifier: DRKS00004519.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Medula Óssea/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Feminino , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Linfócitos T/patologiaRESUMO
Post-acute sequelae of COVID-19 (PASC) is emerging as global problem with unknown molecular drivers. Using a digital epidemiology approach, we recruited 8,077 individuals to the cohort study for digital health research in Germany (DigiHero) to respond to a basic questionnaire followed by a PASC-focused survey and blood sampling. We report the first 318 participants, the majority thereof after mild infections. Of those, 67.8% report PASC, predominantly consisting of fatigue, dyspnea, and concentration deficit, which persists in 60% over the mean 8-month follow-up period and resolves independently of post-infection vaccination. PASC is not associated with autoantibodies, but with elevated IL-1ß, IL-6, and TNF plasma levels, which we confirm in a validation cohort with 333 additional participants and a longer time from infection of 10 months. Blood profiling and single-cell data from early infection suggest the induction of these cytokines in COVID-19 lung pro-inflammatory macrophages creating a self-sustaining feedback loop.
Assuntos
COVID-19 , Citocinas , COVID-19/complicações , COVID-19/imunologia , COVID-19/patologia , Estudos de Coortes , Citocinas/imunologia , Progressão da Doença , Humanos , Testes Imunológicos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Fator de Necrose Tumoral alfa/imunologia , Síndrome de COVID-19 Pós-AgudaRESUMO
The COVID-19 pandemic shows that vaccination strategies building on an ancestral viral strain need to be optimized for the control of potentially emerging viral variants. Therefore, aiming at strong B cell somatic hypermutation to increase antibody affinity to the ancestral strain - not only at high antibody titers - is a priority when utilizing vaccines that are not targeted at individual variants since high affinity may offer some flexibility to compensate for strain-individual mutations. Here, we developed a next-generation sequencing based SARS-CoV-2 B cell tracking protocol to rapidly determine the level of immunoglobulin somatic hypermutation at distinct points during the immunization period. The percentage of somatically hypermutated B cells in the SARS-CoV-2 specific repertoire was low after the primary vaccination series, evolved further over months and increased steeply after boosting. The third vaccination mobilized not only naïve, but also antigen-experienced B cell clones into further rapid somatic hypermutation trajectories indicating increased affinity. Together, the strongly mutated post-booster repertoires and antibodies deriving from this may explain why the third, but not the primary vaccination series, offers some protection against immune-escape variants such as Omicron B.1.1.529.