RESUMO
Memory B cells (MBCs) can persist for a lifetime, but the mechanisms that allow their long-term survival remain poorly understood. Here, we isolated and analyzed human splenic smallpox/vaccinia protein B5-specific MBCs in individuals who were vaccinated more than 40 years ago. Only a handful of clones persisted over such an extended period, and they displayed limited intra-clonal diversity with signs of extensive affinity-based selection. These long-lived MBCs appeared enriched in a CD21hiCD20hi IgG+ splenic B cell subset displaying a marginal-zone-like NOTCH/MYC-driven signature, but they did not harbor a unique longevity-associated transcriptional or metabolic profile. Finally, the telomeres of B5-specific, long-lived MBCs were longer than those in patient-paired naive B cells in all the samples analyzed. Overall, these results imply that separate mechanisms such as early telomere elongation, affinity selection during the contraction phase, and access to a specific niche contribute to ensuring the functional longevity of MBCs.
Assuntos
Memória Imunológica , Células B de Memória , Linfócitos B/metabolismo , Centro Germinativo , Humanos , Imunoglobulina G/metabolismoRESUMO
Lymphoid stromal cells (LSCs) are essential organizers of immune responses. We analyzed tonsillar tissue by combining flow cytometry, in situ imaging, RNA sequencing, and functional assays, defining three distinct human LSC subsets. The integrin CD49a designated perivascular stromal cells exhibiting features of local committed LSC precursors and segregated cytokine and chemokine-producing fibroblastic reticular cells (FRCs) supporting B and T cell survival. The follicular dendritic cell transcriptional profile reflected active responses to B cell and non-B cell stimuli. We therefore examined the effect of B cell stimuli on LSCs in follicular lymphoma (FL). FL B cells interacted primarily with CD49a+ FRCs. Transcriptional analyses revealed LSC reprogramming in situ downstream of the cytokines tumor necrosis factor (TNF) and transforming growth factor ß (TGF-ß), including increased expression of the chemokines CCL19 and CCL21. Our findings define human LSC populations in healthy tissue and reveal bidirectional crosstalk between LSCs and malignant B cells that may present a targetable axis in lymphoma.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Tonsila Palatina/imunologia , Células Estromais/imunologia , Células Cultivadas , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Humanos , Integrina alfa1/metabolismo , Tonsila Palatina/citologia , Transdução de Sinais/imunologia , Células Estromais/citologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
ABSTRACT: Plasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high throughput sequencing (scATAC-seq) to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to activating protein 1 (AP-1) transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription-mediated reprogramming events that sustain PC differentiation.
Assuntos
Diferenciação Celular , Cromatina , Perfilação da Expressão Gênica , Plasmócitos , Análise de Célula Única , Humanos , Diferenciação Celular/genética , Plasmócitos/metabolismo , Plasmócitos/citologia , Análise de Célula Única/métodos , Cromatina/metabolismo , Cromatina/genética , Transcriptoma , Epigênese Genética , Células CultivadasRESUMO
MOTIVATION: Transcriptional regulation is performed by transcription factors (TF) binding to DNA in context-dependent regulatory regions and determines the activation or inhibition of gene expression. Current methods of transcriptional regulatory circuits inference, based on one or all of TF, regions and genes activity measurements require a large number of samples for ranking the candidate TF-gene regulation relations and rarely predict whether they are activations or inhibitions. We hypothesize that transcriptional regulatory circuits can be inferred from fewer samples by (1) fully integrating information on TF binding, gene expression and regulatory regions accessibility, (2) reducing data complexity and (3) using biology-based likelihood constraints to determine the global consistency between a candidate TF-gene relation and patterns of genes expressions and region activations, as well as qualify regulations as activations or inhibitions. RESULTS: We introduce Regulus, a method which computes TF-gene relations from gene expressions, regulatory region activities and TF binding sites data, together with the genomic locations of all entities. After aggregating gene expressions and region activities into patterns, data are integrated into a RDF (Resource Description Framework) endpoint. A dedicated SPARQL (SPARQL Protocol and RDF Query Language) query retrieves all potential relations between expressed TF and genes involving active regulatory regions. These TF-region-gene relations are then filtered using biological likelihood constraints allowing to qualify them as activation or inhibition. Regulus provides signed relations consistent with public databases and, when applied to biological data, identifies both known and potential new regulators. Regulus is devoted to context-specific transcriptional circuits inference in human settings where samples are scarce and cell populations are closely related, using discretization into patterns and likelihood reasoning to decipher the most robust regulatory relations.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Humanos , Regulação da Expressão Gênica/genética , Fatores de Transcrição/metabolismo , Genômica/métodos , Bases de Dados Factuais , Ligação Proteica , Redes Reguladoras de Genes/genéticaRESUMO
The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.
Assuntos
Mieloma Múltiplo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Plasmócitos/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
The terminal differentiation of B cells into antibody-secreting cells (ASCs) is a critical component of adaptive immune responses. However, it is a very sensitive process, and dysfunctions lead to a variety of lymphoproliferative neoplasias including germinal center-derived lymphomas. To better characterize the late genomic events that drive the ASC differentiation of human primary naive B cells, we used our in vitro differentiation system and a combination of RNA sequencing and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC sequencing). We discovered 2 mechanisms that drive human terminal B-cell differentiation. First, after an initial response to interleukin-4 (IL-4), cells that were committed to an ASC fate downregulated the CD23 marker and IL-4 signaling, whereas cells that maintained IL-4 signaling did not differentiate. Second, human CD23- cells also increased IRF4 protein to levels required for ASC differentiation, but they did that independently of the ubiquitin-mediated degradation process previously described in mice. Finally, we showed that CD23- cells carried the imprint of their previous activated B-cell status, were precursors of plasmablasts, and had a phenotype similar to that of in vivo preplasmablasts. Altogether, our results provide an unprecedented genomic characterization of the fate decision between activated B cells and plasmablasts, which provides new insights into the pathological mechanisms that drive lymphoma biology.
Assuntos
Linfócitos B/imunologia , Fatores Reguladores de Interferon/imunologia , Interleucina-4/imunologia , Plasmócitos/imunologia , Receptores de IgE/imunologia , Fator de Transcrição STAT6/imunologia , Células Cultivadas , Humanos , Ativação Linfocitária , Linfoma/imunologia , Transdução de SinaisRESUMO
Follicular lymphoma (FL) originates in the lymph nodes (LNs) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSCs) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been identified. In the current work, we demonstrate that FL B cells produce extracellular vesicles (EVs) that can be internalized by BM-MSCs, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activate transforming growth factor ß-dependent and independent pathways in BM-MSCs and modify their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 and angiopoietin-1. Moreover, we provide the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSCs. This work identifies FL-derived EVs as putative mediators of BM stroma polarization and supports further investigation of their clinical interest for targeting the crosstalk between BM-MSCs and malignant B cells.
Assuntos
Linfócitos B/patologia , Células da Medula Óssea/patologia , Polaridade Celular , Vesículas Extracelulares/patologia , Linfoma Folicular/patologia , Sequência de Bases , Células da Medula Óssea/metabolismo , Comunicação Celular , Diferenciação Celular , Endocitose , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfoma Folicular/genética , Heterotrímero de Linfotoxina alfa1 e beta2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genéticaRESUMO
Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell expansion is challenging. We assessed the value of killer immunoglobulin-like receptor(KIR) phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (n = 46) and a reactive NK group (n = 68). We then developed an NK-cell clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the patients with CLPD-NK, constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK preferentially exhibited a CD16low phenotype, more frequently displayed a lower platelet count, and was associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed whole-exome sequencing of sorted, myeloid, T, and NK cells and found that TET2 mutations were shared by myeloid and NK cells in 3 of 4 cases. Thus, we hypothesized that TET2 alterations occur in early hematopoietic progenitors which could explain a potential link between CLPD-NK and myeloid malignancies. Finally, we analyzed the transcriptome by RNA sequencing of 7 CLPD-NK and evidenced 2 groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including 2 TET2-mutated cases, significantly underexpressed genes known to be downregulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK-cell proliferative disorders and, potentially, new therapeutic opportunities.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Células Matadoras Naturais/metabolismo , Linfoma de Células T/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Receptores KIR/metabolismo , Fator de Transcrição STAT3/metabolismo , Idoso , Doença Crônica , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfoma de Células T/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Receptores KIR/genética , Fator de Transcrição STAT3/genéticaRESUMO
INTRODUCTION: Factor X deficiency is a rare inherited bleeding disorder. To date, 181 variants are reported in the recently updated F10-gene variant database. AIM: This study aimed to describe new F10 variants. METHOD: The F10 gene was analysed in 16 consecutive families with FX deficiency by a targeted high-throughput sequencing approach, including F10, F9, F8 genes, and 78 genes dedicated to haematological malignancies. RESULTS: We identified 19 variants (17 missense, one nonsense and one frameshift) and two copy number variations. Two patients presenting a combined FVII-FX deficiency showed a loss of one F10 gene copy (del13q34) associated with a missense variant on the remaining allele, leading to a FX:C significantly lower than the FVII:C level and explaining their unusual bleeding history. We reported five novel variants. Three missense variants (p.Glu22Val affecting the signal peptide cleavage site, p.Cys342Tyr removing the disulphide bond between the FX heavy and light chains, and p.Val385Met located in FX peptidase S1 domain) were detected at compound heterozygosis status in three patients with severe bleeding symptoms and FX:C level below 10 IU/dL. Two truncating variants p.Tyr279* and p.Thr434Aspfs*13 leading to an altered FX protein were found at heterozygous state in two patients with mild bleeding history. CONCLUSION: This study showed the feasibility and the interest of high-throughput sequencing approach for rare bleeding disorders, enabling the report of F10 gene screening in a 3-weeks delay, suitable for clinical use. The description of five new variants may contribute to a better understanding of the phenotype-genotype correlation in FX deficiency.
Assuntos
Deficiência do Fator X , Humanos , Deficiência do Fator X/genética , Deficiência do Fator X/complicações , Fator X/genética , Variações do Número de Cópias de DNA , Hemorragia/complicações , HeterozigotoRESUMO
B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.
Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/imunologia , Alelos , Animais , Diferenciação Celular/genética , Citidina Desaminase/imunologia , Marcação de Genes , Humanos , Região de Troca de Imunoglobulinas/imunologia , Tecido Linfoide/imunologia , Camundongos , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Sequências Reguladoras de Ácido NucleicoRESUMO
Tumor-associated macrophage and T-cell subsets are implicated in the pathogenesis of diffuse large B-cell lymphoma, follicular lymphoma, and classical Hodgkin lymphoma. Macrophages provide essential mechanisms of tumor immune evasion through checkpoint ligand expression and secretion of suppressive cytokines. However, normal and tumor-associated macrophage phenotypes are less well characterized than those of tumor-infiltrating T-cell subsets, and it would be especially valuable to know whether the polarization state of macrophages differs across lymphoma tumor microenvironments. Here, an established mass cytometry panel designed to characterize myeloid-derived suppressor cells and known macrophage maturation and polarization states was applied to characterize B-lymphoma tumors and non-malignant human tissue. High-dimensional single-cell analyses were performed using dimensionality reduction and clustering tools. Phenotypically distinct intra-tumor macrophage subsets were identified based on abnormal marker expression profiles that were associated with lymphoma tumor types. While it had been proposed that measurement of CD163 and CD68 might be sufficient to reveal macrophage subsets in tumors, results here indicated that S100A9, CCR2, CD36, Slan, and CD32 should also be measured to effectively characterize lymphoma-specific tumor macrophages. Additionally, the presence of phenotypically distinct, abnormal macrophage populations was closely linked to the phenotype of intra-tumor T-cell populations, including PD-1 expressing T cells. These results further support the close links between macrophage polarization and T-cell functional state, as well as the rationale for targeting tumor-associated macrophages in cancer immunotherapies.
Assuntos
Centro Germinativo/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Macrófagos/imunologia , Adulto , Idoso , Feminino , Citometria de Fluxo , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.
Assuntos
Proteínas de Choque Térmico HSP110/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/metabolismo , Estudos de Coortes , Proteínas de Choque Térmico HSP110/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , Estabilidade Proteica , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The benefit of radiotherapy (RT) after chemotherapy in limited-stage diffuse large B-cell lymphoma (DLBCL) remains controversial. We conducted a randomized trial in patients with nonbulky limited-stage DLBCL to evaluate the benefit of RT after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients were stratified according to the modified International Prognostic Index, including lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, age, and disease stage. The patients received 4 or 6 consecutive cycles of R-CHOP delivered once every 2 weeks, followed or not by RT at 40 Gy delivered 4 weeks after the last R-CHOP cycle. All patients were evaluated by fluorodeoxyglucose-positron emission tomography scans performed at baseline, after 4 cycles of R-CHOP, and at the end of treatment. The primary objective of the trial was event-free survival (EFS) from randomization. The trial randomly assigned 165 patients in the R-CHOP arm and 169 in the R-CHOP plus RT arm. In an intent-to-treat analysis with a median follow-up of 64 months, 5-year EFS was not statistically significantly different between the 2 arms, with 89% ± 2.9% in the R-CHOP arm vs 92% ± 2.4% in the R-CHOP plus RT arm (hazard ratio, 0.61; 95% confidence interval [CI], 0.3-1.2; P = .18). Overall survival was also not different at 92% (95% CI, 89.5%-94.5%) for patients assigned to R-CHOP alone and 96% (95% CI, 94.3%-97.7%) for those assigned to R-CHOP plus RT (P = not significant). R-CHOP alone is not inferior to R-CHOP followed by RT in patients with nonbulky limited-stage DLBCL. This trial was registered at www.clinicaltrials.gov as #NCT00841945.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/radioterapia , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/efeitos adversos , Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Feminino , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Prednisona/uso terapêutico , Intervalo Livre de Progressão , Estudos Prospectivos , Rituximab , Resultado do Tratamento , Vincristina/administração & dosagem , Vincristina/efeitos adversos , Vincristina/uso terapêuticoRESUMO
Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.
Assuntos
Mieloma Múltiplo , Proteínas/genética , Proliferação de Células/genética , Metilação de DNA , Epigênese Genética , Epigenômica , Humanos , Mieloma Múltiplo/genéticaRESUMO
Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accumulation of germinal center-derived malignant B cells engaged in a bidirectional crosstalk with their supportive microenvironment in invaded lymph nodes (LNs) and bone marrow (BM). T follicular helper (TFH) cells and infiltrating stromal cells have been shown to favor FL B-cell growth, but the mechanisms of their protumoral effect and how the LN/BM microenvironment is converted into a lymphoma-permissive cell niche remain poorly understood. We demonstrated here that FL-infiltrating LN and BM stromal cells overexpressed CXCL12 in situ. Interleukin-4 high (IL-4hi) FL-TFH cells, unlike FL B cells themselves, triggered CXCL12 upregulation in human stromal cell precursors. In agreement, expression of CXCL12 was associated with IL-4 expression and signaling within the FL BM and LN niches. This IL-4/CXCL12 axis was amplified in activated lymphoid stromal cells as shown in our in vitro model of human lymphoid stroma differentiation and in an inducible mouse model of ectopic lymphoid organ formation. Finally, CXCL12 triggered primary FL B-cell activation, migration, and adhesion, a process antagonized by BTK and PI3K inhibitors. These data identified the IL-4/CXCL12 loop as a previously unrecognized pathway involved in lymphoid stroma polarization and as a potential therapeutic target in FL patients.
Assuntos
Medula Óssea/imunologia , Quimiocina CXCL12/imunologia , Interleucina-4/imunologia , Linfonodos/imunologia , Linfoma Folicular/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Medula Óssea/patologia , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocina CXCL12/genética , Feminino , Humanos , Interleucina-4/genética , Linfonodos/patologia , Linfoma Folicular/genética , Linfoma Folicular/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Células Estromais/imunologia , Células Estromais/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaAssuntos
Leucemia Linfocítica Granular Grande , Receptores de Antígenos de Linfócitos T gama-delta , Neoplasias Esplênicas , Humanos , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patologia , Leucemia Linfocítica Granular Grande/diagnóstico , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfoma de Células T/genética , Linfoma de Células T/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Feminino , Genômica/métodos , IdosoRESUMO
In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid-derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases and in numerous tumors including multiple myeloma, chronic lymphocytic leukemia, and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified, in a cohort of 66 DLBCL patients, an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Index, event-free survival, and number of circulating Tregs. Furthermore, T-cell proliferation was restored after monocyte depletion. Myeloid-dependent T-cell suppression was attributed to a release of interleukin-10 and S100A12 and increased PD-L1 expression. In summary, we identified expanded MDSC subsets in DLBCL, as well as new mechanisms of immunosuppression in DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B/imunologia , Células Supressoras Mieloides/patologia , Linfócitos T/imunologia , Arginase/metabolismo , Antígeno B7-H1/metabolismo , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Imunossupressão , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-10/metabolismo , Linfoma Difuso de Grandes Células B/genética , Monócitos/metabolismo , Células Supressoras Mieloides/metabolismo , Proteína S100A12/metabolismo , Linfócitos T/metabolismo , Transcriptoma/genéticaRESUMO
RATIONALE: Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression, but their clinical impact remains poorly defined. OBJECTIVES: To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in patients with sepsis. METHODS: Peripheral blood transcriptomic analysis was performed on 29 patients with sepsis and 15 healthy donors. A second cohort of 94 consecutive patients with sepsis, 11 severity-matched intensive care patients, and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. MEASUREMENTS AND MAIN RESULTS: Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8, and ARG1, were up-regulated in the peripheral blood of patients with sepsis. CD14posHLA-DRlow/neg monocytic (M)-MDSCs were expanded in intensive care unit patients with and without sepsis and CD14negCD15pos low-density granulocytes/granulocytic (G)-MDSCs were more specifically expanded in patients with sepsis (P < 0.001). Plasma levels of MDSC mediators S100A8/A9, S100A12, and arginase 1 were significantly increased. In vitro, CD14pos- and CD15pos-cell depletion increased T-cell proliferation in patients with sepsis. G-MDSCs, made of immature and mature granulocytes expressing high levels of degranulation markers, were specifically responsible for arginase 1 activity. High initial levels of G-MDSCs, arginase 1, and S100A12 but not M-MDSCs were associated with subsequent occurrence of nosocomial infections. CONCLUSIONS: M-MDSCs and G-MDSCs strongly contribute to T-cell dysfunction in patients with sepsis. More specifically, G-MDSCs producing arginase 1 are associated with a higher incidence of nosocomial infections and seem to be major actors of sepsis-induced immune suppression.
Assuntos
Infecção Hospitalar/imunologia , Células Supressoras Mieloides/imunologia , Sepse/imunologia , Adulto , Idoso , Proliferação de Células , Infecção Hospitalar/sangue , Feminino , Citometria de Fluxo , Granulócitos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sepse/sangueRESUMO
In lymphomas arising from the germinal center, prognostic factors are linked to the myeloid compartment. In particular, high circulating monocyte or myeloid-derived suppressor cell counts are associated with poor prognosis for patients with high-grade B-cell lymphomas. Macrophages with an M2 phenotype are enriched within lymphoma tumors. However, the M1/M2 nomenclature is now deprecated and the clinical impact of this phenotype remains controversial. Across cancer types, myeloid cells are primarily thought to function as immune suppressors during tumor initiation and maintenance, but the biological mechanisms behind the myeloid signatures are still poorly understood in germinal center B-cell lymphomas. Herein, we describe the role and clinical relevance of myeloid cells in B-cell lymphoma and propose innovative approaches to decipher this complex cellular compartment. Indeed, characterization of this heterogeneous cell ecosystem has been largely accomplished with "low-resolution" approaches like morphological evaluation and immunohistochemistry, where cells are characterized using a few proteins and qualitative metrics. High-resolution, quantitative approaches, such as mass cytometry, are valuable to better understand myeloid cell diversity, functions, and to identify potential targets for novel therapies.