RESUMO
Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Transcriptoma , Adulto , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Células Tumorais CultivadasRESUMO
Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of â¼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Neoplasia Residual/patologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos de Boro/uso terapêutico , Bortezomib/uso terapêutico , Aberrações Cromossômicas , Dexametasona/uso terapêutico , Feminino , Citometria de Fluxo , Glicina/análogos & derivados , Glicina/uso terapêutico , Humanos , Lenalidomida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Resultado do TratamentoRESUMO
Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-ß and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.
Assuntos
Anemia de Fanconi , Pancitopenia , Humanos , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Terapia Genética/métodos , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Pancitopenia/metabolismo , Transtornos da Insuficiência da Medula Óssea/metabolismoRESUMO
Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.
Assuntos
Antígenos CD , Mieloma Múltiplo , Células Supressoras Mieloides , Proteínas de Neoplasias , Antígenos CD/sangue , Antígenos CD/genética , Antígenos CD/imunologia , Feminino , Seguimentos , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Transcrição Gênica/imunologiaRESUMO
Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.
Assuntos
Hematopoiese Clonal , Mieloma Múltiplo/patologia , Síndromes Mielodisplásicas/etiologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Terapia Combinada , Feminino , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Mutação , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Transplante Autólogo , Microambiente TumoralRESUMO
BACKGROUND: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-ß signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , RNA-Seq , Análise de Célula Única , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fibroblastos/patologia , Humanos , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologiaRESUMO
Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.
Assuntos
Linfócitos B/imunologia , Antígeno B7-H1/imunologia , Regulação Neoplásica da Expressão Gênica , Ativação Linfocitária , Linfoma Difuso de Grandes Células B/imunologia , Receptor de Morte Celular Programada 1/imunologia , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Animais , Linfócitos B/patologia , Antígeno B7-H1/genética , Feminino , Humanos , Imunoterapia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Masculino , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/genética , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Genoma Humano/genética , Instabilidade Genômica/genética , Células Hep G2 , Humanos , Splicing de RNA/genética , Troca de Cromátide Irmã/genéticaRESUMO
Not available.
Assuntos
COVID-19/imunologia , Neoplasias Hematológicas/imunologia , SARS-CoV-2/imunologia , COVID-19/epidemiologia , Neoplasias Hematológicas/epidemiologia , HumanosRESUMO
Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs.
Assuntos
Amiloidose/genética , Cadeias Leves de Imunoglobulina/genética , Paraproteinemias/genética , Plasmócitos/metabolismo , Transcriptoma , Amiloidose/metabolismo , Amiloidose/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Análise em Microsséries , Paraproteinemias/metabolismo , Paraproteinemias/patologia , Fenótipo , Plasmócitos/patologiaRESUMO
Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered atwww.clinicaltrials.gov as #NCT01237249.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/genética , Idoso , Bortezomib/administração & dosagem , Moléculas de Adesão Celular/metabolismo , Dexametasona/administração & dosagem , Progressão da Doença , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imunofenotipagem , Integrinas/metabolismo , Lenalidomida , Masculino , Melfalan/administração & dosagem , Modelos Genéticos , Mieloma Múltiplo/patologia , Neoplasia Residual/patologia , Fenótipo , Plasmócitos/patologia , Prednisona/administração & dosagem , Prognóstico , Talidomida/administração & dosagem , Talidomida/análogos & derivadosRESUMO
Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25(-)CD22(+) B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25(+)CD22(+low)) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Gamopatia Monoclonal de Significância Indeterminada/genética , Macroglobulinemia de Waldenstrom/genética , Linfócitos B/metabolismo , Transformação Celular Neoplásica/patologia , Células Clonais , Citometria de Fluxo , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imunoglobulina M/análise , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fenótipo , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Plasmacytoid dendritic cells (pDCs) are considered to be the principal type-I IFN (IFN-I) source in response to viruses, whereas the contribution of conventional DCs (cDCs) has been underestimated because, on a per-cell basis, they are not considered professional IFN-I-producing cells. We have investigated their respective roles in the IFN-I response required for CTL activation. Using a nonreplicative virus, baculovirus, we show that despite the high IFN-I-producing abilities of pDCs, in vivo cDCs but not pDCs are the pivotal IFN-I producers upon viral injection, as demonstrated by selective pDC or cDC depletion. The pathway involved in the virus-triggered IFN-I response is dependent on TLR9/MyD88 in pDCs and on stimulator of IFN genes (STING) in cDCs. Importantly, STING is the key molecule for the systemic baculovirus-induced IFN-I response required for CTL priming. The supremacy of cDCs over pDCs in fostering the IFN-I response required for CTL activation was also verified in the lymphocytic choriomeningitis virus model, in which IFN-ß promoter stimulator 1 plays the role of STING. However, when the TLR-independent virus-triggered IFN-I production is impaired, the pDC-induced IFNs-I have a primary impact on CTL activation, as shown by the detrimental effect of pDC depletion and IFN-I signaling blockade on the residual lymphocytic choriomeningitis virus-triggered CTL response detected in IFN-ß promoter stimulator 1(-/-) mice. Our findings reveal that cDCs play a major role in the TLR-independent virus-triggered IFN-I production required for CTL priming, whereas pDC-induced IFNs-I are dispensable but become relevant when the TLR-independent IFN-I response is impaired.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Células Dendríticas/virologia , Interferon Tipo I/biossíntese , Nucleopoliedrovírus/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/classificação , Feminino , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/fisiologia , Transdução de Sinais/imunologia , Receptor Toll-Like 9/fisiologiaRESUMO
Interferon-α is a potent antiviral agent and a vigorous adjuvant in the induction of T-cell responses but its use is limited by hematologic toxicity. Interferon-α alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-α exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-α to achieve sustained high serum levels of the cytokine. The bone marrow of these animals showed diminished long-term and short-term hematopoietic stem cells, reduction of multipotent progenitor cells, and marked decrease of B cells, but significant increase in the proportion of CD8(+) and CD4(+)CD8(+) T cells. Upon adoptive transfer to RAG(-/-) mice, bone marrow cells from interferon-α-treated animals generated CD4(+) and CD8(+) T cells while CD19(+), CD11b(+) and NK1.1(+) lineages failed to develop. These effects are associated with the transcriptional downregulation of transcription factors involved in B-cell differentiation and modulation of key factors for T-cell development. Thus, sustained interferon-α exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Interferon-alfa/farmacologia , Linfopoese/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Interferon-alfa/genética , Contagem de Leucócitos , Leucócitos/citologia , Leucócitos/metabolismo , Linfopoese/genética , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Previous mouse and human studies have demonstrated that direct IFN-α/ß signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α-primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15(-/-) and IL-7(-/-) recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2(+) CD8 T cells with autologous dendritic cells loaded with MART1(26-35) peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/fisiologia , Homeostase/imunologia , Imunização Secundária/métodos , Memória Imunológica , Interferon-alfa/fisiologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva/métodos , Animais , Antígenos/fisiologia , Linfócitos T CD8-Positivos/transplante , Células Cultivadas , Humanos , Interleucina-15/fisiologia , Interleucina-17/fisiologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/transplanteRESUMO
Infection is the leading cause of death in multiple myeloma (MM). However, the cellular composition associated with immune dysfunction is not defined. We analyzed immune profiles in the peripheral blood of patients with MM (n = 28) and B-cell chronic lymphoproliferative disorders (n = 53) vs. health care practitioners (n = 96), using multidimensional and computational flow cytometry. MM patients displayed altered distribution of most cell types (41/56, 73%), particularly within the B-cell (17/17) and T-cell (20/30) compartments. Using COVID-19 as a case study, we compared the immune response to vaccination based on 64,304 data points generated from the analysis of 1099 longitudinal samples. MM patients showed limited B-cell expansion linked to lower anti-RBD and anti-S antibody titers after the first two doses and booster. The percentages of B cells and CD4+ T cells in the blood, as well as the absolute counts of B cells and dendritic cells, predicted vaccine immunogenicity at different time points. In contrast with the humoral response, the percentage and antigen-dependent differentiation of SARS-CoV-2-specific CD8+ T cells was not altered in MM patients. Taken together, this study defined the cellular composition associated with immune dysfunction in MM and provided biomarkers such as the B-cell percentage and absolute count to individualize vaccination calendars.
Assuntos
Linfócitos B , Vacinas contra COVID-19 , COVID-19 , Mieloma Múltiplo , SARS-CoV-2 , Vacinação , Humanos , Mieloma Múltiplo/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Linfócitos B/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologiaRESUMO
Hematological toxicity is a common side effect of CAR-T therapies, particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.7%, and grade>3 thrombocytopenia and neutropenia, one month after infusion, was observed in 57% and 53% of the patients, being still present after one year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on HSPCs differentiation using an ex-vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation, promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers, which could be prevented with a combination of IFNγ, TNFα/ß, TGFß, IL-6 and IL-17 inhibitors. Single-cell RNA-seq demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2, MAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.
RESUMO
Despite the development of novel therapies for acute myeloid leukemia, outcomes remain poor for most patients, and therapeutic improvements are an urgent unmet need. Although treatment regimens promoting differentiation have succeeded in the treatment of acute promyelocytic leukemia, their role in other acute myeloid leukemia subtypes needs to be explored. Here we identify and characterize two lysine deacetylase inhibitors, CM-444 and CM-1758, exhibiting the capacity to promote myeloid differentiation in all acute myeloid leukemia subtypes at low non-cytotoxic doses, unlike other commercial histone deacetylase inhibitors. Analyzing the acetylome after CM-444 and CM-1758 treatment reveals modulation of non-histone proteins involved in the enhancer-promoter chromatin regulatory complex, including bromodomain proteins. This acetylation is essential for enhancing the expression of key transcription factors directly involved in the differentiation therapy induced by CM-444/CM-1758 in acute myeloid leukemia. In summary, these compounds may represent effective differentiation-based therapeutic agents across acute myeloid leukemia subtypes with a potential mechanism for the treatment of acute myeloid leukemia.