RESUMO
Germinal center (GC) B cells undergo proliferation at very high rates in a hypoxic microenvironment but the cellular processes driving this are incompletely understood. Here we show that the mitochondria of GC B cells are highly dynamic, with significantly upregulated transcription and translation rates associated with the activity of transcription factor A, mitochondrial (TFAM). TFAM, while also necessary for normal B cell development, is required for entry of activated GC precursor B cells into the germinal center reaction; deletion of Tfam significantly impairs GC formation, function and output. Loss of TFAM in B cells compromises the actin cytoskeleton and impairs cellular motility of GC B cells in response to chemokine signaling, leading to their spatial disorganization. We show that B cell lymphoma substantially increases mitochondrial translation and that deletion of Tfam in B cells is protective against the development of lymphoma in a c-Myc transgenic mouse model. Finally, we show that pharmacological inhibition of mitochondrial transcription and translation inhibits growth of GC-derived human lymphoma cells and induces similar defects in the actin cytoskeleton.
Assuntos
Linfoma de Células B , Linfoma , Camundongos , Humanos , Animais , Linfócitos B/patologia , Centro Germinativo/patologia , Transcrição Gênica , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos Transgênicos , Microambiente TumoralRESUMO
Our previous study using systems vaccinology identified an association between the sterol regulatory binding protein (SREBP) pathway and humoral immune response to vaccination in humans. To investigate the role of SREBP signaling in modulating immune responses, we generated mice with B cell- or CD11c+ antigen-presenting cell (APC)-specific deletion of SCAP, an essential regulator of SREBP signaling. Ablation of SCAP in CD11c+ APCs had no effect on immune responses. In contrast, SREBP signaling in B cells was critical for antibody responses, as well as the generation of germinal centers,memory B cells and bone marrow plasma cells. SREBP signaling was required for metabolic reprogramming in activated B cells. Upon mitogen stimulation, SCAP-deficient B cells could not proliferate and had decreased lipid rafts. Deletion of SCAP in germinal center B cells using AID-Cre decreased lipid raft content and cell cycle progression. These studies provide mechanistic insights coupling sterol metabolism with the quality and longevity of humoral immunity.
Assuntos
Proteínas de Transporte , Linfoma de Células B , Esteróis , Animais , Humanos , Camundongos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Esteróis/metabolismo , Linfoma de Células B/metabolismoRESUMO
B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.
Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Antígenos CD19/genética , Antígenos CD19/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Humanos , Imunoterapia Adotiva/efeitos adversos , Glicoproteínas de Membrana/metabolismo , Poliadenilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Transativadores/metabolismoRESUMO
The mechanisms that drive normal B cell differentiation and activation are frequently subverted by B cell lymphomas for their unlimited growth and survival. B cells are particularly prone to malignant transformation because the machinery used for antibody diversification can cause chromosomal translocations and oncogenic mutations. The advent of functional and structural genomics has greatly accelerated our understanding of oncogenic mechanisms in lymphomagenesis. The signaling pathways that normal B cells utilize to sense antigens are frequently derailed in B cell malignancies, leading to constitutive activation of prosurvival pathways. These malignancies co-opt transcriptional regulatory systems that characterize their normal B cell counterparts and frequently alter epigenetic regulators of chromatin structure and gene expression. These mechanistic insights are ushering in an era of targeted therapies for these cancers based on the principles of pathogenesis.
Assuntos
Linfoma de Células B/etiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Epigênese Genética , Humanos , Evasão da Resposta Imune , Linfoma de Células B/tratamento farmacológico , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Dosagem de Genes , Centro Germinativo/metabolismo , Imunidade Humoral , Linfoma de Células B/genética , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/deficiência , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Haploinsuficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , CoesinasRESUMO
Most adult B cell lymphomas originate from germinal center (GC) B cells, but it is unclear to what extent B cells in overt lymphoma retain the functional dynamics of GC B cells or are blocked at a particular stage of the GC reaction. Here we used integrative single-cell analysis of phenotype, gene expression and variable-region sequence of the immunoglobulin heavy-chain locus to track the characteristic human GC B cell program in follicular lymphoma B cells. By modeling the cyclic continuum of GC B cell transitional states, we identified characteristic patterns of synchronously expressed gene clusters. GC-specific gene-expression synchrony was lost in single lymphoma B cells. However, distinct follicular lymphoma-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B cell state but might adopt new dynamic modes of functional diversity, which opens the possibility of novel definitions of lymphoma identity.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/fisiologia , Centro Germinativo/fisiologia , Região Variável de Imunoglobulina/genética , Linfoma de Células B/genética , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/patologia , Humanos , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Análise de Célula Única , Transcriptoma/genéticaRESUMO
The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.
Assuntos
Linfócitos B/citologia , Proteína Forkhead Box O1/genética , Centro Germinativo/imunologia , Linfoma de Células B/patologia , Animais , Linfócitos B/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Linfoma de Células B/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Nussenzweig and colleagues evaluate genomic instability and germinal center derived lymphomagenesis in mice infected with Plasmodium to recreate some of the hallmark characteristics of Burkitt lymphoma, a form of cancer more common in parts of Africa where malaria is endemic.
Assuntos
Citidina Desaminase/metabolismo , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Translocação Genética , Animais , HumanosRESUMO
Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt's lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells. PAPERCLIP.
Assuntos
Instabilidade Genômica , Linfoma de Células B/genética , Malária/complicações , Malária/genética , Plasmodium chabaudi/fisiologia , Animais , Linfócitos B/patologia , Doença Crônica , Citidina Desaminase/metabolismo , Replicação do DNA , Genes p53 , Centro Germinativo/parasitologia , Malária/parasitologia , Malária/patologia , Camundongos , Translocação GenéticaRESUMO
We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Cromossomos , DNA/metabolismo , Linfoma de Células B/metabolismo , Proteínas Nucleares/metabolismo , Prófase , Proteoma , Proteômica , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Galinhas , Cromatina/genética , DNA/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas Nucleares/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fatores de TempoRESUMO
Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
Assuntos
Complemento C1q/imunologia , Citotoxicidade Imunológica/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoterapia , Neoplasias/tratamento farmacológico , Fagocitose/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/imunologia , Linhagem Celular Tumoral , Cromatografia em Gel , Cromatografia Líquida , Complemento C1q/metabolismo , Cristalização , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Técnicas In Vitro , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/imunologia , Espectrometria de Massas , Camundongos , Neoplasias/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptores de IgG/metabolismo , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em TandemRESUMO
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
Assuntos
Carbolinas/farmacologia , Morte Celular/efeitos dos fármacos , Glutationa Peroxidase/antagonistas & inibidores , Piperazinas/farmacologia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Xenoenxertos , Humanos , Linfoma de Células B/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Fosfolipídeo Hidroperóxido Glutationa PeroxidaseRESUMO
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
Assuntos
Herpesvirus Humano 4/química , Engenharia de Proteínas , Proteínas/farmacologia , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Biologia Computacional , Cristalografia por Raios X , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Herpesvirus Humano 4/fisiologia , Xenoenxertos , Humanos , Linfoma de Células B/tratamento farmacológico , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas/química , Proteínas/metabolismo , Alinhamento de Sequência , Proteínas Virais/químicaRESUMO
Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.
Assuntos
Engenharia Celular , Imunoterapia Adotiva , Lógica , Neoplasias , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Transdução de Sinais , Linfócitos T , Humanos , Engenharia Celular/métodos , Imunoterapia Adotiva/efeitos adversos , Leucemia de Células B , Linfoma de Células B , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.
Assuntos
Linfócitos B/enzimologia , RNA Helicases DEAD-box/metabolismo , Linfoma de Células B/enzimologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , RNA Helicases DEAD-box/genética , Estresse do Retículo Endoplasmático , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação com Perda de Função , Linfoma de Células B/genética , Linfoma de Células B/patologia , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/genética , Proteínas de Neoplasias/genética , Biossíntese de Proteínas , Proteoma , Proteostase , Proteínas Proto-Oncogênicas c-myc/genética , Adulto JovemRESUMO
Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.
Assuntos
Autoimunidade , Neoplasias/enzimologia , Neoplasias/prevenção & controle , Quinase Syk/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Antígenos CD19/metabolismo , Linfócitos B , Cálcio/metabolismo , Diferenciação Celular , Transformação Celular Neoplásica , Ativação Enzimática , Humanos , Tolerância Imunológica , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Camundongos , Modelos Genéticos , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
High-affinity B cell selection in the germinal center (GC) is governed by signals delivered by follicular helper T (Tfh) cells to B cells. Selected B cells undergo clonal expansion and affinity maturation in the GC dark zone in direct proportion to the amount of antigen they capture and present to Tfh cells in the light zone. Here, we examined the mechanisms whereby Tfh cells program the number of GC B cell divisions. Gene expression analysis revealed that Tfh cells induce Myc expression in light-zone B cells in direct proportion to antigen capture. Conditional Myc haplo-insufficiency or overexpression combined with cell division tracking showed that MYC expression produces a metabolic reservoir in selected light-zone B cells that is proportional to the number of cell divisions in the dark zone. Thus, MYC constitutes the GC B cell division timer that when deregulated leads to emergence of B cell lymphoma.
Assuntos
Linfócitos B/imunologia , Genes myc/genética , Centro Germinativo/imunologia , Linfoma de Células B/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Afinidade de Anticorpos , Diferenciação Celular , Divisão Celular , Proliferação de Células , Seleção Clonal Mediada por Antígeno , Regulação da Expressão Gênica , Humanos , CamundongosRESUMO
Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.