RESUMO
Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Proteômica , Análise de Célula Única , Transcriptoma , Humanos , Análise de Célula Única/métodos , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteômica/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Hematopoese , Nicho de Células-Tronco , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologiaRESUMO
Fibroblastic reticular cells (FRCs) direct the interaction and activation of immune cells in discrete microenvironments of lymphoid organs. Despite their important role in steering innate and adaptive immunity, the age- and inflammation-associated changes in the molecular identity and functional properties of human FRCs have remained largely unknown. Here, we show that human tonsillar FRCs undergo dynamic reprogramming during life and respond vigorously to inflammatory perturbation in comparison to other stromal cell types. The peptidase inhibitor 16 (PI16)-expressing reticular cell (PI16+ RC) subset of adult tonsils exhibited the strongest inflammation-associated structural remodeling. Interactome analysis combined with ex vivo and in vitro validation revealed that T cell activity within subepithelial niches is controlled by distinct molecular pathways during PI16+ RC-lymphocyte interaction. In sum, the topological and molecular definition of the human tonsillar stromal cell landscape reveals PI16+ RCs as a specialized FRC niche at the core of mucosal immune responses in the oropharynx.
Assuntos
Tonsila Palatina , Linfócitos T , Humanos , Fibroblastos , Linfócitos/metabolismo , Inflamação/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismoRESUMO
In development, pioneer transcription factors access silent chromatin to reveal lineage-specific gene programs. The structured DNA-binding domains of pioneer factors have been well characterized, but whether and how intrinsically disordered regions affect chromatin and control cell fate is unclear. Here, we report that deletion of an intrinsically disordered region of the pioneer factor TCF-1 (termed L1) leads to an early developmental block in T cells. The few T cells that develop from progenitors expressing TCF-1 lacking L1 exhibit lineage infidelity distinct from the lineage diversion of TCF-1-deficient cells. Mechanistically, L1 is required for activation of T cell genes and repression of GATA2-driven genes, normally reserved to the mast cell and dendritic cell lineages. Underlying this lineage diversion, L1 mediates binding of TCF-1 to its earliest target genes, which are subject to repression as T cells develop. These data suggest that the intrinsically disordered N terminus of TCF-1 maintains T cell lineage fidelity.
Assuntos
Linfócitos T , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linfócitos T/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Cromatina/metabolismoRESUMO
CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.
Assuntos
Barreira Hematoencefálica/metabolismo , Células Epiteliais/metabolismo , Imunoterapia Adotiva/efeitos adversos , Animais , Anticorpos Biespecíficos/imunologia , Antígenos CD19/imunologia , Linfócitos B/imunologia , Barreira Hematoencefálica/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Imunoterapia Adotiva/métodos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Músculo Liso Vascular/metabolismo , Neoplasias , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Análise de Célula Única/métodos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Assuntos
Anticorpos Neutralizantes/metabolismo , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , SARS-CoV-2/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Cultivadas , Epitopos , Humanos , Ativação Linfocitária , Polissorbatos , RNA Viral/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Esqualeno , Vacinação , Vacinas de mRNARESUMO
Non-neuronal cholinergic signaling, mediated by acetylcholine, plays important roles in physiological processes including inflammation and immunity. Our group first discovered evidence of non-neuronal cholinergic circuitry in adipose tissue, whereby immune cells secrete acetylcholine to activate beige adipocytes during adaptive thermogenesis. Here, we reveal that macrophages are the cellular protagonists responsible for secreting acetylcholine to regulate thermogenic activation in subcutaneous fat, and we term these cells cholinergic adipose macrophages (ChAMs). An adaptive increase in ChAM abundance is evident following acute cold exposure, and macrophage-specific deletion of choline acetyltransferase (ChAT), the enzyme for acetylcholine biosynthesis, impairs the cold-induced thermogenic capacity of mice. Further, using pharmacological and genetic approaches, we show that ChAMs are regulated via adrenergic signaling, specifically through the ß2 adrenergic receptor. These findings demonstrate that macrophages are an essential adipose tissue source of acetylcholine for the regulation of adaptive thermogenesis, and may be useful for therapeutic targeting in metabolic diseases.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/genética , Macrófagos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Gordura Subcutânea/citologia , Animais , Células Cultivadas , Temperatura Baixa , Deleção de Genes , Técnicas de Inativação de Genes , Camundongos , Cultura Primária de Células , Gordura Subcutânea/metabolismo , TermogêneseRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Linfoma Difuso de Grandes Células B , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Ubiquitina , Proteômica , Recidiva Local de Neoplasia/tratamento farmacológico , Rituximab/uso terapêutico , Vincristina , Ciclofosfamida , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Prednisona , Mutação , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptor Notch2/genéticaRESUMO
Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.
Assuntos
Bancos de Espécimes Biológicos , Criopreservação , Humanos , Linfócitos , Linfonodos/patologia , Células EstromaisRESUMO
Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.
Assuntos
Bronquiolite Obliterante , Proteína Potenciadora do Homólogo 2 de Zeste , Centro Germinativo , Doença Enxerto-Hospedeiro , Proteínas , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/metabolismo , Bronquiolite Obliterante/patologia , Doença Crônica , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/farmacologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Proteínas/metabolismo , TranscriptomaRESUMO
Pan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors might also promote cancer because NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction might combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors.
Assuntos
Leucemia/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Receptor Notch1/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Células Jurkat , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Linfócitos T/patologiaRESUMO
Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Animais , Camundongos , Linfócitos T Reguladores , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Intestino Delgado , InflamaçãoRESUMO
Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.
Assuntos
Fator Ativador de Células B/metabolismo , Transplante de Medula Óssea/efeitos adversos , Doença Enxerto-Hospedeiro/patologia , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Receptor Notch2/metabolismo , Quinase Syk/metabolismo , Linfócitos T/imunologia , Animais , Fator Ativador de Células B/genética , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Isoanticorpos/imunologia , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcr/genética , Receptor Notch2/genética , Quinase Syk/genética , Transplante HomólogoRESUMO
The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.
Assuntos
Transplante de Medula Óssea , Ciclopropanos/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Receptores X de Retinoides/agonistas , Animais , Transplante de Medula Óssea/efeitos adversos , Reposicionamento de Medicamentos , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologiaRESUMO
Intensive chemotherapy with cytarabine and anthracycline (7&3) remains the standard therapy for patients medically fit for induction, but the assessment of fitness remains controversial. Venetoclax and hypomethylating agent (ven/HMA) combination therapy has improved outcomes in unfit patients but no prospective study has assessed ven/HMA versus 7&3 as initial therapy in older, fit patients. Given no studies and expectation of ven/HMA use in patients outside of trial criteria, we evaluated retrospective outcomes among newly diagnosed patients. A nationwide electronic health record (EHR)-derived database and the University of Pennsylvania EHR identified 312 patients receiving 7&3 and 488 receiving ven/HMA who were 60-75 years old without history of organ failure. Ven/HMA patients were older and more likely to have secondary AML, adverse cytogenetics, and adverse mutations. Median overall survival (OS) for patients receiving intensive chemotherapy was 22 versus 10 months for ven/HMA (HR 0.53, 95% CI 0.40-0.60). Controlling for measured baseline characteristic imbalances reduced survival advantage by half (HR 0.71, 95% CI 0.53-0.94). A sub-group of patients with equipoise, likelihood at least 30%-70% of receiving either treatment, had similar OS outcomes (HR 1.10, 95% CI 0.75-1.6). Regarding safety outcomes, 60-day mortality was higher for ven/HMA (15% vs. 6% at 60 days) despite higher documented infections and febrile neutropenia for 7&3. In this multicenter real-word dataset, patients selected for intensive chemotherapy had superior OS but a large group had similar outcomes with ven/HMA. Prospective randomized studies, controlling for both measured and unmeasured confounders, must confirm this outcome.
Assuntos
Citarabina , Leucemia Mieloide Aguda , Humanos , Idoso , Pessoa de Meia-Idade , Estudos Retrospectivos , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversosRESUMO
Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.
Assuntos
Alelos , Fator de Transcrição GATA3/genética , Regulação da Expressão Gênica/genética , Linfócitos T/metabolismo , Animais , Medula Óssea/metabolismo , Proliferação de Células/genética , Células Cultivadas , Fator de Transcrição GATA3/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/genética , Timócitos/citologia , Timócitos/metabolismo , Transativadores/genéticaRESUMO
T-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell-mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb-/-) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb-/- donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Leucemia Experimental/complicações , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Tacrolimo/farmacologia , Animais , Doença Crônica , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologiaRESUMO
Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Linfócitos T CD8-Positivos/metabolismo , Doença Enxerto-Hospedeiro/imunologia , N-Acetilglucosaminiltransferases/metabolismo , Receptores Notch/metabolismo , Animais , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Citometria de Fluxo/métodos , Glicosilação/efeitos dos fármacos , Humanos , Leucossialina/metabolismo , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Sensibilidade e Especificidade , Sialomucinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Estromais/imunologia , Células Estromais/metabolismo , Transplante Homólogo/efeitos adversos , Regulação para CimaRESUMO
Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.
Assuntos
Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Mutação em Linhagem Germinativa/genética , Deficiência Intelectual/genética , Microcefalia/genética , Serina Proteases/genética , Adulto , Criança , Pré-Escolar , Disceratose Congênita/patologia , Feminino , Retardo do Crescimento Fetal/patologia , Células HeLa , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Microcefalia/patologia , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Linhagem , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Serina Proteases/química , Complexo Shelterina , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.
Assuntos
Linfócitos B/imunologia , Bronquiolite Obliterante/complicações , Centro Germinativo/efeitos dos fármacos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-6/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Doença Crônica , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD.