RESUMEN
Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.
Asunto(s)
Citrobacter rodentium , Infecciones por Enterobacteriaceae , Glucólisis , Proteínas HMGB , Inmunidad Innata , Linfocitos , Ratones Noqueados , Animales , Ratones , Adaptación Fisiológica/inmunología , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/genética , Interleucina-17/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Transactivadores/metabolismo , Transactivadores/genética , Proteínas HMGB/genética , Proteínas HMGB/inmunología , Proteínas HMGB/metabolismoRESUMEN
During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer's patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 axis as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.
RESUMEN
During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer's patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 pathway as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.
RESUMEN
Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.
Asunto(s)
Neoplasias , Sialiltransferasas , Animales , Ratones , Ratones Noqueados , MicroARNs/genética , MicroARNs/inmunología , Ácido N-Acetilneuramínico/inmunología , Neoplasias/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Sialiltransferasas/genética , Sialiltransferasas/inmunologíaRESUMEN
After antigenic activation, quiescent naive CD4+ T cells alter their metabolism to proliferate. This metabolic shift increases production of nucleotides, amino acids, fatty acids, and sterols. Here, we show that histone deacetylase 3 (HDAC3) is critical for activation of murine peripheral CD4+ T cells. HDAC3-deficient CD4+ T cells failed to proliferate and blast after in vitro TCR/CD28 stimulation. Upon T-cell activation, genes involved in cholesterol biosynthesis are upregulated while genes that promote cholesterol efflux are repressed. HDAC3-deficient CD4+ T cells had reduced levels of cellular cholesterol both before and after activation. HDAC3-deficient cells upregulate cholesterol synthesis appropriately after activation, but fail to repress cholesterol efflux; notably, they overexpress cholesterol efflux transporters ABCA1 and ABCG1. Repression of these genes is the primary function for HDAC3 in peripheral CD4+ T cells, as addition of exogenous cholesterol restored proliferative capacity. Collectively, these findings demonstrate HDAC3 is essential during CD4+ T-cell activation to repress cholesterol efflux.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Colesterol/metabolismo , Histona Desacetilasas/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Animales , Colesterol/genética , Femenino , Histona Desacetilasas/genética , Masculino , Ratones , Ratones MutantesRESUMEN
Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Stimulated ABCB7-deficient splenic B cells from CD23-cre mice also had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.
Asunto(s)
Linfocitos B/fisiología , Cambio de Clase de Inmunoglobulina , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Proliferación Celular , Daño del ADN , Femenino , Hierro/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Bazo/citología , Azufre/metabolismoRESUMEN
Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.
Asunto(s)
Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Vigilancia Inmunológica , Animales , Puntos de Control del Ciclo Celular , Línea Celular , Quimiocinas CXC/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Genes ras , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína de Retinoblastoma/metabolismo , Estrés Fisiológico , Linfocitos T Citotóxicos/inmunología , Transcripción GenéticaRESUMEN
Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.
Asunto(s)
Inmunidad Innata/inmunología , Inmunidad/inmunología , Sialiltransferasas/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Transfección , Microambiente TumoralRESUMEN
The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS.
Asunto(s)
Subgrupos de Linfocitos B/fisiología , Linfocitos B/fisiología , Células de la Médula Ósea/fisiología , Sistema Nervioso Central/inmunología , Duramadre/citología , Linfopoyesis , Meninges/citología , Meninges/inmunología , Cráneo/anatomía & histología , Envejecimiento , Animales , Subgrupos de Linfocitos B/inmunología , Movimiento Celular , Sistema Nervioso Central/fisiología , Duramadre/inmunología , Fibroblastos/fisiología , Homeostasis , Privilegio Inmunológico , Ratones , Células Plasmáticas/fisiología , Análisis de la Célula IndividualRESUMEN
Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.
Asunto(s)
COVID-19/inmunología , COVID-19/virología , Autorrenovación de las Células/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , SARS-CoV-2/inmunología , Biomarcadores , COVID-19/metabolismo , Citocinas/metabolismo , Susceptibilidad a Enfermedades/inmunología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Transducción de SeñalRESUMEN
TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos/metabolismo , Células Germinativas/metabolismo , Análisis por Matrices de Proteínas , Animales , Humanos , Masculino , Ratones , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Testículo/metabolismoRESUMEN
Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The roles of different FA species and their impacts on humoral immunity remain poorly understood. Here, we report that proliferating B cells require monounsaturated FAs (MUFAs) to maintain mitochondrial metabolism and mTOR activity and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Furthermore, B cell-extrinsic stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity.
Asunto(s)
Linfocitos B/fisiología , Ácidos Grasos Monoinsaturados/inmunología , Ácidos Grasos Monoinsaturados/metabolismo , Inmunidad Humoral , Mitocondrias/fisiología , Estearoil-CoA Desaturasa/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia , Estrés del Retículo Endoplásmico , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/inmunología , Serina-Treonina Quinasas TOR/inmunologíaRESUMEN
The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic ß cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.
Asunto(s)
Diabetes Mellitus/inducido químicamente , Diabetes Mellitus/metabolismo , Sialiltransferasas/metabolismo , Estreptozocina/farmacología , Animales , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Diferenciación de Linfocitos B/metabolismo , Autoinmunidad/inmunología , Diabetes Mellitus/inmunología , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Tolerancia Inmunológica/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Ligandos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Sialiltransferasas/inmunologíaRESUMEN
T cell development involves stepwise progression through defined stages that give rise to multiple T cell subtypes, and this is accompanied by the establishment of stage-specific gene expression. Changes in chromatin accessibility and chromatin modifications accompany changes in gene expression during T cell development. Chromatin-modifying enzymes that add or reverse covalent modifications to DNA and histones have a critical role in the dynamic regulation of gene expression throughout T cell development. As each chromatin-modifying enzyme has multiple family members that are typically all coexpressed during T cell development, their function is sometimes revealed only when two related enzymes are concurrently deleted. This work has also revealed that the biological effects of these enzymes often involve regulation of a limited set of targets. The growing diversity in the types and sites of modification, as well as the potential for a single enzyme to catalyze multiple modifications, is also highlighted.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Linfopoyesis , Linfocitos T/inmunología , Linfocitos T/metabolismo , Acetilación , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Histonas , Humanos , Linfopoyesis/genética , Linfopoyesis/inmunología , Metilación , Procesamiento Proteico-Postraduccional , Linfocitos T/citología , Linfocitos T/enzimología , UbiquitinaciónRESUMEN
NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.
RESUMEN
NKAP and HDAC3 are critical for T cell maturation. NKAP and HDAC3 physically associate, and a point mutation in NKAP, NKAP(Y352A), abrogates this interaction. To evaluate the significance of NKAP and HDAC3 association in T cell maturation, transgenic mice were engineered for cre-mediated endogenous NKAP gene deletion coupled to induction of NKAP(Y352A) or a wild type (WT) control transgene, NKAP(WT), in double positive thymocytes or regulatory T cells (Tregs). T cell maturation was normal in mice with endogenous NKAP deletion coupled to NKAP(WT) induction. However, severe defects occurred in T cell and Treg maturation and in iNKT cell development when NKAP(Y352A) was induced, recapitulating NKAP deficiency. Conventional T cells expressing NKAP(Y352A) failed to enter the long-term T cell pool, did not produce cytokines, and remained complement susceptible, whereas Tregs expressing NKAP(Y352A) were eliminated as recent thymic emigrants leading to lethal autoimmunity. Overall, these results demonstrate the significance of NKAP-HDAC3 association in T cells.
Asunto(s)
Diferenciación Celular/fisiología , Histona Desacetilasas/metabolismo , Células T Asesinas Naturales/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T Reguladores/metabolismo , Timocitos/metabolismo , Animales , Autoinmunidad/genética , Células Cultivadas , Activación de Complemento , Complemento C3/inmunología , Citocinas/metabolismo , Femenino , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Peroxidación de Lípido/genética , Masculino , Ratones , Ratones Noqueados , Mutación Puntual , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Timo/citologíaRESUMEN
Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.
Asunto(s)
Diferenciación Celular/inmunología , Complemento C3/inmunología , Lectina de Unión a Manosa/inmunología , Proteínas Represoras/inmunología , Linfocitos T/inmunología , Animales , Linfopenia/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Timocitos/inmunología , Timo/inmunología , Transcripción Genética/inmunologíaRESUMEN
Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4+ T cells directed by the chemoattractant receptor Ebi2. In the absence of Ebi2, CD4+ T cells lose their location bias and are delayed in antigen recognition, proliferative expansion, differentiation, direct effector activity, and provision of help for CD8+ T cell-mediated memory responses, limiting host defense and vaccine responses. These findings demonstrate evolutionary selection for distinct niches within the LN that promote cellular responses, emphasizing the critical link between fine-grained tissue organization and host defense.
Asunto(s)
Inmunidad Adaptativa/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Presentación de Antígeno/inmunología , Antígenos/inmunología , Diferenciación Celular/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/genéticaRESUMEN
NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Histona Desacetilasas/inmunología , Proteínas Represoras/inmunología , Sustitución de Aminoácidos , Animales , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Embrión de Mamíferos/citología , Embrión de Mamíferos/inmunología , Fibroblastos/citología , Fibroblastos/inmunología , Células HEK293 , Células Madre Hematopoyéticas/citología , Histona Desacetilasas/genética , Humanos , Ratones , Ratones Transgénicos , Mutación Missense , Proteínas Represoras/genéticaRESUMEN
Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression.