Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for ...polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.
Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) are key drivers of hematopoietic malignancies. Although these mutations are most commonly associated with myeloid diseases, they also ...occur in malignancies of the T-cell lineage. To investigate their role in these diseases and provide tractable disease models for further investigation, we analyzed the T-cell compartment in a conditional knock-in (KI) mouse model of mutant Idh1. We observed the development of a spontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these animals. The disease was transplantable and maintained expression of mutant IDH1. Whole-exome sequencing revealed the presence of a spontaneous activating mutation in Notch1, one of the most common mutations in human T-ALL, suggesting Idh1 mutations may have the capacity to cooperate with Notch1 to drive T-ALL. To further investigate the Idh1 mutation as an oncogenic driver in the T-cell lineage,we crossed Idh1-KI mice with conditional Trp53 null mice, a well-characterized model of T-cell malignancy, and found that T-cell lymphomagenesis was accelerated in mice bearing both mutations. Because both IDH1 and p53 are known to affect cellular metabolism, we compared the requirements for glucose and glutamine in cells derived from these tumors and found that cells bearing the Idh1 mutation have an increased dependence on both glucose and glutamine. These data suggest that mutant IDH1 contributes to malignancy in the T-cell lineage and may alter the metabolic profile of malignant T cells.
UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. ...Because conditional homozygous deletion ofUvragin mice results in early embryonic lethality, we generated T-cell–specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell–specific loss of UVRAG dampened CD8⁺T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis.
TNF receptor-associated factor 2 (TRAF2) is a key intracellular signaling mediator that acts downstream of not only TNFα but also various members of the TNFα superfamily. Here, we report that, ...despite their lack of TNFα signaling, TRAF2–/–TNFα–/– mice develop an inflammatory disorder characterized by autoantibody accumulation and organ infiltration by T cells with the phenotypes of activated, effector, and memory cells. RAG1–/– mice reconstituted with TRAF2–/–TNFα–/– bone marrow cells showed increased numbers of hyperactive T cells and rapidly developed progressive and eventually lethal inflammation. No inflammation was observed in RAG1–/– mice reconstituted with TRAF2–/–TNFα–/–T-cell receptor β–/– or TRAF2–/–TNFα–/–NFκB-induced kinase+/– bone marrow cells. The pathogenic TRAF2–/–TNFα–/– T cells showed constitutive NFκB2p52 activation and produced elevated levels of T-helper 1 and T-helper 17 cytokines. Our results suggest that a regulatory circuit consisting of TRAF2–NFκB-induced kinase–NFκB2p52 is essential for the proper control of effector T-cell polarization and that loss of T-cell TRAF2 function induces constitutive NFκB2p52 activity that drives fatal autoimmune inflammation independently of TNFα signaling. The involvement of this regulatory circuit in controlling autoimmune responses highlights the delicate balance required to avoid paradoxical adverse events when implementing new targeted anti-inflammatory therapies.
Resident macrophages orchestrate homeostatic, inflammatory, and reparative activities. It is appreciated that different tissues instruct specialized macrophage functions. However, individual tissues ...contain heterogeneous subpopulations, and how these subpopulations are related is unclear. We asked whether common transcriptional and functional elements could reveal an underlying framework across tissues. Using single-cell RNA sequencing and random forest modeling, we observed that four genes could predict three macrophage subsets that were present in murine heart, liver, lung, kidney, and brain. Parabiotic and genetic fate mapping studies revealed that these core markers predicted three unique life cycles across 17 tissues. TLF
(expressing TIMD4 and/or LYVE1 and/or FOLR2) macrophages were maintained through self-renewal with minimal monocyte input; CCR2
(TIMD4
LYVE1
FOLR2
) macrophages were almost entirely replaced by monocytes, and MHC-II
macrophages (TIMD4
LYVE1
FOLR2
CCR2
), while receiving modest monocyte contribution, were not continually replaced. Rather, monocyte-derived macrophages contributed to the resident macrophage population until they reached a defined upper limit after which they did not outcompete pre-existing resident macrophages. Developmentally, TLF
macrophages were first to emerge in the yolk sac and early fetal organs. Fate mapping studies in the mouse and human single-cell RNA sequencing indicated that TLF
macrophages originated from both yolk sac and fetal monocyte precursors. Furthermore, TLF
macrophages were the most transcriptionally conserved subset across mouse tissues and between mice and humans, despite organ- and species-specific transcriptional differences. Here, we define the existence of three murine macrophage subpopulations based on common life cycle properties and core gene signatures and provide a common starting point to understand tissue macrophage heterogeneity.
PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in ...macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called "Cluster Similarity Scoring and Distinction Index" (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6C
patrolling monocytes and the expansion properties of peritoneal macrophages. PirB's effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68
Irf8
"neuronal-primed" subset and an CD68
Ftl1
"iron-loaded" subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.
A hallmark of cancer, including pancreatic ductal adenocarcinoma (PDA), is a massive stromal and inflammatory reaction. Many efforts have been made to identify the anti- or protumoral role of ...cytokines and immune subpopulations within the stroma. Here, we investigated the role of interleukin-17A (IL17A) and its effect on tumor fibroblasts and the tumor microenvironment. We used a spontaneous PDA mouse model (KPC) crossed to IL17A knockout mice to show an extensive desmoplastic reaction, without impaired immune infiltration. Macrophages, especially CD80
and T cells, were more abundant at the earlier time point. In T cells, a decrease in FoxP3
cells and an increase in CD8
T cells were observed in KPC/IL17A
mice. Fibroblasts isolated from IL17A
and IL17A
KPC mice revealed very different messenger RNA (mRNA) and protein profiles. IL17A
fibroblasts displayed the ability to restrain tumor cell invasion by producing factors involved in extracellular matrix remodeling, increasing T cell recruitment, and producing higher levels of cytokines and chemokines favoring T helper 1 cell recruitment and activation and lower levels of those recruiting myeloid/granulocytic immune cells. Single-cell quantitative PCR on isolated fibroblasts confirmed a very divergent profile of IL17A-proficient and -deficient cells. All these features can be ascribed to increased levels of IL17F observed in the sera of IL17A
mice, and to the higher expression of its cognate receptor (IL17RC) specifically in IL17A
cancer-associated fibroblasts (CAFs). In addition to the known effects on neoplastic cell transformation, the IL17 cytokine family uniquely affects fibroblasts, representing a suitable candidate target for combinatorial immune-based therapies in PDA.
Oncogenic isocitrate dehydrogenase (IDH)1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite ...2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.
Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including ...abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl–coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.
•IDH1-mutant mice develop myeloid dysplasia with anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts.•D-2-hydroxyglutarate produced by mutated IDH1 inhibits enzymatic activity of oxoglutarate dehydrogenase, reducing succinyl-CoA production and thereby impairing heme synthesis.
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Mutations in
, and
are recurrently observed in myeloid neoplasms.
and
encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). ...Oncogenic
mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms,
,
, and
mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that
and
loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in
cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of
and
mutations, providing support for the optimization of genotype-specific therapies.
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