Search

Increasing evidence indicates close interaction between immune cells and the brain, revising the traditional view of the immune privilege of the brain. However, the specific mechanisms by which immune cells promote normal neural function are not entirely understood. Mucosal-associated invariant T cells (MAIT cells) are a unique type of innate-like T cell with molecular and functional properties that remain to be better characterized. In the present study, we report that MAIT cells are present in the meninges and express high levels of antioxidant molecules. MAIT cell deficiency in mice results in the accumulation of reactive oxidative species in the meninges, leading to reduced expression of junctional protein and meningeal barrier leakage. The presence of MAIT cells restricts neuroinflammation in the brain and preserves learning and memory. Together, our work reveals a new functional role for MAIT cells in the meninges and suggests that meningeal immune cells can help maintain normal neural function by preserving meningeal barrier homeostasis and integrity.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cell populations formed in response. This encoding emerges from the decisions of lymphocytes to maintain or lose self-renewal and memory potential during a challenge. By tracking responding CD8 T cells at the single-cell and clonal lineage level using time-resolved transcriptomics and quantitative live imaging, we identify a remarkably flexible decision-making strategy, whereby T cells initially choose whether to maintain or lose memory potential early after antigen recognition, but following pathogen clearance may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by acis-epigenetic switch that silences the memory regulator TCF1 in a stochastic and reversible manner in response to stimulatory inputs. Mathematical modeling shows how this strategy allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decision making ensures optimal immune responses against diverse threats.

Innate lymphoid cells (ILCs) are transcriptionally and functionally similar to T cells but lack adaptive antigen receptors. They play critical roles in early defense against pathogens. In this review, we summarize recent discoveries of ILC progenitors and discuss possible mechanisms that separate ILCs from T cells. We consider mechanisms of lineage specification in early ILC development and also examine whether differences exist between adult and fetal ILC development.

In this issue of Cell Stem Cell, Jing et al. inhibit EZH1 expression in a system that supports mature T cell development from iPSCs in vitro. The authors efficiently generate T cells that are highly functional against tumors.

The Global Thymus Network convened virtually for ThymUS 2020 on 3–4 November 2020. Participants shared recent advances in thymus biology and enjoyed a reprieve from 2020 stressors.

Regulatory elements activate promoters by recruiting transcription factors (TFs) to specific motifs. Notably, TF-DNA interactions often depend on cooperativity with colocalized partners, suggesting an underlying cis-regulatory syntax. To explore TF cooperativity in mammals, we analyze ∼500 mouse and human primary cells by combining an atlas of TF motifs, footprints, ChIP-seq, transcriptomes, and accessibility. We uncover two TF groups that colocalize with most expressed factors, forming stripes in hierarchical clustering maps. The first group includes lineage-determining factors that occupy DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprises ∼30 SP, KLF, EGR, and ZBTB family members that recognize overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking reveal that USFs impart accessibility to colocalized partners and increase their residence time. Mammalian cells have thus evolved a TF superfamily with overlapping DNA binding that facilitate chromatin accessibility.

Innate lymphoid cells (ILCs) are a family of immune cells that possess similar functions as T cells. We review steps of central ILC development in the bone marrow of adult mice and discuss recent evidence for peripheral ILC development suggesting extramedullary sites of ILC development. We also assess the contribution of development during different phases of life towards shaping the composition of the adult ILC pool. Finally, we briefly review the local cues that lead to heterogeneity of ILCs between tissues. We propose that tissue-resident ILC progenitors may economically allow tissues to elicit rapid expansion of specific ILC types that are needed based on the nature of antigenic assaults in tissues.

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

The thymus is required for the development of both adaptive and innate-like T cell subsets. There is keen interest in manipulating thymic function for therapeutic purposes in circumstances of autoimmunity, immunodeficiency, and for purposes of immunotherapy. Within the thymus, thymic epithelial cells play essential roles in directing T cell development. Several transcription factors are known to be essential for thymic epithelial cell development and function, and a few transcription factors have been studied in considerable detail. However, the role of many other transcription factors is less well understood. Further, it is likely that roles exist for other transcription factors not yet known to be important in thymic epithelial cells. Recent progress in understanding of thymic epithelial cell heterogeneity has provided some new insight into transcriptional requirements in subtypes of thymic epithelial cells. However, it is unknown whether progenitors of thymic epithelial cells exist in the adult thymus, and consequently, developmental relationships linking putative precursors with differentiated cell types are poorly understood. While we do not presently possess a clear understanding of stage-specific requirements for transcription factors in thymic epithelial cells, new single-cell transcriptomic and epigenomic technologies should enable rapid progress in this field. Here, we review our current knowledge of transcription factors involved in the development, maintenance, and function of thymic epithelial cells, and the mechanisms by which they act.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive (“exhausted” or “dysfunctional”) within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8 + T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8 + CAR + PD-1 high TIM3 high (“exhausted”) tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 ( Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8 + T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

Group 2 innate lymphoid cells (ILC2) are tissue-resident, long-lived innate effector cells implicated in allergy and asthma. Upon activation, mature ILC2 rapidly secrete large amounts of type-2 cytokines and other effector molecules. The molecular pathways that drive ILC2 activation are not well understood. In this study, we report that the transcriptional controller core binding factor β (CBFβ) is required for ILC2 activation. Deletion or inhibition of CBFβ did not impair the maintenance of ILC2 at homeostasis but abolished ILC2 activation during allergic airway inflammation. Treatment with CBFβ inhibitors prevented ILC2-mediated airway hyperresponsiveness in a mouse model of acute Alternaria allergen inhalation. CBFβ promoted expression of key ILC2 genes at both transcriptional and translational levels. CBF transcriptional complex directly bound to Il13 and Vegfa promoters and enhancers, and controlled gene transcription. CBFβ further promoted ribosome biogenesis and enhanced gene translation in activated ILC2. Together, these data establish an essential role for CBFβ in ILC2 activation.

Checkpoint blockade can reverse CD8+ T-cell functional exhaustion, and TCF-1 is essential for this process. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence in checkpoint blockade non-responsive tumors remains a challenge. We demonstrate that targeting Cbx3/HP1γ causes augmented transcription initiation, chromatin remodeling at Lef1 and Il21r leading to increased transcriptional activity at these loci. Mechanistic studies show LEF-1 and IL-21R are required for Cbx3/HP1γ-deficient CD8+ effector T cells to persist resulting in improved control of ovarian cancer, melanoma and neuroblastoma in pre-clinical models. Cbx3/HP1γ-deficient CD8+ T cells enhanced persistence in the TME facilitates remodeling of the chemokine/receptor landscape that ensures their optimal tumor invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by checkpoint blockade, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for resistant, non-responsive solid tumors.

Helper innate lymphoid cells (ILCs) combat pathogen infection by rapid secretion of cytokines whereas killer ILCs (NK cells) eliminate virus-infected cells or tumor cells during early defense. However, the early development of NK cells and lineage relationships between NK cells and helper ILCs remain elusive. In PLZF lineage tracing model, normal NK cells are traced at a much lower ratio than helper ILCs, which indicates most NK cells develop from progenitors that do not express PLZF. However, it is also possible that most NK cells develop from PLZF-expressing ILC progenitors (ILCPs), but PLZF-labelled NK cells may proliferate less than unlabeled NK cells. To address these possibilities, we transferred ILCPs and its upstream progenitors early innate lymphoid progenitors (EILPs) and all lymphoid progenitors (ALPs), and then assessed PLZF labelling in NK cells derived from these progenitors. We found that ILCP-derived NK cells were traced at a ratio as high as in helper ILCs whereas EILP and ALP-derived NK cells were labelled at a much lower ratio, which is similar to the ratio in normal NK cells. These results indicate that NK cells develop from both ILCP-dependent and ILCP-independent pathways. Our findings establish novel lineage relationships between NK cells and helper ILCs. We are currently assessing whether different developmental pathways of NK cells lead to functionally distinct subsets of NK cells.

Innate T cells and innate lymphoid cells in barrier tissues are essential for both tissue fitness and immunity against pathogens. γδTCR+ lymphocytes that secrete IL-17 (Tγδ17) localize to dermis and are critical regulators of skin immune responses. Despite their functional importance, how Tγδ17 cells mediate their unique function in the skin is largely unknown. Tγδ17 cells express the two prototypic G-protein coupled receptors (GPCRs) CCR6 and GPR183 (EBI2): mice lacking both receptors had significantly diminished dermal Tγδ17 cells and were resistant to psoriasis induction. This was due to a block in Tγδ17 cell maturation in the thymus of Ccr6−/−Gpr183−/− double knock-out mice. EBI2 recognizes oxysterols generated by the cholesterol hydroxylase CH25H and functional oxysterols were detected in both the thymus and skin. Analysis of Ch25h-reporter mice showed that interfollicular epithelial cells (IFEs) and a subset of medullary thymic epithelial cells (mTEC) are the depot for oxysterols in skin and thymus respectively. Ch25h+ TECs were also identified by unbiased single cell transcriptome analyses. These results demonstrate that developing Tγδ17 cells must be conditioned by sensing cholesterol byproducts synthesized by a discrete subset of mTECs to position in specific oxysterol rich niches in the skin. Moreover, in the skin, dietary cholesterol increased tissue oxysterol concentration and modulated homeostatic and pathogenic effector function of Tγδ17 cells in a EBI2 dependent fashion. Given that oxysterols can be regulated by infection and inflammation, this sensory circuit directed by cholesterol is predicted to program skin innate T cells’ ability to rapidly survey for perturbations in the skin. Supported by grant from NIH R21AI143225.

Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8(+) T cells that possessed distinct characteristics including expression of CD62L, TCF-1 and Eomes; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1(+) pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8(+) T cells, coupled with elevated expression of multiple inhibitory receptors including PD-1, LAG-3, CTLA-4, CD5, and CD160. Indeed, genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62L(hi) TCF-1(+) subset and subsequent CD8(+) T cell memory. Although central memory phenotype CD8(+) T cells (T(CM)) were formed in the absence of these cells, subsequent memory CD8(+) T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8(+) T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8(+) T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8(+) T cell precursor pool may help reconcile models of the developmental origin of long-term CD8(+) T cell memory.

International audience; The transcription factor TCF-1 (encoded by Tcf7) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7. We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7-expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs.

Bcl11b binds to distinctive genomic regions with different partners and regulates completely different target genes in pro-T and ILC2 cells. Despite these divergences in Bcl11b function, a shared enhancer supports initial Bcl11b locus opening in both pro-T and ILC2 lineages., The zinc finger transcription factor, Bcl11b, is expressed in T cells and group 2 innate lymphoid cells (ILC2s) among hematopoietic cells. In early T-lineage cells, Bcl11b directly binds and represses the gene encoding the E protein antagonist, Id2, preventing pro-T cells from adopting innate-like fates. In contrast, ILC2s co-express both Bcl11b and Id2. To address this contradiction, we have directly compared Bcl11b action mechanisms in pro-T cells and ILC2s. We found that Bcl11b binding to regions across the genome shows distinct cell type–specific motif preferences. Bcl11b occupies functionally different sites in lineage-specific patterns and controls totally different sets of target genes in these cell types. In addition, Bcl11b bears cell type–specific post-translational modifications and organizes different cell type–specific protein complexes. However, both cell types use the same distal enhancer region to control timing of Bcl11b activation. Therefore, although pro-T cells and ILC2s both need Bcl11b for optimal development and function, Bcl11b works substantially differently in these two cell types., Graphical Abstract

Ghaedi et al. identify IL-18R+IL-33R− ILC progenitors, which differentiate into multiple ILC lineages, in the lung of neonatal and adult RORα lineage tracer mice. ILC2s in neonatal mouse lungs are divided into distinct cytokine and amphiregulin-producing effector ILC2s., Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor–related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2− population different from conventional IL-18Rα−ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

Harly et al. show that early innate lymphoid progenitors (EILPs) are a developmental intermediate between all-lymphoid progenitors (ALPs) and ILC precursors (ILCps) and identify requirements for their generation and further differentiation., Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor–successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development.

The mechanisms underlying lymphocyte lineage stability and plasticity remain elusive. Recent work indicates that innate lymphoid cells (ILC) possess substantial plasticity. Whereas natural ILC2 (nILC2) produce type-2 cytokines, plastic inflammatory ILC2 (iILC2) can coproduce both type-2 cytokines and the ILC3-characteristic cytokine, IL-17. Mechanisms that elicit this lineage plasticity, and the importance in health and disease, remain unclear. In this study we show that iILC2 are potent inducers of airway inflammation in response to acute house dust mite challenge. We find that Notch signaling induces lineage plasticity of mature ILC2 and drives the conversion of nILC2 into iILC2. Acute blockade of Notch signaling abolished functional iILC2, but not nILC2, in vivo. Exposure of isolated nILC2 to Notch ligands induced Rorc expression and elicited dual IL-13/IL-17 production, converting nILC2 into iILC2. Together these results reveal a novel role for Notch signaling in eliciting ILC2 plasticity and driving the emergence of highly proinflammatory innate lymphocytes.

Nish et al. report that production of a fully committed Th1 effector cell occurs during an asymmetric cell division wherein the other daughter cell remains memory cell–like. Unequal transmission of metabolic signaling may be the driver of this regenerative behavior., Upon infection, an activated CD4+ T cell produces terminally differentiated effector cells and renews itself for continued defense. In this study, we show that differentiation and self-renewal arise as opposing outcomes of sibling CD4+ T cells. After influenza challenge, antigen-specific cells underwent several divisions in draining lymph nodes (LN; DLNs) while maintaining expression of TCF1. After four or five divisions, some cells silenced, whereas some cells maintained TCF1 expression. TCF1-silenced cells were T helper 1–like effectors and concentrated in the lungs. Cells from earliest divisions were memory-like and concentrated in nondraining LN. TCF1-expressing cells from later divisions in the DLN could self-renew, clonally yielding a TCF1-silenced daughter cell as well as a sibling cell maintaining TCF1 expression. Some TCF1-expressing cells in DLNs acquired an alternative, follicular helper-like fate. Modeled differentiation experiments in vitro suggested that unequal PI3K/mechanistic target of rapamycin signaling drives intraclonal cell fate heterogeneity. Asymmetric division enables self-renewal to be coupled to production of differentiated CD4+ effector T cells during clonal selection.

The transcription factor TCF-1 (encoded by Tcf7) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7. We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7-expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8

Innate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Interestingly, ILCs and T cells express many of the same transcription factors. One such factor is TOX, a sequence non-specific nuclear transcriptional regulator that is crucial for development of both CD4 T cells and ILCs. TOX-deficient mice have significantly reduced number of early innate lymphoid progenitors (EILP) and ILC progenitors (ILCP). Consequently, the mature NK cells, ILC1, ILC2 and ILC3 subsets are greatly reduced in Tox−/− mice. Interestingly ILC3 numbers in gut are not reduced by the absence of TOX. We have discovered that TOX2, another member of the TOX family of HMG-box proteins, is highly expressed in ILC precursors. We generated Tox2−/− mice and found they have reduced ILC3 in gut; however, ILC3 cells in spleen or lymph node are intact. We also observed ILCP in fetal gut and adult gut were significantly reduced in Tox2−/−mice suggesting TOX2 is required for gut ILC3 development. However, ILCP numbers in adult gut of Tox−/− mice were unaffected. We are currently performing single-cell RNA-sequencing on gut ILC3 from Tox2−/− and Tox−/− mice to transcriptionally characterize these cells and to identify the heterogeneity within the ILC3 population. Taken together, our results indicate the existence of at least two pathways of ILC development: a central pathway in bone marrow and fetal liver that is TOX dependent; and a peripheral pathway of development of ILC3 in gut that requires TOX2.

Maui, Hawaii, was the home for the latest Global Thymus Network meeting 5-9 June 2016. Participants gathered together to discuss how one little organ can have such huge effects.

The worldwide spread of invasive Aedes mosquitoes and arboviral disease, have renewed the pressure for effective and sustainable urban mosquito control. We report on the success of a model we are confident will usher in a new era of urban mosquito control. The key innovation is the mobilization of neighbors guided by scientific advisors, an approach we termed Citizen Action through Science (Citizen AcTS). This approach was tested in a NE US town of approximately 1,000 residential yards infested with the invasive Asian tiger mosquito, Aedes albopictus, a major nuisance arboviral vector. We report a highly significant reduction in biting pressure that was maintained over time, and establish the thresholds needed for success. The Citizen AcTS model rejects the top-down approach consistently associated with intervention failures. Instead, it works through respectful exchanges among scientists and residents that lead to trust and individual ‘buy-in’ and transferring program ownership to the community.

Discussion of how Eph-ephrin signaling is required for efficient generation of the ETP.

In this issue of Blood , Rodrigues et al use a conditional knockout mouse model to describe a novel role for p53 in the differentiation and maintenance of medullary thymic epithelial cells (mTECs), tissue-restricted antigen (TRA) expression, and self-tolerance. 1

The mechanisms underlying lymphocyte lineage stability and plasticity remain elusive. Recent work indicates that innate lymphoid cells (ILC) possess substantial plasticity. Whereas natural ILC2 (nILC2) produce type-2 cytokines, plastic inflammatory ILC2 (iILC2) can coproduce both type-2 cytokines and the ILC3-characteristic cytokine, IL-17. Mechanisms that elicit this lineage plasticity, and the importance in health and disease, remain unclear. In this study we show that iILC2 are potent inducers of airway inflammation in response to acute house dust mite challenge. We find that Notch signaling induces lineage plasticity of mature ILC2 and drives the conversion of nILC2 into iILC2. Acute blockade of Notch signaling abolished functional iILC2, but not nILC2, in vivo. Exposure of isolated nILC2 to Notch ligands induced Rorc expression and elicited dual IL-13/IL-17 production, converting nILC2 into iILC2. Together these results reveal a novel role for Notch signaling in eliciting ILC2 plasticity and driving the emergence of highly proinflammatory innate lymphocytes.

How the immune system adapts to malnutrition to sustain immunity at barrier surfaces, such as the intestine, remains unclear. Vitamin A deficiency is one of the most common micronutrient deficiencies and is associated with profound defects in adaptive immunity. Here, we found that type 3 innate lymphoid cells (ILC3s) are severely diminished in vitamin A–deficient settings, which results in compromised immunity to acute bacterial infection. However, vitamin A deprivation paradoxically resulted in dramatic expansion of interleukin-13 (IL-13)–producing ILC2s and resistance to nematode infection in mice, which revealed that ILCs are primary sensors of dietary stress. Further, these data indicate that, during malnutrition, a switch to innate type 2 immunity may represent a powerful adaptation of the immune system to promote host survival in the face of ongoing barrier challenges.

Adoptive cell therapy with chimeric antigen receptor expressing T (CAR T) cells has shown promising therapeutic efficacy against leukemia and lymphoma. However, CAR T cells in solid tumors fail to be as effective as in liquid tumors since they enter into a hyporesponsive (exhausted or dysfunctional) state that is induced by chronic antigen stimulation in cancer. Here, we show that CAR T cells in solid tumors exhibit low effector function and high expression of inhibitory receptors such as PD-1, Tim-3 and Lag 3. We also show that the nuclear factor, Thymocyte selection-associated HMG box protein, TOX - a target of the transcription factor NFAT - is highly expressed in CAR T cells in solid tumors with PD-1 expression. To determine how TOX transcription factors contribute to exhaustion/ dysfunction in CAR T cells, we developed CAR T cells targeting huCD19 and lacking TOX and TOX 2. Surprisingly, treatment of huCD19 expressing melanoma or colorectal tumor-bearing mice with CAR T cells doubly deficient in TOX and TOX2 resulted in the eradication of solid tumors and prolonged survival of the tumor-bearing mice, compared to mice treated with WT CAR T cells or CAR T cells lacking TOX or TOX2 alone. Tumor-infiltrating CAR T cells (CAR TILs) doubly deficient in TOX and TOX2 did not increase their expression of inhibitory receptors such as PD-1, Lag3 and CD160 and showed higher expression of TCF1 compared to WT CAR TILs. Our data identify TOX and TOX2 -high mobility group transcription factors belonging to the TOX family - as critical transcriptional factors for inducing hyporesponsiveness in CAR T cells in solid tumors. Our data suggest that disruption of TOX expression or activity could be a promising strategy for cancer immunotherapy. Citation Format: Hyungseok Seo, Joyce Chen, Arundhoti Das, Avinash Bhandoola, Anjana Rao. Disruption of TOX overcomes CAR T-cell dysfunction function in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 938.

T cells expressing chimeric antigen receptors (CARs) have shown impressive therapeutic efficacy against leukemias and lymphomas, but have not been as effective against solid tumors because chronic stimulation with tumor antigens causes them to enter a hyporesponsive (“exhausted” or “dysfunctional”) state. Here we show that the high-mobility group (HMG)-box transcription factors TOX and TOX2 are highly expressed in CAR-expressing exhausted (PD-1highTim3high) CD8+ tumor-infiltrating lymphocytes (TILs). CAR TILs deficient in both TOX and TOX2 (TOX DKO) suppressed tumor growth and prolonged survival of tumor-bearing mice, compared to wildtype (WT), TOX-deficient or TOX2-deficient CAR TILs; they also showed increased effector function with increased TCF1 expression and decreased expression of the inhibitory receptors PD-1, LAG3 and CD160. Like NR4A transcription factors, TOX and TOX2 are critical for the transcriptional program of hyporesponsiveness downstream of NFAT. Disruption of TOX expression or activity could be a promising strategy for cancer immunotherapy.

Summary Group 2 innate lymphoid cells (ILC2) are innate lymphocytes that confer protective type 2 immunity during helminth infection and are also involved in allergic airway inflammation. Here we report that ILC2 development required T cell factor 1 (TCF-1, the product of the Tcf7 gene), a transcription factor also implicated in T cell lineage specification. Tcf7 −/− mice lack ILC2, and were unable to mount ILC2-mediated innate type 2 immune responses. Forced expression of TCF-1 in bone marrow progenitors partially bypassed the requirement for Notch signaling in the generation of ILC2 in vivo. TCF-1 acted through both GATA-3-dependent and GATA-3-independent pathways to promote the generation of ILC2. These results are reminiscent of the critical roles of TCF-1 in early T cell development. Hence, transcription factors that underlie early steps of T cell development are also implicated in the development of innate lymphoid cells.

The mouse thymus supports T-cell development, but also contains non–T-cell lineages such as dendritic cells, macrophages, and granulocytes that are necessary for T-cell repertoire selection and apoptotic thymocyte clearance. Early thymic progenitors (ETPs) are not committed to the T-cell lineage, as demonstrated by both in vitro and in vivo assays. Whether ETPs realize non–T-cell lineage potentials in vivo is not well understood and indeed is controversial. In the present study, we investigated whether ETPs are the major precursors of any non–T-lineage cells in the thymus. We analyzed the development of these populations under experimental circumstances in which ETPs are nearly absent due to either abrogated thymic settling or inhibition of early thymic development by genetic ablation of IL-7 receptorα or Hes1. Results obtained using multiple in vivo approaches indicate that the majority of thymic granulocytes derive from ETPs. These data indicate that myelolymphoid progenitors settle the thymus and thus clarify the pathways by which stem cells give rise to downstream blood cell lineages.

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML MLL cells (with an MLL-ENL fusion and a constitutively active N-RAS) independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML MLL and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML MLL mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Selected CD8+ T cells must divide, produce differentiated effector cells, and self-renew, often repeatedly. We now show that silencing expression of the transcription factor TCF1 marks loss of self-renewal by determined effector cells, and that this requires cell division. In acute infections, the first three CD8+ T cell divisions produce daughter cells with unequal proliferative signaling but uniform maintenance of TCF1 expression. The more quiescent initial daughter cells resemble canonical central memory cells. The more proliferative, effector-prone cells from initial divisions can subsequently undergo division-dependent production of a TCF1-negative effector daughter cell along with a self-renewing TCF1-positive daughter cell, the latter also contributing to the memory cell pool upon resolution of infection. Self-renewal in the face of effector cell determination may promote clonal amplification and memory cell formation in acute infections, sustain effector regeneration during persistent subclinical infections, and be rate-limiting, but remediable, in chronic active infections and cancer.

BackgroundAsthmatic patients are highly susceptible to air pollution and in particular to the effects of ozone (O3) inhalation, but the underlying mechanisms remain unclear.ObjectiveUsing mouse models of O3-induced airway inflammation and airway hyperresponsiveness (AHR), we sought to investigate the role of the recently discovered group 2 innate lymphoid cells (ILC2s).MethodsC57BL/6 and BALB/c mice were exposed to Aspergillus fumigatus, O3, or both (3 ppm for 2 hours). ILC2s were isolated by means of fluorescence-activated cell sorting and studied for Il5 and Il13 mRNA expression. ILC2s were depleted with anti-Thy1.2 mAb and replaced by means of intratracheal transfer of exvivo expanded Thy1.1 ILC2s. Cytokine levels (ELISA and quantitative PCR), inflammatory cell profile, and AHR (flexiVent) were assessed in the mice.ResultsIn addition to neutrophil influx, O3 inhalation elicited the appearance of eosinophils and IL-5 in the airways of BALB/c but not C57BL/6 mice. Although O3-induced expression of IL-33, a known activator of ILC2s, in the lung was similar between these strains, isolated pulmonary ILC2s from O3-exposed BALB/c mice had significantly greater Il5 and Il13 mRNA expression than C57BL/6 mice. This suggested that an altered ILC2 function in BALB/c mice might mediate the increased O3 responsiveness. Indeed, anti-Thy1.2 treatment abolished but ILC2s added back dramatically enhanced O3-induced AHR.ConclusionsO3-induced activation of pulmonary ILC2s was necessary and sufficient to mediate asthma-like changes in BALB/c mice. This previously unrecognized role of ILC2s might help explain the heightened susceptibility of human asthmatic airways to O3 exposure.