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Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Summary: Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer’s patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

How early-life colonization and subsequent exposure to the microbiota affect long-term tissue immunity remains poorly understood. Here, we show that the development of mucosal-associated invariant T (MAIT) cells relies on a specific temporal window, after which MAIT cell development is permanently impaired. This imprinting depends on early-life exposure to defined microbes that synthesize riboflavin-derived antigens. In adults, cutaneous MAIT cells are a dominant population of interleukin-17A (IL-17A)-producing lymphocytes, which display a distinct transcriptional signature and can subsequently respond to skin commensals in an IL-1-, IL-18-, and antigen-dependent manner. Consequently, local activation of cutaneous MAIT cells promotes wound healing. Together, our work uncovers a privileged interaction between defined members of the microbiota and MAIT cells, which sequentially controls both tissue-imprinting and subsequent responses to injury.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

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-1high TIM3high ("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.

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.

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 ex vivo 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.

Thymic epithelial cells (TEC) are essential for the maturation of functional T cells, while thymus size is proportional to the overall output efficiency. Here the authors show, using transcriptome analyses, that mouse fetal TEC maintain a Myc-dependent genetic program to ensure a rapid increase in thymus size, and thereby expedited T cell generation.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal 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 ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

Thymus involution occurs in all vertebrates. It is thought to impact on immune responses in the aged, and in other clinical circumstances such as bone marrow transplantation. Determinants of thymus growth and size are beginning to be identified. Ectopic expression of factors like cyclin D1 and Myc in thymic epithelial cells (TEC)s results in considerable increase in thymus size. These models provide useful experimental tools that allow thymus function to be understood. In future, understanding TEC-specific controllers of growth will provide new approaches to thymus regeneration.

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.

Abstract 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.

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.