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The mechanisms that guide T helper 2 (TH2) cell differentiation in barrier tissues are unclear. Here we describe the molecular pathways driving allergen-specific TH2 cells using temporal, spatial and single-cell transcriptomic tracking of house dust mite-specific T cells in mice. Differentiation and migration of lung allergen-specific TH2 cells requires early expression of the transcriptional repressor Blimp-1. Loss of Blimp-1 during priming in the lymph node ablated the formation of TH2 cells in the lung, indicating early Blimp-1 promotes TH2 cells with migratory capability. IL-2/STAT5 signals and autocrine/paracrine IL-10 from house dust mite-specific T cells were essential for Blimp-1 and subsequent GATA3 upregulation through repression of Bcl6 and Bach2. Spatial microniches of IL-2 in the lymph node supported the earliest Blimp-1+TH2 cells, demonstrating lymph node localization is a driver of TH2 initiation. Our findings identify an early requirement for IL-2-mediated spatial microniches that integrate with allergen-driven IL-10 from responding T cells to drive allergic asthma.

Modern multiomic technologies can generate deep multiscale profiles. However, differences in data modalities, multicollinearity of the data, and large numbers of irrelevant features make analyses and integration of high-dimensional omic datasets challenging. Here we present Significant Latent Factor Interaction Discovery and Exploration (SLIDE), a first-in-class interpretable machine learning technique for identifying significant interacting latent factors underlying outcomes of interest from high-dimensional omic datasets. SLIDE makes no assumptions regarding data-generating mechanisms, comes with theoretical guarantees regarding identifiability of the latent factors/corresponding inference, and has rigorous false discovery rate control. Using SLIDE on single-cell and spatial omic datasets, we uncovered significant interacting latent factors underlying a range of molecular, cellular and organismal phenotypes. SLIDE outperforms/performs at least as well as a wide range of state-of-the-art approaches, including other latent factor approaches. More importantly, it provides biological inference beyond prediction that other methods do not afford. Thus, SLIDE is a versatile engine for biological discovery from modern multiomic datasets.

CD8+T cells are critical for elimination of cancer cells. Factors within the tumor microenvironment (TME) can drive these cells to a hypofunctional state known as exhaustion. The most terminally exhausted T (tTex) cells are resistant to checkpoint blockade immunotherapy and might instead limit immunotherapeutic efficacy. Here we show that intratumoral CD8+tTexcells possess transcriptional features of CD4+Foxp3+regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tTexcell suppression requires CD39, which generates immunosuppressive adenosine. Restricted deletion of CD39 in endogenous CD8+T cells resulted in slowed tumor progression, improved immunotherapy responsiveness and enhanced infiltration of transferred tumor-specific T cells. CD39 is induced on tTexcells by tumor hypoxia, thus mitigation of hypoxia limits tTexsuppression. Together, these data suggest tTexcells are an important regulatory population in cancer and strategies to limit their generation, reprogram their immunosuppressive state or remove them from the TME might potentiate immunotherapy.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4–6. At the same time, Tregcells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Tregcells possess a distinct metabolic profile from effector T cells9–11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Tregcells are linked. Here we show that Tregcells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Tregcells in vitro. Tregcells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Tregcells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1—a lactate transporter—in Tregcells reveals that lactate uptake is dispensable for the function of peripheral Tregcells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Tregcells are metabolically flexible: they can use ‘alternative’ metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell–intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.

Regulatory T cells (Tregcells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Tregcells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here, we found that the cytokines IL-10 and IL-35 (Ebi3–IL-12α heterodimer) were divergently expressed by Tregcell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating several inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8+TILs. While expression of BLIMP1 (encoded by Prdm1) was a common target, IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for Tregcell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+TILs that limits effective anti-tumor immunity.

Lymph-node (LN) stromal cell populations expand during the inflammation that accompanies T cell activation. Interleukin-17 (IL-17)-producing helper T cells (TH17 cells) promote inflammation through the induction of cytokines and chemokines in peripheral tissues. We demonstrate a critical requirement for IL-17 in the proliferation of LN and splenic stromal cells, particularly fibroblastic reticular cells (FRCs), during experimental autoimmune encephalomyelitis and colitis. Without signaling via the IL-17 receptor, activated FRCs underwent cell cycle arrest and apoptosis, accompanied by signs of nutrient stress in vivo. IL-17 signaling in FRCs was not required for the development of TH17 cells, but failed FRC proliferation impaired germinal center formation and antigen-specific antibody production. Induction of the transcriptional co-activator IκBζ via IL-17 signaling mediated increased glucose uptake and expression of the gene Cpt1a, encoding CPT1A, a rate-limiting enzyme of mitochondrial fatty acid oxidation. Hence, IL-17 produced by locally differentiating TH17 cells is an important driver of the activation of inflamed LN stromal cells, through metabolic reprogramming required to support proliferation and survival.

Th17 cells drive autoimmune disease but also control commensal microbes. A common link among antigens from self-proteins or commensal microbiota is relatively low activation of T cell receptor (TCR) and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naive T cells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17 regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. Interleukin (IL)-23 and IL-1β promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together, these data propose that, in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1β.

Interleukin (IL)-22–producing innate lymphoid cells (ILCs; ILC22) comprise a heterogeneous population of cells that are dependent on the transcription factor retinoid-related orphan γt (RORγt) and are critical for barrier function of the intestinal mucosa. A distinct ILC22 subset expresses the natural cytotoxicity receptor NKp46 (NKp46+ ILC22); however, the factors that contribute to the generation of this population versus other subsets are largely unknown. Herein, we show that T-bet (encoded by Tbx21) was highly expressed in NKp46+ ILC22, a feature shared by all NKp46+ cells present in the intestine but not by other IL-22–producing populations. Accordingly, the absence of T-bet resulted in loss of NKp46+ ILC22 in the intestinal lamina propria. The residual NKp46+ ILC22 present in Tbx21−/− mice showed a marked reduction of Rorγt expression and impairment in IL-22 production. Generation and functions of gut NK1.1+ cells were also altered. Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets. Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

Myeloid leukemias in AKXD23 mice contain proviral insertions at Evi1, resulting in transcriptional activation. Although Evi1 is clearly involved in leukemia, gene transfer studies in mice with Evi1 fail to cause leukemia, arguing that cooperating events are necessary. We reanalyzed AKXD-23 tumors for cooperating proviral insertion and found that each tumor had a proviral insertion in Sox4, which encodes an HMG-box transcription factor. RNA analysis revealed these insertions cause increased Sox4 expression. Overexpression of Sox4 in 32Dcl3 cells markedly inhibited cytokine-induced granulocyte maturation, as documented by morphologic and mRNA analysis. Sox4-expressing cells had higher levels of transcripts associated with proliferation, including Evi1. Conversely, in leukemic cells that express Sox4 and bear provirally activated Evi1, suppression of Sox4 with short hairpin RNAs resulted in down-regulation of both Sox4 and Evi1. By cotransfection studies, Sox4 is able to transactivate the AKV long terminal repeat, which likely explains how Sox4 transcriptionally up-regulates provirally activated Evi1; however, Sox4 does not appear to regulate the native Evi1 promoter. We propose that Sox4 proviral activation is selected for in the setting of prior proviral activation of Evi1, because it transactivates the relatively weak LTR of AKV leading to higher Evi1 expression and consequent block to differentiation. (Blood. 2006;107:733-741)

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51–66]. Kv1 channel S1–S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1–S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1–S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1–S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1 K+ channels. The S3–S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.

The multiligand receptors megalin and cubilin and their endocytic adaptor protein Dab2 play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells, and have complex and poorly understood roles in the development of chronic kidney disease. Here we used RNA-Seq and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin (Lrp2), cubilin (Cubn), or Dab2 (Dab2) expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in the Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggest changes in pathways with immunomodulatory functions that might trigger the pathologic changes observed in KO mice and in Donnai-Barrow patients. Additionally, differences in transcription patterns between Lrp2 and Dab2 KO cells suggest the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon disruption of PT endocytic function. A reduction in transcripts encoding Sglt2, was confirmed in Lrp2 KO mouse kidney lysates by qPCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.

The STAT3 signaling pathway is required for early Th17 cell development, and therapies targeting this pathway are used for autoimmune disease. However, the role of STAT3 in maintaining inflammatory effector Th17 cell function has been unexplored. Th17ΔSTAT3 mice, which delete STAT3 in effector Th17 cells, were resistant to experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Th17 cell numbers declined after STAT3 deletion, corresponding to reduced cell cycle. Th17ΔSTAT3 cells had increased IL-6–mediated phosphorylation of STAT1, known to have antiproliferative functions. Th17ΔSTAT3 cells also had reduced mitochondrial membrane potential, which can regulate intracellular Ca2+. Accordingly, Th17ΔSTAT3 cells had reduced production of proinflammatory cytokines when stimulated with myelin antigen but normal production of cytokines when TCR-induced Ca2+ flux was bypassed with ionomycin. Thus, early transcriptional roles of STAT3 in developing Th17 cells are later complimented by noncanonical STAT3 functions that sustain pathogenic Th17 cell proliferation and cytokine production.

A Th2 immune response is central to allergic airway inflammation, which afflicts millions worldwide. However, the mechanisms that augment GATA3 expression in an antigen-primed developing Th2 cell are not well understood. Here, we describe an unexpected role for Blimp-1, a transcriptional repressor that constrains autoimmunity, as an upstream promoter of GATA3 expression that is critical for Th2 cell development in the lung to inhaled but not systemically delivered allergens but is dispensable for TFH function and IgE production. Mechanistically, Blimp-1 acts through Bcl6, leading to increased GATA3 expression in lung Th2 cells. Surprisingly, the anti-inflammatory cytokine IL-10, but not the pro-inflammatory cytokines IL-6 or IL-21, is required via STAT3 activation to up-regulate Blimp-1 and promote Th2 cell development. These data reveal a hitherto unappreciated role for an IL-10–STAT3–Blimp-1 circuit as an initiator of an inflammatory Th2 response in the lung to allergens. Thus, Blimp-1 in a context-dependent fashion can drive inflammation by promoting rather than terminating effector T cell responses.