CAR T cells are in it [CD]4 the long haul.

In a recent issue of Nature, Melenhorst et al. perform an extensive analysis of CAR T cells that persist for ten years in CLL patients. They find a dominant cytotoxic CD4+ population, raising the possibility that CD4+ T cells play an important role in durable CAR T cell therapy.

Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

Hypoxia drives CD39-dependent suppressor function in exhausted T cells to limit antitumor immunity.

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+ tTex cells possess transcriptional features of CD4+Foxp3+ regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tTex cell 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 tTex cells by tumor hypoxia, thus mitigation of hypoxia limits tTex suppression. Together, these data suggest tTex cells 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.

Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.

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.

To loop or not to loop? CNS-28 is the answer.

Regulation of interferon-γ is critical to constrain inflammation and mount effective responses to infection and anti-tumor immunity. In this issue of Immunity, Cui et al. identify a distal silencer element that disrupts promoter-enhancer looping, regulating IFN-γ expression and preventing inappropriate inflammation.

IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival.

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.

Adaptive plasticity of IL-10+ and IL-35+ Treg cells cooperatively promotes tumor T cell exhaustion.

Regulatory T cells (Treg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Treg cells 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 Treg cell 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 Treg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+ TILs that limits effective anti-tumor immunity.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

SLIDE: Significant Latent Factor Interaction Discovery and Exploration across biological domains.

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.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

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, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells 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 Treg cells are linked. Here we show that Treg cells 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 Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells 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 Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells 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.

Innovative super-resolution in spatial transcriptomics: a transformer model exploiting histology images and spatial gene expression.

Spatial transcriptomics technologies have shed light on the complexities of tissue structures by accurately mapping spatial microenvironments. Nonetheless, a myriad of methods, especially those utilized in platforms like Visium, often relinquish spatial details owing to intrinsic resolution limitations. In response, we introduce TransformerST, an innovative, unsupervised model anchored in the Transformer architecture, which operates independently of references, thereby ensuring cost-efficiency by circumventing the need for single-cell RNA sequencing. TransformerST not only elevates Visium data from a multicellular level to a single-cell granularity but also showcases adaptability across diverse spatial transcriptomics platforms. By employing a vision transformer-based encoder, it discerns latent image-gene expression co-representations and is further enhanced by spatial correlations, derived from an adaptive graph Transformer module. The sophisticated cross-scale graph network, utilized in super-resolution, significantly boosts the model's accuracy, unveiling complex structure-functional relationships within histology images. Empirical evaluations validate its adeptness in revealing tissue subtleties at the single-cell scale. Crucially, TransformerST adeptly navigates through image-gene co-representation, maximizing the synergistic utility of gene expression and histology images, thereby emerging as a pioneering tool in spatial transcriptomics. It not only enhances resolution to a single-cell level but also introduces a novel approach that optimally utilizes histology images alongside gene expression, providing a refined lens for investigating spatial transcriptomics.

Regulation and Immunotherapeutic Targeting of the Epigenome in Exhausted CD8 T Cell Responses.

Exhaustion is a state of CD8 T cell differentiation that occurs in settings of chronic Ag such as tumors, chronic viral infection, and autoimmunity. Cellular differentiation is driven by a series of environmental signals that promote epigenetic landscapes that set transcriptomes needed for function. For CD8 T cells, the epigenome that underlies exhaustion is distinct from effector and memory cell differentiation, suggesting that signals early on set in motion a process where the epigenome is modified to promote a trajectory toward a dysfunctional state. Although we know many signals that promote exhaustion, putting this in the context of the epigenetic changes that occur during differentiation has been less clear. In this review, we aim to summarize the epigenetic changes associated with exhaustion in the context of signals that promote it, highlighting immunotherapeutic studies that support these observations or areas for future therapeutic opportunities.

Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.

The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (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-sequencing 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, cubilin, or 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 Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR 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 the 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.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

Shear stress and oxygen availability drive differential changes in opossum kidney proximal tubule cell metabolism and endocytosis.

Kidney proximal tubule (PT) cells have high-metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT functions are impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96 hours showed significant time- and culture-dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis vs development of ion transport and endocytic capacities in our culture system that highlight the complexity of O2 -dependent and mechanosensitive crosstalk to regulate PT cell function.

Thymic self-reactivity selects natural interleukin 17-producing T cells that can regulate peripheral inflammation.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells, the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung, gut and liver. In the liver, these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus, T(H)-17 cells, like T(reg) cells, can be selected by self antigens in the thymus.

Differential expression of Ly6C and T-bet distinguish effector and memory Th1 CD4(+) cell properties during viral infection.

CD4(+) T cells differentiate into multiple effector types, but it is unclear how they form memory T cells during infection in vivo. Profiling virus-specific CD4(+) T cells revealed that effector cells with T helper 1 (Th1) or T follicular helper (Tfh) cell characteristics differentiated into memory cells, although expression of Tfh cell markers declined over time. In contrast to virus-specific effector CD8(+) T cells, increased IL-7R expression was not a reliable marker of CD4(+) memory precursor cells. However, decreased Ly6C and T-bet (Tbx21) expression distinguished a subset of Th1 cells that displayed greater longevity and proliferative responses to secondary infection. Moreover, the gene expression profile of Ly6C(lo)T-bet(int) Th1 effector cells was virtually identical to mature memory CD4(+) T cells, indicating early maturation of memory CD4(+) T cell features in this subset during acute viral infection. This study provides a framework for memory CD4(+) T cell development after acute viral infection.

Transcriptional and epigenetic networks of helper T and innate lymphoid cells.

The discovery of the specification of CD4(+) helper T cells to discrete effector 'lineages' represented a watershed event in conceptualizing mechanisms of host defense and immunoregulation. However, our appreciation for the actual complexity of helper T-cell subsets continues unabated. Just as the Sami language of Scandinavia has 1000 different words for reindeer, immunologists recognize the range of fates available for a CD4(+) T cell is numerous and may be underestimated. Added to the crowded scene for helper T-cell subsets is the continuously growing family of innate lymphoid cells (ILCs), endowed with common effector responses and the previously defined 'master regulators' for CD4(+) helper T-cell subsets are also shared by ILC subsets. Within the context of this extraordinary complexity are concomitant advances in the understanding of transcriptomes and epigenomes. So what do terms like 'lineage commitment' and helper T-cell 'specification' mean in the early 21st century? How do we put all of this together in a coherent conceptual framework? It would be arrogant to assume that we have a sophisticated enough understanding to seriously answer these questions. Instead, we review the current status of the flexibility of helper T-cell responses in relation to their genetic regulatory networks and epigenetic landscapes. Recent data have provided major surprises as to what master regulators can or cannot do, how they interact with other transcription factors and impact global genome-wide changes, and how all these factors come together to influence helper cell function.

CCAAT/Enhancer-binding protein ß promotes pathogenesis of EAE.

The CCAAT/Enhancer Binding Protein ß (C/EBPß) transcription factor is activated by multiple inflammatory stimuli, including IL-17 and LPS, and C/EBPß itself regulates numerous genes involved in inflammation. However, the role of C/EBPß in driving autoimmunity is not well understood. Here, we demonstrate that Cebpb-/- mice are resistant to EAE. Cebpb-/- mice exhibited reduced lymphocyte and APC infiltration into CNS following EAE induction. Furthermore, MOG-induced Th17 cytokine production was impaired in draining LN, indicating defects in Th17 cell priming. In vitro Th17 polarization studies indicated that T cell responses are not inherently defective, instead supporting the known roles for C/EBPß in myeloid lineage cell activation as the likely mechanism for defective Th17 priming in vivo. However, we did uncover an unexpected role for C/EBPß in regulating ll23r expression in APCs. ChIP assays confirmed that C/EBPß binds directly to the Il23r gene promoter in dendritic cells and Th17 cells. These data establish C/EBPß as a key driver of autoimmune inflammation in EAE, and propose a novel role for C/EBPß in regulation of IL-23R expression.

B cells in T follicular helper cell development and function: separable roles in delivery of ICOS ligand and antigen.

B cells are required for follicular Th (Tfh) cell development, as is the ICOS ligand (ICOS-L); however, the separable contributions of Ag and ICOS-L delivery by cognate B cells to Tfh cell development and function are unknown. We find that Tfh cell and germinal center differentiation are dependent on cognate B cell display of ICOS-L, but only when Ag presentation by the latter is limiting, with the requirement for B cell expression of ICOS-L overcome by robust Ag delivery. These findings demonstrate that Ag-specific B cells provide different, yet compensatory, signals for Tfh cell differentiation, while reconciling conflicting data indicating a requirement for ICOS-L expression on cognate B cells for Tfh cell development with those demonstrating that the latter requirement could be bypassed in lieu of that tendered by noncognate B cells. Our findings clarify the separable roles of delivery of Ag and ICOS-L by cognate B cells for Tfh cell maturation and function, and have implications for using therapeutic ICOS blockade in settings of abundantly available Ag, such as in systemic autoimmunity.

Type I IFN induces binding of STAT1 to Bcl6: divergent roles of STAT family transcription factors in the T follicular helper cell genetic program.

CD4(+) T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. The factors that contribute to the differentiation of this helper cell subset are incompletely understood, although several cytokines including IL-6, IL-21, and IL-12 can promote TFH cell formation. Yet, none of these factors, nor their downstream cognate STATs, have emerged as nonredundant, essential drivers of TFH cells. This suggests a model in which multiple factors can contribute to the phenotypic characteristics of TFH cells. Because type I IFNs are often generated in immune responses, we set out to investigate whether these factors are relevant to TFH cell differentiation. Type I IFNs promote Th1 responses, thus one possibility was these factors antagonized TFH-expressed genes. However, we show that type I IFNs (IFN-α/ß) induced B cell lymphoma 6 (Bcl6) expression, the master regulator transcription factor for TFH cells, and CXCR5 and programmed cell death-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by STAT1, which bound to the Bcl6, Cxcr5, and Pdcd1 loci. These data suggest that type I IFNs (IFN-α/ß) and STAT1 can contribute to some features of TFH cells but are inadequate in inducing complete programming of this subset.