Starfysh integrates spatial transcriptomic and histologic data to reveal heterogeneous tumor-immune hubs.

Spatially resolved gene expression profiling provides insight into tissue organization and cell-cell crosstalk; however, sequencing-based spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for rigorous interpretation of cell states, mostly do not use associated histology images and are not capable of inferring shared neighborhoods across multiple tissues. Here we present Starfysh, a computational toolbox using a deep generative model that incorporates archetypal analysis and any known cell type markers to characterize known or new tissue-specific cell states without a single-cell reference. Starfysh improves the characterization of spatial dynamics in complex tissues using histology images and enables the comparison of niches as spatial hubs across tissues. Integrative analysis of primary estrogen receptor (ER)-positive breast cancer, triple-negative breast cancer (TNBC) and metaplastic breast cancer (MBC) tissues led to the identification of spatial hubs with patient- and disease-specific cell type compositions and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC.

ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.

The identification of cell-type-specific 3D chromatin interactions between regulatory elements can help to decipher gene regulation and to interpret the function of disease-associated non-coding variants. However, current chromosome conformation capture (3C) technologies are unable to resolve interactions at this resolution when only small numbers of cells are available as input. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps and regulatory interactions from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility profiles across metacells, and predicted CTCF motif tracks as input features and employs a lightweight architecture to enable training on standard GPUs. Once trained on paired scATAC-seq and Hi-C data in human cell lines and tissues, ChromaFold can accurately predict both the 3D contact map and peak-level interactions across diverse human and mouse test cell types. In benchmarking against a recent deep learning method that uses bulk ATAC-seq, DNA sequence, and CTCF ChIP-seq to make cell-type-specific predictions, ChromaFold yields superior prediction performance when including CTCF ChIP-seq data as an input and comparable performance without. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations. ChromaFold thus achieves state-of-the-art prediction of 3D contact maps and regulatory interactions using scATAC-seq alone as input data, enabling accurate inference of cell-type-specific interactions in settings where 3C-based assays are infeasible.

Drivers of heterogeneity in synovial fibroblasts in rheumatoid arthritis.

Inflammation of non-barrier immunologically quiescent tissues is associated with a massive influx of blood-borne innate and adaptive immune cells. Cues from the latter are likely to alter and expand activated states of the resident cells. However, local communications between immigrant and resident cell types in human inflammatory disease remain poorly understood. Here, we explored drivers of fibroblast-like synoviocyte (FLS) heterogeneity in inflamed joints of patients with rheumatoid arthritis using paired single-cell RNA and ATAC sequencing, multiplexed imaging and spatial transcriptomics along with in vitro modeling of cell-extrinsic factor signaling. These analyses suggest that local exposures to myeloid and T cell-derived cytokines, TNF, IFN-γ, IL-1ß or lack thereof, drive four distinct FLS states some of which closely resemble fibroblast states in other disease-affected tissues including skin and colon. Our results highlight a role for concurrent, spatially distributed cytokine signaling within the inflamed synovium.

Conserved transcriptional connectivity of regulatory T cells in the tumor microenvironment informs new combination cancer therapy strategies.

While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell 'connectivity' to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Spatiotemporal regulation of peripheral T cell tolerance.

The incomplete removal of T cells that are reactive against self-proteins during their differentiation in the thymus requires mechanisms of tolerance that prevent their effector function within the periphery. A further challenge is imposed by the need to establish tolerance to the holobiont self, which comprises a highly complex community of commensal microorganisms. Here, we review recent advances in the investigation of peripheral T cell tolerance, focusing on new insights into mechanisms of tolerance to the gut microbiota, including tolerogenic antigen-presenting cell types and immunomodulatory lymphocytes, and their layered ontogeny that underlies developmental windows for establishing intestinal tolerance. While emphasizing the intestine as a model tissue for studying peripheral T cell tolerance, we highlight overlapping and distinct pathways that underlie tolerance to self-antigens versus commensal antigens within a broader framework for immune tolerance.

Cold Spring Harbor Laboratory 2022: emerging insights and viewpoints in immunology.

Some of the current and former organizers of the Cold Spring Harbor Laboratory (CSHL) 'Gene Expression and Signaling in the Immune System' (GESIS) meeting offer opinions on emerging questions in immunology, discussing the strong value of this recurring scientific meeting in the field.

Principles of regulatory T cell function.

Regulatory T (Treg) cells represent a distinct lineage of cells of the adaptive immune system indispensable for forestalling fatal autoimmune and inflammatory pathologies. The role of Treg cells as principal guardians of the immune system can be attributed to their ability to restrain all currently recognized major types of inflammatory responses through modulating the activity of a wide range of cells of the innate and adaptive immune system. This broad purview over immunity and inflammation is afforded by the multiple modes of action Treg cells exert upon their diverse molecular and cellular targets. Beyond the suppression of autoimmunity for which they were originally recognized, Treg cells have been implicated in tissue maintenance, repair, and regeneration under physiologic and pathologic conditions. Herein, we discuss the current and emerging understanding of Treg cell effector mechanisms in the context of the basic properties of Treg cells that endow them with such functional versatility.

Novel antigen-presenting cell imparts Treg-dependent tolerance to gut microbiota.

Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1-4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5-8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-ß-activating integrin αvß8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.

The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis.

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

CXCR4+ Treg cells control serum IgM levels and natural IgM autoantibody production by B-1 cells in the bone marrow.

Regulatory T (Treg) cells represent a specialized lineage of suppressive CD4+ T cells whose functionality is critically dependent on their ability to migrate to and dwell in the proximity of cells they control. Here we show that continuous expression of the chemokine receptor CXCR4 in Treg cells is required for their ability to accumulate in the bone marrow (BM). Induced CXCR4 ablation in Treg cells led to their rapid depletion and consequent increase in mature B cells, foremost the B-1 subset, observed exclusively in the BM without detectable changes in plasma cells or hematopoietic stem cells or any signs of systemic or local immune activation elsewhere. Dysregulation of BM B-1 B cells was associated with a highly specific increase in IgM autoantibodies and total serum IgM levels. Thus, Treg cells control autoreactive B-1 B cells in a CXCR4-dependent manner. These findings have significant implications for understanding the regulation of B cell autoreactivity and malignancies.

Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4+ T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells suppress autoimmunity, pTreg cells enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms supporting their differentiation remain poorly understood. Here, we used genetic tracing to identify microbiota-induced pTreg cells and found that many of their distinguishing features were Foxp3 independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3. While Foxp3 was critical for the suppression of a Th17 cell program, colitis, and mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-ß (TGF-ß) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

Hierarchical regulation of the resting and activated T cell epigenome by major transcription factor families.

T cell activation, a key early event in the adaptive immune response, is subject to elaborate transcriptional control. In the present study, we examined how the activities of eight major transcription factor (TF) families are integrated to shape the epigenome of naive and activated CD4 and CD8 T cells. By leveraging extensive polymorphisms in evolutionarily divergent mice, we identified the 'heavy lifters' positively influencing chromatin accessibility. Members of Ets, Runx and TCF/Lef TF families occupied the vast majority of accessible chromatin regions, acting as 'housekeepers', 'universal amplifiers' and 'placeholders', respectively, at sites that maintained or gained accessibility upon T cell activation. In addition, a small subset of strongly induced immune response genes displayed a noncanonical TF recruitment pattern. Our study provides a key resource and foundation for the understanding of transcriptional and epigenetic regulation in T cells and offers a new perspective on the hierarchical interactions between critical TFs.

Expression of Foxp3 by T follicular helper cells in end-stage germinal centers.

Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3+ T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (TFH) cells. Ectopic expression of Foxp3 in TFH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by TFH cells as a potential regulator of GC lifetimes.

Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss of function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of rescued Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad-spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells.

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

Inflammatory adaptation in barrier tissues.

Surface epithelia provide a critical barrier to the outside world. Upon a barrier breach, resident epithelial and immune cells coordinate efforts to control infections and heal tissue damage. Inflammation can etch lasting marks within tissues, altering features such as scope and quality of future responses. By remembering inflammatory experiences, tissues are better equipped to quickly and robustly respond to barrier breaches. Alarmingly, in disease states, memory may fuel the inflammatory fire. Here, we review the cellular communication networks in barrier tissues and the integration between tissue-resident and recruited immune cells and tissue stem cells underlying tissue adaptation to environmental stress.

Assembly of a spatial circuit of T-bet-expressing T and B lymphocytes is required for antiviral humoral immunity.

Effective antiviral immunity requires generation of T and B lymphocytes expressing the transcription factor T-bet, a regulator of type 1 inflammatory responses. Using T-bet expression as an endogenous marker for cells participating in a type 1 response, we report coordinated interactions of T-bet-expressing T and B lymphocytes on the basis of their dynamic colocalization at the T cell zone and B follicle boundary (T-B boundary) and germinal centers (GCs) during lung influenza infection. We demonstrate that the assembly of this circuit takes place in distinct anatomical niches within the draining lymph node, guided by CXCR3 that enables positioning of TH1 cells at the T-B boundary. The encounter of B and TH1 cells at the T-B boundary enables IFN-γ produced by the latter to induce IgG2c class switching. Within GCs, T-bet+ TFH cells represent a specialized stable sublineage required for GC growth but dispensable for IgG2c class switching. Our studies show that during respiratory viral infection, T-bet-expressing T and B lymphocytes form a circuit assembled in a spatiotemporally controlled manner that acts as a functional unit enabling a robust and coherent humoral response tailored for optimal antiviral immunity.

Immunotherapy breaches low-sugar dieting of tumor Treg cells.

Glycolysis supports effector T cell function but is detrimental to the immunosuppressive activity of regulatory T cells. In a recent issue of Nature, two papers address a role for glucose and lactate availability within the tumor microenvironment for the balance of pro- and anti-tumoral effects of T cells and the efficacy of neoadjuvant cancer immunotherapy.

A distal Foxp3 enhancer enables interleukin-2 dependent thymic Treg cell lineage commitment for robust immune tolerance.

Activation of the STAT5 transcription factor downstream of the Interleukin-2 receptor (IL-2R) induces expression of Foxp3, a critical step in the differentiation of regulatory T (Treg) cells. Due to the pleiotropic effects of IL-2R signaling, it is unclear how STAT5 acts directly on the Foxp3 locus to promote its expression. Here, we report that IL-2 - STAT5 signaling converged on an enhancer (CNS0) during Foxp3 induction. CNS0 facilitated the IL-2 dependent CD25+Foxp3- precursor to Treg cell transition in the thymus. Its deficiency resulted in impaired Treg cell generation in neonates, which was partially mitigated with age. While the thymic Treg cell paucity caused by CNS0 deficiency did not result in autoimmunity on its own, it exacerbated autoimmune manifestations caused by disruption of the Aire gene. Thus, CNS0 enhancer activity ensures robust Treg cell differentiation early in postnatal life and cooperatively with other tolerance mechanisms minimizes autoimmunity.