Thetis cells induce food-specific Treg cell differentiation and oral tolerance.

The intestinal immune system must establish tolerance to food antigens to prevent onset of allergic and inflammatory diseases. Peripherally generated regulatory T (pTreg) cells play an essential role in suppressing inflammatory responses to allergens; however, the antigen-presenting cell (APC) that instructs food-specific pTreg cells is not known. Here, we show that antigen presentation and TGF-ß activation by a subset of RORγt + antigen-presenting cells (APC), Thetis cells IV (TC IV), is required for food-induced pTreg cell differentiation and oral tolerance. By contrast, antigen presentation by dendritic cells (DCs) was dispensable for pTreg induction but required for T H 1 effector responses, highlighting a division of labor between tolerogenic TCs and pro-inflammatory DCs. While antigen presentation by TCs was required for food-specific pTreg generation both in early life and adulthood, the increased abundance of TCs in the peri-weaning period was associated with a window of opportunity for enhanced pTreg differentiation. These findings establish a critical role for TCs in oral tolerance and suggest that these cells may represent a key therapeutic target for the treatment of food-associated allergic and inflammatory diseases.

Early life imprinting of intestinal immune tolerance and tissue homeostasis.

Besides its canonical role in protecting the host from pathogens, the immune system plays an arguably equally important role in maintaining tissue homeostasis. Within barrier tissues that interface with the external microenvironment, induction of immune tolerance to innocuous antigens, such as commensal, dietary, and environmental antigens, is key to establishing immune homeostasis. The early postnatal period represents a critical window of opportunity in which parallel development of the tissue, immune cells, and microbiota allows for reciprocal regulation that shapes the long-term immunological tone of the tissue and subsequent risk of immune-mediated diseases. During early infancy, the immune system appears to sacrifice pro-inflammatory functions, prioritizing the establishment of tissue tolerance. In this review, we discuss mechanisms underlying early life windows for intestinal tolerance with a focus on newly identified RORγt+ antigen-presenting cells-Thetis cells-and highlight the role of the intestinal microenvironment in shaping intestinal immune system development and tolerance.

Thymic mimicry: The art of imitation.

Display of tissue self-antigens within the thymus is critical for the regulation of self-reactive T cells. In this issue of JEM, Michelson et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230461) continue to advance our understanding of self-antigen representation by medullary thymic epithelial cells, identifying a new role for Hnf4γ in the regulation of thymic mimetic cells as well as their peripheral counterparts.

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.

SEACells infers transcriptional and epigenomic cellular states from single-cell genomics data.

Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here we present single-cell aggregation of cell states (SEACells), an algorithm for identifying metacells that overcome the sparsity of single-cell data while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying comprehensive, compact and well-separated metacells in both RNA and assay for transposase-accessible chromatin (ATAC) modalities across datasets with discrete cell types and continuous trajectories. We demonstrate the use of SEACells to improve gene-peak associations, compute ATAC gene scores and infer the activities of critical regulators during differentiation. Metacell-level analysis scales to large datasets and is particularly well suited for patient cohorts, where per-patient aggregation provides more robust units for data integration. We use our metacells to reveal expression dynamics and gradual reconfiguration of the chromatin landscape during hematopoietic differentiation and to uniquely identify CD4 T cell differentiation and activation states associated with disease onset and severity in a Coronavirus Disease 2019 (COVID-19) patient cohort.

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.

Retinoic acid signaling acts as a rheostat to balance Treg function.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

Volume control: Turning the dial on regulatory T cells.

Treg-mediated immunosuppression must be tightly regulated to support immunity while limiting tissue damage. In this issue of Cell, Wong et al. and Marangoni et al. use high-resolution imaging to define feedback circuits that quantitatively control local Treg expansion and function.

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.

PD-L1 Blockade Therapy: Location, Location, Location.

The mechanisms by which PD-1/PD-L1 inhibition elicits anti-tumor immunity are not fully understood. In this issue of Cancer Cell, Dammeijer et al. address the role of PD-L1 inhibition specifically within the tumor-draining lymph node, identifying a potential role for PD-L1 expressing dendritic cells within the lymph node in regulation of anti-tumor immune responses.

Single-cell sequencing reveals clonal expansions of pro-inflammatory synovial CD8 T cells expressing tissue-homing receptors in psoriatic arthritis.

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis.

Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity.

Dendritic cells (DCs) play a critical role in orchestrating adaptive immune responses due to their unique ability to initiate T cell responses and direct their differentiation into effector lineages. Classical DCs have been divided into two subsets, cDC1 and cDC2, based on phenotypic markers and their distinct abilities to prime CD8 and CD4 T cells. While the transcriptional regulation of the cDC1 subset has been well characterized, cDC2 development and function remain poorly understood. By combining transcriptional and chromatin analyses with genetic reporter expression, we identified two principal cDC2 lineages defined by distinct developmental pathways and transcriptional regulators, including T-bet and RORγt, two key transcription factors known to define innate and adaptive lymphocyte subsets. These novel cDC2 lineages were characterized by distinct metabolic and functional programs. Extending our findings to humans revealed conserved DC heterogeneity and the presence of the newly defined cDC2 subsets in human cancer.

CD161+ Tconv and CD161+ Treg Share a Transcriptional and Functional Phenotype despite Limited Overlap in TCRß Repertoire.

Human regulatory T cells (Treg) are important in immune regulation, but can also show plasticity in specific settings. CD161 is a lectin-like receptor and its expression identifies an effector-like Treg population. Here, we determined how CD161+ Treg relate to CD161+ conventional T cells (Tconv). Transcriptional profiling identified a shared transcriptional signature between CD161+ Tconv and CD161+ Treg, which is associated with T helper (Th)1 and Th17 cells, and tissue homing, including high expression of gut-homing receptors. Upon retinoic acid (RA) exposure, CD161+ T cells were more enriched for CCR9+ and integrin α4+ß7+ cells than CD161- T cells. In addition, CD161+ Tconv and CD161+ Treg were enriched at the inflamed site in autoimmune arthritis, and both CD161+ and CD161- Treg from the inflamed site were suppressive in vitro. CD161+ T cells from the site of autoimmune arthritis showed a diminished gut-homing phenotype and blunted response to RA suggesting prior imprinting by RA in the gut or at peripheral sites rather than during synovial inflammation. TCRß repertoires of CD161+ and CD161- Tconv and Treg from blood showed limited overlap whereas there was clear overlap between CD161+ and CD161- Tconv, and CD161+ and CD161- Treg from the inflamed site suggesting that the inflamed environment may alter CD161 levels, potentially contributing to disease pathogenesis.

Seeing through the dark: New insights into the immune regulatory functions of vitamin A.

The importance of vitamin A for host defense is undeniable and the study of its mechanisms is paramount. Of the estimated 250 million preschool children who are vitamin A-deficient (VAD), 10% will die from their increased susceptibility to infectious disease. Vitamin A supplementation was established in the 1980s as one of the most successful interventions in the developing world. Understanding how vitamin A controls immunity will help curb the mortality and morbidity associated with vitamin A deficiency and exploit the immune-enhancing capacity of vitamin A to heighten host resistance to infectious disease. The discoveries that retinoic acid (RA) imprints the homing of leukocytes to the gut and enhances the induction of regulatory T cells, highlighted a potential role for RA in mucosal tolerance. However, more recently emerging data tell of a more profound systemic impact of RA on leukocyte function and commitment. In animal models using genetic manipulation of RA signaling, we learned when and how RA controls T cell fate. Here, we review the role for RA as a critical checkpoint regulator in the differentiation of CD4(+) T cells within the immune system.

Retinoic acid is essential for Th1 cell lineage stability and prevents transition to a Th17 cell program.

CD4(+) T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4(+) T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells.

Clinical significance of isolated v lesions in paediatric renal transplant biopsies: muscular arteries required to refute the diagnosis of acute rejection.

Intimal vascular lesions are considered features of acute T-cell-mediated rejection yet can occur in the absence of tubulointerstitial inflammation, termed isolated 'v' lesions. The clinical significance of these lesions is unclear. The diagnosis requires a biopsy with the presence of arteries. The frequency of adequate biopsies was analysed in 89 renal transplant biopsies from 57 paediatric renal allograft recipients, and the incidence of isolated endarteritis was determined. 60 (67%) biopsies contained an artery and of these, isolated 'v' lesions occurred in 6 (10%). 5 (83%) biopsies with isolated 'v' lesions were associated with positive DSA, suggesting that these lesions may represent acute antibody-mediated rejection. Patients with vessel-negative biopsies had an increased decline in eGFR (median -20.5, IQR -24.4 to 1.2 ml/min/1.73 m(2) vs. -9.6, IQR -78.7 to -6.8 ml/min/1.73 m(2) ; P = 0.01). Patients with vessel-negative biopsies were more likely to have repeat biopsy for ongoing allograft dysfunction, (25.0% vs. 2.4%; P < 0.01). The data suggest that isolated 'v' lesions are more common than previously thought. A significant proportion of biopsies classified as 'normal' or 'borderline change' in the absence of a large vessel may represent undiagnosed acute rejection. This may result in suboptimal therapy with possible adverse effects on renal outcome.

Making your way as an academic paediatric trainee in the UK.

A career path in academic paediatric medicine is an extremely rewarding one, and while not traditionally considered an academic specialty, it offers a wealth of exciting research opportunities. Developing academic paediatrics is becoming increasingly important, as recently reviewed in the Royal College of Paediatrics and Child Health (RCPCH) Turning the Tide report, and developing future leaders in academic paediatrics is a key goal of the academic training pathways. Strategies are being implemented to ensure that the enthusiasm of academic trainees is maintained, and their development into future leaders is secured.