Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells.

Unprimed mice harbor a substantial population of 'memory-phenotype' CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.

The transcriptional regulator Aire binds to and activates super-enhancers.

Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells.

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Thymus aging and immune reconstitution, progresses and challenges.

Thymus is a primary lymphoid organ essential for the development of T lymphocytes. Age-related thymic involution is a prominent feature of immune senescence. The thymus undergoes rapid growth during fetal and neonatal development, peaks in size before puberty and then begins to undergo a decrease in cellularity with age. Dramatic changes occur with age-associated thymic involution. The most prominent features of thymic involution include: (i) epithelial structure disruption, (ii) adipogenesis, and (iii) thymocyte development arrest. There is a sex disparity in thymus aging. It is a multifactorial process controlled and regulated by a series of molecules, including the transcription factor FOXN1, fibroblast and keratinocyte growth factors (FGF and KGF, respectively), sex steroids, Notch signaling, WNT signaling, and microRNAs. Nevertheless, there is still no satisfactory evolutionary or physiological explanation for age-associated thymic involution, and understanding the precise mechanism(s) for thymus aging remains challenging. Sustained thymic regeneration has yet to be achieved by sex steroid ablation. Recent preclinical studies indicate that long-term thymic reconstitution can be achieved via adoptive transfer of in vitro-generated progenitor T (proT) cells, and improvements in the methods for the generation of human proT cells make this an attractive approach. Future clinical applications may rely on new applications integrating proT cells, cytokine support and sex-steroid inhibition treatments.

Disorganization of secondary lymphoid organs and dyscoordination of chemokine secretion as key contributors to immune aging.

Aging is characterized by progressive loss of organ and tissue function, and the immune system is no exception to that inevitable principle. Of all the age-related changes in the body, reduction of the size of, and naïve T (Tn) cell output from, the thymus occurs earliest, being prominent already before or by the time of puberty. Therefore, to preserve immunity against new infections, over much of their lives, vertebrates dominantly rely on peripheral maintenance of the Tn cell pool in the secondary lymphoid organs (SLO). However, SLO structure and function subsequently also deteriorate with aging. Several recent studies have made a convincing case that this deterioration is of major importance to the erosion of protective immunity in the last third of life. Specifically, the SLO were found to accumulate multiple degenerative changes with aging. Importantly, the results from adoptive transfer and parabiosis studies teach us that the old microenvironment is the limiting factor for protective immunity in old mice. In this review, we discuss the extent, mechanisms, and potential role of stromal cell aging in the age-related alteration of T cell homeostatic maintenance and immune function decline. We use that discussion to frame the potential strategies to correct the SLO stromal aging defects - in the context of other immune rejuvenation approaches, - to improve functional immune responses and protective immunity in older adults.

Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

Brain-Thymus Connections in Chagas Disease.

BACKGROUND: The brain and the immune systems represent the two primary adaptive systems within the body. Both are involved in a dynamic process of communication, vital for the preservation of mammalian homeostasis. This interplay involves two major pathways: the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. SUMMARY: The establishment of infection can affect immunoneuroendocrine interactions, with functional consequences for immune organs, particularly the thymus. Interestingly, the physiology of this primary organ is not only under the control of the central nervous system (CNS) but also exhibits autocrine/paracrine regulatory circuitries mediated by hormones and neuropeptides that can be altered in situations of infectious stress or chronic inflammation. In particular, Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), impacts upon immunoneuroendocrine circuits disrupting thymus physiology. Here, we discuss the most relevant findings reported in relation to brain-thymic connections during T. cruzi infection, as well as their possible implications for the immunopathology of human Chagas disease. KEY MESSAGES: During T. cruzi infection, the CNS influences thymus physiology through an intricate network involving hormones, neuropeptides, and pro-inflammatory cytokines. Despite some uncertainties in the mechanisms and the fact that the link between these abnormalities and chronic Chagasic cardiomyopathy is still unknown, it is evident that the precise control exerted by the brain over the thymus is markedly disrupted throughout the course of T. cruzi infection.

Aire regulates chromatin looping by evicting CTCF from domain boundaries and favoring accumulation of cohesin on superenhancers.

Aire controls immunological tolerance by driving promiscuous expression of a large swath of the genome in medullary thymic epithelial cells (mTECs). Its molecular mechanism remains enigmatic. High-resolution chromosome-conformation capture (Hi-C) experiments on ex vivo mTECs revealed Aire to have a widespread impact on higher-order chromatin structure, disfavoring architectural loops while favoring transcriptional loops. In the presence of Aire, cohesin complexes concentrated on superenhancers together with mediator complexes, while the CCCTC-binding factor (CTCF) was relatively depleted from structural domain boundaries. In particular, Aire associated with the cohesin loader, NIPBL, strengthening this factor's affiliation with cohesin's enzymatic subunits. mTEC transcripts up-regulated in the presence of Aire corresponded closely to those down-regulated in the absence of one of the cohesin subunits, SA-2. A mechanistic model incorporating these findings explains many of the unusual features of Aire's impact on mTEC transcription, providing molecular insight into tolerance induction.

Age Specifics of Homeostasis Response to Cold Stress in Wistar Rats: Endurance Improvement or Disorganization?

Cold exposure, especially in combination with exercise, is a common procedure to fortify the body. However, its effects in old age are poorly understood. Using the homeostasis stability coefficient as an indicator, the body response to a 20-min swim in cold water was for the first time studied comprehensively in old and young Wistar rats. Effects on the hormonal, hematological, and morphofunctional systems of the liver and thymus were assessed. Unfavorable age-related changes in the stability of homeostasis were observed in old rats. The changes complicated the recovery after cold stress and required the involvement of a greater number of homeostatic mechanisms than in young rats. The liver was found to be the most vulnerable to cold stress. It was concluded that fortifying the body by cold exposure is possible to use in old age, but with due regard to age-related restrictions.

Generation of functional human thymic cells from induced pluripotent stem cells.

BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.

Aging is accompanied by T-cell stiffening and reduced interstitial migration through dysfunctional nuclear organization.

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.

MHC-Independent Thymic Selection of CD4 and CD8 Coreceptor Negative αß T Cells.

It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4-CD8-TCRαß+, double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5high DN thymocytes that preferentially yield TCRαß+ CD8αα intestinal lymphocytes, we find that mature CD5low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4+CD8+ (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells.

UHRF1 Controls Thymocyte Fate Decisions through the Epigenetic Regulation of EGR1 Expression.

Thymocyte differentiation is a highly complex process that is accompanied by epigenetic changes. Ubiquitin-like containing PHD ring finger 1 (UHRF1) is a critical epigenetic modifier involved in various cellular processes. In this study, we demonstrated that it is highly expressed in T cell precursors of the thymus. Further, its deficiency results in significantly reduced thymocyte cellularity and thymus size in mice. Through systematic analysis based on single-cell RNA sequencing, we found that UHRF1 deficiency thwarts αß T cell lineage development, whereas biasing γδ T lineage differentiation dampens the progression of immature single-positive cells. UHRF1 deficiency promotes the IL-17 secreting and RORγt expression in γδ T cell, indicating a Tγδ17 phenotype. Further, the analysis of gene-regulatory networks demonstrated that UHRF1 controls the expression of early growth response 1 (EGR1). UHRF1 interacts with DNA methyltransferase 1 (DNMT1) at the CpG promoter region of Egr1 loci and affects the nearby chromatin modifications of H3K9me3 and H3K4me3. Taken together, our results demonstrate that UHRF1 is a key factor that mediates the epigenetic regulation of EGR1 and, consequently, thymocyte fate decisions.

Light-Induced Functional Pinealectomy. Effect on the Thymus of C57BL/6 Mice.

Light-induced functional pinealectomy was simulated in C57BL/6 mice by 14-day exposure to constant lighting. Immunophenotyping of CD3hi and CD3low thymocytes was performed by staining with CD3-APC antibodies followed by flow cytofluorometry. To study the cell cycle distribution of thymus cells, the content of intracellular DNA was measured by the level PI inclusion. In animals with light-induced functional pinealectomy, blood leukocyte content, the relative number of CD3low and CD3hi T cells in the thymus, and the ratio of CD3low/CD3hi thymocytes decreased. The number of G0/G1-phase thymus cells (non-dividing cells) increased and the content of S-phase cells (division phase) decreased. Continuous lighting stimulated the development of thymocyte apoptosis. The results obtained indicate that prolonged 24-h illumination inhibits differentiation and maturation of young CD3low thymocytes into mature CD3hi forms and leads to the development of T-cell apoptosis in the thymus and, as a consequence, to leukopenia.

Setting Up the Perimeter of Tolerance: Insights into mTEC Physiology.

Medullary thymic epithelial cells (mTECs) play a central role in T cell tolerance. However, how the mTEC compartment is maintained remains elusive. We review recent discoveries on new transcription factors involved in mTEC homeostasis and discuss the possibility that their actions might be facilitated by the unique biology of mTECs.

Thymic Development of a Unique Bone Marrow-Resident Innate-like T Cell Subset with a Potent Innate Immune Function.

Mainstream CD8+ and CD4+ T cells of αß lineage are developed in the thymus through TCR-mediated selection in the context of MHC class I and MHC class II in association with self-peptides, respectively. In addition, minor αßT cells bearing invariant TCRs, NKT cells, and mucosal-associated invariant T cells are selected via MHC-like molecules, CD1d, and MR1 complexed with nonpeptide Ags, respectively, parts of which express neither CD4 nor CD8. In this study, we indicate that bone marrow (BM), but barely other lymphoid tissues, harbors CD4/CD8 double-negative αßT cells with an apparently diverse TCR repertoire at considerable proportions in healthy adult mice. The BM-resident double-negative αßT (BMDNT) cells are developed in the thymus in a Notch and IL-7-dependent manner but independently of known restriction elements, including MHC class I, MHC class II, CD1d, and MR1. These cells are sustained in BM throughout the adult stage with "homeostatic" proliferation via IL-1ß derived from normal myeloid cells dominating the BM environment. Although BMDNT cells secrete a unique set of cytokines, including IL-17, GM-CSF, IL-3, and CCL chemokines on TCR stimulation, these T cells also express a series of NK receptors and exhibit a potent NK-like cytotoxic activity. Furthermore, BMDNT cells show robustly accelerated proliferation and activation following systemic administration of TLR ligands likely through the enhanced production of IL-1ß by myeloid cells in situ. Our results suggest that αßT lineage cells that are developed in the thymus by default of TCR-mediated selection are maintained and differentiated to innate-like T cells in BM and may play a role in innate immunity in the hematopoietic environment.

Antenatal thymus volumes in fetuses that delivered <32 weeks' gestation: An MRI pilot study.

INTRODUCTION: Infection and inflammation have been implicated in the etiology and subsequent morbidity associated with preterm birth. At present, there are no tests to assess for fetal compartment infection. The thymus, a gland integral in the fetal immune system, has been shown to involute in animal models of antenatal infection, but its response in human fetuses has not been studied. This study aims: (a) to generate magnetic resonance imaging (MRI) -derived fetal thymus volumes standardized for fetal weight; (b) to compare standardized thymus volumes from fetuses that delivered before 32 weeks of gestation with fetuses that subsequently deliver at term; (c) to assess thymus size as a predictor of preterm birth; and (d) to correlate the presence of chorioamnionitis and funisitis at delivery with thymic volumes in utero in fetuses that subsequently deliver preterm. MATERIAL AND METHODS: Women at high-risk of preterm birth at 20-32 weeks of gestation were recruited. A control group was obtained from existing data sets acquired as part of three research studies. A fetal MRI was performed on a 1.5T or 3T MRI scanner: T2 weighted images were obtained of the entire uterine content and specifically the fetal thorax. A slice-to-volume registration method was used for reconstruction of three-dimensional images of the thorax. Thymus segmentations were performed manually. Body volumes were calculated by manual segmentation and thymus:body volume ratios were generated. Comparison of groups was performed using multiple regression analysis. Normal ranges were created for thymus volume and thymus:body volume ratios using the control data. Receiver operating curves (ROC) curves were generated for thymus:body volume ratio and gestation-adjusted thymus volume centiles as predictors of preterm birth. Placental histology was analyzed where available from pregnancies that delivered very preterm and the presence of chorioamnionitis/funisitis was noted. RESULTS: Normative ranges were created for thymus volume, and thymus volume was standardized for fetal size from fetuses that subsequently delivered at term, but were imaged at 20-32 weeks of gestation. Image data sets from 16 women that delivered <32 weeks of gestation (ten with ruptured membranes and six with intact membranes) and 80 control women that delivered >37 weeks were included. Mean gestation at MRI of the study group was 28+4  weeks (SD 3.2) and for the control group was 25+5  weeks (SD 2.4). Both absolute fetal thymus volumes and thymus:body volume ratios were smaller in fetuses that delivered preterm (P < .001). Of the 16 fetuses that delivered preterm, 13 had placental histology, 11 had chorioamnionitis, and 9 had funisitis. The strongest predictors of prematurity were the thymus volume Z-score and thymus:body volume ratio Z-score (ROC areas 0.915 and 0.870, respectively). CONCLUSIONS: We have produced MRI-derived normal ranges for fetal thymus and thymus:body volume ratios between 20 and 32 weeks of gestation. Fetuses that deliver very preterm had reduced thymus volumes when standardized for fetal size. A reduced thymus volume was also a predictor of spontaneous preterm delivery. Thymus volume may be a suitable marker of the fetal inflammatory response, although further work is needed to assess this, increasing the sample size to correlate the extent of chorioamnionitis with thymus size.

Thymic involution and rising disease incidence with age.

For many cancer types, incidence rises rapidly with age as an apparent power law, supporting the idea that cancer is caused by a gradual accumulation of genetic mutations. Similarly, the incidence of many infectious diseases strongly increases with age. Here, combining data from immunology and epidemiology, we show that many of these dramatic age-related increases in incidence can be modeled based on immune system decline, rather than mutation accumulation. In humans, the thymus atrophies from infancy, resulting in an exponential decline in T cell production with a half-life of ∼16 years, which we use as the basis for a minimal mathematical model of disease incidence. Our model outperforms the power law model with the same number of fitting parameters in describing cancer incidence data across a wide spectrum of different cancers, and provides excellent fits to infectious disease data. This framework provides mechanistic insight into cancer emergence, suggesting that age-related decline in T cell output is a major risk factor.

Neonatal thymectomy in children-accelerating the immunologic clock?

The thymus is critical for central tolerance and diverse T-lymphocyte repertoire development, to provide lifelong defense against pathogens while maintaining self-tolerance. Peak thymic output occurs in utero, during infancy, and in early childhood, diminishing throughout life. Infants with congenital heart disease requiring sternotomy often undergo thymectomy to clear the surgical field. The long-term effects of early thymectomy are just being appreciated. Many patients remain asymptomatic despite immunologic findings mirroring those of immunosenescence. Few develop increased infection or lymphoreticular malignancy risk. When considering the effects of infant thymectomy, patients with partial DiGeorge syndrome or hypomorphic recombination-activating gene (RAG) mutations may be instructive. These patients are lymphocytopenic, with increased early-onset infection and autoimmunity risk that is not seen in most patients who underwent thymectomy during infancy. The thymic structure of patients with partial DiGeorge syndrome or hypomorphic RAG is abnormal, with disrupted architecture inclining to perturbation of central tolerance. Similar findings may be seen in patients with myasthenia gravis, although disrupted peripheral tolerance may play a greater role in autoimmunity development. In conclusion, thymectomy during infancy may increase future risk of infection or autoimmunity, with premature immunosenescence mediated through disruption of central and peripheral tolerance mechanisms initiated by early cessation or diminution of thymic output. Ideally, some thymic tissue should be preserved at the time of surgery.