Cell Antigen Receptor Repertoire Research Articles

A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer’s patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Single-cell transcriptomic and T cell antigen receptor analysis of human cytomegalovirus (hCMV)-specific memory T cells reveals effectors and pre-effectors of CD8+- and CD4+-cytotoxic T cells.

Latent human cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals. Although T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in hCMV immunity is lacking. Here, in an unbiased manner, we characterized over 8000 hCMV-reactive peripheral memory T cells isolated from seropositive human donors, at a single-cell resolution by analysing their single-cell transcriptomes paired with the T cell antigen receptor (TCR) repertoires. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental and functional memory T cell subsets such as, long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs) of both CD4 and CD8 origin. The hCMV-specific TREGs, in addition to being enriched for molecules known for their suppressive functions, showed enrichment for the interferon response signature gene sets. The hCMV-specific CTLs were of two types, the pre-effector- and effector-like. The co-clustering of hCMV-specific CD4-CTLs and CD8-CTLs in both pre-effector as well as effector clusters suggest shared transcriptomic signatures between them. The huge TCR clonal expansion of cytotoxic clusters suggests a dominant role in the protective immune response to CMV. The study uncovers the heterogeneity in the hCMV-specific memory T cells revealing many functional subsets with potential implications in better understanding of hCMV-specific T cell immunity. The data presented can serve as a knowledge base for designing vaccines and therapeutics.

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and β repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and β of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

A Beginner's Guide to T Cell Development.

T lymphocytes (T cells) are essential components of the adaptive immune system; they serve multiple functions in responses to pathogens and to ensure immune homeostasis. Written for readers first entering this field of study, this chapter is a brief overview of the development of T cells in the thymus, from the entry of thymus-settling bone marrow-derived precursors to the egress of mature T cells. Surveyed topics include the differentiation and expansion of early precursors, the generation of the T cell antigen receptor repertoire, the selection of αβ T cell precursors, and their acquisition of functional competency.

Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.

Immunological Correlates of Treatment with the CTLA-4 Inhibitor Ipilimumab in Patients with Refractory Myelodysplastic Syndromes (MDS)

Introduction: Failure of hypomethylating agents (HMAs) in patients with myelodysplastic syndromes (MDS) is associated with dismal survival and there are no currently approved therapies in this setting. We recently presented the results of a multi-center phase 1b study of the immune checkpoint blocker (ICB) ipilimumab given as monotherapy after HMA failure in 29 MDS patients. Eight doses of 3mg/kg administered in induction and maintenance phases were safe and well tolerated. Very limited data has been reported on immunologic correlates of response after ICB therapy among patients with hematologic malignancies. Here we present a detailed analysis of the immunological correlates of achieving clinical benefit with ipilimumab.Methods: We defined patients who achieved marrow complete response (mCR) or prolonged stable disease (PSD) for ≥46 weeks as having achieved meaningful clinical benefit (MCB) for analyses of immunologic studies. 11 patients were treated during the dose escalation phase (6 patients at 3mg/kg dose and 5 patients at 10 mg/kg), while another 18 patients were treated in the dose expansion part at the 3 mg/kg dose. Correlative immunologic studies were performed on mononuclear cells from both peripheral blood (PBMC) and bone marrow (BMMC) samples that were collected at baseline and at pre-determined timepoints after ipilimumab treatment (after induction cycle 2 and 4; after each maintenance cycle 1-4, and end of treatment or study discontinuation). Correlative assays included immunophenotyping of PBMCs and BMMCs by multicolor flow cytometry, as well as high throughput sequencing of the T cell antigen receptor (TCR) repertoire. T cell percentages, absolute numbers, and ratios were summarized by time point for patients and overall for healthy controls using descriptive statistics (mean, SD). Differences in log-transformed T cell values were compared between healthy controls and patients at pre-treatment using two-sample t tests. Differences from pre-treatment to cycle 2 and cycle 4 were estimated for patients and tested for positive or negative changes with paired t tests. Analyses were completed using R version 3.3.2.Results: MCB was observed in 28% of patients, 2 (7%) patients with mCR and 6 (21%) patients with PSD, respectively. Immunologic studies were performed on 16 patients who had samples available from at least two time points. Compared to solid tumor literature, we did not observe a correlation between early dynamic changes in serial absolute lymphocyte count and probability of achieving MCB. Expression of co-stimulatory molecules (ICOS - Inducible T Cell CO-Stimulator) and co-inhibitory molecules (PD-1 and CTLA-4) showed that a higher fraction of CD4+ and CD8+ T-cells in PB expressed ICOS in patients who achieved MCB following ipilimumab treatment (P=0.05 and P=0.01, respectively, Figure 1]. Similarly, expression of the immune co-inhibitory receptor CTLA-4 followed a similar pattern showing a trend of increased expression in both CD4+ and CD8+ T-cells (average increase of 6% and 2.4% respectively, P=ns.) over the first 4 cycles of ipilimumab treatment among patients who achieved MCB, followed by a gradual decrease. In contrast to CTLA-4, there was a wide range in frequencies of CD4+ and CD8+ T-cells expressing PD-1 in both patients and HC and PD-1 levels remained stable throughout ipilimumab treatment suggesting that the lack of T-cell activation in non-responders was not due to increased expression of other co-inhibitory molecules. There was a significantly higher frequency of CD3+CD4+Foxp3+ and activated effector Tregs (eTregs) prior to treatment which persisted throughout the examined time points and neither their levels, nor the CD8 T-cell to Treg (CD8/Treg) ratio were impacted by ipilimumab treatment.Conclusions: These data represent, to our knowledge, the first detailed analysis of immunologic correlates of response to ICB therapy in MDS patients. Our data suggest that ICOS expression is positively correlated with achievement of MCB with single agent ipilimumab in patients with MDS and should be further explored as a biomarker for response. The results of the high throughput sequencing of the T cell antigen receptor (TCR) repertoire are currently being analyzed and will be presented at the meeting. [Display omitted] DisclosuresSmith:Celgene: Other: DSMB member; Jazz: Membership on an entity's Board of Directors or advisory committees. Zeidner:Tolero Pharmaceuticals: Honoraria; Agios: Honoraria; Tolero Pharmaceuticals: Research Funding; Takeda: Research Funding; Merck: Research Funding; Celgene: Honoraria. Frattini:Astellas: Honoraria; Lin Bioscience: Consultancy. Levy:Actinium Pharmaceuticals: Equity Ownership; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Takeda: Consultancy, Speakers Bureau. Zeidan:AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Takeda: Speakers Bureau; Otsuka: Consultancy.

Cytoskeletal control of the developing and mature B cell antigen receptor repertoire

Abstract The existence of diverse B cell antigen receptor (BCR) specificities is essential for development of protective antibody responses against a wide variety of pathogens. In developing B cells, the BCR repertoire is crafted through sequential rearrangements of heavy and light chain genes, and involves both combinatorial and junctional diversification catalyzed by recombinases and nucleases. Mature B cells undergo further diversification in peripheral lymphoid organs through isotype switching and somatic hypermutations in response to foreign antigens; although these events are not known to alter BCR antigen specificity. Cytoskeletal proteins modulate BCR signal strength and thereby antibody responses, but it is not known if they play a role in the generation of BCR diversity. Using next generation sequencing, and iRepertoire, Inc and ImMunoGeneTics (IMGT) software platforms we examined BCR repertoires in developing and mature B cells that lack the expression of Ezrin, a prominent cytoskeletal regulator of BCR organization, signaling and activation. We show that Ezrin deficiency impacts the generation of BCR repertoire diversity during B cell development, as well as the mature BCR repertoire. Our analysis provides a window into the major processes involved in diversifying the B cell repertoire in control and Ezrin-deficient B cells, including V gene usage, number, frequency and length of unique CDR3s, nucleotide additions and trimming, and shared and unique clonotypes. Our data suggest that the Ezrin cytoskeleton in B cells impacts humoral immunity not only through modulation of B cell activation upon foreign antigen encounter, but also through qualitative and quantitative modulation of the available BCR repertoire.

'Reverse evolution' in T cell biology.

Comprehensive analysis of CD8+ T cell populations specific to cytomegalovirus reveals that the evolution of the T cell antigen receptor repertoire during chronic infections is characterized by the expansion of low-affinity clones.

Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells.

The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318, which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88-nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

The biology and functional importance of MAIT cells.

In recent years, a population of unconventional T cells called 'mucosal-associated invariant T cells' (MAIT cells) has captured the attention of immunologists and clinicians due to their abundance in humans, their involvement in a broad range of infectious and non-infectious diseases and their unusual specificity for microbial riboflavin-derivative antigens presented by the major histocompatibility complex (MHC) class I-like protein MR1. MAIT cells use a limited T cell antigen receptor (TCR) repertoire with public antigen specificities that are conserved across species. They can be activated by TCR-dependent and TCR-independent mechanisms and exhibit rapid, innate-like effector responses. Here we review evidence showing that MAIT cells are a key component of the immune system and discuss their basic biology, development, role in disease and immunotherapeutic potential.

Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.

Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA-FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.

Thymic regulatory T cells arise via two distinct developmental programs.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non–self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP) and Foxp3lo Treg cell progenitors (Foxp3lo TregP). The CD25+ TregP had higher rates of apoptosis and interacted with thymic self-antigens with higher affinity than Foxp3lo TregP, and had a T cell antigen receptor (TCR) repertoire and transcriptome distinct from that of Foxp3lo TregP. The development of CD25+ TregP and Foxp3lo TregP was controlled by distinct signaling pathways and enhancers. Transcriptomic and histocytometric data suggested that CD25+ TregP and Foxp3lo TregP arose by coopting negative and positive selection programs, respectively. Treg cells derived from CD25+ TregP, but not Foxp3lo TregP, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immune tolerance.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101.

Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

Differing roles of CD1d2 and CD1d1 proteins in type I natural killer T cell development and function

MHC class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of natural killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. Here, we report that CD1d2 molecules are expressed in the thymus of some mouse strains, where they select functional type I NKT cells. Intriguingly, the T cell antigen receptor repertoire and phenotype of CD1d2-selected type I NKT cells in CD1D1-/- mice differed from CD1d1-selected type I NKT cells. The structures of CD1d2 in complex with endogenous lipids and a truncated acyl-chain analog of α-galactosylceramide revealed that its A'-pocket was restricted in size compared with CD1d1. Accordingly, CD1d2 molecules could not present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chain antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby impacting the development of invariant NKT cells.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1.

Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4+CD8+ double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1S557A) impairs generation of CD4+ thymocytes. These results suggest that the PKD–SHP-1 axis positively regulates TCR signalling to promote CD4+ T cell development.

Reconstructing and mining the B cell repertoire with ImmunediveRsity

The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/

Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.

A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla.

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.