The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Circulating Tumor Reactive KIR+CD8+ T cells Suppress Anti-Tumor Immunity in Patients with Melanoma.

Effective anti-tumor immunity is largely driven by cytotoxic CD8+ T cells that can specifically recognize tumor antigens. However, the factors which ultimately dictate successful tumor rejection remain poorly understood. Here we identify a subpopulation of CD8+ T cells which are tumor antigen-specific in patients with melanoma but resemble KIR+CD8+ T cells with a regulatory function (Tregs). These tumor antigen-specific KIR+CD8+ T cells are detectable in both the tumor and the blood, and higher levels of this population are associated with worse overall survival. Our findings therefore suggest that KIR+CD8+ Tregs are tumor antigen-specific but uniquely suppress anti-tumor immunity in patients with melanoma.

KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo.

Nuclear factor-κB (NF-κB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-κB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-κB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-κB, the latter have the potential to allow the analysis of NF-κB activity at single-cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knock-in NF-κB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-κB transcriptional activity at the single-cell level of various cell types during inflammatory and infectious diseases.

High-resolution profiling of MHC II peptide presentation capacity reveals SARS-CoV-2 CD4 T cell targets and mechanisms of immune escape.

Understanding peptide presentation by specific MHC alleles is fundamental for controlling physiological functions of T cells and harnessing them for therapeutic use. However, commonly used in silico predictions and mass spectroscopy have their limitations in precision, sensitivity, and throughput, particularly for MHC class II. Here, we present MEDi, a novel mammalian epitope display that allows an unbiased, affordable, high-resolution mapping of MHC peptide presentation capacity. Our platform provides a detailed picture by testing every antigen-derived peptide and is scalable to all the MHC II alleles. Given the urgent need to understand immune evasion for formulating effective responses to threats such as SARS-CoV-2, we provide a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of several HLA class II alleles. We show that several mutations arising in viral strains expanding globally resulted in reduced peptide presentability by multiple HLA class II alleles, while some increased it, suggesting alteration of MHC II presentation landscapes as a possible immune escape mechanism.

LCMV induced downregulation of HVEM on antiviral T cells is critical for an efficient effector response.

T-cell responses against tumors and pathogens are critically shaped by cosignaling molecules providing a second signal. Interaction of herpes virus entry mediator (HVEM, CD270, TNFRSF14) with multiple ligands has been proposed to promote or inhibit T-cell responses and inflammation, dependent on the context. In this study, we show that absence of HVEM did neither affect generation of effector nor maintenance of memory antiviral T cells and accordingly viral clearance upon acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, due to potent HVEM downregulation during infection. Notably, overexpression of HVEM on virus-specific CD8+ T cells resulted in a reduction of effector cells, whereas numbers of memory cells were increased. Overall, this study indicates that downregulation of HVEM driven by LCMV infection ensures an efficient acute response at the price of impaired formation of T-cell memory.

Protection against autoimmunity is driven by thymic epithelial cell-mediated regulation of Treg development.

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote the development of thymic regulatory T cells (tTregs) that mediate peripheral tolerance. The relative importance of these two mechanisms of thymic education to prevent autoimmunity is unclear. We generated a mouse model to specifically target the development and function of mTECs by conditional ablation of the NF-κB­inducing kinase (NIK) in the TEC compartment. In contrast to germline-deficient NIK−/− mice, Foxn1CreNIKfl/fl mice rapidly developed fatal T cell­dependent multiorgan autoimmunity shortly after birth. Thymic transplantation and adoptive transfer experiments demonstrated that autoimmunity arises specifically from the emergence of dysfunctional tTregs. Thus, Treg function, rather than negative selection, enforces the protection of peripheral tissues from autoimmune attack.

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αß T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

Single-Cell Transcriptomics Identifies the Adaptation of Scart1+ Vγ6+ T Cells to Skin Residency as Activated Effector Cells.

IL-17-producing γδ T cells express oligoclonal Vγ4+ and Vγ6+ TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6+ T cells as a highly homogeneous Scart1+ population in contrast to their Scart2+ IL-17-producing Vγ4+ T cell counterparts. Parabiosis demonstrates that Vγ6+ T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1+ Vγ6+ T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1+ Vγ6+ T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.

A spontaneous leptin receptor point mutation causes obesity and differentially affects leptin signaling in hypothalamic nuclei resulting in metabolic dysfunctions distinct from db/db mice.

OBJECTIVE: Leptin (Lep) plays a crucial role in controlling food intake and energy expenditure. Defective Lep/LepRb-signaling leads to fat accumulation, massive obesity, and the development of diabetes. We serendipitously noticed spontaneous development of obesity similar to LepR-deficient (db/db) mice in offspring from a C57BL/6J breeding and transmittance of the phenotype in a Mendelian manner. Candidate gene sequencing revealed a spontaneous point mutation in the LepRb gene. We investigated leptin responsiveness, leptin receptor signaling and metabolic phenotype of this novel LepRb mutant mouse variant. METHODS: Overexpression and functional tests of the mutant LepRb in 3T3 cells. Measurement of leptin responsiveness in hypothalamic nuclei, glucose tolerance, food uptake and energy expenditure in the mutant mice. RESULTS: The mutation results in the exchange of a glycine for serine (G506S) and introduces an alternative splice acceptor which, when used, encodes for a protein with a 40aa deletion that is retained in the cytoplasm. LepRb signaling was abrogated in the hypothalamic ventromedial nucleus (VMN) and dorsomedial nucleus (DMN), but only partially reduced in the hypothalamic arcuate nucleus (ARC) of LepRbG506S/G506S mice, most likely due to differential splicing in neurons located in the respective regions of the hypothalamus. Extensive metabolic characterization of these mice revealed interesting differences in the control of food intake, glucose tolerance, energy expenditure, and fat accumulation in LepRbG506S/G506S compared with LepRb-deficient db/db mice. CONCLUSIONS: This study provides further insight into differences of the leptin responsiveness in VMN, DMN, and ARC and its metabolic consequences.

Author Correction: Deciphering CD4+ T cell specificity using novel MHC-TCR chimeric receptors.

In the version of this article initially published, a reference (23) was cited incorrectly and two references were not included in the second sentence of the first paragraph of the second Results subsection ('Screening for gp61 mimotopes with different functional properties'). The correct citation is as follows: "... we replaced the very stable GFP with a slow fluorescent timer (FT)27,28." Full details on the added references can be found in the correction notice. The errors have been corrected in the print, PDF and HTML versions of the paper.

Deciphering CD4+ T cell specificity using novel MHC-TCR chimeric receptors.

αß T cell antigen receptors (TCRs) bind complexes of peptide and major histocompatibility complex (pMHC) with low affinity, which poses a considerable challenge for the direct identification of αß T cell cognate peptides. Here we describe a platform for the discovery of MHC class II epitopes based on the screening of engineered reporter cells expressing novel pMHC-TCR (MCR) hybrid molecules carrying cDNA-derived peptides. This technology identifies natural epitopes of CD4+ T cells in an unbiased and efficient manner and allows detailed analysis of TCR cross-reactivity that provides recognition patterns beyond discrete peptides. We determine the cognate peptides of virus- and tumor-specific T cells in mouse disease models and present a proof of concept for human T cells. Furthermore, we use MCR to identify immunogenic tumor neo-antigens and show that vaccination with a peptide naturally recognized by tumor-infiltrating lymphocytes efficiently protects mice from tumor challenge. Thus, the MCR technology holds promise for basic research and clinical applications, allowing the personalized identification of T cell-specific neo-antigens in patients.

Interleukin-17-Producing γδ T Cells Originate from SOX13+ Progenitors that Are Independent of γδTCR Signaling.

Lineage-committed αß and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2+ γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13+ progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13+ progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals.

The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4-CD8- thymocyte population, whereas Txnrd1 deletion in CD4+CD8+ thymocytes does not affect further maturation and peripheral homeostasis of αßT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

The origin and fate of γδT cell subsets.

Recent experiments indicate that in contrast to αßT cells, γδT cell effector functions are largely preprogrammed in the thymus during fetal life. However the thymus also exports juvenile γδT cells that can mature and be polarized in the periphery. How these developmental pathways are regulated and how much they contribute to the γδT cell effector pool is unclear. Here we discuss recent advances in the understanding of γδT cell subset development, with particular focus on IL-17-producing γδT cells and their beneficial and pathogenic roles in immunity.

Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk.

Psoriasis is a chronic inflammatory disorder of the skin affecting approximately 2% of the world's population. Accumulating evidence has revealed that the IL-23/IL-17/IL-22 pathway is key for development of skin immunopathology. However, the role of keratinocytes and their crosstalk with immune cells at the onset of disease remains poorly understood. Here, we show that IL-36R-deficient (Il36r-/-) mice were protected from imiquimod-induced expansion of dermal IL-17-producing γδ T cells and psoriasiform dermatitis. Furthermore, IL-36R antagonist-deficient (Il36rn-/-) mice showed exacerbated pathology. TLR7 ligation on DCs induced IL-36-mediated crosstalk with keratinocytes and dermal mesenchymal cells that was crucial for control of the pathological IL-23/IL-17/IL-22 axis and disease development. Notably, mice lacking IL-23, IL-17, or IL-22 were less well protected from disease compared with Il36r-/- mice, indicating an additional distinct activity of IL-36 beyond induction of the pathological IL-23 axis. Moreover, while the absence of IL-1R1 prevented neutrophil infiltration, it did not protect from acanthosis and hyperkeratosis, demonstrating that neutrophils are dispensable for disease manifestation. These results highlight a central and unique IL-1-independent role for IL-36 in control of the IL-23/IL-17/IL-22 pathway and development of psoriasiform dermatitis.

Evidence for the divergence of innate and adaptive T-cell precursors before commitment to the αß and γδ lineages.

In addition to adaptive T cells, the thymus supports the development of unconventional T cells such as natural killer T (NKT) and CD8αα intraepithelial lymphocytes (IELs), which have innate functional properties, particular antigenic specificities, and tissue localization. Both conventional and innate T cells are believed to develop from common precursors undergoing instructive, TCR-mediated lineage fate decisions, but innate T cells are proposed to undergo positive instead of negative selection in response to agonistic TCR signals. In the present study, we show that, in contrast to conventional αßT cells, innate αßT cells are not selected against functional TCRγ rearrangements and express TCRγ mRNA. Likewise, in contrast to the majority of γδT cells, thymic innate γδT cells are not efficiently selected against functional TCRß chains. In precursors of conventional T cells, autonomous TCR signals emanating from the pre-TCR or γδTCR in the absence of ligand mediate selection against the TCR of the opposite isotype and αß/γδ lineage commitment. Our data suggest that developing innate T cells ignore such signals and rely solely on agonistic TCR interactions. Consistently, most innate T cells reacted strongly against autologous thymocytes. These results suggest that innate and adaptive T-cell lineages do not develop from the same pool of precursors and potentially diverge before αß/γδ lineage commitment.

Nrf2 is essential for cholesterol crystal-induced inflammasome activation and exacerbation of atherosclerosis.

Oxidative stress and inflammation--two components of the natural host response to injury--constitute important etiologic factors in atherogenesis. The pro-inflammatory cytokine interleukin (IL)-1 significantly enhances atherosclerosis, however, the molecular mechanisms of IL-1 induction within the artery wall remain poorly understood. Here we have identified the oxidative stress-responsive transcription factor NF-E2-related 2 (Nrf2) as an essential positive regulator of inflammasome activation and IL-1-mediated vascular inflammation. We show that cholesterol crystals, which accumulate in atherosclerotic plaques, represent an endogenous danger signal that activates Nrf2 and the NLRP3 inflammasome. The resulting vigorous IL-1 response critically depended on expression of Nrf2, and Nrf2-deficient apolipoprotein E (Apoe)-/- mice were highly protected against diet-induced atherogenesis. Importantly, therapeutic neutralization of IL-1α and IL-1ß reduced atherosclerosis in Nrf2+/- Apoe-/- but not in Nrf2-/- Apoe-/- mice, suggesting that the pro-atherogenic effect of Nrf2-signaling was primarily mediated by its permissive role in IL-1 production. Our studies demonstrate a role for Nrf2 in inflammasome activation, and identify cholesterol crystals as disease-relevant triggers of the NLRP3 inflammasome and potent pro-atherogenic cytokine responses. These findings suggest a common pathway through which oxidative stress and metabolic danger signals converge and mutually perpetuate the chronic vascular inflammation that drives atherosclerosis.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

BACKGROUND: Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. METHODS: We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. RESULTS: Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E. coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. CONCLUSION: Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

IL-21 induces death of marginal zone B cells during chronic inflammation.

Interleukin-2 (IL-2) and IL-21 share activities in the control of T- and B-cell maturation, proliferation, function, and survival. However, opposing roles for IL-2 and IL-21 have been reported in the development of regulatory T cells. To dissect unique, redundant, and opposing activities of IL-2 and IL-21, we compared T- and B-cell development and function in mice lacking both IL-2 receptor α (IL-2Rα) and IL-21R (double knockouts [DKO]) with single knockout and wild-type (WT) mice. Similarly to il2ra(-/-) mice, DKO showed reduced numbers of regulatory T cells and, consequently, hyper-activation and proliferation of T cells associated with inflammatory disease (ie, colitis), weight loss, and reduced survival. The absence of IL-2Rα resulted in overproduction of IL-21 by IFN-γ-producing CD4(+) T cells, which induced apoptosis of marginal zone (MZ) B cells. Hence, MZ B cells and MZ B-cell immunoglobulin M antibody responses to Streptococcus pneumoniae phosophorylcholine were absent in il2ra(-/-) mice but were completely restored in DKO mice. Our results highlight key roles of IL-2 in inhibiting IL-21 production by CD4(+) T cells and of IL-21 in negatively regulating MZ B-cell survival and antibody production.