Eomes cannot replace its paralog T-bet during expansion and differentiation of CD8 effector T cells.

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.

IFNL4 rs368234815 polymorphism does not predict risk of BK virus associated nephropathy after living-donor kidney transplant: A case-control study.

BACKGROUND: BK polyoma virus (BKPyV) associated nephropathy (BKPyVAN) is a major cause of kidney graft loss in renal transplant patients. Interferons (IFNs) are an important innate immune response against viral infections and genetic polymorphisms of the IFN-pathways can affect susceptibility and mortality during viral infection. Here, we investigated whether the dinucleotide polymorphism rs368234815 (ΔG/TT) in the IFNL4 gene contributed to BKPyV reactivation or BKPyVAN after living-donor kidney transplantation. METHODS: This retrospective case-control study determines the prevalence of IFNL4 variants in a Caucasian population of living-donor kidney transplant recipients and donors and explores its association with BKPyV infection and BKPyVAN development. We included 28 recipients with BKPyV reactivation, 10 of which developed BKPyVAN and 30 BKPyV negative controls. Targeted sequencing of the IFNL4 gene from both recipients and their respective donors was performed. RESULTS: We found IFNL4 rs368234815 ΔG allele frequencies of 41.7% in BKPyV negative and 39.3% in BKPyV positive recipients (P = .85), and 41.7% and 40.4% (P>.99) in their respective donors. IFNL4 rs368234815 ΔG allele frequencies in BKPyVAN developing recipients and their respective donors were 50% and 43.7% (P = .60 and P>.99). CONCLUSIONS: Our results indicate that the IFNL4 rs368234815 ΔG allele is not associated with BKPyV reactivation, nor the manifestation of BKPyVAN.

A dual-fluorescence reporter in the Eomes locus for live imaging and medium-term lineage tracing.

The T-box transcription factor Eomes (also known as Tbr2) shows short-lived expression in various localized domains of the embryo, including epiblast cells during gastrulation and intermediate progenitor cells in the cerebral cortex. In these tissues Eomes fulfills crucial roles for lineage specification of progenitors. To directly observe Eomes-dependent cell lineages in the living embryo, we generated a novel dual-fluorescence reporter allele that expresses a membrane-bound tdTomato protein for investigation of cell morphology and a nuclear GFP for cell tracing. This allele recapitulates endogenous EOMES protein expression and is suitable for live imaging. We found that the allele can also be used as a short-to-medium-term lineage tracer, as GFP persists in cells longer than EOMES protein and marks Eomes-dependent lineages with a timeframe of days to weeks depending on the proliferation rate. In summary, we present a novel genetic tool for investigation of Eomes-dependent cell types by live imaging and lineage tracing.

Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation.

Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Clustering Approaches for Gait Analysis within Neurological Disorders: A Narrative Review.

Background: The prevalence of neurological disorders is increasing, underscoring the importance of objective gait analysis to help clinicians identify specific deficits. Nevertheless, existing technological solutions for gait analysis often suffer from impracticality in daily clinical use, including excessive cost, time constraints, and limited processing capabilities. Summary: This review aims to evaluate existing techniques for clustering patients with the same neurological disorder to assist clinicians in optimizing treatment options. A narrative review of thirteen relevant studies was conducted, characterizing their methods, and evaluating them against seven criteria. Additionally, the results are summarized in two comprehensive tables. Recent approaches show promise; however, our results indicate that, overall, only three approaches display medium or high process maturity, and only two show high clinical applicability. Key Messages: Our findings highlight the necessity for advancements, specifically regarding the use of markerless optical tracking systems, the optimization of experimental plans, and the external validation of results. This narrative review provides a comprehensive overview of existing clustering techniques, bridging the gap between instrumented gait analysis and its real-world clinical utility. We encourage researchers to use our findings and those from other medical fields to enhance clustering techniques for patients with neurological disorders, facilitating the identification of disparities within groups and their extent, ultimately improving patient outcomes.

Tissue-resident memory T cells break tolerance to renal autoantigens and orchestrate immune-mediated nephritis.

Immune-mediated nephritis is a leading cause of acute kidney injury and chronic kidney disease. While the role of B cells and antibodies has been extensively investigated in the past, the advent of immune-checkpoint inhibitors has led to a reappraisal of the role of T cells in renal immunology. However, it remains elusive how T cells with specificity for renal autoantigens are activated and participate in immune-mediated nephritis. Here, we followed the fate and function of pathogen-activated autoreactive CD8 T cells that are specific for a renal autoantigen. We demonstrate that recently activated splenic CD8 T cells developed a hybrid phenotype in the context of renal autoantigen cross-presentation, combining hallmarks of activation and T cell dysfunction. While circulating memory T cells rapidly disappeared, tissue-resident memory T cells emerged and persisted within the kidney, orchestrating immune-mediated nephritis. Notably, T cells infiltrating kidneys of patients with interstitial nephritis also expressed key markers of tissue residency. This study unveils how a tissue-specific immune response can dissociate from its systemic counterpart driving a compartmentalized immune response in the kidneys of mice and man. Consequently, targeting tissue-resident memory T cells emerges as a promising strategy to control immune-mediated kidney disease.

Kidins220 and Aiolos promote thymic iNKT cell development by reducing TCR signals.

Development of T cells is controlled by the signal strength of the TCR. The scaffold protein kinase D-interacting substrate of 220 kilodalton (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell-specific Kidins220 knockout (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stages 2 and 3 shows that Kidins220 down-regulates TCR signaling at these stages. scRNA-seq indicated that the transcription factor Aiolos is down-regulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.

The Caspase-Activated DNase drives inflammation and contributes to defense against viral infection.

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection.

Eomes is sufficient to regulate IL-10 expression and cytotoxic effector molecules in murine CD4+ T cells.

The T-box transcription factors T-bet and Eomesodermin regulate type 1 immune responses in innate and adaptive lymphocytes. T-bet is widely expressed in the immune system but was initially identified as the lineage-specifying transcription factor of Th1 CD4+ T cells, where it governs expression of the signature cytokine IFN- γ and represses alternative cell fates like Th2 and Th17. T-bet's paralog Eomes is less abundantly expressed and Eomes+ CD4+ T cells are mostly found in the context of persistent antigen exposure, like bone marrow transplantation, chronic infection or inflammation as well as malignant disorders. However, it has remained unresolved whether Eomes executes similar transcriptional activities as T-bet in CD4+ T cells. Here we use a novel genetic approach to show that Eomes expression in CD4+ T cells drives a distinct transcriptional program that shows only partial overlap with T-bet. We found that Eomes is sufficient to induce the expression of the immunoregulatory cytokine IL-10 and, together with T-bet, promotes a cytotoxic effector profile, including Prf1, Gzmb, Gzmk, Nkg7 and Ccl5, while repressing alternative cell fates. Our results demonstrate that Eomes+ CD4+ T cells, which are often found in the context of chronic antigen stimulation, are likely to be a unique CD4+ T cell subset that limits inflammation and immunopathology as well as eliminates antigen-presenting and malignant cells.

Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1- T-bet+ intraepithelial lymphocyte precursors.

Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4-CD8αß-TCRαß+NK1.1- IEL precursors (NK1.1- IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1- IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1- IELPs and deepen our understanding of this still elusive cell type.

A committed postselection precursor to natural TCRαß+ intraepithelial lymphocytes.

The intestine is a major immune organ with several specialized lymphoid structures and immune cells. Among these are thymus-derived natural intraepithelial lymphocytes (IELs) that lack expression of the classical co-receptors CD4 or CD8αß (double negative (DN)). Natural IELs are both αß+ and γδ+ T cells that play important roles in the maintenance of the epithelial barrier at steady state and during inflammation. The transcription factor T-bet is essential for the peripheral development of natural IELs, but its role during thymic development has remained less clear. Here we show that a T-bet gradient in DN TCRαß+NK1.1- thymocytes (IEL precursors (IELPs)) determines IEL fate in natural TCRαß+ IELs. Employing T-bet ZsGreen reporter mice in in vitro cultures and in vivo transfer experiments, we demonstrate that with increasing expression of T-bet, DN TCRαß+NK1.1- thymocytes are gradually restricted to a DN IEL fate. Furthermore, we show that the natural TCRαß+ IELs seed the intestine within the first month of life. This in turn is preceded by the appearance of T-bet- and T-bet+ IELPs that egress from the thymus in a sphingosine-1-phosphate (S1P)-dependent manner. In summary, the use of T-bet reporter mice has enabled us to identify and refine an immediate and clearly committed postselection precursor of natural TCRαß+ IELs.