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 9 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 hours, and were no longer detectable at 24 hours. Bortezomib therefore appears promising for treating MG and possibly other antibody-mediated autoimmune or allergic disorders, especially if given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

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 envbinding 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.Keywords: DCs r DC/T-cell conjugates r HERV r Immune tolerance r Immunological synapse r T-cell activation Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionHuman endogenous retroviruses (HERVs) are estimated to comprise about 8% of the human genome [1]. They are mainly germline transmitted. HERVs are typically defective as a result of multiple mutations in their genomes, and it is therefore only on exception that viral particles can be produced [1,2]. Endogenous retroviruses (ERVs) can substantially regulate host cell gene expression and functions. In addition to viral transcripts, ERV encoded proteins translated from intact ORFs were implicated Correspondence: Dr. Sibylle Schneider-Schaulies e-mail: s-s-s@vim.uni-wuerzburg.de in both pathological and physiological processes [3]. The latter has for instance been revealed for the envelope (env) regions of three HERVs (ERV-3, HERV-W, and HERV-FRD). These are highly expressed in early and late placentae, and their importance in successful accomplishment of pregnancy has been suggested [4][5][6][7][8][9][10], not least because spontaneous abortions or complications of gestation like preeclampsia are associated with reduced levels of HERV env proteins [11,12]. In fact, their expression has been found crucial in the intrauterine fetal development in promoting syncytiotrophoblast formation, while the HERV-FRD env was also associated with fetomaternal tolerance to prevent rejection of the fetus [13][14][15][16][17].Immunodeficiency is commonly associated with retroviral infections (for a recent review see [18] effector mechanisms studied, immunosuppressive domains (ISD) we...

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 CD8αβ (double negative (DN)). Natural IELs are both αβ 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 TCRαβNK1.1 thymocytes (IEL precursors (IELPs)) determines IEL fate in natural TCRαβ 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 TCRαβNK1.1 thymocytes are gradually restricted to a DN IEL fate. Furthermore, we show that the natural TCRαβ 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 TCRαβ IELs.

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.

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 − CD8αβ − TCRαβ + 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 highresolution molecular framework for thymic IEL development of NK1.1 − IELPs and deepen our understanding of this still elusive cell type.

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 Tbet) 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.

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.

In the autoimmune disease myasthenia gravis (MG), autoantibodies against the muscle AChR are mainly produced by both short- and long-lived plasma cells, which are resistant to standard immunosuppressive drugs (i.e. glucocorticoids). A novel therapy to eliminate plasma cells is the proteasome inhibitor bortezomib, which is used to treat patients with multiple myeloma (MM, a plasma cell malignancy). Previously, we demonstrated that bortezomib also reduced autoantibody titers in an animal model of MG (Gomez, A. M. J. Immunol. 2011). The thymus of MG patients is frequently enriched in germinal centers and contains plasma cells that produce autoantibodies in vitro, even after irradiation (which depletes B and T lymphocytes). We studied the in vitro effects of bortezomib in cultured thymus cells from MG patients undergoing therapeutic thymectomy. Treatment with a single dose of bortezomib blocked the production of these pathogenic autoantibodies, reduced the total IgG levels and eliminated plasma cells. Ultrastructural signs of apoptosis were detected in plasma cells as early as 8 h after addition of bortezomib; at 24 h, no plasma cells could be detected. Moreover, we demonstrated that the minimum concentration of bortezomib that eliminates plasma cells in vitro is 60-fold lower than the peak concentration found in MM patients treated with bortezomib, suggesting that low doses might be effective for eliminating plasma cells in patients with antibody-mediated autoimmune diseases.