Search

countries. In the past decade evidence for a clinical relevance of GPCR-autoimmunity in human HF has substantially increased. Stimulating autoantibodies targeting the second extracellular loop (ECII) of the cardiac beta1-adrenoceptor (beta1-aabs) have been claimed to be involved in the pathogenesis of HF and to increase the risk of cardio-vascular death by three-fold. Still, the events triggering the formation of beta1-aabs and their impact on HF-progression are unknown. Methods: Based on the rationale that presentation of beta1-adrenoceptors and other cardiac membrane antigens following inflammation or necrosis may induce the formation of beta1-aabs, in total 13 University Hospitals (12 in Germany, 1 in Serbia) prospectively recruited 226 patients with a first acute myocardial infarction (FAMI), and 140 pts with acute (biopsy- or cMRI-proven) myocarditis (AMitis) into the Etiology, Titer-Course and effect on Survival of cardiac autoantibodies-study (ETiCS-study). At baseline (BL) and three follow-up visits (Fup1-3) blood was sampled to follow the time-course of various GPCR-aabs. Beta1-aabs were assessed by using Dynabeads® M-270-Epoxy coated with increasing amounts of beta1-ECII-peptides (2.5-100 μg/ml). As control, beads were coated with a peptide comprising same amino-acids, but in a scrambled order. After reacting with the beads (15min on ice) the samples were measured on a FACScan flow-cytometer. Data were analyzed using FlowJo (Treestar); when half-maximal binding was calculable the serum was classified beta1-aab-positive. Results: From n=366 pts (226 FAMI/140 AMitis) recruited into the ETiCS-study 45 pts had to be excluded because of unperformed cMRI’s; 46 pts stopped the study before Fup-1 (month 3). Only 180/226 FAMI- and 98/140 AMitis-pts had complete Fup1-3 (after 3, 6, and 12 months, including clinical evaluation, echocardiograms, and cMRI’s at BL & Fup-3). All valid ETiCS-pts (197 FAMI-/123 AMitis-pts) were assessed for the formation of beta1-aabs, and then compared focussed on the development of echo-LVEF. In the course of the study relevant (high-affinity) beta1-aab-titres were detected in ~31% (37/123) of the AMitis-pts compared to only ~21% (42/197) of the FAMI-pts. In aab-positive AMitis-pts echo-LVEF did not recover and was always significantly inferior to aab-negative AMitis-pts (BL: 38 vs. 49% LVEF; Fup-3: 49 vs. 64% LVEF) whereas such a difference was not noted in FAMI-pts. In addition, aab-positive AMitis-pts had higher NT pro-BNP-, renin-, and aldosterone-levels than aab-negative AMitispts. Conclusion: These first results from the ETiCS-study suggest that acute microbial-induced rather than post-infarction myocardial inflammation triggers the formation of clinically relevant beta1-aabs. AAb-positive AMitis-patients might profit from early intensification of standard HF-therapy (including early beta-blockade) and/or novel antibody-directed experimental therapies which are currently under development., Advances in RAB Research, Vol 1 No 1 (2018)

In rodents, low doses of CD28-specific superagonistic monoclonal antibodies (CD28 superagonists, CD28SA) selectively activate regulatory T cells (Treg). This observation has recently been extended to humans, suggesting an option for the treatment of autoimmune and inflammatory diseases. However, a mechanistic explanation for this phenomenon is still lacking. Given that CD28SA amplify T cell receptor (TCR) signals, we tested the hypothesis that the weak tonic TCR signals received by conventional CD4\(^{+}\) T cells (Tconv) in the absence of cognate antigen require more CD28 signaling input for full activation than the stronger TCR signals received by self-reactive Treg. We report that in vitro, the response of mouse Treg and Tconv to CD28SA strongly depends on MHC class II expression by antigen-presenting cells. To separate the effect of tonic TCR signals from self-peptide recognition, we compared the response of wild-type Treg and Tconv to low and high CD28SA doses upon transfer into wild-type or H-2M knockout mice, which lack a self-peptide repertoire. We found that the superior response of Treg to low CD28SA doses was lost in the absence of self-peptide presentation. We also tested if potentially pathogenic autoreactive Tconv would benefit from self-recognition-induced sensitivity to CD28SA stimulation by transferring TCR transgenic OVA-specific Tconv into OVA-expressing mice and found that low-dose CD28SA application inhibited, rather than supported, their expansion, presumably due to the massive concomitant activation of Treg. Finally, we report that also in the in vitro response of human peripheral blood mononuclear cells to CD28SA, HLA II blockade interferes with the expansion of Treg by low-dose CD28SA stimulation. These results provide a rational basis for the further development of low-dose CD28SA therapy for the improvement of Treg activity.

Supplemental material for Fecal SCFAs and SCFA-producing bacteria in gut microbiome of human NAFLD as a putative link to systemic T-cell activation and advanced disease by Monika Rau, Ateequr Rehman, Marcus Dittrich, Albert K. Groen, Heike M. Hermanns, Florian Seyfried, Niklas Beyersdorf, Thomas Dandekar, Philip Rosenstiel and Andreas Geier in United European Gastroenterology Journal

International audience; Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2+) and then either blocked CD28-ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4+ T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4+ T cells.

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4\(^{+}\) conventional (CD4\(^{+}\)CD25\(^{-}\)Foxp3\(^{-}\), Tconv) and regulatory (CD4\(^{+}\)CD25\(^{+}\)Foxp3\(^{+}\), Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4\(^{+}\) Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4\(^{+}\) Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.

Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4\(^{+}\) T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4\(^{+}\) T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn\(^{2+}\) ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn\(^{2+}\). In vitro stimulation assays using highly purified mouse CD4\(^{+}\) T cells showed that Pra1 increased proliferation of CD4\(^{+}\) T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4\(^{+}\) T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4\(^{+}\) T cell responses as shown in our in vitro assays.

Breakdown of sphingomyelin as catalyzed by the activity of sphingomyelinases profoundly affects biophysical properties of cellular membranes which is particularly important with regard to compartmentalization of surface receptors and their signaling relay. As it is activated both upon TCR ligation and co-stimulation in a spatiotemporally controlled manner, the neutral sphingomyelinase (NSM) has proven to be important in T cell activation, where it appears to play a particularly important role in cytoskeletal reorganization and cell polarization. Because these are important parameters in directional T cell migration and motility in tissues, we analyzed the role of the NSM in these processes. Pharmacological inhibition of NSM interfered with early lymph node homing of T cells in vivo indicating that the enzyme impacts on endothelial adhesion, transendothelial migration, sensing of chemokine gradients or, at a cellular level, acquisition of a polarized phenotype. NSM inhibition reduced adhesion of T cells to TNF-α/IFN-γ activated, but not resting endothelial cells, most likely via inhibiting high-affinity LFA-1 clustering. NSM activity proved to be highly important in directional T cell motility in response to SDF1-α, indicating that their ability to sense and translate chemokine gradients might be NSM dependent. In fact, pharmacological or genetic NSM ablation interfered with T cell polarization both at an overall morphological level and redistribution of CXCR4 and pERM proteins on endothelial cells or fibronectin, as well as with F-actin polymerization in response to SDF1-α stimulation, indicating that efficient directional perception and signaling relay depend on NSM activity. Altogether, these data support a central role of the NSM in T cell recruitment and migration both under homeostatic and inflamed conditions by regulating polarized redistribution of receptors and their coupling to the cytoskeleton.

Einleitung: Regulatorische T-Zellen (Treg) spielen in der Immunregulation eine bedeutende Rolle: Bei verschiedenen Autoimmunerkrankungen konnte bereits eine Fehlregulation durch Treg gezeigt werden. Hier soll nun ihr Einfluss auf die Ätiologie der chronisch polypösen Sinusitis untersucht werden.[zum vollständigen Text gelangen Sie über die oben angegebene URL], 87. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

Introduction: For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co‐transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)—a potentially life‐threatening complication. Methods: Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD. Results: Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4\(^{+}\) T cell transplantation with the Hsp90 inhibitor 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia‐bearing mice after transplantation of allogeneic CD4\(^{+}\) and CD8\(^{+}\) T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4\(^{+}\) T cells with a relative resistance of CD4\(^{+}\) regulatory and CD8\(^{+}\) T cells toward Hsp90 inhibition. Conclusions: Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.

Niklas Beyersdorf, Thomas Kerkau, Thomas Hünig Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany Abstract: T-cells play a key role within the adaptive immune system mediating cellular immunity and orchestrating the immune response as a whole. Their activation requires not only recognition of antigen/major histocompatibility complexes by the T-cell receptor but in addition co-stimulation via the CD28 molecule through binding to CD80, CD86, or as recently discovered, inducible co-stimulator ligand expressed by antigen-presenting cells. Apart from tight control of the co-stimulatory signal by the T-cell receptor complex, expression of the inhibitory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) sharing its ligands with CD28 is required to avoid inappropriate or prolonged T-cell activation. CD4+ Foxp3+ regulatory T (Treg) cells, which are crucial inhibitors of autoimmunity, add another level of complexity in that they differ from conventional non-regulatory CD4+ T-cells by strongly depending on CD28 signaling for their generation and homeostasis. Moreover, CTLA-4 is constitutively expressed by Treg cells where it serves as a key mediator of suppression, while conventional CD4+ T-cells express CTLA-4 only after activation. Here, we discuss recent insights into the molecular events underlying CD28-mediated co-stimulation, its impact on gene regulation, and the differential role of CD28 expression on Treg cells versus conventional CD4+ and CD8+ T-cells. Moreover, we summarize the exciting therapeutic options which have arisen from our current understanding of T-cell co-stimulation. Some of these have already been translated into the clinic, while others are expected to follow soon due to promising preclinical results. In particular, we discuss the failed 2006 trial of the CD28 superagonist TGN1412, and the return of this potent T-cell activator to clinical development. Keywords: CTLA-4, mAbs, rheumatoid arthritis, Treg cells, CD28 superagonist, TGN1412

Peripheral T cell lymphomas (PTCLs) are associated with a poor prognosis due to often advanced disease at the time of diagnosis and due to a lack of efficient therapeutic options. Therefore, appropriate animal models of PTCL are vital to improve clinical management of this disease. Here, we describe a monoclonal CD8\(^+\) CD4\(^−\) αβ T cell receptor Vβ2\(^+\) CD28\(^+\) T cell lymphoma line, termed T8-28. T8-28 cells were isolated from an un-manipulated adult BALB/c mouse housed under standard pathogen-free conditions. T8-28 cells induced terminal malignancy upon adoptive transfer into syngeneic BALB/c mice. Despite intracellular expression of the cytotoxic T cell differentiation marker granzyme B, T8-28 cells appeared to be defective with respect to cytotoxic activity as read-out in vitro. Among the protocols tested, only addition of interleukin 2 in vitro could partially compensate for the in vivo micro-milieu in promoting growth of the T8-28 lymphoma cells.

Die Reifung, Differenzierung und Funktion von Lymphozyten wird maßgeblich von aktivierenden und inhibitorischen Zelloberflächenrezeptoren reguliert. "Killer cell Lectin-like receptor G1" (KLRG1) ist ein Typ-II-Transmembranprotein, dessen Expression auf Subpopulationen von T-Lymphozyten und Natürlichen Killerzellen beschränkt ist. Die vorliegende Arbeit hatte zum Ziel, die differentielle Expression und mögliche Funktion von KLRG1 auf diesen Zellen im Maussystem zu charakterisieren. Mit Hilfe zellbiologischer Untersuchungsmethoden konnte gezeigt werden, dass die KLRG1-Expressionsfrequenz mit dem Reifegrad der Zellen korreliert. Frühere Beobachtungen, wonach KLRG1 durch Erkennung eigener Klasse-I-Moleküle des Haupthistokompatibilitätskomplexes (MHC) über inhibitorische Rezeptoren der Ly49-Familie induziert wird, konnten auf klassische Klasse-I-Moleküle und neue Ly49-Familienmitglieder ausgeweitet werden. Ferner belegen Daten dieser Arbeit, dass reife NK-Zellen die KLRG1-Expressionsfrequenz in verschiedenen lymphoiden Organen dem Klasse-I-Niveau der umgebenden Zellen anpassen können und dass T- und B-Lymphozyten möglicherweise eine zentrale Rolle hierbei spielen. Somit stellt KLRG1 einen NK-Zellrezeptor dar, dessen Expression durch Ly49-Rezeptorengagement dynamisch reguliert wird. Es ist möglich, dass KLRG1 kompensatorische (ko-)inhibitorische Eigenschaften besitzt, die für die Aufrechterhaltung der Selbsttoleranz von NK-Zellen von Bedeutung sind. Unter den CD8-T-Zellen identifiziert ein polyklonales abT-Zellrezeptorrepertoire und die Expression von CD8 als ab-Heterodimer den 2-3%igen Anteil an KLRG1+ Zellen als konventionelle T-Zellen thymischen Ursprungs. Umfangreiche phänotypische und funktionelle Analysen ergaben, dass KLRG1-exprimierende CD8-Zellen sich aus ca. 20% proinflammatorischer Effektorzellen und ca. 80% Gedächtniszellen zusammensetzen. Aufgrund von Daten der Arbeitsgruppe Pircher scheint das Zellteilungsvermögen letzterer ausgeschöpft zu sein. Demzufolge markiert KLRG1 eine neue Subpopulation von CD8-T-Zellen, der sowohl Effektorzellen, als auch "replikativ seneszente" Gedächtniszellen angehören. Abschließende Untersuchungen ergaben, dass KLRG1 interessanterweise auch von 1-2% der CD4+ T-Zellen exprimiert wird und dass die KLRG1+ CD4-T-Zellen zum Großteil CD25 koexprimieren. Funktionelle Anschlußexperimente zeigten, dass es sich bei diesen Zellen um regulatorische T-Zellen handelt. Zusammenfassend kennzeichnet KLRG1-Expression eine Subpopulationen von NK-Zellen, die körpereigene Zellen über Klasse-I erkennen können, eine Untergruppe von Effektor- und seneszenten CD8-T-Zellen sowie neuartige regulatorische CD4-T-Zellen., The maturation, differentiation and function of lymphocytes is crucially regulated by activating and inhibitory cell surface receptors. "Killer cell lectin-like receptor G1" (KLRG1) is a type-II transmembrane protein, which is expressed on subpopulations of T cells and Natural Killer cells. The aim of this work was to characterise the differential expression and possible function of KLRG1 on these cells in the mouse. Cell biological assays revealed that the expression frequency of KLRG1 and the maturity of the cells are positively correlated. The earlier finding that KLRG1 expression is induced after recognition of self class I molecules of the Major Histocompatibility Complex (MHC) by inhibitory receptors of the Ly49 family could be extended to classical class I molecules and to novel members of the Ly49 family. Moreover, the data contained in this work provide evidence that mature NK cells are capable of adapting KLRG1 expression frequencies to the level of class I expression on surrounding cells in the respective lymphoid organ and that T and B lymphocytes probably play a central role in this process. KLRG1 expression on NK cells, thus, is subject to dynamic regulation via engagement of Ly49 receptors. Moreover, it is possible that KLRG1 is endowed with compensatory (co-)inhibitory properties which are important for maintaining self-tolerance of NK cells. Among CD8 T cells it is the polyclonal ab T cell receptor repertoire and the expression of CD8 as an ab heterodimer which indicates thymic origin of the 2-3% KLRG1+ cells. Extensive phenotypical and functional analyses revealed that KLRG1-expressing CD8 cells are a mixture of about 20% pro-inflammatory effector cells and about 80% memory cells. According to data generated in the group of Hanspeter Pircher the latter have lost the potential to undergo further cell divisions. Therefore, KLRG1 is a marker for a novel subpopulation of CD8 T cells consisting of, both, effector cells and "senescent" memory cells. In final experiments, KLRG1 expression was detected on 1-2% of CD4+ T cells with the majority of KLRG1+ CD4 T cells co-expressing CD25. Subsequent functional studies revealed that these cells are regulatory T cells. In summary, KLRG1 expression marks a subpopulation of NK cells, which are capable of recognising other cells via class I molecules, a subset of effector and senescent CD8 T cells as wells as novel regulatory CD4 T cells.