T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross- sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder

Neuronal vulnerability and multilineage diversity in multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.Includes NIHR, ERC and Wellcome Trust

a dynamic equilibrium

Obgleich der Systemische Lupus Erythematodes (SLE) gewissermaßen als Prototyp der systemischen Autoimmunerkrankungen gilt, ist seine Pathogenese noch weitgehend unklar. Von Bedeutung scheint im Rahmen des Auftretens von Autoantikörpern gegen dsDNA auch eine mangelnde Kontrolle autoreaktiver CD4+ T -Helfer-Zellen zu sein. Diese vor allem gegen nukleäre Peptidantigene gerichteten T-Helfer-Zellen können in einer immunologischen Kreuzreaktion die Produktion von Anti-dsDNA-Antikörpern induzieren. Die Untersuchung autoreaktiver T-Helfer-Zellen gestaltete sich in der Vergangenheit jedoch methodisch schwierig. Hauptgrund hierfür ist ihre sehr geringe Frequenz im periphervenösen Blut. Dies machte lange Stimulationszeiten von mehreren Tagen erforderlich, um die T-Helfer-Zellen anhand ihrer gesteigerten Proliferation identifizieren zu können. Mit steigender Stimulationsdauer ergab sich allerdings die Schwierigkeit, eine ausreichende Spezifität der Messungen sicherzustellen. Durch Zelluntergang eingetragene nukleäre Antigene (endogene Kontamination) und der Einfluss von außen eingetragener Verunreinigungen (exogene Kontamination) brachten die vorhandenen Methoden an die Grenze ihrer spezifischen Aussagekraft. In dieser Arbeit wird eine leicht zu handhabende zytometrische Nachweismethode autoreaktiver T-Helfer-Zellen im periphervenösen Blut von SLE-Patienten vorgestellt. Als Marker für die antigenspezifische Aktivierung wurde CD40L verwandt. Die Stimulationszeit konnte mit 6 Stunden wesentlich kürzer gewählt werden als bei den bisherigen Proliferationsassays. Dies garantiert eine Messung der präformierten autoaggressiven Immunantwort und umgeht den Spezifitätsverlust durch eine mehrtägige Stimulation. Die Methode erlaubt im selben Arbeitsgang eine genaue zytometrische Phänotypisierung der autoaggressiven Zellen, die zudem einer gezielten Anreicherung und Kultivierung mittels MACS zur Verfügung stehen. Mittels der CD40L-Expression wurde die autoaggressive T-Helfer-Zell-Antwort in einem repräsentativen SLE-Kollektiv (n= 41 Blutproben) untersucht. Gegen Nukleosomen gerichtete T-Helfer-Zellen konnten bei 6/19 (31,6%) der untersuchten Proben festgestellt werden. Autoreaktive T-Helfer-Zellen gegen das nukleäre Peptidantigen SmD1(83-119) ließen sich demgegenüber wesentlich seltener nachweisen 2/41 (4,9%). Weiterhin wurde der Einfluss der regulatorischen T-Zellen auf die autoaggressive Immunantwort bei 11 Proben mittels einer in vitro-CD25-Depletion untersucht. Durch die Depletion der Treg-Zellen konnte eine Steigerung der T-Helfer-Zell-Antwort gegen das SmD1(83-119) Peptid um 46,6% erreicht werden (p=0,05), die sich damit signifikant vom Kontrollniveau abhebt (p=0,026). Diese demaskierte SmD1(83-119)-Antwort zeigt zudem eine exzellente Korrelation mit der Krankheitsaktivität gemessen im SLEDAI-Score (p=0,005; r=0,779) und ist in dieser Untersuchung der beste Krankheitsaktivitätsmarker. Die Ergebnisse dieser Arbeit unterstreichen die Bedeutung des dynamischen Gleichgewichtes autoreaktiver und regulatorischer T-Zellen für die Pathogenese des SLE. Wird die im Rahmen eines Krankheitsschubs verstärkte autoreaktive Immunantwort nicht effektiv durch die regulativen T-Zellen beherrscht, kommt es zur autoreaktiven Selbstschädigung. Die entscheidende Rolle der regulatorischen T-Zellen für die Krankheitsdynamik im humanen SLE konnte hier erstmals in dieser Form gezeigt werden. Regulative T-Zellen und das Autoantigen SmD1(83-119)-Peptid stellen vielversprechende Ansatzpunkte für zukünftige spezifische Immuntherapien dar. Die hier etablierte Methode zur zytometrischen Messung autoreaktiver T-Helfer-Zellen im Patientenblut könnte auch in anderen Autoimmunerkrankungen einen direkten Zugang zur autoaggressiven T-Zell-Antwort ermöglichen.Although systemic lupus erythematosus (SLE) is considered to be a prototype of a systemic autoimmune disease, its pathogenesis still remains largely unclear. However, an impaired control of autoreactive CD4+ T helper cells seems to be important for the tolerance break down as autoreactive T helper cells specific for nuclear peptide antigens have been shown to induce the production of anti- dsDNA antibodies. The detection of these autoreactive T helper cells is limited by their very low frequency in the peripheral venous blood, though. Accordingly, several days of antigenic stimulation have been required to efficiently identify T helper cells due to their increased proliferation. Since long term stimulations are prone to unspecific effects caused by exogenous culture contamination and endogenous contamination with nuclear autoantigens liberated in the course of naturally occurring cell death the significance of these measurements was limited. In this work, a recently established method of measuring T cell activation by flow cytometry is advanced for the detection of autoreactive T helper cells in the peripheral venous blood of SLE patients. As a marker of antigen-specific activation CD40L (CD154) was used. The considerably shorter stimulation time of six hours guarantees a detection of the preformed autoaggressive immune response and avoids the loss of specificity of a long term stimulation. At the same time, this method enables an exact cytometric phenotyping of the autoaggressive cells, which are further accessible to their enrichment by MACS. The autoaggressive T helper cell response was determined according to the intracellular expression of CD40L in CD4+ T helper cells after stimulation with SLE-associated autoantigens in a representative SLE collective (n = 41 blood samples). While a autoreactive response to human nucleosomes could be observed in 6 of 19 (31.6%) samples, autoreactive T helper cells specific for the SmD1(83-119) peptide were detectable only in 2 of 41 (4.9%) samples. To determine the influence of regulatory T cells (Tregs) on the activation of autoaggressive T helper cells, Tregs were removed by depleting CD25+ cells prior to antigenic stimulation in 11 samples. Due to the removal of Tregs a significant increase of the SmD1(83-119)-specific T helper cell response could be achieved (+46.6%, p=0.05). This unmasked SmD1(83-119)-response laid significantly above the level of the unstimulated controls (p = 0.026). Furthermore, it strongly correlated with the disease activity as assessed by the SLEDAI score (p=0.005, r=0.779). The results of this study emphasize the importance of a dynamic balance of autoreactive and regulatory T cells in the pathogenesis of SLE. Thus, an ineffective control of the autoreactive immune response by Tregs can lead to autoreactive self-defeating. Regulatory T cells and the autoantigen SmD1(83-119) peptide provide promising starting points for future specific immunotherapies. The direct assessment of autoantigen-specific CD4+ T helper cells according to the antigen-induced CD154 expression in combination with a depletion of CD25+ Tregs may be of further use in other autoimmune diseases

G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis

Neuroinflammation leads to neuronal stress responses that contribute to neuronal dysfunction and loss. However, treatments that stabilize neurons and prevent their destruction are still lacking. Here, we identify the histone methyltransferase G9a as a druggable epigenetic regulator of neuronal vulnerability to inflammation. In murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS), we found that the G9a-catalyzed repressive epigenetic mark H3K9me2 was robustly induced by neuroinflammation. G9a activity repressed anti-ferroptotic genes, diminished intracellular glutathione levels, and triggered the iron-dependent programmed cell death pathway ferroptosis. Conversely, pharmacological treatment of EAE mice with a G9a inhibitor restored anti-ferroptotic gene expression, reduced inflammation-induced neuronal loss, and improved clinical outcome. Similarly, neuronal anti-ferroptotic gene expression was reduced in MS brain tissue and was boosted by G9a inhibition in human neuronal cultures. This study identifies G9a as a critical transcriptional enhancer of neuronal ferroptosis and potential therapeutic target to counteract inflammation-induced neurodegeneration

Prenatal Administration of Betamethasone Causes Changes in the T Cell Receptor Repertoire Influencing Development of Autoimmunity

Prenatal glucocorticoids are routinely administered to pregnant women at risk of preterm delivery in order to improve survival of the newborn. However, in half of the cases, birth occurs outside the beneficial period for lung development. Glucocorticoids are potent immune modulators and cause apoptotic death of immature T cells, and we have previously shown that prenatal betamethasone treatment at doses eliciting lung maturation induce profound thymocyte apoptosis in the offspring. Here, we asked if there are long-term consequences on the offspring’s immunity after this treatment. In the non-obese diabetic mouse model, prenatal betamethasone clearly decreased the frequency of pathogenic T cells and the incidence of type 1 diabetes (T1D). In contrast, in the lupus-prone MRL/lpr strain, prenatal glucocorticoids induced changes in the T cell repertoire that resulted in more autoreactive cells. Even though glucocorticoids transiently enhanced regulatory T cell (Treg) development, these cells did not have a protective effect in a model for multiple sclerosis which relies on a limited repertoire of pathogenic T cells for disease induction that were not affected by prenatal betamethasone. We conclude that prenatal steroid treatment, by inducing changes in the T cell receptor repertoire, has unforeseeable consequences on development of autoimmune disease. Our data should encourage further research to fully understand the consequences of this widely used treatment

Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor interactome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications

Prenatal administration of Betamethasone causes changes in the T cell receptor repertoire influencing Development of autoimmunity

Prenatal glucocorticoids are routinely administered to pregnant women at risk of preterm delivery in order to improve survival of the newborn. However, in half of the cases, birth occurs outside the beneficial period for lung development. Glucocorticoids are potent immune modulators and cause apoptotic death of immature T cells, and we have previously shown that prenatal betamethasone treatment at doses eliciting lung maturation induce profound thymocyte apoptosis in the offspring. Here, we asked if there are long-term consequences on the offspring's immunity after this treatment. In the non-obese diabetic mouse model, prenatal betamethasone clearly decreased the frequency of pathogenic T cells and the incidence of type 1 diabetes (T1D). In contrast, in the lupus-prone MRL/lpr strain, prenatal glucocorticoids induced changes in the T cell repertoire that resulted in more autoreactive cells. Even though glucocorticoids transiently enhanced regulatory T cell (Treg) development, these cells did not have a protective effect in a model for multiple sclerosis which relies on a limited repertoire of pathogenic T cells for disease induction that were not affected by prenatal betamethasone. We conclude that prenatal steroid treatment, by inducing changes in the T cell receptor repertoire, has unforeseeable consequences on development of autoimmune disease. Our data should encourage further research to fully understand the consequences of this widely used treatment