Transcriptomic and immunohistochemical analysis of T cell activity in PCD model L7-HA

Paraneoplastische neurologische Degeneration ist eine schwere Erkrankung begleitet von massivem Verlust von Purkinje-Neuronen und einer hohen Sterblichkeit. Patienten mit dieser Krankheit leiden an einem Tumor, welcher Antigene produziert, die gleichzeitig im Cerebellum exprimiert werden. Das Immunsystem erkennt diese Antigene und induziert eine Immunantwort, welche zur Schädigung des cerebellaren Gewebes führt. Diese Schädigung wird vorwiegend durch zytotoxische DC8+ T-Zellen verursacht. Während dieser cerebellaren Entzündung konnten aktivierte CD8+ T-Zellen in der Nähe von Purkinje-Zellen detektiert werden, wobei diese schädliche Mengen IFN-γ produzierten. IFN-γ förderte widerum die Entzündungskaskade. Mit der Einführung von Immuncheckpoint-Inhibitoren (ICIs), erlangte PCD wieder mehr Aufmerksamkeit. ICIs streben grundsätzlich eine effektive Immunantwort gegen den Tumor an, allerdings inhibieren sie auch wichtige Kontrollmechanismen, welche normalerweise eine Überreaktion des Immunsystems verhindern. In seltenen Fällen, treten durch die Anwendung von ICIs paraneoplastische Symptome aus, wie zum Beispiel PCD. Auf Grund der schwerewiegenden neurologischen Symptome, ist es von großer Bedeutung, die immunologischen Mechanismen, insbesondere die Rolle von IFN-γ sowie sein Potenzial als therapeutisches Zielmolekül, weiter zu erforschen. In diesem Projekt wurde das Mausmodell L7-HA mit PCD-Pathologie verwendet, um die T-Zell-Aktivität, sowie deren Korrelation mit der Präsenz von IFN-γ untersucht. Dabei wurde der Fokus auf den Effekt von Anti-IFNγ-Antikörpern, welche IFN-γ im peripheren Gewebe des Mausmodells. neutralisieren sollten, gelegt. Verschieden Gruppen des Mausmodells erhielten verschiedene Behandlungen. Post mortem wurde mit dem cerebellaren Gewebe der Tiere eine transkriptomische Analyse durchgeführt, um Genexpressionswege der Cerebella der verschiedenen Behandlungsgruppen zu vergleichen. Des Weiteren wurden T-Zellen und pSTAT-positive Zellen quantifiziert, um die transkriptomischen Resultate zu bestätigen. Die konfokale Mikroskopie wurde verwendet, um Korrelationen zwischen pSTAT1-, MHCI- und Calbindin-positiven Zellen in den verschiedenen Behandlungsgruppen zu analysieren. Die Resultate ergaben, dass Anti-IFN-γ-behandelte Tiere im Vergleich zu unbehandelten Tieren eine Aufregulierung von Genexpressionswegen, welche an der neuronalen Homöostase beteiligt sind, zeigen. Unbehandelte Tiere wiesen eine Aufregulierung von inflammatorischen Genexpressionswegen auf. Diese waren in Funktionen, wie die IFN-γ- Antwort, involviert und zeigten eine hohe T-Zell- und pSTAT1-positive Zell-Zahl im Cerebellum. Bilder der konfokalen Mikroskopie unterstützen diese Beobachtungen. Anti-IFN-γ-behandelte Tiere wiesen meistens eine Abwesenheit oder minimale Erhöhung von MHC-I-Molekülen an der Zelloberfläche auf. Die Menge an Calbindin war in diesen Tieren näher an physiologischen Niveaus, verglichen zu unbehandelten Tieren. Diese Beobachtungen bestätigen das große Potenzial des Anti-IFN-γ-Antikörpers als mögliche anti-inflammatorische Therapie gegen PCD.Paraneoplastic neurological degeneration is a severe disorder characterized by an extensive loss of Purkinje cells in the cerebellum which is accompanied by high mortality. In this disorder, patients suffer from a tumour which produces an oncoprotein that is simultaneously expressed in the cerebellum. The immune system recognizes the antigens and induces a response resulting in cerebellar tissue damage predominantely caused by the cytotoxicity of CD8+ T cells. During cerebellar inflammation, activated CD8+ T cells have been detected in close proximity to Purkinje cells and found to produce harmful amounts of IFN-γ which further promotes inflammation. 1 Since, the introduction of immune checkpoint inhibitors (ICIs), PCD has gained increasing attention. ICIs aim to elicit an effective immune response against the tumour, however, they also inhibit the control mechanisms that prevent an overreaction of the immune system. In rare cases, the application of ICIs is accompanied by paraneoplastic syndromes, including PCD. Due to the severe neurological symptoms, immunological events, especially the role of IFN-γ and its potential as a therapeutic target, have to be further investigated2,3 In this project L7-HA mouse model exhibiting the pathology of PCD was used to investigate T cell activity and its correlation with IFN-γ presence. The focus was put on the effect of anti-IFN-γ antibodies which aimed to neutralize IFN-γ in the peripheral tissue of this mouse-model.1 Different groups of this mouse model received distinct treatments. Upon sacrifice of the subjects, transcriptomics were used to perform a comparative analysis of pathways differentially expressed in the cerebellum of the distinct groups. Moreover, immunohistochemical detection of T cell numbers and pSTAT-positive cells helped to validate the results. In addition, confocal microscopy was used to analyse the presence of pSTAT1, MHC class I and calbindin positive cells in the cerebellum of the differently treated animals. Results revealed that anti-IFN-γ treated animals show an upregulation of pathways involved in neuronal homeostasis compared to untreated animals with PCD pathology. Untreated animals showed an upregulation of proinflammatory pathways involved in functions, such as IFN-γ response and exhibited high numbers of T cells and pSTAT1-positive cells in the cerebellum. Confocal microscopy further confirmed these findings. Anti-IFN-γ treated animals mostly showed absence or only slight increase of MHC class I molecules on cell surface and calbindin levels were closer to physiological conditions compared to untreated animals. These findings further confirm the great potential of anti-IFN-γ antibody as useful anti-inflammatory therapy against PCD

Temporal lobe epilepsy with GAD antibodies: neurons killed by T cells not by complement membrane attack complex

Troscher AR, Mair KM, de Juan LV, et al. Temporal lobe epilepsy with GAD antibodies: neurons killed by T cells not by complement membrane attack complex. Brain: A Journal of Neurology. 2022: awac404.Temporal lobe epilepsy (TLE) is one of the syndromes linked to antibodies against glutamic acid decarboxylase (GAD). It has been questioned whether 'limbic encephalitis with GAD antibodies' is a meaningful diagnostic entity. The immunopathogenesis of GAD-TLE has remained enigmatic. Improvement of immunological treatability is an urgent clinical concern. We retrospectively assessed the clinical, MRI and CSF course as well as brain tissue of 15 adult patients with GAD-TLE who underwent temporal lobe surgery. Brain tissue was studied by means of immunohistochemistry, multiplex fluorescent microscopy and transcriptomic analysis for inflammatory mediators and neuronal degeneration. In 10 patients, there was a period of mediotemporal swelling and T2 signal increase; in nine cases this occurred within the first 6 years after symptom onset. This resulted in unilateral or bilateral hippocampal sclerosis (HS); three cases developed HS already within the first 2 years. All CSF studies done within the first year (n=6) revealed intrathecal synthesis of immunoglobulin G. Temporal lobe surgeries were done after a median disease duration of 9 years (range 3 weeks to 60 years). Only two patients became seizure free. Brain parenchyma collected during surgery in the first 6 years revealed high numbers of plasma cells but no signs of antibody-mediated tissue damage. Even more dense was the infiltration by CD8+cytotoxic T lymphocytes (CTLs) that were seen to locally proliferate. Further, a portion of these cells revealed an antigen-specific resident memory T cell phenotype. Finally, CTLs with cytotoxic granzyme B+ granules were also seen in microglial nodules and attached to neurons, suggesting a CTL-mediated destruction of these cells. With longer disease duration, the density of all lymphocytes decreased. Whole transcriptome analysis in early/active cases (but not in late/inactive stages) revealed 'T cell immunity' and 'Regulation of immune processes' as the largest overrepresented clusters. To a lesser extent, pathways associated with B cells and neuronal degeneration also showed increased representation. Surgically treated patients with GAD-TLE go through an early active inflammatory, 'encephalitic' stage (≤ 6 years) with CTL-mediated, antigen-driven neuronal loss and antibody-producing plasma cells but without signs of complement-mediated cell death. Subsequently, patients enter an apparently immunologically inactive or low-active stage with ongoing seizures, probably caused by the structural damage to the temporal lobe. 'Limbic encephalitis' with GAD antibodies should be subsumed under GAD-TLE. The early tissue-damage explains why immunotherapy does usually not lead to seizure-freedom. © The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used