Suspension MC Session

Affiliation

Abstract

Biosketch

Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain

CyTOF harmonization for multicenter immune monitoring studies

Background: 3TR is a multicenter consortium studying common molecular signatures across 7 chronic diseases. Among various OMICs technologies, cytomics data will be obtained and whole blood samples will be analyzed using mass cytometry. Due to high numbers of individuals (>1000) and limited CyTOF throughput, various instruments need to be involved.

Objectives: Optimize samples acquisition protocol and verify if multiple CyTOF instruments can be used.

Methods: For acquisition solution selection, identical aliquots of blood samples were acquired using CAS, CASPLUS, and water. FlowSOM and PCA analysis was performed. To study spillover matrices stability, 12-plex stained compensation beads were analyzed in fresh or after freezing for up to 3 months. To test multiple CyTOF instruments, barcoded blood from 3 donors was stained (22-plex panel), aliquoted and frozen. Aliquots were acquired using 4 HELIOS and 1 CyTOF XT. Coefficient of variation (CV) was calculated for cell frequencies and median signal intensities.

Results: Increasing background was detected using CAS. Water introduced artefacts during clustering. CASPLUS presented the highest sample stability, if acquired in aliquots for no longer than 1h each. Spillover compensation was consistent during 3 different experiments up to 3 months. Center-based effect was reduced with bead normalization, however CVs >20% were detected in some populations. CytoNorm normalization reduced center enrichment in the clustering analysis, additionally yielding CVs <20%.

Conclusions: Multicenter CyTOF experiments are feasible, however special sample acquisition protocol and additional normalization steps are needed to align the centers.

Berlin Institute of Health (BIH), Charité – Universitätsmedizin Berlin, Berlin, Germany

High-parameter immunophenotyping using cytometry by time of flight (CyTOF) and unbiased bioinformatical analysis identifies biomarkers of Post-Covid Syndrome ME/CFS and of response to immunoadsorption therapy

COVID-19 infections can lead to symptoms of chronic fatigue and exertion intolerance, persisting for months and even years post-infection – known as Post-Covid Syndrome (PCS) or Long-Covid. The estimated incidence is about 400.000 new cases in Germany every year and no prescribable therapies exist. The heterogeneous clinical appearance points towards multiple etiologies. A subset of patients develops myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) a severe postinfectious sequela of various infections. Clinical studies provided evidence for a role of autoantibodies in ME/CFS. Recent reports connect severe COVID-19 and PCS with an aberrant immune response with some patients developing autoantibodies. These might benefit from IG removal (immunoadsorption, IA), but biomarkers for patient stratification and mechanistic insights are lacking.

Cytometry by time of flight (CyTOF) was used to profile leukocytes in blood samples from PCS-ME/CFS patients (PCS patients meeting ME/CFS criteria) pre- and post-treatment and from age- and sex-matched controls. PCS-ME/CFS patients divided into IA-responder and non-responder. IA-responders showed heightened B cell activation across the entire B cell compartment, whereas non-responders did not, indicating a potential biomarker for PCS-ME/CFS and autoantibody-targeting treatment. This normalized after IA-treatment, suggesting a causal link.

This study presents those B cell alterations as potential biomarkers for PCS-ME/CFS and autoantibody-targeting treatment. Currently, we perform single-cell transcriptome, plasma proteomics and mechanistic in vitro analyses to understand which factors might drive the dysregulation of the B cell compartment and its contribution to PCS development. However, the study also highlights the complexity of PCS and the need to identify biomarkers predicting therapy response.

Neuro-SysMed, Department of Neurology, Haukeland University Hospital and Department of Clinical Medicine, University of Bergen, Norwey

Immune Reconstitution After Hematopoietic Stem Cell Transplantation in Multiple Sclerosis

Patients with relapsing-remitting multiple sclerosis (RRMS) may experience persistent disease activity despite treatment with disease-modifying therapies (DMTs) or present with an aggressive, rapidly progressing disease. For these patients, autologous hematopoietic stem cell transplantation (aHSCT) is an alternative treatment option. This procedure has demonstrated effectiveness in halting inflammatory disease progression, achieving long-term disease remission, and restoring immune tolerance by eliminating autoreactive immune cells. However, the mechanisms underlying its efficacy remain incompletely understood.

This study explores how G-CSF mobilization, immunoablation with cyclophosphamide and anti-thymocyte globulin, and subsequent immune reconstitution affect the immune system in RRMS patients undergoing aHSCT. Data were sourced from Arm A of the RAM-MS clinical trial, which compares aHSCT to alemtuzumab, cladribine, or ocrelizumab in RRMS patients. Over 100 million single cells from stabilized whole blood of 25 patients were analyzed using mass cytometry (CyTOF) at several time points – on inclusion and at 3, 6, 12, and 24 months following aHSCT.

Following aHSCT, naïve CD4 and CD8 T cells and memory B cells were nearly entirely depleted, while CD4 and CD8 memory T cells and naïve B cells were rapidly reconstituted. Some of these changes persisted over time. Functional alterations in chemokine and adhesion markers were also observed, suggesting profound immunological reprogramming.

This study provides valuable insights into the dynamic immune changes following aHSCT and enhances our understanding of its mechanisms in treating RRMS, offering a potential for refining future therapeutic approaches.