Objective: Mass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA)... Show moreObjective: Mass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA) patients enrolled in the Tight Control SpondyloArthritis (TiCoSpA) trial. Methods: Fresh, longitudinal PBMCs samples (baseline, 24, and 48 weeks) from 9 early, untreated axSpA patients and 7 HLA-B27+ controls were analyzed using a 35-marker panel. Data were subjected to HSNE dimension reduction and Gaussian mean shift clustering (Cytosplore), followed by Cytofast analysis. Linear discriminant analyzer (LDA), based on initial HSNE clustering, was applied onto week 24 and 48 samples. Results: Unsupervised analysis yielded a clear separation of baseline patients and controls including a significant difference in 9 T cell, B cell, and monocyte clusters (cl), indicating disrupted immune homeostasis. Decrease in disease activity (ASDAS score; median 1.7, range 0.6-3.2) from baseline to week 48 matched significant changes over time in five clusters: cl10 CD4 Tnai cells median 4.7 to 0.02%, cl37 CD4 T-em cells median 0.13 to 8.28%, cl8 CD4 Tcm cells median 3.2 to 0.02%, cl39 B cells median 0.12 to 2.56%, and cl5 CD38+ B cells median 2.52 to 0.64% (all p<0.05). Conclusions: Our results showed that a decrease in disease activity in axSpA coincided with normalization of peripheral T- and B-cell frequency abnormalities. This proof of concept study shows the value of MC immuno-monitoring in clinical trials and longitudinal studies in axSpA. MC immunophenotyping on a larger, multi-center scale is likely to provide crucial new insights in the effect of anti-inflammatory treatment and thereby the pathogenesis of inflammatory rheumatic diseases. Show less
Koppejan, H.; Beyrend, G.; Hameetman, M.; Abdelaal, T.; Toes, R.E.M.; Gaalen, F.A. van 2023
ObjectiveMass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA)... Show moreObjectiveMass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA) patients enrolled in the Tight Control SpondyloArthritis (TiCoSpA) trial.MethodsFresh, longitudinal PBMCs samples (baseline, 24, and 48 weeks) from 9 early, untreated axSpA patients and 7 HLA-B27+ controls were analyzed using a 35-marker panel. Data were subjected to HSNE dimension reduction and Gaussian mean shift clustering (Cytosplore), followed by Cytofast analysis. Linear discriminant analyzer (LDA), based on initial HSNE clustering, was applied onto week 24 and 48 samples.ResultsUnsupervised analysis yielded a clear separation of baseline patients and controls including a significant difference in 9 T cell, B cell, and monocyte clusters (cl), indicating disrupted immune homeostasis. Decrease in disease activity (ASDAS score; median 1.7, range 0.6–3.2) from baseline to week 48 matched significant changes over time in five clusters: cl10 CD4 Tnai cells median 4.7 to 0.02%, cl37 CD4 Tem cells median 0.13 to 8.28%, cl8 CD4 Tcm cells median 3.2 to 0.02%, cl39 B cells median 0.12 to 2.56%, and cl5 CD38+ B cells median 2.52 to 0.64% (all p<0.05).ConclusionsOur results showed that a decrease in disease activity in axSpA coincided with normalization of peripheral T- and B-cell frequency abnormalities. This proof of concept study shows the value of MC immuno-monitoring in clinical trials and longitudinal studies in axSpA. MC immunophenotyping on a larger, multi-center scale is likely to provide crucial new insights in the effect of anti-inflammatory treatment and thereby the pathogenesis of inflammatory rheumatic diseases. Show less
Koppejan, H.; Hameetman, M.; Beyrend, G.; Unen, V. van; Kwekkeboom, J.C.; Helm-van Mil, A.H. van der; ... ; Gaalen, F.A. van 2021
Background Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more... Show moreBackground Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA- RA. Methods Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA-) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA-) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. Results MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L(+) basophils (p = 0.33) and a subset of CD16(-) NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L(+) basophil frequency when comparing ACPA+ to ACPA- patients (mean 0.32% vs. 0.13%; p = 0.01). Conclusions Although no differences in major lineages were found between early ACPA+ and ACPA- RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status. Show less
Koppejan, H.; Jansen, D.T.S.L.; Hameetman, M.; Thomas, R.; Toes, R.E.M.; Gaalen, F.A. van 2019
CD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the... Show moreCD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the existence multiple CD8+ T-cell subsets that may differently impact the progression of disease. Here we aim to characterize the CD8+ T-cell population in human atherosclerotic lesions using a combination of histology, flow cytometry and mass cytometry (Cytof).Fresh carotid or femoral endarterectomy and matching blood samples were collect from a local hospital. Part of the sample was fixed for histology and the remainder was digested to obtain single cell suspensions. Cells were labelled with fluorescently labelled antibodies for flowcytometry and isotope labelled antibody for mass cytometry. Results were analyzed with Flowjo (flowcytometry and mass cytometry) and Cytosplore (mass cytometry) software.CD8+ T-cells were detected in every lesion (n=42) analyzed and on average constituted 23% of the total leukocyte. The percentage of CD8+ T-cells inversely correlated with the percentage of macrophages in the lesion, suggesting a role for CD8+ T-cells in preventing macrophage accumulation in the lesion. Mass cytometry revealed at least 13 phenotypically distinct CD8+ T-cells in the lesions, terminally differentiated CD27-/CD28- representing the largest CD8+ section.Human atherosclerotic lesions contain various CD8+ T-cell populations that may differentially affect disease progression and lesion stability. Although the overall effect of CD8+ T-cell presence in the lesion appear to be beneficial, identifying the protective subsets and expanding them may open new avenues for treatment. Show less
Jansen, D.; Klinken, E.; Nel, H.; Law, S.C.; Koppejan, H.; Hameetman, M.; ... ; Thomas, R. 2017
