Candidate markers for stratification and classification in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease, characterized by synovitis in small- and medium-sized joints and, if not treated early and efficiently, joint damage, and destruction. RA is a heterogeneous disease with a plethora of treatment options. The pro-inflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA, and TNF inhibitors effectively repress inflammatory activity in RA. Currently, treatment decisions are primarily based on empirics and economic considerations. However, the considerable interpatient variability in response to treatment is a challenge. Markers for a more exact patient classification and stratification are lacking. The objective of this study was to identify markers in immune cell populations that distinguish RA patients from healthy donors with an emphasis on TNF signaling. We employed mass cytometry (CyTOF) with a panel of 13 phenotyping and 10 functional markers to explore signaling in unstimulated and TNF-stimulated peripheral blood mononuclear cells from 20 newly diagnosed, untreated RA patients and 20 healthy donors. The resulting high-dimensional data were analyzed in three independent analysis pipelines, characterized by differences in both data clean-up, identification of cell subsets/clustering and statistical approaches. All three analysis pipelines identified p-p38, IkBa, p-cJun, p-NFkB, and CD86 in cells of both the innate arm (myeloid dendritic cells and classical monocytes) and the adaptive arm (memory CD4+ T cells) of the immune system as markers for differentiation between RA patients and healthy donors. Inclusion of the markers p-Akt and CD120b resulted in the correct classification of 18 of 20 RA patients and 17 of 20 healthy donors in regression modeling based on a combined model of basal and TNF-induced signal. Expression patterns in a set of functional markers and specific immune cell subsets were distinct in RA patients compared to healthy individuals. These signatures may support studies of disease pathogenesis, provide candidate markers for response, and non-response to TNF inhibitor treatment, and aid the identification of future therapeutic targets.publishedVersio

Titrating complex mass cytometry panels

We describe here a simple and efficient antibody titration approach for cell‐surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well‐characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre‐optimized “backbone” antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies.publishedVersio

Exploration of Cellular Signaling Patterns for the Stratification of Patients with Rheumatoid Arthritis

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by synovial inflammation that, if not treated early and efficiently, causes joint damage. The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA and is the target of treatment with TNF inhibitors. TNF inhibitors are generally effective and well-tolerated. However, up to one-third of patients are primary non-responders, and responses in up to one-third of initial responders abate over time. Currently, there are no predictive biomarkers for treatment with TNF inhibitors. TNF inhibitor drug levels and anti-drug antibodies (ADAb) are indicative of secondary treatment responses, but these markers are not standardized for clinical application. Previous studies have indicated the potential of single-cell profiling by flow or mass cytometry for patient stratification in RA and in other autoimmune conditions. Distinct signaling patterns have been found in leukocytes of RA patients before and during treatment with TNF inhibitors in exploratory and proof-of-principle studies. The aim of this thesis was to explore and compare existing markers for TNF inhibitor drug responses, to set up a methodological background for mass cytometry experiments and finally to explore signaling signatures in immune cell subsets from RA patients compared to healthy individuals, with an emphasis on TNF signaling. Material, methods and results: First, we explored existing assays for TNF inhibitor drug levels and for ADAb in sera from 107 patients with inflammatory diseases treated with the TNF inhibitor infliximab. We found that the assays measured on different scales and that the agreement between quantitative results was limited. However, inter-assay differences could partially be overcome by assay-individualized translations of quantities into categories, which is also necessary for meaningful clinical application (paper 1). Second, we established a basis for mass cytometry experiments, including the extensive collection of biobank material and patient data. Methodological work in the design and titration of antibody panels for mass cytometry was carried out to provide a hierarchical titration method for complex mass cytometry panels, which takes in account abundancies, sources of signal spillover and non-specific antibody binding (paper 2). Last, we explored signaling patterns in heterogeneous immune cells from 20 newly diagnosed RA patients and from 20 healthy donors, with a focus on TNF signaling. In an automated data analysis pipeline, 18 of 20 RA patients and 17 of 20 healthy donors were correctly classified based on their signaling patterns (paper 3). Conclusion: RA is a heterogeneous disease with a plethora of treatment options, and patients might profit from more exact classification and stratification. This thesis highlights the lack of classification and stratification markers, and shows, how single cell profiling by mass cytometry may contribute to the search for such markers. Methodological aspects such as antibody panel design and approaches for the analysis of high-dimensional data are emphasized. The core results of the thesis show that newly diagnosed RA patients can be classified correctly with relatively high precision based on signaling patterns in single cells, when compared to healthy donors. The mass cytometry platform adds many dimensions to “cytomics” of heterogenous cell suspensions and tissues. While studies on malignancies as well as physiology and development of the immune system dominate the field, rheumatic diseases are currently relatively underrepresented. The door for further and deeper study of rheumatic diseases and signaling far beyond the TNF pathway is wide open

Exploration of Cellular Signaling Patterns for the Stratification of Patients with Rheumatoid Arthritis

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by synovial inflammation that, if not treated early and efficiently, causes joint damage. The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA and is the target of treatment with TNF inhibitors. TNF inhibitors are generally effective and well-tolerated. However, up to one-third of patients are primary non-responders, and responses in up to one-third of initial responders abate over time. Currently, there are no predictive biomarkers for treatment with TNF inhibitors. TNF inhibitor drug levels and anti-drug antibodies (ADAb) are indicative of secondary treatment responses, but these markers are not standardized for clinical application. Previous studies have indicated the potential of single-cell profiling by flow or mass cytometry for patient stratification in RA and in other autoimmune conditions. Distinct signaling patterns have been found in leukocytes of RA patients before and during treatment with TNF inhibitors in exploratory and proof-of-principle studies. The aim of this thesis was to explore and compare existing markers for TNF inhibitor drug responses, to set up a methodological background for mass cytometry experiments and finally to explore signaling signatures in immune cell subsets from RA patients compared to healthy individuals, with an emphasis on TNF signaling. Material, methods and results: First, we explored existing assays for TNF inhibitor drug levels and for ADAb in sera from 107 patients with inflammatory diseases treated with the TNF inhibitor infliximab. We found that the assays measured on different scales and that the agreement between quantitative results was limited. However, inter-assay differences could partially be overcome by assay-individualized translations of quantities into categories, which is also necessary for meaningful clinical application (paper 1). Second, we established a basis for mass cytometry experiments, including the extensive collection of biobank material and patient data. Methodological work in the design and titration of antibody panels for mass cytometry was carried out to provide a hierarchical titration method for complex mass cytometry panels, which takes in account abundancies, sources of signal spillover and non-specific antibody binding (paper 2). Last, we explored signaling patterns in heterogeneous immune cells from 20 newly diagnosed RA patients and from 20 healthy donors, with a focus on TNF signaling. In an automated data analysis pipeline, 18 of 20 RA patients and 17 of 20 healthy donors were correctly classified based on their signaling patterns (paper 3). Conclusion: RA is a heterogeneous disease with a plethora of treatment options, and patients might profit from more exact classification and stratification. This thesis highlights the lack of classification and stratification markers, and shows, how single cell profiling by mass cytometry may contribute to the search for such markers. Methodological aspects such as antibody panel design and approaches for the analysis of high-dimensional data are emphasized. The core results of the thesis show that newly diagnosed RA patients can be classified correctly with relatively high precision based on signaling patterns in single cells, when compared to healthy donors. The mass cytometry platform adds many dimensions to “cytomics” of heterogenous cell suspensions and tissues. While studies on malignancies as well as physiology and development of the immune system dominate the field, rheumatic diseases are currently relatively underrepresented. The door for further and deeper study of rheumatic diseases and signaling far beyond the TNF pathway is wide open

Signaling effects of sodium hydrosulfide in healthy donor peripheral blood mononuclear cells

Hydrogen sulfide (H2S) is an endogenous gasotransmitter in human physiology and inflammatory disease, however, with limited knowledge of how signal transduction pathways are involved in immune cells. To examine the effects of sulfide on relevant intracellular signaling in human peripheral blood mononuclear cells (PBMCs), we stimulated healthy donor PBMCs with sodium hydrosulfide (NaHS, 1–1000 μM) to mimic H2S stimulation, and analyzed phosphorylation of p38 mitogen activated protein kinase (MAPK) (pT180/pY182), NF-κB p65 (pS529), Akt (pS473) and CREB/ATF1 (pS133/pS63) with flow and mass cytometry. In contrast to transient effects in subsets of lymphocytes, classical monocytes demonstrated sustained phosphorylation of p38, Akt and CREB/ATF1. NaHS induced calcium dependent phosphorylation of p38, Akt and CREB, but not NF-κB, and the phosphorylation of Akt was partly dependent on p38, indicative of p38-Akt crosstalk. Attenuation of these effects by molecules targeting p38 and Hsp90 indicated Hsp90 as a possible target for H2S-induced activation of p38. These results provide a description of a NaHS-induced signal transduction pathway in human primary immune cells that may have relevance for the role of sulfides in inflammation

Candidate markers for stratification and classification in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease, characterized by synovitis in small- and medium-sized joints and, if not treated early and efficiently, joint damage, and destruction. RA is a heterogeneous disease with a plethora of treatment options. The pro-inflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA, and TNF inhibitors effectively repress inflammatory activity in RA. Currently, treatment decisions are primarily based on empirics and economic considerations. However, the considerable interpatient variability in response to treatment is a challenge. Markers for a more exact patient classification and stratification are lacking. The objective of this study was to identify markers in immune cell populations that distinguish RA patients from healthy donors with an emphasis on TNF signaling. We employed mass cytometry (CyTOF) with a panel of 13 phenotyping and 10 functional markers to explore signaling in unstimulated and TNF-stimulated peripheral blood mononuclear cells from 20 newly diagnosed, untreated RA patients and 20 healthy donors. The resulting high-dimensional data were analyzed in three independent analysis pipelines, characterized by differences in both data clean-up, identification of cell subsets/clustering and statistical approaches. All three analysis pipelines identified p-p38, IkBa, p-cJun, p-NFkB, and CD86 in cells of both the innate arm (myeloid dendritic cells and classical monocytes) and the adaptive arm (memory CD4+ T cells) of the immune system as markers for differentiation between RA patients and healthy donors. Inclusion of the markers p-Akt and CD120b resulted in the correct classification of 18 of 20 RA patients and 17 of 20 healthy donors in regression modeling based on a combined model of basal and TNF-induced signal. Expression patterns in a set of functional markers and specific immune cell subsets were distinct in RA patients compared to healthy individuals. These signatures may support studies of disease pathogenesis, provide candidate markers for response, and non-response to TNF inhibitor treatment, and aid the identification of future therapeutic targets

Optimization of receptor occupancy assays in mass cytometry: Standardization across channels with QSC beads

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high‐parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody‐binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry‐based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4‐integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal‐conjugated antibodies for detection of natalizumab and α4‐integrin. QSC beads with known antibody binding capacity were stained with the same metal‐conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization

Titrating complex mass cytometry panels

We describe here a simple and efficient antibody titration approach for cell‐surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well‐characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre‐optimized “backbone” antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies

Optimization of receptor occupancy assays in mass cytometry: Standardization across channels with QSC beads

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high‐parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody‐binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry‐based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4‐integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal‐conjugated antibodies for detection of natalizumab and α4‐integrin. QSC beads with known antibody binding capacity were stained with the same metal‐conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization