scholarly journals FRI0011 DEVELOPMENT OF A HIGH-DIMENSIONAL FLOW CYTOMETRY PANEL TO ANALYSE NATURAL KILLER CELLS IN SLE

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 576.2-576
Author(s):  
N. Fewings ◽  
S. Schibeci ◽  
F. Mckay ◽  
S. Swaminathan ◽  
M. W. Lin
Keyword(s):  
Gene Expression ◽  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Lupus Erythematosus ◽  
Nk Cell ◽  
High Dimensional ◽  
Inhibitory Receptors ◽  
Systemic Lupus

Background:Natural Killer (NK) cells are an innate immune cell type that has somewhat been overlooked in the context of systemic lupus erythematosus (SLE). SLE patients display a reduced number of NK cells with an activated phenotype and increased capacity to produce IFN-γ, decreased antibody-dependent cellular cytotoxicity (ADCC), and altered natural cytotoxicity (1). NK cell activation is determined by the integration of input from a myriad of activating and inhibitory receptors. Previously, using Nanostring® gene expression technologies, we found our SLE cohort showed decreased gene expression of a number of these receptors (KLRC2, KLRC1, KLRB1, KLRF1, KLRG1, PRF1 and IL2RB) leading us to explore NK cells in SLE in more depth.Objectives:Our aim was to develop a high-dimensional flow cytometry panel to characterise dysregulation of NK cell in SLE, with particular reference to the activating and inhibitory receptors found to be dysregulated in SLE at the gene expression level.Methods:Markers for NK panel were selected to include canonical phenotyping/functional molecules of NK cells with a particular emphasis on receptors found to be lower in our SLE cohort’s gene expression findings. NK panel was designed to minimise spectral overlap, expression and co-expression of markers was taken into consideration. Antibodies were titrated, and voltages optimised to achieve the best separation index for each of the antibodies. The 24-marker panel was run on 52 SLE patients of various disease manifestations, treatments and disease severity. 20 healthy controls were also run for comparison.Results:A 24-marker flow cytometry panel including 19 NK cell antigens was optimised, including basic phenotype (CD3/CD56/CD16/NKp46) and NK differentiation markers (CD57/CD94), activating and inhibitory receptors (NKG2A/NKG2C/NKG2D), costimulatory receptors (CD244/CD226), transcription factors (Eomes/Tbet) and effector molecules (granzyme/perforin). Immunophenotypic high-parameter analysis of SLE and control samples is in progress and results will be presented.Conclusion:Our development of a high-dimensional immunophenotypic panel allows identification of changes in NK cells in SLE including antigen expression levels, subset percentages and potentially of novel subsets. This panel will be used to investigate NK cell changes with disease course/activity, therapeutic response, and to discover potential drug targets for SLE.References:[1]Spada R, Rojas JM, Barber DF. Recent findings on the role of natural killer cells in the pathogenesis of systemic lupus erythematosus. J Leukocyte Biol. 2015;98(4):479-487. doi:10.1189/jlb.4ru0315-081rrAcknowledgments:Westmead Institute for Medical Research Genomics FacilityWestmead Institute for Medical Research Flow Cytometry FacilityStaff Specialists’ TESL and Trust Fund CommitteeDisclosure of Interests:None declared

scholarly journals Single-cell multi-omics analysis reveals IFN-driven alterations in T lymphocytes and Natural Killer cells in systemic lupus erythematosus

2021 ◽  
Author(s):  
Dominik Trzupek ◽  
Mercede Lee ◽  
Fiona Hamey ◽  
Linda S. Wicker ◽  
John A. Todd ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cell ◽  
Nk Cells ◽  
Single Cell ◽  
Natural Killer ◽  
Immune Activation ◽  
Lupus Erythematosus ◽  
Nk Cell ◽  
Type I ◽  
Systemic Lupus

AbstractBackgroundThe characterisation of the peripheral immune system in the autoimmune disease systemic lupus erythematosus (SLE) at the single-cell level has been hampered by the reduced sensitivity of current whole-transcriptomic technologies. Here we employ a targeted single-cell multi-omics approach, combining protein and mRNA quantification, to generate a high-resolution map of the T lymphocyte and Natural Killer (NK) cell populations in blood from SLE patients.MethodsWe designed a custom panel to quantify the transcription of 534 genes in parallel with the expression of 51 surface protein targets using the BD Rhapsody single-cell system. We applied this technology to profile 20,656 T and NK cells isolated from peripheral blood from an SLE patient with a type I interferon (IFN) signature (IFNhi), and an age- and sex-matched IFNlow SLE patient and healthy donor.ResultsWe confirmed the presence of a rare cytotoxic CD4+ T cell (CTL) subset, which was exclusively present in the IFNhi patient. Furthermore, we identified additional alterations consistent with increased immune activation in this patient, most notably a shift towards terminally differentiated CD57+ CD8+ T cell and CD16+ NKdim phenotypes, and the presence of a subset of naïve CD4+ T cells recently activated by cytokines.ConclusionsOur results identify IFN-driven changes in the composition and phenotype of T and NK cells that are consistent with a systemic immune activation within the IFNhi patient, and underscore the power of this multi-omics approach to identify rare immune subsets, which could be missed using lower parametric or less sensitive single-cell technologies. Consequently, we were able to find evidence for novel cellular peripheral biomarkers of SLE disease activity, including a subset of CD57+ CD4+ CTLs.

scholarly journals Tim-3 associated with apoptotic NK cells and disease activity in SLE

2021 ◽  
Vol 19 ◽  
pp. 205873922110005
Author(s):  
Di Zhao ◽  
Xiao Yang ◽  
Jie Zhang ◽  
Yi Zhang
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
T Cell ◽  
Disease Activity ◽  
Nk Cells ◽  
Lupus Erythematosus ◽  
Healthy Controls ◽  
Systemic Lupus ◽  
Potential Target

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been found to play important roles in systemic lupus erythematosus (SLE), however, whether Tim-3 is involved in apoptosis of NK cells in SLE remains unknown. The proportion of CD3−CD56+ NK cells and the percentage of AnnexinV+ NK cells were analyzed by flow cytometry in SLE patients and healthy controls. Tim-3 expression on NK cells was also evaluated by flow cytometry. We firstly observed a decreased proportion of NK cells and an increased proportion of apoptotic NK cells in SLE patients. The proportion of apoptotic NK cells was positively correlated with anti-dsDNA and SLEDAI. Tim-3 expression on NK cells was up-regulated in SLE patients. Further analysis showed that Tim-3 expression on NK cells was negatively correlated with the proportion of apoptotic NK cells, anti-dsDNA and SLEDAI, while positively correlated with the proportion of NK cells. The present results suggest that Tim-3 might play roles in SLE by regulating the apoptosis of NK cells and Tim-3 might serve as a potential target for the treatment of SLE.

scholarly journals Single-cell multi-omics analysis reveals IFN-driven alterations in T lymphocytes and natural killer cells in systemic lupus erythematosus

2021 ◽  
Vol 6 ◽  
pp. 149
Author(s):  
Dominik Trzupek ◽  
Mercede Lee ◽  
Fiona Hamey ◽  
Linda S. Wicker ◽  
John A. Todd ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cell ◽  
Nk Cells ◽  
Single Cell ◽  
Natural Killer ◽  
Immune Activation ◽  
Lupus Erythematosus ◽  
Gene Expression Signature ◽  
Type I ◽  
Systemic Lupus

Background: The characterisation of the peripheral immune system in the autoimmune disease systemic lupus erythematosus (SLE) at the single-cell level has been limited by the reduced sensitivity of current whole-transcriptomic technologies. Here we employ a targeted single-cell multi-omics approach, combining protein and mRNA quantification, to generate a high-resolution map of the T lymphocyte and natural killer (NK) cell populations in blood from SLE patients. Methods: We designed a custom panel to quantify the transcription of 534 genes in parallel with the expression of 51 surface protein targets using the BD Rhapsody AbSeq single-cell system. We applied this technology to profile 20,656 T and NK cells isolated from peripheral blood from an SLE patient with a type I interferon (IFN)-induced gene expression signature (IFNhi), and an age- and sex- matched IFNlow SLE patient and healthy donor. Results: We confirmed the presence of a rare cytotoxic CD4+ T cell (CTL) subset, which was exclusively present in the IFNhi patient. Furthermore, we identified additional alterations consistent with increased immune activation in this patient, most notably a shift towards terminally differentiated CD57+ CD8+ T cell and CD16+ NKdim phenotypes, and the presence of a subset of recently-activated naïve CD4+ T cells. Conclusions: Our results identify IFN-driven changes in the composition and phenotype of T and NK cells that are consistent with a systemic immune activation within the IFNhi patient, and underscore the added resolving power of this multi-omics approach to identify rare immune subsets. Consequently, we were able to find evidence for novel cellular peripheral biomarkers of SLE disease activity, including a subpopulation of CD57+ CD4+ CTLs.

scholarly journals Rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren’s syndrome shared megakaryocyte expansion in peripheral blood

2021 ◽  
pp. annrheumdis-2021-220066
Author(s):  
Yukai Wang ◽  
Xuezhen Xie ◽  
Chengpeng Zhang ◽  
Miaotong Su ◽  
Sini Gao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
Lupus Erythematosus ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Primary Sjögren’S Syndrome ◽  
Rna Seq ◽  
Systemic Lupus

ObjectivesRheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) share many clinical manifestations and serological features. The aim of this study was to identify the common transcriptional profiling and composition of immune cells in peripheral blood in these autoimmune diseases (ADs).MethodsWe analysed bulk RNA-seq data for enrichment of biological processes, transcription factors (TFs) and deconvolution-based immune cell types from peripheral blood mononuclear cells (PBMCs) in 119 treatment-naive patients (41 RA, 38 pSS, 28 SLE and 12 polyautoimmunity) and 20 healthy controls. The single-cell RNA-seq (scRNA-seq) and flow cytometry had been performed to further define the immune cell subsets on PBMCs.ResultsSimilar transcriptional profiles and common gene expression signatures associated with nucleosome assembly and haemostasis were identified across RA, SLE, pSS and polyautoimmunity. Distinct TF ensembles and gene regulatory network were mainly enriched in haematopoiesis. The upregulated cell-lineage-specific TFs PBX1, GATA1, TAL1 and GFI1B demonstrated a strong gene expression signature of megakaryocyte (MK) expansion. Gene expression-based cell type enrichment revealed elevated MK composition, specifically, CD41b+CD42b+ and CD41b+CD61+ MKs were expanded, further confirmed by flow cytometry in these ADs. In scRNA-seq data, MKs were defined by TFs PBX1/GATA1/TAL1 and pre-T-cell antigen receptor gene, PTCRA. Cellular heterogeneity and a distinct immune subpopulation with functional enrichment of antigen presentation were observed in MKs.ConclusionsThe identification of MK expansion provided new insights into the peripheral immune cell atlas across RA, SLE, pSS and polyautoimmunity. Aberrant regulation of the MK expansion might contribute to the pathogenesis of these ADs.

scholarly journals Effect of Interleukin-15 on CD11b, CD54, and CD62L Expression on Natural Killer Cell and Natural Killer T-Like Cells in Systemic Lupus Erythematosus

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Syh-Jae Lin ◽  
Ji-Yih Chen ◽  
Ming-Ling Kuo ◽  
Hsiu-Shan Hsiao ◽  
Pei-Tzu Lee ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Lupus Erythematosus ◽  
Killer Cell ◽  
Healthy Controls ◽  
Systemic Lupus ◽  
Cd56 Expression ◽  
Interleukin 15

Adhesion molecules may play an important role in systemic lupus erythematosus (SLE) pathogenesis. We investigated the effect of interleukin- (IL-) 15 on CD11b, CD54, and CD62L expression on natural killer (NK) cells, T cells, and CD56+CD3+NKT-like cells from SLE subjects and healthy controls. SLE patients had decreased circulating NK cells and NKT-like cells compared to controls. NK cells from SLE patients showed higher CD11b and CD62L expression compared to controls. IL-15 enhanced CD11b and CD54 but downregulated CD62L expression on NK cells from SLE patients. Similar observations were found for T cells and NKT-like cells. NK cells from SLE patients expressed higher CD56 than controls; both could be further enhanced by IL-15. IL-15 also enhanced CD56 expression of NKT-like cells from SLE patients. A greater degree of IL-15 induced downregulation of CD62L on NKT-like cells noted in SLE patients compared to controls. The percentage of CD11b expressing NK cells and the % inhibition of CD62L expression on NKT-like cells by IL-15 correlated with serum anti-dsDNA levels in SLE patients, respectively. Taken together, we demonstrated the dysfunctional NK and NKT-like cells in SLE patients with regard to CD11b and CD62L expression and their response to IL-15.

scholarly journals POS0680 BELIMUMAB ADD-ON THERAPY MOBILISES MEMORY B CELLS INTO THE CIRCULATION OF PATIENTS WITH SLE

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 585.1-585
Author(s):  
E. J. Arends ◽  
M. Zlei ◽  
C. M. Tipton ◽  
Z. Osmani ◽  
S. Kamerling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Memory B Cells ◽  
Lymphocyte Trafficking ◽  
Systemic Lupus ◽  
Research Support

Background:Belimumab (BLM), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active severe systemic lupus erythematosus (SLE) and lupus nephritis (LN). There is clinical evidence that combining BLM with B cell depleting therapy can ameliorate disease activity in severe, refractory SLE patients1. Although BLM is a B cell directed therapy and has been shown to significantly decrease total B cells, flow cytometry observations suggest a rapid increase of circulating memory B cells (MBC)2.Objectives:To investigate dynamics of B-cell subsets in SLE patients treated with or without BLM, with a focus on assessing MBC characteristics.Methods:Extensive B cell subset phenotyping was performed by high-sensitivity (HS) flow cytometry (acquisition of 107 leukocytes; per EuroFlow protocols3) on samples from active LN or SLE patients with major organ involvement treated with standard of care (SOC) consisting of high dose steroids and mycophenolate mofetil combined with or without the addition of BLM. MBC gene expression profiles were characterized with single-cell RNA and V(D)J sequencing (ScRNA-SEQ).Results:By employing HS flowcytometry, we established that the absolute increase in circulating MBC in SLE and LN patients was significant for patients who initiated BLM (Figure 1). The increase was observed in a broad range of MBC subsets (Unswitched, IgG1+, IgG2+, IgA1+, IgA2+) at 2 and 4 weeks following initiation of BLM treatment. This rise in MBC could hypothetically be attributed to either proliferation of blood MBC, BLM induced migration of tissue-resident MBCs or BLM related retention of tissue-destined MBC in the blood. ScRNA-SEQ analysis of cell cycle gene-expression was performed and established in both groups a non-proliferating phenotype [in approximately ~94%] of MBC post-treatment, including absence of MKI67 as active proliferation marker. Clonal diversity analysis comparing week 2 with baseline revealed an unexpected decrease of the largest MBC clones in BLM, whereas no change in clonality was observed with SOC alone. Together these data indicate that proliferation is unlikely to be responsible for the observed increase in MBC by BLM. Furthermore, a clear difference was found in gene-expression levels between both treatment groups: BLM was responsible for the upregulation of 72 vs 10 genes in SOC, likewise 162 vs 32 genes were downregulated. Most importantly, a significant downregulation of the migration genes SELL (CD62L), CCR7, ITGB1, RAC2 and ICAM2, were specifically seen in BLM treated patients. This may reflect disrupted lymphocyte trafficking, preventing MBCs from transmigrating from the blood into tissue owing to reduced migration molecules, or preventing MBCs from being retained at the tissue level owing to reduction in tissue adhesion proteins.Conclusion:The addition of BLM to SOC significantly increases MBCs in patients with SLE independently of proliferation, accompanied by a strong modulation of gene expression, including reduced expression of migration markers pointing towards disrupted lymphocyte trafficking. These data may have important implications for improving treatment strategies in patients with LN or severe SLE, as a deeper depletion of autoreactive MBCs could be established by adding B-cell-depleting therapy after the initiation of BLM.Figure 1.Change in pre-germinal center and memory B cell counts from baseline to week 4 of patients with SLE or LN treated with SOC (n=8) or SOC+BLM (n=11).References:[1]Arends EJ et al. Long-term effects of combined B cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results. Nephrol Dial Transplant. 2020 Jun 27 gfaa117.[2]Stohl W et al. Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with SLE. Arthritis Rheum. 2012;64(7):2328-2337.[3]Blanco et al, Age-associated distribution of B and plasma cell subsets in peripheral blood - J Allergy Clin Immunol 2018 141 2208-2219.Disclosure of Interests:Eline J. Arends: None declared, Mihaela Zlei: None declared, Christopher M. Tipton: None declared, Zgjim Osmani: None declared, Sylvia Kamerling: None declared, Ton Rabelink: None declared, Ignacio Sanz: None declared, Jacques J.M. van Dongen Paid instructor for: BD Biosciences: Educational Services (fees for LUMC), Consultant of: BD Biosciences and Cytognos (fees for LUMC), Grant/research support from: GSK (flow cytometry studies for GSK BLISS-BELIEVE study NCT03312907), Cees van Kooten: None declared, Y.K. Onno Teng Consultant of: Aurinia provided financial compensation for consultancy, Grant/research support from: GSK provided belimumab for free for the Synbiose-2 clinical trial and provided an unrestricted grant to conduct the study.

scholarly journals The enrichment of neutrophil extracellular traps impair the placentas of systemic lupus erythematosus through accumulating decidual NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Jiang ◽  
Nan Shen ◽  
Haibo Zhou ◽  
You Wang ◽  
Sihan Lin ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Nk Cells ◽  
Lupus Erythematosus ◽  
Neutrophil Extracellular Traps ◽  
Clinical Samples ◽  
Systemic Lupus ◽  
Extracellular Traps ◽  
Fetal Birth Weight ◽  
Immunological Disorder ◽  
Adverse Pregnancy

AbstractDespite the advances made in the management of pregnancies in women with systemic lupus erythematosus (SLE), the rate of adverse pregnancy outcomes is still higher than that in the general population. In the last few years, neutrophil extracellular traps (NETs) were proven to be detrimental in both autoimmune diseases and placental injury. We investigated whether NETs could be detected in the placentas of pregnant individuals with SLE and explored the relationship between NETs and decidual natural killer cells (dNKs), which comprise the majority of immune cells at the maternal–fetal interface, using clinical samples and animal models. In this study, we found that the infiltration of NETs and dNKs, especially CD56+CD16+ NK cells, was significantly increased in pregnant individuals with SLE with placental insufficiency. In the murine models of SLE, the number of dNKs was significantly decreased due to the decreased formation of NETs affected by Ly6G. Moreover, the histopathological placental injury was reduced, with a remarkable increase in fetal birth weight. This study shows that NETs may contribute to immunological disorder in the placenta and the pathological changes in pregnancies with SLE, which provides a research basis for further explorations of the mechanism of SLE in placental impairment.

scholarly journals THU0231 IL-2 DRIVES THE CONVERSION OF T FOLLICULAR HELPER TO T FOLLICULAR REGULATORY CELLS THROUGH EPIGENETIC MODIFICATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 343.2-343
Author(s):  
H. Hao ◽  
S. Nakayamada ◽  
Y. Kaoru ◽  
N. Ohkubo ◽  
S. Iwata ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
B Cells ◽  
Histone Modifications ◽  
Lupus Erythematosus ◽  
Regulatory Cells ◽  
Systemic Lupus ◽  
Research Support ◽  
Tfh Cells ◽  
Follicular Helper

Background:Systemic lupus erythematosus (SLE) is a complex polygenic autoimmune disease characterized by immune-system aberrations. Among several types of immune cells, T follicular helper (Tfh) cells promote autoantibody production, whereas T follicular regulatory (Tfr) cells suppress Tfh-mediated antibody responses.(1)Objectives:To identify the characteristics of Tfr cells and to elucidate the mechanisms of conversion of Tfh cells to Tfr cells, we probed the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors.Methods:Peripheral blood mononuclear cells from SLE patients (n=44) and healthy donors (HD; n=26) were analyzed by flow cytometry. Memory Tfh cells were sorted and cultured under stimulation with T cell receptor and various cytokines. Expression of characteristic markers and phosphorylation of STATs (p-STATs) were analyzed by flow cytometry and quantitation PCR. Histone modifications were evaluated by chromatin immunoprecipitation.Results:The proportion of CXCR5+FoxP3+Tfr cells in CD4+T cells tended to increase (2.1% vs 1.7%, p=0.17); however, that of CD4+CD45RA-FoxP3hiactivated Tfr cells in Tfr cells was decreased (4.8% vs 7.1%, p<0.05), while CD4+CD45RA-FoxP3lownon-suppressive Tfr cells was increased (50.1% vs 38.2%, p<0.01) in SLE compared to HD. The percentage of PD-1hiactivated Tfh cells was significantly higher in SLE compared to HD (15.7% vs 5.9%, p<0.01). Furthermore, active patients had a higher ratio of activated Tfh/Tfr cells compared to inactive patients. In vitro study showed that IL-2, but not other cytokines such as TGF-β1, IL-12, IL-27, and IL-35, induced the conversion of memory Tfh cells to functional Tfr cells characterized by CXCR5+Bcl6+Foxp3hipSTAT3+pSTAT5+cells. The loci ofFOXP3at STAT binding sites were marked by bivalent histone modifications. After IL-2 stimulation, STAT5 directly bound on FOXP3 gene loci accompanied by suppressing H3K27me3. Finally, we found that serum level of IL-2 was decreased in SLE and that stimulation with IL-2 suppressed the generation of CD38+CD27+B cells by ex vivo coculture assay using Tfh cells and B cells isolated from human blood.Conclusion:Our findings indicated that the regulatory function of Tfr cells is impaired due to the low ability of IL-2 production and that IL-2 restores the function of Tfr cells through conversion of Tfh cells to Tfr cells in SLE. Thus, the reinstatement of the balance between Tfh and Tfr cells will provide important therapeutic approaches for SLE.References:[1]Deng J, Wei Y, Fonseca VR, et al. T follicular helper cells and T follicular regulatory cells in rheumatic diseases. Nat Rev Rheumatol. 2019; 15 (8): 475-90.Disclosure of Interests: :He Hao: None declared, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yamagata Kaoru: None declared, Naoaki Ohkubo: None declared, Shigeru Iwata: None declared, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin

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