SUPPLEMENTAL INFORMATION. Impaired TLR9 responses in B cells from patients with systemic lupus erythematosus
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1 1 SUPPLEMENTAL INFORMATION Impaired TLR9 responses in B cells from patients with systemic lupus erythematosus Vincent Gies, 1,2 Jean-Nicolas Schickel, 3* Sophie Jung, 1,4* Aurélie Joublin, 1 Salomé Glauzy, 3 Anne- Marie Knapp, 1 Anne Soley, 1 Vincent Poindron, 2 Aurélien Guffroy, 1,2 Jin-Young Choi, 3 Jacques-Eric Gottenberg, 1,5,6 Jennifer H. Anolik, 7 Thierry Martin, 1,2,5 Pauline Soulas-Sprauel, 1,2,8 Eric Meffre, 3** Anne-Sophie Korganow, 1,2,5** 1 CNRS UPR 3572 "Immunopathology and Therapeutic Chemistry"/Laboratory of Excellence Médalis, Institute of Molecular and Cellular Biology (IBMC), Strasbourg, France. 2 Department of Clinical Immunology and Internal Medicine, National Reference Center for Autoimmune Diseases, University Hospital, Strasbourg, France. 3 Departement of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA. 4 Department of Oral Medicine and Oral Surgery, University Hospital - Faculty of Dentistry, University of Strasbourg, Strasbourg, France. 5 UFR Medicine, University of Strasbourg, Strasbourg, France. 6 Department of Rheumatology, National Reference Center for Autoimmune Diseases, University Hospital, Strasbourg, France. 7 Department of Medicine, Division of Allergy, Immunology and Rheumatology, University of Rochester Medical Center, Rochester, New York, 14642, USA. 8 UFR Pharmaceutical Sciences, Strasbourg University, Illkirch, France. *These authors contributed equally to the work **These authors contributed equally to the work Corresponding authors: Eric Meffre, PhD Yale University School of Medicine, 3 George Street, Room 353F, New Haven, CT 6511, USA Phone: ; Fax: , eric.meffre@yale.edu Prof. Anne-Sophie Korganow, MD PhD CNRS UPR 3572 Immunopathology and Therapeutic Chemistry Institute of Molecular and Cellular Biology (IBMC) 15 rue René Descartes, 6784 Strasbourg Cedex, FRANCE Phone: ; Fax: korganow@unistra.fr Conflict of interest: The authors have declared that no conflict of interest exists. 1
2 IgM MFI ratio 41 Supplemental Figures 42 A Clone B-ly4 HD Quiescent SLE B 25 2 **P= CD21 HD Quiescent SLE Activ e SLE Clone BL13 43 CD Supplemental Figure 1. Validation of decreased CD21 and increased IgM expression on SLE B cells. (A) Histograms represent CD21 MFI on CD19 + B cells of SLE patients (black) and healthy donors (HD; grey) using 2 different anti-human CD21 antibodies (clones B-ly4 and BL13). (B) IgM MFI ratio on B cells from HD (n=41), quiescent (n=22) and active (n=1) SLE patients. MFI ratio was determined as follows: [MFI of the considered marker]/[mean of the MFI of the considered marker in HD group]. MFI: mean of fluorescence intensity. *P<.5, **P<.5, ***P<.1. Twotailed Mann-Whitney U-test. 51 2
3 CD19 MFI CD19 MFI HD SLE 8 6 R=.55 ***P= R=.5 **P= CD21 MFI Supplemental Figure 2 Correlation between CD19 and CD21 cell surface expression. MFI of CD19 and CD21 staining on CD19 + B cells from healthy donors (HD; n=41, left) and SLE patients (n=32, right). MFI: mean of fluorescence intensity. *P<.5, **P<.5. Two-tailed Spearman rank correlation test CD21 MFI 2 3
4 Relative mrna expression Relative mrna expression A 1 8 Quantitative RT-qPCR [Total B cells] HD (n=21) Quiescent SLE (n=16) Active SLE (n=6) B 5 4 Quantitative RT-qPCR [Mature naïve] HD (n=4) Quiescent SLE (n=4) 6 **P= CD19 CD21 CD81 PAX5 CD19 CD21 CD81 PAX Supplemental Figure 3. Normal CD19, CD21, CD81 and PAX5 gene expression in B cells from quiescent SLE patients. (A) Real-time quantitative RT-qPCR of CD19, CD21, CD81 and PAX5 expression in sorted B cells from quiescent (n 12) and active (n=6) SLE patients compared to healthy donors (HD; n 16). (B) Real-time quantitative RT-qPCR of CD19, CD21, CD81 and PAX5 expression in mature naïve B cells from quiescent (n=4) SLE patients compared to HD (n=4). *P<.5, **P<.5. Two-tailed Mann-Whitney U-test. 4
5 CD19 MFI ratio CD21 MFI ratio CD19 MFI ratio CD21 MFI ratio CD19 and CD21 MFI ratio CD19 and CD21 MFI ratio A *P=.1 **P=.6 *P=.2 ***P=.3 ***P=.1 ***P<.1 B HD (n=19) SLE(n=12) CD19 HD (n=38) Untreated SLE (n=9) CD21 SLE without steroïds (n=22) SLE without HCQ (n=11). CD19 CD21 C 3. Serum of HD (control) Serum of SLE 3. Serum of HD (control) Serum of SLE /1 1/5 1/2. 1/1 1/5 1/2 D 3. NS 1 ng/ml 1 ng/ml 1 ng/ml 3. NS 1 ng/ml 1 ng/ml 1 ng/ml IFNγ IFNα IFNγ IFNα Supplemental Figure 4. Normalization in vitro of CD19 and CD21 expression on SLE B cells. (A) CD19 and CD21 MFI ratios of B cells from SLE patients without steroïds (n=22), without hydroxychlroroquine (HCQ; n=11) or without any treatments (n=9) compared to healthy donors (HD; n=38). MFI ratio was determined as follows: [MFI of the considered marker]/[mean of the MFI of the considered marker in HD group]. (B) CD19 and CD21 MFI ratios of B cells from SLE patients (n=12) compared to HD (n=19) after 2 days in culture with medium only. (C) CD19 and CD21 MFI ratios of sorted B cells from HD (n=4) after in vitro stimulation with pooled HD or SLE patients serum for 48 hours. MFI ratio was determined as follows: [MFI of the considered marker]/[mean of the MFI of the considered marker at the same dilution with pooled HD serum] (D) CD19 and CD21 MFI ratios of sorted B cells from HD (n=4) after no stimulation (NS) or in vitro stimulation with interferon γ (IFNγ) or IFNα at different concentrations for 48 hours. MFI ratio was determined as follows: [MFI of the considered marker]/[mean of the MFI of the considered marker in NS samples]. MFI: mean of fluorescence intensity. *P<.5, **P<.5, ***P<.1. Two-tailed Mann-Whitney U-test. 5
6 CD19 and CD21 MFI ratio CD19 and CD21 MFI ratio A HD (n=6) pss(n=6) B HD (n=38) SLE without anti-dsdna (n=9) SLE with anti-dsdna (n=23) ***P=.3 ***P< CD19 CD21. CD19 CD Supplemental Figure 5. Decreased CD19 and CD21 expression observed on SLE B cells is not found on B cells from patients with Sjögren syndrom and correlates with serum anti-dsdna autoantibodies. (A) CD19 and CD21 MFI ratios of B cells from primary Sjögren s syndrome (pss) patients (n=6) compared to healthy donors (HD; n=6). (B) CD19 and CD21 MFI ratios of B cells from SLE patients with (n=23) or without (n=9) anti-dsdna compared to HD (n=38). MFI ratio was determined as follows: [MFI of the considered marker]/[mean of the MFI of the considered marker in HD group]. *P<.5, **P<.5, ***P<.1. Two-tailed Mann-Whitney U-test. 87 6
7 % CD86+ B cells Nonstimulated Imiquimod 1 HD: B cells only (n=6) HD: B cells + hydroxychloroquine (n=6) 8 **P=.2 **P=.2 6 HD: B cells only HD: B cells + hy droxy chloroquine CpG Pokeweed CD86 NS Imiquimod CpG Pokeweed Supplemental Figure 6. Hydroxychloroquine inhibits TLR7- and TLR9-induced B cell activation. Histograms represent CD86 molecules MFI on B cells from healthy donors (HD), in the presence or absence of hydroxychloroquine, after no stimulation (NS) or in vitro stimulation with imiquimod (TLR7 ligand), CpG (TLR9 ligand) or pokeweed for 2 days. The graphic represent the frequency of CD19 + CD86 + B cells from HD, in the presence (n=6) or absence (n=6) of hydroxychloroquine, after no stimulation or in vitro stimulation with imiquimod, CpG or pokeweed for 2 days. *P<.5, **P<.5. Two-tailed Mann-Whitney U-test
8 % of CD69 + B cells CD69 + B cells 1 HD (n=19) SLE (n=12) NS F(ab ) 2 IgM Gardiquimod CpG CD4L Supplemental Figure 7. Normal CD69 upregulation in SLE B cells after TLR9 stimulation. Frequency of CD69 + in gated CD2 + CD27 - naïve B cells from healthy donors (HD; n=19) or SLE patients without hydroxychloroquine (HCQ) (n=12) after no stimulation (NS) or in vitro stimulation of B cells with polyclonal F(ab ) 2 anti-human IgM, gardiquimod (TLR7 ligand), CpG (TLR9 ligand) or CD4 ligand (CD4L) for 2 days. Two-tailed Mann-Whitney U-test. 8
9 Relative mrna expression Quantitative RT-qPCR 12 HD (n 13) SLE (n 9) TLR9 TLR7 MYD88 UNC93B BTK Supplemental Figure 8. Normal TLR9, TLR7, MYD88, UNC93B and BTK gene expresstion in B cells from quiescent SLE patients. Real-time quantitative RT-qPCR of TLR9, TLR7, MYD88, UNC93B and BTK expression in sorted total B cells of quiescent SLE patients (n 9) and healthy donors (HD; n 13) compared to HD (n 13). Two-tailed Mann-Whitney U-test. 9
10 19 Supplemental Tables Supplemental Table 1. Clinical features and disease activity index of the 49 SLE patients. Patient n Age Sex SLEDAI* Ig levels (g/l) ACAN Anti-dsDNA Steroids Hydroxy chloroquine 1 36 F ND 1/ F 13 1/ F / F / F / F / F / F / F 12 1/ F / F / F / F / F 2 ND 1/ F 2 ND 1/ F 2 ND 1/ F 19 1/ F / F 2 ND 1/ F / F / M / F 8.1 1/ F / F / F ND 1/ F / F / M / F / F / F 8 ND 1/ F / F /
11 F / M 8 ND 1/ M / F 11 ND 1/ M / F 8.8 ND F / F / F 9.5 1/ M / F / M 17.9 ND F ND ND F / F / *SLEDAI: Systemic Lupus erythematosus disease activity index 11
12 113 Supplemental Table 2. Clinical features and serological parameters of the 49 SLE patients. Patient n Cutaneous disorder Joint disorder Renal disorder Neurological disorder Other systemic disorder Haematological disorder APL syndrome* antinuclear Abs anti- DNA Abs / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / APL
13 / / / / / / / / / / / / *APL syndrome: antiphospholipid syndrome according to ACR criteria 13
14 115 Supplemental Table 3. Biological features of the 6 patients with primary Sjögren syndrome. Patient n Age Sex Ig levels (g/l) ACAN F / F / F / F / M / F 4.4 1/128 14
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