Regulatory B cells in autoimmunity. Liwei Lu University of Hong Kong, China

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1 Regulatory B cells in autoimmunity Liwei Lu University of Hong Kong, China

2 Multiple functions of B cells in immunity LeBien and Tedder, Blood (2008)

3 B cell functions in autoimmune pathogenesis

4 The Immunopathogensis of Rheumatoid Arthritis Nature 423 (2003)

5 Role of BAFF in B cell maturation and function B cell activating factor (BAFF, BLyS) * A member of TNF family cytokines * A survival factor for B cell maturation Increased BAFF in DC from CIA mice Serum BAFF levels: increased in SLE & RA

6 BAFF enhances the survival of plasma cells Viable plasma cells (%) Control BAFF BCMA:Fc Days in culture BAFF overproduction correlates with elevated autoab in CIA OD * Auto-Ab * BAFF (ng/ml) BAFF 0 Control Acute CIA Chronic CIA 0 Control Acute CIA Chronic CIA

7 Effect of BAFF on B cell maturation and function BAFF-silenced DCs are defective in B cell maturation and Ab production BAFF gene silencing by lentivirus expressing shrna in joint tissue LV-shActin LV-shBAFF

8 BAFF silencing suppresses CIA development Intra-articular injection of lentivirus expressing shrna-baff Reduced CIA incidence Delayed CIA onset Long term, localized effect Bone erosion Joint damage

9 BAFF silencing inhibits Th17 cells in the joint and LN Detection of IL-17 producing cells by ELISPOT assays * *

10 A pathogenic role of BAFF in autoimmune arthritis Validation of BAFF as a therapeutic target in experimental arthritis Lam Q, et al, PNAS (2010)

11 BAFF induces IL-10 production in B cells

12 BAFF induces IL-10 production in MZ B cells Lymphoid follicle IL-10-producing B cells suppress T cell function I: B:T II: Induced B (IL-10-/-):T III: Induced B:T IV: Induced B+a-IL-10:T Yang M, et al, J Immunol (2010)

13 Existence of IL-10-producing B cells in vivo Spleen CIA Joint tissue IL-10-producing B cells

14 Kinetic changes of Breg and Th17 cells during CIA induction Breg cells DBA/1 CIA Th17

15 Localization of transferred Breg cells In vivo In vitro expansion of Breg cells B10 cells

16 Transfer of Breg cells suppresses CIA development

17 Transfer of Breg cells inhibits Th17 response in vivo

18 Breg cells inhibit Th17 expansion in vitro

19 Breg cells inhibit Th17-mediated CIA in IL-17 -/- mice Yang M et al. A J Pathol ( 2012)

20 Role of Breg cells in Sjögren Syndrome Henrik Sjögren ( ) Dry eyes / dry mouth CD4 CD20 Adamson TC et al. J Immunol (1983)

21 Available mouse models for Sjögren Syndrome Spontaneous SS models: NOD, Fas(lpr), (NZB+NZW)F1 Gene-modified animal models: IL-12-Tg, BAFF-Tg,Id3-KO, IL-14a-Tg Induction of Experimental Sjögren Syndrome in normal mice C57BL/6 SG protein Immunization

22 Enhanced Th17 responses during ESS development A Serum Auto-antibodies (OD Value at 450 nm) IL-17 IFN-γ IFN-γ B 0d 3d 10d 17d 24d CD19 30wk D E Ctrl-peptide 8 M3R-peptide 6 Anti-ANA F CD4 + CD8 + CD wk 5 wk 30 wk CD4 20 mm IL-17 CD4 Hoechst 4 SSA-peptide *** *** Ctrl 5wk 0.3 3d 10d 17d 24d 30wk ESS 10wk 2.1 CD4 15wk wk wk 12.9 IL-17 *** CD4

23 B A Saliva Flow Rate (µl/15min) IL-17KO mice are resistant to ESS induction ** * ** ** WT IL-17 KO Days post-immunization C WT IL-17 D KO WT 4 IL-17 KO E 20 µm WT IL-17 KO B IL-17A WT 20 µm Histological Score IL-17 KO WT IL-17A KO Cell Number (x 10 6 ) Histological Score WT *** IL-17A 10 IL 5 W ** CD4 Th1 T

24 Th17 cell transfer restores disease pathology in IL-17KO mice 98.6 C CD4 + T cells Transfer Th17 cells Transfer CD3 20 µm CD4 Hoechst L-17 KO mice mice 20 µm Tunnel Hoechst 20 µm

25 REVIEWS A critical role of Th17 cells in ESS development Epithelial cell Autoantigen ICs Virus? Plasma cell pdc BAFF B cell IFN-α B-cell activation GC-like structure Other activator of innate immunity? NK-cell activation T FH cell NK cell IFN-γ DC IL-12R Th17 IL-12 T-cell activation Figure 1 Schematic representation of the pathophysiology processes hypothesized to underlie pss, based on our current understanding. Environmental factors such a virus or other activators of innate immunity cause epithelial cell and DC activation (see Box 1 for a description of how viruses might instigate T H 1 cell infiltrates, but interestingly without any d in salivary glands at the onset of auto imm potential explanation for the elusiveness pss could be that overexpression of som retroviral sequences has been overlook be considered instead of searching for a responsible for disease-associated activa system. For example, epigenetic abnorm expression of endogenous retroviral ele responsible for activation of the type I IF How about macrophages? NFκB pathway dysfunction in pss pa One of the consequences of the stimu immunity is the activation of nuclear fac which can occur in a number of differe salivary gland epithelial cells from pat hyperactivation of NFκB has been a decreased expression of one of the regu activation, A20 (also known as TNF-α-in [TNFAIP3]). 28 Interestingly, defective another gene involved in feedback regu NFκB inhibitor α (also known as IκBα), l ment of a pss phenotype in mice. 29 Fu first GWAS in pss 14 identified an assoc this disease and a gene (TNIP1) encod interacting protein 1, a protein that int and is involved in the regulation of N Together, these observations suggest (Lin X, at al Ann Rheum Dis. 2015)

26 Functional defects of Breg cells in ESS development

27 Transfer of Breg cells suppresses ESS progression

28 Breg transfer suppresses Th1/Th17 cells in ESS mice

29 Breg transfer suppresses inflammatory cytokine production Serum level (ng/ml) IL-17 ** *** Ctrl PBS Breg Bve Serum level (ng/ml) IL-12 Ctrl PBS Breg Bve Cell therapy? ESS ESS Serum level (ng/ml) IL-21 *** Ctrl PBS Breg Bve * Serum level (ng/ml) IL-6 * * Ctrl PBS Breg Bve ESS ESS (Manuscript in preparation)

30 Mechanisms of action for Bregs in immune responses Yang M et al, Cell Mol Immunol (2014)

31 Acknowledgements Queenie Lam Min Yang Jun Deng Xiang Lin Xiaohui Wang Qian Chen Kongyang Ma Otis Ko This project was funded by Hong Kong Research Grants Council, National Science Foundation of China, 973 Program of MOST, and Hong Kong Croucher Foundation.

32 BAFF in regulating B cell maturation and function Proliferation BAFF TACI Survival p52 RelB Ig Class switching IKKa NIK TBK1 BAFF-R BAFF NEMO IKKb p50 RelA Differentiation