2017 Arizona United Rheumatology Alliance Annual Meeting. Overview of the Immune System and Memory B-cell Activity in RA

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1 2017 Arizona United Rheumatology Alliance Annual Meeting Overview of the Immune System and Memory B-cell Activity in RA Gregg J. Silverman, MD Professor of Medicine and Pathology Director, Laboratory of B-cell Immunobiology NYU School of Medicine New York, NY

2 Learning Objectives Discuss how the layers of the immune system are assembled and interconnected. Briefly review the ancient innate system, components and properties. Discuss the special abilities of the adaptive immune system. Review B lymphogenesis. Discuss how antigenic selection molds the B cell repertoire. Brief overview of the immunology of Rheumatoid Arthritis and associated ectopic lymphoid tissue and GCs. Discuss the effect of therapy on disease-associated B cell memory and circulating ACPA.

3 Organization of the Immune System Bad outer world Anatomical and Physiological Barriers Lysozyome in tears and saliva Low stomach ph Ciliary clearance Intact skin Neutrophils TLR s Complement Innate Immunity Host defense peptides Adaptive Immunity B cells T cells NLR s 0-4 hours 4-96 hours Late > 96 hours Dendritic cells

4 Synchronous Functioning: Features of the Innate and Adaptive Immune Systems 1,2 INNATE Rapid Antigen-nonspecific pathogen recognition Activates the adaptive immune system ADAPTIVE Delayed Antigen-specific pathogen recognition Confers long-lasting immunity Macrophage Dendritic cell IL-1β T cell B cell TNF-α Neutrophil IL-6 Complement Plasma cell Antibodies Abbreviations: IL, interleukin; TNF, tumor necrosis factor. References: 1. Murphy K, ed. Janeway s Immunobiology Chaplin DD. J Allergy Clin Immunol. 2010;125(2 suppl 2):S3-S23.

5 What is the Function of Innate Immunity? 1. Limit the extent of the initial microbial infection a) Phagocytosis, macrophage activation b) Neutrophil activation, degranulation c) Release of antimicrobial peptides d) Cytokine, chemokine production 2. Prime the adaptive immune system a) Antigen presentation b) Expression of costimulatory molecules c) Cytokine, chemokine production 3. Wound repair and tissue remodeling a) DAMPs = Danger-associated Molecular Patterns

6 Immune Response - Innate All of these defenses are without MEMORY Early response ( minutes to 3-5 days) consisting of CELLULAR and SOLUBLE elements Expressed by GERMLINE encoded receptors Does not recognize every antigen but rather highly conserved structures DAMP

7 The Roles of Innate Cell Types: Neutrophils, Monocytes, Macrophages, and Dendritic Cells 1,2 INNATE Macrophage Inflammatory stimulus Neutrophil IL-6 Antimicrobial Dendritic cell functions: IL-12 NET generation TNF-α IL-1β Granule release Complement Phagocytosis IL-15 Abbreviation: NET, neutrophil extracellular trap. References: 1. Murphy K, ed. Janeway s Immunobiology Kolaczkoska E, Kubes P. Nat Rev Immunol. 2013:13(3): Neutrophils - Phagocytose and kill extracellular pathogens - Release proinflammatory factors and antibacterial proteins Macrophages - Secrete proinflammatory cytokines, antimicrobial peptides, and degradative enzymes - Phagocytose pathogens - Present antigen Dendritic cells - Phagocytose pathogens - Present antigen - Secrete cytokines such as IL- 12, IL-15, IL-23

8 Pattern Recognition Molecule (PRMs) Toll-like Receptors (TLRs) TOLL 1996 NOD-like Receptors (NLRs) inflammation, apoptosis RIG-I-like Receptors (RLRs) Pentraxins opsonization, complement cascade Collectins Ficollins C-type lectins phagocytosis Scavenger receptors

9 History of TLRs Christiane Nusslein-Vollhard 1985/1995* Janeway linked TLR to NFkB 1997 Jules Hoffman immune defense connection 95/2011 Bruce Beutler Role of the LPS receptor 2011

10 Capsule DNA (nucleoid) Plasma Membrane Cell Wall Mesosome Ribosomes Cytoplasm Few species have an adaptiive immune system Adaptive Immunity innate immunity

11 Key properties of the adaptive immune Diverse repertoire of clonally distributed antigen receptors Assembled by combinatorial assembly from an immense number of germline genes Antigens can induce clonal selection to address a specific threat Memory of a past exposure is banked away for more rapid responses Memory at a cellular level is not passed to the next generation Bad self-reactive memory can drive an autoimmune disease

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14 Basic B cell biology

15 Somatic recombination generates functional Ig heavy and light chain genes Antigen receptor genes move during B- and T-cell differentiation

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17 Acquired tolerance necessitates alternative fates driven by the same antigen receptor Two signal hypothesis (Bretscher and Cohn) Science (3950): Signal 1 only = death/inactivation 1 Signal 1+ Signal 2 = activation 2 Courtesy of M. Cancro, U Penn

18 Acquired tolerance necessitates alternative fates driven by the same antigen receptor Two signal hypothesis (Bretscher and Cohn) Science (3950): Signal 1 only = death/inactivation 1 Signal 1+ Signal 2 = activation 2 Signal 3 molds the immune bias of the lymphocyte Courtesy of M. Cancro, U Penn

19 B-cell development: Three types of Mature B cells B1 lineage Central and peripheral tolerance Natural antibodies (defense from infection, Inflammation and autoimmunity) Bone marrow or foetal liver HSC HSC Central tolerance Mature B1 cell Self renewal Peripheral tolerance Short-lived plasma cell Mature B2 cell Antigen T cell Activated B cell Short-lived plasma cell GC B cell Extrafollicular response GC reaction (after T-cell reaction) Bone marrow Immature B cell T1 B cell T2 B cell Memory B cell HSC = haemopoietic stem cell; GC = germinal centre MZ = marginal zone MZ B cell Short-lived plasma cell Plasma cell Long-lived plasma cell Adapted from Dörner T, et al. Nature Rev Rheumatol 2009;5:433 41

20 Antigen-specific clonal selection Humans have ~ B cells and T cells distributed into ~ 10 6 B-cell and T-cell clones Optimal T-dependent responses require coordination (physical interactions) of complementary B and T cell clones For optimal kinetics of responses, this cannot come from random encounters

21 Spatiotemporal organization of B-T cell interactions Frequencies of antigen-specific B cells and complementary and antigen-specific T cells are generally low How do the B and T cells find each other in the body? Each should provide what the other needs.

22 INNATE

23 Germinal centers: Arenas for -cell clonal selection

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26 Properties of T Follicular Helper (TFH) cells IL-21- pleotropic cytokine activates most lymphocytes induces proliferation of CD40L activated B cells induces Activation Induced Deaminase (AID) for somatic hypermutation With downregulation of T cell zone homing CC receptor 7 (CCR7) and IL-7 receptor-a (IL-7Ra) TFH differentiation also affected by CD40L, CD26, STAT3, SLAM associated protein that all induce Bcl6. Induce B cell differentiation at B-T border into extrafollicular plasmablasts From Tangye et al. Nat Rev Immunol 2013

27 Germinal center reactions provide benefits for the mammalian adaptive immune system 1. Specialized anatomic site for coordinated spatiotemporal interactions of antigen-specific B cells and T cells. 2. Antigen-specific T cells (TFH) are required for second signals (soluble and membrane) inducing: - hypermutation machinery (e.g., AID) - survival (e.g., BAFF/BLyS) - differentiation (to plasma cell) - class-switch recombination (CSR). 3. Follicular dendritic cells help to provide a well furnished arena for competition between clonal daughters based on stregth of BDR signal and availability of second signals

28 Ag specific B cells and cognate peptide-antigen specific T cells: The perfect couple! As each may represent < 1:10,0000 Lymphocyte clones, How do they find each other? Don t forget ICOS/ICOS-L and modulating inhibitory paired R-L systems

29 BLyS/BAFF play fundamental roles in ensuring the survival of B cells Potentially expressed by multiple immune cells 1,2 Neutrophils Monocytes Dendritic Cells activated T cells Plasma cells B cells BLyS (TNFSF13B) exists membrane-bound and soluble forms 1 Related to A Proiferation Inducing Ligand (APRIL,TNFSF13) Three molecules bind together to form the trimeric soluble protein 1,3 BLyS is important in ensuring that new B cells mature, survive and differentiate 1,3 1. Cancro MP, et al. J Clin Invest 2009;119: Chu T, et al. Arthritis Rheum 2009;60: Miller JP, et al. J Immunol 2006;176:

30 Mechanisms of B-cell tolerance for self-antigens in health many steps that can be dysregulated causing autoimmune responses Peripheral lymphoid organs Fas-mediated apoptosis GC response T T T T B Extrafollicular B lymphoblast Short-lived PC Follicular naïve T T T T T T T Absence of T-cell help Autoantigen-mediated apoptosis Memory Anergy Absence of T-cell help Competitive disadvantage for survival factors (BAFF) Somatic hypermutation Long-lived PC Increase avidity for foreign antigen Eliminate autoreactivity Create autoreactivity de novo Courtesy of Jen Anolik U of Rochester

31 Human Autoimmune Diseases associated with aberrant TFH cells From Tangye et al. Nat Rev Immunol 2013

32 Ectopic Lymphoid Structures: Powerhouse of Autoimmunity Corsiero Pitzalis 2016 Front Immunol :430.

33 Long lived plasma cells provide persistent high level Ab defenses Current Opin Immunol 20(2) 2008, Pages

34 Plasma cells are antibody producing factories Lots of endoplasmic reticulum (ER)!

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36 Autoimmune Disease: Seropositive Rheumatoid Arthritis is an archetype

37 Autoimmunity and Pathogenetic B-cell mechanisms Bone Marrow Mature Activation Blast Immature T1 T2 Plasma cell Pro B Pre B Spleen FO MZ Plasmablast Memory GC B FDC T DC B T DC TNF-α T B IL- 6 LT Mφ? Lymphoid organogenesis Multiple sclerosis Sjögren s syndrome RA Antigen presentation and costimulation Multiple sclerosis SLE RA Inflammatory cytokines All autoimmune disorders Autoantibodies Graves disease Myasthenia gravis Pemphigus vulgaris SLE RA Immune complexes SLE RA Abbreviations: DC, dendritic cell; FDC, follicular dendritic cell; FO, follicular; GC, germinal center; IL, interleukin; LT, lymphotoxin; Mφ, macrophage; MZ, marginal zone; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; TNF, tumor necrosis factor. Adapted from Martin Annu Rev Immunol 2006;24:

38 HLA and Non-HLA Genes Are Linked in APCA-Positive Genome-Wide Association Studies (GWAS)* Log 10 P Value Chromosome The grey horizontal line indicates SNPs that are significant at a genomewide level *SNPs plotted according to chromosomal location, with the log10 P values corrected with the use of genomic control. SNPs, single nucleotide polymorphism; MHC, major histocompatibility complex; TRAF1, TNF signal transduction gene; C5, complement 5 gene. Plenge RM et al. N Engl J Med. 2007;357(12):

39 Antigen Presentation -- First Step in the development of Rheumatoid arthritis -- Genetic Susceptibility- Certain Class II MHC alleles ie HLA-DR4 (DRB1) (*0401,*0404) -- Antigen Presentation to T Lymphocyte (CD4+)

40 Citrullination by Peptidylarginine Deiminase in Rheumatoid Arthritis Annals of the New York Academy of Sciences 41 Vol 1108, Issue 1, pp , 29 AUG 2007 DOI: /annals

41 Development of ACPA Repertoire Prior to Clinical Onset ACPA spreading occurs years before disease Number of peptides recognized Reactivity to different peptides The number of citrullinated peptides recognized increased over time The reactivity to different citrullinated peptides increased over time van der Woude 42 D et al. Ann Rheum Dis. 2010;69(8): Abstract 1091.

42 RHEUMATOID ARTHRITIS: HYPOTHESIS FOR STAGES OF DISEASE ACPA Epitope Spreading + Titer Elevation Genetics RF - HLA-DRB1 (shared epitope)* - GWAS (PTPN22, others) Cytokines Healthy Autoimmune Early Synovitis OVERT Rheumatoid Arthritis - Smoking - Hormones - Bacteria* - Diet? Pre-Clinical Phase - Arthritis - Radiographic Changes - Remicade * Environmental Factors Treatment Often merited Klareskog and others

43 Theoretical Pathway for Breaking Tolerance in RA Anti-CCP B cells capture any citrullinated protein, giving rise to expanded T-cell responses against selfantigens More Autoreactive T Cells IgG anti-citrulline immune complexes are antigens for RF B cells RF B cell Autoreactive T Cells T Cell Anti-citrulline B Cell Anti-citrulline Plasma cell PADI RF and anti-ccp antibodies lead to immune complexes, macrophage secretion of proinflammatory cytokines Citrullinated antigens Arginine-containing antigens Adapted from: Niewold TB, et al. QJM. 2007;100(4):

44 Citrullination, ACPA Formation, and IL-8 Expression Are Key Processes in Osteoclastogenesis in RA Catrina AI, et al. Nat Rev Rheumatol. 2017;13:79-86.

45 Osteoclastogenesis May Occur Before Clinical Disease Onset Schett G, Gravallese E. Nat Rev Rheumatol. 2012;8:

46 Bone Volume and Density Are Reduced in ACPA+ But Non-Arthritic Individuals Arrows show cortical thinning, cortical fenestration as well as small bone erosions. ACPA, anti-citrullinated protein antibody. Kleyer A, et al. Ann Rheum Dis. 2014;73:

47 B cells and antibodies produced in rheumatoid asynovium drive pathogenesis Catrina I I, et al. Nature Reviews in Rheum 10, 645 (2014) 48

48 Diverse Recirculating Cells (Including Memory B Cells) Traffic Through Rheumatoid Joints macrophage TFN-a T Cell TFN-a Neutrophil TFN-a Immature B Cell TFN-a TFN-a T Cell B Cell Dendritic cell BAFF BAFF APRIL BAFF BAFF Synoviocytes B Cell Silverman GJ. Arthritis Rheum. 2006;54:

49 Bad B cells in RA ACPA and RF are used as serological markers and aid diagnosis. Serum ACPA and RF + patients have worse prognosis Where are bad autoantibodies ACPA & RF made? 1. In affected synovium (ectopic lymphoid tissue), tht should respond to treatment 2. From long-lived plasma cells in the bone marrow? 3. Continuously produced by short-lived plasmablasts in peripheral lymphoid tissue (and affected joints)

50 Functional B cell abnormalities in RA: ACPA levels are proportional to levels of recirculating Bad ACPA memory Where are serum ACPA made? Bone Marrow Plasma cells? Synovial Plasmablasts Relationship to recirculating ACPA bearing B cells? Pelzek et a. Silverman Arth Rheum 2017

51 Clinical treatment to reduce inflammation (DAS remission) does not eradicate bad ACPA memory Pelzek et a. Silverman Arth Rheum 2017

52 Concluding remarks In the adaptive immune system, Germinal center (GC) responses facilitate highly coordinated B-T cell interactions With breach of immune tolerance to self antigens, GC responses may drive pathogenic immune responses There are several stages at which immune dysregulation can contribute to autoimmune disease Understanding B-T cell interactions has highlighted new therapeutic targets that include: Antigen-specific B cells and T cells Chemokine receptors/ligands Factors that regulate TFH cells B cell survival factors Factors that support long-lived plasma cells and others

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