11/12/2012

Production of Citrullinated Filaggrin-Specific IgG in Rheumatoid Arthritis Financial Disclosures: Nothing to disclose Patients is Associated with an Expansion of Citrullinated Filaggrin Tetramer-Binding Switched-Memory Blood B Cells

Philip Titcombe, Laura O. Barsness, Lauren Giacobbe, Emily Baechler Gillespie, Erik J. Peterson and Daniel L. Mueller

Division of Rheumatic and Autoimmune Diseases Center for Immunology

University of Minnesota Medical School, Minneapolis, MN

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Evidence-Based Medicine: References B cells in Rheumatoid Arthritis (RA)

• Samuels J, Ng YS, Coupillaud C, Paget D, Meffre E. Impaired early B cell tolerance in • RA patients demonstrate defects in central and peripheral B cell patients with rheumatoid arthritis. J Exp Med. 2005 May 16;201(10):1659-67. self-tolerance checkpoints (Samuels J et al. J Exp Med. 2005) PubMed PMID: 15897279; PubMed Central PMCID: PMC2212916. • RA is highly associated with rheumatoid factors (RF) and anti- • Snir O, Widhe M, Hermansson M, von Spee C, Lindberg J, Hensen S, Lundberg K, citrullinated protein antibodies (ACPA) Engström A, Venables PJ, Toes RE, Holmdahl R, Klareskog L, Malmström V. Antibodies to several citrullinated antigens are enriched in the joints of • RA synovial tissues form tertiary lymphoid follicles and germinal rheumatoid arthritis patients. Arthritis Rheum. 2010 Jan;62(1):44-52. PubMed centers, with in situ production of ACPAs (Snir O et al. Arthritis PMID: 20039432. Rheum. 2010) • Taylor JJ, Martinez RJ, Titcombe PJ, Barsness LO, Thomas SR, Zhang N, Katzman SD, • Key RA disease-associated gene alleles in ACPA+ patients are Jenkins MK, Mueller DL. Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen. J Exp Med. 2012 Oct 22;209(11):2065- expressed by B cells (e.g., MHCII, PTPn22, CD40) 77. doi: 10.1084/jem.20112272. Epub 2012 Oct 15. PubMed PMID: 23071255; PubMed Central PMCID: PMC3478923. • Rituximab anti-CD20 mAb therapy depletes CD20+ B cells and reduces RA disease activity

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Use of ‘Antigenic Tetramers’ to Explore Self Antigen- Specific Polyclonal B cell Tolerance in Mice B cells in Autoimmune Arthritis Model Systems • No evidence for efficient negative selection or anergy induction for low affinity polyclonal responders to glucose-6-phosphate isomerase (GPI) • Immunization against synovial target antigens in genetically susceptible mice can lead to destructive inflammatory arthritis • Physically sorted naïve GPI tetramer-binding B cells transfer arthritis to B g7 (e.g., type II collagen, aggrecan, human cartilage GP39) cell-deficient mice in the presence of GPI/I-A -specific KRN CD4+ T cells • Similar B cell tolerance (ignorance) for a soluble transgenic ovalbumin • Spontaneous production of IgG1 anti-GPI in K/BxN transgenic mice is strongly associated with the development of arthritis • A membrane form of transgenic ovalbumin leads to partial deletion (~50%) of high affinity ovalbumin tetramer-binding B cells at the T1 to T2 peripheral self tolerance checkpoint, and clonal anergy in the rest • Transfer of K/BxN arthritic mouse serum (containing anti-GPI) to healthy recipients causes arthritis • Therefore, the avoidance of B cell reactivity to several soluble (but not membrane) self antigens appears to rely mainly on tolerance in the helper CD4+ T cell compartment Taylor JJ et al. J. Exp. Med. (2012)

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1 11/12/2012

Hypotheses

• Low affinity B cells specific for monomeric citrullinated proteins do exist in Goals for Human Investigation healthy individuals – the absence of a high frequency of citrullinated peptide/DR4-specific CD4+ T helper cells maintains these B cells at low frequency in a naïve state, and prevents the development of RA • Develop tetrameric antigen probes to identify and characterize • In the secondary and tertiary lymphoid tissues of RA patients, citrullinated human pathogenic B cells by making use of knowledge about peptide/DR4-specific CD4+ T cells provide cognate help to naïve autoantigen targets in RA citrullinated protein-specific B cells

• Design antigen-specific therapeutic strategies to enhance B cell – B cell activation tolerance to relevant autoantigens, while maintaining the full – clonal expansion responsiveness of the non-pathogenic and protective B cell – isotype switch repertoire – memory cell differentiation • Expanded and differentiated (CD27+ IgD-) autoreactive B cells circulate between synovial tissues and can be found in the blood

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Human B cell Antigenic Tetramer Development Based on 1st Generation CCP Autoantibody Test Human B cell Antigenic Tetramer Development Based on 1st Generation CCP Autoantibody Test • First generation cyclic citrullinated peptide antibody (CCP) test, with 37% sensitivity and 98% specificity • Cfc1-biotin-SA-PE tetrameric complex • Association with rheumatoid factor production, rheumatoid nodules, – Arginine312—>Citrulline substituted filaggrin306-324 cyclic peptide boney erosions, and deformity • Cf0-biotin-SA-PE-AF647 tetrameric decoy complex • Association with smoking, and with the presence of HLA-DRB1*0101, – Native filaggrin306-324 cyclic peptide *0401, and *0404 alleles

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Citrullinated Filaggrin Peptide Tetramer Set Enrichment of Tetramer-Binding B cells

Decoy-Specific

Filaggrin Filaggrin PE PE SA SA Dual-Binding Filaggrin AF647 Filaggrin

Multiparameter flow • Cyclic Citrullinated Filaggrin Peptide • Cyclic Native Filaggrin Peptide cytometry of bound (Cfc1) Tetramer (Cf0) Decoy Tetramer and flow-through column fractions – Biotinylated Cfc1 attached to SA-PE core – Biotinylated Cf0 attached to AF647- conjugated SA-PE core – Citrulline substitutes for arginine Cfc1-Specific

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Cfc1-Specific Antibody Is Detected in 43% of ACPA+ RA Patients But Not in Healthy Controls

30 Flow Cytometric Peripheral Bood Mononuclear Cell (PBMC) ACPA+ RA Patients Healthy Controls + + +

0.4 0.4 Ig CD19 CD20 B cell Gating Strategy s

l ACPA+ Patients

a

n

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i 20

t d t 0.3

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a a

v Healthy Controls

r

r i

t + + + t

d Viable, single Ig CD19 CD20 n

n resting, n

e + –

e I

Ig Dump

c c

f PBMC B cells

n

n

o

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0.2 0.2 r

e

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C

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m 10

d

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n 0.1 0.1

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0 CD20

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5

3 2 1

0.0 0.0 4

0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3

0 0 0 0

......

. . . .

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0

0

- - - 1 f0 1 f0 - fc c fc c c c Difference between cfc1 and cf0 antibody concentration Switched- + • Over a period of one year, ACPA (2nd/3rd Gen. CCP test) RA patients were Dump(CD3, CD14, CD16) Memory newly enrolled into a University of Minnesota Rheumatic and Autoimmune Intracellular H+L Ig CD19 CD27 Diseases ACPA+ RA cohort (ongoing treatment with rituximab being the only exclusion criteria) • 33 out of 76 enrolled ACPA+ RA patients (43%) tested positive for anti-Cfc1 Ab 60 mL blood sample in a tetramer-based ELISA (with 95% CI cut-off) University of Minnesota Rheumatic and University of Minnesota Rheumatic and Monday, November 12, 2012 13 Monday, November 12, 2012 14 Autoimmune Diseases Autoimmune Diseases

Cfc1–Tetramer Binding B cells in the Cfc1 Tetramer–Binding B cells in the Blood

Blood of a Healthy Donor of a Cfc1 Ab– RA Patient

AF647 AF647

- -

PE PE

- -

SA SA

- -

Cfo Cfo

Cfc1-SA-PE Cfc1-SA-PE

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Cfc1-Specific B cells are Increased in the PBMC Switched and Activated Cfc1–Binding B cells from anti-Cfc1 Ab+ RA Patients in the Blood of a Cfc1 Ab+ RA Patient

p=0.0069 3 10 p=0.0031

ns

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Increased Frequency of Switched-Memory (CD27+ IgD-) Switched-Memory B cell Differentiation is Strongly Cfc1-Specific B cells in Cfc1 Ab+ RA Patients Correlated with Cfc1-Specific B cell Clonal Expansion

Percent Switched-Memory Frequency Switched-Memory

p<0.0001 100

c

i 2

p=0.0020 f R =0.6588

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g RA Patients (n = 21)

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Total Cfc1-Specific B cell Frequencies Correlate with anti-Cfc1 Ab Levels in Individual RA Patients Generation of Switched-Memory Cfc1-Specific B cells Correlates with anti-Cfc1 Ab Levels in RA patients

Cfc1-Specific B cells Cf0-Specific B cells Cfc1-Specific B cells Cf0-Specific B cells Total Polyclonal B cells 3 4 10 10 2 3 6 s 3 R =0.0443 10 l 10

r 10

l 2 l

2 e 2 R =0.4821 r

R =0.0130 a 2

e

t e

R =0.4341 p R =0.00592

c

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1 c f cfc1 Antibody cfc1 Antibody cfc1 Antibody c 101 102 0.01 0.1 1 0.01 0.1 1 cfc1 Antibody cfc1 Antibody

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Conclusions

• Potentially pathogenic citrullinated protein-specific B cells can now be Summary prospectively monitored in the blood of patients with ACPA+ RA using antigenic tetramer sets • A citrullinated filaggrin (Cfc1) tetramer identified autoreactive B cells in both healthy individuals (n = 9) and a cohort of ACPA+ RA patients (n = 43) • The development of citrullinated filaggrin autoantibody is not a consequence of defective central and peripheral B cell tolerance checkpoints (clonal deletion) in RA • Development of ACPA+ RA in the absence of Cfc1 Ab (n = 22) was not associated patients, since the frequency of Cfc1-specific B cells is similar in both Cfc1 Ab- RA with an increase in the frequency of total Cfc1 tetramer-binding B cells p = ns); patients and healthy controls nevertheless, increased differentiation to a Switched-Memory phenotype was observed for these autoreactive B cells (p = 0.02) • Our results are more consistent with the hypothesis that naïve citrullinated protein-specific B cells exist within the normal human polyclonal repertoire, but do • Blood CD20+ CD19+ Cfc1 tetramer-binding B cells in Cfc1 Ab+ RA patients (n = 21) not undergo clonal expansion and differentiation (class switch, memory cell demonstrated significant clonal expansion (p = 0.0069) and differentiation to a Switched-Memory cell phenotype (p < 0.0001) generation) due to a lack of CD4+ T cell help

• Similar results were obtained with a citrullinated enolase peptide tetramer (not • Finally, RA development with ACPA autoantibody production is now shown to be shown today) associated with an expansion of Switched-Memory citrullinated protein-specific B cells in the blood

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4 11/12/2012

Acknowledgements

University of Minnesota University of Minnesota Rheumatic and Autoimmune Department of Microbiology Diseases Marc Jenkins Mueller Lab Justin Taylor Philip Titcombe Ryan Martinez Laura Barsness Stephanie Thomas Na Zhang Mayo Clinic Lokesh Kalekar Shirdi Schmiel Shreyasee Amin Lauren Giacobbe Ann Reed

Erik Peterson Funding provided by the NIAID/NIH, Lupus Foundation of Minnesota, and Emily Gillespie an ACR-REF Within Our Reach award Joe Wilson

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