Cristina Croia, Michele Bombardieri, Barbara Serafini, Eliana Marina Coccia, Stephen Kelly, Paola Migliorini, Francesca Aloisi, Costantino Pitzalis

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1 Anti-VCA and EBNA1 antibodies are produced in the rheumatoid synovium in the presence of ectopic lymphoid structures and correlate with ACPA production. Cristina Croia, Michele Bombardieri, Barbara Serafini, Eliana Marina Coccia, Stephen Kelly, Paola Migliorini, Francesca Aloisi, Costantino Pitzalis

2 - Human γ-herpesvirus; genome is 172 Kb ds DNA Epstein-Barr Virus (EBV) - It infects 98% of the world population, mainly establishing an asymptomatic latent infection. Rarely causes malignancies like Burkitt s lymphoma, nasopharyngeal carcinoma etc. - If primary infection occurs during adolescence it cause infectious mononucleosis EBV LIFE CYCLE Epithelial cell BZLF1 Gp350/220 CR2 EBV B cell Muscle smooth cell monocyte Tatsuya Tsumuri et al, Rev. Med. Virology, 2005

3 Evidences linking EBV-RA 1. Serological data 2. EBV viral load 3. Molecular mimicry 4. Cell-mediated control of EBV in the joint and peripheral blood Very heterogeneous results searching EBV nucleic acids or proteins in the RA synovia Absence of EBV proteins/rna in RA synovium Evidences of EBV proteins/rna in RA synovium Niedobiteck et al, Arthritis Rheum, 2000 Takey et al, Int. Immunol No LMP1 No BZLF1 EBER 20% EBER+ EBER LMP1 20% EBER+ 50% LMP1 + CD21 - EBNA2 - Brousset et al, Ann Rheum Dis No EBER No BHFR1 Takeda et al, Arthritis Rheum, 2000 EBER BZLF1 Gp350/220 55% EBER+ 100% BZLF1+ 77% gp350/220+ LMP1-

4 Link EBV- ectopic lymphoid neogenesis v Ectopic B cell follicles in brain of MS patients are the major site of EBV reactivation; in the same sites there are signs of an ongoing anti-viral immune response v Serafini et al, J Exp Med, 2007 v EBV infection is associated with ectopic B cells follicle in MG thymus; signs of antiviral immune response. v Cavalcante P, Ann Neurol, 2010 Is EBV infection also implicated in the auto-immune dysregulation which characterized RA synovium in the presence of such ectopic lymphoid structures?

5 Ectopic Lymphoid structures in RA Morphological features typical of SLO Functionality typical of SLO T/B cells segregation Networks of CD21+ follicular dendritic cells CD3 CD21 AID HEVs differentiation expression of lymphoid chemokines CXCL13 and CCL21 CD3 CD21 CXCL13 CCL21 Manzo A, Eur J Immunol, 2005 In situ production of ACPA Humby F, PloS Med, 2009

6 Study design Patients and samples 50 RA patients (paraffin samples, frozen samples, RNA) 11 OA patients (paraffin samples, RNA) Histological grading Analysis of EBV proteins and RNA and gene expression Link ACPA-producing cells and EBV reactivation Samples with aggregates and FDC network (CD21+), with aggregates without FDC network (CD21-) and with diffuse inflammation By IHC and IF; LMP1, LMP2A, EBNA2, BMFRF1, BFRF1, p160 and gp350/220 By ISH: EBERs Double IF EBV proteins with /CD138 Quantitative real time RT-PCR on pre-amplified cdna (CD19, LMP2A, EBERs) By double IF citrullinated and biotinylated fibrinogen /BFRF1 or CD138 Synovial humoral immune response against unmodified and citrullinated EBV antigens CD8+ T cell and EBV By ELISA on serum of SCID mice transplanted with RA synovium By double IF CD8/; CD8/CD138; CD8/GranzymeB; CD8/BFRF1; GranzymeB/BFRF1

7 50 paraffin RA synovia 1. Histological characterization of ELS of RA synovial samples Sample with aggregates and FDC network Sample with aggregates without FDC network Sample with diffuse inflammation CD3 CD3 CD3 CD3 CD138 CD138 CD138 CD138 CD68 CD68 CD21 CD68 CD68

8 2. The expression of latent EBV antigens correlates with infiltration in RA synovium GC-like structures with B cells LMP2A+ LMP2A CD138 CD3 CD21

9 LMP2A is associated with the presence of B cell follicles; it is near always expressed by B cells LMP2A merge LMP2A merge RA synovia N LMP2A+ ELS+/FDC % ELS+/FDC % diffuse 20 0% From 8,2% to 54,7% of the + cells express LMP2A

10 3. EBV latent gene expression in RA synovia Real time RT-PCR after a selective pre amplification of target genes (13 RA; 4 OA) LMP2A EBERs EBER+ cells correlate with B-cell content in the RA synovial tissue EBER

11 4. EBV reactivation in RA synovia correlates with plasma cell infiltration By IHC analysis of BFRF1 and BMRF1 CD138 BFRF1 a B cell follicle (+ cells) surrounded by CD138+ plasma cells; BFRF1+ cells distribution match with plasma cell area CD138 BFRF1 gp250/320 p160 Markers of productive viral infection found in association of ELS and matching plasma cells distribution Strong association BFRF1and CD138 score RA synovia N BFRF1+ ELS+ / FDC % ELS+/FDC % diffuse 13 0% and 100% of RA samples FDC+ showed EBV reactivation (in 3%-23% of plasma cells)

12 5. Lytic reactivation of EBV occurs in auto-reactive plasma cells CD138 CD138 cfb CD138 cfb Cb-Fib BFRF1 Cb-Fib BFRF1 EBV could play a role in the survival and in situ differentiation of autoreactive plasma cells in the RA synovium through the expression of its lytic program

13 6. Unmodified and citrullinated anti-ebv antibodies are locally produced in RA synovia 30 SCID mice transplanted with ELS+ vs ELS- RA synovium 1. EBNA1 igg (past infections) 2. VCA igg (primary and recent infections) 3. EA igg (acute infections or reactivations) Anti-EBNA1 Anti-VCA Anti-EA ACPA Human anti-ebna1, anti-vca but not anti-ea igg were significantly higher in sera of mice transplanted with ELS+ compared to ELS- RA synovia Anti-EBV antibodies production correlates with local ACPA production in RA synovia

14 7. Unmodified and citrullinated anti-ebv antibodies are locally produced in RA synovia Serum of SCID mice transplanted with ELS+ RA synovium analysed for VCP1 and VCP2 (citrullinated EBV antigens derived from EBNA1 and EBNA2) Anti-VCP1 and -VCP2 antibodies are locally produced in RA synovia and correlate with ACPA production Citrullinated EBV antigens contribute to stimulate cross-reactive antibodies in the RA synovium

15 8. Ongoing cytotoxic activity in sites where EBV-lytically infected cells accumulate CD8+ cells do not enter B cell follicles /CD8 CD8/GranzymeB A subset of CD8+ cells are Granzyme B+ CD8/GranzymeB CD138 CD8+ and GranzymeB+ cells accumulate in association with EBV virally infected plasma cells BFRF1/CD8 BFRF1/CD8 Granzyme B BFRF1 Viral reactivation in locally differentiated PCs could be implicated in local chronic inflammation upon activation of a cytotoxic T cell response

16 Summary 1. EBV LATENCY IN RA SYNOVIA: - The expression of EBV latent proteins correlate with ectopic lymphoid neogenesis - LMP1, LMP2A and EBERs expression is mostly restricted to B cell follicles 2. EBV REACTIVATION IN RA SYNOVIA: - EBV reactivation in RA synovia (BFRF1, BMRF1) correlates with ectopic lymphoid neogenesis - Reactivation is restricted to plasma cells surrounding ELS 3. LYTIC REACTIVATION OF EBV OCCURS IN AUTOREACTIVE PLASMA CELLS - Colocalization of BFRF1 and citrullinated fibrinogen positive PCs 4. UNMODIFIED AND CITRULLINATED ANTI-EBV ANTIBODIES ARE LOCALLY PRODUCED IN THE RA SYNOVIA 5. ONGOING CYTOTOXIC ACTIVITY IN SITES WHERE EBV-LYTICALLY INFECTED CELLS ACCUMULATE

17 Acknowledgments: Dr. Barbara Serafini Dr. Eliana Coccia Dr. Michele Bombardieri Dr. Francesca Aloisi Dr Stephen Kelly Prof. Costantino Pitzalis Dr. Paola Migliorini

2011 Update on the ECIL-3 guidelines for EBV management in patients with leukemia and other hematological disorders

UPDATE ECIL-4 2011 2011 Update on the ECIL-3 guidelines for EBV management in patients with leukemia and other hematological disorders Jan Styczynski, Hermann Einsele, Rafael de la Camara, Catherine Cordonnier,