Anti-Ly9 (CD229) antibody treatment reduces marginal zone B cell numbers and salivary gland inflammation in a mouse model of Sjögren s Syndrome

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1 Anti-Ly9 (CD229) antibody treatment reduces marginal zone B cell numbers and salivary gland inflammation in a mouse model of Sjögren s Syndrome Joan Puñet-Ortiz, Manuel Sáez Moya, Marta Cuenca, Adriana Lázaro, Pablo Engel Immunology Unit, Department of Biomedical Sciences, Medical School, University of Barcelona, C/ Casanova 143, 836 Barcelona, Spain. Barcelona, November 218

2 Ly9 (CD229) IS AN IMPORTANT IMMUNE-MODULATOR OF AUTOIMMUNIT AND B CELLS Ly9 (CD229) Unique SLAM family molecule with 4 Ig-like domains. V Ly9 interacts in a homophilic manner. C 2 V C 2 S S S S Expression restricted to lymphocytes. Highly expressed on innatelike lymphocytes, such as NKT cells and MZ B cells. Contains two immunoreceptor tyrosine-based switch motifs (ITSM) in its cytoplasmic tail. Recruits SAP adaptor molecule or phosphatases (SHP2 or SHIP-1) ITSM=Immunoreceptor tyrosine-basedswitchmotif Aged Ly9-deficient mice spontaneously develop features of systemic autoimmunity (de Salort et al., Front Immunol 213) Ly9 is a negative regulator for the development of marginal zone B cells (Cuenca et al., J Immunol 215)

3 SJÖGREN S SNDROME (SjS) MA BE A POTENTIAL TARGET FOR Ly9 TARGETING SjS is a common chronic autoimmune disease affecting salivary/lacrimal glans (dryness) but also extraglandular tissues Presence of autoantibodies (anti-dsdna, anti- Ro, Rheumatoid Factor) are key markers B-cell hyperactivity induces MZ B cell expansion that can lead to MZ-B cell lymphoma B cells play a key role in both humans and mice SjS All current therapies have failed Murine models are essential to understand its pathophysiology Salivary gland tissue with lymphocytic infiltrate. Voulgarelis, M. & Tzioufas, A. G. Nat. Rev. Rheumatol. 6, (21). Lymphocytic CD3 and CD2 cell infiltrate. x2

4 THE NOD.H-2 h4 MICE: AN SPONTANEOUS MODEL OF SJS-LIKE DISEASE The NOD.H-2 h4 mice spontaneously develope SjS-like disease but not diabetes At 24 weeks-old all female mice have SjS-related autoantibodies and salivary gland infiltrates The NOD.H-2 h4 mice show abnormal MZ B cell pool enlargment Spleen Balb/c MZ 3,37% NOD.H2 h4 MZ 24.6% Salivary Gland infiltrateof NODH2h4 mice. 1x. CD21 CD23

5 Previous results demonstrated that treatment of mice with, an agonistic mab against moue Ly9, had a dramatic effect on splenic MZ and B1 B cells. Since these two subsets of innate-like B cells have been postulated to be essential players in SjS in both humans and mice, we decided to test the hypothesis that injection with may be able to hinder the development of autoimmunity in NOD.H-2 h4 mice Specific Objectives 1. To test the ability of to deplete the MZ B cells and other subsets in the murine model of SjS NOD.H-2 h4. 2. To check if treatment with anti-ly9 mab have an impact on salivary gland infiltrates of d NOD.H-2 h4 mice. 3. To assess if treatment is able to reduce the levels of autoantibodies in NOD.H-2 h4 mice. 4. To prove if anti-ly9 mab is able to protect NOD.H-2 h4 mice from developing SjS disease. NOD.H-2 h4 Autoantibodies Salivary Gland infiltrate 8w 24w 27w 3w 25µg/mouse 25µg/mouse End-point evaluation Read-out: Organs - Spleen - Draining LN - Salivary Gland - Kidney Plasma

6 Results: MZ, B1 and GC B cells are depleted in different lymphoid organs CD21 CD95 Spleen % of lymphocytes CD23 Gated on B22 + MZ: 32.9% GL FO: 56.8% Gated on B22 + GC:.52% MZ: 1.45% **** **** ** B1 B1a B1b FO: 86.8% GC:.82% Control x1 6 lymphocytes MZ B cells (% of B22 + ) GC B cells (% of B22 + ) **** 8 Control MZ B cells (x1 6 ) ***.2 GC B cells (x1 6 ) Control ** **** **** ** B1 B1a B1b Draining LN CD21 CD95 CD23 Gated on B22 + MZ: 4.16% GL7 FO: 87.9% Gated on B22 + GC: 1.59% MZ: 1.29% FO: 93.2% GC:.15% MZ B cells (% of B22 + ) GC B cells (% of B22 + ) ***.2 MZ B cells (x1 6 ) Control Control GC B cells (x1 6 ) **

7 Results: Salivary gland area of infiltrate is reduced in anti-ly9 treated NOD.H-2 h4 mice Salivary Gland 1x 1x B22 + CD3 B22 + CD3 1x 1x B22 + CD3 4x B22 + CD3 4x B22 + CD4 B22 + CD4 1x 1x B22 + CD8 B22 + CD8

8 Results: Treatment with is able to affect autoantibodies levels Serum pre-treatment 1/32 4x 1/32 4x ANAs titers (dilution) 1, * anti-dsdna IgG (µg/ml) * post-treatment 1/32 4x 1/32 4x Rheumatoid Factor (IgM) (O.D.) ** anti-ro52 IgG (O.D.)

9 Results: Early treatment with is able to protect from salivary gland infiltration Salivary Gland Autoantibodies Salivary Gland infiltrate 8w 12w 14w 24w 27w 3w NOD.H-2 h4 25µg/mouse End-point evaluation A 1 B.5 Foci Number Total Area of Infiltrate (mm 2 ) Untreated Untreated 12-weeks-old 14-weeks-old 12-weeks-old 14-weeks-old

10 CONCLUSIONS Ly9 (CD229) targeting is able to deplete MZ, B1 and GC B cells in the spleen and draining lymph nodes Mice treated with the mab anti-ly9 show reduced area of lymphocyte infiltrates in the salivary glands The administration of is capable to affect autoantibody levels in mouse sera Early anti-ly9 mab treatment before disease onset impedes salivary gland infiltration In contrast with the B cell depletion therapies, the selective deletion of MZ, B1 and GC B cells should be regarded as an attractive new therapeutic approach

11 THANKS! Pablo Engel Manu Sáez Adriana Lázaro Marta Cuenca