10/29/2013

Disclosure

• No Disclosure The NKp30/B7H6 axis contributes to pathogenesis in primary Sjögren’s syndrome (pSS)

Gaetane Nocturne, Sylvie Rusakiewicz, Damien Sene, Gunnel Nordmark, Maija-Leena Eloranta, Per Eriksson, Elke Theander, Helena Forsblad-d'Elia, Roald Omdal, Marie Wahren-Herlenius, Roland Jonsson, Lars Rönnblom, Joanne Nititham, Kimberly E. Taylor, Christopher J. Lessard, Kathy L. Sivils, Jacques-Eric Gottenberg, Lindsey A. Criswell, Corinne Miceli-Richard, Laurence Zitvogel and Xavier Mariette

Why addressing the role of NK cells in Strategies of NK cell recognition: pSS?

- NK cells: innate anti viral and anti tumor immune response • NK cells: crucial components of the innate immune - NK Cell activation is controlled by the balance of activating and system inhibitory signals • NK cells and IFN signature • Type I IFN: cross-talk pDC/NK • Type II IFN: IFN-γ producing cells • What is known? • Murine models • Regulatory role of NK cells in mouse CMV-induced sialadenitis • Early NK cell infiltration in salivary glands of NZB/W F1 mouse model • Controversial data concerning the role of NK cells in pSS Nandula SR, Oral Dis, 2011 Hagberg N., J Immunol, 2011 Izumi et al, J Lab Clin Med, 2006 Vivier et al., Nat Immunol, 2008 Tessmer MS, Plos Pathog, 2011 Szodoray et al, Clin Exp Immunol. 2009

NCR3/NKp30 Objectives

• Killing cell activating mainly To investigate the role of NK cells in pSS expressed by NK cells pathogeny focusing on NCR3/NKp30 effector • NCR3 is encoded on 6 functions (6p21.3)-MHCIII region 1. Genetic association of NKp30 polymorphisms with pSS susceptibility • : -H6 2. expression: Phenotypic characteristics • Function : of peripheral NK cells - Tumor target recognition and lysis 3. Functional consequences within salivary glands - NK / (DC) cross-talk

Ferlazzo, JEM, 2002 Brandt, JEM, 2009

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Genetic polymorphisms within the NKp30 promoter region are associated with decreased Single-SNP logistic regression results susceptibility to pSS

•Exploratory cohort:

Meta-analysis of 2 •632 pSS vs 522 healthy volunteers: 2 Groups Exploratory (French) cohort Replication (Scandinavian) cohort •pSS patients vs HV from KB cohorts •Patients from ASSESS cohort vs HV from St Louis’ hospital 574 pSS cases (340 SSAB+, 233 SSAB-) and 451 436 pSS cases (329 SSAB+, 105 SSAB-) and fixed effect controls 441 controls •Replication cohort: MAF MAF MAF MAF SNP OR P OR P OR P •436 pSS cases vs 441 HV cases controls cases controls •Scandinavian cohort (Norway + Sweden) rs11575837 0.017 0.041 0.41 0.0021 0.0080 0.010 0.80 0.66 0.48 0.0039

•European ancestry (AIMs, self declarative) All pSS cases versus healthy rs2736191 0.025 0.058 0.46 0.0013 0.025 0.032 0.75 0.31 0.56 0.0019 controls

•Genotyping of rs2187668* 0.24 0.12 2.46 1.73E-11 0.33 0.13 4.91 2.9E-27 3.36 1.7E-34 •9 SNPs within NCR3 (6p21.3) •3 additional SNP proxies for HLA–DR2, HLA-DR3 and TNF given the extensive linkage disequilibrium within the MHC region Replication cohort: - rs11575837 associated with disease characterized by anti-SSA and/or -SSB positivity [p=0.011, OR= 0.060]

Functional consequences of disease Multi-SNP logistic regression associated promoter variant

• rs11575837 is located

Replication (Scandinavian) Meta-analysis of 2 within NCR3 promoter Exploratory (French) cohort cohort cohorts 8 *

574 pSS cases (340 SSAB+, 233 SSAB-) and 451 436 pSS cases (329 SSAB+, 105 fixed effect 6 controls SSAB-) and 441 controls • Investigation of NKp30 mRNA levels in 78 pSS 4 SNP OR P OR P OR P patients from the French rs11575837 0.38 0.001 1.22 0.7 0.5 0.008 2

ASSESS cohort NCR3/TBP ratio All pSS cases versus healthy rs2736191 0.43 0.001 1.05 0.9 0.62 0.014 0 – 12 carried the minor allele G/A G/G controls rs2187668* 2.47 2.0E-11 4.93 8.2E-27 3.37 5.0E-34 (rs11575837A) – 66 carried the major allele (rs11575837G) Replication cohort: - rs11575837 associated with disease characterized by anti-SSA and/or -SSB positivity [p=0.007, OR= 0.37]

Elevated NKp30 expression and Elevated NKp30 expression and function in pSS function in pSS

• Comparison between 30 pSS patients and 30 healthy

volunteers A

• Eight color multiparametric flow cytometry analyses on freshly isolated peripheral blood NK cells

• Frequency of different NK cell sub populations (CD56 B C dim vs CD56 bright) • Expression of activating receptor such as NCR (NKp46, NKp44, NKp30), NKG2D (C lectin family), KIR S. • Expression of inhibitory receptor (KIR L, NKG2A)

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Elevated NKp30 expression and Functional relevance of high expression function in pSS levels of NKp30 on NK cells • Receptor cross-linking assay to analyze – Degranulation (CD-107a) – IFN-γ secretion • Anti NKp30 agonist • Controls – isotype – anti-CD16 antibody (Ab) as controls

Degranulation IFNγ secretion

Cross-linking NKp30 Cross-linking CD16 Cross-linking NKp30 Cross-linking CD16 2.5 µg/ml 0.8 µg/ml 0.3 µg/ml 2.5 µg/ml 2.5 µg/ml 0.8 µg/ml 0.3 µg/ml 2.5 µg/ml

30 30 30 30 30 30 * ns 30 ** 30 ns ns ns 2 ns ns 2 r =0,282 2 2 2 2 2 2 r =0,350 r =0,640 r =0,050 20 r =0,032 20 r =0,072 20 r =0,005 20 r =0,105 20 20 20 20

10 10 10 10 10 10 10 10

0 0 0 0 0 0 0 0

0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

30 30 30 30 30 30 30 30 ns ns ns ns ns ns ns 2 ns 20 r =0,001 r2=0,001 2 2 2 2 2 2 %IFNγ+ NK 20 20 r =0,036 20 r =0,111 20 r =0,004 20 r =0,012 20 r =0,004 20 r =0,111 10 %CD107a+ %CD107a+ NK 10 10 10 10 10 10 10

0 0 0 0 0 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

%NKp30+ NK %NKp30+ NK N = 16 patients, among them 4 harbor the rs11575837 minor allele (in red)

Accumulation of NK cells in minor salivary gland biopsy NKp30 expression in salivary glands?

2 500 2 500 r2=0,425 r2=0,002

400 *** 400 ns

cells per mm per cells

cells per mm per cells + 300 + 300

200 200

inside the foci the inside  NKp30 is expressed within salivary glands outside the the foci outside

100 100 Number of NKp46 of Number 0 NKp46 of Number 0 0 2 4 6 8 0 2 4 6 8 Focus score Focus score

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Consequences of NKp30 expression within Consequences of NKp30 expression within salivary gland salivary gland

500 NKp30A

200 * 20 * NKp30B )

n 400

o

L i

s NKp30C s

150 15 m

/

e 6 r Vecteur Control

g 300

p

H

p

x

7

( e

HSG

10

B 100

e

2 f

- 200

v

o

i

t

L

a I l 50 5

e 100 R 0 0 Medium TNF Poly I:C 0 Medium TNF Poly I:C Medium TNF Medium TNF HSG SGEC HSG SGEC NKp30-transduced Jurkat cells HSG + NKp30-transduced Jurkat cells

Consequences of NKp30 expression within salivary gland To conclude

Implication of NKp30 in the pathogenesis of pSS:

* Genetic susceptibility  increased mRNA level

HSG SGEC NKp30-transduced Jurkat cells HSG + NKp30-transduced Jurkat cells High level of protein  High IFN-γ production

*** ns ns *** *** 600

) 500

L Medium

m 400

/ Anti-PVR (IgG1) Blocking Ab

g p

( 300 Anti-NKp44 (IgM) Blocking Ab

In salivary glands? 2

- Anti-NKp30 (IgG1) Blocking Ab

L 200

I Anti-NKp30 (IgM) Blocking Ab 100 0 Medium TNF

Published in Sci Transl Med. 2013 Jul

Virus? Other activator of innate immunity? Acknowledgments

IFN-γ

NK NK Epith cell NKp30/B7H6 IC pDC

o To all my co-authors IFN-α Auto-Ag o To investigators of the ASSESS cohort supported by a grant from the French Ministry of Health (PHRC) o To patients BAFF

IFN-γ T

T TFH T T T

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