10/29/2013

Dendritic cell receptors for nucleic acids and damaged cells by WEHI has patents for Clec9A Dr Irina Caminschi

Introducing “dendritic cells (DC)” Decision between immunity and tolerance is guided by the detection of “anomalies” indicative of infection

• DC are the sentinel cells of the immune system

DC decision • DC are considered the “professional” antigen presenting DC have receptors that recognize: cells - uniquely equipped to activate naive T cells pathogen associated molecular patterns (PAMPs) • DC can present antigen in an “immunogenic” or damage associated molecular “tolerogenic” manner tolerance patterns (DAMPs) Immunity DC decision:

tolerance Immunity

Introducing Clec9A (DNGR1)

Clec9A – a (unusual) DAMP * C-type

* Mouse - Human Clec9A: 53%aa identity

* Expression is restricted to certain DCs; - mouse CD8+, CD103+ DCs, and pDC - human BDCA3+ DC

* Function conserved between species: Clec9A binds filamentous actin (exposed on dead cells) Zhang et al. Immunity 2012 and Ahrens et al. Immunity 2012.

hemITAM

* Clec9A facilitates cross-presentation Caminschi & Lahoud.2008 Blood of dead cell-associated antigen Sancho et al. 2008 J Clin Invest Sancho D, et al. Nature 2009. 16:899-903. Huysamen et al. 2008 J Biol Chem

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Clec9A facilitates cross-presentation Clec9A recognizes filamentous actin (exposed on necrotic/dead cells) of dead cell-associated antigen Sancho D, et al. Nature 2009. 16:899-903.

Zhang et al. Immunity 2012. Ahrens et al. Immunity 2012.

The Clec9A mediates cross-priming of CTL during vaccinia virus infection in mice Iborra et al. J Clin Invest. 2012

Taylor et al. Nat Rev Microbiol. 2011

Dendritic cell surface receptors that can be exploited to deliver antigen to DC Anti-tumor responses elicited by targeting antigen to Clec9A

α-Receptor mAb OR isotype mAb

Measure anti-cargo Immune response Cargo: antigen

Sancho et al. 2008. JCI 118: 2098

Targeting Clec9A induces potent Ab Primate trials responses without adjuvant

RAT α-Receptor mAb OR bleed Measure RAT isotype mAb Anti-RAT Ig

Stephen Kent Robert De Rose

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Clec9A is a DAMP receptor TLR9 (DEC205?) – a PAMP receptor

• Conserved between mouse and man

• Binds filamentous actin (exposed on dead cells)

• Facilitates cross-presentation (and normal MHCII presentation)

• Can be exploited to deliver antigen to DC and elicit potent immune responses  vaccine target

DC recognise bacterial DNA as an indicator An outstanding question: of invasion.... How does extracellular DNA reach intracellular TLR9?

Biology: Unmethylated CpG is rare in mammalian DNA but frequent • Receptors are involved in the uptake of CpG-ODN in bacterial DNA therefore it provides a means to detect “foreign DNA” Toll-like receptor 9 (TLR9) recognizes unmethylated CpG motifs in DNA

Application:  Synthetic CpG oligonucleotides (ODN) mimic bacterial DNA  CpG-ODN are used as adjuvants to activate DC and promote vaccine efficacy.

DEC-205 DEC-205 is a receptor for CpG oligonucleotides DEC-205 Expression pattern Cysteine-rich domain Fibronectin type II domain DEC-205 is on: • Splenic CD8+DC • Lymph node DC • Thymic epithilium, B cells, 10 C-type lectin-like activated macrophage domain Function: unknown Binds necrotic + apoptotic cells Binds plasminogen activator of Yersinia pestis

FSSVRY motif Lahoud…Caminschi Proc.Natl.Acad.Sci.USA 2012 (clathrin coated pit endocytosis)

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Expression of DEC-205 DEC-205 binds CpG ODN by ELISA

Expression pattern ELISA: DEC-205 is on: • Splenic CD8+DC • Lymph node DC • Thymic epithilium, B cells, activated macrophage

DEC-205

DEC-205 facilitates the uptake of CpG ODN Consequence of lacking DEC-205

Inject CpG – Cy3 30 min Purify DC

Extract spleen

CpG (i) Impaired DC and B cell maturation to CpG ODN CpG MHC II

Michel Nussenzweig Simone Meuter

DEC-205 is required for optimal DC DEC-205 is required for optimal B cell maturation in response to CpG maturation in response to CpG

5 hr post injection In vitro activation Purified B cells Culture o/n +CpG Analyze B cells

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Consequence of lacking DEC-205 DEC-205 is required for optimal serum cytokine production in response to CpG ODN

Chin-Nien Lee (ii) Impaired cytokine production to CpG ODN

Difference significant: IL-6, MIP-1a, MIP-1b, IL-1a, TNF-a, MCP-1, IL-10

DEC-205 is required for optimal IL-6 from B Consequence of lacking DEC-205 cells in vitro

In vitro activation Purified B cells Culture o/n +CpG Analyze B cell SNF

(iii) Impaired induction of cytotoxic T cells

DEC-205 is required for the priming of CTL in Surface receptor for DNA response to CpG Direct binding knock-out Reference

Inject CpG+ anti-Clec12A-OVA DEC205 ✔ ✔ Lahoud et al. PNAS 2012 Day 6 Day 7 OVA-targets Measure killing DEC-205-/- or B6 mice RAGE ✔ ✔ Sirois et al. JEM 2013

CD14 ✗ ✔ Baumann et al. JEM indirect 2010

KIR3DL2 ✔ ✗ Sivori et al. Blood 2010

CXCL16 ✔ ✗ Gursel et al. JI 2006 low affinity?

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Introducing CpG ODN: structure

Phosphorothioation: What are the requirements for DEC-205 to bind ODN? 1668 1826

Krieg 2006. Nat. Rev. 5: 471.

DEC-205 prefers phosphorothioated B-ODN and DEC-205 prefers B-ODN does not require CpG motif

Mouse DEC-205

1.8

1.6

1.4

1.2 class 1

1668 B OD 0.8 1826 B

0.6 2006 B

2216 0.4 A 2395 0.2 C

0 0.1 1 10 100 1000 10000 CpG conc (ng/ml)

Approach Human DEC-205 binds CpG ODN

• Identify a CpG ODN that binds DEC205 poorly and improve binding by adding a DNA motif that binds DEC205 = ‘combo  Human DEC-205 binds human B-ODN better ODN’

original ODN

combo ODN 2006 B-CpG ODN + = DEC205 binding motif 1668 B-CpG ODN • Compare stimulatory capacity of combo ODN with original ODN and binding motif – Cytokine production – Expansion of Ag specific CD8+ T cells

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Induction of cytokines Induction of cytokines

3 h Collect plasma – measure 3 h Collect plasma – measure cytokines cytokines

IL-6 plus July Serum-column G-CSF plus July Serum-column MCP1 plus July Serum-column

400 IL-6 IL-6 15000 G-CSF MCP1

8000

300 10000 6000

200 4000 5000

100 2000

Cytokines Cytokines (pg/ml) Cytokines (pg/ml)

0 0 0 d d te te o o d d te te o o d d te te o o e e a a b b e e a a b b te te a a b b t t r r t t r r c c r r m m Genotype: cWT cK/O a WT aK/O mWT K/Om Genotype: WT K/O WT K/O WT K/O cWT cK/O aWT aK/O mWT K/Om e WT e K/O a WT a K/Oo WTo K/O je je p p o o je je p p o o j j p p c c n n e e c c n n e e c c in in e e i i s s 6 i i s s 6 n n s s 6 C n n C n n C u u 6 c B E u u 6 c B E u u 6 c B E B e Treatment: B e D Treatment: B e D 6 C D D 6 C none D combo ODN none combo ODN 6 C none D combo ODN B E none combo ODN B E B E D D + + D + +

Expansion of OVA-specific Expansion of OVA-specific CD8+ T cells CD8+ T cells

d6 Measure OVA-specific d6 Measure OVA-specific

4,5,6 column CD8+ T cells CD8+ T cells s

l 2.5

l

e

c

T

2.0

8

D C

1.5

e

v

+

r 1.0

e

m

a r

t 0.5

r

e

t

% 0.0 d d 4 4 o o p p Genotype: WTe K/Oe WTB K/OB WTb K/Ob WTe K/Oe Genotype: WT K/O WT K/O WT K/O WT K/O 0 0 s s im im 1 1 m m r noner No CpG o comboo ODN 6 C none No CpG combo ODN Treatment: p p 6 C c c B E Treatment: n n B E 6 C D + + u u D B E 6 C D B E D

Expansion of OVA-specific Expansion of OVA-specific CD8+ T cells CD8+ T cells

d6 Measure OVA-specific d6 Measure OVA-specific CD8+ T cells CD8+ T cells

Genotype: WT K/O WT K/O WT K/O WT K/O Genotype: WT K/O WT K/O WT K/O WT K/O Treatment: none No CpG combo ODN Treatment: none No CpG combo ODN + +

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DEC-205 binds CpG ODN and facilitates What does the recognition of B-ODN up-take represent?

Some of the bacteria known to phosphorothioate - E.coli spp - Vibro spp  Prefers: - Pseudomonas spp  Class B ODN - Streptomyces spp  Phosphorothioated DNA  Enhances immune responses Phosphorothioation protects against restriction enzyme cleavage

 Clinically relevant, but is there biological relevance?

PNAS 108: 2963, 2011

How frequently do bacteria Biofilms are made of exogenous DNA phosphorothioate?

Ma et al. PLoS Pathogens, 2009: 5

PNAS 108: 2963, 2011

This frequency implies ~6 base cut site

How much thioation is required for DEC-205 Summary binding?

DEC-205 binds CpG ODN:  Facilitates the uptake of CpG ODN  Binding conserved between mouse and man, but there are species-preferences

1668 DEC-205 recognition of CpG ODN has biological outcome: Diester 1668 G-Ps-diester 1668  DC and B cell maturation impaired  Cytokine production impaired e.g. IL-12  Impaired CTL

DEC-205 recognizes modified DNA and may serve to distinguish self from bacterial DNA

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Acknowledgements Acknowledgements Melbourne University WEHI/Burnet Bill Heath Ken Shortman Jessica Li Yu (Alex) Kato Fatma Ahmet Melbourne University WEHI Gayle Davies Yu (Alex) Kato Ken Shortman Lucy Sullivan Gayle Davey David Vremec Andrew Brooks Scott Mueller Hae Young Park Mireille Lahoud David Tarlinton Jose Villadangos Antonia Policheni Jose Villadangos Jian-Guo Zhang Simone Meuter Soo San Wan Simone Meuter Nick Nicola Justine Mintern Susie Kitsoulis Burnet Institute Justine Mintern Chin-Nien Lee Steve Kent IRENE CAMINSCHI Steve Nutt Rob de Rose Burnet Institute Fatma Ahmet Andrew Brooks Dimitra Zotos Meredith O’Keffee Mireille Lahoud Lucy Sullivan Monash University Antonia Policheni WEHI Monash University Richard Berry Michel Nussenzweig Soo San Wan Steve Kent Jian-Guo Zhang Richard Berry Jamie Rossjohn Hidde Ploegh Susie Kitsoulis Rob de Rose Nick Nicola Jamie Rossjohn Meredith O’Keffee Michel Nussenzweig Chin-Nien Lee Hidde Ploegh

DEC205 is a receptor for CpG ODN DEC-205 prefers ssDNA

But not all CpG ODNs bind equally – the most potent CpG ODN in

humans also binds human DEC205 1

to the greatest degree 0.9

0.8

0.7 5'1668-bio

0.6 5'1668 plus Reverse-bio + reverse O.D. 0.5 5'1668 plus Forward-bio + forward 0.4

0.3

0.2

0.1

Hypothesis: Ability to bind DEC205 0 1 10 100 1000 enhances potency of ODN ng/ml 1668-bio

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