核酸アジュバントによる 樹状細胞活性化の分子メカニズム

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1 ワクチンフォーラム 2010 September 14, 2010 核酸アジュバントによる 樹状細胞活性化の分子メカニズム 理研 免疫 アレルギー科学総合研究センター生体防御研究チーム改正恒康 Homepage: index.html

2 Nucleic acid-sensing receptors Virus Plasma membrane Endosome Virus Virus infected cell dsdna ssrna dsrna dsrna TLR7 TLR8 (human) TLR9 Toll-like receptor(tlr) CpG DNA CpG TLR3 AIM2 DAI/Zbp1 MDA5 LGP2 RIG-I-like receptor (RLR) 5 -triphosphate RNA PPP-5 RIG-I

3 Conventional and plasmacytoid DC Spleen (whole) Bone marrow B220 Spleen (CD11c+ by MACS) Plasmacytoid DC Conventional DC CD11c

4 Plasmacytoid DC PDC expresses nucleic acid sensing TLRs, TLR7 and TLR9 exclusively among pathogen sensors. In response to TLR7/9 stimuli, pdc can produce vast amounts of type I IFNs including IFN- and IFN-b. PDC (B220 + CD11c + ) Endosome TLR7/9 Conventional DC (cdc) (B220 - CD11c + ) TLR7/9 IFN- IL-12 IFN- IL-12

5 In human Human DC subsets TLR4 TLR8 TLR7 TLR9 MDC PDC IL-12 IFN-

6 Type I IFN induction through TLR7/9 signaling is critical not only for antiviral immunity but also for pathogenesis of autoimmune diseases. Virus DC CpG Self DNA with CpG motif TLR7/9 Anti-DNA Ab Self RNA Type I IFN Anti-RNP Ab Antiviral immunity Immune cell activation Autoimmunity

7 PDC PDCspecific pathway Endosome IRF7 TLR7/9 MyD88 NF-kB Nucleus P P IFN- IL-12

8 IKK is critical for TLR7/9-induced type I IFN production through the association with and activation of IRF-7 in pdc. (Hoshino et al. nature 2006.) PDC PDCspecific pathway Endosome IRF7 IKK TLR7/9 MyD88 IKKb NF-kB IkB Nucleus P P IFN- IL-12

9 DOCK2 The Rac activating molecule at the downstream of neutrophil GPCR Chemotactic factor GPCR Rac Activation DOCK2 Translocation To the membrane PIP3 Gbg Gbg G GDP G GTP Actin reorganizatiion DOCK2 PI3K

10 DOCK2 is required for TLR7/9-mediated type I IFN induction in pdc Serum cytokine level after injection of TLR7/9 agonists Cytokine production from TLR7/9-stimulated pdc Gotoh K et al. J Exp Med 2010,207:

11 DOCK2-mediated Rac activation is critical for IKKα activation in pdcs. Impairment of IRF-7 nuclear translocation and IKK phosphorylation induced by TLR9 stimuli Akt, ERK, JNK, p38, and IRAK-1 activation is intact. Gotoh K et al. JEM 2010,207:

12 PDC DOCK2 regulates TLR7/9-induced IRF-7 activation through IKK DOCK2 TLR7/9 IRF7 IKK MyD88 IKKb NF-kB IkB Nucleus P P IFN- IL-12

13 Effects of IKK inhibitor on human pdc 関西医科大学尼川龍一先生 伊藤量基先生との共同研究 R. Miyamoto, et al. Arthritis Research Therap, in press.

14 In human Human DC subsets TLR4 TLR8 TLR7 TLR9 MDC PDC IL-12 IFN-

15 IKK 阻害剤 (BAY-11) は TLR7/9 刺激による pdc からの I 型 IFN 産生誘導を阻害する CpG 2216 (TLR9) Loxoribine (TLR7) 3000 IFN- (pg/ml) <10 TNF- (pg/ml) Cf. IC 50 (for IkB phosphorylation)=10mm (M) IKK inhibitor (concentration) (M)

16 BAY-11/ スタチンは pdc の IRF7 の核内移行を阻害する

17 B220 CD4 樹状細胞サブセットとその特性 Plasmacytoid DC (pdc) CD8 - CD4 + cdc CD8 - CD4 - cdc CD8+ conventional DC (cdc) 47.1 CD11c TLR7/9 expression Type I IFN production 22.2 CD8 9.9% TLR3 expression Crosspresentation In vivo splenic DC

18 Xcr1 is selectively expressed in CD8 + DCs Common name Probe ID pdc CD8 + DC CD8 - DC Ccr _at _at _at Ccr _at _at _at Ccr _at Ccr _a_at _at Ccr _a_at _x_at Ccr _a_at Ccr _at Ccr _at _a_at _a_at Ccr _x_at _at _at Cx3cr _at _at Cxcr _at Cxcr _at Cxcr _a_at _at Xcr _at

19 Human peripheral blood DCs are divided into three subsets BDCA3 Human DCs from peripheral blood Lineage (CD3, CD14, CD16, CD19, CD56) negative & HLA-DR positive BDCA3 + DC BDCA3 + DC pdc pdc BDCA3 - DC BDCA3 - DC BDCA4 CD11c

20 ND ND Human XCR1 is selectively expressed in BDCA3 + DC 1x10-3 1x10-4 1x10-5 1x10-6 1x10-7 1x10-8 1x10-9 XCR1 TLR3 CLEC9A 1x10-5 1x10-6 1x10-7 1x10-8 1x10-3 1x10-4 1x10-5 1x10-6 1x10-7 1x10-8 RT-PCR

21 BDCA3 BDCA3 + DCs dominantly migrate to XCL1 (%) of input cells Input Migrated cells Bulk DCs XCL1 (ng/ml) CCL3 (100ng/ml) CXCL12 (500ng/ml) 4.2% 1.7% 3.4% 19% 46% 1.4% 1.9% 40% 55% 1.3% 96% 0.9% 92% 0.5% 79% 0.3% 53% 0.3% 98% 38% 58% CD11c Migrated cells BDCA3 + DC BDCA3 - DC pdc CCL3 CXCL12 XCL1 (ng/ml)

22 XCL1 is mainly produced by NK cell CD11c - CD11c + B220 + CD11c + B220-CD8 + CD11c + B220-CD8 - CD3e - CD19 - CD11c - CD11c + B220 + Gr-1 - Gr-1 + DX5 - DX5 + NK1.1 - NK1.1 + CD11b - CD11b low CD11b high NK1.1 - PDCA1 - NK1.1 - PDCA1 + NK1.1 + PDCA Relative expression

23 XCL1 (ng/ml) Serum XCL1 levels were decreased in NK cell depleted mice 2.0 Serum XCL1 levels Normal rabbit serum Anti-asialo GM1

24 Relative expression CD8 +, but not CD4 +, T cells increase Xcl1 expression upon activation 2000 NK IL-2 CD4 + T IL-2 CD4 + T anti-cd3e/cd28 CD8 + T anti-cd3e/cd Hours after stimulation

25 T B CD8 + T Human CD8 + T cell and NK cell also can express XCR1 ligands Xcl1 XCL1 (pg/ml) CD4 + T CD8 + T B NK CD4 + T CD8 + T Xcl2 NK CD4 + T medium Anti-CD3/28 PMA+IONO medium Anti-CD3/28 PMA+IONO medium IFN- IL-12 IL-2 (10) IL-2 (100) >1500 B NK Relative expression medium IL-4+anti-IgM LPS n.d. n.d

26 NK XCL1/XCR1 in innate and adaptive CTL responses IL-2 CD8 + T CD3/28 XCL1 C. Yamazaki et al. BBRC, 397:756,2010. XCR1 CD8 + DC BDCA3 + DC