Role of Toll-like Receptors in the Activation of Natural Killer T (NKT) lymphocytes HOST PATHOGEN CROSS TALK Annecy, Les Pensières September 24-26, 2007 Institut Pasteur de Lille Inserm 547 (Prof M. Capron) «Innate Immunity and Immunoregulation»
Ag MHC I/II peptide TCR Ag presenting cell (APC) (dendritic cell, ) Conventional T lymphocyte (CD4 +, CD8 + ) Ag CD1 (glyco)lipid TCR APC Lipid-reactive T lymphocyte
Invariant Natural Killer T cells (inkt cells) NK1.1 Ly49 NKT * Express markers found on NK cells and on T lymphocytes TCR CD1d * Very conserved TCR Invariant TCR :V 14-J 1818 segment/ chains (inkts) APC * Recognized exogenous or self lipid Ags restricted by the CD1d molecules alpha-galactosylceramide l l ( -GalCer) as a canonical lli ligand ceramide Polar head (carbohydrate(s)) Glycosphingolipide * Rapid production of IFN- and/or IL-4 Strong impact on innate and acquired immune responses Benefical : infectious diseases, cancer Detrimental : auto-immune and inflammatory diseases
Factors involved in the activation/orientation of auto-reactive inkt cells during stress conditions IL-4 Flexibility IFN- APC/iNKT cell cross-talk NKT TCR CD1d Nature of the endogenous lipids APC Nature of the co-factors produced Stress (infection, inflammation)
Mechanisms by which inkt cells become activated during infection Exogenous (microbial) vs self lipid Ags Mycobacteria Leishmania Sphingomonas Ehrlichia Borrelia Exogenous Ag Il-12 (1) CD1d TcR Innate and adaptive responses pathogen (2) Innate sensors (TLR4) DC CD40 CD86 Endogenous Ag (still unknown)
Do diverse TLR signals in conventional DCs differentially stimulate inkt cells? 4 4 2, 5 7/8, 9 3 TLRs? TRIF APC NKT 0 peptidoglycan dsrnas ssrnas dscpg-rich DNA Cell surface Endosome/lysosome
TLR4, TLR7 and TLR9-stimulated DCs activate inkt cells TLR agonists + washing 48 hrs ELISA BMDC Sorted CD5 + NK1.1 + liver cells TLR member 9 7/8 4 2/1 2/6 3 5
inkt cell activation requires CD1d and soluble co-factors CpG stimulation and fixation with paraformaldehyde Co-factors? TLR7 TLR9 CD1d TCR Lipid(s)? inkt
IL-12 is not involved in inkt cell activation in response to DC/CpG agonists + Neutralizing Abs washing BMDC WT or IL-12p40 -/-
inkt cell activation in response to CpG ODN-treated DCs requires type I IFN Neutralizing Abs BMDC NKT cells (WT, IFNAR -/- )
IFN I TLR9? CD1d TCR inkt De novo synthesis of self ligands following TLR9 stimulation?
NB-DGJ N-butyl-deoxygalactonojirimycin GalNAcT Ceramide Cer Glucosyl Tf Glc-Cer Lactosylcer synthase Lac-Cer GlcNAcT Cer Glc-Cer Gal-Glc-Cer (Lac Cer) GA2 Gal T GM3 GM3 S igb3 Gb3 Lc3 o-series a-series Gangliosides (iso)globo-series (Neo)lacto-series inkt cell activation requires de novo synthesis of -linked GlcCer N-butyl-deoxygalactonojirimycin 48 hrs before CpG ODN stimulation
Lipids are generated in CpG ODN-stimulated DCs to activate inkt cells NS or CpG ODN + BMDC Lipid extraction Total lipids + BMDC 12 hrs Washing +/- IFN type I Same data with R848/DC
Charged glucosylseramide (s) is (are) involved in inkt cell activation NS or CpG ODN + BMDC Lipid extraction Neutral Total lipids + Charged BMDC 12 hrs Washing +/- IFN type I +/- base treated (NaOH): eliminates all lipids excepted GSLs +/- ceramide glycanase treated: eliminates GSLs
What happens in vivo? Can DC/CpG activate inkt cells in vivo? Functional relevance?
CpG ODN/DCs activate inkt cells in vivo NS/DCs -GC/DCs CpG ODN/DCs Gated TCR + /TT + liver cells NS/DCs -GalCer/DCs CpG ODN/DCs CD4 Isotype control IFN-
CpG ODN/DCs activate inkt cells in vivo to protect mice against B16 melanoma B16F10 (5 x10 5 ) Day 0 Day 14 NS/DCs -GalCer/DCs CpG ODN/DCs Sacrifice
Self lipids (charged -linked glucosylceramides) are generated in CpG ODN-stimulated DCs to activate inkt cells, in concert with IFN type I IFN I TLR9 CD1d TCR Charged GSL(s) inkt «NKT1» phenotype Paget et al. Immunity (in Press) Relevant during infection? Nature of the self glycolipid?
Relevance during viral/bacterial infection? DNA viruses (HSV, MCMV, ) Intracellular bacteria DAI cytosol DNA sensors Invading dsdna TLR9 endosome Type I INF Glycolipid synthesis Activation of inkt/nk cell pathway
Yellow Fever (YF) virus 17D activates inkt cells through DCs YF17D 1 MOI + washing 48 hrs ELISA BMDC Sorted inkt cells * *
Impact of TLR activation on glycolipid synthesis n=6 Ceramide (Cer) GlcCer Glc 1-1Cer Ugcg B4galnt1 GA2 GalNAc 1-4Gal 1-4Glc 1-1Cer LacCer B4galt6 Gal 1-4Glc 1-1Cer B3gnt5 LC3 GlcNAc 1-3Gal 1-4Glc 1-1Cer St3gal5 A3galt2 A4galt GM3 NeuNAc 2-3Gal 1-4Glc 1-1Cer igb3 Gal 1-3Gal 1-4Glc 1-1Cer Gb3 Gal 1-4Gal 1-4Glc 1-1Cer Ganglio-series (o, a, b,c) Charged GSLs Isoglobo-series Globo-series (Neo)lacto series Neutral GSLs
Ganglioside pathway St3gal5 St8sia1 St8sia5 LacCer GM3 GD3 GT3 Cer Cer Cer Cer B4galnt1 GA2 GM2 GD2 GT2 Cer Cer Cer Cer B3galt4 GA1 GM1a GD1b GT1c Cer Cer Cer Cer St3gal1/2/4 GM1b GD1a GT1b GQ1c Cer Cer Cer Cer St8sia5 GD1c GT1a GQ1b GP1c Cer Cer Cer Cer n = 6 o-series a-series b-series c-series St: sialyltransferase Glucose 1,3Gal 2,3NeuAc 2,8NeuAc 1,4GalNAc Towards the quest for the ligand(s)
Inserm 547 Oxford Univ F. Platt A. Speak Hôpital Necker/CNRS ML Leite de Moraes CNRS, Orléans C. Paget B. Ryffel T. Mallevaey J. Fontaine D. Torrès IPP/IPL C. Vendeville G. Barba-Spaeth C. Faveeuw J. Dubuisson