Mechanisms of extracorporeal photochemotherapy-induced tolerance : Mouse model of CHS
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1 Mechanisms of extracorporeal photochemotherapyinduced tolerance : Mouse model of CHS Olivier HEQUET Marc VOCANSON Aurélie GUIRONNET PAQUET Deborah ARNAUD INSERM U1111 CIRI Team leader: Prof. JF. NICOLAS SFH : ECP symposium Vendredi 12 décembre 214 APHERESIS UNIT Centre Hospitalier LyonSud LYON France
2 CHS pathophysiology Mouse Ear Swelling Test (MEST) CD8+ Teff cells are mandatory for the development of skin allergy Sensitization + 5d Challenge Skin inflammation (ear swelling, histology, PCR,FACS) Specific immune responses (LN, Facs, PCR, functional assays) Strong haptens (DNFB, OXA) Strong haptens Ear swelling (µm) CD4+ T celldeficient mice MHC class II/ (C57BL/6) anticd4 mab treatment Normal C57BL/6 or BALB/c mice CD8+ T celldeficient mice MHC class I/ (C57BL/6) anticd8 mab treatment Days after challenge CD8+ T cells are effector cells CD4+ T cells comprise regulatory T cells Major production of IFNg mainly CD8+ T cells Cytokine IFNg IL4 IL17 SFC / 16 cells
3 Infusion of ECPtreated DC before DNFB sensitization hampers the priming of CD8+Teff and the development of CHS reaction ECPDNBSDC UVA/8MOP hapten DC infusion 5x16 i.v. +5 days Sensitization + 5d Challenge Ear swelling UVADC DNBSDC = = DNFBspecific T cells (IFNg ELISPOT ) DNFB Infusion Infusion CHS response (48 hrs) DNBSDC ECP DNBSDC UVADC Ear swelling (%) ns DNBSDC ECPDNBSDC UVADC CD8+T cell response (IFNg) IFNg SFC/ 16 CD8+ T cells ns ECP treatment allows for a robust tolerance in the model of CHS to DNFB 3
4 Infusion of ECPtreated DC confer tolerance in Tregdeficient mice ECPDNBSDC UVA/8MOP hapten DC infusion 5x16 i.v. Sensitization + 1 day +5 days + 5 days DNFBspecific T cells (ELISPOT) AntiCD4+ mab Depletion C57BL/6 mice Abeta/ mice DNFB Infusion ECPDNBS DC ECPDNBSDC ECP DNBSDC Recipient wt wt wt + wt + MHC II / MHC II / AntiCD4+ mab CD8+T cell response (IFNg) IFNg SFC/ 16 CD8+ T cells Potent tolerance in mice deficient in CD4+ Tregs
5 CD8+TECP cells complement CD4+TECP cell subset to confer ECP tolerance UVA/8MOP hapten ECPDNBSDC DC infusion 5x16 i.v. Purified CD4+ or CD8+T cells (spleen) +5 days Adoptive transfert 51x16 i.v. +1 day Sensitization + 5d DNFBspecific T cells (ELISPOT) Recipient C57BL/6 DNFB Transfert to 2nd recipient CD8+ cells CD4+ cells CD4+ and CD8+ cells CD8+T cell response (IFNg) IFNg SFC/ 16 CD8+ T cells CD8+ T cells collected from the spleen of infusedanimals confer Agspecific tolerance upon adoptive transfer, demonstrating that they are endowed with suppressive activities
6 CD8+ECP T cells hampered the priming of CD8CHS effector cells upon adoptive transfer DC infusion 5 days 1st recipient wt CD45.2+ CD8+T cell purification (spleen) iv transfer 2nd recipient Congenic Ly5a CD45.1+ CD8+ T cell response: DNFB 5 days CD8+TECP reactivation + sensitization CD8+T effchs primary activation dlns FACS analysis ECPDNBSDC UVA/8MOP hapten CD8+ T cell response to DNFB CD8+ TeffCHS CD8+TECP DNBSDC = number of KI+ IFN + CD8 T cells p<.1 p<.1 DNBSDC ECPDNBSDC p<.1 DNBSDC ECPDNBSDC
7 Phenotypic analysis of CD8+ T cell response upon infusion of ECPtreated DC Infusion DC infusion 5x16 i.v. + 5d DNFBspecific T cells (Proliferation (FACS) DNBSDC 33% ECPDNBSDC 25% Generation of CD8+ T cells (FACS) UVA/8MOP DNBS ECPDNBSDC Activation Ki67 Cytotoxicity DNBSDC = 1 CD122 1 Icos 1 PD1 1 CD44 1 CD17a < < ECPDNBSDC DNBSDC < <
8 Conclusions ECPtreament hinders T cell priming and promotes CD8+ T ECP cells endowed with apreciable effector properties (cytotoxicity). CD8+ T ECP cells confer tolerance in CHS model by preventing the priming of new T eff CHS cells and their differenciation in TEM. CD8+ T ECP persist 1 month after generation and reactivation, suggesting potential maintenance of ECPinduced tolerance. Perspectives Important issues to uncover: Which mechanisms for tolerance? à à No IL1, negative costimulation (PD1, RANK ), Qa1restricted CD8+Tsupp?, Trail (sepsis model)?, transfer of regulatory mirna? Characterise phenotype and function of ECP DC Mechanisms are common to other strategies of tolerance? Feasibility of strategies aiming at reinducing tolerance in patients
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