Modified IFN-  subtypes production and chemokine networks in the thymus during acute SIV infection, impact on thymopoiesis Jacques DUTRIEUX PhD. DUTRIEUX J. et al., AIDS Published online march 7th 2014 Institut Cochin INSERM U1016 Team « Cytokines and viral infections » Rémi CHEYNIER

HIV infection impacts on thymopoiesis  Thymopoiesis is rapidly affected during primary HIV infection (Hatzakis et al. 2000; Richardson et al. 2000; Douek et al. 2001; Dion et al. 2004)  Diminished thymocyte proliferation (Dion et al., 2004)  Consequence of IFN-  response ? 2 From Dion et al Intra-thymic proliferation

Acute HIV/SIV infection 3 VL IFN-  HumanRhesus macaque  In both humans and macaques : Plasma IFN-  peaks around day 10 post-infection (Stacey et al., 2009; Khatissian et al. 1996; Giavedoni et al. 2000; Abel et al. 2002; Bruel et al., 2014, Dutrieux et al. 2014). From Stacey et al. 2009

Acute SIV infection study SIVmac 251 Infection (50AID 50 ) IV Blood sampling D0, D3, D5, D7, D10, D12, D14 T-cell subsets analysis Thymic function analysis Plasma IFN-  measurement 24 rhesus macaques Necropsy D0 (3) and in acute phase (D3, D7, D10, D14) Thymus sampling IFN-  and chemokines mRNA quantifications and identification 4

Thymic modification in vivo /1014 Days post-infection * * Intra-thymic proliferation Intra-thymic proliferation decreased at day 10 post-infection Diminished intra-thymic proliferation correlates with plasma IFN-  concentration Intra-thymic proliferation decreased at day 10 post-infection Diminished intra-thymic proliferation correlates with plasma IFN-  concentration Intra-thymic proliferation (Fold change) p=0.017 r= Plasma IFN- 

Interferon  diversity 6 From Woelk et al IFN 

IFN  mRNA copies / 10 3 HPRT mRNA copies Days post-infection Number of expressed IFN-  subtypes ** Thymic production of IFN-  subtypes Day 0 IFN-  1 IFN-  2 IFN-  3 IFN-  4-6 IFN-  5 IFN-  7 IFN-  8 IFN-  9 IFN-  13 IFN-  14 IFN-  Day 3 IFN-  1 IFN-  2 IFN-  3 IFN-  4-6 IFN-  5 IFN-  7 IFN-  8 IFN-  9 IFN-  13 IFN-  14 IFN-  Day 14 IFN-  1 IFN-  2 IFN-  3 IFN-  4-6 IFN-  5 IFN-  7 IFN-  8 IFN-  9 IFN-  13 IFN-  14 IFN-  Diversified thymic IFN-  expression

In vitro differentiation of simian thymocytes Acidic purification 12 simian IFN-  Simian IFN-  subtypes transfected in CHO Flt3L OP9-hDL1 Frequency of mature cells Thymocyte proliferation 14 days R-sIL-7 DN thymocytes Healthy macaque thymus 8

IFN-  subtypes differentially inhibit thymopoiesis # Medium IFN-  1IFN-  2IFN-  3IFN-  4IFN-  5IFN-  6IFN-  7IFN-  8IFN-  9 IFN-  13IFN-  14 IFN-  12/15 Divided cells (%) Day 5 Day 9 Day 12 * * * * * * * * * IFN-  subtypes differentially inhibit thymocytes proliferation without affecting their differentiation capability 9

IFN-  subtypes differentially inhibit thymopoiesis # Medium IFN-  1IFN-  2IFN-  3IFN-  4IFN-  5IFN-  6IFN-  7IFN-  8IFN-  9 IFN-  13IFN-  14 IFN-  12/15 Divided cells (%) Day 5 Day 9 Day 12 In vivo-produced IFN-  subtypes demonstrate major anti-proliferative effect In vivo-produced IFN-  subtypes demonstrate major anti-proliferative effect 10 * * * * * * * * *

CCL25 CXCL12 CCL19 Chemokine-driven thymocyte migrations CCL19 11

CCL25 transcription in the thymus Days post-infection CCL25 mRNA copies / 10 2 HPRT mRNA copies * * 200µm C C M C Cortical expression Increased transcription by day 3 Cortical expression Increased transcription by day 3 CCL25mRNA DR 12

CCL19 transcription in the thymus Medullar expression Increased transcription by day 3 Medullar expression Increased transcription by day Days post-infection CCL19 mRNA copies / 10 2 HPRT mRNA copies ** 200µm CC M 13 CCL19mRNA DR

CXCL12 transcription in the thymus Expressed in subcapsular region Decreased transcription by day 3 Expressed in subcapsular region Decreased transcription by day 3 CXCL12 mRNA copies / 10 2 HPRT mRNA copies Days post-infection * 200µm C C M C C C CXCL12 DR 14

Diversified IFN-  production Thymic IFN-  subtypes Inhibit thymocytes proliferation Modified thymic chemokine network Diversified IFN-  production Thymic IFN-  subtypes Inhibit thymocytes proliferation Modified thymic chemokine network 15 CCL25 CXCL12Proliferation CCL19 In the thymus

16 CCL25 CXCL12Proliferation CCL19 In the thymus Diversified IFN-  production Thymic IFN-  subtypes Inhibit thymocytes proliferation Modified thymic chemokine network Diversified IFN-  production Thymic IFN-  subtypes Inhibit thymocytes proliferation Modified thymic chemokine network Accelerated thymopoiesis Increased thymic function Increased naive T-cell diversity Accelerated thymopoiesis Increased thymic function Increased naive T-cell diversity Eventually lead to the characteristic HIV-associated thymic exhaustion Eventually lead to the characteristic HIV-associated thymic exhaustion

Team Cytokines & Viral Infections Rémi CHEYNIER Véronique FABRE-MERSSEMAN Bénédicte DE MUYLDER Suzanne MORGADO Anne COUEDEL COURTEILLE Isabelle BOURGAULT-VILADA Magali RANCEZ Stéphanie BEQ Sandra ROZLAN Rosalie PONTE Vélislava TERZIEVA Amandine BERNARD Sandrine LOGEROT Raphaëlle PARKER Primate center Institut Pasteur Céline GOMMET Xavier MONTAGUTELLI Acknowledgements Team Antigen presentation by dendritic cells Agnès LEBON Facilities