1. Impact of glycosylated simian IL-7 on T cell homeostasis and thymic function in healthy Rhesus macaque          

2. sj/  TREC fold increase 1,0 2,0 3,0 -3,0 -2,0 -1,0 D63 Vs baselineD118 Vs D63 Thymic function Viral Load (Log 10 copies/  l) 0 1 2 3 4 5 6 7 8 020406080100120140160180 ARV IL-7 CD4 + T cells /  020406080100120140160180 ARV IL-7 Naïve CD4 + T cells /  ARV IL-7 0 1000 2000 3000 4000 35557595115 135155175195 0 1000 2000 3000 4000 5000 Recombinant IL-7 induces transient T cell count increase in SIV-infected Rhésus macaques under ART (Beq et al. J.I. 2006) Viral LoadCD4 Memory CD4 35557595115 135155 175195 0 0.2 0.4 0.8 1.0 0.6 ARV IL-7 HLA-DR HLA-DR+ CD4 + T cells

3. Macaques IL-7 C-C- 36C - 45 Non-glycosylated recombinant IL-7 is strongly immunogenic in Rhésus macaques (Beq et al. J.I. 2006)

4. Identification of RTE T cells in Rhesus macaque by flow cytometry SSC FSC CD3 CD4 CD95 CD28 Naives Memory effector CD31 RTE CD31-CD31+CD31high Naives CD8 sjTREC frequency CD3 CD8 CD31 RTE CD4 CD31-CD31+CD31high Naives CD95 CD28 Naïves Memory Effector sjTREC frequency

5. SSC FSC CD3 CD4 CD3 CD8 CD95 CD28 Naïves Memory Effector Caspase 3CD95 CD28 Naïves Memmory Effector CD31 Bcl-2 RTE CD31 Ki67 CD31 Bcl-2 CD31 Bcl-2 CD31 Effector CD4 deathNaïves CD4 survivalEffector CD8 survival Naïves CD4 ProliferationNaïves CD8 Proliferation Naive CD8 survival Memory CD8 survival CD31 RTE Identification of T cells subpopulation using a CyAn™ ADP 9 parameters, 7 colors, 6 on the same cell

6. 0 50 100 150 200 03691224487296168336 hours post injection 0 40 80 120 20 60 100 140 03691224487296168336 hours post injection 0 20 40 60 80 100 120 Single dose injections of r-sIL-7 gly induce the loss of CD127 expression on both CD4 and CD8 T cell CD8 Controls 0 50 100 150 200 0 50 100 150 200 % of initial CD127 frequency CD4

7. Single dose injections of r-sIL-7 gly induce repeated and sustained CD4 T cell counts increases * Undetectable anti-IL-7 antibodies Days R-sIL-7gly (ng/ml) 0 2 4 6 8 10 020406080100 1/2 life ~ 14h Fold increase Days post injection CD4 0 0,5 1 1,5 2 2,5 05101520250204060 * * IL-7 0,5 ng

8. Single dose injections of r-sIL-7 gly induce repeated and sustained CD4 T cell counts increases Naïve CD4 0 0,5 1 1,5 2 2,5 05101520250204060 Fold increase IL-7 Memory CD4 Days post injection 0 1 2 3 4 05101520250204060 Fold increase IL-7 Fold increase RTE CD4 0 1 2 3 4 5 0204060 IL-7 Days post injection

9. Single dose injection of r-sIL-7gly induces transient CD4 proliferation Ki-67 in RTE CD4 0 10 20 30 40 50 0 204060 % Ki-67 Cells IL-7 Ki-67 in Memory CD4 0 5 10 15 20 25 30 35 40 0204060 % Ki-67 Cells IL-7 Ki-67 in Naive CD4 0 5 10 15 20 25 0204060 % Ki-67 Cells IL-7 Days post injection

10. Single dose injection of r-sIL-7gly transiently induces T cell survival BCl-2 MFI 0 Naive CD4 20 40 60 80 100 120 0204060 Memory CD4 Days post injection 0 20 40 60 80 100 120 0204060 BCl-2 MFI IL-7 Days post injection RTE CD4 0 20 40 60 80 100 120 140 160 0204060 BCl-2 MFI IL-7

11. Single dose injection of glycosylated recombinant simian IL-7 induces thymic function SjTRECs per 10 5 cells 0 500 1000 1500 2000 2500 3000 D0D1D2D3D4D7D14D45 sjTRECs Days post injection IL-7 Ratio 0 5 10 15 20 D0D1D2D3D4D7D14D45 Days post injection sj/  TRECs IL-7

12. 0 200 400 600 800 1000 1200 1400 1600 CD4 /mm3 time post injection D0 h6h9h0h6h9h0h6h9 D7D14 D9 D16D2D4D18 D22 * Undetectable anti-IL-7 antibodies 0 20 40 60 80 100 120 time post injection IL-7 CD127 Fold increase D0 h0h6h9h0h6h9h0h6h9 D7D14 D9 D16D2D4D18 D22 * * * Repeated injection of r-sIL-7gly induces a transient loss of CD127 expression and an increased T cell count time post injection 0 2000 4000 6000 8000 10000 12000 14000 pg/ml D0 h0h6h9h6h9h0h6h9 D7D14 D9 D16D2D4D18 IL-7 D22 IL-7 CD127 CD4 counts

13. Conclusions As in human, CD31 expression on naive T cell defines Recent Thymic Emigrant subset Glycosylated r-sIL-7 is not immunogenic in vivo Glycosylation increases r-sIL-7 in vivo 1/2 life (14h) Single dose injections of r-sIL-7gly induce transient internalization of the IL-7R with rapid recovery (4-7 days) r-sIL-7gly induces sustained increase of all T cell subsets by at least 3 mechanisms: -Increased Proliferation (Ki-67) -Increased Survival (Bcl-2) -Increased Thymic output (sj/  TREC ratio)     

14. CYTHERIS S.A. Brigitte Assouline Iann Rancé Michel Morre Animalerie Centrale (I.P.) Pascal Lavedan Xavier Montagutelli Unité des Virus Lents (I.P.) David Gautier Clémentine Schilte Rémi Cheynier