Ligand Screens in RAW Cells – Cytokine Release

1. Ligand Screens in RAW Cells – Cytokine Release Ron Taussig AfCS Annual Meeting Dallas, TX May, 2004

2. Cytokine Release Assay • Outputs for Macrophages – “End point” assay. • What are the responses to individual ligands? • How rich is the combinatorial response data set? • 23 ligands (look at all 23x23 combinations). • Approach: BioRad Luminex technology – assay release of 18 cytokines.

3. Toll-like Receptor Signaling

4. P3C P2C LPS 848 Toll-like Receptor Ligands

5. IFN-g Receptor Signaling

6. IL-6 Receptor Signaling

7. Time Course Data Analysis Excel On Site Database (Dallas) Web Display (AfCS) Cytokine Release Assay From Assay Development to Double Ligand Screen Single Ligand Screen Ligand Survey Double Ligand Screen January February March April May June

8. 137 134 134 137 Combination Ligand SurveyExperimental plan (cytokine release) • 4 experiments • 23 Ligands tested • All individually tested • All pairwise combinations • 1 Time point: 4 hours • 24 well plate • ~ 4 x105 cells/sample • Completion Time: 3 weeks

9. Cytokine Release AssayLigand Survey • 6 of the 18 cytokines evaluated in the assay gave no ligand-induced responses. • Only one of the 23 ligands tested could be eliminated from the analysis (all revealed some interaction with another ligand). • >4000 data points: • ~1.5% (5% with relaxed criteria) of the combinations reveal inhibitory interactions • ~0.5% (1.5% with relaxed criteria) of the combinations reveal synergistic interactions • Finalization of the protocol still needed (time course, dilutions, culture conditions).

10. Time Course of IL-1a Secretion in Response to Ligand Stimulation

11. Time Course of MIP-1α Secretion in Response to Ligand Stimulation

12. Cytokine Release – Standards

13. Cytokine Release – Standards

14. Cytokine Release – Single Responses (TNFa)

15. Time Course of TNFα Secretion in Response to LPS Stimulation

16. Time Course of TNFα Secretion in Response to 2MA Stimulation

17. GMCSF IFNg I10 IL12p40 IL12p70 IL17 IL1a IL1b IL2 IL3 IL4 IL5 IL6 KC MIP1a RANTES TNFa GCSF 2MA C5A EST GMF HIS IFA IFB IFG I1B I10 IL4 IL6 ISO LPA LPS MCF M1A MC1 P2C P3C PAF PGE 848 S1P TGF UDP UTP

18. Cytokine Release – Single Ligand Screen Summary

19. Single Ligand Screen Summary Stimulation Stimulation (small) Inhibition Ligand = Cytokine

20. Cytokine Release – Single Ligand Screen Summary Complete • In the week following the meeting: • Curate the data • Display on the public website • Additional ligands

21. Ligand Screens Experimental Plan

22. Cytokine Release – Interactions Among Ligand Pairs

23. Cytokine Release – Interactions Among Ligand Pairs

24. Cytokine Release – Interactions Among Ligand Pairs

25. Cytokine Release – Interactions Among Ligand Pairs TNFa IL-10

26. Cytokine Release – Interactions Among Ligand Pairs

27. Cytokine Release – Interactions Among Ligand Pairs

28. Cytokine Release – Summary of Interactions Among Ligand Pairs G-CSF

29. Cytokine Release – Summary of Interactions Among Ligand Pairs IL-6

30. Cytokine Release – Summary of Interactions Among Ligand Pairs IL-10

31. Cytokine Release – Summary of Interactions Among Ligand Pairs Mip1a

32. Cytokine Release – Summary of Interactions Among Ligand Pairs Rantes

33. Cytokine Release – Summary of Interactions Among Ligand Pairs TNFa

34. Cytokine Release – Double Ligand Screen Summary • Incubations are complete (Cell Lab) • Cytokine assays near complete (Antibody Lab) • First level data analysis • Quality of data • Identify and prioritize repeat assays • Curate the data • Display on the public website • Higher data analysis • Plan next generation of experiments