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

2. Cytokine Release Assay 1)Outputs for Macrophages – “End point” assay. 2)What are the responses to individual ligands? 3)How rich is the combinatorial response data set? 4)23 ligands (look at all 23x23 combinations). 5)Approach: BioRad Luminex technology – assay release of 18 cytokines.

3. Toll-like Receptor Signaling

4. Toll-like Receptor Ligands P3C P2C LPS 848

5. IFN-  Receptor Signaling

6. IL-6 Receptor Signaling

7. Cytokine Release Assay From Assay Development to Double Ligand Screen Ligand SurveyDouble Ligand Screen Single Ligand Screen JanuaryFebruaryMarchAprilMayJune Time Course Excel On Site Database (Dallas) Web Display (AfCS) Data Analysis

8. Combination Ligand Survey Experimental plan (cytokine release) 134 137 4 experiments 23 Ligands tested All individually tested All pairwise combinations 1 Time point: 4 hours 24 well plate ~ 4 x10 5 cells/sample Completion Time: 3 weeks

9. Cytokine Release Assay Ligand Survey 1)6 of the 18 cytokines evaluated in the assay gave no ligand- induced responses. 2)Only one of the 23 ligands tested could be eliminated from the analysis (all revealed some interaction with another ligand). 3)>4000 data points: a)~1.5% (5% with relaxed criteria) of the combinations reveal inhibitory interactions b)~0.5% (1.5% with relaxed criteria) of the combinations reveal synergistic interactions 4)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

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. 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 GCSF GMCSFIFNgI10IL12p40IL12p70IL17IL1aIL1bIL2IL3IL4IL5IL6KCMIP1aRANTESTNFa

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: 1)Curate the data 2)Display on the public website 3)Additional ligands

21. Ligand Screens Experimental Plan

22. Cytokine Release – Interactions Among Ligand Pairs

25. IL-10 TNF 

26. Cytokine Release – Interactions Among Ligand Pairs

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

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

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

31. Mip1  Cytokine Release – Summary of Interactions Among Ligand Pairs

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

33. TNF  Cytokine Release – Summary of Interactions Among Ligand Pairs

34. Cytokine Release – Double Ligand Screen Summary 1)Incubations are complete (Cell Lab) 2)Cytokine assays near complete (Antibody Lab) 3)First level data analysis a)Quality of data b)Identify and prioritize repeat assays 4)Curate the data 5)Display on the public website 6)Higher data analysis 7)Plan next generation of experiments