Ligand Screens in RAW Cells –

Cytokine Release

Ron TaussigAfCS Annual Meeting

Dallas, TX May, 2004

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.

Toll-like Receptor Signaling

Toll-like Receptor Ligands

P3C P2C LPS 848

IFN- Receptor Signaling

IL-6 Receptor Signaling

Cytokine Release Assay From Assay Development to Double Ligand Screen

Ligand Survey Double Ligand Screen

Single Ligand Screen

January February March April May June

TimeCourse

Excel

On Site Database (Dallas)

Web Display (AfCS)

Data Analysis

Combination Ligand SurveyExperimental plan (cytokine release)

134

134

137

137

• 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

Cytokine Release AssayLigand 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 interactionsb) ~0.5% (1.5% with relaxed criteria) of the combinations reveal

synergistic interactions4) Finalization of the protocol still needed (time course, dilutions,

culture conditions).

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

0.0

30.0

60.0

90.0

0 1 2 3 4 5

Time (Hours)

pg

/ml

Untreated

LPS

IFG

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

0.0

40000.0

80000.0

120000.0

160000.0

0 1 2 3 4 5

Time (Hours)

pg

/ml

Untreated

LPS

EST

P2C

IFG

EST + LPS

LPS + P2C

IFG + LPS

EST + P2C

EST + IFG

IFG + P2C

Cytokine Release – Standards

Cytokine Release – StandardsCytokine Lower Limit Upper LimitMo G-CSF (54) 7.8 32000Mo GM-CSF (73) 31.25 2000Mo IFN-g (34) 31.25 8000Mo IL-10 (56) 31.25 32000Mo IL-12(p40) (76) 31.25 32000Mo IL-12(p70) (58) 31.25 32000Mo IL-17 (72) 31.25 8000Mo IL-1a (53) 7.8 2000Mo IL-1b (19) 31.25 32000Mo IL-2 (36) 1.95 2000Mo IL-3 (18) 1.95 32000Mo IL-4 (32) 1.95 8000Mo IL-5 (52) 1.95 2000Mo IL-6 (38) 31.25 8000Mo KC (57) 1.95 2000Mo MIP-1a (77) 500 32000Mo RANTES (55) 31.25 2000Mo TNF-a (21) 31.25 32000

Dynamic Range

16,000-fold

64-fold

2,000-fold

Cytokine Release – Single Responses (TNFa)

Time Course of TNFα Secretion in Response to LPS Stimulation

Time Course of TNFα Secretion in Response to 2MA Stimulation

2MAC5AESTGMFHISIFAIFBIFGI1BI10IL4IL6ISOLPALPSMCFM1AMC1P2CP3CPAFPGE848S1PTGFUDPUTP

GC

SF

GM

CS

F

IFN

g

I10

IL12

p40

IL12

p70

IL17

IL1a

IL1b

IL2

IL3

IL4

IL5

IL6

KC

MIP

1a

RA

NT

ES

TN

Fa

Cytokine Release – Single Ligand Screen Summary

Single Ligand Screen Summary

StimulationStimulation (small)InhibitionLigand = Cytokine

GC

SF

GM

CS

F

IFN

IL1a

IL1b

IL2

IL3

IL4

IL5

IL6

IL1

0

IL1

2(p

40

)

IL1

2(p

70

)

IL1

7

KC

MIP

1a

RA

NT

ES

TN

Fa

ISOPGEC5ALPA2MAPAFUDPUTPS1PMCFTGFGMFI04I06I10IFAIFBIFGI1BLPSP2C848NL T T T

P3C ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Cytokine Release – Single Ligand Screen Summary

CompleteIn the week following the meeting:1) Curate the data 2) Display on the public website3) Additional ligands

2MALPS ISOUDPP2C I1B C5A I04 GMF I10 IFA IFB IFG I06 LPATGFMCFP3CPAFPGE2MALPSISOUDPP2CI1BC5AI04

GMFI10IFAIFBIFGI06

LPATGFMCF848PAFPGE

S1P

EYC040512 EYC040518 EYC040519

EYC040414 EYC040423 EYC040518

EYC040406 EYC040421 EYC040430 EYC040513

EYC040326 EYC040416 EYC040503

EYC040318 EYC040331 EYC040422

EYC040325 EYC040415 EYC040429 EYC040507

EYC040315 EYC040330 EYC040506

TrianglesCombinations = 36Single ligand = 9Control = 2

RectanglesCombinations = 32Single ligand = 12Control = 2

Ligand Screens Experimental Plan

Cytokine Release – Interactions Among Ligand Pairs

Cytokine Release – Interactions Among Ligand Pairs

Cytokine Release – Interactions Among Ligand Pairs

Cytokine Release – Interactions Among Ligand Pairs

IL-10

TNFa

Cytokine Release – Interactions Among Ligand Pairs

Cytokine Release – Interactions Among Ligand Pairs

G-CSF

Cytokine Release – Summary of Interactions Among Ligand Pairs

IL-6

Cytokine Release – Summary of Interactions Among Ligand Pairs

IL-10

Cytokine Release – Summary of Interactions Among Ligand Pairs

Mip1a

Cytokine Release – Summary of Interactions Among Ligand Pairs

Rantes

Cytokine Release – Summary of Interactions Among Ligand Pairs

TNFa

Cytokine Release – Summary of Interactions Among Ligand Pairs

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 datab) Identify and prioritize repeat assays

4) Curate the data 5) Display on the public website6) Higher data analysis7) Plan next generation of experiments