Antibody Conjugated Bead Stimulation of Toll-like Receptors

Thiru Sivakumaran Joaquin Madrenas

SRTP Final Presentation

Outline

I) A Primer on Toll-like Receptorsa) What are they? b) Why are they important?

II) The Experimental Question

II) A Novel Approach to TLR Stimulation in PBMCsa) Motivation and Methodologyb) Advantages and Disadvantages

III) What we founda) Results

IV) Implications and Future Work

A Primer on Toll-like Receptors

Source: BS Park et al. Nature 000, 1-5 (2009)

TLR2/6 Heterodimer:Diacylated Lipopeptides

TLR2/10 Heterodimer:Unknown

TLR2/1 Heterodimer:Triacylated Lipopeptides

TLR2/6 Heterodimer:Diacylated Lipopeptides

TLR2/10 Heterodimer:Unknown

TLR2/1 Heterodimer:Triacylated Lipopeptides

TLR2/6 Heterodimer:Diacylated Lipopeptides

TLR2/10 Heterodimer:Unknown

TLR2/1 Heterodimer:Triacylated Lipopeptides

Outline

I) A Primer on Toll-like Receptorsa) What are they? b) Why are they important?

II) The Experimental Question

II) A Novel Approach to TLR Stimulation in PBMCsa) Motivation and Methodologyb) Advantages and Disadvantages

III) What we founda) Results

IV) Implications and Future Work

TLR2/6 Heterodimer:Diacylated Lipopeptides

TLR2/10 Heterodimer:Unknown

TLR2/1 Heterodimer:Triacylated Lipopeptides

The Experimental Question

2 1 2 2 2 6 2 10

?

Can TLR2 signal independently of TLRs 1, 6, and 10?

A Change in Approach

Approach #1

CD4 extracellular domainTLR intracellular domain

Cytokines

Pam3Csk4

Approach #2

Approach #3

Our Approach:Antibody Conjugated Immunobeads

2 1 2 2 2 6 2 10

?

Can TLR2 signal independently of TLRs 1, 6, and 10?

2 1 2 2 2 6 2 10

We believe that it does

Results

1) Confirmation of Antibody Bead Conjugation2) Immunobead Stimulation of PBMCs3) Dual Immunobead Stimulation of PBMCs4) Receptor Signaling

Confirmation of Antibody-Bead Conjugation

Immunobead Stimulated PBMCs: IL-6 Production

IL-6(pg mL-1)

Immunobead Configuration

Immunobead Stimulated PBMCs: IL-6 Production

TNF-a(pg mL-1)

Immunobead Configuration

Immunobead Stimulated PBMCs: IL-10 Production

IL-10(pg mL-1)

Immunobead Configuration

Immunobead Stimulated PBMCs: IL-1beta Production

IL-1B(pg mL-1)

Immunobead Configuration

Stimulation of TLR2 by Immunobeads is ReceptorEpitope Independent

TNF-a(pg mL-1)

Immunobead Configuration

Signaling: TLR2 immunobead stimulation in PBMC Results in phosphorylation of ERK

Dual Immunobead Stimulation of PBMCs: IL-6 Production

IL-6(pg mL-1)

Immunobead Configuration

Dual Immunobead Stimulation of PBMCs: IL-10 Production

Immunobead Configuration

IL-10(pg mL-1)

Dual Immunobead Stimulation of PBMCs: TNF-a Production

Immunobead Configuration

TNF-a(pg mL-1)

Dual Immunobead Stimulation of PBMCs: TNF-a

Cytokine Production by TLR10 Immunobead is not due to Endotoxin Contamination

Signaling: pERK induced by TLR10 ImmunoBeads

Summary

1) Established a Novel Method for Studying Toll-Like Receptors in Human Cells.

2) Demonstrated that TLR2 is Capable of Signaling Independently of TLR1, 6, and 10

3) demonstrated that this Signaling is TLR2 Receptor Epitope Independent

4) Shown that Heterodimer Binding in This system is in a cis configuration, in agreement with the literature

5) Shown that ligation of tlr2 complexes induces pERK

We Have:

Summary

6) Demonstrated Preliminary evidence that TLR10 is capable of signaling independently

7) Ruled out endotoxin contamination of TLR10 immunobeads

We Have:

implications

Receptors that modulate innate immunity are excellentTherapeutic targets

Example: use of Helminth Derivative Downregulates TLR4 and halts Sepsis! Nature Immunology 2011

Not only infection: neoplastic processes, autoimmune Disease

Knowledge of receptor signaling is the first step to Formulating efficacious treatments for diseases with High morbidity and/or mortality

Future work

Publish a paper

Acknowledgements

Madrenas Lab:

Luan ChauThu Chau

Darah ChristieTheresa Fernandes

Holly LemonCynthia Liu

Peter Mitsopolous

SRTP COMMITTEECIHR

Schulich School of Medicine and Dentistry

…And of Course

Thank you, Dr. Madrenas