thiru sivakumaran - srtp 2011
TRANSCRIPT
![Page 1: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/1.jpg)
Antibody Conjugated Bead Stimulation of Toll-like Receptors
Thiru Sivakumaran Joaquin Madrenas
SRTP Final Presentation
![Page 2: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/2.jpg)
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
![Page 3: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/3.jpg)
A Primer on Toll-like Receptors
![Page 4: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/4.jpg)
Source: BS Park et al. Nature 000, 1-5 (2009)
![Page 5: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/5.jpg)
TLR2/6 Heterodimer:Diacylated Lipopeptides
TLR2/10 Heterodimer:Unknown
TLR2/1 Heterodimer:Triacylated Lipopeptides
![Page 6: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/6.jpg)
TLR2/6 Heterodimer:Diacylated Lipopeptides
TLR2/10 Heterodimer:Unknown
TLR2/1 Heterodimer:Triacylated Lipopeptides
![Page 7: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/7.jpg)
TLR2/6 Heterodimer:Diacylated Lipopeptides
TLR2/10 Heterodimer:Unknown
TLR2/1 Heterodimer:Triacylated Lipopeptides
![Page 8: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/8.jpg)
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
![Page 9: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/9.jpg)
TLR2/6 Heterodimer:Diacylated Lipopeptides
TLR2/10 Heterodimer:Unknown
TLR2/1 Heterodimer:Triacylated Lipopeptides
![Page 10: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/10.jpg)
The Experimental Question
![Page 11: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/11.jpg)
2 1 2 2 2 6 2 10
?
Can TLR2 signal independently of TLRs 1, 6, and 10?
![Page 12: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/12.jpg)
A Change in Approach
![Page 13: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/13.jpg)
Approach #1
CD4 extracellular domainTLR intracellular domain
Cytokines
Pam3Csk4
Approach #2
![Page 14: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/14.jpg)
Approach #3
![Page 15: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/15.jpg)
Our Approach:Antibody Conjugated Immunobeads
![Page 16: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/16.jpg)
2 1 2 2 2 6 2 10
?
Can TLR2 signal independently of TLRs 1, 6, and 10?
![Page 17: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/17.jpg)
2 1 2 2 2 6 2 10
We believe that it does
![Page 18: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/18.jpg)
Results
1) Confirmation of Antibody Bead Conjugation2) Immunobead Stimulation of PBMCs3) Dual Immunobead Stimulation of PBMCs4) Receptor Signaling
![Page 19: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/19.jpg)
Confirmation of Antibody-Bead Conjugation
![Page 20: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/20.jpg)
Immunobead Stimulated PBMCs: IL-6 Production
IL-6(pg mL-1)
Immunobead Configuration
![Page 21: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/21.jpg)
Immunobead Stimulated PBMCs: IL-6 Production
TNF-a(pg mL-1)
Immunobead Configuration
![Page 22: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/22.jpg)
Immunobead Stimulated PBMCs: IL-10 Production
IL-10(pg mL-1)
Immunobead Configuration
![Page 23: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/23.jpg)
Immunobead Stimulated PBMCs: IL-1beta Production
IL-1B(pg mL-1)
Immunobead Configuration
![Page 24: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/24.jpg)
Stimulation of TLR2 by Immunobeads is ReceptorEpitope Independent
TNF-a(pg mL-1)
Immunobead Configuration
![Page 25: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/25.jpg)
Signaling: TLR2 immunobead stimulation in PBMC Results in phosphorylation of ERK
![Page 26: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/26.jpg)
Dual Immunobead Stimulation of PBMCs: IL-6 Production
IL-6(pg mL-1)
Immunobead Configuration
![Page 27: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/27.jpg)
Dual Immunobead Stimulation of PBMCs: IL-10 Production
Immunobead Configuration
IL-10(pg mL-1)
![Page 28: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/28.jpg)
Dual Immunobead Stimulation of PBMCs: TNF-a Production
Immunobead Configuration
TNF-a(pg mL-1)
![Page 29: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/29.jpg)
Dual Immunobead Stimulation of PBMCs: TNF-a
![Page 30: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/30.jpg)
Cytokine Production by TLR10 Immunobead is not due to Endotoxin Contamination
![Page 31: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/31.jpg)
Signaling: pERK induced by TLR10 ImmunoBeads
![Page 32: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/32.jpg)
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:
![Page 33: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/33.jpg)
Summary
6) Demonstrated Preliminary evidence that TLR10 is capable of signaling independently
7) Ruled out endotoxin contamination of TLR10 immunobeads
We Have:
![Page 34: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/34.jpg)
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
![Page 35: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/35.jpg)
Future work
Publish a paper
![Page 36: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/36.jpg)
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
![Page 37: Thiru Sivakumaran - SRTP 2011](https://reader037.vdocuments.us/reader037/viewer/2022102806/5576386ed8b42a015c8b5167/html5/thumbnails/37.jpg)
Thank you, Dr. Madrenas