CaP Particle Presentation
- 1. CaP particle stimulation of dendritic cells for vaccine development Stephen Braunewell Wake Forest University 2014 College Intern Immunotope, Inc.
- 2. Cell mediated immunity begins with antigen processing • Endogenously biosynthesized antigen • CD8 T Cell Activation • Exogenous/extracellular antigen • CD4 T Cell Activation Parham 2006
- 3. DC activating T cell O’Hagan and Valiante 2003
- 4. My Project • The goal: Determine if Calcium phosphate nanoparticles are capable of inducing dendritic cell maturation • Readout: Expression of cell surface proteins on dendritic cell that are key to T cell activation. • MHC Class I, Class II, CD80, and CD86 molecules on cell surface. • Importance: Up regulation of these molecules are vital in the production of potent vaccines.
- 5. Procedure Harvest DC’s (DC2.4 or Primary) Feed into 12 well plate DC2.4 (M ouse) Prim ary (H um an) Add CaP Add CaP Flow cytometry and cytokine analysis (MagPix) Flow cytometry 2hrs, 4 hrs and 24 hrs later24 hrs later 1 x 10e6 cells per well 0.5 x 10e6 cells per well
- 6. CaP Induced Maturation of DC 2.4s DC 2.4: Ab 0 10 20 30 40 50 60 70 80 90 100 None 80uL 160uL LPS MFI DC 2.4: Kb 0 10 20 30 40 50 60 70 None 80uL 160uL LPS MFI DC 2.4: CD80 0 10 20 30 40 50 60 70 80 90 None 80uL 160uL LPS MFI DC 2.4: CD86 0 50 100 150 200 250 300 350 400 450 None 80uL 160uL LPS MFI
- 7. Primary DC: Class II 0 20 40 60 80 100 120 140 160 None 80uL of CaP Only 80uL of CaP +Peptide LPS MFI CaP Induced Maturation of Primary DCs Primary DC: Class I 0 20 40 60 80 None 80uL ofCaP Only 80uL ofCaP +Peptide LPS MFI Primary DC: CD86 0 20 40 60 80 100 120 None 80uL of CaP Only 80uL of CaP +Peptide LPS MFI
- 8. CaP Induced Maturation of Primary DCs
- 9. CaP Induced Cytokine Secretion
- 10. Conclusion/Next Steps • CaP nano particles have potential to be a useful adjuvant in the creation of more potent vaccines. • Next step would be to repeat this assay and see if there is statistical significance in the data. • Test nanoparticles using T cell activation assays. Lessons/Techniques Learned • Cell culture • Flow Cytometry • MagPix • Immune system information
- 11. Acknowledgments A huge thank you to everyone who made this experience possible: Immunotope Ramilia Philip, PhD Joseph Comber, PhD Aykan Karabudak Xiaofang Huang, PhD Tulin Morcol, PhD Hepatitis B Foundation Timothy Block, PhD Pamela Norton, PhD Peggy Farley, MBA Joan Block, RN, BSN