The document summarizes a 2014 college intern's project to determine if calcium phosphate (CaP) nanoparticles can induce dendritic cell maturation. The goal was to see if CaP upregulates expression of cell surface proteins (MHC class I, II, CD80, CD86) on dendritic cells, which is important for T cell activation and producing potent vaccines. The results showed that CaP induced increased expression of these proteins on both mouse and human dendritic cells. Next steps would be to repeat the assays and test the nanoparticles' ability to activate T cells.

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