A universal nanoparticle cell-secretion capture assay for the study of HIV-1 infected tissues.

1. A universal nanoparticle cell-secretion capture assay for the study of HIV-1 infected tissues. A new tool to identify cytokine secreting cells Wendy Fitzgerald and Jean-Charles Grivel The Eunice Kennedy-Shriver National Institute of Child Health and Human Development, Bethesda, MD.

2. Identification of secreting cells Intracellular cytokine staining ELISPOT Exocytosis assay These assays have flaws: They kill the cells being studied. They do not distinguish between pre-stored cytokines and secreted cytokine. Cell fixation and permeabilization interfere with downstream molecular work. Elispot, doesn’t provide information on cell phenotype. Exocytosis is limited to certain cell types and doesn’t reveal the nature of the cytokine secreted.

3. The ideal assay: Captures the secreted cytokine before it diffuses away from the producing cells. Is compatible with cell isolation and culture. Some assays have been developed: Agarose cell encapsulation (One cell systems) Requires dedicated instrumentation to encapsulate the cells Bi-specific antibody affinity matrix (Miltenyi Biotech)

4. Cell surface anchor Cytokine of interest Cytokine detection antibody Nanoparticle-based affinity matrix Cell surface specific antibody Nanoparticule Cytokine capture antibody Functionalized Magnetic Nano Particles: MNPs

5. 20 minutes Magnetic separation Principle for the construction of an affinity matrix Cell surface targeting Ab Secreted protein capture Ab + 10-20 minutes ! Cell and cytokine bi-specific affinity matrix

6. Nanoparticles can be used to form an affinity matrix on the cells surface CD3+ Isotype-MNPs CD3-MNPs Anti-CD3 labeled NC Isotype control CD3 surface label CD3 surface label CD3 Surface staining CD3+ IsotypeMNPs CD3-MNPs

7. Comparison of capture assay, intracellular cytokine staining and commercial secretion assay Capture assay Intracellular assay Commercial assay 12% 9.5% 20% IL-2 14.7% 18.7% 19.4% IFN-g CD8

8. Capture of cytokine secretion: Non-commercially available assays MIP-1a MIP-1b RANTES 16.4% 13.7% 19.4% Cytokine CD8

9. Separation on centrifugal concentration units Pall Nanosep 300K : Pore opening ± 35nm Underivatized nanoparticles are not retained IgG Antibodies are not retained Complexed nanoparticles are retained Allows the use of non-magnetic nanoparticles, such as Qdots

10. Comparison of several nanoparticles Qdots GAM CD45 Targeted 15 nm MNPs CD45 Targeted Labeled anti-IL-2 Ab Labeled anti-IL-2 Ab CD45+ IL-2 Qdot CD45+ IL-2 MNPs 15 nm MNPs ON Qdots Invitrogen Qdots IL-2 CD8 Non-Activated cells Activated cells

11. Qdots allow simultaneous cell labeling and cytokine capture Cell labeling Cytokine capture 11% IL-2 CD45 (surface) CD8 QDot 620 (IL-2/CD45 QDot 620)

12. The cytokine capture assay can be multiplexed IL-2 IFN-g

13. Conclusions We have designed a simple, fast and inexpensive method to capture and analyze cell secretion by flow cytometry. We have measured Il-1a, Il-1b,IL-2, IL-17, MIP-1a, MIP-1b, RANTES, TNF-a, TGF-b, IFN-g This method allows the detection on virtually any secreted protein on any cell provided that antibodies are available. Flexibility in the choice of cell targeting reagents. Qdots can be used to form the affinity matrix allowing the simultaneous detection of the targeted population and the secreted protein. The capture assay can be multiplexed for the detection of several cytokines at once.

14. Thanks to Leonid Margolis Wendy Fitzgerald