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.

Identification of secreting cells ELISPOT Intracellular cytokine staining Exocytosis assay These assays have flaws: They kill the cells being studied. Cell fixation and permeabilization interfere with downstream molecular work. They do not distinguish between pre-stored cytokines and secreted cytokine. 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.

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)

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

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

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

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

19.4% MIP-1  13.7% RANTES 16.4% MIP-1  Capture of cytokine secretion: Non-commercially available assays CD8 Cytokine

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

15 nm MNPs CD45 Targeted Qdots GAM CD45 Targeted Comparison of several nanoparticles CD45 + IL-2 MNPs Labeled anti-IL-2 Ab CD45 + IL-2 Qdot Non-Activated cells Activated cells ON QdotsInvitrogen Qdots15 nm MNPs CD8 IL-2

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

The cytokine capture assay can be multiplexed IL-2 IFN- 

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

Thanks to Wendy Fitzgerald Leonid Margolis