1; Nikon). The light source was a 488 nm solid-state laser (Sapphire 488-30; Coherent, Dieburg, Germany). Between 2 × 105 and 5 × 105 CHO cells were seeded on glass cover slips 2–3 days before the experiments. MK-1775 research buy Immediately before Ca2+ imaging, the cells were incubated with the particular concentration of fusion proteins in 50 μl culture medium and washed afterwards with culture medium with 10 mm HEPES added. Glass cover slips were mounted on the stage of an Olympus IX 70 microscope equipped with a 20 × (UApo/340, N.A. 0·75) objective in a self-made

recording chamber, which allowed a complete solution exchange < 1 second. In parallel, T cells were loaded at 22–23° for 30 min with 2 μm fura-2/acetoxymethyl ester (AM) (Invitrogen) in culture medium with 10 mm HEPES added, washed with fresh medium, and immediately used. T cells

were then added and cells were alternately illuminated at 340 and 380 nm with the Polychrome IV monochromator (TILL Photonics, Gräfelfing, Germany) and with an infrared light source using SP 410 as excitation filter and DCLP 410 as dichroic mirror. The fluorescence emissions at λ > 440 nm (LP 440) were captured with CH5424802 in vitro a CCD camera (TILL Imago), digitized, and analysed using TILL Vision software. Ratio images were recorded at intervals of 5 seconds. In some experiments thapsigargin (TG, 1 μm) was used to completely empty the stores. Excel, Igor Pro and TILL Vision were used for data analysis. An unpaired, two-sided Students t-test was used to test for significance. All fusion proteins were generated as single chain molecules to prevent any false pairing or degradation (Fig. S1). The extracellular domains of CD80 and CD86 were cloned at the N-terminal end of the scFv anti-CD33 to ensure correct binding to their respective receptors.52

Soluble proteins were produced in HEK-293 cells by transient gene expression with a yield of 0·5–2 mg total protein/l of cell culture supernatant, purified by IMAC and checked by Coomassie and Western blot analysis for purity and integrity. Proper binding for all fusion proteins was tested by enzyme-linked immunosorbent assay on recombinant CD33 antigen (data not shown) and flow cytometry Evodiamine (Fig. S2) on either CD33-transfected CHO or Jurkat T cells. Binding of the scFv anti-CD33 was not altered in any of the fusion proteins when compared with the parental scFv anti-CD33. The scFv anti-CD3 and the extracellular domains of CD80 and CD86 showed a moderate to weak binding affinity to their respective receptors. The dscFv anti-CD3/anti-CD19 were used as control. The dscFv anti-CD33/anti-CD3 construct induced proliferation of naïve T cells in the presence of the CD33 antigen in a dose-dependent manner (Fig. 1).