Cignal lenti nfat luciferase virus

Example 55

The ability of soluble, full-length anti-TREM2 antibodies Ab9, Ab14, Ab22, Ab45, and Ab65 to inhibit TREM2-dependent genes was evaluate using a luciferase reporter gene under the control of an NFAT (nuclear factor of activated T-cells) promoter.

A cell line derived from mouse thymus lymphoma T lymphocytes BW5147.G.1.4 (ATCC® TIB48™) was infected with mouse Trem2 and Dap12, and with Cignal Lenti NFAT-Luciferase virus (Qiagen). Soluble, full-length anti-TREM2 antibodies were added at increasing concentration to the cells. Cells were incubated for 6 hours at 37° C. and luciferase activity was measured using OneGlo Reagent (Promega).

The cells display tonic TREM2-dependent signaling due to either the presence of an endogenous ligand or to spontaneous receptor aggregation, which leads to TREM2 signaling.

The dotted line in FIG. 32 indicates the levels of TREM2 activity without stimulation.

As shown in FIG. 32, soluble, full-length anti-TREM2 antibodies Ab9, Ab14, Ab45, and Ab65 were able to inhibit tonic, TREM2-dependent gene expression. In contrast, soluble, full-length anti-TREM2 antibody Ab22 did not appear to block tonic TREM2 signaling (FIG. 32).

Example 33

The ability of antagonistic anti-Siglec-9 antibodies activate NFAT-dependent genes is evaluate using a luciferase reporter gene under the control of an NFAT (nuclear factor of activated T cells) promoter.

A cell line derived from mouse T lymphocytes BW5147.G.1.4 (ATCC® TIB48™) that express the ITAM motif containing co-receptor DAP12 and its ligand binding partner TREM2 is infected with human Siglec-9, and with Cignal Lenti NFAT-Luciferase virus (Qiagen). Luciferase signaling is activated by plate bound anti-TREM2 antibodies. Full-length and Fab fragment anti-Siglec-9 antibodies are either co-plated with the TREM2 antibodies or applied in solution. For plate binding, antibodies are applied at 10 μg/ml in DPBS on tissue-culture treated clear bottom white 96 well plates (100 ul/well), overnight at 4° C. Wells are rinsed three times with DPBS and subsequently plated at 100,000 cells/well in media with 1% serum. As a positive control for signaling, PMA (0.05 ug/ml) and ionomycin (0.25 uM) are added together. Cells are incubated for 6 hours and luciferase activity is measured by adding ONE-Glo™ reagent (Promega) to each well and incubating 3 min at RT on a plate shaker. Luciferase signal is measured using a BioTek plate reader.

Example 34

The ability of antagonistic anti-CD33 antibodies activate NFAT-dependent genes is evaluate using a luciferase reporter gene under the control of an NFAT (nuclear factor of activated T cells) promoter.

A cell line derived from mouse T lymphocytes BW5147.G.1.4 (ATCC® TIB48™) that express the ITAM motif containing co-receptor DAP12 and its ligand binding partner TREM2 is infected with human CD33, and with Cignal Lenti NFAT-Luciferase virus (Qiagen). Luciferase signaling is activated by plate bound anti-TREM2 antibodies. Full-length and Fab fragment anti-CD33 antibodies are either co-plated with the TREM2 antibodies or applied in solution. For plate binding, antibodies are applied at 10 μg/ml in DPBS on tissue-culture treated clear bottom white 96 well plates (100 μL/well), overnight at 4° C. Wells are rinsed three times with DPBS and subsequently plated at 100,000 cells/well in media with 1% serum. As a positive control for signaling, PMA (0.05 μg/ml) and ionomycin (0.25 uM) are added together. Cells are incubated for 6 hours and luciferase activity is measured by adding ONE-Glo™ reagent (Promega) to each well and incubating 3 min at RT on a plate shaker. Luciferase signal is measured using a BioTek plate reader.