Anti tigit antibodies

Sorted CD4⁺CD45RA^–CXCR5⁺ cTfh cells were stimulated with anti-CD3/anti-CD28 antibodies (each 0.5 μg/mL) for total 4 days. Sorted autologous CD20⁺CD27⁺ memory B cells were stimulated with CD40L (0.1 μg/mL) plus IL-21 (20 ng/mL) and anti-TIGIT antibodies (catalog 15-9500-82, MBSA43) or an equivalent amount of mouse IgG1 kappa isotype control (catalog 16-4714-82, P3.6.2.8.1) (all from eBioScience). After 2 days, B cells were washed twice and cocultured with cTfh cells at a ratio of 1:1 for 2 days. In some experiments, nucleofected B cells with siRNAs instead of anti-TIGIT antibodies were used to knock down TIGIT expression. T cells were labeled using a CellTrace Violet Proliferation Kit (Invitrogen, Thermo Fisher Scientific), and their proliferation was determined on a BD LSR Fortessa flow cytometer. Supernatants were collected, and IFN-γ and IL-17 production was measured by ELISA (R&D Systems).

The transplanted cardiac grafts were collected and fixed in 4% paraformaldehyde, embedded in paraffin, sliced into 4-mm-thick sections, and then stained by hematoxylin and eosin (H&E) to assess the severity of rejection. The slices are photographed under a light microscopy (Olympus Corporation, Hachioji, Tokyo, Japan).
The spleen samples of B6 recipient mice were collected, grinded, and filtered through a 100-mesh screen to obtain a homogeneous cell suspension. The red blood cells were lysed by red blood cell lysis solution (Beyotime Biotechnology, Shanghai, China), and then the splenocytes were washed, centrifuged, suspended, and analyzed with fluorescent-conjugated anti-CD4, anti-CD25, anti-Foxp3, and anti-TIGIT antibodies (eBioscience, San Diego, California, USA) by flow cytometry.
The expression levels of IFN-γ; tumor necrosis factor (TNF)-α; interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12; and transforming growth factor (TGF)-β1 in the supernatants of recipients’ serum were detected by the ELISA kits (Dakewe Bioengineering, China) according to the manufacturer’s instructions. The absorbance was measured at 450 nm by using a Microplate Reader (Tecan, Männedorf, Switzerland).