Measuring T Cell Proliferation Inhibition

Splenocytes from 4C mice (C57BL/6 transgenic for a TCR recognizing the BALB/c MHC I-A^d) (23 (link)) were used as responders against irradiated BALB/c splenocytes as stimulators in a standard MLR assay (19 (link)). Briefly, 4C splenocytes were cultured in a Petri dish for 45 minutes at 37°C to enrich for T cells. Non-adherent cells were collected, washed, and incubated with 2000 cGy irradiated BALB/c splenocytes in 96-well U-bottom plates (10⁵ cells/well/each) in complete MLR medium (19 (link)). After 48 hrs of incubation, cultures were supplemented with varying concentrations of SA-PDL1 protein or equimolar concentrations of control SA protein. Cultures were incubated for a total of 72 hrs, with the last 16 hrs pulsed with [³H] thymidine (1 μCi/well). Cultures were then harvested with Tomtec cell harvester to assess DNA-associated radioactivity [counts per minute (cpm)] as the measure of cell proliferation using a Beta plate counter. The percentage inhibition of T cell proliferation was calculated using the formula of 1- [cpm in test proliferation / cpm in control proliferation] x 100.