The TIM-3 extracellular region is required for co-stimulatory effects on T cell signaling.
(A) Schematic representation of PD-1/TIM-3 chimeric constructs. PTT contains the PD-1 extracellular region (ECR) plus transmembrane (TM) and intracellular regions of TIM-3. In PPT, only the intracellular region is replaced with that from TIM-3. (B) A GFP reporter was used to measure NF-κB transcriptional activity downstream of TCR activation in parental NF-κB reporter Jurkat cells (black) and the same cells engineered to stably express TIM-3 (red curve), PTT (purple solid curve), PPT (purple dotted curve), or PD-1 (blue curve), stimulated with 1 μg/ml αCD3 plus 1 μg/ml αCD28. Representative histograms of NF-κB-driven GFP expression are shown. (C) Relative mean GFP fluorescence intensity (MFI) from three biological repeats of the experiment shown in (B) is plotted, expressed as fold change above that measured for parental NF-κB GFP reporter cells. Bars are color coded as in (B) and represent mean MFI value (± SD) — with P-values determined by two-tailed, unpaired Student's t-test.