PD-L2 transiently inactivates T cells which can reactivate when pro-inflammatory conditions persist 4426

Description Hypothesis Given its distinct expression pattern relative to PD-L1, we hypothesize that PD-L2 engages PD-1 in a manner which propagates a unique downstream inhibitory signature in T cells. Methods Jurkat T cells with a NFAT luciferase reporter were cocultured with CHO cells transduced to overexpress huPD-L1 or huPD-L2 and an anti-CD3 scFv, resulting in suppression of the bioluminescent signal if T cell activation is suppressed. Western blots were used to assess the dampening of the TCR phosphoproteomic signatures, and microarrays along with GSEA helped elucidate the ensuing transcriptomic effects. Recovery assays, in the form of anti-CD3/CD28 activation post PD-L1/PD-L2 inhibition, allowed for the assessment of marked differences in functional cytokine production. Results PD-L1 and PD-L2 equally inhibit NFAT activation yet differ in the temporal dephosphorylation of membrane proximal proteins in the CD3ζ signaling axis. GSEA shows PD-L1 having a greater effect on the mTOR pathway while PD-L2 negatively affects microtubule spindle formation. Propidium iodide studies reveal that PD-L1 and PD-L2 preferentially accumulate T cells in distinct phases of the cell cycle, and unlike PD-L1, PD-L2 inhibited T cells respond to anti-CD3/CD28 restimulation by recovering high-level IL-2 production. Conclusions We speculate that during primary T cell responses, induction of PD-L2 may transiently inactivate T cells which can reactivate if needed when pro-inflammatory conditions persist. Funding Sources This work was supported by CPRIT Grant RP170067 and a generous donation from the Big Easy Ranch Foundation. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)