Differential signaling through the PD-1 receptor allows for a distinct inhibitory signature and unique signal dichotomy between PD-L1 and PD-L2 2261044

Abstract Introduction Checkpoint blockade therapies, especially those targeting the PD-1/PD-L1 axis, have revolutionized cancer therapy. Despite these advances, numerous patients fail to respond or relapse. Poor detection reagents and lack of equivalence between mouse and human function fostered a lack of appreciation for the role of PD-L2 in tumor immune suppression. Given its distinct expression pattern and greater binding affinity 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 an anti-CD3 scFv and huPD-L1 or huPD-L2, resulting in depression 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 differ in the temporal dephosphorylation of membrane proximal proteins in the TCR signaling cascade. RPPA and GSEA corroborate PD-L1 having a greater effect on the Ras/Raf/MEK/ERK/MAPK pathway while PD-L2 negatively affects the PI3K/Akt/mTOR pathway. Despite the consistent use of the PD-1 ITSM canonical motif, differential recruitment of downstream phosphatases appears to drive the differential signaling. Moreover, unlike PD-L1, PD-L2 inhibited T cells respond to anti-CD3/CD28 restimulation by recovering high-level IL-2 production. Conclusion 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, and actively screening for PD-L2 in tumor biopsies would help improve patient screening for immune checkpoint blockade. Funding Source 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)