Background Checkpoint blockade therapies, especially those targeting the PD-1/PD-L1 axis, have revolutionized cancer therapy.¹ Despite these advances, numerous patients fail to respond or relapse following an initial response.^{2 3} Poor detection reagents and lack of equivalence between mouse and human function fostered a lack of appreciation for the significant role of PD-L2 in tumor immune suppression.⁴ Given its unique expression pattern relative to PD-L1,⁵ we hypothesize that PD-L2 engages PD-1 in a distinct manner which propagates a unique downstream inhibitory signature in T cells.

Methods Jurkat T cells with a nuclear factor of activated T cells (NFAT) luciferase reporter were cocultured with CHO cells transduced to overexpress huPD-L1 or huPD-L2 and an anti-CD3 scFv. Co-culture of the Jurkat cells with these CHO cells results 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 gene set enrichment analysis (GSEA) helped elucidate the ensuing transcriptomic effects. Similar experiments were performed on primary human T cells as well as therapeutic chimeric antigen receptor (CAR) T cells. Recovery assays, in the form of anti-CD3/CD28 activation post PD-L1/PD-L2 inhibition, also allowed for the assessment of marked differences in functional cytokine production (IL-2, IFNγ, and TNFα).

Results When compared head-to-head, we discovered that PD-L2 provides a distinct and more readily reversible inhibitory signal compared to PD-L1. While both PD-Ligands equally inhibited NFAT activation and cytokine production, PD-L1 and PD-L2 differed in the temporal dephosphorylation of membrane proximal proteins in the CD3ζ signaling axis. GSEA showed PD-L1 having a greater effect on the mTOR pathway and PD-L2 negatively affecting microtubule spindle formation. Propidium iodide studies revealed that PD-L2:PD-1 engagement preferentially accumulates T cells in the S phase of the cell cycle, while PD-L1:PD-1 arrests T cells in the G1/G0 phase. PD-L2 inhibited T cells responded to CD3/CD28 restimulation by restoring high-level IL-2 production, whereas those inhibited with PD-L1 could not.

Conclusions Gene duplication events allow for sub-functionalization and we demonstrate that PD-L2 confers an inhibitory signature through PD-1 distinct from PD-L1. Like PD-L1, PD-L2 drives T cells to an inhibited state, however, PD-L2 inhibited T cells are capable of recovering cell-intrinsic IL-2 production following restimulation while those exposed to PD-L1 are not. We speculate that during primary T cell responses, induction of PD-L2 may set aside a ‘ready reserve’ of T cells that can reactivate if needed if pro-inflammatory conditions persist.

References

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