PD-L1 couples with LTR signaling to accelerate regulatory T cell lymphatic migration and tumor metastasis

Abstract Introduction: Tregs in the tumor environment inhibit anti-tumor immunity and are critical targets for anti-tumor strategies. We previously showed that Tregs use surface lymphotoxin (LTαβ) and PD-1 to signal LTβR and PD-L1 on lymphatic endothelial cells, thereby promoting Treg lymphatic migration. Most tumor cells express LTβR and PD-L1, yet tumor interactions with LTαβ and PD-1 on Tregs are poorly studied. We hypothesized that PD-L1 couples with LTβR signaling on tumors to regulate migration and tumor metastasis. Methods: Wild type and CRISPR/Cas9 LTβR or PD-L1 knockout B16F10 melanoma were used for signaling and cell migration. Specific LTβR classical and nonclassical NFκB blocking peptides were used. Results: PD-L1 bound to LTβR in resting B16F10, and blocking LTβR-nonclassical NFκB signaling increased melanoma PD-L1 expression. RNASeq analysis of melanoma revealed that gene expression regulated by LTβR-nonclassical NFκB signaling was increased by PD-L1 depletion, while genes driven by PD-L1 signaling remained unaltered by LTβR depletion. Melanoma cocultured with Tregs had enhanced migration, while combined blockade of PD-L1 and LTβR nonclassical NFκB pathways synergistically blocked tumor migration. In vivo, PD-L1 blockade combined with LTβR classical or nonclassical NFκB blocking peptides inhibited tumor growth and metastases, and enhanced host survival. Conclusions: PD-L1 couples with LTβR-nonclassical NFκB signaling to regulate tumor growth and migration. Blocking both arms of tumor LTβR-NFκB-signaling enhanced immune checkpoint blockade efficacy and mouse survival. These observations provide a rational strategy to modulate Treg activities to prevent tumor spread. supported by NIH R37 AI062765 and Emerald grant 2022