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PD-1/PD-ligand-axis immunotherapy-mediated activation of T-cells for cancer cell elimination is a promising treatment of nonsmall cell lung cancer (NSCLC). However, the effect of immunotherapy on intracellular signaling pathways in cancer cells still needs further delineation. Repulsive Guidance Molecule b (RGMb), a regulator of Bone Morphogenetic Proteins (BMPs) signaling, interacts with the PD-ligand, PD-L2, at cancer cell membranes. Accordingly, a clarification of the functions of RGMb and its relation to PD-L2 might provide insight into NSCLC cell signaling responses to PD-1/PD-ligand-axis immunotherapy. In this study, the functions of RGMb and PD-L2 were examined using the two NSCLC cell lines HCC827 and A549. CRISPR/Cas9 was used to decrease the expression of RGMb and PD-L2, while lentiviral vectors were used to increase their expression. Downstream effects were examined by RT-qPCR and immunoassays. Ectopic expression of RGMb impacted BMP2-induced expression of ID1 and ID2 messenger RNA (mRNA) independently of PD-L2, while RGMb depletion by CRISPR/Cas9 did not affect the BMP2-mediated induction of ID1, ID2, and ID3 mRNA. However, depletion of RGMb resulted in a partial epithelial-mesenchymal transition (EMT) gene expression profile in HCC827 cells, which was not mimicked by PD-L2 depletion. The results show that RGMb is a coregulator of BMP signaling and hence, ID mRNA expression and that RGMb can control the EMT balance in NSCLC cells. However, RGMb appears to exert these functions independently of PD-L2, and accordingly, the PD-1/PD-ligand axis for immune surveillance in NSCLC cells., (© 2023 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)