![]() The deletion of CDK6 or CDK4 in splenic T cells isolated from CDK6 or CDK4 KO mice was further confirmed by immunoblotting ( Figure S2P). In addition, we confirmed that CDK4 and CDK6, together with cyclin D2 and cyclin D3, were highly expressed in activated T cells in which cyclin D1 was barely expressed ( Figure S2O). Pre-depleting CD4 + T cells also reversed the host anti-tumor effects of CDK6 KO ( Figures 3E and 3F). We similarly depleted CD4 + T cells with anti-CD4 mAbs and examined tumor growth in CDK6 KO mice ( Figure 3D). These data clearly demonstrate that the antitumor activity seen in CDK6 KO mice is CD8 + T cell-dependent. Pre-depleting CD8 + T cells completely reversed the host anti-tumor effects of CDK6 KO ( Figure 3C). Anti-CD8 monoclonal Abs (mAbs) were injected intraperitoneally to deplete CD8 + T cells ( Figure 3A), as confirmed by fluorescence-activated cell sorting (FACS) prior to tumor inoculation ( Figure 3B). We next addressed the anti-tumor contribution of CD8 + T cells in CDK6 KO mice. CDK6 depletion in the TME reshapes the tumor immune microenvironment and increases tumor-infiltrating T cell cytotoxicity These results indicate that expression of CDK6 and cyclin D3 in the TME play important roles in facilitating tumor growth. Tumor growth was significantly inhibited in cyclin D3 but not in D1 or D2 KO mice ( Figures S1F–S1H). Because the cell-cycle kinase activity of CDK4/6 depends on their associated cyclin D partners, we also tested tumor growth in cyclin D KO mice ( Figure S1C, right panels). Thus, we conclude that the absence of CDK6 in the TME inhibited tumor growth. ![]() Tumor growth was slowed in CDK6 KO mice, significantly prolonging their survival compared with WT mice ( Figures 1G and 1H). The inhibition of tumor growth in CDK6 KO mice was further confirmed using B16F10 cells ( Figure S1E). Tumor-bearing CDK6 KO mice showed prolonged survival with slower tumor growth compared with either WT mice or mice with heterozygous deletion of CDK6 ( Figures 1E, 1F, and S1D). Notably, MC38 tumor growth in CDK6 but not CDK4 KO mice was significantly inhibited compared with WT littermates ( Figures 1C and 1D). A similar analysis was performed in parallel with CDK4 KO mice. Syngeneic mouse models were established by subcutaneously implanting MC38 cells into CDK6 KO mice. We applied a CDK6 knockout (KO) mouse model to examine wild-type (WT) tumor growth in mice lacking CDK6 in cells of the TME ( Figure S1C, left panel). Because CDK6 expression in tumors is positively correlated with resistance to immunotherapy ( Figures 1B and S1A), we asked whether CDK6 plays a role in the TME to promote tumor growth and decrease the efficacy of immunotherapy. Expression levels of CDK6, cyclin D1, and cyclin D3 are nearly equal between the tumor and TME ( Figure S1B). The results showed that CDK4 is more abundantly expressed in tumor cells than in the TME, while the reverse occurs for cyclin D2. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8 + and CD4 + T cells. ![]() Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. ![]()
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