BackgroundThe receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells.MethodsMelanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3+CD8+ cytotoxic T cells, CD49b+NK cells, CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+ MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8+ T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-β1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively.ResultsDownregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4+ and CD8+ T cells. The percentage of CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-β1.ConclusionThese findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-β1.

Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent‐cell‐based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple‐negative breast cancer. However, the programmed death receptor‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor‐1 (sPD1)‐expressing senescent cells to overcome PD‐L1/PD‐1‐mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T‐cell activation. In the present study, sPD1‐expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1‐expressing senescent tumor cell vaccine (STCV/sPD‐1) treatment attracted more mature DC and fewer exhausted‐PD1+ T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD‐1 than for control treatments. STCV/sPD‐1 pre‐injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD‐1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD‐1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention.

Background Lymphopenic condition after radiotherapy or chemotherapy might be able to create an environment to mount an efficient antitumor immunity through the homeostatic proliferation of residual or adoptive lymphocytes. However, the antitumor immunity seems to decrease rapidly with the growth of tumor, and the mechanism remain elusive. Methods In this study, we first examined the dynamic change of PD-1 expression on T cells and analyzed the connection between PD-1 expression and the exhaustion of CTLs when the number of T cells recovered from lymphopenic conditions. Anti-PD-1/PD-L1 therapies, including anti-PD-1 antibodies, PD-1 or PD-L1 knockout by Crispr-Cas9 system, were used in the study to evaluate the role of PD-1/PD-L1 block on the antigen recognition, differentiation, activation, killing, survival and prognosis of HP cells. Results Our study found that in the mouse model of homeostatic proliferation, although T cells proliferated continuously in lymphopenic host, the number of IFN-γ-releasing CD8+T cells rapidly decreased in course of homeostatic proliferation, indicating the immunosuppressive state in tumor microenvironment. The expression of PD-1 on T cells, increased gradually in course of homeostatic proliferation, were significantly negatively related to the cytotoxicity of effector T cell. Blocking of PD-1/PD-L1 axis by PD-1 antibody promoted HP cells to recognize tumor associated antigen (TAAs) and resulted in the activation of DC cells. It can also enhanced the number of IFN-γ-releasing CD8+ T cells and the cytotoxicity of effector T cell in lymphopenic mice. Furthermore, PD-1 antibody induced a portion of tumor-specific CTL convert to central memory T cells (TCM). Finally, we found that Crispr/Cas9-mediated knockout of PD-1 in T cells or PD-L1 in melanoma cells significantly enhanced homeostatic proliferation–driven antitumor responses and inhibited tumor growth. Conclusion These findings suggested PD-1/PD-L1 played an important role in the formation of immune tolerance during the period of homeostatic proliferation. Anti-PD-1/PD-L1 therapies may be used to enhance antitumor immunity during recovery from lymphopenic condition after chemotherapy or radiotherapy.