Regulation Of Human Memory T Cells And The Effect Of Regulatory T Cells In Bone Marrow Microenvironment

Partâ… . EZH2 regulates human memory T cells through Notch/Bcl signaling pathway[Objective] To study the effect of EZH2 in human memory T cell regulation and the corresponding mechanisms.[Methods] Isolate CD3+ T cells from peripheral blood and stimulate T cells in vitro with anti-CD3/CD28 antibodies. Inhibit EZH2 expression with DZNep and specifically knock down EZH2 expression on T cells with lentivirus encoding with shEZH2. Investigate T cell phenotype and cytokine production with FACS. Examine cell apoptosis with Annexinâ…¤and 7-AAD staining and cell cycle with BrdU and 7-AAD staining with FACS. Examine gene expression with PCR and protein expression with Western Blot. Examine the regulation of EZH2 on the Notch suppressors with ChIP. Investigate the effect of EZH2 on T cell-mediated antitumor immunity with murine lung metastasis model. Examine EZH2 expression on T cells in tissue with immunofluorescent staining.[Results] EZH2 expression on human primary T cells is limited. EZH2+ T cells have higher proliferative potential and more memory characteristics. Inhibition of EZH2 leads to suppressed proliferation, increased apoptosis, decreased cytokine production and impaired effector function. EZH2 suppresses Notch suppressors by H3K27me3 and indirectly regulates Notch, which in turn regulates Bcl-2.[Conclusion] EZH2 regulates proliferation, apoptosis and cytokine production of T cells through Notch/Bcl signaling pathway and plays an important role in T cell-mediated antitumor immunity. It might be therapeutically beneficial by modifying EZH2 expression on memory T cells in treating human diseases. Part II. Effect of Treg in Bone Marrow Microenvironment in Patients with Prostate Cancer[Objective] To investigate the effect of Treg in prostate cancer-associated bone marrow.[Methods] Human peripheral blood and bone marrow aspiration were collected for this study. Phenotypes and cytokine profile of immune cells were analyzed with flow cytometry analysis (FACS). CD4+CD25high T cells were sorted and applied for migration and immunosuppressive assay and coculture with DCs. Cytokine production was examined by ELISA and mRNA level was detected by PCR. Mouse bone marrow cells were used for osteoclast differentiation assay. Bone mineral density and content were measured and in vivo bioluminescent and radiographic imaging were performed. Mouse bone metastatic models with mouse or human prostate cancer cell lines were employed to investigate the in vivo effect of Treg cells.[Results] In this study we show that functional Treg cells are increased in the bone marrow microenvironment in prostate cancer patients with bone metastasis, and that CXCR4/CXCL12 signaling pathway contributes to Treg cell bone marrow trafficking. Treg cells exhibit active cell cycling in the bone marrow, and bone marrow dendritic cells express high levels of receptor activator of NF-kB (RANK), and promote Treg cell expansion through RANK and RANKL signals. Furthermore, Treg cells suppress osteoclast differentiation induced by activated T cells and M-CSF, adoptive transferred Treg cells migrate to bone marrow, and increase bone mineral density in the xenograft mouse models. In vivo depletion of Treg cells results in reduced BMD in tumor bearing mice.[Conclusion] The data indicates that bone marrow Treg cells may form an immunosuppressive niche to facilitate cancer bone metastasis and contribute to bone deposition, the major bone pathology in prostate cancer patients with bone metastasis. These findings mechanistically explain why Treg cells accumulate in the bone marrow, and demonstrate a previously unappreciated role for Treg cells in patients with prostate cancer. Thus, targeting Treg cells may not only improve anti-tumor immunity, but also ameliorate bone pathology in prostate cancer patients with bone metastasis.