Fig 2: Low-dose decitabine therapy enhances NF-?B activation in CD4+ T cells and associates with clinical response in solid tumor patients. (A,B) Human CD4+ T cells were sorted from peripheral blood mononuclear cells of healthy donors, stimulated with anti-CD3 for 24 h, and treated with PBS or 10 nM decitabine for 3 days. After PBS or decitabine treatment, IFN-? and Ki67 levels were detected by flow cytometry (A). PBS or decitabine-pretreated CD4+ T cells were co-cultured with HCT116 cells, the cytotoxicity of human CD4+ T cells against HCT116 cells was determined by using DELFIA time-resolved fluorescence (TRF) assays at the indicated E:T ratios, and CD107a level was detected by flow cytometry (B). (C) CD4+ T cells were sorted from solid tumor patients (P1, P2, P4, and P5) before treatment. Cells were treated with PBS or 10 nM decitabine for 3 days followed by 24 h-anti-CD3 antibody activation, and stimulated with PMA/Ionomycin for 15 min before collecting. The total proteins were extracted and the expression levels of the indicated proteins were detected by western blot. (D,E) CD4+ T cells were sorted from solid tumor patients (P1, P2, P4, and P5) before and after 5 day decitabine therapy, the frequencies of IFN-?+ cells or Ki67+ cells as in CD4+ T cells were analyzed using flow cytometry. All results from three independent experiments are shown. *P < 0.05. (F) The proposed model for decitabine-mediated NF-?B activation and immune response. **P < 0.01.

Fig 3: Low-dose decitabine promotes the activation and viability of sorted CD4+ T cells in vitro. (A) To detect T cell early activation markers, mouse CD4+ T cells were sorted from splenocytes, activated with immobilized anti-CD3/CD28 for 24 h, and treated with different concentrations of decitabine (DAC, 0, 1, 10, 100, and 1,000 nM) plus IL-2 for 2 days. The surface marker CD69 was detected by flow cytometry. (B,C) After anti-CD3/CD28 activation, CD4+ T cells were treated with PBS (CON) or 10 nM DAC for 3 days, the expressions of CD25 (B) and CD28 (C) were detected by flow cytometry. (D) Sorted CD4+ T cells were activated with anti-CD3/CD28, the CFSE-labeled (2 µM) T cells were then treated with PBS or different concentrations of decitabine for 3 days, and detected by flow cytometry. (E) Ki67 was detected by flow cytometry on day three after PBS or decitabine treatment. All results from three independent experiments are shown. *P <0.05 and **P < 0.01. ns, not significant.

Fig 4: HSCs critically require Cited2 to reconstitute hematopoiesis upon transplantation(A) Transplantation assay: 100 HSCs or 2,000 LSK cells sorted from Cited2CTL and Cited2CKO mice were transplanted into lethally irradiated recipients together with 200,000 support CD45.1+ total BM cells.(B and C) Percentage of donor-derived CD45.2+ cells in (B) PB and (C) the LSK cell compartment of recipient mice following transplantation of 100 HSCs (n = 5 recipients per donor; 2 donors per genotype).(D and E) Percentage of donor-derived CD45.2+ cells in (D) PB and (E) the LSK compartment of the recipient mice following transplantation of 2,000 LSK cells (n = 4 recipients per donor; 4 donors per genotype).(F) Homing assay: 160,000 LSK cells were injected into irradiated CD45.1+-recipient mice and analyzed 18 h later.(G) Percentage of donor-derived CD45.2+ cells in BM (n = 4–5).(H) Percentage of HSCs from 8- to 10-week-old Cited2CKO and Cited2CTL mice in the G0 (DAPI-Ki67-), G1 (DAPI-Ki67+), and S/G2/M (DAPI+Ki67+) phases of the cell cycle (n = 3).(I) Percentage of Annexin V+ cells in the HSC, MPP, HPC-1, and HPC-2 cell compartments in BM of Cited2CKO and Cited2CTL mice (n = 3). For (B)–(E) and (G)–(I), data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Fc?RIIB Contributes to IC Binding in 564Igi Mixed BM Chimeras(A) Confocal micrographs of FDC networks in splenic cryosections from passively immunized 564Igi mixed BM chimeras. The recipients of 564Igi mixed BM chimeras were passively immunized with pooled serum from WT mice immunized with PE in complete Freund’s adjuvant (CFA), followed by i.v. PE injection. The left panels show representative FDC networks from a WT and Fc?RIIB-/- recipient with indicated staining. The right panels show the same networks with the mask outline (based on CD35 staining) used for quantification and the PE signal only. Only FDC networks associated with clusters of Ki67+ GC were included in the analysis. The arrowhead in the left panel indicates non-FDC-associated PE, likely derived from a dendritic cell (DC) or macrophage. Scale bar, 100 µm. All of the panels are scaled equally.(B) Quantification of PE on FDCs in passively immunized 564Igi chimeras. B6, C57BL/6 recipients of 564Igi mixed BM and Fc?RIIB-/- (KO, knockout) recipients of 564Igi mixed BM, n = 3 mice per group; each point represents 1 FDC network. No IgG and no PE are negative controls, with B6 564Igi mixed BM chimeras receiving PE only or no IgG and PE, respectively.(C) Area of each FDC network mask in number of pixels. NS, not significant, ****p < 0.0001, ANOVA with Tukey post hoc test, B6 versus all other groups.