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: Systemic chronic oxygen restriction inhibits CD8+ T cell activation and response.(A) Schematic of the experiment presented in panels B-H. C57BL6 mice were primed intradermally in the ear pinna with 5 × 106 transduction units (TU) of Lv-OVA or left untreated. Twenty-four hours following the viral challenge, mice were transferred to chambers for additional 6 days and kept under either 8% or 21% oxygen pressure. Extracted cells from the deep cervical lymph nodes were then analyzed by flow cytometry as follows: TCR Va2+, CD8+ T cells from naïve mice (left/gray) or Lv-OVA challenged mice that were kept under either atmospheric oxygen pressure (21% O2) (middle/black) or 8% oxygen pressure (right/red). (naïve n = 3 biological replicates, activated n = 6 in each group). (B) Representative flow cytometry plots of CD44 vs. CD62L, numbers indicate the frequencies of CD44+ CD62L- cells. (C) Bar graph quantification of TCR Va2+, CD8+, CD44+, CD62L- cells (P value * 0.0238, ** 0.0095). (D) Same as in B, focusing on CD25 vs. CD62L. (E) Same as in C focusing on TCR Va2+, CD8+, CD62L- CD25+ cells. (P value * 0.0238, ** 0.0095). (F) Same as in C, focusing on TCR Va2+, CD8+, CD62L- CD25+, CD44+ cells. (P value * 0.0238, ** 0.0095). (G) Representative flow cytometry plots of Ki67 vs. FSC gated on TCR Va2+, CD8+, CD25+ T cells. (H) Bar graph quantification of TCR Va2+, CD8+, CD25+, Ki67+ cells. (P value * 0.0357, * 0.0159). (I-L) CellTrace-labeled splenocytes were activated with anti-CD3/28 for 72 hr under either 1% or 21% of oxygen. (n = 5 biological replicates). (I) Representative flow cytometry plots of FSC vs. CD25 gated on CD8+ T cells. Numbers indicate the frequencies of CD25+ cells. (J) Bar graph summarizes results in I. (P value * 0.0022). (K) Representative Flow cytometry overlay histogram of CellTrace intensity gated on CD8+ T cells. (L) Bar graph summarizes results in K as Proliferation Index (P. Index). (P value * 0.0022). Statistical method, non-parametric Mann–Whitney test, mean ± s.e.m.

Fig 5: Treatment response of 6 different groups (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) in an orthotopic mouse model. (A) CMT167 cells were transplanted into the lungs of C57BL/6 mice. Follow-up treatment (injected with DDP, anti-PD-1, anti-TGF-ß, SHR-1701, or isotype control antibody) was then performed in six groups as shown. (B) Images and quantification of in vivo bioluminescence imaging of the six groups (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) at the indicated time points. The P-value represents a comparison of bioluminescence intensity on the 15th day. (C) Effect of different treatments (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) on survival of CMT167 orthotopic tumor-bearing mice. (D-E) Representative images of IHC staining for tumor-infiltrating Foxp3+ Treg cells (D) and CD8+ T cells (E). Scale bars: 100 µm, upper panel; 40 µm, lower panel. (F) Orthotopic tumors were analyzed by flow cytometry for GITR (left panel), IFN-? (middle panel), and CD69 (right panel) expression on CD8+ T cells after stimulation with CD3 and CD28 (both 1 mg/mL) for 48 h. (G) IFN-? (left panel) and CD69 (right panel) expression on CD8+ T cells in spleens were analyzed. Bar graphs represent the means ± SEM for each group. B-G: n = 5 for each group. One-way ANOVA statistical tests were adopted for more than two groups (B, D, E, F, and G). Log-rank tests were used in C. *, P < 0.050; **, P < 0.010; ***, P < 0.001. Abbreviations: n.s., not significant; PD-1, programmed cell death-protein 1; DDP, cisplatin; TGF-ß, transforming growth factor-beta; Treg, regulatory T cell; IFN-?, interferon-?; SEM, standard error of the mean; BL, bioluminescence