Fig 2: Low-dose decitabine therapy enhances NF-κB 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-γ 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-γ+ 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-κB 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 Vα2+, 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 Vα2+, 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 Vα2+, CD8+, CD62L- CD25+ cells. (P value * 0.0238, ** 0.0095). (F) Same as in C, focusing on TCR Vα2+, CD8+, CD62L- CD25+, CD44+ cells. (P value * 0.0238, ** 0.0095). (G) Representative flow cytometry plots of Ki67 vs. FSC gated on TCR Vα2+, CD8+, CD25+ T cells. (H) Bar graph quantification of TCR Vα2+, 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‐β, 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‐β, 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‐β, 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‐β, 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‐γ (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‐γ (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‐β, transforming growth factor‐beta; Treg, regulatory T cell; IFN‐γ, interferon‐γ; SEM, standard error of the mean; BL, bioluminescence