Fig 2: LIF blockade induces CD8+ T cell tumor infiltration and promotes tumor regression when combined with anti-PD1. a Schematic representation of GBM patient-derived xenografts and human organotypic models. b Organotypic specimens were treated with anti-LIF for 72 h and then cultured with PBMCs for 24 h. CXCL9 and CCL2 mRNA expression levels are shown. Representative images (patient 4, 5, 6) of CFSE-stained PBMCs into Matrigel containing GBM specimens and IF of the indicated factors in organotypic tissues are displayed. Bars represent quantification of five different fields of each condition. Data are presented as mean ± SD. Scale bar, 20 µm. c, d FC of CD8+ T infiltrating cells detected by flow cytometry in organotypic tissues treated with anti-LIF (c, d) and/or anti-CXCL9 (1.5 µg/ml) (d) for 72 h and then cultured with PBMCs for 48 h (patient 4, 5, 6). e CD8+ T infiltrating cells into subcutaneously engrafted GBM specimens in NSG mice. Bar graph represents the ratio of CD8+ T cells detected by flow cytometry in the tissue vs. CD8+ T cells detected in the blood of the same animal. Four patients (7, 8, 9, 10) with their corresponding PBMCs were evaluated and are represented with different colors. f Representative images of treated or untreated patient-derived xenografts (PDX) bearing mice 48 h after luciferase CD3+ T cells inoculation. T cells infiltration is measured as total flux (p/s) within the tumor. Data are mean ± SEM. g Scheme represents the experimental procedure of GL261N mice treated with anti-LIF, anti-PD1 or the combination. Overall survival determined by Kaplan-Meier curves is shown. h Tumor growth of the treated GL261N model represented as a fold change of tumor size between 13 and 6 dpi. Representative images are shown. i Scheme representing the experimental procedure for regression study. Images of mice and tumor size measured by total flux (p/s) at 13 dpi are shown. j Schematic representation shows the effect of LIF in CD8+ T cell tumor infiltration. Statistical analyses by Mann–Whitney T-test or Log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 3: Anti-PD1/chidamide shows promising efficiency in clinical and its mechanism scenario. a, Case 1 is a female patient with NK-T cell lymphoma who received more than 2-year treatment of anti-PD1 and chidamide after refractory from first-line chemotherapy. b, Case 2 is a female patient with NK-T cell lymphoma who received anti-PD1 and chidamide as maintenance treatment after first-line chemotherapy finished. c, anti-PD1/Chidamide combination induces chemokine expression in multiple cell types resulting in enhanced immune cell recruitment, trafficking and infiltration. Chemokine ligands and receptors binding include Cxcr3-Cxcl9/10/11 in CD8 T-tumour cells or NK-tumour cells and Ccr5-Ccl4/Ccl5/Ccl20 in dendritic-tumour cells. Besides, the combination enhances IFN-γ expression and acetylated level in multiple cell types resulting in enhanced antigen presentation, T cell activation, immune cell infiltration, and tumour cell killing. Cd274 is also upregulated by IFN-γ as a feedback loop and tumour cells become more responsive to anti-PD1 treatment. By contrast, there was no additive anti-tumour effect when romidepsin combined with anti-PD1, including no changes in chemokines and IFN-γ responses.

Fig 4: LIF represses CXCL9 through epigenetic silencing. a, b qRT-PCR analysis of the indicated genes in BMDMs. BMDMs were pre-incubated with 20 ng/ml LIF for 72 h and then stimulated with 5 ng/ml IFN? or 10 µg/ml IL4 during 6 h (a) or with 0.1, 0.5, 1 and 5 ng/ml IFN? for 24 h (b). c CXCL9 ELISA from BMDMs pre-incubated with 20 ng/ml LIF and then stimulated with 0.1 ng/ml IFN? for 24 h. d CXCL9 ELISA from human CD11b+sorted cells (77% CD11b+ CD14+, see Supplementary Fig. 7b, c) from human GBM cultured with 20 ng/ml LIF for 72 h and then with 0.1 ng/ml IFN? for 24 h. e ChIP of Tri-methyl-histone H3 (H3K27me3), EZH2 and acetyl-histone4 (H4ac) was performed in BMDMs treated with 20 ng/ml LIF for 72 h. Scheme shows the analysed CXCL9 promoter region. Representative data are presented as mean ± SD. f Representative images of IF of the indicated markers in human GBM organotypic slices (patients 1, 2, 3) incubated with 10 µg/ml anti-LIF for 3 days. Scale bar, 20 µm. (see Supplementary Fig. 8). Bottom, percentage of double positive cells relative to Iba1+ cells and percentage of CXCL9+ cells relative to the total number of cells. Data are mean of all patients ± SEM. Statistical analyses by Student’s t-test or Mann–Whitney T-test. *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 5: Anti-PD1/chidamide shows an immune response with epigenetic regulation. a, Western blot of Ifn-γ, Cxcl9, Cxcr3, Acetylated histone H3, histone H3, Pd-l1, and β-actin in anti-PD1/chidamide and each monotherapy. b, Western blot of Ifn-γ, Cxcl9, Cxcr3, Acetylated histone H3, histone H3, Pd-l1, and β-actin in anti-PD1/romidepsin and each monotherapy. c, ELISA of tumour lysis in anti-PD1/chidamide. d, ELISA of tumour lysis in anti-IFN-γ neutralization. e, Heatmap of ChIP-seq in high-dose, low-dose anti-PD1/chidamide, high-dose anti-PD1/romidepsin. f, Acetylated level of immune responses factor Ifn-γ, Gzmb, Gzmk, and Cxcr3 (left to right). g, ChIP-qPCR validation of Ifn-γ and Cxcr3 in different groups of anti-PD1/chidamide and anti-PD1/romidepsin. h, immunohistochemistry staining of Cd8, Cxcl9, Ifn-γ, and Pd-l1 in anti-PD1/chidamide. i, immunohistochemistry staining of Cd8, Cxcl9, Ifn-γ, and Pd-l1 in anti-PD1/romidepsin. j, immunohistochemistry staining of Cd8, Cxcl9, Ifn-γ, and Pd-l1 in anti-PD1/romidepsin or anti-PD1/chidamide ± anti- Ifn-γ (∗P＜0.05，∗∗P＜0.001, ∗∗∗P＜0.0001, Mann–Whitney U-test and one-way ANOVA).