Lactylation-driven TNFR2 expression in regulatory T cells promotes the progression of malignant pleural effusion

Abstract

Background: Although tumor necrosis factor receptor 2 (TNFR2) has been recognized as an attractive next-generation candidate target for cancer immunotherapy, the factors that regulate the gene expression and their mechanistic effects on tumor-infiltrating regulatory T cells (Treg cells) remain poorly understood.

Methods: Single-cell RNA sequencing analysis was employed to analyze the phenotypic and functional differences between TNFR2⁺ Treg cells and TNFR2^- Treg cells. Malignant pleural effusion (MPE) from humans and mouse was used to investigate the potential mechanisms by which lactate regulates TNFR2 expression.

Results: Treg cells with high TNFR2 expression exhibited elevated levels of immune checkpoint molecules. Additionally, the high expression of TNFR2 on Treg cells was positively correlated with a poor prognosis in MPE patients. Moreover, we revealed that lactate upregulated TNFR2 expression on Treg cells, thereby enhancing their immunosuppressive function in MPE. Mechanistically, lactate modulated the gene transcription of transcription factor nuclear factor-κB p65 (NF-κB p65) through histone H3K18 lactylation (H3K18la), subsequently upregulating the gene expression of TNFR2 and expediting the progression of MPE. Notably, lactate metabolism blockade combined with immune checkpoint blockade (ICB) therapy effectively enhanced the efficacy of ICB therapy, prolonged the survival time of MPE mice, and improved immunosuppression in the microenvironment of MPE.

Conclusions: The study explains the mechanism that regulates TNFR2 expression on Treg cells and its function in MPE progression, providing novel insights into the epigenetic regulation of tumor development and metabolic strategies for MPE treatment by targeting lactate metabolism in Treg cells.

Keywords: Immunotherapy; Lung Cancer; T regulatory cell - Treg; Tumor microenvironment - TME.

Figure 1. TNFR2^high Treg cells exhibit stronger immunosuppressive functions in MPE (A, B) Treg cells from different MPE patients with lung cancer were stratified into two subgroups: TNFR2^high Treg cells (TNFR2 positivity for ≥60% of Treg cells, n=19) or TNFR2^low Treg cells (TNFR2 positivity for <60% of Treg cells, n=20). The expression level of TNFR2 on Tregs in MPE from patients with lung cancer was measured using flow cytometry Representative plots (A) and a representative histogram (B) are shown. (C) Correlation between TNFR2^high Treg cells and survival time in MPE was analyzed using Kaplan-Meier analysis (n=18–22). The median survival time of TNFR2^high group was 9 months, while the median survival time of TNFR2^low group was 17 months. (D) MPE specimens of untreated MPE patients with lung cancer were collected. CD45⁺ immune cells were isolated using magnetic activated cell sorting for scRNA-seq. A t-distributed stochastic neighbor embedding (t-SNE) plot based on scRNA-seq clustered the CD4⁺CD25⁺FOXP3⁺ Treg cells. (E, F) Treg cells from the same MPE patients with lung cancer were stratified into two subgroups: TNFR2⁺ Treg cells or TNFR2^- Treg cells. Differentially expressed genes were identified between TNFR2⁺ and TNFR2⁻Treg cells by scRNA-seq data of MPE (E). including inhibitory markers and Treg activity markers were shown (F). (G, H) Four immune checkpoint molecules were identified to be significantly different in MPE and NSCLC, including GITR, ICOS, CTLA4 and TIGIT. A Venn diagram was shown by integrating RNA-seq data from MPE and public databases of NSCLC (GSE1311907, GSE99254, GSE146771) (G). Violin plot analyzed the differential genes enriched between TNFR2⁺ and TNFR2⁻Treg cells in MPE (H). (I) The relative mRNA expression levels of immune checkpoint molecules between TNFR2^high and TNFR2^low Treg cells were measured by q-PCR assay. (J, K) The CD4⁺CD25⁺FOXP3⁺ Treg cells were isolated from human MPE. The immune checkpoint molecules of TNFR2^high and TNFR2^low Treg cells were measured by flow cytometry. Representative plots (J) and representative histograms (K) are shown. (L, M) Suppression assays with human CD8⁺ T cells were performed (L). TNFR2^high and TNFR2^low Treg cells were isolated from corresponding human MPE by magnetic-activated cell sorting (MACS), CD8⁺ T cells were isolated from peripheral blood of healthy donors by MACS. CFSE assay was performed to detect the proliferation of CD8⁺ T cells after co-cultured with Treg cells. The tumor-killing activities (INF-γ, TNF-α, granzyme B and perforin) of CD8⁺ T cells were analyzed by flow cytometry after co-culturing with Treg cells for 48 hours (M). Statistical analysis was performed using unpaired two-tailed Student’s t-test (B, I, J, M). Data represent mean±SD of independent experiments (n=3 biological replicates). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CFSE, carboxyfluorescein succinimidyl ester; MPE, malignant pleural effusion; ns, not significant; NSCLC, non-small cell lung cancer; TNFR2, tumor necrosis factor receptor type II.

Figure 2. Lactate increases TNFR2 expression and enhances the immunosuppressive function of Treg cells. (A) Gene-set enrichment analysis was performed on gene sets of glycolysis signaling pathway. Positive NES indicates higher expression in TNFR2⁺ Treg cells. (B) Comparison of the concentration of lactate in MPE and PB from untreated patients with lung cancer (n=45–46). Lactate concentration was measured by a Lactate Colorimetric/Fluorometric Assay Kit. (C) Correlation between the proportions of TNFR2⁺cells present in CD4⁺CD25⁺FOXP3⁺ Treg cells and lactate levels in MPE. (D) Correlation between lactate level and survival in MPE was analyzed using Kaplan-Meier analysis (n=18–24). The median survival time of patients with high lactate level (≥4 mM) was 10 months. While the median survival time of patients with low lactate level (<4 mM) was 20 months. (E) The expression of MCT-1 and LDHB in TNFR2^high and TNFR2^low Treg cells were analyzed using flow cytometry. (F, G) Treg cells from human MPE individuals were stimulated with the indicated concentration of LA for 24 hours. The relative expression levels of TNFR2 genes were measured by qRCR in sorted Treg cells isolated from human MPE (F). The expression level of TNFR2 was assessed by flow cytometry (n=3–7) (G). (H) The ratio of FOXP3 was assessed by flow cytometry after being treated with the indicated concentration of LA for 24 hours (n=5). (I, J) Immune inhibitory molecules (GITR, ICOS, CTLA4, TIGIT) in Treg cells of human MPE treated with 10 mM LA or α-TNFR2 for 24 hours were detected by flow cytometry. Representative plots (I) and representative histograms (J) are shown (n=3–5). (K) Suppression assays with human CD8⁺ T cells were performed. (K) Treg cells were isolated from corresponding human MPE by MACS, CD8⁺ T cells were isolated from peripheral blood of healthy donors by MACS. CFSE assay was performed to detect the proliferation of CD8⁺ T cells after co-cultured with Treg or Treg (LA) cells. Data shown in (A−K) are representative of at least three independent experiments (mean±SD). Statistical analysis was performed using unpaired two-tailed Student’s t-test (B, D, E) or one-way ANOVA (F, G, H, J, K). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; LA, lactate; LDHB, lactate dehydrogenase B; NES, Normalized Enrichment Score; NS, not significant.

Figure 3. Blocking lactate metabolism pathway downregulates TNFR2 expression on Treg cells. (A) Regulation of glycolysis and lactate production by diverse metabolic modulators. (B–D) CD4⁺CD25⁺FOXP3⁺Treg was separated from human MPE by Miltenyi Biotec Kit. Intracellular lactate levels were measured in Treg cells treated with different concentrations of AZD3965 for 24 hours (n=3) (B). The expression level of TNFR2 of Treg cells was measured by flow cytometry after being treated with different concentrations of AZD3965 for 24 hours (n=3) (C). The expression level of TNFR2 of Treg cells was measured by flow cytometry after being treated with 10 mM LA combined with 10 mM AZD3965 for 24 hours (n=3) (D). (E, F) Intracellular lactate levels were measured in Treg cells treated with different concentrations of Oxamate (E) or 2-DG (F) for 24 hours (n=3). (G, H) The expression level of TNFR2 of Treg cells was measured by flow cytometry after being treated with different concentrations of Oxamate (G) or 2-DG (H) for 24 hours (n=4–6). Representative plots (left) and representative histograms (right) are shown. (I) Intracellular lactate levels were measured in Treg cells treated with different concentrations of Rotenone for 24 hours (n=3). (J) The expression level of TNFR2 of Treg cells was measured by flow cytometry after being treated with different concentrations of Rotenone for 24 hours (n=4). Data shown in (B–J) are representative of at least three independent experiments (mean±SD). Statistical analysis was performed using one-way ANOVA (B–J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; 2-DG, 2-deoxy-d-glucose; MPE, Malignant pleural effusion; ns, not significant.

Figure 4. Elevated histone lactylation regulates expression of TNFR2 on Treg cells. (A) Lactylation levels were detected in Treg cells sorted from PBMC and PEMC by Western Blot and quantified by using Image J software. The expression level of TNFR2 on CD4⁺CD25⁺FOXP3⁺Treg cells sorted from PBMC and PEMC was examined by flow cytometry. The lactylation levels at different sites in Treg cells were assessed by Western Blot analysis following treatment with 10 mM LA and quantified by using Image J software. Lactylation and H3K18la levels were detected in Treg cells isolated from human MPE by Western Blot following treatment with the indicated concentration of LA for 24 hours and quantified by using Image J software. Lactylation and H3K18la levels were detected in Treg cells by Western Blot following treatment of 10 mM AZD3965 (a MCT1 inhibitor) for 24 hours and quantified by using Image J software. (F–H) Lactylation and H3K18la levels were detected in Treg cells by Western Blot treated with indicated glycolysis modulators for 24 hours and quantified by using Image J software. (I, J) The relative expression levels of TNFR2 genes were measured by qRCR in sorted Treg cells isolated from human MPE following treatment 10uM C646 (an acetyltransferase p300 inhibitor) and 10 mM LA for 24 hours (n=3) (I). The expression level of TNFR2 was assessed by flow cytometry following treatment 10uM C646 and 10 mM LA for 24 hours (n=5) (J). Data shown in (B–J) are representative of at least three independent experiments (mean±SD). Statistical analysis was performed using one-way ANOVA (I, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; MPE, malignant pleural effusion; ns, not significant; PBMC, peripheral blood mononuclear cell; PEMC, pleural effusion mononuclear cell.

Figure 5. NF-κB p65 regulates TNFR2 expression on Treg cells. (A) Distribution of H3K18la sites relative to translation start site (TSS). (B) A Venn diagram was shown by integrating Chip-seq data and RNA-seq data from MPE. (C) Bioinformatics analysis filtered NF-kB p65 as a downstream target of H3K18la (D) IGV tracks for NF-kB p65 (RELA) from Chip-seq analysis. (E) The binding of H3K18la to the promoter regions of NF-kB p65 was determined by Chip-qPCR in Treg cells from human MPE (n=3). (F, G) Alterations in the NF-kB p65 ratio on Treg cells are observed by flow cytometry following treatment with LA (F) or C646 (G). The binding of NF-kB p65 to the promoter regions of RELA was determined by Chip-qPCR in Treg cells from human MPE (n=3) (I and J) Alterations in the NF-kB p65 and TNFR2 ratio on Treg cells are observed by flow cytometry following treatment with regulators of the glycolytic pathway as well as NF-kB activators (PMA) (I) and NF-kB inhibitors (PDTC) (J) (n=3). Statistical analysis was performed using unpaired two-tailed Student’s t-test (E–H) or one-way ANOVA (I, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not statistically significant. ANOVA, analysis of variance; Chip-qPCR, chromatin immunoprecipitation-PCR; MPE, malignant pleural effusion.

Figure 6. Targeting lactate metabolism alleviates MPE immunosuppression and potentiates response to ICB therapy (A–E) Schematic diagram of MPE mouse model was drawn by FigDraw (www.figdraw.com; ID: TPORYeaf76). The MPE mice were randomly assigned to four groups: mice with PBS, mice with AZD3965, mice with anti-PD-1 antibody (α-PD-1), mice with AZD2965 and α-PD-1. LUC cells (2X10⁵) were injected subcutaneously into wild-type C57BL/6 mice on day 0. (n=6 per group). AZD3965 and α-PD-1 were injected on days 0, 6, 9,12,15 (A). Representative images of mouse MPE and pleural cavity tumors in different groups (B) Representative in vivo bioluminescence images of the growth of mice MPE (n=4) (C). MPE volume (D) and Kaplan-Meier survival plot of MPE mice (E) were observed (n=9–11). (F–J) The expression level of TNFR2 on Treg cells (F）the percentage of Treg cells (G) in immune cells, IFN-γ⁺CD8⁺ (H), NK cells (I) and TAM (J) were analyzed by flow cytometry (n=5–8). (K, L) Schematic diagram of MPE mouse model was drawn by FigDraw (www.figdraw.com; ID: TPORYeaf76). The MPE mice were randomly assigned to four groups: mice with PBS, mice with Oxamate, mice with anti-PD-1 antibody (α-PD-1), mice with Oxamate and α-PD-1. LUC cells (2×10⁵) were injected subcutaneously into wild-type C57BL/6 mice on day 0. (n=6 per group). Oxamate and α-PD-1 were injected on days 0, 6, 9, 12, 15 (K). Representative images of mouse MPE and pleural cavity tumors in different groups (L). MPE volume (M) and Kaplan-Meier survival plot of MPE mice were observed (n=9–11) (N). (O–S) The expression level of TNFR2 on Treg cells, the percentage of Treg cells (O) in immune cells (P), IFN-γ⁺CD8⁺ (Q), NK cells (R) and TAM (S) were analyzed by flow cytometry (n=5–8). Statistical analysis was performed using unpaired two-tailed Student’s t-test (E, F, G, H) or one-way ANOVA (I, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; LLC, Lewis lung cancer cells; MPE, malignant pleural effusion; ns: not significant; Oxa, oxamate; PBS, phosphate-buffered saline; TAM, tumor-associated macrophage.