Eubacterium rectale Improves the Efficacy of Anti-PD1 Immunotherapy in Melanoma via l-Serine-Mediated NK Cell Activation

Abstract

Natural killer (NK) cells, as key immune cells, play essential roles in tumor cell immune escape and immunotherapy. Accumulating evidence has demonstrated that the gut microbiota community affects the efficacy of anti-PD1 immunotherapy and that remodeling the gut microbiota is a promising strategy to enhance anti-PD1 immunotherapy responsiveness in advanced melanoma patients; however, the details of the mechanism remain elusive. In this study, we found that Eubacterium rectale was significantly enriched in melanoma patients who responded to anti-PD1 immunotherapy and that a high E. rectale abundance was related to longer survival in melanoma patients. Furthermore, administration of E. rectale remarkably improved the efficacy of anti-PD1 therapy and increased the overall survival of tumor-bearing mice; moreover, application of E. rectale led to a significant accumulation of NK cells in the tumor microenvironment. Interestingly, conditioned medium isolated from an E. rectale culture system dramatically enhanced NK cell function. Gas chromatography-mass spectrometry/ultrahigh performance liquid chromatography-tandem mass spectrometry-based metabolomic analysis showed that l-serine production was significantly decreased in the E. rectale group; moreover, administration of an l-serine synthesis inhibitor dramatically increased NK cell activation, which enhanced anti-PD1 immunotherapy effects. Mechanistically, supplementation with l-serine or application of an l-serine synthesis inhibitor affected NK cell activation through Fos/Fosl. In summary, our findings reveal the role of bacteria-modulated serine metabolic signaling in NK cell activation and provide a novel therapeutic strategy to improve the efficacy of anti-PD1 immunotherapy in melanoma.

Fig. 1.

Abundance of E. rectale predicts patient responsiveness to anti-PD1 therapy. (A and B) Results of LEfSe analysis for the R and NR groups. (A) Taxonomic cladogram from LEfSe showed different taxa enriched in the R and NR group (LDA ≥ 4; P < 0.05). (B) LEfSe identified significantly differentially abundant taxa in the R and NR groups (LDA ≥ 4; P < 0.05). (C) Heatmap of relative abundance differences between the R and NR groups at the species level (Wilcoxon test). (D) The Kaplan–Meier method with log-rank test estimates the mPFS for patients with higher or lower abundance of E. rectale. (E) A prediction model was constructed by E. rectale in the training set, and predictive performance on the testing set was evaluated by ROC curves. R (n = 14), NR (n = 11). Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 2.

E. rectale enhances the efficacy of anti-PD1 treatment. (A) Experimental model of treatment of B16F10-bearing mice with E. rectale in combination with an anti-PD1 mAb. (B) Tumor samples isolated from C57BL/6 mice at the end of the treatment according to the administration method shown in the figure. (C) Tumor volume of mice measured every other day during the indicated treatment regimen. (D) Body weight of mice recorded every other day during the indicated treatment regimen. (E) Survival time (days) of mice beginning at tumor implantation (endpoints: the time at which the tumor volume reached 2,000 mm³ or death). (F to N) Flow cytometry detection of the CD3⁺CD8⁺ T cell proportion (F), CD3⁺CD4⁺ T cell proportion (G), F4/80⁺CD11B⁺ macrophage proportion (H), Gr1⁺CD11B⁺MDSC cell proportion (I), CD4⁺CD25⁺FOXP3⁺ T_reg cell proportion (J), IFN-γ⁺CD8⁺ T cell proportion (K), IFN-γ⁺CD4⁺ T cell proportion (L), NK1.1⁺ NK cell proportion (M), and IFN-γ⁺NK1.1⁺ NK cell proportion (N) in tumor tissue (N = 5). E. rectale is represented by Er in the figure. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 3.

E. rectale-conditioned medium enhances the tumor-killing activity of NK cells. (A and B) Proportions of NK cells in mouse spleen peripheral blood mononuclear cells (PBMCs) before (A) and after (B) NK cell magnetic bead sorting. (C to F) RT-PCR analysis of differentiation and functional molecular indices (PFN2, CCL2, IL-13, and IL-21) of NK cells under vehicle, medium, and E. rectale-conditioned medium culture conditions. (G) Cytotoxic activity of NK cells against tumor cells under coculture conditions. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 4.

E. rectale reduced the l-serine in PD1 mAb-treated mice. (A) Heatmap of differential metabolites associated with l-serine detected by GC-MS metabolomics analysis. (B) KEGG pathway enrichment analysis of differential metabolites detected by GC-MS metabolomics analysis. (C) KEGG functional enrichment analysis of differential metabolites detected by GC-MS metabolomics analysis. (D) GC-MS metabolomics detected l-serine abundance in the vehicle and E. rectale plus anti-PD1 treatment groups. (E) Results for ROC analysis of l-serine in GC-MS metabolomics analysis. (F) UHPLC-MS/MS detected l-serine abundance in the serum of melanoma tumor-bearing mice treated with vehicle or E. rectale. E. rectale is represented by Er in the figure. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 5.

The l-serine synthesis inhibitor NCT503 enhances the efficacy of anti-PD1 treatment by affecting NK cells. (A) Model for the experimental method of using NCT503 combined with an anti-PD1 mAb to treat B16F10-bearing C57BL/6 mice. (B) Tumor tissues of mice collected at the end of the treatment regimen according to the administration method shown in the figure. (C) Tumor volume of mice measured every other day during the treatment regimen. (D) Body weight of mice recorded every other day during the treatment regimen. (E to J) Flow cytometry detection of the GZMB⁺CD4⁺ T cell proportion (E), IFN-γ⁺CD8⁺ T cell proportion (F), IFN-γ⁺CD4⁺ T cell proportion (G), NK1.1⁺ NK cell proportion (H), GZMB⁺NK1.1⁺ NK cell proportion (I), and IFN-γ⁺NK1.1⁺ NK cell proportion (J) in tumor tissues (N = 4). (K) Enzyme-linked immunosorbent assay (ELISA) detection of the serum l-serine concentration in vehicle- and NCT503-treated mice with melanoma. (L to O) RT-PCR detection of differentiation and functional molecular indices of NK cells under l-serine and NKT503 treatment. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 6.

l-Serine inhibits NK cell function via the transcription factors FOS/FOSL2. (A) RNA-seq results shown in a differential gene expression heatmap for melanoma cells after serine or NCT503 treatment. (B) Western blotting detection of P-p38 MAPK expression in NK92 cells after l-serine (1 mM/l) treatment. (C and D) RT-PCR detection of FOS (C) and FOSL2 (D) mRNA expression in NK92 and primary NK cells under l-serine or NKT503 treatment. (E) Cytotoxic activity of NK92 cells against tumor cells after FOS knockdown and/or E. rectale-conditioned medium treatment. (F and G) RT-PCR detection of FOS, PFN1, CCL2, IL-13, and IL-21 mRNA expression after FOS knockdown in NK92 cells. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.

Fig. 7.

Correlation of l-serine synthesis pathway genes with anti-PD1 treatment efficacy. (A and B) Expression of the serine-metabolizing enzymes PHGDH (A) and PSPH (B) in anti-PD1-treated melanoma patients in the anti-PD1 treatment-nonresponsive (PD) group and anti-PD1 treatment-responsive (PRCR) group. (C to F) Expression of PAST1 (C), PSPH (D), SHMT1 (E), and SHMT2 (F) in anti-PD1-treated melanoma patients. (G to I) Correlations of PSPH (G), SHMT1 (H), and SHMT2 (I) with the immune score in PD1-treated patients. (J to L) Correlations of PSPH (J), PSAT1 (K), and SHMT2 (L) with tumor purity. (M) E. rectale promote NK cell function by reducing l-serine in the environment, which enhances the efficacy of PD1 immunotherapy. Multiple experimental data were counted and are presented according to the statistical method, and an asterisk (*) indicates the P value.