Faecalibacterium prausnitzii strain EXL01 boosts efficacy of immune checkpoint inhibitors

Figures & data

Figure 1. F. prausnitzii level predicts clinical response to PD-L1 in patients with advanced non-small-cell lung cancer.

Kaplan – Meier curves and Cox regression analyses of the OS of all patients (n = 337) according to F. prausnitzii level (low, medium and high tertiles) (a), in patients who received ABX (n = 68) (b), and in patients who did not receive ABX (n = 269) (c). F. prausnitzii levels were compared using the stratified log-rank test. (d) Cox logistic regression multivariate analysis of progression-free survival in 337 patients according to F. prausnitzii level and other relevant clinical parameters. (e) Survival probability at 18 months in patients according to PD-L1 expression (0, 1–49% and >50%) and F. prausnitzii level.

Figure 2. F. prausnitzii level predicts clinical response to ICI in a dominant group of patients with advanced melanoma.

(a) Kaplan – Meier curves and Cox regression analyses of OS of all patients (n = 192) according to baseline F. prausnitzii levels (low and medium & high tertiles). F. prausnitzii levels were compared using the stratified log-rank test. (b) La Place model fit, with two clusters identified as an optimal fit. (c) The 40 most significantly abundant bacterial species (best qval) differentially represented between the two identified clusters according to the multivariate analysis (MaAsLin2). (d) Cox logistic regression multivariate analysis of progression-free survival in the two identified clusters according to F. prausnitzii baseline level and other relevant clinical parameters. (e) Kaplan–Meier curves and Cox regression analyses of OS of patients in the first (n = 127) and the second (n = 65) cluster according to F. prausnitzii baseline level.

Figure 3. F. prausnitzii EXL01strain restores anti-tumor response to ICI in presence of ABX-induced gut microbiota perturbation.

(a) Schematic representation of the MCA205 tumor-bearing mouse model. After a 2-weeks ABX treatment, C57BL/6 wildtype mice were inoculated subcutaneously with sarcoma MCA205 tumor cell line at Day 0. On Days 6, 9, 12, and 15 post-tumor inoculation, mice were treated with or without intraperitoneal administration of anti-PD-L1 antibody (5 mg/kg) and starting from Day 6, received either E×L01 strain or vehicle alone, daily by oral gavage (10¹⁰ TCC per dose) until Day 30. (b) Tumor growth overtime in vehicle (n = 20), anti-PD-L1 (n = 20), anti-PD-L1+ABX (n = 25) and anti-PD-L1+ABX+EXL01 (n = 25) groups. Two-way ANOVA tests with correction for FDR according to BH (p < .05, q < 0.1) were used to compare the groups. *p < .05; **p < .01; ***p < .001; ****p < .0001. (c) Mean tumor volume at day 26 post-inoculation. Kruskal Wallis tests with BH correction for multiple comparison were used to compare the groups. *p < .05, ***p < .001. (d) Kaplan–Meier curves and Cox regression analyses of the overall survival (OS) of the different groups. Data are mean±SEM of two independent experiments.

Figure 4. F. prausnitzii EXL01 strain administration does not impact fecal microbiota composition and diversity.

(a) Alpha diversity indices (Shannon) calculated from the raw taxonomic tables of the mouse groups at different time points during the course of the experiment. Kruskal–Wallis tests with Dunn’s test post-hoc (Benjamini–Hochberg p-value correction method) were used to compare the three groups. *p < .05; **p < .01; ***p < .001. (b) Relative abundance of prokaryotic taxa identified at the different time points in microbiota of the mouse groups at family level. (c) PCoA built from the Bray-Curtis dissimilarity matrices constructed from the normalized abundance of species of each microbiota. Ellipses were drawn around the centroids of each emerging community (Treatment with or without ABX; PERMANOVA: F = 85.667, df = 1, P = .001) at 95% (inner) and 97% (outer) confidence intervals. (d) Distance of each group from its baseline at day 14, obtained using Bray-Curtis dissimilarity matrices.

Figure 5. F. prausnitzii EXL01strain administration restores normal response to ICI at the tumor transcriptomics level in gut microbiota perturbation context.

(a) PCA built from normalized gene counts. Ellipses were drawn around the centroids of each mice group. (b) Number of genes significantly differentially expressed between the indicated groups. (c) Bubble plot built from KEGG pathways that were significantly down- and up-regulated in the pairwise comparison of the mouse groups. (d) Significant HALLMARK pathways down- and up-regulated (best P adj) in the pairwise comparison. (e) Schematic representation of the results of the RNA-Seq analysis.

Figure 6. F. prausnitzii EXL01 boost activation of dendritic cells and T cells in the presence of anti-PD-L1.

(a) Production of IFN$\mathit{\gamma }$ by CD4+ T cells prepared from the PBMCs of healthy human donors (n = 3), after stimulation with increasing doses of EXL01 strain (MOI 0, 1, 10, 100). (b-d) Expression of PD-L1, CD80 (CD28 ligand) and CD209 by mDC differentiated from the PBMCs of healthy human donors (n = 3), after 1-d maturation in the presence of LPS and increasing doses of EXL01 strain. (e) Schematic representation of the mixed lymphocyte reaction (MLR) assay performed from three independent healthy donor pairs. (f) Production of IFN$\mathit{\gamma }$ by CD4+ T cells in co-culture with mDC (MLR assay, n = 3) after stimulation with increasing doses of EXL01 strain. (g) Percentage of proliferative CD4+ T cells in co-culture with mDC (MLR assay, one representative donor) after stimulation with increasing doses of EXL01 strain. Kruskal Wallis tests with BH correction for multiple comparisons were used to compare the groups. *p < .05, **p < .01, ***p < .001.

Data availability statement

Raw sequence RNAseq data are accessible in Gene Expression Omnibus (accession number GSE234818), and 16S data are accessible in the European Nucleotide Archive (accession number PRJNA975093).