Lactobacillus gallinarum-derived metabolites boost anti-PD1 efficacy in colorectal cancer by inhibiting regulatory T cells through modulating IDO1/Kyn/AHR axis

Abstract

Objective: Gut microbiota is a key player in dictating immunotherapy response. We aimed to explore the immunomodulatory effect of probiotic Lactobacillus gallinarum and its role in improving anti-programmed cell death protein 1 (PD1) efficacy against colorectal cancer (CRC).

Design: The effects of L. gallinarum in anti-PD1 response were assessed in syngeneic mouse models and azoxymethane/dextran sulfate sodium-induced CRC model. The change of immune landscape was identified by multicolour flow cytometry and validated by immunohistochemistry staining and in vitro functional assays. Liquid chromatography-mass spectrometry was performed to identify the functional metabolites.

Results: L. gallinarum significantly improved anti-PD1 efficacy in two syngeneic mouse models with different microsatellite instability (MSI) statuses (MSI-high for MC38, MSI-low for CT26). Such effect was confirmed in CRC tumourigenesis model. L. gallinarum synergised with anti-PD1 therapy by reducing Foxp3⁺ CD25⁺ regulatory T cell (Treg) intratumoural infiltration, and enhancing effector function of CD8⁺ T cells. L. gallinarum-derived indole-3-carboxylic acid (ICA) was identified as the functional metabolite. Mechanistically, ICA inhibited indoleamine 2,3-dioxygenase (IDO1) expression, therefore suppressing kynurenine (Kyn) production in tumours. ICA also competed with Kyn for binding site on aryl hydrocarbon receptor (AHR) and antagonised Kyn binding on CD4⁺ T cells, thereby inhibiting Treg differentiation in vitro. ICA phenocopied L. gallinarum effect and significantly improved anti-PD1 efficacy in vivo, which could be reversed by Kyn supplementation.

Conclusion: L. gallinarum-derived ICA improved anti-PD1 efficacy in CRC through suppressing CD4+Treg differentiation and enhancing CD8+T cell function by modulating the IDO1/Kyn/AHR axis. L. gallinarum is a potential adjuvant to augment anti-PD1 efficacy against CRC.

Keywords: colorectal cancer; immunotherapy; probiotics.

Figure 1

Lactobacillus gallinarum improved anti-PD1 efficacy in MC38 and CT26 syngeneic mouse models. (A) Schematic diagram of experimental design for syngeneic mouse model. L. gallinarum, in combination with anti-PD1 therapy, significantly inhibited tumour growth in MC38 syngeneic mouse model (microsatellite instability-high model), as evidenced by (B) representative tumour pictures, (C) tumour weight and (D) tumour volume. L. gallinarum also promoted anti-PD1 efficacy in CT26 syngeneic mouse model, an microsatellite instability-low model, as supported by (E) representative tumour pictures, (F) tumour weight and (G) tumour volume. (H) Schematic diagram of experimental design of germ-free mouse model. L. gallinarum mono-colonisation improved anti-PD1 efficacy in germ-free MC38 tumour-bearing mice, as shown by (I) representative tumour picture and (J) tumour weight. Statistical significance was determined by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. Statistical significance of tumour growth curve over time was determined by two-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001. BHI, brain heart infusion; E.c., E. coli; L.g., L. gallinarum; PBS, phosphate-buffered saline; PD1, programmed cell death protein 1; αPD1, anti-PD1.

Figure 2

Lactobacillus gallinarum reduced Foxp3⁺CD25⁺ Treg infiltration and increased IFNγ⁺CD8⁺ T cells in the tumour microenvironment. (A) Representative flow cytometry plots of Foxp3⁺ CD25⁺ Treg suggested a significant reduction of Treg in tumour tissues of L. gallinarum-treated mice compared with that of BHI and E. coli controls. L. gallinarum significantly reduced Foxp3⁺ CD25⁺ Treg infiltration in MC38 tumours and CT26 tumours. (B) Representative histogram of IFNγ⁺ CD8⁺ T cells. Increased IFNγ⁺ CD8⁺ T cells were shown. in L. gallinarum, in combination with anti-PD1-treated MC38 tumours and CT26 tumours. (C) IHC staining confirmed a significant reduction of Foxp3⁺ cells in L. gallinarum+anti-PD1-treated mice. The quantification of Foxp3⁺ cells (the brown spot) was shown as the mean value of three independent HPF in tumours. Scale bar=50 µm. (D) Representative tumour pictures from the Treg depletion model. The administration of anti-CD25 monoclonal antibody abolished the effect of L. gallinarum in promoting anti-PD1 efficacy, as evidenced by (E) tumour weight and (F) tumour volume. Statistical significance was determined by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. Statistical significance of tumour growth curve over time was determined by two-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001. E.c., E. coli; L.g., HPF, high-power fields; L. gallinarum; PD1, programmed cell death protein 1; Tregs, regulatory T cells; αCD25, anti-CD25; αPD1, anti-PD1.

Figure 3

Lactobacillus gallinarum improved anti-PD1 efficacy in AOM/DSS-induced CRC mouse model. (A) Schematic diagram of experimental design for AOM/DSS-induced CRC model. (B) Representative colonoscopy images and (C) representative colon images of AOM/DSS-induced CRC tumourigenesis mouse model. L. gallinarum, in combination with anti-PD1, reduced (D) tumour number, (E) tumour load and (F) number of large tumours (diameter larger than 2 mm). (G) Percentage of Foxp3⁺CD25⁺ Tregs in colonic tumour tissues. (H) Percentage of IFNγ⁺ CD8⁺ T cells in colonic tumour tissues. Statistical significance was determined by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. AOM, azoxymethane; BHI, brain heart infusion; CRC, colorectal cancer; DSS, dextran sulfate sodium; E.c., E. coli; L.g., L. gallinarum; PD1, programmed cell death protein 1; Tregs, regulatory T cells; αPD1, anti-PD1.

Figure 4

Lactobacillus gallinarum produced tryptophan metabolites in vitro and in vivo. (A) Untargeted metabolomics showing the heatmap analysis of BHI, E. coli and L. gallinarum culture supernatant in vitro. (B) Untargeted metabolomics showing heatmap analysis of stool samples from MC38 syngeneic mouse model revealed a differential abundance of metabolites in BHI-treated, E. coli-treated and L. gallinarum-treated mice. E. coli mutant expressing aromatic amino acid aminotransferase (E. coli-ArAT) improved ant-PD1 efficacy in CT26 syngeneic mouse model, as evidenced by (C) representative tumour picture, (D) tumour weight and (E) tumour volume. (F) No significant difference of IAld was observed in serum samples between groups. An elevated level of ICA was detected in serum after L. gallinarum gavage. (G) No significant difference of IAld was observed in tumour tissues between groups. ICA was enriched in tumour tissues of L. gallinarum-treated mice in MC38 syngeneic mouse model. Statistical significance was determined by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. Statistical significance of tumour growth curve over time was determined by two-way analysis of variance. *p<0.05, **p<0.01. ArAT, aromatic amino acid aminotransferase; E.c., E. coli; IAld, indole-3-carboxaldehyde; ICA, indole-3-carboxylic acid. L.g., L. gallinarum; WT, wild-type; αPD1, anti-PD1.

Figure 5

Lactobacillus gallinarum and ICA inhibited IDO1 expression and Kyn production in tumour. L. gallinarum reduced (A) serum Kyn level, (B) tumour Kyn level, (C) Kyn/Trp ratio in tumours in MC38 syngeneic mouse model. (D) RNA sequencing revealed a differential gene expression between BHI plus anti-PD1 group versus L. gallinarum plus anti-PD1 group. (E) Enrichment plot of Trp metabolism pathway. (F) Immunohistochemical staining of IDO1 in tumour tissues of CT26 syngeneic mouse model. Scale bar=50 µm. (G) ICA (5 µM) significantly inhibited IDO1 expression in HCT116 and LoVo cell lines. IFNγ (100 ng/mL) was added 1 day after ICA/DMSO treatment to induce IDO1 expression. (H) ICA (5 µM) reduced Kyn level in HCT116 and LoVo cell lysates, as detected by Liquid chromatography-mass spectrometry, after normalisation of protein concentration. IFNγ (100 ng/mL) was added to induce IDO1 expression. Statistical significance was determined by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. E.c., E. coli; ICA, indole-3-carboxylic acid; IDO1, indoleamine 2,3-dioxygenase; Kyn, kynurenine; L.g., L. gallinarum; Trp, tryptophan.

Figure 6

ICA outcompeted Kyn and inhibited Kyn-mediated AHR activation on CD4+T cells. (A) ICA did not directly affect Treg differentiation in vitro. (B) Effect of ICA and Kyn (50 µM) on Foxp3⁺Treg differentiation. ICA antagonised Kyn-mediated Treg differentiation in a dose-dependent manner. (C) CYP1B1 expression of CD4+T cells treated with different doses of ICA and Kyn (50 µM). ICA significantly inhibited Kyn-mediated upregulation of CYP1B1 expression. (D) CH-233191 (AHR antagonist) (10 µM) abolished the effect of ICA and Kyn. (E) Molecular docking analysis of ICA and Kyn on LBD of human AHR protein. ICA bound to the LBD of AHR protein with a binding energy of −6.654 kcal/mol. Kyn bound to the LBD of AHR protein with a binding energy of −6.985 kcal/mol. (F) SPR assay of ICA and Kyn on human AHR protein. ICA has a higher receptor affinity to AHR compared with Kyn (KD: 39.4 µM vs 61.5 µM). (G) Lineweaver-Burk plot was constructed by treating mouse T cells with various dosages of ICA and Kyn. Reaction velocity (V) is defined as the relative change of CYP1B1 expression per day. Statistical significance was determined by Kruskal-Walli test, followed by Dunn’s multiple comparison test. ***p<0.001, ****p<0.0001. AHR, aryl hydrocarbon receptor; ICA, indole-3-carboxylic acid; KD, dissociation constant; Kyn, kynurenine; LBD, ligand-binding domain; SPR, surface plasmon resonance.

Figure 7

ICA improved anti-PD1 efficacy and was rescued by Kyn supplementation. ICA in combination with anti-PD1 impeded tumour growth, as evidenced by (A) representative tumour picture, (B) tumour weight and (C) tumour volume in CT26 syngeneic mouse model. (D) ICA reduced Foxp3⁺ CD25⁺ infiltration in tumour tissues, which was reversed by Kyn supplementation. (E) ICA increased the proportion of IFNγ⁺ CD8⁺ T cells in tumour tissues, which was reversed by Kyn supplementation. (F) ICA reduced Kyn level in serum and tumour tissues, and decreased Kyn/Trp ratio in tumour tissues, which was reversed by Kyn supplementation. (G) Immunohistochemistry staining revealed that ICA reduced IDO1 expression in tumour tissues. Scale bar=50 µm. (H) Schematic diagram outlining the mechanistic pathway of how L. gallinarum improved anti-PD1 efficacy in colorectal cancer. Statistical significance was determined by Mann-Whitney U test or Kruskal-Wallis test, followed by Dunn’s multiple comparison test, where appropriate. Statistical significance of tumour growth curve over time was determined by two-way analysis of variance. *p<0.05, **p<0.01, ****p<0.0001. ICA, indole-3-carboxylic acid; Kyn, kynurenine; Tregs, regulatory T cells; Trp, tryptophan; αPD1, anti-PD1.