Helicobacter pylori infection has a detrimental impact on the efficacy of cancer immunotherapies

Abstract

Objective: In this study, we determined whether Helicobacter pylori (H. pylori) infection dampens the efficacy of cancer immunotherapies.

Design: Using mouse models, we evaluated whether immune checkpoint inhibitors or vaccine-based immunotherapies are effective in reducing tumour volumes of H. pylori-infected mice. In humans, we evaluated the correlation between H. pylori seropositivity and the efficacy of the programmed cell death protein 1 (PD-1) blockade therapy in patients with non-small-cell lung cancer (NSCLC).

Results: In mice engrafted with MC38 colon adenocarcinoma or B16-OVA melanoma cells, the tumour volumes of non-infected mice undergoing anticytotoxic T-lymphocyte-associated protein 4 and/or programmed death ligand 1 or anti-cancer vaccine treatments were significantly smaller than those of infected mice. We observed a decreased number and activation status of tumour-specific CD8⁺ T cells in the tumours of infected mice treated with cancer immunotherapies independent of the gut microbiome composition. Additionally, by performing an in vitro co-culture assay, we observed that dendritic cells of infected mice promote lower tumour-specific CD8⁺ T cell proliferation. We performed retrospective human clinical studies in two independent cohorts. In the Dijon cohort, H. pylori seropositivity was found to be associated with a decreased NSCLC patient survival on anti-PD-1 therapy. The survival median for H. pylori seropositive patients was 6.7 months compared with 15.4 months for seronegative patients (p=0.001). Additionally, in the Montreal cohort, H. pylori seropositivity was found to be associated with an apparent decrease of NSCLC patient progression-free survival on anti-PD-1 therapy.

Conclusion: Our study unveils for the first time that the stomach microbiota affects the response to cancer immunotherapies and that H. pylori serology would be a powerful tool to personalize cancer immunotherapy treatment.

Keywords: Helicobacter pylori; cancer; cancer immunobiology; immunotherapy.

Figure 1

Helicobacter pylori infection decreases the effectiveness of cancer immunotherapies in preclinical models. (A) Mice were injected with MC38 colon adenocarcinoma cells and intraperitoneally injected with anti-CTLA4 (αCTLA4) or IgG2b isotype as control. At day 19, tumour volumes of non-infected (NI) and infected (INF) mice treated with anti-CTLA4 therapy were significantly different (p<0.01, two-way analysis of variance (ANOVA)). Experimental groups included seven mice. (B) MC38 tumour growth kinetics of NI and INF mice treated with anti-CTLA4/PD-L1 (αCTLA4/αPD-L1) combination therapy. Anti-CTLA4/PD-L1 treatment resulted in tumour rejection in seven of eight NI mice, whereas anti-CTLA4/PD-L1-treated INF mice showed tumour rejection in two of six mice. At day 21, the tumour volumes of NI and INF mice treated with anti-CTLA4/PD-L1 antibodies were statistically different (p=0.009, two-way ANOVA). Experimental groups included five to eight mice. (C) B16-OVA tumour growth kinetics of NI and INF mice treated with an anti-cancer vaccine (VAC) or phosphate-buffered saline (PBS) as a control. Vaccination resulted in decreased tumour growth in NI mice, however, did not efficiently limit tumour growth in INF mice. At day 20, tumour volumes of vaccinated NI and INF mice were statistically different (p<0.05, two-way ANOVA). Experimental groups included five to eight mice. (D) B16-OVA tumour growth kinetics of NI and H. felis (H.f)-infected mice treated with an anti-cancer vaccine or PBS as a control. Vaccination resulted in statistically significant decrease in tumour growth in NI and H.f-infected mice (p<0.001, two-way ANOVA). Experimental groups included nine to ten mice. (E) MC38 tumour growth kinetics of NI and H.f-infected mice treated with anti-CTLA4 (αCTLA4) therapy. Anti-CTLA4 treatment resulted in decreased tumour growth in both NI and H.f mice. At day 19, the tumour volumes of NI and H.f mice treated with anti-CTLA4 antibodies were not statistically different (p=0.65, two-way ANOVA). Experimental groups included six mice. (F) Number of colon tumours of NI and H. pylori INF-infected mice treated with anti-CTLA4 (αCTLA4) or IgG2b isotype as control. Anti-CTLA4 injection resulted in decreased tumour number in NI mice but not in INF mice. At sacrifice, tumour numbers of anti-CTLA4-injected NI and INF mice were statistically different (p<0.0013, Mann-Whitney test). Experimental groups included ten mice. For the experiments described in figure 1A–F, the infectious status of each individual mouse was confirmed at sacrifice by performing rapid urease tests and/or colony forming units on the stomach (see online supplemental figures S2A–F). CTLA4, cytotoxic T-lymphocyte-associated protein 4; PD-L1, programmed death ligand 1.

Figure 2

Helicobacter pylori-mediated immunosuppression of cancer immunotherapies is independent of the H. pylori-induced modification of the faecal microbiota. (A) Co-housing experiments, non-infected (NI) (n=9) and H. pylori-infected (INF) (n=10) mice were subcutaneously injected with B16-OVA tumour cells and vaccinated. Vaccination (VAC) showed very limited tumour growth inhibition in INF mice. At days 27 and 30, the tumour volumes of vaccinated NI and INF mice were statistically different (p<0.001, two-way analysis of variance (ANOVA)). At sacrifice, we performed rapid urease tests on the stomach of NI and INF co-housed mice. We only detected H. pylori infection in INF and not in NI mice, confirming that co-housing does not allow for H. pylori transmission (see online supplemental figures S2A). (B) Shannon, Inverse Simpson and Bray-Curtis based Non-metric Multidimensional Scaling (NMDS) analysis of 16S rRNA gene sequencing of the intestinal microbiota before the initiation of the vaccine-based immunotherapy. 16S rRNA analyses were performed in non-co-housing conditions. At steady state, INF mice displayed higher alpha diversity compared with NI mice (p=0.02, Mann-Whitney test). (C) 16S rRNA gene sequencing of the intestinal microbiota before the initiation of the vaccine-based immunotherapy. Differential abundance analysis performed at the genus taxonomic level comparing 16S rRNA sequencing of INF vs NI mice at steady state. Only bacteria with an adjusted p value <0.05 are displayed (Benjamini-Hochberg method). (D) Shannon, Inverse Simpson and Bray-Curtis based Non-metric Multidimensional Scaling (NMDS) analyses of 16S rRNA gene sequencing of the intestinal microbiota during vaccine-based immunotherapy. The faecal microbiota of INF and NI mice were similar on day 8 post CpG/OVA vaccination (no statistical difference, Mann-Whitney test). (E) Faecal transplantation of INF faeces into NI or INF recipient mice. Recipient mice were treated with antibiotics and orally administered with 100 mg of faeces from INF mice on a daily basis for 5 days. Two weeks after the last oral gavage, vaccine-based immunotherapy was initiated on B16-OVA tumour-bearing mice (online supplemental figures S1E). Remarkably, vaccinated NI mice transplanted with INF faeces (n=9) are still capable to very efficiently control tumour growth compared with non-vaccinated (PBS) counterparts (n=7) (p<0.001, two-way ANOVA). Additionally, vaccinated NI mice transplanted with INF faeces (n=9) show better tumour growth control compared with vaccinated INF mice transplanted with INF faeces (n=4) (p<0.001, two-way ANOVA). We observed that faecal transplantation of INF faeces to NI recipient mice do not allow for H. pylori infection (online supplemental figures S2C). (F) Antibiotherapy (ATB) does not increase the efficacy of anti-cancer vaccination. Mice were infected with H. pylori during the neonatal period and treated with ATB at 6 weeks of age. One-month post ATB, mice were engrafted with B16-OVA melanoma cells and vaccinated (online supplemental figures S1F). We observed that the eradication of H. pylori infection by ATB (online supplemental figure S2B) did not substantially increase the efficacy of cancer vaccination (p<0.05, two-way ANOVA). For panels A–D and F, data shown are representative of two independent experiments. For the experiments described in figure 2A–D, the infectious status of each individual mouse was confirmed at sacrifice by performing rapid urease tests and/or colony forming unit on the stomach (see online supplemental figures S2A–C).

Figure 3

Helicobacter pylori jeopardises tumour-specific immune responses. (A) Absolute cell number and activation status of OT-1 CD8⁺ T cells isolated from the tdLN, ndLN and tumour(s) of non-infected (NI) and infected (INF) vaccinated B16-OVA tumour-bearing mice (days 10 and 15 post B16 inoculation, respectively, for LNs and tumours). Activated OT-1 cells that were defined has CD44⁺CD62L⁻. (B) Left panel: percentage of proliferating OT-1 cells in the tdLN of NI and INF mice evaluated by carboxyfluorescein succinimidyl ester staining. Right panel: in vivo cytotoxic activities of OT-1 cells in vaccinated NI and INF mice. (C) The presence of H. pylori affects the functionality of DCs. Left panel: expression of the activation marker CD86 on CD11c⁺CD11b⁻CD8a⁺ DCs in the tdLN of NI and INF mice. Right panel: frequency of proliferating OT-1 cells on ex vivo stimulation with DCs isolated from the spleen of NI or INF mice. Data are representative of three independent experiments (NI, n=9; INF, n=10) (p<0.05, unpaired t-test). (D) The presence of H. pylori affects innate immune responses, as determined by inflammatory cytokine (TNFα, IFNγ, IL-6 and IL-17) levels in the serum of NI and INF mice. NI and INF B16-OVA tumour-bearing mice were injected with OT-1 cells and vaccinated with OVA in CpG. The serums were recovered on days 1, 2 and 3 postvaccination. For panels A–C, data shown are representative of three independent experiments. For panel D, data shown are representative of two independent experiments. For the experiments described in figure 3A–D, the infectious status of each individual mouse was confirmed at sacrifice by performing rapid urease tests and/or colony forming unit on the stomach (see online supplemental figures S2E). DCs, dendritic cells; MFI, mean fluorescence intensity; ndLN, non-draining lymph nodes; tdLN, tumour-draining lymph node.

Figure 4

Helicobacter pylori seropositivity is associated with reduced effectiveness of anti-PD-1 immunotherapy in patients with non-small cell lung cancer (NSCLC). (A) Dijon cohort: overall survival and overall progression-free survival of patients with NSCLC from the Dijon cohort (60 patients). Kaplan-Meier curves depicting the overall survival of patients with NSCLC treated with nivolumab or pembrolizumab (anti-PD-1 monoclonal antibodies). H. pylori seropositivity was found to be associated with a clearly defined decrease of NSCLC patient survival on anti-PD-1 therapy (p=0.001, Wald test as part of a univariate of Cox model). (B) Montreal cohort: overall survival and overall progression-free survival of patients with NSCLC from the Montreal cohort (29 patients). Kaplan-Meier curves depicting the overall survival of patients with NSCLC treated with nivolumab or pembrolizumab. Log-rank (Mantel-Cox) test *p<0.05. (C) H. pylori infection substantially affects myeloid cells in the tumour(s) of patients with NSCLC. RNAseq analysis was performed on the tumours of patients from the discovery cohort. MCP-counter software was used to determine the tumour infiltration of CD3⁺ T cells, CD8⁺ T cells, cytotoxic lymphocytes, NK cells, B lymphocytes, cells originating from monocytes (monocytic lineage), myeloid-derived dendritic cells, neutrophils, endothelial cells and fibroblasts. We observed that in the tumours of H. pylori seronegative patients, there was a substantially higher expression of genes expressed by cells originating from monocytes compared with H. pylori seropositive patients (p=0.01, Wilcoxon test). (D) Gene-set enrichment analyses (GSEA) were performed on the RNAseq data and revealed that H. pylori infection substantially decreases the expression of genes that are controlled by type I IFN, IFNγ and IL-6 in the tumours of patients with NSCLC undergoing immunotherapy. PD-1, programmed cell death protein 1.