The frequency of circulating integrin α4β7+ cells correlates with protection against Helicobacter pylori infection in immunized mice

Abstract

Background: Chronic Helicobacter pylori infection is the cause of peptic ulcers in a subpopulation of individuals and a risk factor for the development of gastric cancer. A vaccine against H pylori infection can prevent the acquisition of the infection and protect against reinfections. Clinical trials to date evaluating the efficacy of H pylori vaccines in human challenge models have shown moderate to poor protection with difficulties in predicting efficacy. Thus, while further studies are needed to design an effective vaccine, we also need to find relevant correlates for vaccine efficacy.

Objective: To find immune correlates to vaccine efficacy, the frequencies of neutrophils, eosinophils and inflammatory monocytes and CD4⁺ T-cell memory and mucosa homing integrin α4β7⁺ cells were assessed by flow cytometry in the blood of mice after vaccination.

Materials and methods: H pylori antigens and cholera toxin or the multiple mutant CT (mmCT) were administered via the sublingual (SL) and intragastric route (IG). The vaccinated mice were infected with H pylori strain SS1 bacteria, and colonization in the stomach and immune responses were evaluated.

Results: The H pylori vaccine was effective in reducing bacterial load in the stomach of mice and enhancing immune responses compared to unvaccinated infection controls. In the blood of mice after SL or IG route of vaccination, we observed changes in frequencies of innate and adaptive immune cell subsets compared to infection controls. Remarkably, the frequency of circulating mucosal homing α4β7⁺ CD4⁺ T cells after vaccination correlated with low bacterial load in the stomach of individual mice irrespective of the immunization route.

Conclusions: Our study shows that the innate and adaptive immune cell subsets can be measured in the blood after vaccination and that increased frequency of α4β7⁺ CD4⁺ in the blood after immunization could be used as a predictive marker for the efficacy of vaccine against H pylori infection.

Keywords: Helicobacter pylori; CD4+T cells; IL-17A and IFNγ; inflammatory monocytes; neutrophils; α4β7.

Figure 1

Sublingual immunization induced strong upregulation of neutrophils and inflammatory monocytes in circulation. A, Representative flow cytometry plots of leukocytes, Siglec‐F+ (eosinophils) CD11b⁺Ly6G⁺ (neutrophils) and CD11b⁺Ly6G⁻Ly6CintMHCII⁻ (inflammatory monocytes among CD11b⁺Ly6G⁻ cells). The numbers denote the frequency of cells within the gate. Blood was collected from mice at day 0 (D0), day (D4), and day 7 (D7) after both 1st and 2nd immunizations, and analyzed by flow cytometry the frequency of circulating (B) CD11b⁺Ly6G⁺ neutrophils (C) SSC^highSiglec‐F⁺ eosinophils and (D) CD11b⁺Ly6G^negLy6C^int.MHCII^neg inflammatory monocytes. Data combined are shown from three independent experiments having similar results with n = 3‐5 mice per group per experiment. Bars indicate mean frequencies, and error bars represent standard errors of the mean (SEM) values. Significant upregulation in the frequency of cells at post‐immunizations compared to pre‐immunization was assessed by one‐way ANOVA with Dunnett's posttest, as denoted by *P < .05; **P < .001; ***P < .0001

Figure 2

Upregulation in the frequency of CD4⁺TcRβ⁺ memory CD62L^‐CD4⁺T and gut homing α₄β₇ ⁺ CD4⁺ T cells in the peripheral blood after intragastric immunization. A, Flow cytometry plots of CD4⁺T‐cell populations (TcRβ⁺) and CD62L and α₄β₇ ⁺. In peripheral blood of mice at day 0 (D0), day (D4), and day 7 (D7) after both 1st and 2nd immunizations and frequency of the immunization induced (B) effector memory CD62L⁻CD4⁺T cells and (C) gut homing α₄β₇ ⁺ CD4⁺ T cells. Combined data are shown from three independent experiments giving similar results with n = 3‐5 mice per group per experiment. Bars indicate mean frequencies, and error bars represent standard errors of the mean (SEM) values. Significant upregulation in frequencies of cells at post‐immunizations compared to pre‐immunization was assessed by one‐way ANOVA with Dunnett's posttest, as denoted by *P < .05; **P < .001; ***P < .0001

Figure 3

Protection against H pylori infection induced by intragastric or sublingual immunization with H pylori antigens and CT or mmCT. A, Groups of mice were immunized via the SL or IG route as indicated. Immunized and unimmunized mice were infected with a high dose of live H pylori SS1 bacteria, and the stomachs were collected to assess the protection against H pylori infection. B, Number of bacteria enumerated from the stomach of mice. Each dot represents one mouse, and the line indicates geometric mean. Combined data shown from three independent experiments giving similar results with n = 3‐5 mice per group per experiment. Statistical analysis of the reduction in gastric bacterial loads compared to inf. control was assessed by one‐way ANOVA with Dunnett's posttest, as denoted by ***P < .0001. C, Spearman correlation analysis between the frequency of α₄β₇ ⁺ CD4⁺ T cells in circulation days 4 and 7 after the 1st and 2nd immunization via the SL (left panel) or IG (right panel) route and the number of bacteria in the stomach in the same mice

Figure 4

Cellular and cytokine responses specific to H pylori antigens in response to sublingual (SL) and intragastric (IG) immunization following infection. Splenocyte proliferative responses to H pylori antigens (0.1 or 1 μg/mL) by immunized infected and unimmunized infected mice, determined in vitro by the incorporation of radioactive thymidine indicated as mean counts per minute (cpm) + SEM and IL‐2 levels secreted by the splenocytes in cell culture supernatants (A). Combined data from two independent experiments giving similar results with n = 3‐5 mice per group. Supernatants from stimulation cultures analyzed using the Luminex assay for IL‐17A and GM‐CSF (B) and IFNγ (C). Data are shown from one experiment with n = 3‐5 mice per group. Each dot represents one mouse, and line indicates geometric mean. Statistical significance calculated using the one‐way ANOVA with Dunnett's multiple comparisons test, as denoted by *P < .05; **P < .001; ***P < .0001

Figure 5

Multivariate analysis to elucidate the relationship between SL and IG immunized mice based on analysis of multiple immune parameters in the same mice. OPLS discriminant analysis (OPLS‐DA) displaying the separation between groups mice immunized via the SL (red) or IG (blue) route and with adjuvant CT (square symbol) or mmCT (circle). The X‐variables in the OPLS‐DA analysis were the frequency of α₄β₇ ⁺ CD4⁺ T cells, counts per minute of proliferation of spleen cells and cytokines IL‐17A, IFNγ, GM‐CSF, and IL‐2 in supernatant. R2 denotes the goodness of the fit of the model (values of ≥0.5 defines good discrimination), and Q2 denotes the goodness of prediction of the model (values of no more than 0.2‐0.3 less than the R2 value defines predictive ability)