Immunodominant epitope-specific Th1 but not Th17 responses mediate protection against Helicobacter pylori infection following UreB vaccination of BALB/c mice

Abstract

Helicobacter pylori (H. pylori) infects more than half of the world's population, causing chronic gastritis, peptic ulcers and gastric cancer. Urease B subunit (UreB), a conserved protein of H. pylori, is capable of inducing specific CD4(+) T-cell responses and provides protection against this infection. Previous studies have confirmed the effectiveness of rUreB subunit vaccines in generating CD4(+) T-cell-mediated protection, but less is known regarding the roles of different subtypes of T-cell immunity, such as Th1, Th2 and Th17, particularly the immunodominant epitopes inducing specific CD4(+) T-cell responses, in vaccine-mediated protection. In this study, we demonstrated that the vaccination of BALB/c mice with rUreB resulted in significant antigen-specific Th1 and Th17 immune responses. Importantly, two novel Th epitopes, UreB317-329 and UreB409-421, which are recognized by a major population of CD4(+) T cells, were identified in immunized mice. Our results demonstrated that two novel epitopes can simultaneously induce Th1 and Th17 immune responses; however, only the epitope vaccine-induced CD4(+) T-cells secreting IFN-γ mediated the protection against H. pylori; cells secreting IL-17A did not. Taken together, our results suggest that two novel immunodominant epitopes can induce Th1 and Th17 immune responses, but only the induced Th1 lymphocytes mediate protection against H. pylori.

Figure 1. Types of CD4+ T-cell responses elicited via UreB immunization.

(a) H. pylori colonization of the gastric mucosa of UreB-immunized mice at 4 weeks after the last infection. (b) Proliferation of CD4⁺ and CD8+ T cells isolated from the spleens of UreB-immunized mice was assessed using the ³H-TdR incorporation assay. (c) The proliferation of CD4⁺ and CD8+ T cells isolated from stomach-draining lymph nodes of UreB-immunized mice was assessed using the ³H-TdR incorporation assay. (d) IFN-γ, IL-17A and IL-4 mRNA levels in CD4⁺ T cells isolated from UreB-immunized mice were analyzed. (e) Splenic lymphocytes from UreB-immunized mice or PBS controls were cultured in vitro in the presence of recombinant UreB. IFN-γ-producing and IL-17A-producing CD4⁺ T cells were assessed using the UreB peptide pool on day 7. (f) IFN-γ-producing CD4⁺ T-cell responses of all immunized mice were assayed. (g) IL-17A-producing CD4⁺ T-cell responses of all immunized mice were assayed. All results were repeated more than three times. The data are expressed as the mean ± S.D (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Mapping of UreB immunodominant epitopes.

(a) UreB-specific T cells expanded from immunized mice were screened for their specific IFN-γ response to 93 overlapping 18-mer UreB peptides at a final concentration of 5 μmol/l in an ICS assay. The locations of the identified 18-mer sequences are shown. The embedding data showed the responses of immunized mice and PBS control mice to the UreB peptide pool. (b) The 13-mer overlapping peptides within the UreB_313–330 18-mers were screened (left). The 13-mer peptides within UreB_403–420 and UreB_409–426 were screened (middle). Peptides were titrated to compare their activities (right). (c) Th17 epitopes within the 93 overlapping 18-mer UreB peptides were identified using UreB-specific T cells expanded from immunized mice. The embedding data showed the responses of immunized mice and PBS control mice to the UreB peptide pool. (d) 13-mer peptides within UreB_313–330 18-mers were screened as described in b (left). The 13-mer peptides within UreB_403–420 and UreB_409–426 were screened (middle). Peptides were titrated under serum-free conditions to compare their activities (right). The results are representative of five independent experiments. The data are expressed as the mean ± S.D. (n = 10). ***P < 0.001.

Figure 3. Detailed characterization of immunodominant anti-UreB CD4⁺ T-cell responses.

(a) MHC antibodies were used to identify the restriction profile of the Th1 epitope UreB_317–329 (left). Natural processing and presentation of the novel Th1 epitope UreB_317–329 by DCs (right). (b) Information of the three Th1 peptides predicted using a bioinformatics approach, including the location, amino acid sequences, length and MHC restriction profiles confirmed in our previous study (left). IFN-γ-producing CD4⁺ T-cell responses induced by UreB_317–329 and the predicted peptides were analyzed (right). (c) MHC antibodies were used to identify the restriction profile of UreB_317–329 epitope-specific Th17 responses (up). Natural processing and presentation of the epitope UreB_317–329 inducing CD4+ T cells secreting IL-17A (down). (d) The MHC restriction profile for UreB_409–421 inducing Th1 responses was determined using MHC antibodies (left). Natural processing and presentation of the epitope UreB_409–421 inducing CD4+ T cells secreting IFN-γ (right). (e) Information of the three Th1 peptides predicted using a bioinformatics approach (left). IFN-γ-producing CD4⁺ T-cell responses induced by UreB_409–421 and the predicted peptides were analyzed (right). (f) The MHC restriction profile for UreB_409–421 inducing Th17 responses was determined using MHC antibodies (up). The natural processing and presentation of the epitope UreB_409–421 induced CD4+ T cell secretion of IL-17A (down). The results were repeated five times. The data are expressed as the mean ± S.D (n = 10).

Figure 4. Distinguishing between epitope-specific Th1 and Th17 cells.

Splenic lymphocytes of mice immunized with UreB antigen were isolated and cultured in the presence of UreB in vitro and then harvested. The responses of the cultured CD4⁺ T cells to peptides UreB_317–329/UreB_409–421 and homing receptors expressed by epitope specific T cells were detected via flow cytometry. (a) The expanded CD4⁺ T cells secreting IFN-γ and IL-17A were determined simultaneously using the ICS assay after the stimulation of peptides UreB_317–329 and UreB_409–421. All cells were identified under CD3⁺ and CD4⁺ gates. The results are representative of five independent experiments. (b) The kinetics of epitope-specific T-cell responses, Th1 (middle) and Th17 (right), and non-responsive cells (left) were assessed. (c) UreB_317–329 and UreB_409–421 epitope-specific Th1 and Th17 cells expressing α4β7 homing receptors were analyzed using flow cytometry. Representative flow cytometry plots of five independent experiments for α4β7 expression on the surface of specific T lymphocytes are shown. Data are shown under CD4⁺ IFN-γ⁺ and CD4⁺ IL-17⁺ gates. (d) UreB_317–329 and UreB_409–421 epitope-specific Th1 and Th17 cells expressing L-selectin chemokine receptors were analyzed using flow cytometry, and representative flow cytometric plots of five independent experiments for L-selectin expression on the surface of specific T lymphocytes are shown. Data are shown under CD4⁺ IFN-γ⁺ and CD4⁺ IL-17⁺ gates. The data are expressed as the mean ± S.D (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Effect of two novel immunodominant epitopes against H. pylori infection.

(a) H. pylori colonization in the stomachs of mice immunized with peptides was determined using real-time quantitative PCR. (b) The number of gastric mucosal CD4⁺ T lymphocytes was analyzed using flow cytometry (left). Gastric mucosal CD4⁺ T cells expressing α4β7 homing receptors were quantified (middle). Gastric mucosal CD4⁺ T cells expressing L-selectin chemokine receptors were determined (right). (c) Gastric inflammation scores of mice immunized with peptides were identified 4 weeks after infection and representative gastric histopathology of immunized mice and PBS controls are shown (H&E staining, original magnification × 200). (d) The epitope-specific CD4⁺ T-cell responses, including Th1, Th17 and Th2, of mice immunized with UreB_317–329 peptide were analyzed. (e) The epitope-specific CD4⁺ T-cell responses, including Th1, Th17 and Th2, of mice immunized with UreB_409–421 peptide were determined. All data were repeated more than three times. The data are expressed as the mean ± S.D (n = 10). **P < 0.01, ***P < 0.001.

Figure 6. Determination of the protective effects of epitope-specific Th1 and Th17 cells.

(a) Splenic lymphocytes of mice immunized with the peptides, UreB_317–329 and UreB_409–421, were cultured in vitro in the presence of the peptides. Then, epitope-specific Th1 and Th17 cells were isolated using a cell enrichment and detection kit, and their purities were assessed via flow cytometry. The results are representative of five independent experiments (b) After adoptive transfer with epitopes-specific Th1 or Th17 cells, H. pylori colonization of the stomachs of mice was determined. (c) UreB_317–329-specific Th1 responses of mice transferred with specific CD4⁺ T cells secreting IFN-γ were analyzed (left). UreB_317–329-specific Th17 responses of mice transferred with specific CD4⁺ T cells secreting IL-17A were analyzed (right). (d) UreB_409–421 specific Th1 and Th17 responses of mice transferred with specific CD4⁺ T cells secreting IFN-γ and IL-17A were analyzed as described in c. (e) IL-17^−/− mice were immunized with peptides (UreB_317–329 combined with UreB_407–419) or UreB. Four weeks after H. pylori challenge, the bacterial colonization in the stomachs of mice immunized with peptides (left) and UreB (right) was assessed. The results of (b–e) were repeated three times. The data are expressed as the mean ± S.D (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.