Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals

Abstract

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.

FIG. 1.

Responses of CD4⁺ T cells after stimulation with H. pylori MP. CD4⁺ T cells from Hp+ and Hp− volunteers were stimulated with MP-pulsed DCs, and the resulting proliferation (A) and IFN-γ production (B) were determined. Each symbol represents the response of one individual after subtraction of the background response. Median values are indicated by horizontal bars.

FIG. 2.

Responses of memory and naive CD4⁺ T cells after stimulation with MP or TT. (A) CD4⁺ cells were labeled with anti-CD45RA-FITC and anti-L-selectin-PE antibodies and sorted into memory (CD45RA⁻ L-selectin^+/− or CD45RA⁺ L-selectin⁻) and naive (CD45RA⁺ L-selectin⁺) populations. The contour plots show 1 representative experiment out of 22. The percentages of memory and naive cells are shown inside the gates. (B to E) Memory and naive cells were stimulated with DCs pulsed with MP (B and C) or TT (D and E), and the resulting proliferation and IFN-γ production were determined. Each symbol represents the response of one individual after subtraction of the background response. Median values are indicated by horizontal bars. The Mann-Whitney test was used for statistical evaluation. n.s., not significant.

FIG. 3.

Effects of IL-2 on MP- and TT-induced memory CD4⁺ T-cell responses. Memory CD4⁺ T cells from Hp+ (closed symbols) and Hp− (open symbols) volunteers were stimulated with DCs pulsed with MP or TT in the presence or absence of 25 U of IL-2/ml, and proliferation (A) and IFN-γ production (B) were determined. Values are shown after subtraction of background responses. Median values are indicated by horizontal bars. A paired t test was used for statistical evaluation.

FIG. 4.

Effects of CD4⁺ CD25^high Treg cells on MP- and TT-induced memory T-cell responses. (A) CD4⁺ cells were labeled with anti-CD25- allophycocyanin, anti-CD45RA- FITC, and anti-L-selectin- PE antibodies. CD45RA⁻ L-selectin^+/− or CD45RA⁺ L-selectin⁻ memory cells were then sorted into CD25^high and CD25^low/− cells. The histograms show one representative experiment out of nine. The percentages of CD25^high cells are shown inside the gates. (B to D) Memory cells and CD25^low/− memory cells from Hp+ (closed symbols) and Hp− (open symbols) volunteers were stimulated with DCs pulsed with MP (B and C) or TT (D), and proliferation and IFN-γ production were determined. Values are shown after subtraction of background responses. Median values are indicated by horizontal bars. A paired t test was used for statistical evaluation. n.s., not significant. (E and F) CD25^high and CD25^low/− memory cells from Hp+ volunteers were cultured together (cell ratio, 1:1) and stimulated with DCs pulsed with MP, and proliferation and IFN-γ production were determined. The responses of memory cells were set to 100% in each experiment. Values shown are arithmetic means and standard errors of the means of three independent experiments.

FIG. 5.

Flow cytometric analysis of proliferation induced in CD4⁺ memory cells by stimulation with MP and PHA. CD4⁺ memory and CD4⁺ CD25^low/− memory cells were labeled with CFSE and stimulated with MP-pulsed DCs or PHA. The proliferation of CD3⁺ cells after 6 days of culturing was determined by flow cytometry. The histograms shown are from one experiment out of two. The percentages of proliferating cells are shown inside the gates. ag, antigen.