Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients

Abstract

Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4(+) CD25(high) T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4(+) CD25(low) and CD4(+) CD25(-) cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4(+) CD25(high) T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4(+) CD25(high) cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.

FIG. 1.

Gating approach for discrimination of CD25^high, CD25^low, and CD25⁻ cells. Cells were labeled with antibodies against CD4, CD8, and CD25 and then analyzed by FCM. For this example, the CD25 expression on CD4⁺ and CD8⁺ T cells from the antral mucosa of an H. pylori-positive individual was analyzed in a single plot. The gates for the CD25^high and CD25^low populations were set by comparing the CD25 expression levels of CD4⁺ and CD8⁺ cells, as described in Materials and Methods.

FIG. 2.

Expression of CD25 and CTLA-4 on T cells isolated from the antral and duodenal lamina propriae and peripheral blood of H. pylori-positive asymptomatic individuals and H. pylori-negative controls. (A) CD25 expression detected by FCM on CD4⁺ and CD8⁺ T cells. CD25^high cells were gated as indicated in the figure, and the frequencies of CD4⁺ cells expressing CD25^high are shown by the numbers outside the gates. The results shown are for one representative experiment of seven experiments with H. pylori-positive individuals and six experiments with H. pylori-negative individuals. (B) Presence of CD4⁺ CD25^high cells in the antral and duodenal lamina propriae and peripheral blood of H. pylori-positive and -negative individuals. Each symbol represents the percentage of CD25^high cells among the CD4⁺ lymphocytes for one individual, and median values are indicated by horizontal bars. (C) Intracellular expression of CTLA-4 on CD4⁺ CD25^high, CD25^low, and CD25⁻ cells from an H. pylori-positive individual. The results shown are from one representative experiment of three total experiments. Five thousand or more CD4⁺ cells and ≥250 CD4⁺ CD25^high cells were analyzed for each sample.

FIG. 3.

Expression of FOXP3 and CD25 by CD4⁺ T cells from peripheral blood. Peripheral blood T cells were sorted into CD4⁺ CD25^high, CD4⁺ CD25^low, and CD4⁺ CD25⁻ cell fractions, and the expression of FOXP3 and CD25 before and after stimulation with anti-CD3 and anti-CD28 antibodies was determined. (A and C) FOXP3 mRNA as well as mRNA for the housekeeping gene β-actin was detected by RT-PCR, and the PCR products were visualized in silver-stained polyacrylamide gels. Results for one representative experiment of three total experiments are shown. (B and D) Expression levels of FOXP3 were determined by real-time PCR and were related to the level of the housekeeping gene GAPDH. The values shown are arithmetic means plus standard errors of the means (SEM) (n = 3). (E) CD25 expression on sorted cells, as determined by FCM. The results shown are from one representative experiment of three total experiments.

FIG. 4.

FOXP3 expression by CD4⁺ T cells isolated from antral and duodenal lamina propriae and peripheral blood from asymptomatic H. pylori-positive individuals. T cells were sorted into CD4⁺ CD25^high, CD4⁺ CD25^low, and CD4⁺ CD25⁻ cell fractions, and the expression of FOXP3 mRNA was determined. (A) FOXP3 mRNA as well as mRNA for the housekeeping gene β-actin was detected by RT-PCR, and the PCR products were visualized in silver-stained polyacrylamide gels. Results for one experiment of two total experiments are shown. (B) Expression levels of FOXP3 were determined by real-time PCR and were related to the level of the housekeeping gene GAPDH. The values shown are arithmetic means plus SEM (n = 2).

FIG. 5.

CD25 expression in the antrum of an H. pylori-positive asymptomatic individual, as detected by IHC. Cryosections were stained for CD25 and analyzed under a microscope. Arrows indicate CD25⁺ MNCs. Original magnification, ×400.

FIG. 6.

Expression of CD25 and FOXP3 by lamina propria T cells isolated from histologically normal and cancer-affected stomach mucosae of H. pylori-positive gastric adenocarcinoma patients. (A) CD25 expression on gated CD4⁺ and CD8⁺ T cells. The results shown are from one representative experiment each of seven experiments with healthy tissue and five experiments with cancer tissue. (B) Presence of CD4⁺ CD25^high cells in healthy and cancer-affected stomach mucosae. Each symbol represents the percentage of CD25^high cells among the CD4⁺ lymphocytes from one individual, and median values are indicated by horizontal bars. For FCM, ≥3,500 CD4⁺ cells and ≥150 CD4⁺ CD25^high cells were analyzed for each sample. (C) Expression levels of FOXP3 in sorted CD4⁺ CD25^high, CD4⁺ CD25^low, and CD4⁺ CD25⁻ cells isolated from histologically normal stomach mucosa were determined by real-time PCR and then related to the level of the housekeeping gene GAPDH. The values shown are arithmetic means plus SEM (n = 2).