Helicobacter pylori -Speciﬁc CD4 (cid:1) T Cells Home to and Accumulate in the Human Helicobacter pylori -Infected Gastric Mucosa

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori -induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori -infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4 (cid:1) but similar numbers of CD8 (cid:1) lamina propria T cells were isolated from stomach biopsies from H. pylori -positive compared to H. pylori -negative individuals. CD4 (cid:1) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4 (cid:1) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4 (cid:1) T cells with similar receptor expression were isolated from the duodenum of H. pylori -positive and H. pylori negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori -speciﬁc T-cell responses were detected in stomach CD4 (cid:1) T cells from H. pylori -positive but not H. pylori -negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori -positive individuals, suggesting limited H. pylori speciﬁcity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori -speciﬁc CD4 (cid:1) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment. We of and T cells are present in metaplastic and normal areas of the were separation of mucosa in the present In 2 from were subjected to via their forward and side scatter properties, and T cells were identiﬁed based on their expression of CD4 and/or CD8. Control samples showed that the contamination of CD3 (cid:5) cells in the CD4 and CD8 gates was negligible ( (cid:7) 0.5%). Unstained cells and cells stained with isotype-matched control antibodies served as controls. The numbers of different lymphocyte subsets isolated from 8.5 pooled biopsies from each site and individual were estimated by multiplying the frequencies of CD4 (cid:1) , CD8 (cid:1) , and CD19 (cid:1) cells detected by ﬂow cytometry with the total numbers of isolated LPLs. A membrane preparation (MP) was prepared from H. pylori strain Hel 305 isolated from a duodenal ulcer patient. This strain carries an intact cag pathogenicity island vacuolating cytotoxin s1/m1) and Lewis X antigen. The bacteria were grown on blood agar plates under microaerobic conditions for 3 days, and the MP was prepared by sonication of the bacteria followed by differential centrifugation as previously described (5). Gel electro-phoresis of MP showed that it contained more than 20 different proteins, among which urease, the neutrophil activating protein, the H. pylori adhesin A, and ﬂagellin were identiﬁed by Western blotting using monoclonal antibodies speciﬁc for the different antigens 43).