Pd1(+) Regulatory T Cells Are Increased in Helicobacter pylori-Associated Gastritis

INTRODUCTION: Regulatory T cells (Treg) (CD4+CD25+FoxP3+) play a fundamental role in modulating the balance between inflammation and immune tolerance and are identified as a factor that contributes to bacterial persistence and to infection chronicity. It is emerging that Treg include different functional and phenotypic subpopulations according to the expression intensity of surface CD25 molecule (IL-2 receptor alpha-chains). Moreover, more recently it has been described that they can be subclassified based on the surface expression of programmed death receptor 1 (PD1). PD-1 is a negative co-stimulatory molecule that plays an important role in the balance and regulation of adaptive immune responses. PD1/PDL1 engagement can down-regulate dramatically T cell activation. AIM: To evaluate Treg cell subtypes in relation with phenotype CD25 and PD1 in H. pylori(+) and H. pylori () biopsies specimens from patients suffering gastritis. METHODS: We analyzed CD4(+) lymphocytes from 16 gastric biopsy specimens of patients with gastritis. H. pylori infection was diagnosed with histology and rapid urease test. Afterwards, it was confirmed by RT PCR assay with SYBR Green I fluorochrome, performed on gastric biopsies using a set of primers for the ureC gene specific for the bacteria. 12 cases of H. pylori(+)-gastritis and 4 of gastritis where H. pylori was not detected were studied. After disintegrating biopsies with a needle, lymphocytes were isolated stirring the remaining tissue in a collagenase-DNase solution (100 U collagenase/mL and 0.1 mg DNase/mL) at 37°C for 2h. The cell suspension was then filtered through mesh and the staining for flow cytometry was performed. The following antibodies were used: anti-PD1-FITC, anti-CD4-PECy5 and anti-CD25-APC. For intracellular labeling with anti-Foxp3-PE, cells were previously permeabilized using Cytofix/ Cytoperm solution. Cells were classified in CD25+ and CD25++ according to the fluorescence of this antibody (cut-off was determined with CD8(+) fluorescence). RESULTS: The percentage of CD4+CD25++Foxp3+ was elevated 5-fold in H. pylori(+) compared to H. pylori(-) samples. The number of CD4+CD25+Foxp3+ was similar in both cases. The subpopulation of CD4+CD25++Foxp3+PD1+ cells in biopsies H. pylori(+) were increased 1.7-fold compared to those found in biopsies H. pylori(-). CONCLUSIONS: H. pylori infection is associated with a marked increase of Treg subpopulation expressing PD1 in gastric mucosa. The use of antibodies anti-PD1 inhibiting only such subset of cells should be investigated as a potential new therapy to reduce gastric inflammation associated with H. pylori infection