Treg cells play a crucial role in immune system tolerance but systems that creates Treg cells are poorly recognized. harmful antigens such as for example pathogens and deceased or aberrant sponsor cells[1 2 At the same time immune CGP 60536 system tolerance is necessary to avoid damaging normal host tissues and to allow the presence CGP 60536 of harmless antigens such as commensal bacteria and food antigens in the intestinal tract. Regulatory T (Treg) cells play a crucial role in generation and maintenance of immune tolerance. It has been shown that transforming growth factor-beta (TGF-β) stimulates na?ve CD4+CD25? T cells to differentiate into either CD4+CD25+Foxp3+ Treg cells or Th17 cells[5 6 while all-trans retinoic acid (ATRA) from intestinal dendritic cells (DC) induces the differentiation of na?ve T CGP 60536 cells into Treg cells in presence of TGF-β1 but suppress the differentiation of Th1 Th2 and Th17 cells[7-9]. The activation of aldehyde dehydrogenase (ALDH) a unique rate-limiting enzyme during ATRA synthesis has been considered as the signal for cells to produce ATRA. The mucosal immune system rather than systemic immune system acts as the main sensor and effector in responses to exogenous antigens. Gut-associated lymphoid tissue (GALT) the largest lymphoid organ in the mucosal immune system is comprised of Peyer’s patches interdigitating lymphocytes plasma cells and lymphocytes in the LP and mesenteric lymph nodes in which LP is the loci for the highest frequency of Treg cells expansion. In LP CD103+ DC and CD11b+ F4/80+ CD11c? macrophages can induce the generation of Treg cells while CD11c+ CD11b+ CD103? DC induce the differentiation of Th17 cells[13 14 However except for DC and macrophages no other immune cells have been reported to induce the differentiation of na?ve CD4+CD25? T cells. While investigating the function of DC and macrophages from murine LP we identified eosinophils that displayed a high activity of ALDH as well as producing high levels of ATRA and expressing TGF-β1 mRNA. In the present study we provided evidence that this subset of eosinophils (LP eosinophils) represents a novel inducer of the differentiation of na?ve T cells into CGP 60536 Treg cells. Materials and Methods Mice Female wild-type or OT-II transgenic C57BL/6 mice of 6-10 weeks of age were kindly provided by Joint Venture SIPPR-BK Experimental Animal Company (Shanghai China) or Dr. Jian-Li Wang (Department of Immunology Zhejiang University School of Medicine China). All mice were maintained in a specific pathogen-free animal facility with a standardized light (12 h light/dark cycle) temperature (22±1°C) and humidity (55±15%). Animals were fed food and water freely. Cages were changed weekly. At this study mice were sacrificed by cervical dislocation. All of animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Zhejiang University. Cells To isolate LP cells small intestines were removed and their Peyer’s patches were cleaned then opened along the mesenteric side and washed of fecal contents. Intestines were cut into 5 mm in length and incubated for 30 min at 37°C with PBS containing 10% CGP 60536 FCS 10 mM EDTA 20 mM HEPES 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco USA) to remove the epithelium. Tissues were washed twice AFX1 with PBS minced and digested for 60 min with continuous stirring at 37°C with 1 mg/ml collagenase D (Roche Germany) and 0.1 mg/ml Dnase (Sigma USA) in CGP 60536 RPMI 1640 plus 10% FCS. Tissues were filtered through 40 μm and 70 μm cell strainer (BD Biosciences USA) and washed in PBS twice. Cells were resuspended into FACS buffer and stained with biotin-conjugated monoclonal anti-mouse CD11c (N418;) anti-mouse CD11b (M1/70) (eBioscience USA) rat anti-mouse Siglec-F (E50-2440; BD Pharmingen USA) anti-mouse MHC-II (AF6-120.1) anti-mouse DEC-205 (205yekta) anti-mouse CD103 (2E7) anti-mouse CD40 (1C10) anti-mouse CD80 (16-10A1) anti-mouse CD86 (GL-1) and anti-mouse F4/80 (BM8)(eBioscience). The data were analyzed using CELLQuest (BD Biosciences) and FlowJo (TreeStar USA) software. In the four sorted cell subsets (P1-P4) the P4 subset shown an eosinophil-specific phenotype such as for example positive Siglec-F; this cell subset was consequently isolated through the single-cell suspension system using Compact disc11b-covered microbeads (Miltenyi Biotec Germany) as well as the Compact disc11b+cells were after that sorted utilizing a FACSAria II movement cytometer (BD Biosciences) with FITC-conjugated rat anti-mouse CCR3.