Oliver AM, Martin F, Kearney JF. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by antigenic stimuli. INTRODUCTION Human B cells can be subdivided into virgin and memory cells, which generally can be identified by the expression of CD27 (1,2) and FcRL-4 (3,4) antigens by memory B cells. B cells also can be classified based on the anatomic areas where they seed, that is, follicular (FO) and marginal zone (MZ) B cells. FO B cells can be in turn separated into follicular mantle (FM) and germinal center (GC) B cells depending on their location within secondary lymphoid follicles (5C7). The splenic MZ is usually defined as the outermost portion of the white pulp, the structure of which permits the transit of B cells from and to the bloodstream and facilitates encounters between blood-borne pathogens with B cells and macrophages (8C10). While FO B cells are believed to be capable of generating plasma cells secreting high-affinity antibodies and switched memory (SM) B cells (7), MZ B cells are thought to produce IgM antibodies in a T cellCindependent manner, particularly to polysaccharide antigens of encapsulated bacteria (11C13). This response represents a first defense line to protect the host from bacterial infection spread until an efficient FO response can develop. Special areas of other lymphoid organs, including subepithelial areas of tonsils, the dome region of Peyer patches, the subcapsular Rabbit polyclonal to ACSF3 areas of lymph nodes and the mucosa-associated lymphoid tissue (MALT) tissue, are believed to be the equivalent of the splenic MZ (14). Studies in mice have decided that MZ B cells have a sIgMhighsIgDlow phenotype with high CD21 and low CD23 expression and respond to T cellCindependent antigens and in the MZ, as suggested by both the imprint of somatic hypermutation (SHM) and the presence of clonal families exhibiting an ongoing process of diversification. GPR40 Activator 1 MATERIALS GPR40 Activator 1 AND METHODS Samples Five spleens, free GPR40 Activator 1 of neoplastic cells at histological inspection, were obtained at surgery for malignancy (three patients had pancreatic malignancy, one metastatic breast malignancy and one liposarcoma). Mononuclear cells were isolated by Ficoll-Hypaque (Seromed; Biochrom KG, Berlin, Germany) density gradient centrifugation. Four tonsils were obtained from 5- to 12-year-old children undergoing program tonsillectomies, and their cells were purified as previously reported (29). The study was approved by the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Azienda Ospedaliera Universitaria (AOU) San Martino-IST Institutional Review Table and knowledgeable consent was obtained from patients. Circulation Cytometry and Cell Sorting Mononuclear cells were stained and sorted with antibodies by FacsAria 2 (BD, Milan, Italy) to a purity of >98%. A list of all antibodies used is usually reported in the supplementary materials. Isotypic negative controls were acquired for each fluorochrome. Data were analyzed and normalized by using FlowJo software (TreeStar, Ashland, OR, USA). Immunohistochemical Staining Formalin-fixed paraffin-embedded sections (3 m solid) were subjected to antigen retrieval with citrate buffer at high pH and immunostained with polyclonal anti- antibodies (Abs) and anti-CD38 monoclonal antibody (mAb) (Diapath SRL, Martinengo, Italy), with a BenchMark XT automated stainer (Ventana Medical Systems, Strasbourg, France). For activation-induced cytidine deaminase (AID) staining, a mouse anti-AID mAb (Invitrogen/Life Technologies, Carlsbad, CA, USA) was used. Slides were revealed with the ultraView Universal Alkaline Phosphatase Red Detection Kit (Ventana Medical Systems Inc./Roche, Basel, Switzerland) and counterstained with modified Gills hematoxylin. The slides were examined with an Olympus microscope (Olympus Italia, Segrate, Italy). Immunofluorescence Microscopy Serial splenic OCT cryosections (5C6 m) were slice, laid on glass cover-slips and stored GPR40 Activator 1 at ?80C. Sections were thawed briefly and incubated with the primary.