Addition of IL-10 to the IL-4 + anti-CD40 antibody cultures further up-regulated IgG4 in both groups. IL-10 or IL-10 + TGF- in combination, induced IgA production, albeit lower than found in B cells from controls. The B cells from the IgA-deficient subjects were less effective in differentiating into CD138+ X-box binding protein 1 (XBP-1)+ plasma cells when stimulated with TGF-, IFN- or IL-10. 3,5-Diiodothyropropionic acid Interestingly, when adding IL-4 to TGF- alone or in combination with IL-10, the immunoglobulin production in B cells from IgA-deficient donors was comparable with those of normal controls. These data show that in healthy subjects IgA production can be up-regulated by addition of IL-10 to CD40-stimulated B cells, whereas a similar B cell differentiation does not occur in IgA-deficient subjects. Addition of IL-4, 3,5-Diiodothyropropionic acid however, reverts this abnormality. Keywords: IgA deficiency, class-switch recombination, human, B cells Introduction Immunoglobulin A (IgA) is quantitatively the most important isotype in humans, playing a key role in host protection of respiratory and intestinal mucosal surfaces. IgA deficiency is one of the most common forms of primary immunodeficiency, and although most IgA-deficient subjects are asymptomatic, some are affected by repeated upper respiratory tract and/or gastrointestinal infections. IgA expression in B cells is controlled by cytokines and interactions with T cells. Cross-linking of CD40 with anti-CD40 antibody mimics the cross-linking of the B cell CD40 exerted by the T cell CD40 ligand. Stimulating IgM/IgD-bearing B cells with anti-CD40 antibody in combination with transforming growth factor (TGF)- or interleukin (IL)-10 induces class-switch recombination (CSR) from IgM to IgA [1C4]. Upon stimulation B cells differentiate into IgA-positive cells through a DNA recombination process that joins the region to the region with a deletion of the intervening sequence, a process that 3,5-Diiodothyropropionic acid is initiated by production of germline transcripts (GLT) [3]. After switching, B cells normally differentiate from membrane IgA-bearing activated cells to IgA-producing plasma cells. In this study we set out to investigate IgA-deficient subjects by examining the ability of their naive B cells to transform into IgA-producing cells, and we attempted to delineate whether a particular step in the B cell differentiation process is flawed. An explanation of the malfunction in IgA-deficient subjects could be that the processes of CSR or plasma cell differentiation are defect. To test these hypotheses, we used four molecular markers: (1) activation-induced cytidine deaminase (AID), a ssDNA deaminase essential for CSR and strictly induced in stimulated B cells undergoing class switching [5C7]; (2) germline transcripts (GLT), the production of which is required for CSR [8C10]; (3) Ig production, to test for a possible defect in the transcription/translation of the IgA and (4) the plasma cell transcription factor, X-box binding protein 1 (XBP-1), which is essential 3,5-Diiodothyropropionic acid for the differentiation into plasma cells [11]. Materials and methods Subjects Four IgA-deficient subjects with Mef2c serum IgA below the detection limit (< 05 ng/ml) were identified among blood donors and included anonymously in this study (one male and three females, mean age 28 years, range 22C33 years). Four age-matched healthy subjects were used as controls (all females, mean age 268 years, range 23C32 years). Cell preparation and cultures The murine CD32-transfected fibroblastic cell line was kindly provided by Eva Steen (Lund University, Sweden) and was used in all the experiments alone or in the presence of anti-CD40 antibody to mimic the effect of T cells carrying CD40L. Using the Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient method, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized buffy-coat preparations of blood donations. Naive IgD-positive cells were purified and cultured. Briefly, naive IgD+ B cells were isolated by positive selection using specific anti-IgD monoclonal antibody (MoAb)-coated magnetic beads. Purity was.