When HCMV-DBspecific IgG was measured between times 10 and 60 postchallenge, simply no difference in antibody titers was observed between animals that had received GK1

When HCMV-DBspecific IgG was measured between times 10 and 60 postchallenge, simply no difference in antibody titers was observed between animals that had received GK1.5 as well as the control mice (Fig. was resilient and antigen particular totally, excluding polyclonal B cell activation. Compact disc4+T cells weren’t included since (a) additional depletion of Compact disc4+T cells within the receiver mice didn’t alter the antibody response and (b) receiver mice included no detectable Compact disc4+T cells 90 d posttransfer. Storage B cells cannot be turned on by way of a soluble viral proteins without T cell help. Transfer of storage B cells into immunocompetent pets indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is usually impartial of cognate or bystander T cell help. Keywords:antigen-specific immunity, immunological memory, B lymphocyte memory, cytomegalovirus, adoptive transfer == Introduction == During a main humoral immune response, conversation between different cell forms of the adaptive and innate immune system is usually essential. In peripheral lymphoid organs, APCs such as DCs and macrophages make contact with antigen-specific T cells, resulting in T cell activation. B cells that have encountered their antigen in the blood, the lymph nodes, or on follicular DCs (FDCs) present antigen on MHC class II molecules and migrate to the borders of the T cellrich areas of the lymphoid organs where cognate TB cell conversation occurs (1,2). This conversation can result either in differentiation of activated B cells into low affinity IgM-secreting plasma cells or into memory B cell precursors (3). These precursors are recruited into the germinal center (GC) reactions. They form GCs where proliferation, somatic mutation of the Ig genes, and antigenic selection by FDCs takes place (4). Betamipron The B cells differentiate into high affinity IgG-, IgA-, or IgE-secreting plasma cells or they enter into the memory B cell pool, transporting high affinity immunoglobulin as surface receptors (4). A second encounter with the Betamipron same antigen leads to a much quicker activation of memory B cells, resulting in the appearance of high affinity IgG-secreting plasma cells within a few days (3). The ITGAM requirements for the memory B cell activation upon this second antigen encounter and the involvement of T cell help are not clear. A second round of TB cell conversation seemed to be essential for the activation of memory B cells into Betamipron IgG-secreting plasma cells in experimental protocols including either immunization with soluble proteins and hapten-carrier conjugates or viral contamination of mice (5,6). Here, we analyzed the memory B cell activation in response to two different enveloped viruses using adoptive transfer of highly purified B cells from immunized donors into recombinase-activating gene (RAG)-1/mice. We show that memory B cells can be activated Betamipron by viral particles to produce specific IgG with virus-neutralizing capacity in the absence of helper T cells. == Materials and Methods == == Mice. == 812-wk-old C57BL/6 mice were obtained from Charles River Laboratories. C57BL/6-RAG-1/mice were obtained from Irmgard Frster. C57BL/6-TCR//mice were obtained from The Jackson Laboratory. C57BL/6-TNF/, C57BL/6-TNF/LT/mice (7) and C57BL/6 Ly5.1 mice were a gift from H. Krner (Nikolaus Fiebiger Center). All mice were maintained free of specific pathogens in isolated ventilated cages. == Antigens and Immunizations. == Human cytomegalovirus (HCMV) strain AD169 was propagated in main human foreskin fibroblasts produced in MEM supplemented with 5% FCS, glutamine (100 mg/L), and gentamycine (350 mg/L). Virions and dense body (DBs; enveloped particles missing the viral core) were isolated Betamipron via glycerol-tartrate gradient centrifugation as explained (8). Tick-born encephalitis computer virus (TBEV) particles and HCMV glycoprotein B (gB) were gifts from Chiron Behring and Aventis Pasteur, respectively. Both preparations corresponded to the antigen used for vaccination in humans. C57BL/6 mice were immunized twice with 20 g HCMV-DBs, 2 g TBEV particles, or 10 g soluble gB in aluminium hydroxide i.p. at intervals of 4 wk and with 10 g HCMV-DBs, 2 g TBEV, and 10 g gB, respectively, in PBS i.v., again after 4 wk. B cells for adoptive transfer were isolated at least 6 wk after the last immunization. == Circulation Cytometry and Adoptive Transfer of B Lymphocytes. == Single cell suspensions of spleens from immunized mice were stained with PE-conjugated anti-CD19 and FITC-conjugated anti-CD8 and anti-CD4 antibodies (all antibodies were obtained from BD Biosciences). CD19+cells were isolated by two rounds of cell sorting using a MoFlocell sorter (Cytomation) and analyzed for purity by circulation cytometry using a FACSCalibur(Becton Dickinson). For unfavorable selection of B lymphocytes, single cell suspensions of spleens from HCMV-DBimmunized mice were stained with FITC-conjugated antibodies against CD4, CD8, TCR, TCR /, NK1.1, CD11b, CD11c, and c-kit and propidium iodine. Positive cells were removed by two rounds of cell sorting. In general, a purity >99.8% was achieved by either process. 5 106purified CD19+B cells were adoptively transferred into the tail vein of C57BL/6RAG-1/mice. For adoptive cotransfer of primed helper T cells, 5 106purified CD4+T cells from spleens and lymph nodes of C57BL/6 mice previously immunized with.