The immune system of aged mice produces antibodies that are characterized by low affinity, diminished protection against infections and autoreactivity. It has been shown that these antibodies may be encoded by different immunoglobulin V genes and that the mechanism of somatic hypermutation in the V genes is inefficient. Studies on scid mice reconstituted with B and T cells from donors of different ages suggested that both lymphocyte subsets may contribute to the age-related changes in antibody repertoire. With help provided by T cells from young mice, the response to a hapten, nitrophenyl(acetyl), became gradually dominated by B-cell clones that rearranged a particular germline VH gene (V186.2). However, help from the aged T cells resulted in a heterogeneous response of B cells expressing many different V segments. Analysis of discrete foci of primary antibody-forming cells suggested that the aged T-helper cells are unable to govern the normally-occurring competition between the B-cell clones that have different affinities for the hapten. It is proposed that a signaling disequilibrium from the aged T cells, which provide less efficient help in quantitative terms, supports the growth of low-affinity B cells. This process may be exacerbated due to the apparent hyperactivity of aged B cells to CD40-mediated mitogenic signal.

A significant proportion of memory B cells home to bone marrow (BM) which is a major site of anamnestic antibody responses in mice. We hypothesized that memory T cells likewise accumulate in BM perhaps to provide help for antibody production, and that the compartment of CD4+ T cells in BM of unimmunized mice would be enriched for memory phenotype cells that might have been activated by environmental antigens. The phenotype of activated/memory CD4+ lymphocytes has been defined as CD44hi CD45RBlo CD62L-. Conversely, the phenotype of immunologically naive cells is CD44lo CD45RBhi CD62L+. Flow cytrometric analysis of tissue from normal, adult C57BL/6 mice identified 1-2 % CD3+CD4+ cells in BM. Up to 40 % of CD3+CD4+ cells in the BM expressed the activated/memory phenotype compared with < or = 10% in the spleen and lymph nodes. Analysis of TCR Vbeta repertoire revealed that expression of Vbeta3 and Vbeta7 genes was increased as much as fourfold in BM compared to the periphery; most of this increase was within the CD44hi T cells. The accumulation of activated/memory T cells and clonotypic expansion(s) was not seen in the BM of germ-free mice, indicating that it reflects the history of the animal's exposure to antigens. Finally, immunization of mice which express a transgenic T cell receptor specific for ovalbumin peptide resulted in appearance of antigen-specific T cells with activated/memory phenotype in the BM.

Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igαand Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating an order of magnitude increase in BCR surface expression. Surface Ig levels correlated with transgenic Igα expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis. V ESICULAR organelles in eukaryotic cells frequently maintain an acidic pH (reviewed by Paroutis et al. 2004) that is created by the vacuolar H+-ATPase (V-ATPase). The V-ATPase is a large (910-kDa) molecular machine that couples ATP hydrolysis to the movement of protons across biological membranes. The V-ATPase has a V 0 -sector that creates the pore-for-proton translocation through the lipid bilayer and a V 1 -sector, located in the cytoplasm, that is the site of ATP hydrolysis. Each V-ATPase holoenzyme is composed of 14 different subunits, some of which are present in multiple copies (reviewed by Toei et al. 2010). In yeast, there is one gene for each V-ATPase subunit, except for the "a" subunit, which is encoded by two genes (reviewed by Kane 2006). The physiological properties of the V-ATPase are in part determined by which of these two "a" subunits it contains (Kawasaki-Nishi et al. 2001). In humans and other animals, the "a" and other V-ATPase subunits are encoded by more than one gene (reviewed by Toei et al. 2010). Subunit diversity presumably allows the V-ATPase to be either customized for a specific function or utilized in a tissuespecific fashion.In this article, we use pH-sensitive vital dyes and specific inhibitors to show that sperm-specific MOs use the V-ATPase to acidify their interior a...

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