SummaryThe effects ofinterleukin 7 (IL-7) on the growth and differentiation of murine B cell p~ogenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations. In the adult mouse, B lymphocytes develop from progenitor cells in the bone marrow. This development proceeds in an ordered fashion and can be characterized by the sequential acquisition of Ig gene rearrangements and cell surface markers (1, 2). The principal marker of the B lineage in murine bone marrow is the CD45R isoform identified by the 6B2 mAb and is designated B220 (3). The earliest cells committed to the B lineage, however, are B220-(4). A number of other cell surface antigens have been described whose expression is characteristic of particular stages in the sequence orb cell development (1) which culminates in the expression of functional surface IgM.The early development of B cells in the marrow is dependent on stromal cells and is mediated by cell contact and secreted cytokines (5). The role of cytokines in the development of B lymphocytes has been characterized primarily using in vitro methods in which particular cytokines such as IL-7 (6), mast cell growth factor (kit ligand) (7) or its antagonist (8), or insulin-like growth factor 1 (9) can be shown to regulate the proliferation of B cell progenitors. In vitro data suggest that as Ig genes rearrange, B cell progenitors progress from a stage in which they are stromal cell dependent and IL-7 independent to a stage in which they require IL-7 (10, 11). Very little information has been available on the actual role of these molecules in vivo, although treatment of normal mice with recombinant IL-7 has been shown to greatly augment B lymphopoiesis (12).The extent to which the B cells in the peripheral lymphoid organs are derived from IL-7-dependent precursors remains unclear. Whereas pre-B cells in the marrow turn over very rapidly, the rate at which cells turn over in the periphery appears to be much slower. This view is based on studies using bromodeoxyuridine administration (13) which indicate that the majority of peripheral B cells are long-lived. A small proportion of peripheral B cells turns over more rapidly and these are presumably replaced by newly developed marrowderived immature cells. B cells of the B-1 lineage, found primarily in the peritoneum, self-renew in the adult and are derived from immature progeni...

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8+ T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen’s developmental cycle. In addition to their use as candidate Ags for stimulating CD8+ T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8+ T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8+ T cells specific for an H-2Db-restricted epitope from CrpA are elicited at a significant level (∼4% of splenic CD8+ T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.

During infection with Chlamydia trachomatis, CD8 ؉ T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8 ؉ T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8 ؉ T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8 ؉ T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

Dependent on the viral vector and the specific assay used, viral titers produced from commonly used retroviral packaging cell lines have an upper limit in the range of 10(5) to 10(7) infectious units/ml. We have developed a generally applicable method, using hollow-fiber filtration technology, which allows for the concentration of infectious virus derived from packaging lines. This method resulted in a reproducible 10- to 30-fold increase in viral titer and can readily be scaled to accommodate larger input volumes. Over 80% of the input virus is recovered in an infectious form in the concentrate. Concentrated virus containing media was seen to produce higher infection frequencies in Jurkat T cells as compared to unconcentrated virus containing media; however, this was not proportional to the differences in viral titer observed by limiting dilution analysis on NIH-3T3 cells. These results are discussed in relation to the importance of factors other than viral titer in determining transduction frequencies.

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