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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The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8+ T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8+ T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8+ T cell responses in vivo. These CD8+ T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8+ T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8+ T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8+ T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.

BackgroundSipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that enhances both B and T cell development and proliferation, would augment and prolong antigen-specific immune responses against both PA2024 (the immunogen for sip-T) and prostatic acid phosphatase (PAP).MethodsFifty-four patients with mCRPC treated with sip-T were subsequently enrolled and randomized 1:1 into observation (n=26) or IL-7 (n=28) arms of a phase II clinical trial (NCT01881867). Recombinant human (rh) IL-7 (CYT107) was given weekly×4. Immune responses were evaluated using flow cytometry, mass cytometry (CyTOF), interferon (IFN)-γ ELISpot, 3H-thymidine incorporation, and ELISA.ResultsTreatment with rhIL-7 was well tolerated. For the rhIL-7-treated, but not observation group, statistically significant lymphocyte subset expansion was found, with 2.3–2.6-fold increases in CD4+T, CD8+T, and CD56bright NK cells at week 6 compared with baseline. No significant differences in PA2024 or PAP-specific T cell responses measured by IFN-γ ELISpot assay were found between rhIL-7 and observation groups. However, antigen-specific T cell proliferative responses and humoral IgG and IgG/IgM responses significantly increased over time in the rhIL-7-treated group only. CyTOF analyses revealed pleiotropic effects of rhIL-7 on lymphocyte subsets, including increases in CD137 and intracellular IL-2 and IFN-γ expression. While not powered to detect clinical outcomes, we found that 31% of patients in the rhIL-7 group had prostate specific antigen (PSA) doubling times of >6 months, compared with 14% in the observation group.ConclusionsTreatment with rhIL-7 led to a significant expansion of CD4+ and CD8+ T cells, and CD56bright natural killer (NK) cells compared with observation after treatment with sip-T. The rhIL-7 treatment also led to improved antigen-specific humoral and T cell proliferative responses over time as well as to increased expression of activation markers and beneficial cytokines. This is the first study to evaluate the use of rhIL-7 after sip-T in patients with mCRPC and demonstrates encouraging results for combination approaches to augment beneficial immune responses.

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.