Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1.

Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Mechanisms of B and T lymphocyte accumulation in tumor-draining lymph nodes

Thesis (Master's)--University of Washington, 2016-06

Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines

Human Valpha24(+)Vbeta11(+) NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using alpha-galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4(+)CD8(-), CD4(-)D8(+) and CD4(-)CD8(-)) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-1 2 or IL-15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4(+) and CD4(-) NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1-biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autommume encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4(+) T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4(+), CD8(+), CD14(+) (monocytes) and CD56(+) NK cells but not to CD19(+) B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn 10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn 10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

Analysis of the effect of different NKT cell subpopulations on the activation of CD4 and CD8 T cells, NK cells, and B cells

Objective. NKT cells have diverse immune regulatory functions including activation of cells involved in Th1- and Th2-type immune activities. Most previous studies have investigated the functions of NKT cells as a single family but more recent evidence indicates the distinct functional properties of NKT cell subpopulation. This study aims to determine whether NKT cell subpopulations have different stimulatory activities on other immune cells that may affect the outcome of NKT cell-based immunotherapy. Methods. NKT cells and NKT cell subpopulations (CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(+)) were cocultured with PBMC and their activities on immune cells including CD4(+) and CD8(+) T cells, NK cells, and B cells were assessed by flow cytometry. The production of cytokines in culture was measured by enzyme-linked immunsorbent assay. Results. The CD4(+)CD8(-) NKT cells demonstrated substantially greater stimulatory activities on CD4(+) T cells, NK cells, and B cells than other NKT cell subsets. The CD4(-)CD8(+) NKT cells showed the greatest activity on CD8(+) T cells, and were the only NKT cell subset that activated these immune cells. The CD4(-)CD8(-) NKT cells showed moderate stimulatory activity on CD4(+) T cells and the least activity on other immune cells. Conclusion. The results here suggest that NKT cell subpopulations differ in their abilities to stimulate other immune cells. This highlights the potential importance of manipulating specific NKT cell subpopulations for particular therapeutic situations and of evaluating subpopulations, rather than NKT cells as a group, during investigation of a possible role of NKT cells in various disease settings. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocircullate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HILA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathollogy in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytolkines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.