Protection of mice against lethal coxsackievirus B3 infection by using DNA immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.     

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.     

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.     

Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.     

T-Cell-independent immunoglobulin G responses in vivo are elicited by live-virus infection but not by immunization with viral proteins or virus-like particles

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor beta chain [TCR-beta] -/- or TCR-betaxdelta -/-) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires alpha(v)beta5 integrin, rather than alpha(v)beta3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both alpha(v)beta3 and alpha(v)beta5 integrins, only alpha(v)beta3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of alpha(v)beta5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, alpha(v)beta5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas alpha(v)beta3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, alpha(v)beta5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for alpha(v)beta5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of alpha(v)beta5 integrin with internalized ROS. Control experiments showed that blocking alpha(v)beta3 function with antibodies did not inhibit ROS phagocytosis and that alpha(v)beta3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor alpha(v)beta5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-alpha(v)beta5 complex. Alpha(v)beta5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.     

Neonatal injection of Lewis rats with recombinant V beta 8.2 induces T cell but not B cell tolerance and increased severity of experimental autoimmune encephalomyelitis

In Lewis rats with experimental autoimmune encephalomyelitis (EAE) mediated by V beta 8.2 effector cells, anti-idiotypic T cells and antibodies could be boosted by injection of V beta 8.2 peptides, inducing both T cells and antibodies that reduced the severity and shortened the course of disease. However, EAE in Lewis rats is self-limiting, and we sought to determine if the anti-idiotypic response contributed to the natural recovery process. In a previous study, we found that adult tolerance induced to one of the regulatory idiotopes, V beta 8.2-44-54, caused worsening of EAE, implicating response to this epitope in recovery from EAE. However, neonatally-induced tolerance to V beta 8.2-44-54 did not alter the course of EAE, suggesting either compensation by additional V beta 8.2 determinants, or mechanistic differences in tolerization protocols. In this report, we reevaluate the role of V beta 8.2 determinants in recovery from EAE, using two recombinant V beta 8.2 constructs to induce neonatal tolerance to the comprehensive set of V beta 8.2 epitopes prior to adult induction of EAE. We found that neonatal exposure to either of the recombinant V beta 8.2 molecules induced "split" tolerance-specific T cell tolerance but enhanced antibody responses- and a more severe course of EAE. In contrast, neonatal exposure to a V beta 8.2 + T cell hybridoma or a control protein did not induce T cell tolerance to V beta 8.2 determinants and did not alter the EAE disease course. These results are consistent with those obtained by inducing adult tolerance, and suggest that our previous result (normal recovery from EAE in rats neonatally tolerized to V beta 8.2-44-54) was probably due to a compensatory response to other V beta 8.2 determinants. In both studies, the data clearly implicate T cell recognition of V beta 8.2 determinants in the natural EAE recovery process.     

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.     

Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function

Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b+ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation.     

Ligation of CD5 on resting B cells, but not on resting T cells, results in apoptosis

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5+ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5+ B cells were more sensitive to IgM-induced apoptosis than CD5 B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.     

Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.     

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.     

Protection against lethal Japanese encephalitis virus infection of mice by immunization with the highly attenuated MVA strain of vaccinia virus expressing JEV prM and E genes

Genes encoding the glycosylated precursor of the membrane (prM) and envelope (E) proteins of a Korean strain of Japanese encephalitis virus (JEV) were inserted into the genome of the host-range restricted, highly attenuated, and safety-tested MVA strain of vaccinia virus. MVA recombinants containing the JEV genes, under strong synthetic or modified H5 vaccinia virus promoters, were isolated. Synthesis of JEV prM and E proteins was detected by immunofluorescence microscopy, flow cytometry, and polyacrylamide gel electrophoresis. Mice inoculated and boosted by various routes with either of the MVA recombinants produced JEV neutralizing antibodies, that had titres comparable with those induced by an inactivated JEV vaccine, as well as haemagglutination-inhibiting antibodies. Mice immunized with 2 x 10(6) infectious units of MVA/JEV recombinants by intramuscular or intraperitoneal routes were completely protected against a 10(5) LD50 JEV challenge at 9 weeks of age.