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Biomedical subjects

H Tighe

Publications and source records attributed to H Tighe.

At least 19 recordsLinked to original sources
PubMed2000

Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity.

Immunostimulatory DNA sequences (ISS) are a potent Th1 adjuvant. We hypothesized that conjugation of ISS to protein antigens would strongly enhance their immunogenicity because both antigen and adjuvant (ISS) would be delivered to the same locale/antigen-presenting cell. To test this hypothesis, we conjugated a 22-mer immunostimulatory oligodeoxynucleotide (ISS-ODN) to two test antigens of differing intrinsic immunogenicity, namely Escherichia coli beta-galactosidase and the HIV-1 envelope glycoprotein gp120. We show that the antigen-ISS conjugates rapidly induce Th1 cells secreting high levels of IFN-gamma, strong CTL activity, and high titer IgG2a and HIV-neutralizing antibodies, exceeding gene and protein vaccination alone or immunization with mixtures of antigen and ISS-ODN. The data suggest that this procedure generates a novel and unique vaccine that rapidly triggers strong humoral and cell-mediated immunity.

Animals↗
PubMed2000

Conjugation of immunostimulatory DNA to the short ragweed allergen amb a 1 enhances its immunogenicity and reduces its allergenicity.

BACKGROUND: Allergen immunotherapy is inconvenient and associated with the risk of anaphylaxis. Efforts to improve the safety of immunotherapy by means of chemical modification of allergens have not been successful because it greatly reduced their antigenicity. Recently, immunostimulatory DNA sequences (ISS or CpG motifs) have been shown to act as strong T(H)1 response-inducing adjuvants. OBJECTIVE: We sought to determine whether conjugation of ISS to the major short ragweed allergen Amb a 1 results in enhanced immunotherapeutic potential in mice and decreased allergenicity in human subjects. METHODS: A 22-mer ISS oligodeoxynucleotide (ISS-ODN) was coupled to Amb a 1 and used for immunization of mice, rabbits, and monkeys. RESULTS: In mice the Amb a 1-ISS conjugate induced a T(H)1 response (IFN-gamma secretion), whereas Amb a 1 induced a T(H)2 response (IL-5 secretion). The T(H)1 response was not observed with an Amb a 1-non-ISS conjugate. Coinjection of Amb a 1 with ISS-ODN was much less effective in inducing a T(H)1 response. In mice primed for a T(H)2 response, injection with Amb a 1-ISS conjugate induced a de novo T(H)1 response and suppressed IgE antibody formation after challenge with Amb a 1. Amb a 1-ISS conjugate induced high-titer anti-Amb a 1 IgG antibodies in rabbits and cynomolgus monkeys, whereas Amb a 1 alone or Amb a 1 coinjected with ISS-ODN did not induce a detectable response. Amb a 1-ISS conjugate was less allergenic than Amb a 1 alone, as shown by a 30-fold lower histamine release from human basophils of patients with ragweed allergy, whereas mixing ISS-ODN with Amb a 1 did not reduce histamine release. CONCLUSION: Amb a 1-ISS conjugate has an enhanced T(H)1-biased immunogenicity and reduced allergenicity. It may offer a more effective and safer approach for allergen immunotherapy than currently available methods.

Allergens↗
PubMed1999

In vivo priming by DNA injection occurs predominantly by antigen transfer.

DNA vaccines can stimulate both humoral and cytolytic immune responses. Although bone marrow-derived elements present the expressed Ag, the mechanisms for acquiring immunogenic peptides have yet to be fully elucidated. APCs may become directly transfected by plasmid DNA or process extracellular proteins produced by other transfected cells. Using a transactivating plasmid system and bone marrow chimeras, we show that both mechanisms appear to be involved; however, the bulk of the immune response is dependent on expression of Ag by nonlymphoid tissues and transfer to APCs. These in vivo studies are the first to define the role of transfected nonlymphoid cells in generating Ag for presentation by bone marrow-derived APCs after needle injection with plasmid DNA.

Animals↗
PubMed1999

Human rheumatoid factor production is dependent on CD40 signaling and autoantigen.

High-affinity pathologic rheumatoid factor (RF) B cells occur in autoimmune diseases such as rheumatoid arthritis, but are deleted in healthy individuals. The reasons for the survival and differentiation of these autoreactive B cells in rheumatoid arthritis are not known. Previous studies in mice transgenic for a human IgM RF have shown that peripheral encounter with soluble human IgG leads to deletion of high-affinity RF B cells; however, deletion can be prevented when concomitant T cell help is provided. This study aimed to further discern the minimal factors necessary not only for the in vivo survival of RF B cells, but also for their differentiation into Ab-secreting cells. The combination of MHC class II-reactive T cells and Ag induced the production of RF in human IgM RF transgenic mice, while either stimulus alone was ineffective. Neutralizing Abs against CD40 ligand (CD40L), but not against IL-4 or IL-15, abrogated IgM-RF production. Moreover, blockade of CD40L-CD40 allowed IgG to delete the RF precursor cells. Most importantly, activating Abs to CD40 could substitute entirely for T cell help in promoting the survival of RF precursors and in stimulating RF synthesis in T cell deficient animals. The data indicate that CD40 signaling alone can prevent deletion of RF B cells by Ag and in the presence of IgG is sufficient to trigger RF synthesis. The results suggest that selective induction of apoptosis in high-affinity RF B cells may be achieved by blockade of CD40L-CD40 interaction.

Animals↗
PubMed1999

Rheumatoid factor B cell tolerance via autonomous Fas/FasL-independent apoptosis.

Normal individuals do not express the high-affinity autoantibodies specific for self-IgG (rheumatoid factors, RF) that are commonly seen in rheumatoid arthritis patients. Studies of transgenic mice expressing a human IgM rheumatoid factor have shown that one mechanism by which higher affinity RF B cells are tolerized to IgG is through abortive RF B cell activation followed by deletion in the absence of T cell help. We show that RF B cell deletion occurs through an intrinsic apoptotic mechanism that is independent of the Fas/FasL pathway and does not involve active killing by T cells, as it occurs in RAG-1-deficient RF transgenic mice to the same extent as in the parental RF transgenic line.

Animals↗
PubMed1999

Inhibition of allergic inflammation in the lung by plasmid DNA allergen immunization.

The nature of the immune response (Th1/Th2) in mice to protein antigens or allergens was compared to that of immunization with pDNA encoding the same antigens. pDNA immunization induced a Th1 response and no IgE antibodies whereas the proteins induced a Th2 response and IgE antibodies. Furthermore, the pDNA induced Th1 response dominated over the protein elicited Th2 response in a secondary immune response. In particular, a preexisting Th2 response (as is the case in allergic patients) did not prevent a new Th1 response to an allergen-pDNA booster injection. The major reason why pDNA immunization induced a Th1 response to allergens was the presence of immunostimulatory non-coding DNA sequences (ISS) in the plasmid constructs having a CpG motif. These ISS caused antigen presenting cells to secrete INF-alpha, INF-beta and IL-12, all cytokines that induce naive T cells to differentiate into CD4+ Th1 cells and CD8+ Tc1 cells. Passive transfer of both Th1 and Tc1 cells from pDNA immunized mice into naive mice inhibited a Th2 response and IgE antibody formation to a subsequent injection of allergen in alum. pDNA immunization or ISS-oligonucleotide injection prior to allergen challenge reduced both immediate type airway sensitivity and late phase allergen induced eosinophil filtration of the lung. Allergen-pDNA immunization may provide a novel type of immunotherapy for the treatment of allergic diseases in man. Since only small amounts of allergen are secreted by the allergen-pDNA transformed cells, allergen-pDNA immunotherapy will unlikely carry the risk of the anaphylactic reactions that are associated with classical allergen injection immunotherapy.

Allergens↗
PubMed1998

Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice.

We have used a mouse model of allergen-induced airway hyperresponsiveness to demonstrate that immunostimulatory DNA sequences (ISS) containing a CpG DNA motif significantly inhibit airway eosinophilia and reduce responsiveness to inhaled methacholine. ISS not only inhibited eosinophilia of the airway (by 93%) and lung parenchyma (91%), but also significantly inhibited blood eosinophilia (86%), suggesting that ISS was exerting a significant effect on the bone marrow production of eosinophils. The inhibition of the bone marrow production of eosinophils by 58% was associated with a significant inhibition of T cell-derived cytokine generation (IL-5, granulocyte-macrophage CSF, and IL-3). ISS exerted this inhibitory effect on T cell cytokine production indirectly by stimulating monocytes/macrophages and NK cells to generate IL-12 and IFNs. The onset of the ISS effect on reducing the number of tissue eosinophils was both immediate (within 1 day of administration) and sustained (lasted 6 days), and was not due to ISS directly inducing eosinophil apoptosis. ISS was effective in inhibiting eosinophilic airway inflammation when administered either systemically (i.p.), or mucosally (i.e., intranasally or intratracheally). Interestingly, a single dose of ISS inhibited airway eosinophilia as effectively as daily injections of corticosteroids for 7 days. Moreover, while both ISS and corticosteroids inhibited IL-5 generation, only ISS was able to induce allergen-specific IFN-gamma production and redirect the immune system toward a Th1 response. Thus, systemic or mucosal administration of ISS before allergen exposure could provide a novel form of active immunotherapy in allergic diseases.

Adjuvants, Immunologic↗
PubMed1998

The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen.

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcgamma binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.

Animals↗
PubMed1997

Costimulation provided by DNA immunization enhances antitumor immunity.

The interaction of the TCR with MHC class I-bound Ag is insufficient for the priming of CTL unless secondary costimulatory signals are provided. To ascertain the minimum elements required to activate an Ag-specific CTL response in vivo, we injected mice intradermally or i.m. with plasmid DNA encoding a MHC class I-restricted peptide Ag (minigene) and different membrane-bound costimulatory ligands. The minigene-encoded epitope only primed a specific CTL response if injected in the vicinity of an ectopically expressed costimulatory ligand. Vector encoding B7-1 was repeatedly more potent at stimulating a cytolytic response than vector encoding B7-2. In contrast the B7-2-encoding plasmid preferentially enhanced Ag-specific Ab responses when injected with either protein or a cDNA expression vector. Gene vaccination with plasmids encoding OVA and B7-1, but not B7-2, prolonged survival in mice challenged with an OVA-transfected tumor. These results show that functional B7-1 transfection can be achieved in vivo and induces the selective induction of CTL. The data suggest that B7-1 plasmids should be coadministered with naked DNA vaccines that aim to induce tumor-specific cellular immunity.

Animals↗
PubMed1997

Peripheral deletion of rheumatoid factor B cells after abortive activation by IgG.

Rheumatoid factor (RF) B cells proliferate during secondary immune responses to immune complexed antigen and antigen specific T cells, but higher affinity RFs are not detected except in patients with rheumatoid arthritis and other autoimmune diseases. Consequently, there must exist highly efficient mechanisms for inactivation of these higher-affinity RF B cell clones under normal circumstances. Exposure of transgenic mice expressing a human IgM RF to soluble human IgG in the absence of T cell help causes antigen specific B cell deletion in 2-3 days. The deletion is independent of the Fas/Fas ligand (FasL) pathway of apoptosis and is preceded by a phase of partial activation involving increase in cell size and expression of B7 and ICAM-1, and transient release of low levels of immunoglobulin. Complete B cell activation involving the formation of germinal centers and sustained high level RF secretion only occurs if T cell help is provided simultaneously. RF B cells exposed to tolerogen remain competent to secrete RF in vitro if provided with an appropriate antigenic stimulus and T cell help. Consequently, death of these cells is not preceded by anergy. Abortive activation/deletion of B cells by antigen in the absence of T cell-derived survival signals may represent the major mechanism for maintaining peripheral tolerance in B cells expressing higher affinity RF. The lack of anergy, and the potential for reactivation before death, provide a means for maintaining RF production under pathologic circumstances, such as may occur in the inflamed rheumatoid synovium.

Animals↗
PubMed1996

Gene vaccination with naked plasmid DNA: mechanism of CTL priming.

The injection of naked plasmid DNA directly into the muscle cells of mice has been shown to induce potent humoral and cellular immune responses. The generation of a cytotoxic T lymphocyte (CTL) response after plasmid DNA injection may involve the presentation of the expressed antigen in the context of the injected myocytes' endogenous major histocompatibility (MHC)-encoded class I molecules or may use the MHC molecules of bone marrow-derived antigen presenting cells (APC) which are capable of providing co-stimulation as well. To resolve which cell type provides the specific restricting element for this method of vaccination we generated parent-->F1 bone marrow chimeras in which H-2bxd recipient mice received bone marrow that expressed only H-2b or H-2d MHC molecules. These mice were injected intramuscularly with naked plasmid DNA that encoded the nucleoprotein from the A/PR/8/34 influenza strain, which as a single antigen has epitopes for both H-2Db and H-2Kd. The resulting CTL responses were restricted to the MHC haplotype of the bone marrow alone and not to the second haplotype expressed by the recipient's myocytes. The role of somatic tissues that express protein from injected plasmids may be to serve as a reservoir for that antigen which is then transferred to the APC. Consequently, our data show that the mechanism of priming in this novel method for vaccination uses the MHC from bone marrow-derived APC, which are efficient at providing all of the necessary signals for priming the T cell.

Animals↗
PubMed1996

Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Amino Acid Sequence↗
PubMed1996

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Animals↗
PubMed1995

Human immunoglobulin (IgG) induced deletion of IgM rheumatoid factor B cells in transgenic mice.

The singular ability of immunoglobulin genes to hypermutate their variable regions, while permitting the generation of high-affinity antibodies against foreign antigens, poses a problem in terms of maintenance of immunological self-tolerance. Immunoglobulin gene hypermutation driven by a foreign antigen has the potential to generate antibodies that cross-react with self-components. Consequently, there must exist a mechanism in the periphery for inactivation of mature autoreactive B cell clones. The classical experimental system used to address this problem is the induction of tolerance to soluble, deaggregated human IgG. We have analyzed the mechanism of induction of tolerance to human IgG using transgenic mice that express a human IgM rheumatoid factor (IgM RF) on a large proportion of their B cells. Injection of deaggregated human IgG caused a specific deletion of those B cells that express an intact IgM RF on their cell surface. The degree of RF B cell deletion was proportional to the reduction in the proliferative response of splenocytes to antigen (aggregated human IgG), or to F(ab')2 fragments of anti-human IgM antibodies. Control experiments showed that IgG administration had little effect on the numbers of mouse Ig-bearing cells or their ability to proliferate to a nonspecific mitogen. Thus, the effects of IgG on the human IgM RF B cell are antigen specific and are not due to nonspecific toxic effects of the human IgG preparation. These experiments demonstrate that peripheral exposure to IgG induces deletion of reactive B cells, without any evidence for anergy, and differ from data obtained by other investigators studying tolerance to soluble protein antigens. The results imply that human Igs have distinct properties as soluble antigens, and that peripheral nonresponsiveness to IgG may be due to lymphocyte deletion.

Animals↗
PubMed1993

Induction of antibodies to a kappa V region by gene immunization.

Direct gene transfer into muscle can lead to sustained gene expression at the injected sites. Here we tested the ability of in vivo gene transfection to immunize mice against an isolated human IgV region. The Humkv325 germline kappa L chain V gene encodes the kappa L chain in V regions of several human IgM autoantibodies and is used frequently in chronic lymphocytic leukemia. This gene was inserted into a mammalian expression vector, pREP7, to produce pREVk3. Mice injected i.m. with pREVk3 produced antibodies against the V region of Glo, a human monoclonal IgM paraprotein whose kappa L chain is encoded by Humkv325. Co-injection of an expression vector encoding IL-2 enhanced anti-Glo antibody production fivefold and induced a localized delayed hypersensitivity reaction. Antibody production also was induced when vectors encoding Humkv325 and IL-2 were injected s.c. These experiments demonstrate that gene immunization vectors can stimulate immune responses to antibody V region determinants.

Animals↗