Academic literature on the topic 'Ova-transgenic'

Abstract T cells recognize a complex of peptide Ag bound within the groove of MHC-encoded molecules. Although many studies have attempted to correlate TCR gene expression with specificity for particular Ag/MHC combinations, it is still not clear exactly how the TCR physically interacts with its cognate ligand. We have analyzed transgenic mice that carry a rearranged gene encoding a V beta 5.2+ TCR beta-chain derived from the CD8+ CTL clone B3, which is specific for chicken OVA+H-2Kb. Surprisingly, we have found that peripheral lymphocytes isolated from naïve V beta 5.2 transgenic mice can generate a strong primary anti-OVA CTL response when stimulated in vitro with OVA+H-2b, whereas generation of even a weak anti-OVA response from nontransgenic littermates requires in vivo priming. This response is Ag specific, because the transgenic mice are unable to respond with or without priming to vesicular stomatitis virus, which contains a dominant epitope presented in the context of H-2Kb. The precursor frequency of OVA-specific CTL in unprimed V beta 5.2 transgenic mice is approximately 30-fold higher than that in nontransgenic littermate controls. Reverse transcription-PCR analyses demonstrate that OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice express a variety of TCR V alpha elements, indicating that the transgenic anti-OVA response is not solely due to the reconstitution of the original B3 TCR. In fact, our data suggest that even a nontransgenic V beta 5+ TCR is intrinsically OVA specific. First, five separate OVA-specific oligoclonal CTL lines derived from individual nontransgenic mice demonstrate dramatic skewing toward expression of V beta 5.1+ or V beta 5.2+ TCR over the course of several in vitro stimulations. Second, sorting for V beta 5+CD8+ nontransgenic cells enriches for OVA-specific CTL. However, peptide antagonism experiments using mutant forms of the Kb-restricted OVA peptide reveal distinct differences between the recognition patterns of two individual OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice. These experiments support the notion that a discrete portion of the responding TCR can heavily influence but not necessarily be solely sufficient for the recognition of a peptide Ag presented in the cleft of an MHC-encoded molecule.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M2 muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

Recombinant influenza viruses that bear the single immunodominant CD8+ T cell epitopeOVA257−264or the CD4+ T cell epitopeOVA323−339of the model antigen ovalbumin (OVA) have been useful tools in immunology. Here, we generated a recombinant influenza virus,WSN-OVAI/II, that bears both OVA-specific CD8+ and CD4+ epitopes on its hemagglutinin molecule. Live and heat-inactivatedWSN-OVAI/IIviruses were efficiently presented by dendritic cellsin vitroto OT-I TCR transgenic CD8+ T cells and OT-II TCR transgenic CD4+ T cells.In vivo,WSN-OVAI/IIvirus was attenuated in virulence, highly immunogenic, and protected mice from B16-OVA tumor challenge in a prophylactic model of vaccination. Thus,WSN-OVAI/IIvirus represents an additional tool, along with OVA TCR transgenic mice, for further studies on T cell responses and may be of value in vaccine design.

ABSTRACT The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4+ T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4+ T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-γ). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-γ−/− mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4+-T-cell responses during toxoplasmosis.

Abstract BALB/c mice immunized with protein Ags such as OVA in adjuvant mount a Th1-type response. Inhibition of Th1 and development of Th2 cells can be induced by pretreating BALB/c mice with soluble OVA before priming. To investigate some aspects of this immune deviation in vivo, naive TCR transgenic T cells specific for the chicken OVA peptide 323-339 presented by I-A(d) molecules were adoptively transferred into normal BALB/c mice. The frequency and fate of the transferred T cells can be followed with an anti-clonotypic Ab. In response to priming with OVA in CFA, the transferred transgenic T cells expand and differentiate into Th1 cells producing IL-2 and IFN-gamma. If recipient mice are injected with soluble OVA before priming, the frequency of transgenic T cells is not affected, but their expansion in response to Ag priming is inhibited. Yet, the fewer transgenic T cells recovered are not anergic, they proliferate as control cells when restimulated in vitro by plate-bound anticlonotypic Ab or by Ag. Analysis of Th phenotype indicates that pretreatment with soluble OVA has suppressed Th1 cell differentiation in favor of the generation of Th2 cells producing IL-4 and IL-5. Pretreatment with soluble peptide 323-339 also inhibits Th1 cell development, but fails to induce Th2 cell differentiation. Thus, pretreatment with soluble protein Ag or with synthetic peptide inhibits Th1 cell development, but only protein, not peptide, administration can deviate the in vivo response of a clonal T cell population from the Th1 to the Th2 pathway.

Abstract To study how peripheral B cell tolerance against non-lymphoid tissue autoantigens is maintained, we generated transgenic RIP-OVA/HEL (ROH) mice expressing the model antigens, OVA and HEL, in pancreatic islet beta cells. Immunization with OVA/HEL/aluminiumhydroxide induced IgG auto-Ab titers that were much lower than in non-transgenic controls. Depletion of CD25+ cells during immunization completely restored auto-Ab production but did not affect titers against foreign antigens, indicating regulatory tolerance. Purified CD25+ FoxP3+ CD4+ T cells from ROH mice transferred B cell suppression into non-transgenic recipients. CD25+ cells also suppressed naïve transgenic HEL-specific B cells adoptively transferred into ROH mice, confirming peripheral B cell tolerance. B cell suppression mechanistically involved inhibiting the proliferation of autoreactive B cells, inducing their apoptosis after immunization with autoantigen, suppressing antibody secretion per B cell and downregulating IgMa and MHC II B cell surface expression in the autoantigen-draining lymph node. We conclude that CD25+ FoxP3+ regulatory T cells were necessary and sufficient to specifically suppress auto-Ab production against pancreatic islet cell antigens.

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4+ T cells specific for an OVA peptide–I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

ABSTRACT CD4+ and CD8+ T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4+ or OT-I CD8+ T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3′ nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8+ T cells. The ability of L. major transgenic parasites to activate OT-I CD8+ T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level.

Abstract To date mechanisms of T-cell mediated immunity and immune-escape in high grade lymphomas are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven in parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt’s lymphoma, we developed a syngeneic model to investigate the anti-lymphoma activity of lymphoma specific T-cells. By retroviral transduction of a lymphoma specific antigen (chicken ovalbumin-IRES-GFP vector) into primary cell lines from c-myc transgenic lymphomas we established a model that would allow us to investigate the contribution of interferon gamma signalling in rejection of high grade lymphoma. All lymphomas established displayed low MHC class I and II levels on the surface when compared to wildtype B-cells. This expression could be enhanced by treatment with interferon gamma (100U/ml) up to 10 fold. When retrovirally transduced lymphoma cells were injected into wildtype or GFP transgenic C57BL/6 recipients, animals displayed a significant delay in lymphoma growth compared to IRES-GFP transduced control cell lines. 50% of the recipient mice rejected OVA containing lymphomas whereas we observed a 100% lymphoma growth of IRES-GFP transduced lymphomas in GFP transgenic recipients. Developing OVA containing lymphomas displayed a loss of GFP expression indicating a selection for non transduced cells. In spleens from mice successfully rejecting OVA-containing lymphomas we found up to 1.5% (±0.12%) SIINFEKL specific T-cells. To gain mechanistic insights of lymphoma rejection, we transferred OVA transduced lymphoma cells to Stat1−/− and IFNg−/− recipients. Lack of STAT1−/− on the recipient side or inability to secrete interferon gamma was associated with fast lymphoma progression and was not different when compared to IRES-GFP transduced cell lines injected into GFP transgenic hosts. Although we found 1.6% (±0.52%) SIINFEKL T cells in spleens of lymphoma bearing STAT1−/− animals, interferon gamma production was significantly decreased. We could also show that in wild type recipients, OVA containing lymphomas displayed high MHC class I and II expression which is completely absent in lymphomas from Stat1−/− recipients. Our results suggest that rejection of high grade lymphoma by a specific minor antigen is possible and Stat1 signaling and interferon gamma production by the host is crucial for lymphoma rejection.

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ Eosinophils obtained from inflammatory tissues and secretions display an altered phenotype in comparison to peripheral blood eosinophils, with increased surface expression of major histocompatibility complex (MHC) proteins and adhesion molecules (Hansel et al., 1991), and migration across the microvascular endothelium may also increase their capacity to generate an oxidative burst (Walker et al., 1993; Yamamoto et al., 2000). Eosinophils are phagocytic cells, and have been shown to present simple (no requirement for intracellular processing) and complex antigens to MHC-restricted, antigen-specific T lymphocytes (Del Pozo et al., 1992; Weller et al., 1993). Furthermore, eosinophils express the costimulatory molecules required for effective antigen presentation (Tamura et al., 1996), and ligation of costimulatory molecules on the eosinophil cell surface can induce the release of eosinophil derived cytokines (Woerly et al., 1999; Woerly et al., 2002). Therefore the eosinophil may also regulate immune responses.¶ To date, no studies have demonstrated the ability of eosinophils to modulate activated T lymphocyte function via presentation of relevant antigen in the context of MHC class II (MHC-II), concomitant with Th2 cytokine release. In the experiments described in this thesis, murine eosinophils have been observed to rapidly migrate to sites of antigen deposition within the airways mucosa of naïve mice, suggesting a potential role for this granulocyte in the primary response to inhaled antigen. However, human allergic diseases are often diagnosed after the establishment of allergic responses, and symptom development. Therefore, a murine model of allergic airways disease (AAD) was used to investigate the ability for eosinophils to participate as antigen presenting cells (APCs), and thereby modulate activated T lymphocyte function both in vitro and in vivo. Detailed histological analysis of the pulmonary draining lymph nodes following antigen challenge in sensitised mice revealed a rapid infiltration of eosinophils into this tissue, which preceded the accumulation of eosinophils in bronchoalveolar lavage fluid (BALF). This suggested that eosinophils were preferentially translocating to the draining lymph nodes following antigen challenge, and that the subsequent accumulation of these cells in the BALF was a consequence of continued antigen delivery to the lower airways.¶ Eosinophil trafficking to lymphoid tissue via the afferent lymphatics was substantiated using electron microscopy of lymph node sections and the intravenous (i.v.) transfer of fluorescently labeled eosinophils, which did not traffic to lymph nodes via the blood. During the resolution of AAD, eosinophils were noted for their persistence in the pulmonary draining lymph nodes. These observations suggested a continued modulation of T cell function by lymph node dwelling eosinophils during AAD resolution, particularly in light of recent observations for draining lymph node T cell proliferation following instillation of antigen-pulsed eosinophils into the allergic mouse lung (Shi et al., 2000).¶ To further investigate the antigen presenting capacity, eosinophils were obtained from the BALF of mice with AAD, and their surface expression of MHC class II (MHC-II) proteins and costimulatory molecules confirmed using flow cytometric analysis. The ability to acquire and process complex antigen both in vitro and in vivo was also confirmed using naturally quenching fluorescenated ovalbumin (OVA), which is degraded into fluorescent peptides by the action of intracellular proteases. Thus, eosinophil expression of the surface molecules necessary for effective antigen presentation was confirmed, as was their ability to process complex antigen. Further investigations revealed that eosinophils can present complex OVA antigen to CD4+ T lymphocytes obtained from the allergic mouse, and to in vitro derived OVA-specific Th2 cells. In the presence of exogenous antigen, eosinophils co-cultured with T lymphocytes were able to induce Th2 cytokine production, and demonstrated an ability for eosinophils to modulate T lymphocyte function in vitro.¶ The ability for eosinophils to act as antigen presenting cells in vivo was also investigated. Eosinophils obtained from the antigen-saturated lungs of OVA sensitised and challenged mice were transferred to the peritoneal cavities of naïve host mice. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a pulmonary eosinophilia similar to that of OVA sensitised and challenged mice. To validate this finding, the experimental procedure was altered to accommodate the use of non-allergy derived eosinophils, which were pulsed with OVA in vitro, prior to transfer into naïve recipients. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a peripheral blood and pulmonary eosinophilia, and stimulation with OVA induced IL-5 and IL-13 cytokine production from pulmonary draining lymph node cells. Notably, the AAD induced by transfer of antigen pulsed eosinophils did not induce detectable OVA-specific IgG1, which may be attributed to the lack of soluble antigen required for B cell antibody production.¶ During the course of these investigations, an OVA T cell receptor (TCR) transgenic mouse (OT-II) was procured with a view to defining the interaction between eosinophils and activated T lymphocytes (Barnden et al., 1998). Despite having specificity for the OVA323-339 peptide, an immunodominant epitope that skews naïve T cell responses towards Th2 cytokine release (Janssen et al., 2000), T lymphocytes from the OT-II mouse preferentially secreted IFN-γ in response to stimulation with either OVA peptide or OVA. These mice were further characterised in a mouse model of AAD, and found to be refractory to disease induction and progression, which may be attributed to significant IFN-γ secretion by transgenic CD4+ T lymphocytes during antigen sensitisation. Indeed, these cells were noted for their ability to attenuate pulmonary eosinophilia when transferred to OVA sensitised and challenged wild type mice, although serum OVA-specific IgG1, peripheral blood eosinophilia levels and airways response to methacholine challenge remained intact.¶ Knowledge of the biased Th1 phenotype in naïve OT-II provided a unique opportunity to investigate the fate of T lymphocytes bearing high affinity OVA-specific TCRs following neonatal antigen exposure to soluble OVA. In a previous study, subcutaneous (s.c.) administration of soluble OVA to wild type neonatal mice was suspected to have deleted OVA-specific T cells from the T cell repertoire (Hogan et al., 1998a). Using flow cytometry and TCR specific antibody, the delivery of s.c. OVA to OT-II neonates did not alter transgenic T cell populations in adult mice. Instead, it was surprising to find a skewing towards the Th2 phenotype and loss of IFN-γ secretion following OVA sensitisation and challenge in adult mice. A mechanism for this reprogramming of the transgenic T cell from the Th1 to a Th2 phenotype following OT-II neonatal exposure to soluble OVA is proposed, and further experimentation may validate this hypothesis.¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ ...¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia