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Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 253 records · Page 14Linked to original sources
PubMed1995

Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi OspA or B.

The evolution of Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface proteins (Osps) A or B was assessed to investigate the role of immunity to OspA or B in infection and pathogenesis of Lyme disease. Antibodies to OspA or B protect immunocompetent C3H/HeJ or C.B.17 severe combined immunodeficient (scid) mice from challenge with B. burgdorferi. Moreover, arthritis in infected C3H mice resolves with the rise of high titers of B. burgdorferi specific antibodies, including OspA and B, whereas disease persists in scid mice--suggesting that the regression of arthritis may be due to the development of borreliacidal OspA or B antibodies. To evaluate the course of Lyme borreliosis in OspA or B tolerant mice we developed transgenic mice that expressed OspA or B under control of the major histocompatibility complex (MHC) class I promoter. Mice carrying OspA or B transgenes on a C3H/HeJ (C3H, disease-susceptible) or C57BL/6 (B6, disease-resistant) background, immunized with OspA or B, did not mount a humoral or cellular immune response to OspA or B, respectively, but responded normally to other B. burgdorferi antigens. The evolution of Lyme borreliosis, including infection and the development of arthritis and carditis, was similar in transgenic and nontransgenic littermates suggesting that an OspA or B immune response is not singularly involved in either the genesis or regression of Lyme disease in C3H or B6 mice.

Animals↗
PubMed1995

Expression of the co-stimulator molecule B7-1 in pancreatic beta-cells accelerates diabetes in the NOD mouse.

B7-1 is a co-stimulatory molecule that signals T-cells that recognize antigen to proliferate and differentiate into effector T-cells. The same cell must present antigen and express co-stimulatory molecules, such as B7-1, to activate naive T-cells. Thus, tissues that do not express co-stimulatory molecules would not be expected to induce immune responses, while expression of a co-stimulator on tissue cells may convert them into effective antigen-presenting cells and induce autoimmunity. To test this, transgenic mice have been generated that express B7-1 on the beta-cells of the pancreatic islets of Langerhans. On a B6 genetic background, B7-1 expression on beta-cells does not predispose to diabetes. B6 mice are resistant to diabetes. However, when B7-1 is expressed on the beta-cells of B6 mice backcrossed once to the genetically susceptible NOD strain, the onset of diabetes is accelerated and the autoimmune attack intensified. This illustrates that B7-1 is a very potent co-stimulatory molecule in vivo and that its presence on the surface of tissue cells can potentiate the autoimmune process.

Aging↗
PubMed1995

Costimulation in tolerance and autoimmunity.

The two signal model of T cell activation predicts that a second costimulatory signal provided by antigen presenting cells (APC) is required in conjunction with the antigenic signal to trigger T cell activation. Considerable evidence indicates that indeed, T cell activation requires such a costimulatory signal which results, at least in part, from the interaction of CD28 with its ligands B7 expressed on all antigen-presenting cells (APC). The second prediction of the two signal model is that T cell receptor engagement in the absence of such a costimulatory signal would lead to specific inactivation of antigen reactive cells. Thus, tissue cells that do not express costimulatory signals would not trigger T cell activation but rather lead to specific inactivation of auto-reactive T cells. By such a model, tolerance to peripheral antigens would be permanently re-established. We review here the evidence suggesting that the CD28-B7 costimulatory pathway might play an important role in T cell tolerance and in the development of autoimmune responses.

Animals↗
PubMed1995

Reactivity of white-footed mouse and raccoon sera to whole cell and recombinant antigens of Borrelia burgdorferi.

Serum samples were collected from white-footed mice (Peromyscus leucopus) and raccoons (Procyon lotor) during 1983, 1984, and 1990 through 1993 in Connecticut (USA) and were tested in enzyme-linked immunosorbent assays (ELISA) against whole cell Borrelia burgdorferi sensu stricto (strain 2591) and the following recombinant antigens of this spirochete: p41-G (an immunogenic epitope of flagellin), outer surface protein (OSP)A, and OSPB. Antibodies were most frequently detected when whole cell antigen was used in the analyses. Reactivity to highly specific recombinant antigens also occurred and was particularly helpful in verifying B. burgdorferi infection. Geometric mean antibody titers for assays with whole cell antigen ranged from 453 to 2,363 and were at least two-fold higher than geometric means calculated for tests with recombinant antigens, which ranged from 226 to 640. With greater sensitivity, an ELISA with whole cell antigen is preferred for determining presence of antibody in sites enzootic for Lyme borreliosis. However, use of highly specific recombinant antigens, particularly OSPA and OSPB, in an ELISA can provide supportive information in ecological studies of this disease.

Animals↗
PubMed1995

Regulation of CD4+ T cell differentiation.

Naive T cells can be induced to differentiate from an uncommitted precursor to T helper 1 (Th1) and Th2 cells. During this differentiation, genes for transcription factors are activated, and transcription factors such as AP-1 accumulate. To study this activation, we have developed reporter transgenic mice for a number of factors, including AP-1. Naive T cells require two signals to activate AP-1. However, upon becoming effector cells, activation through diacylglycerol analogues is sufficient. Rested effector cells lose accumulated AP-1, and the induction of AP-1 synthesis requires both Ca2+ diacylglycerol signals, but not co-stimulation.

Animals↗
PubMed1994

Thymic development in human CD4 transgenic mice. Positive selection occurs after commitment to the CD8 lineage.

Developing T cells are positively selected on the basis of their recognition of polymorphic MHCs (major histocompatibility molecules). The CD4 and CD8 co-receptors, together with a compatible TCR-alpha beta, bind class II and class I MHC, respectively, delivering a signal that allows T cell maturation to proceed along the appropriate co-receptor lineage. During development, thymocytes first co-express CD4 and CD8 and subsequently commit to a single co-receptor phenotype. Debate exists over the stage at which positive selection occurs. Positive selection may precede or accompany co-receptor lineage commitment or occur subsequent to the acquisition of a single co-receptor phenotype. We developed transgenic (TG) mice expressing human CD4 (huCD4) on T cells. To study differentiation of CD8+ cells, huCD4 TG mice were bred with mice lacking class I MHC (class I-). This enabled us to assess maturation of mCD8+mCD4+huCD4+ thymocytes to mCD8+mCD4-huCD4+ cells after interaction between huCD4 and mclass II MHC. In the absence of huCD4, class I- mice failed to produce mCD8+ cells. However, expression of huCD4 allowed development of mCD8+mCD4-huCD4+ T cells, suggesting that selection of cells precommitted to the mCD8+mCD4- lineage occurred. Importantly, CD8+ T cells were increased in both the thymus and the periphery of huCD4 TG x class I- animals, indicating their intrathymic origin. V beta 14+ CD8+ T cells were also increased, as predicted for positive selection of CD8+ cells bearing class II-reactive TCRs. These data provide evidence that positive selection occurs at a late stage of thymocyte differentiation, after commitment to the CD8 lineage.

Animals↗
PubMed1994

Class II transactivator (CIITA) is sufficient for the inducible expression of major histocompatibility complex class II genes.

The class II transactivator (CIITA) has been shown to be required for major histocompatibility complex (MHC) class II gene expression in B cells and its deficiency is responsible for a hereditary MHC class II deficiency. Here we show that CIITA is also involved in the inducible expression of class II genes upon interferon gamma (IFN-gamma) treatment. The expression of CIITA is also inducible with IFN-gamma before the induction of MHC class II mRNA. In addition, CIITA mRNA expression does not require new protein synthesis, although new protein synthesis is necessary for the transcription of class II. This suggests that synthesis of new CIITA protein may be essential to induce class II gene expression. We also showed that the JAK1 protein tyrosine kinase activity is required to induce the expression of CIITA upon IFN-gamma stimulation. This finding indicates that CIITA is part of the signaling cascade from the IFN-gamma receptor to the activation of class II genes. In addition, the expression of CIITA is sufficient to activate class II genes in the absence of IFN-gamma stimulation suggesting that CIITA is the major regulatory factor for the inducible expression of class II genes. Together, these data suggest that CIITA is the IFN-inducible cycloheximide sensitive factor previously shown to be required for the induction of MHC class II gene expression.

Base Sequence↗
PubMed1994

AP-1 transcriptional activity requires both T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes.

The transcription factor AP-1 contributes significantly to the regulation of interleukin-2 gene transcription during T-cell activation and may play a role in thymocyte development. To study the regulation of AP-1 transcriptional activity in primary T-cells, reporter transgenic mice were generated that express luciferase gene under the control of AP-1 binding sites. Here, we demonstrate that while protein kinase C activation is sufficient to induce DNA-binding activity, an additional intracellular calcium increase is required to induce transcriptional activity of AP-1 in primary mouse T-cells. Furthermore, transcriptional, but not DNA-binding, activity of AP-1 is cyclosporin sensitive and requires tyrosine phosphorylation. This dissociation between DNA-binding and transcriptional activity is likely due, at least partially, to post-translational modifications of the AP-1 complex required for transcriptional activity. Moreover, in addition to these two signals delivered by ligand binding to the T-cell receptor (TcR) AP-1 transcriptional activity absolutely requires the presence of a co-stimulatory signal that can be mediated by the interaction of CD28 with its ligands B7-1 and B7-2. Thus, TcR-mediated and co-stimulatory signals required for T-cell activation appear to be integrated, in part, at the level of the regulation of transcriptional activity of AP-1.

Animals↗
PubMed1994

Costimulator B7-1 confers antigen-presenting-cell function to parenchymal tissue and in conjunction with tumor necrosis factor alpha leads to autoimmunity in transgenic mice.

Tolerance to peripheral antigens is thought to result from the inability of parenchymal tissue to stimulate T cells--an inability that is believed to relate to the lack of expression of the costimulatory signal(s) required for T-cell activation. To test this model, we generated transgenic mice expressing costimulatory molecule B7-1 on the B cells of the pancreas. We find that islets from these transgenic mice are immunogenic for naive T cells in vitro and in vivo. Nonetheless, mice expressing the costimulator B7-1 specifically on beta cells do not develop diabetes, suggesting that expression of the B7-1 costimulator is not sufficient to abrogate the tolerance to peripheral antigens. We have reported that tumor necrosis factor alpha subunit (TNF-alpha) expressed by beta cells leads to a local inflammation but no islet destruction. Strikingly, however, the combination of a local inflammation due to the expression of the cytokine TNF-alpha and the expression of B7-1 results in tissue destruction and diabetes.

Animals↗
PubMed1994

Human CD4 restores normal T cell development and function in mice deficient in murine CD4.

The ability of a human coreceptor to function in mice was investigated by generating human CD4 (hCD4)-expressing transgenic mice on a mouse CD4-deficient (mCD4-/-) background. From developing thymocyte to matured T lymphocyte functions, hCD4 was shown to be physiologically active. By examining the expansion and deletion of specific V beta T cell families in mutated mice with and without hCD4, it was found that hCD4 can participate in positive and negative selection. Mature hCD4 single positive cells also were found in the periphery and they were shown to restore MHC class II-restricted alloreactive and antigen-specific T cell responses that were deficient in the mCD4 (-/-) mice. In addition, these hCD4 reconstituted mice can generate a secondary immunoglobulin G humoral response matching that of mCD4 wild-type mice. The fact that human CD4 is functional in mice and can be studied in the absence of murine CD4 should facilitate studies of human CD4 activity in general and human immunodeficiency virus 1 gp120-mediated pathogenesis in acquired immune deficiency syndrome specifically.

Animals↗
PubMed1994

The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is thought to act as a chaperone that assists class II during folding, assembly, and transport. To define more precisely the role of Ii chain in regulating class II function, we have investigated in detail the biosynthesis, transport, and intracellular distribution of class II molecules in splenocytes from mice bearing a deletion of the Ii gene. As observed previously, the absence of Ii chain caused significant reduction in both class II-restricted antigen presentation and expression of class II molecules at the cell surface because of the intracellular accumulation of alpha and beta chains. Whereas much of the newly synthesized MHC molecules enter a high molecular weight aggregate characteristic of misfolded proteins, most of the alpha and beta chains form dimers and acquire epitopes characteristic of properly folded complexes. Although the complexes do not bind endogenously processed peptides, class II molecules that reach the surface are competent to bind peptides added to the medium, further demonstrating that at least some of the complexes fold properly. Similar to misfolded proteins, however, the alpha and beta chains are poorly terminally glycosylated, suggesting that they fail to reach the Golgi complex. As demonstrated by double label confocal and electron microscope immunocytochemistry, class II molecules were found in a subcompartment of the endoplasmic reticulum and in a population of small nonlysosomal vesicles possibly corresponding to the intermediate compartment or cis-Golgi network. Thus, although alpha and beta chains can fold and form dimers on their own, the absence of Ii chain causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.

Animals↗
PubMed1994

Prominent T lymphocyte response to Borrelia burgdorferi from peripheral blood of unexposed donors.

The proliferative response of peripheral blood T cells to the spirochete, Borrelia burgdorferi, can be as pronounced in unexposed normal individuals as it is in Lyme disease patients. This finding was observed using three geographically distinct isolates of B. burgdorferi. The response is not due to a lipopolysaccharide effect of the spirochete, is sensitive to Proteinase K, and requires antigen processing. It does not result from cross-reactivity of memory T cells that may be reactive to another antigen; the proliferative response to B. burgdorferi is equally distributed between naive (CD29-, CD45RO-) and memory (CD29+, CD45RO+) T cells, whereas the tetanus response is confined to the memory subset. In support of this notion, cord blood specimens that contain almost entirely naive T cells, respond as vigorously to B. burgdorferi as T cells from normal adult peripheral blood. A large panel of CD4+ T cell clones has been derived that are specific for B. burgdorferi. The majority of these clones are reactive to B. burgdorferi in the presence only of autologous HLA-DR molecules. Collectively, these data suggest that the T cell response from normal individuals is more likely due to multiple antigenic epitopes within Borrelial proteins than a superantigen response.

Antigen-Presenting Cells↗
PubMed1994

T-cell tolerance and autoimmunity in transgenic models of central and peripheral tolerance.

Experiments with transgenic mice expressing genes encoding both antigens in defined tissues and T-cell receptor genes of known specificities have enhanced our understanding of the mechanisms involved in the pathogenesis of autoimmune states. They have also shed light on the means by which potentially autoreactive cells may be prevented from exerting their autoaggressive potential. The value of the transgenic approach is that it can overcome the low frequency of peptide-specific T cells occurring in normal animals, and also provide a tissue-specific, cognate antigen that is absent in controls. These factors allow reactive T cells to be isolated or quantified by flow cytometry and their responses to antigen in vitro and in vivo be defined.

Animals↗
PubMed1994

Mice deficient for the CD40 ligand.

To study the potential roles of CD40L in immune responses, we generated CD40L-deficient mice by gene targeting. Similar to the effects of CD40L mutations in humans (hyper-IgM syndrome), CD40L-deficient mice have a decreased IgM response to thymus-dependent antigens, fail altogether to produce an antigen-specific IgG1 response following immunization, yet respond normally to a T-independent antigen, TNP-Ficoll. Moreover, these mice do not develop germinal centers in response to thymus-dependent antigens, suggesting an inability to develop memory B cell responses. Although CD40L-deficient mice have low levels of most circulating immunoglobulin isotypes, they do not exhibit the spontaneous hyper-IgM syndrome seen in humans, at least up to 12 weeks of age. In summary, our study confirms the important role of CD40-CD40L interactions in thymus-dependent humoral immune responses and germinal center formation.

Animals↗
PubMed1994

Sera from patients with chronic Lyme disease protect mice from Lyme borreliosis.

Sera from selected patients with Lyme disease in different stages were used to passively immunize mice against Borrelia burgdorferi challenge to determine if human antibodies could protect the animals from infection. Sera from 2 patients with late-stage Lyme disease that contained strong antibody reactivity to proteins in B. burgdorferi lysates, including antibodies to the outer surface proteins (Osps) A and B, partly protected mice from infection after challenge with a small inoculum (10(2)) of B. burgdorferi. Mice immunized with sera from either of these 2 patients developed significantly fewer infections from the borreliae (patient 1 serum, 5%; patient 2 serum, 25%) relative to control mice (patient 1 serum, 90%; patient 2 serum, 74%). In contrast, sera from 2 patients with early or late Lyme disease that lacked antibodies reactive to OspA and OspB did not confer protection. Immunity appeared to be related, at least in part, to the presence of a strong humoral response to the Osps. These results suggest that during prolonged infection, some patients develop an immune response that may be partly protective against reinfection with B. burgdorferi. Therefore, although most patients do not mount a strong humoral response to the Osps during natural infection, vaccination with an Osp may elicit protective immunity.

Animals↗
PubMed1994

Transgenic mice expressing constitutive levels of IL-2 in islet beta cells develop diabetes.

IL-2 plays an important role in the clonal expansion of T cells during an immune response and it has been implicated in autoimmune disease. To examine the role of IL-2 in the regulation of peripheral tolerance we produced transgenic mice in which the expression of murine IL-2 was directed by the rat insulin II promoter. The IL-2 transgene was expressed specifically in the pancreas. Islets from transgenic mice synthesized biologically active IL-2. Expression of IL-2 in the pancreas resulted in a massive inflammatory response directed at the beta cells of the pancreas. The infiltrate consisted primarily of B cells and CD4+ and CD8+ T cells. The infiltrate resulted in destruction of the insulin-producing beta cells and diabetes, but there was no evidence for antigen specificity. The results suggest that local IL-2 production elicits the recruitment and activation of cells capable of destroying beta cells by non-antigen-specific mechanisms.

Animals↗
PubMed1994

Local production of human IL-6 promotes insulitis but retards the onset of insulin-dependent diabetes mellitus in non-obese diabetic mice.

We produced transgenic mice which overexpress human IL-6 in pancreatic beta cells of C57BL/6 x CBA and non-obese diabetic (NOD) mice. Transgenic C57BL/6 x CBA mice back-crossed onto a C57BL/6 background do not develop insulitis or diabetes. In contrast, NOD/F1 transgenic mice develop a lymphocytic infiltrate of pancreatic islets which is not seen in negative littermates. Immunohistochemistry reveals these cells to be predominantly CD4+, CD8+, B220+ cells. Despite the insulitis, these mice do not develop diabetes. Transgenic rat insulin promoter-IL-6 mice were therefore also made on an inbred NOD background. These mice showed no difference in the onset or extent of insulitis when compared with non-transgenic NOD mice and no difference was found in the phenotype of the infiltrating cells. However, transgenic NOD mice had lower average fasting glucose levels and delayed onset of diabetes compared with age and sex matched littermate negative NOD mice. As a consequence, transgenic NOD mice also had longer survival than littermate negative NOD mice. We conclude that the expression of IL-6 by beta cells does not cause insulitis or diabetes in C57BL/6 x CBA mice, but that the interaction of IL-6 and diabetes susceptibility genes causes insulitis in NOD/F1 mice. Since IL-6 delays the onset of diabetes and prolongs survival of NOD mice, it is possible that the protective effect is caused by local IL-6 action on the islets, by the infiltrating lymphocytes or both.

Animals↗