Showing papers by "Richard A. Flavell published in 1994"
TL;DR: The study confirms the important role of CD40-CD40L interactions in thymus-dependent humoral immune responses and germinal center formation and indicates an inability to develop memory B cell responses.
768 citations
TL;DR: The 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.
Abstract: 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.
313 citations
TL;DR: It is documented that peripheral deletion is accompanied by the induction of abortive T cell activation followed by the disappearance of transgene-positive T cells, likely to be a general clearance pathway for T cells that are programmed to undergo apoptosis in vivo.
300 citations
TL;DR: 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, indicating that TcR‐mediated and co‐stimulatory signals required for T‐cell activation appear to be integrated, in part, at the level of the regulation of transcriptionalactivity ofAP‐1.
Abstract: 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.
285 citations
TL;DR: It is concluded that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine.
Abstract: We produced transgenic mice which overexpress human IL-6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II+ cells, and B220+ cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47 +/- 0.06 vs 0.43 +/- 0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45 +/- 0.07 vs 0.43 +/- 0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose of methacholine to produce a 100% increase in respiratory system resistance as compared with non-transgenic littermates (1.34 +/- 0.24 vs 0.34 +/- 0.05 mg/ml, P < or = 0.01). We conclude that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6-induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology.
212 citations
TL;DR: It is found that islets from these transgenic mice are immunogenic for naive T cells in vitro and in vivo, and mice expressing the costimulator B7-1 specifically on beta cells do not develop diabetes, suggesting that expression of the B 7-1 costimulators is not sufficient to abrogate the tolerance to peripheral antigens.
Abstract: 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.
195 citations
TL;DR: The role of Ii chain is defined as a chaperone that assists class II during folding, assembly, and transport and causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.
Abstract: 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.
191 citations
TL;DR: Cl cloning and characterization of two outer surface proteins (Osps) from strain N40 of Borrelia burgdorferi, the spirochetal agent of Lyme disease, reveal a hydrophobic domain and a consensus cleavage sequence recognized by signal peptidase II, and [3H]palmitate labeling shows that OspE and OspF are lipoproteins.
Abstract: We report the cloning and characterization of two outer surface proteins (Osps), designated OspE and OspF, from strain N40 of Borrelia burgdorferi, the spirochetal agent of Lyme disease. The ospE and ospF genes are structurally arranged in tandem as one transcriptional unit under the control of a common promoter. The ospE gene, located at the 5' end of the operon, is 513 nucleotides in length and encodes a 171-amino-acid protein with a calculated molecular mass of 19.2 kDa. The ospF gene, located 27 bp downstream of the stop codon of the ospE gene, consists of 690 nucleotides and encodes a protein of 230 amino acids with a calculated molecular mass of 26.1 kDa. Pulsed-field gel electrophoresis showed that the ospE and ospF genes are located on a 45-kb plasmid. Comparison of the leader sequences of OspE and OspF with those of the four known B. burgdorferi Osps (OspA, OspB, OspC, and OspD) reveals a hydrophobic domain and a consensus cleavage sequence (L-X-Y-C) recognized by signal peptidase II, and [3H]palmitate labeling shows that OspE and OspF are lipoproteins. Immunofluorescence studies demonstrated that both the OspE and OspF proteins are surface exposed. These features are consistent with the finding that OspE and OspF are B. burgdorferi surface lipoproteins.
186 citations
TL;DR: Evidence is presented indicating that the level of major histocompatibility complex (MHC) antigen expressed by islet cells plays a critical role in the development of in vivo tolerance to islets antigen.
171 citations
TL;DR: OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity, and antibodies to OspF were rarely detected in patients.
Abstract: We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.
84 citations
TL;DR: Results suggest that during prolonged infection, some patients develop an immune response that may be partly protective against reinfection with B. burgdorferi, and although most patients do not mount a strong humoral response to the Osps during natural infection, vaccination with an Osp may elicit protective immunity.
Abstract: 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.
TL;DR: 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.
TL;DR: It is concluded 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 ofIL-6 and diabetes susceptibility genes causesinsulitis in NOD/F1 mice.
Abstract: 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.
01 Jan 1994
TL;DR: These findings demonstrate an uncoupling of IL-6induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology.
Abstract: We produced transgenic mice which overexpress human IL6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II + cells, and B220 + cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47+0.06 vs 0.43±0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45±0.07 vs 0.43±0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose ofmethacholine to produce a 100% increase in respiratory system resistance as compared with nontransgenic littermates (1.34±0.24 vs 0.34±0.05 mg/ml, P s 0.01). We conclude that the expression ofhuman IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology. (J. Clin. Invest. 1994. 94:2028-2035.) Key words: cytokines * airway physiology * lung biology * methacholine. airway hyperresponsiveness
TL;DR: The fact that human CD4 is functional in mice and can be studied in the absence of murine CD4 should facilitate studies of humanCD4 activity in general and human immunodeficiency virus 1 gp120-mediated pathogenesis in acquired immune deficiency syndrome specifically.
Abstract: 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.
TL;DR: In this article, the authors describe the isolation of the gene encoding a 22-kDa antigen from Borrelia burgdorferi, the etiologic agent of Lyme disease.
Abstract: We describe the isolation of the gene encoding a 22-kDa antigen from Borrelia burgdorferi, the etiologic agent of Lyme disease. The p22 gene is 582 nucleotides in length and encodes a protein of 194 amino acids with a predicted molecular mass of 21.8 kDa. The leader signal sequence of P22 consists of a positively charged short amino terminus, a central hydrophobic domain, and at the carboxyl terminus, a cleavage site that is presumably recognized and cleaved by a B. burgdorferi signal peptidase. P22 has 98.5% homology with the recently described B. burgdorferi protein IpLA7. P22 is processed as a lipoprotein, as demonstrated by [3H]palmitate labeling. Pulsed-field gel electrophoresis showed that p22, like LA7, is localized to the linear chromosome of B. burgdorferi. Examination of sera from patients with Lyme disease revealed that antibodies to P22 are rarely detected in patients with early-stage disease characterized by erythema migrans (2 of 20), and 35% of the patients with late-stage disease characterized by arthritis (9 of 26) developed antibodies to P22. Sera from patients with syphilis did not react with P22. When patients with late-stage disease were tested for their antibody reactivities to four other outer surface proteins (OspA), OspB, OspE, and OspF), 75% of these patients responded to P22 or to one or more outer surface proteins.
TL;DR: The T cell response from normal individuals is more likely due to multiple antigenic epitopes within Borrelial proteins than a superantigen response, and cord blood specimens that contain almost entirely naive T cells, respond as vigorously to B. burgdorferi as T cells from normal adult peripheral blood.
Abstract: 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.
TL;DR: The presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response are investigated and confirm a recent report that Jak1 plays a critical early signaling role for both IFN -alpha/ beta and IFn-gamma systems.
Abstract: The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.
TL;DR: 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.
Abstract: 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.
01 Jan 1994
TL;DR: Although expression of a co-stimulator appears to be required for the activation of islet-specific T cells in the experimental system, it does not seem to be sufficient because none of the RIP-B7 mice develop diabetes.
Abstract: Publisher Summary During the life-span of a mouse, T cells are generated that are specific for antigens uniquely expressed in the periphery. These potentially autoreactive cells circulate in the periphery but normally remain silent. Recruitment of these cells to the tissue can occur under the influence of cytokines but normally this event does not lead to tissue destruction. If circumstances favoring the activation and clonal expansion of the autoreactive T cells exist in the tissue, it results in autoimmunity. In the case of SV-40 T antigen transgenic mice, autoimmunity is the consequence of local antigen presentation mediated by hyperplasia, accompanying necrosis, and concomitant tissue remodeling. On expression of the B7/BB1 molecule, islets become effective antigen presenting cells (APCs) and can stimulate autoreactive T cells to destroy these islets. Although expression of a co-stimulator appears to be required for the activation of islet-specific T cells in the experimental system, it does not seem to be sufficient because none of the RIP-B7 mice develop diabetes.
01 Jan 1994
TL;DR: The initial tick bite and skin rash can be difficult to identify, therefore, an effective vaccine should prove useful in endemic areas.
Abstract: Lyme borreliosis is a tick borne infection caused by the spirochete, Borrelia burgdorferi (1). Disease is often marked by a skin rash (erythema migrans), and can progress through several stages to cause arthritis, carditis or neurologic symptoms (2). The initial tick bite and skin rash can be difficult to identify, therefore, an effective vaccine should prove useful in endemic areas.