Showing papers by "Richard A. Flavell published in 1995"

Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme

Abstract: The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.

Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand.

Abstract: LACK of functional expression of CD40 ligand (CD40L) on T cells results in hyper-IgM syndrome (HIGMl), a human immunodeficiency associated with a severely impaired humoral immune response that is consistent with defects in B-cell responses1–3. Patients also succumb to recurrent opportunistic infections such as Pneumocystis carinii and Cryptosporidial diarrhoea4,5, suggesting that T-cell functions are also compromised in these individuals, but so far this has not been explained. We have previously shown that mice deficient for CD40L, like HIGMl patients, show grossly abnormal humoral responses6. Here we report that CD40L-deficient mice are defective in antigen-specific T-cell responses. Adoptively transferred antigen-specific CD4+ T cells lacking CD40L failed to expand upon antigen challenge of the recipients, showing that expression of CD40L on T cells is required for in vivo priming of CD4+ T cells and therefore for the initiation of specific T-cell immune responses.

Class II transactivator regulates the expression of multiple genes involved in antigen presentation.

Abstract: CIITA (a major histocompatibility complex [MHC] class II transactivator) has been shown to be required for the expression of MHC class II genes in both B cells and interferon gamma-inducible cells. Here we demonstrate that CIITA not only activates MHC class II genes but also genes required for antigen presentation. Mutant HeLa cells, defective in the expression of classic MHC class II genes, invariant chain, and the newly described human histocompatibility leukocyte antigen-DM genes, were used to study the role of CIITA in the regulation of these genes. Upon transfection with CIITA cDNA, the mutant cells expressed all three genes, suggesting that CIITA is a global regulator for the expression of genes involved in antigen presentation.

Borrelia burgdorferi genes selectively expressed in the infected host

Abstract: An immunological screening strategy was used to select microbial genes expressed only in the host. Differential screening of a Borrelia burgdorferi (the Lyme disease agent) expression library identified a gene (p21) encoding a 20.7-kDa antigen that reacted with antibodies in serum from actively infected mice but not serum from mice immunized with heat-killed B. burgdorferi. Selective expression of p21 in the infected host was confirmed by Northern blot analysis and RNA PCR. Further differential screening of the expression library identified at least five additional B. burgdorferi genes are selectively expressed in vivo. This screening method can be used to identify genes induced in vivo in a wide variety of pathogenic microorganisms for which a gene transfer system is not currently available.

An essential role for gp39, the ligand for CD40, in thymic selection.

Abstract: The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

Peptide and protein antigens require distinct antigen-presenting cell subsets for the priming of CD4+ T cells.

Abstract: Priming of naive CD4+ T cells to Ag requires an antigen-presenting cell (APC) that can take up the Ag and present peptide bound to MHC class II molecules. We have used both in vivo and in vitro approaches to demonstrate that the APC used to prime naive CD4+ T cells depends on the initial form in which an Ag is administered. Although Ag delivered as a peptide was presented most efficiently to CD4+ T cells by DC, these APC were poor at priming to a protein form of the same Ag. In contrast, the presence of B cells was a requisite for priming to protein Ag.

Mechanisms of immune tolerance induction through the thymic expression of a peripheral tissue-specific protein.

Abstract: A major process through which the immune system becomes tolerant to self proteins involves the deletion of self reactive cells in the thymus. However, T cells reactive to peripheral tissue-specific proteins can escape this deletion and become tolerized in the periphery by a variety of mechanisms. We report here, contrary to expectation, that the pancreas-specific protein, elastase I, is also expressed at a low level in the thymus, and that this thymic expression contributes to tolerance induction. To study the mechanism of this tolerance induction, we utilized a double transgenic mouse model. In these mice the expression of a model protein, SV40 T antigen, is directed by the elastase I promoter and hence parallels elastase I expression in the pancreas and thymus. These mice were crossed with mice transgenic for a TCR specific for T antigen, so the majority of thymocytes and T cells in these mice express the transgene. In double transgenic mice we find that thymic expression of T antigen results in anergic thymocytes which also show a reduction of Th1 activity with no decrease in Th2 activity. These functional characteristics persist in peripheral T cells, but there is also a depletion in the number of T antigen reactive T cells in lymph nodes. Chimeras were constructed which directly demonstrated that the thymus is the site of tolerance induction and that the tolerizing element is thymic epithelium. We propose that the loss of Th1 activity as a consequence of the thymic epithelium being encountered by tissue-specific proteins results in the functional tolerization of CTL in vivo, despite the fact that CTL are fully functional in vitro. In this way autoimmune destruction is contained. Thymic expression of peripheral proteins may therefore be an additional way in which tolerance to peripheral proteins can be achieved.

CIITA activates the expression of MHC class II genes in mouse T cells.

Abstract: It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively.

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

Abstract: 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.

B7 costimulation is necessary for the activation of the lytic function in cytotoxic T lymphocyte precursors.

Abstract: B7-CD28 costimulation is essential for the activation of CD4+ T helper cells, mainly by regulating IL-2 and other cytokine production. The requirement for this costimulatory pathway in the activation of CD8+ T cells, however, is still poorly understood. Here we analyzed the role of B7-CD28 costimulation in the differentiation of Ag-specific CTL precursor. We found that the activation of not only IL-2 production but also cytotoxic function in CD8 T cells requires B7 costimulation. The costimulatory signal, which cannot be replaced by exogenous IL-2, is directly implicated in the activation of the lytic machinery in CD8 T cells. Moreover, B7-CD28 costimulation appears to play a critical role in the accumulation of mRNA encoding at least one of the granzymes required for cytolytic function, granzyme B or CTLA-1. The production of IFN-gamma by CD8 T cells, however, does not appear to require costimulation.

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Abstract: Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Breaking immunologic ignorance to an antigenic peptide of simian virus 40 large T antigen.

Abstract: Expression of antigenic proteins in the periphery may result in immunologic tolerance, immunologic ignorance, or autoimmunity. It is not clear why some Ags induce tolerance, whereas others activate Ag-reactive T cells. The physical nature of the Ag, the developmental timing of Ag expression, the priming of reactive lymphocytes, and the level of Ag expression are possible factors determining the immunologic response to extra-thymic Ags. Expression of the whole SV40 large T Ag (SV40-T) induces transformation of T antigen-expressing cells in vivo, and this phenomenon has been postulated to be the triggering event that leads to autoimmunity in some transgenic mouse models. Here we present a model in which a nononcogenic, yet antigenic, fragment of the SV40-T (SV40-Tfrag) is expressed specifically in pancreatic islet beta-cells. In contrast to whole SV40-T transgenic mice, SV40-Tfrag mice that are also transgenic for a TCR specific for the SV40-T are ignorant of Ag in vivo. They do not respond in vivo to the tissue-specific SV40-Tfrag Ag even after priming, but are fully responsive in vitro. This immunologic ignorance cannot be broken after activated SV40-T reactive T cells are transferred into sublethally irradiated mice expressing the islet-specific SV40-Tfrag. However, similar adoptive transfer experiments in mice co-expressing B7-1 and SV40-Tfrag on islet cells specifically lead to Ag recognition and diabetes. This demonstrates that in some circumstances the presence of (primed) reactive T cells is not sufficient to break tolerance; rather, costimulation is additionally required to elicit an autoimmune response. This also suggests that SV40-T-induced cellular transformation is important for the autoimmune response directed against SV40-T in other tissue-specific transgenic models.

Oral vaccination with an attenuated Salmonella typhimurium strain expressing Borrelia burgdorferi OspA prevents murine Lyme borreliosis.

Abstract: Borrelia burgdorferi is the causative agent of Lyme disease. In the mouse model, protection is correlated with the development of antibodies to a major outer surface protein, OspA. In this study, we expressed OspA in an attenuated strain of Salmonella typhimurium and tested the efficacy of the transformed strain in protecting against disease. We show that mice inoculated by gavage developed high titers of anti-OspA antibodies and were protected against an intradermal challenge with the spirochete.

Efficacy of human Lyme disease vaccine formulations in a mouse model.

Abstract: Although immunization with recombinant outer surface protein A (OspA) appears to protect mice against infection by the agent of Lyme disease, all reported experiments have involved formulations that would not be suitable for use in humans or have not used realistic challenges. This study was designed to determine whether vaccines prepared and used in a phase I human trial, including one currently being used for a phase II trial in sites with endemic Borrelia burgdorferi, conferred protection in the C3H/HeJ mouse model. The challenge was ticks collected from a major site of the trial. None of the vaccinated mice became infected or developed disease, whereas 60% of unvaccinated mice became infected. Spirochetes were destroyed within the guts of virtually all recovered challenge ticks. These preparations of recombinant OspA effectively induced immunity to protect mice from Lyme disease when bitten by ticks collected from a field trial site.

B7-1 expression by a non-antigen presenting cell-derived tumor.

Abstract: The existence of a naturally occurring immunosurveillance against neoplastic cells is controversial. A difficulty with this concept is that tumor-specific antigen-reactive T cells would not be expected to become activated after encountering tumor cells, since T cells that bind to antigen in the absence of the costimulation provided by antigen-presenting cells may be inactivated. We studied a transgenic model of tumorigenesis where T cells reactive to a particular tumor-specific antigen are lost prior to the development of non-antigen-presenting cell-derived tumors; therefore, the tumors that develop are not subjected to immunosurveillance. We found that a tumor cell line derived from one such tumor expresses the T-cell costimulatory molecule B7-1, the expression of which is normally restricted to antigen-presenting cells. In addition, we found that several immortalized cell lines, which are nontumorigenic and thus have suffered only early genetic events in the tumorigenesis process, express B7. This suggests that a host cell can be induced to express surface B7-1 molecules after suffering an oncogenic insult, which might possibly be a primary mechanism of immunosurveillance against tumors.

Costimulation in Tolerance and Autoimmunity

Abstract: 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.

Selection of variant Borrelia burgdorferi isolates from mice immunized with outer surface protein A or B.

Abstract: A nonclonal population of Borrelia burgdorferi N40 (passage 3) that survived protective immunity following challenge inoculation of outer surface protein (Osp) A- or B-hyperimmunized mice were characterized for the molecular basis of evasion of immunity. Two of six B. burgdorferi isolates, cultured from OspA-immunized mice, had antigenic diversity in the carboxyl terminus of OspA and did not bind to the protective OspA monoclonal antibody designated IXDII. However, OspA-immunized mice challenged with these variants were fully protected. Moreover, B. burgdorferi isolates with a point mutation in ospB, which results in a truncated OspB that does not bind to protective OspB monoclonal antibody 7E6C, were frequently enriched after infection of OspB-immunized mice. These studies suggest that the incomplete efficacy of an OspA- or OspB-based vaccine may be partly due to immunomediated in vivo selective pressure, resulting in the persistence of some spirochetes that do not bind to protective antibodies.

The hook protein of Borrelia burgdorferi, encoded by the flgE gene, is serologically recognized in Lyme disease.

Abstract: The periplasmic flagellum of Borrelia burgdorferi consists of a unipeptide flagellar filament, a hook, and a basal body. Here, we report the cloning and expression of the hook gene, flgE, of B. burgdorferi N40. The flgE gene is 1,119 nucleotides long and is located on the 950-kb linear chromosome of B. burgdorferi. The primary protein sequence of FlgE shows 73% similarity to the FlgE protein of Treponema phagedenis and approximately 50% similarity to the FlgG proteins of both gram-positive and gram-negative bacteria. The flgE gene was cloned into an Escherichia coli expression plasmid, pMX, to produce FlgE protein. Subsequently, FlgE murine antiserum was prepared by immunizing mice with the partially purified B. burgdorferi FlgE protein. By Western blot (immunoblot) analysis, the antiserum was found to react with a 40-kDa peptide in the whole-cell lysates, confirming the expression of the flgE gene in B. burgdorferi. Additionally, antibodies to FlgE were found in serum specimens from 19 of 42 patients with Lyme disease. Moreover, when other antigens, including 41G (the immunodominant domain of flagellin), OspE, OspF, and p22, were used to test for the development of corresponding antibodies in these patients, 67% of these patients (28 of 42) reacted to at least one of these five antigens, suggesting that a combination of FlgE with other available B. burgdorferi recombinant proteins is a good candidate for substrates in assays to aid in the diagnosis of Lyme disease.

A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease.

Abstract: We have identified a 55-kDa antigen encoded by a gene on a 49-kb plasmid of Borrelia burgdorferi. The screening of a B. burgdorferi DNA expression library (N40 strain) with rabbit anti-B. burgdorferi serum and then with serum from a patient with Lyme disease arthritis revealed a clone that synthesized an antigen that was reactive with both sera. DNA sequence analysis identified an operon with two genes, s1 and s2 (1,254 and 780 nucleotides), that expressed antigens with the predicted molecular masses of 55 and 29 kDa, respectively. Pulsed-field gel electrophoresis showed that the s1-s2 operon was located on the 49-kb plasmid. Recombinant S1 was synthesized as a glutathione S-transferase fusion protein in Escherichia coli. Antibodies to recombinant S1 bound to a 55-kDa protein in lysates of B. burgdorferi, indicating that cultured spirochetes synthesized S1. Thirty-one of 100 Lyme disease patients had immunoglobulin G (IgG) and/or IgM antibodies to S1. IgG antibodies to S1 were detected by enzyme-linked immunosorbent assay and immunoblots in the sera of 21 (21%) of 100 patients with Lyme disease; 11 (27.5%) of the S1-positive samples were from patients (40) with early-stage Lyme disease, and 10 (16.7%) were from patients (60) with late-stage Lyme disease. Fifteen (38.5%) of 40 serum samples from patients with early-stage Lyme disease had IgM antibodies to S1. These data suggest that the S1 antigen encoded by a gene on the 49-kb plasmid is recognized serologically by a subset of patients with early- or late-stage Lyme disease.

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

Abstract: 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.

An ospA frame shift, identified from DNA in Lyme arthritis synovial fluid, results in an outer surface protein A that does not bind protective antibodies.

Abstract: Passive immunization with murine or human Abs to outer surface protein A (OspA) can protect mice against Borrelia burgdorferi, but OspA Abs elicited during natural infection in mice or humans are unable to clear the spirochete from the infected host. To examine Ab binding by OspA during the course of human infection, we amplified the operon encoding full-length ospA and ospB from synovial fluids of a patient with chronic Lyme arthritis, the first such recoveries from human material, at four separate time points over 4.5 mo, and expressed OspA in Escherichia coli. OspA mAbs that passively protected mice from infection did not bind one of the expressed OspAs, because of a deletion in ospA that resulted in a frame shift and premature stop codon near the carboxyl terminus. However, expressed OspA from a later synovial fluid sample did not contain this deletion. Thus, although altered forms of OspA, which potentially can influence host immune effectiveness, do occur in the human host, they cannot be the only factors responsible for microbial persistence.

Regulation of CD4+ T cell differentiation.

Abstract: 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.

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

Abstract: 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 Ly...