Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 199 records · Page 11Linked to original sources

PubMed1996

Tissue immunogenicity: the role of MHC antigen and the lymphocyte costimulator B7-1.

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. One approach to circumvent the requirement for recipient immune suppression is to reduce or eliminate the immunogenicity of the donor graft prior to transplantation. In this study, we have examined the relative contributions of graft MHC Ag expression and donor-derived costimulatory (CoS) activity to the rejection of islet allografts. Depletion of donor hemopoietic APCs from islet tissue facilitated long-term allograft survival even when donor class I MHC Ag expression was greatly increased by IFN-gamma treatment prior to grafting. Conversely, islet allografts from transgenic mice expressing the CoS molecule B7-1 (CD80) on islet beta cells were acutely rejected even when hemopoietic APCs were eliminated. Thus, B7-1 is sufficient to confer the capacity of islet parenchymal cells to stimulate allorejection. Taken together, these results point towards donor-derived CoS activity as a primary target of intervention therapy to modulate tissue immunogenicity.

Animals↗

PubMed1996

Double knockout of the MRP gene leads to increased drug sensitivity in vitro.

Overexpression of the multidrug resistance-associated protein (MRP) gene has been implicated in the resistance of tumor cell lines to a wide array of chemotherapeutic agents, but its normal physiological function(s) remains unknown. We have compared the sensitivity to chemotherapeutic drugs and toxins of wild-type W9.5 embryonic stem cells (ES) and of single and double MRP gene knockout cells derived therefrom. MRP expression was totally abrogated in the double knockout cell line and partially abrogated in the single knockout cell line. Reverse transcription-PCR analyses demonstrated that the MDR1, MDR2, and MDR3 genes were not expressed in either wild-type or MRP knock-out cells. The cytotoxic activities of etoposide, teniposide, vincristine, doxorubicin, daunorubicin, and sodium arsenite were significantly greater in double knockout cells than in parental wild-type ES cells; single knockout ES cells displayed an intermediate level of sensitivity. In contrast, no difference in sensitivity to colchicine and 1-beta-D-arabinofuranosylcytosine existed between the cell lines. Etoposide accumulation in double knockout ES cells was 2-fold higher than in wild-type ES cells. These findings indicate that baseline MRP expression has the capacity to exert a protective role against the toxicity of multiple chemotherapeutic agents and natural toxins.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1996

Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.

Programmed cell death (apoptosis) is a prominent feature of the development of the immune and nervous systems. The identification of the Caenorhabditis elegans cell death gene, ced-3, as a prototype of the interleukin-1beta converting enzyme (ICE) protease family has led to extensive evidence implicating these enzymes in apoptosis. Among the ten or more members of the ICE protease family, CPP32/yama/apopain exhibits the highest similarity to CED-3 in both sequence homology and substrate specificity. To analyse its function in vivo, we generated CPP32-deficient mice by homologous recombination. These mice, born at a frequency lower than expected by mendelian genetics, were smaller than their littermates and died at 1-3 weeks of age. Although their thymocytes retained normal susceptibility to various apoptotic stimuli, brain development in CPP32-deficient mice was profoundly affected, and discernible by embryonic day 12, resulting in a variety of hyperplasias and disorganized cell deployment. These supernumerary cells were postmitotic and terminally differentiated by the postnatal stage. Pyknotic clusters at sites of major morphogenetic change during normal brain development were not observed in the mutant embryos, indicating decreased apoptosis in the absence of CPP32. Thus CPP32 is shown to play a critical role during morphogenetic cell death in the mammalian brain.

Animals↗

PubMed1996

Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells.

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.

Adoptive Transfer↗

PubMed1996

Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor.

Signals from CD4+ T cells induce two opposite fates in B cells: clonal proliferation of B cells that bind specifically to foreign antigens and clonal deletion of equivalent B cells that bind self-antigens. This B cell fate decision is determined by the concerted action of two surface proteins on activated T cells, CD40-and Fas-ligands (CD40L and FasL), whose effects are switched by signals from the B cell antigen receptor (BCR). Foreign antigens that stimulate the BCR acutely cause CD40L and FasL to promote clonal proliferation. CD40L and FasL trigger deletion, however, when the BCRs become desensitized by chronic stimulation with self-antigens or when BCRs have not bound an antigen. The need for both Fas and CD40L to correctly regulate self-reactive B cell fate may explain the severe autoantibody disorders in Fas- or CD40L-deficient children.

Animals↗

PubMed1996

Requirement for invariant chain in B cell maturation and function.

Previously the role of invariant chain (Ii) had been described only as a chaperone that facilitates folding and transport of major histocompatability complex class II molecules; here it is shown that Ii is required for B cell development. B cells from mice lacking Ii were found to have a low response to T-independent type II antigen and could not proliferate after the mice were injected with antigen. Study of cell surface markers revealed a developmental arrest that prevented immature virgin B cells from becoming mature B cells in the periphery. This block was independent of major histocompatability complex class II expression and was an intrinsic feature of B cells that correlated with the amount of Ii. Thus, Ii participates by an unknown mechanism in B cell maturation.

Animals↗

PubMed1996

Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis.

The mechanism of CD40 ligand (CD40L)-mediated in vivo activation of CD4(+) T cells was examined by investigation of the development of experimental allergic encephalomyelitis (EAE) in CD40L-deficient mice that carried a transgenic T cell receptor specific for myelin basic protein. These mice failed to develop EAE after priming with antigen, and CD4(+) T cells remained quiescent and produced no interferon-gamma (IFN-gamma). T cells were primed to make IFN-gamma and induce EAE by providing these mice with B7.1(+) antigen-presenting cells (APCs). Thus, CD40L is required to induce costimulatory activity on APCs for in vivo activation of CD4(+) T cells to produce IFN-gamma and to evoke autoimmunity.

Animals↗

PubMed1996

Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.

Animals↗

PubMed1996

Protective antibodies in murine Lyme disease arise independently of CD40 ligand.

Borrelia burgdorferi-infected mice develop acute arthritis that undergoes Ab-mediated resolution. To further investigate the role of B. burgdorferi-specific Abs in Lyme borreliosis, CD40 ligand-deficient (CD40L-deficient) mice were infected with B. burgdorferi. The development and regression of arthritis were similar in CD40L-deficient and control mice. Although CD40L-deficient mice have defects in Ig class switching, infected CD40L-deficient mice developed B. burgdorferi-specific IgG2b Abs. Moreover, the transfer of serum from B. burgdorferi-infected CD40L-deficient animals prevented infection in severe combined immunodeficient mice. These data show that B. burgdorferi-infected CD40L-deficient mice are capable of producing Abs that are protective, despite the inability of these mice to mediate T-dependent immune responses.

Animals↗

PubMed1996

Autoimmune lpr/lpr mice deficient in CD40 ligand: spontaneous Ig class switching with dichotomy of autoantibody responses.

Fas-deficient MRL/Mp-lpr/lpr mice develop a syndrome that resembles human systemic lupus erythematosus, including production of IgG autoantibodies against small nuclear ribonucleoproteins (snRNPs), dsDNA, and self IgG (rheumatoid factor). To investigate the necessity for T-B cell contact in MRL autoimmunity, mice deficient in CD40 ligand (CD40L) were backcrossed onto this background, and Ab synthesis was assessed. In comparison to their CD40L-intact lpr/lpr counterparts, CD40L-deficient lpr/lpr mice had elevated levels of serum IgM and lower levels of IgG; however, a subset of animals had IgG2a, and to a lesser extent, IgG2b levels similar to those found in wild-type lpr/lpr mice. Levels of both isotypes in CD40L-deficient lpr/lpr mice were significantly greater than those found in nonautoimmune CD40L-deficient animals. IgG autoantibodies, including those directed against small nuclear ribonucleoproteins, also arose in CD40L-deficient lpr/lpr mice; however, they did not develop IgG rheumatoid factors or anti-dsDNA, and lacked histologic evidence of overt glomerulonephritis at age 3 mo, in contrast to CD40L-intact lpr/lpr animals. These results indicate that isotype switching occurs in lpr/lpr mice deficient in CD40L, and that production of IgG autoantibodies to ribonucleoproteins is at least partially preserved. They also suggest that different mechanisms may be responsible for eliciting autoantibody responses in lpr/lpr mice.

Animals↗

PubMed1996

Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2.

The retinoic acid-inducible transcription factor AP-2 is expressed in epithelial and neural crest cell lineages during murine development. AP-2 can regulate neural and epithelial gene transcription, and is associated with overexpression of c-erbB-2 in human breast-cancer cell lines. To ascertain the importance of AP-2 for normal development, we have derived mice containing a homozygous disruption of the AP-2 gene. These AP-2-null mice have multiple congenital defects and die at birth. In particular, the AP-2 knockout mice exhibit anencephaly, craniofacial defects and thoraco-abdominoschisis. Skeletal defects occur in the head and trunk region, where many bones are deformed or absent. Analysis of these mice earlier in embryogenesis indicates a failure of cranial neural-tube closure and defects in cranial ganglia development. We have shown that AP-2 is a fundamental regulator of mammalian craniofacial development.

Animals↗

PubMed1996

CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response.

The ligand for CD40 (CD40L) is expressed on the surface of activated CD4+ T cells and its role in T-B cell collaborations and thymus-dependent humoral immunity is well established. Recently, by generating CD40L-knockout mice, we have confirmed its previously described role in humoral immunity and defined another important function of this molecule in the in vivo clonal expansion of antigen-specific CD4+ T cells. Here, we investigated the potential in vivo role of CD40L in antiviral immunity by examining the immune response mounted by CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV), Pichinde virus, or vesicular stomatitis virus. Humoral immune responses of CD40L-deficient mice to these viruses were severely compromised, although moderate titres of antiviral IgM and some IgG2a were produced by virus-infected CD40L-deficient mice by a CD4+ T cell-independent mechanism. By contrast, CD40L-deficient mice made strong primary CTL responses to all three viruses. Interestingly however, although memory CTL activity was detectable in CD40L-deficient mice two months after infection with LCMV, the memory CTL response was much less efficient than in wild-type mice. Together, the results show that CD40-CD40L interactions are required for strong antiviral humoral immune responses, and reveal a novel role for CD40L in the establishment and/or maintenance of CD8+ CTL memory.

Animals↗

PubMed1996

Impaired primary T cell responses in L-selectin-deficient mice.

L-selectin is a homing receptor that mediates the selective attachment of leukocytes to specialized high endothelial venules. To study the potential role of L-selectin in immune responses in intact mice, we generated L-selectin-deficient mice by gene targeting. L-selectin-deficient mice are defective in cutaneous delayed-type hypersensitivity (DTH) responses when tested after conventional intervals of immunization (4 d). Primary T cell proliferative responses and cytokine production (interleukin [IL] 2, IL-4, and interferon gamma) were also compromised when tested after 5 d of immunization, indicating that L-selectin is important for the immune response to antigens. In contrast, after more prolonged immunization protocols (9 d), normal responses were observed, suggesting that L-selectin-independent compensatory mechanisms exist. Interestingly, humoral responses of L-selectin-deficient mice to keyhole limpet hemocyanin are indistinguishable from wild-type control mice, implying that L-selectin plays no rate-limiting role in T cell help of B cell function. Thus, our results suggest that L-selectin plays an important role in the generation of primary T cell responses but may not be essential for humoral and memory T cell responses. L-selectin does not appear to be rate limiting for the events leading to antigen-driven neutrophil recruitment, since normal DTH responses are obtained at late time points after immunization.

Animals↗

PubMed1996

Thymus-leukemia antigen interacts with T cells and self-peptides.

The functional role of the class Ib thymus-leukemia (TL) Ag expressed within the thymic cortex and intestinal mucosa of the mouse remains unknown. In an approach to elucidate the potential functionality of TL, we developed transgenic mice that ectopically express the H-2T18d gene product on essentially all nucleated cells through the control of a heterologous H-2Kb gene promoter. Transgenic mice demonstrated an increase in the number of CD4+ lymphocytes within the thymus and lymph nodes; these cells displayed an altered T cell receptor repertoire possibly suggesting a role for the ectopically expressed TL protein. The TL protein additionally displayed the characteristics of a bona fide transplantation Ag, because skin grafts from transgenic animals onto MHC- and minor histocompatibility Ag-matched nontransgenic recipient mice resulted in a rapid and vigorous immunologic rejection of the allograft. In MLR studies, transgenic stimulator cells induced the proliferation of responders to a level intermediate between genetically identical and H-2-disparate responder-stimulator combinations. The TL protein was also capable of stimulating cytotoxic T lymphocytes, thereby resulting in specific lysis of TL+ target cells. Further data demonstrated that the TL protein assembles with peptides that are modified at the amino terminus, and that TL retains these molecules at the cell surface. Together, these data suggest that H-2T18d is capable of interacting with T cells via a bound peptide. These data further support the possibility that TL may subserve a specialized function within the immunologic system.

Animals↗

PubMed1996

CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.

T cells play an important role in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse. CD8 cytotoxic T cell lines and clones were generated from the lymphocytic infiltrate in the islets of Langerhans of young (7-wk-old). NOD mice by growing them on (NOD x B6-RIP-B7-1)F1 islets. These cells proliferate specifically to NOD islets and kill NOD islets in vitro. The cells are restricted by H-2Kd, and all bear T cell antigen receptor encoded by V beta 6. When these CD8 T cell lines and clones are adoptively transferred to irradiated female NOD, young NOD-SCID, and CB17-SCID mice, diabetes occurs very rapidly, within 10 d of transfer and without CD4 T cells.

Amino Acid Sequence↗

PubMed1996

Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice.

The expression of the ligand for CD40 (CD40L) is critical for induction of T cell-dependent Ab responses. To examine how critical the expression of CD40L is for induction of cell-mediated immune responses, the ability of T cells from CD40L knockout mice to activate macrophage effector function was assessed. CD4+ T cells from CD40L-knockout mice were fourfold less effective than +/+ T cells in activating the nitric oxide response in allogeneic macrophages. CD40L-knockout T cells that were fixed with paraformaldehyde after a 6-h activation period, a time point at which CD40L dominates the macrophage-activating capability of the T cell, could activate neither macrophage production of inflammatory cytokines (TNF-alpha) nor generation of reactive nitrogen intermediates. After 24 h of activation, however, both CD40L-knockout and +/+ T cells could induce similar but weak responses from the macrophages. This study demonstrates that animals deficient in CD40L expression display a deficiency in T cell-dependent macrophage-mediated immune responses.

Animals↗

PubMed1996

Glycosylphosphatidylinositol-anchored H-2Db molecules are defective in antigen processing and presentation to cytotoxic T lymphocytes.

Glycosylphosphatidylinositol-anchored (GPI)-Db molecules are defective in mediating cytotoxic T lymphocytes (CTL) lysis of transfected lymphoma cells, compared to their transmembrane (TM) counterpart. This defect is manifest when antigenic peptide must be processed and presented through the endogenous pathway. These same transfectants can be lysed by allospecific CTL, or by antigen-specific Db-restricted CTL when pulsed with appropriate exogenous synthetic peptide, demonstrating that they can bind and present peptide for CTL-mediated lympholysis. The defect apparently results from differences between GPI-Db and TM-Db assembly and transport, or from differences in membrane topology that affect CD8+ CTL recognition of major histocompatibility complex/peptide complex.

Animals↗

PubMed1996

The contribution of insulitis to diabetes development in tumor necrosis factor transgenic mice.

The inflammatory response mediated by cytokines such as TNF can promote recruitment of lymphocytes to a tissue. Moreover, if other conditions are met, this can provide a predisposing role to autoimmune disease. TNFs induce the appearance of adhesion molecules (and presumably, therefore, extravasation of lymphocytes into tissue from the vasculature) and increase the levels of MHC class I on tissue. However, it is not clear which of these effects plays the key role in induction of disease. This should be the subject of further study. The data substantiate the hypothesis that chronic inflammation might play a precipitating role in autoimmunity and could be one of the environmental factors of importance in the development of so many autoimmune diseases.

Animals↗