Biomedical subjects

Steven J Burakoff

Publications and source records attributed to Steven J Burakoff.

15 recordsLinked to original sources

PubMed2006

A CD8/Lck transgene is able to drive thymocyte differentiation.

Efficient development of thymocytes requires participation of a CD8 or CD4 coreceptor in the TCR:MHC interaction. Both CD8 and CD4 coreceptor cytoplasmic domains associate with Lck. In this study, we attempted to delineate the role of CD8alpha-associated Lck in driving CD8 single positive (SP) thymocyte development. We used a chimeric molecule encoding the extracellular and transmembrane domains of CD8alpha fused to full-length Lck. In mice deficient for CD8alpha and transgenic for 2C, a MHC class I-restricted TCR, robust reconstitution of CD8 SP thymocytes occurred both centrally and peripherally. The reconstituted CD8 SP population was phenotypically and functionally comparable to 2C wild-type counterparts expressing endogenous CD8alpha. A CD8alpha/Lck kinase-dead chimera also resulted in reconstitution of CD8 SP thymocytes. Our results suggest that CD8alpha-associated Lck is sufficient to drive CD8 SP thymocyte development. Furthermore, this CD8 SP development may not necessarily depend on Lck kinase activity.

Animals↗

PubMed2005

HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.

Adaptor Protein Complex 2↗

PubMed2004

CD4 phosphorylation partially reverses Nef down-regulation of CD4.

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (approximately 95%), whereas that induced by PMA was partial (approximately 40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.

CD4 Antigens↗

PubMed2004

Nicotine activates nuclear factor of activated T cells c2 (NFATc2) and prevents cell cycle entry in T cells.

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.

Animals↗

PubMed2004

SOD2-deficiency anemia: protein oxidation and altered protein expression reveal targets of damage, stress response, and antioxidant responsiveness.

SOD2 is an antioxidant protein that protects cells against mitochondrial superoxide. Hematopoietic stem cells (HSCs) lacking SOD2 are capable of rescuing lethally irradiated hosts, but reconstituted animals display a persistent hemolytic anemia characterized by increased oxidative damage to red cells, with morphologic similarity to human "sideroblastic" anemia. We report further characterization of this novel SOD2-deficiency anemia. Electron micrographs of SOD2-deficient reticulocytes reveal striking mitochondrial proliferation and mitochondrial membrane thickening. Peripheral blood smears show abundant iron-stainable granules in mature red cells (siderocytes). Fluorescence-activated cell sorting (FACS) analysis of cells labeled with oxidation-sensitive dyes demonstrates enhanced production of superoxide and hydrogen peroxide by SOD2-deficient cells. Oxidative damage to proteins is increased in SOD2-deficient cells, with much of the damage affecting membrane/insoluble proteins. Red cell proteome analysis demonstrates that several proteins involved in folding/chaperone function, redox regulation, adenosine triphosphate (ATP) synthesis, and red cell metabolism show altered expression in SOD2-deficient cells. This data, combined with information on how protein expression levels change upon antioxidant therapy, will aid in identification of proteins that are sensitive to oxidative damage in this model, and by extension, may have a role in the regulation of red cell lifespan in other hemolytic disorders.

Adenosine Triphosphate↗

PubMed2004

Fratricide of CD8+ cytotoxic T lymphocytes is dependent on cellular activation and perforin-mediated killing.

CD8+ CTL mediate the destruction of cells displaying foreign peptides in association with class I MHC molecules. Since CD8+ CTL themselves express class I MHC molecules, a phenomenon known as "fratricide" can be elicited by T cells presenting antigens to other CTL. To gain insight into this mechanism, fratricide was induced in a clone of class I-restricted CD8+ CTL by incubating the T cells with their agonist ligand, an octamer peptide derived from chicken ovalbumin. Our results indicate that agonist peptide not only stimulates proliferation and cytolysis of CTL but also initiates signaling pathways that are pertinent to T cell activation, including the mobilization of transcription factors. Also consistent with T cell activation, fratricide induced the transcription and translation of the pro-inflammatory cytokines TNF-alpha and IFN-gamma. Finally, the essential role of perforin, as opposed to Fas/FasL, in fratricide was demonstrated by the selective inhibition of cytolysis with an inhibitor of the perforin pathway, the absence of FasL expression on T cells and the presence of lytic granules visible by electron microscopy. Collectively, these findings reveal that fratricide is mediated by T cell activation and perforin-mediated cytolysis. These results may have implications for the regulation of CD8+ CTL in immune responses.

Cytotoxicity, Immunologic↗

PubMed2004

Increasing T-cell age reduces effector activity but preserves proliferative capacity in a murine allogeneic major histocompatibility complex-mismatched bone marrow transplant model.

Aging of T cells is characterized by a series of alterations in surface antigen expression and a concomitant decline in functional activity in many assays. We have extended this analysis by comparing the ability of T cells from mice of different ages to cause graft-versus-host disease (GVHD) by using a parent into F(1) model (C57BL/6 T cells into C57BL/6 x C3H host animals). Young (3-5 months), adult (12-14 months), or old (19-24 months) T cells were introduced into irradiated F(1) hosts. Animals that had undergone transplantation were assessed for clinical and pathologic evidence of GVHD and for survival. At a given T-cell dose (2 x 10(6) cells), there was a T-cell (donor) age-dependent decline in severity of GVHD, with all recipients of young T cells succumbing to lethal GVHD, 75% of recipients of adult T cells succumbing, and no deaths occurring among recipients of old T cells. In vivo CD4 T-cell expansion was greater for young than old T-cell groups after transplantation, whereas old CD8 cells showed enhanced in vivo expansion compared with young cells. Among CD4 and CD8 cells, the T-cell receptor repertoire, surface antigen expression on activated cells, and homing receptor function were similar for all ages after expansion in vivo. The progeny of old T cells reisolated after transplantation expressed type 1 cytokines (interferon-gamma and tumor necrosis factor-alpha) at a lower frequency than young cells and had decreased cytolytic function against H-2(k)-bearing target cells. This provides a partial explanation for the decreased GVHD. Carboxyfluorescein diacetate succinimidyl ester labeling of transplanted cells showed comparable rates of proliferation when comparing GVHD-competent (12 months) and GVHD-incompetent (19 months) T cells in both syngeneic and F(1) host animals. We suggest that the lack of effector activity demonstrated by old T cells in vivo is a reflection of a cell-autonomous defect downstream of signals required for antigen-driven proliferation.

Aging↗

PubMed2003

Rapamycin-resistant proliferation of CD8+ T cells correlates with p27kip1 down-regulation and bcl-xL induction, and is prevented by an inhibitor of phosphoinositide 3-kinase activity.

Rapamycin inhibits the proliferation of many mammalian cell types, including lymphocytes, making the compound useful as an immunosuppressant. Rapamycin has also been a useful tool for studying signaling mechanisms regulating cellular proliferation. However, the effects of rapamycin remain poorly understood, and the precise mechanisms of clinical action remain elusive. Previously, we found that, depending on the strength of the signal delivered to the T cell via both the T cell receptor and the costimulatory molecule CD28, CD8+ T cells are capable of rapamycin-resistant proliferation. Here, we have further elucidated the mechanism of rapamycin-resistant proliferation of human CD8+ T cells. Under conditions where rapamycin inhibited proliferation, p27kip1 down-regulation was prevented, whereas under conditions resulting in rapamycin-resistant proliferation, p27kip1 was down-regulated. Further, T cell receptor/CD28-dependent induction of bcl-xL expression was not inhibited by rapamycin, which correlated with both rapamycin-resistant proliferation and increased cell survival. Moreover, an inhibitor of phosphoinositide 3-kinase activity was able to eliminate rapamycin-resistant proliferation of freshly isolated CD8+ human cells, strongly suggesting that phosphoinositide 3-kinase activity was required for the rapamycin-resistant proliferation of CD8+ T cells. The selective immunosuppressive effect of rapamycin in human CD8+ T cell populations could be predictive of a selective effect allowing cytotoxic responses during microbial infections where there are strong strengths of signals associated with high affinity T cell receptors and strong costimulatory second signals. In contrast, the weaker autoimmune and perhaps allogeneic responses can be selectively inhibited by the actions of rapamycin.

Annexin A5↗

PubMed2003

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling.

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.

Amino Acid Motifs↗

PubMed2003

Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2-induced fos gene transcription.

Prostaglandin E(2) (PGE(2)) is the predominant eicosanoid product released by macrophages at the site of inflammation. Binding of PGE(2) to its cognate 7 transmembrane-spanning G protein-coupled receptors (GPCRs) activates signaling pathways, leading to the synthesis of the Fos transcription factor. Because the Ste20 serine/threonine protein kinase (S/TPK) is a critical signal transducer for the G protein-coupled pheromone receptor in Saccharomyces cerevisiae, we postulated that the PGE(2) GPCRs may activate one of the Ste20 mammalian orthologs. We demonstrate here that the catalytic activity of a hematopoietic cell-restricted, Ste20-related S/TPK, HPK1, is positively regulated by exposure to physiological concentrations of PGE(2). Furthermore, ectopic expression studies implicated HPK1 as a negative regulator of PGE(2)-induced transcription of the fos gene. Our data suggest that PGE(2)-induced activation of HPK1 may represent a novel negative regulatory pathway capable of modulating PGE(2)-mediated gene transcription.

Catalysis↗

PubMed2002

Wnt-1 and Wnt-4 regulate thymic cellularity.

Thymic primordium, formed by cells derived from the endoderm, the ectoderm and the neural crest-derived mesenchyme, receive fetal liver derived lymphoid precursors. Reciprocal cell-cell interactions between thymic stromal cells and lymphoid precursors are critical in the expansion and maturation of thymocytes. Transcription factor TCF-1 is critical for the expansion of thymocytes because deletion of TCF-1 results in a significant decrease in the number of thymocytes without affecting the developmental pattern. In this report we show that Wnt-1 and Wnt-4 are expressed in the thymus and the deletion of Wnt-1 or Wnt-4 result in a substantial decrease in the number of thymocytes without affecting the pattern of maturation. Wnt-1 and Wnt-4 both regulate developing thymocytes because a double deficiency results in a significantly greater decrease of immature and mature thymocytes compared to deficiency in either Wnt-1 or Wnt-4.

Amphibian Proteins↗

PubMed2002

NFATc2-mediated repression of cyclin-dependent kinase 4 expression.

The calcineurin-regulated transcription factor, nuclear factor of activated T cells (NFAT), controls many aspects of T cell function. Here, we demonstrate that the calcineurin/NFAT pathway negatively regulates the expression of cyclin-dependent kinase 4 (CDK4). A canonical NFAT binding site was identified and found to be sensitive to calcium signals, FK506/CsA, and histone deacetylase activity and to not require AP-1. Ectopic expression of NFATc2 inhibited the basal activity of the human CDK4 promoter. Additionally, both calcineurin Aalpha(-/-) and NFATc2(-/-) mice had elevated protein levels of CDK4, confirming a negative regulatory role for the calcineurin/NFAT pathway. This pathway may thus regulate the expression of CDK4 at the transcriptional level and control how cells re-enter a resting, nonproliferative state.

Animals↗

PubMed2002

Effect of anti-IL-2Ralpha antibody on IL-2-induced Jak/STAT signaling.

Acute allograft rejection is driven by production of cytokines such as interleukin-2 (IL-2) that activate and expand alloreactive T cells by ligating high-affinity IL-2 receptors composed of three subunit chains: alpha, beta, gamma The alpha chain, expressed only on activated T cells, has become an important therapeutic target. Monoclonal antibodies (mAbs) that bind IL-2Ralpha chains significantly decrease transplant rejection. We examined the ability of the humanized anti-IL-2Ralpha antibody daclizumab to block high-affinity IL-2Rs and interrupt T-lymphocyte signaling. Our evaluation focused on a pathway critical for T-cell proliferation, the Jak/STAT pathway. Daclizumab markedly inhibited phosphorylation of the Jak1, Jak3 and STAT5a/b components of the IL-2R-dependent pathway. Suppression by daclizumab was associated with internalization of IL-2Ralpha but not IL-2Rbetagamma chains. High IL-2 doses overcame daclizumab-induced blockade of Jak/STAT phosphorylation despite absent cell surface highaffinity IL-2Rs. Under these circumstances, IL-2-mediated Jak/STAT pathway activation might be generated through residual intermediate affinity IL-2Rbetagamma receptors, and this was demonstrated by complete blockade of signaling when anti-IL-2Rbeta monoclonal antibody was added. Humanized antibodies are an important part of strategies to induce alloantigen tolerance. Understanding the molecular events associated with their beneficial clinical effect is critical to design of future immunosuppressive strategies.

Antibodies, Monoclonal↗

PubMed2002

Cytolytic pathways in haematopoietic stem-cell transplantation.

The remarkable activity of donor T cells against malignant cells in the context of an allogeneic haematopoietic stem-cell transplantation (HSCT) is arguably, at present, the most potent clinical immunotherapy for cancer. However, alloreactive donor T cells are also important effector cells in the development of graft-versus-host disease (GVHD), which is a potentially lethal complication for recipients of an allogeneic HSCT. Therefore, the separation of the GVHD and graft-versus-tumour (GVT) activity of donor T cells has become a topic of great interest for many investigators. Recent studies have shown that donor T cells make differential use of their cytolytic pathways in mediating GVHD and GVT effects. Therefore, the selective blockade or enhancement of cytolytic pathways provides an intriguing therapeutic opportunity to separate the desired GVT effect from the potentially devastating GVHD.

Animals↗

PubMed2002

Enhanced allostimulatory activity of host antigen-presenting cells in old mice intensifies acute graft-versus-host disease.

Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-alpha and IL-12 after LPS stimulation. In a B6 --> B6D2F1 model, CD4(+) donor T cells but not CD8(+) T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.

Aging↗