Biomedical subjects

E A Greenfield

Publications and source records attributed to E A Greenfield.

15 recordsLinked to original sources

PubMed2001

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation.

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.

Animals↗

PubMed2001

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.

Amino Acid Sequence↗

PubMed2000

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells.

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.

Amino Acid Sequence↗

PubMed2000

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Adjuvants, Immunologic↗

PubMed2000

The BCR/ABL tyrosine kinase induces production of reactive oxygen species in hematopoietic cells.

The BCR/ABL oncogene causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and myeloid cells. It is shown here that transformation of the hematopoietic cell lines Ba/F3, 32Dcl3, and MO7e with BCR/ABL results in an increase in reactive oxygen species (ROS) compared with quiescent, untransformed cells. The increase in ROS was directly due to BCR/ABL because it was blocked by the ABL-specific tyrosine kinase inhibitor STI571. Oxidative stress through ROS is believed to have many biochemical effects, including the potential ability to inhibit protein-tyrosine phosphatases (PTPases). To understand the significance of increased production of ROS, a model system was established in which hydrogen peroxide (H(2)O(2)) was added to untransformed cells to mimic the increase in ROS induced constitutively by BCR/ABL. H(2)O(2) substantially reduced total cellular PTPase activity to a degree approximately equivalent to that of pervanadate, a well known PTPase inhibitor. Further, stimulation of untransformed cells with H(2)O(2) or pervanadate increased tyrosine phosphorylation of each of the most prominent known substrates of BCR/ABL, including c-ABL, c-CBL, SHC, and SHP-2. Treatment of the BCR/ABL-expressing cell line MO7/p210 with the reducing agents pyrrolidine dithiocarbamate or N-acetylcysteine reduced the accumulation of ROS and also decreased tyrosine phosphorylation of cellular proteins. Further, treatment of MO7e cells with H(2)O(2) or pervanadate increased the tyrosine kinase activity of c-ABL. Drugs that alter ROS metabolism or reactivate PTPases may antagonize BCR/ABL transformation.

Animals↗

PubMed1999

B7-2 expressed on EL4 lymphoma suppresses antitumor immunity by an interleukin 4-dependent mechanism.

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the "second signal" pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4-B7-1 cells are completely rejected in syngeneic mice. Unlike EL4-B7-1 cells, we find that EL4-B7-2 cells are not rejected but progressively grow in the mice. A B7-1- and B7-2-EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4-B7-1 tumor line that regressed in vivo. The EL4-B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti-B7-1 antibodies, anti-B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti-B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4-B7-2 and EL4-B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.

Animals↗

PubMed1999

Ox40-ligand has a critical costimulatory role in dendritic cell:T cell interactions.

The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.

3T3 Cells↗

PubMed1998

The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells.

Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18. In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells. Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen. We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane. Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells. These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule. They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.

3T3 Cells↗

PubMed1998

CD28/B7 costimulation: a review.

The current model of T cell activation requires two signals. The first signal is specific, requiring T cell receptor recognition and binding to MHC/Antigen presented by an antigen-presenting cell. The second signal is nonspecific, resulting from the binding of B7 ligand on the antigen-presenting cell with its receptor, CD28, on the T cell. If both signals are provided, the T cell will proliferate and secrete cytokines. Recently, it has been shown that CTLA4, another receptor for B7 that is upregulated following T cell after activation, can deliver an inhibitory signal, downregulating T cell proliferation. The B7 family of ligands has two family members, B7-1 and B7-2. They both bind to CD28 and CTLA4, but they differ in their binding affinity, structure, and temporal expression. Considerable research has been done on the CD28/B7 costimulatory pathway. Different ways of manipulating this pathway could provide insights into the mechanism and treatment of opposing pathological states. Blocking the CD28/B7 pathway could result in immunosuppression, with implications for the treatment of autoimmune diseases, organ transplantation, and graft vs. host disease. Activating the CD28/B7 pathway could be useful for including the immune system to recognize and eliminate tumors that evade the immune system. Finally, the CD28/B7 pathway could be involved with maintaining immune tolerance, as recent studies suggest the preferential binding of the B7-CTLA4 pathway results in the down-regulation of the responding T cells. Thus, the B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses.

Abatacept↗

PubMed1997

Expression of a hypoglycosylated form of CD86 (B7-2) on human T cells with altered binding properties to CD28 and CTLA-4.

CD80 (B7-1) and CD86 (B7-2) on APC provide a major costimulatory signal through interactions with CD28 on T cells. Absent from resting human T cells, CD86 is up-regulated early upon T cell activation, whereas CD80 expression appears later. Whereas T cell expression of CD80 has been implicated in costimulation, the functional significance of CD86 expression on T cells is unclear. We now demonstrate that CD86 expressed on human CD4+ T cell clones does not provide a costimulatory signal for other CD4+ T cell clones. Binding studies using CD28-Ig and CTLA-4-Ig fusion proteins demonstrate that CD86 expressed on T cells has significantly reduced binding affinity for CTLA-4 and no detectable binding to CD28. Biochemical analysis demonstrates that post-translational modifications of CD86 in human T cells are different from those of CD86-transfected Chinese hamster ovary cells or EBV-transformed B cells, in that T cells express a hypoglycosylated form of CD86 on the surface membrane. Thus, our results suggest that while CD86 is expressed on a number of different cell types, its costimulatory function and affinity for its ligands may be regulated by cell type-specific post-translational modifications.

Abatacept↗

PubMed1997

B7.2 expressed by T cells does not induce CD28-mediated costimulatory activity but retains CTLA4 binding: implications for induction of antitumor immunity to T cell tumors.

The B7 family of costimulatory molecules provides the second signal necessary for activation of T cells. In the absence of the second signal, responding T cells become anergic. Although predominantly expressed on professional APCs, recent evidence shows that the B7 molecules are also expressed on T cells. To study the functions of B7 molecules on T cells, we transfected murine B7.1 (CD80) and B7.2 (CD86) cDNAs into the EL4 T cell thymoma cell line and examined the transfectants for their ability to costimulate T cell proliferation in vitro and to induce antitumor immunity in vivo. Here we show that although EL4-B7.1 cells costimulate T cells and induce tumor regression, EL4-B7.2 transfectants failed to costimulate T cell proliferation or induce tumor regression. To understand the cellular basis for this difference, we examined the binding of EL4-B7.1 and EL4-B7.2 to CTLA4 and CD28. Whereas EL4-B7.1 cells bound both CTLA4-Ig and CD28-Ig, EL4-B7.2 transfectants preferentially bound CTLA4-Ig, but not CD28-Ig. Similar binding data were obtained with freshly isolated murine T cells, which have been shown to constitutively express B7.2. Our data suggest, therefore, that B7.2 expressed on T cells may not costimulate but instead inhibit the T cell response by preferential binding to CTLA4.

Abatacept↗

PubMed1996

The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells.

Related adhesion focal tyrosine kinase (RAFTK), also known as proline-rich tyrosine kinase 2 and cellular adhesion kinase beta, has been recently cloned and characterized as a member of the focal adhesion kinase (FAK) subfamily. RAFTK has an overall 48% amino acid homology to p125(FAK) and contains a kinase domain but lacks a transmembrane region, myristylation sites, and Src homology region 2 and 3 domains. By Northern blot analysis, RAFTK is expressed in myeloid, lymphoid, and megakaryocytic hematopoietic cells. Like p125(FAK), we found that RAFTK interacts with the focal adhesion protein paxillin. In the lymphoid cell line BaF3 and the myeloid cell line 32Dcl3, RAFTK coprecipitates with paxillin. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly, through a segment of paxillin that required amino acids 100-227 and a domain in the C terminus of RAFTK. In vitro, RAFTK could phosphorylate paxillin on tyrosine residues. These results suggest that RAFTK, as well as p125(FAK), may be important in phosphotyrosine-signaling events within the focal adhesion.

Animals↗

PubMed1996

Activation of human T cell lymphotropic virus type I-infected T cells is independent of B7 costimulation.

Two distinct signals are required to activate T cells: an Ag-specific signal and a costimulatory signal mediated primarily by B7-1 (CD80) and B7-2 (CD86) through interactions with CD28. Costimulation appears to be critical in regulating autoreactive T cell responses. Here, we demonstrate that, in contrast to the parental uninfected T cell clone, a T cell clone infected by human T cell lymphotropic virus type I (HTLV-I) displays a remarkably enhanced response to Ag in the absence of B7 costimulation. Chinese hamster ovary cells either transfected with DRB1*1501 (t-DR2) alone or cotransfected with DR2 and either B7-1 or B7-2 were fixed, pulsed with myelin basic protein peptide 84-102 (MBPp84-102), and used as APCs. The MBPp84-102-reactive T cell clone Ob1A12.8 required costimulation with either B7-1 or B7-2 molecules, as the response to Ag was reduced by 90% in the absence of B7 costimulation. However, this requirement for B7 costimulation was abrogated after productive infection by HTLV-I. Stimulation of HTLV-I-infected T cells by MBPp84-102/t-DR2 induced the secretion of IL-5 and IFN-gamma, which approached the level induced in the presence of B7 costimulation, whereas IL-4 was induced to one third of its maximal level. Consistently, the secretion of IL-5 and IFN-gamma was not significantly inhibited by anti-B7-1 and B7-2 Abs, whereas IL-4 was inhibited by approximately 50%. In contrast, uninfected T cells required either B7-1 or B7-2 costimulation for significant cytokine secretion, and this response was inhibited by anti-B7-1 and B7-2 Abs. These findings suggest that HTLV-I-infected autoreactive T cells have the potential to induce an autoimmune response in the absence of B7 expression in the target organ. This may be of particular interest in the elucidation of HTLV-I pathogenicity given the association of HTLV-I infection with autoimmune-like diseases.

Animals↗

PubMed1988

Comparative immunocytochemistry of Pelizaeus-Merzbacher disease, the jimpy mouse, and the myelin-deficient rat.

Patients with Pelizaeus-Merzbacher disease (PM), hemizygous mice with the jimpy mutation (jp/Y), and hemizygous rats with X-linked myelin deficiency (md/Y) share a profound lack of proteolipid protein (PLP) in their central nervous systems (CNS). The peripheral nervous system is normal. These X-linked disorders are associated with or actually caused by the lack of normal oligodendrocytes. Vibratome sections of brain were incubated with antisera to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), 2':3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37), PLP, a synthetic PLP-peptide, glial fibrillary acidic protein (GFAP), and transferrin. Reaction product was developed by sequential incubation with biotinylated second antibodies, the avidin-biotin-peroxidase complex (ABC), and diaminobenzidine (DAB) plus hydrogen peroxide as chromogenic substrates. In PM, jp/Y and md/Y, islands of myelin-like structures were revealed by antisera to MBP, MAG, and CNP. Reaction product after application of anti-PLP was absent. Reaction product after anti-PLP-peptide was restricted to infrequent bizarre cells possibly representing abnormal oligodendroglia. The lack of oligodendrocytes in jp/Y and md/Y could also be confirmed by immunocytochemistry for transferrin.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1987

Defective biosynthesis of proteolipid protein in Pelizaeus-Merzbacher disease.

The brain of an 18-year-old patient with Pelizaeus-Merzbacher disease was examined by standard neuropathological and biochemical methods and by immunocytochemical and immunochemical techniques. Analysis revealed a lack of myelin-specific lipids, but showed a residual immunoreactivity for myelin basic protein, myelin-associated glycoprotein, and 2',3'-cyclic nucleotide-3'-phosphodiesterase. Examination by immunocytochemistry and enzyme-linked immunosorbent assay showed an absence of proteolipid apoprotein (lipophilin). The peripheral nervous system was normal. Pelizaeus-Merzbacher disease in humans shares many neuropathological and biochemical features with X-linked mutations in animals, e.g., the jimpy mouse and myelin-deficient rat. The specificity of this protein deficiency in Pelizaeus-Merzbacher disease gains additional support from the recent mapping of the lipophilin gene to the human X chromosome.

Adolescent↗