Altered gene expression upon BCR cross-linking in Burkitt's lymphoma B cell line.

Burkitt's lymphoma cell line, BL2 was stimulated by surface BCR cross-linking and altered gene expression was analyzed by RDA methodology. Consistent with previous reports, we detected up-regulated MDC, IL6R and adhesion molecule LFA1. We also detected gene expression of SIRPalpha, anti-apoptotic A-20, signal regulatory SLP76 and BCAR3, DNA binding proteins EGR2 and DEC1 in addition to some new genes.

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression.

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-kappaB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Dominant negative suppression of major histocompatibility complex genes occurs in trophoblasts.

BACKGROUND: Polymorphic class I and II major histo-: compatibility complex (MHC) genes are not transcribed in trophoblasts although many immune system cells express these genes constitutively. To study the molecular biology of MHC suppression for the purposes of potential transgenic animal development, we examined the effect on MHC expression in B cells by fusing them with trophoblasts. METHODS: Trophoblasts and B cells with separate selection markers were fused with polyethylene glycol. After growth in double selection media, the hybrids were analyzed for HLA-A, -B, -C, -DR, -DP, and -DQ expression by fluorescence-activated cell scanning and class I and II mRNA by Northern blotting. Class II promoter activity in trophoblasts was then analyzed by transfection of a lethal reporter construct and subsequently, the class II transactivator. RESULTS: Class I and II surface antigens and their corresponding mRNA were completely suppressed in the hybrids. The lethal reporter construct demonstrated that class II suppression resulted from lack of activation of the class II promoter. This in turn was caused by lack of functional class II transactivator. CONCLUSIONS: These data indicate that dominant negative trophoblast factors, either directly or indirectly, suppress expression of the MHC genes. If these factors can be cloned, the potential exists for developing transgenic animals that cannot express MHC or peptide antigen to T cell receptors through the MHC system.

Structure/function characterization of porcine CD59: expression, chromosomal mapping, complement-inhibition, and costimulatory activity.

BACKGROUND: Complement regulatory proteins have become important targets to potentially modulate inflammatory reactions or transplant rejection. Since pig into human xenotransplantation could potentially overcome the enormous shortage of donor organs and tissues, characterization of porcine complement regulatory proteins is critical. METHODS: The porcine CD59 cDNA has been isolated from porcine aortic endothelial cells and its structure determined. In addition, a molecular genetic analysis of the gene and its transcriptional properties and a functional analysis have been performed utilizing the transfected cDNA. RESULTS: The most prominent mRNA species is 1.8 kilobases but cloned reverse transcriptase polymerase chain reaction products suggest that multiple polyadenylation sites are utilized. Gene mapping was performed utilizing a polymorphism identified in the 3' UT, and the gene was localized to within 3 cM of follicle-stimulating hormone, beta polypeptide in the middle of the chromosome 2 linkage map. RNA expression was equivalent in endothelial, kidney, and testis cell lines. Comparisons have been made with CD59 sequences from other species to identify possible important domains of the protein. The cDNA has been utilized to express an epitope-tagged or wild-type protein either transiently on COS-7 cells or stably in Chinese hamster ovary cells. The porcine CD59 protein effectively inhibited the antibody-mediated lytic activity of both porcine and human complement. In contrast to human CD59, porcine CD59 is incapable of providing costimulation to human T cells. CONCLUSIONS: These data suggest that overexpression of porcine CD59 might be more effective than human CD59 in prolonging xenograft survival with transgenic pig organs because of reduced immunoreactivity.

Porcine endothelial CD86 is a major costimulator of xenogeneic human T cells: cloning, sequencing, and functional expression in human endothelial cells.

Porcine aortic endothelial cells (PAECs), unlike human endothelial cells, express a surface protein recognized by human CTLA4Ig fusion protein that costimulates human T cells through CD28. We have cloned porcine CD86 (pCD86) from an immortalized porcine endothelial cell line, PEC-A, that expresses high levels of this CTLA-4-binding protein. pCD86 mRNA is expressed in PEC-A and PAECs but not in human endothelial cells. Expression of stably transfected pCD86 in CHO cells modestly costimulates human T cell proliferation and IL-2 secretion. Expression of transiently transfected pCD86 in human umbilical vein endothelial cells strongly costimulates IL-2 production by human T cells, comparable to costimulation by PAECs. Costimulation of human T cells by pCD86 in both systems is as effective as costimulation by human CD80 or CD86, and can be blocked by human CTLA4Ig. We conclude that pCD86 contributes to the strong xenoreactivity of porcine endothelium.

p59fyn tyrosine kinase regulates p56lck tyrosine kinase activity and early TCR-mediated signaling.

To study the role of p59fyn in T cell activation, we used antisense RNA to inhibit p59fyn expression in a T cell clone. Transfectants with reduced levels of p59fyn were functionally impaired in their responses to antigen, Con A+recombinant IL-1 and cross-linking with anti-TCR mAb. Induction of tyrosine phosphorylation on most intracellular substrates was greatly reduced. We also noted that the lck kinase activity was greatly reduced even though the amount of lck protein was equivalent to that present in parental D10 cells. Our results suggest that the protein tyrosine kinase p59fyn is critical in TCR-mediated signaling and also suggests that p59fyn may regulate p56lck tyrosine kinase activity.

T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity.

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity.

Induction of the Ly-6A/E gene by interferon alpha/beta and gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds.

The murine Ly-6A/E gene is transcriptionally induced in cells exposed to interferon alpha/beta or gamma (IFN-alpha/beta or IFN-gamma). Analysis of the 5' flanking sequence using reporter plasmids that contain upstream elements of the Ly-6E gene has previously identified an approximately 850-base-pair IFN-responsive region that lacked an IFN-alpha-stimulated response element (ISRE), the element present and required for an IFN-alpha response of a number of genes. Analysis by deletion and stable transfection of the IFN-responsive region of the Ly-6E promoter has defined an 80-base-pair region containing an IFN-gamma activation site (GAS) but no ISRE that allows IFN-gamma and IFN-alpha inducibility of the Ly-6E gene. As tested by specific antiserum, a 91-kDa protein known to be activated in IFN-alpha- or IFN-gamma-treated cells binds to the GAS element from the Ly-6E promoter. The 91-kDa protein exists as an inactive cytoplasmic precursor and depends on tyrosine phosphorylation for its activation. Thus the same 91-kDa protein appears to act in the signal transduction pathways of both types of IFN for the Ly-6-A/E gene.

Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain.

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.

Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement.

Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21-24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell-surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose-dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement.

Characterization of promoter elements of an interferon-inducible Ly-6E/A differentiation antigen, which is expressed on activated T cells and hematopoietic stem cells.

The Ly-6E/A antigen is expressed on activated murine T cells. Using probes made from the previously characterized cDNA, we have isolated a genomic DNA clone encoding the Ly-6A antigen. We determined the DNA sequence of the genomic clone and conducted a functional analysis of the promoter region. Mouse fibroblast BALB/3T3 cells transfected with this genomic clone constitutively expressed Ly-6A antigen on their cell surface. This expression was inducible by alpha/beta and gamma interferons. The Ly-6E 5'-flanking region was analyzed by chloramphenicol acetyltransferase assays in fibroblast cells for cis-acting elements. At least two positive elements were found to be needed for maximum constitutive promoter activity in L cells. One of the positive elements was specifically bound by a CCAAT box-binding protein from crude nuclear extract, as shown by electrophoretic mobility shift assays and footprinting. The other element, which contains a GGAAA motif and has homology to various known enhancers, also showed a specific binding activity. This second positive element when multimerized became a very powerful enhancing element. Interferon treatment could enhance expression of the chloramphenicol acetyltransferase gene fused to the Ly-6E 5'-flanking region in stably transfected BALB/3T3 cells. The elements responsible for this enhancement lie, at least in part, between positions -1760 and -900 of the gene. Surprisingly, there is no sequence homology between this region of Ly-6E and the established consensus for the interferon-stimulated response element, which has been shown functionally important to all previously characterized alpha/beta interferon-inducible promoters. The Ly-6E gene may prove to be a novel system for the study of interferon induction.

A recombination event in the 5' flanking region of the Ly-6C gene correlates with impaired expression in the NOD, NZB and ST strains of mice.

The murine alloantigen, Ly-6C, is found on 45% of bone marrow cells, 25% of splenocytes and 15% of lymph node cells in all inbred strains of mice tested, with the exception of NOD, NZB and ST. In these three strains, Ly-6C expression can be detected on only 5% of bone marrow cells and not at all on cells from spleen or lymph node. NOD and NZB, which are models for the autoimmune diseases, diabetes and lupus, respectively, also exhibit a depressed syngeneic mixed lymphocyte reaction. Southern blot analysis reveals a restriction fragment length polymorphism involving the Ly-6C gene which is unique to these three strains. Cloning of the affected genomic segment from the NOD mouse indicates the presence of an interruption in the flanking region of the Ly-6C gene at a point 475 bp upstream of the transcription initiation site and the consequent separation of distal 5' sequences from the body of the gene by at least 10 kb. Inspection of the recombination borders reveals a set of inverted copies of a mouse repetitive R element. Transfection of the Ly-6C genes from NOD and BALB/c into a murine carcinoma line indicates relative functional impairment of the NOD gene, thus paralleling performance in vivo.

The CD59 antigen is a structural homologue of murine Ly-6 antigens but lacks interferon inducibility.

A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K-562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross-hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly-6E and Ly-6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol-glycan modification site for Ly-6 molecules. Unlike Ly-6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well-characterized mouse Ly-6 proteins.