Showing papers by "Richard A. Flavell published in 1988"

HIV infection is blocked in vitro by recombinant soluble CD4

Abstract: The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome1–4. HIV recognition of CD4 is prob-ably mediated through the virus envelope glycoprotein (gp!20) as shown by co-immunoprecipitation of CD4 and gp!20 (ref. 5) and by experiments using recombinant gp!20 as a binding probe6. Here we demonstrate that recombinant soluble CD4 (rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.

Transcriptional activation of HLA-DR alpha by interferon gamma requires a trans-acting protein

Abstract: Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.

Conversion of a PI-anchored protein to an integral membrane protein by a single amino acid mutation

Abstract: Qa-2, a cell-surface glycoprotein anchored by phosphatidylinositol (PI), is structurally related to the class I transplantation antigens H-2 K, D, and L, which are integral membrane glycoproteins. The predicted transmembrane segment of Qa-2 differs from those of H-2 K, D, and L by the presence of an aspartate in place of a valine at position 295. A single base change that replaced this aspartate with valine resulted in cell-surface Qa-2 molecules that were insensitive to hydrolysis by a PI-specific phospholipase C and more resistant to papain cleavage, properties shared by H-2D. Cells expressing Asp----Val mutant Qa-2 proteins were still able to attach a PI anchor to endogenous proteins such as Thy-1 and J11D. It therefore appears that this single amino acid change converts Qa-2 from a PI-linked form into an integral membrane protein.

Molecular mapping of signals in the Qa-2 antigen required for attachment of the phosphatidylinositol membrane anchor

Abstract: Proteins anchored in the membrane by covalent linkage to phosphatidylinositol (PtdIns) can be released by treatment with purified PtdIns-specific phospholipase C (Ptd-Ins-PLC). A recent survey of leukocyte antigens using flow cytometry has shown that staining of certain Qa antigens was diminished after PtdIns-PLC treatment, but staining of structurally related H-2 antigens was not affected. Therefore, in this study, the sensitivity of cell-surface Qa-2, H-2Kb, and H-2Db to hydrolysis by PtdIns-PLC was investigated biochemically by immunoprecipitation of radioiodinated molecules from cell lysates or supernatants. Qa-2, but not H-2Kb, was released from the surface of PtdIns-PLC-treated C57BL/10 mouse spleen cells and recovered in the cell supernatants. Similar analysis of thymoma cells transfected with cloned C57BL/10 genes showed that cell-surface Qa-2 molecules encoded by a Q7b cDNA and the Q7b or Q9b gene were sensitive to hydrolysis by PtdIns-PLC, whereas the H-2Kb and H-2Db gene products were resistant. Using thymoma cells transfected with hybrid genes constructed by exchanging exons between Q7b and H-2Db, the signals for PtdIns modification were localized to a defined region of Qa-2. This region differs from H-2Db most significantly by the presence of a central aspartate residue in the transmembrane segment and in the length of the cytoplasmic portion.

Dna sequences, recombinant dna molecules and processes for producing soluble t4 proteins

Abstract: This invention relates to DNA sequences, recombinant DNA molecules and processes for producing soluble T4 protein. More particularly, this invention relates to DNA sequences that are characterized in that they code on expression in an appropriate unicellular host for soluble forms of T4, the receptor on the surface of T4+ lymphocytes, or derivatives thereof. In accordance with this invention, the DNA sequences, recombinant DNA molecules and processes of this invention may be employed to produce soluble T4 essentially free of other proteins of human origin. This soluble protein may then advantageously be used in the immunotherapeutic and diagnostic compositions and methods of this invention. The soluble T4-based immunotherapeutic compositions and methods of this invention are useful in treating immunodeficient patients suffering from diseases caused by infective agents whose primary targets are T4+ lymphocytes. According to a preferred embodiment, this invention relates to soluble T4-based compositions and methods which are useful in preventing, treating or detecting acquired immune deficiency syndrome, AIDS related complex and HIV infection.

Cycloheximide, an inhibitor of protein synthesis, prevents γ-interferon-induced expression of class II mRNA in a macrophage cell line

Abstract: To characterize the mechanisms by which interferon γ (IFN-γ) upregulates major histocompatibility complex class II mRNA levels in mouse macrophages, we studied the effect of IFN-γ on the transcription rate of class II genes and investigated the requirement for ongoing protein synthesis for the induction of class II mRNA expression. Nuclear run-off assays demonstrate that IFN-γ induces class II mRNA at the transcriptional level. Treatment with cycloheximide, an inhibitor of protein synthesis, prevented the IFN-γ-mediated accumulation of E α mRNA in the mouse macrophage cell line P388 D.1, indicating that induction of E α mRNA in P388 D.1 cells requires de novo synthesis of a protein intermediate. Our studies suggest that this putative protein factor is labile and required throughout the induction period.

Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells.

Abstract: Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.

Inhibition of IFN‐γ‐Induced Transcriptional Activation of HLA‐DRα by Cycloheximide

Abstract: The human major histocompatibility (MHC) class I1 complex consists of three class I1 antigens: HLA-DR, DP, and DQ. These molecules are expressed primarily in cells of lymphoid lineage, namely, B cells, macrophages/monocytes, and some activated T cells. In addition, many nonlymphoid cell types are able to express class I1 antigens.' Certain reports have indicated that these cells, once induced, are capable of presenting antigen to T helper cells.'

Conversion of a PI-Anchored Protein to an Integral Membrane Protein by a Single Amino Acid Mutation Conversion of a PI-Anchored Protein to an Integral Membrane Protein by a Single Amino Acid Mutation

Abstract: Qa-2, a cell-surface glycoprotein anchored by phosphatidylinositol (PI), is structurally related to the class I transplantation antigens H-2 K, D, and L, which are integral membrane glycoproteins. The predicted transmembrane segment of Qa-2 differs from those of H-2 K, D, and L by the presence of an aspartate in place of a valine at position 295. A single base change that replaced this aspartate with valine resulted in cellsurface Qa-2 molecules that were insensitive to hydrolysis by a PI-specific phospholipase C and more resistant to papain cleavage, properties shared by H-2D. Cells expressing Asp -> Val mutant Qa-2 proteins were still able to attach a PI anchor to endogenous proteins such as Thy-i and JllD. It therefore appears that this single amino acid change converts Qa-2 from a PI-linked form into an integral membrane Qa-2, a cell-surface glycoprotein anchored by phosphatidylinositol (PI), is structurally related to the class I transplantation antigens H-2 K, D, and L, which are integral membrane glycoproteins. The predicted transmembrane segment of Qa-2 differs from those of H-2 K, D, and L by the presence of an aspartate in place of a valine at position 295. A single base change that replaced this aspartate with valine resulted in cellsurface Qa-2 molecules that were insensitive to hydrolysis by a PI-specific phospholipase C and more resistant to papain cleavage, properties shared by H-2D. Cells expressing Asp -> Val mutant Qa-2 proteins were still able to attach a PI anchor to endogenous proteins such as Thy-i and JllD. It therefore appears that this single amino acid change converts Qa-2 from a PI-linked form into an integral membrane

Regulation of Ia Gene Expression in Murine Macrophages

Abstract: The murine Ia antigens, encoded by a family of genes (E,, E,, A,, and A@) that reside in the major histocompatibility complex (MHC), regulate the immune response through their role as restriction elements in antigen presentation.' In antigenpresenting cells, such as macrophages, the expression of Ia is not constitutive, but is regulated by gamma-interferon (IFN--,).' Thus, treatment with IFN--, has been

Sequences d'adn, molecules d'adn recombinant et procedes de production de proteines t4 solubles

Abstract: Cette invention concerne des sequences d'ADN, des molecules d'ADN recombinant et procedes de production de proteine T4 soluble, et plus particulierement, des sequences d'ADN caracterisees en ce qu'elles codent lors de l'expression dans un hote unicellulaire approprie pour des formes solubles de T4, le recepteur sur la surface des lymphocytes T4+, ou des derives de ceux-ci. Selon cette invention, les sequences d'ADN, les molecules d'ADN recombinant et les procedes de cette invention peuvent etre employes pour produire des T4 solubles virtuellement exemptes d'autres proteines d'origine humaine. On peut alors utiliser avantageusement cette proteine soluble dans les compositions et procedes immunotherapeutiques et de diagnostic de cette invention. Les compositions et procedes immunotherapeutiques a base de T4 soluble de cette invention, sont utiles au traitement de patients immunodeficients souffrant de maladies provoquees par des agents infectieux dont les premieres cibles sont les lymphocytes T4+. Selon un mode de realisation prefere, cette invention a trait a des compositions et a des procedes a base de T4, utiles a la prevention, au traitement et a la detection du syndrome d'immunodeficience acquis, du complexe relatif au SIDA et de l'infection du VIH.