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WO1990007009A1 - Nouveaux genes de lymphokine/cytokine - Google Patents

Nouveaux genes de lymphokine/cytokine Download PDF

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Publication number
WO1990007009A1
WO1990007009A1 PCT/US1989/005603 US8905603W WO9007009A1 WO 1990007009 A1 WO1990007009 A1 WO 1990007009A1 US 8905603 W US8905603 W US 8905603W WO 9007009 A1 WO9007009 A1 WO 9007009A1
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WIPO (PCT)
Prior art keywords
pat
protein
cells
human
cytokine
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PCT/US1989/005603
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English (en)
Inventor
Ulrich Siebenlist
Peter F. Zipfel
Kathleen R. Kelley
Steven G. Irving
Monica Napolitano
Warren J. Leonard
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Application filed by The United States Of America, Represented By The Secretary, United States Department Of Commerce filed Critical The United States Of America, Represented By The Secretary, United States Department Of Commerce
Publication of WO1990007009A1 publication Critical patent/WO1990007009A1/fr
Priority to NO91912189A priority Critical patent/NO912189L/no
Priority to DK113491A priority patent/DK113491A/da

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to genes which are activated by mitogenic stimulation of immune cells, particularly to those encoding polypeptides similar to a family of secreted factors including lymphokines and cytokines. This invention also relates to synthesis of product ' s' of such immune activation genes by recombinant cells, and to the manufacture and use of certain novel products enabled by the identification and cloning of DNAS encoding these factors. "
  • Quiescent non-dividing T lymphocytes respond to antigen and mitogen stimulation by the activation of specific cellular " genes which are thought to control events that lead to cell proliferation and the expression of a differentiated phenotype.
  • T cells produce a large variety of factors which are important for the growth and differentiation of many cells participating in an immune reaction.
  • lymphocyte proliferation and effector functions are regulated by antigen/mitogen and lymphokine binding to cell surface receptors.
  • the bind ⁇ ing of such extracellular ligands results in a cascade of intracellular biochemical events (see references 11-44, 11-45 in Experimental Section II, below) that ultimately set in motion a genetic program for activation-related growth and expression of differentiated functions.
  • quiescent T cells undergo a series of sequentially dependent, ordered transcriptional events following acti- . vation of quiescent cells that culminates in the initi ⁇ ation of DNA synthesis after about 24 hours (II-5, II- 17).
  • Primary gene transcriptional events defined by their early kinetics and lack of dependence on prior protein synthesis, include protooncogenes (e.g., c-myc and c-fos), lymphokines (e.g. IL-2, IFN, GM-CSF), and a cell surface lymphokine receptor (IL-2 receptor) (II-6, II-9, 11-12, 11-17, 11-27, 11-36, 11-44), all of which are thought to have significant effects upon T cell proliferation and/or regulation of an immune response- It is expected that some of the immediately induced .genes play a role in; initiating and thus _.regu- lating the cascade of molecular events that.-.follow mito- genie activation.
  • protooncogenes e.g., c-myc and c-fos
  • lymphokines e.g. IL-2, IFN, GM-CSF
  • IL-2 receptor cell surface lymphokine receptor
  • Such genes are also likely targets of tumorigenic events.
  • a variety of data supports the notion that uncontrolled growth occurs when the? ⁇ mitogen inducible gene c-myc is expressed, inappro ⁇ priately (11-11, 11-26).
  • c-myc a gene that is expressed, inappro ⁇ priately (11-11, 11-26).
  • a limited number of primary induced genes in T cells have been described.
  • genes induced in activated T cells, IL-2 and IL-2 receptor for example, often have been identified on the basis of their encoded proteins.
  • the transcriptional response of T cells is likely more complex than has been reported to date (II-2, II-4, 11-15, 11-23, 11-28, 11-32, II- 41).
  • PB T cells Human peripheral blood (PB) T Cells were used in the development .of this invention, as the model system in which to study activation, for several reasons.
  • PB T cells are primary quiescent cells that have not been selected for adaption to growth in culture, and they can be polyclonally stimulated ⁇ n_ vitro.
  • T cells upon activation, express a variety of differenti- ated functions that play critical roles in the pro ⁇ gression of an immune response. For example, novel lymphokines/cytokines would be expected to be included within the family of inducible genes, since a primary function of PB T cells following activation is to secrete soluble factors that affect ultimately a number of physiological functions (II-5, II-6, II-9, 11-13).
  • CSA immunosuppressive drug cyclosporin A
  • T cell system allows one.-to dissect and contrast the * activation* requirements mediated through various distinct surface molecules that are thought to play roles in T cell development and function (see Experimental Section III, below).
  • peripheral blood T cells Treatment of human, peripheral blood T cells with the mitogenic combination of PHA (phyto- hemagglutinin) and PMA (phorbol myristate acetate) appears to mimic the effects of antigen and antigen presenting cells jLr ⁇ vivo.
  • the two agents are known to induce the expression of genes which are important for progression through the cell cycle, including the c-fos and the c-myc oncogenes, the IL-2 growth factor gene and the IL-2 receptor gene (see references IV-1, IV-2 in Experimental Section IV, below).
  • T cells activated in this manner elaborate many lymphokines/cytokines which exert their effect on a variety of cells.
  • f ctors as IL-3, IL-4, IL-5 and IL-6, GM-CSF, -IFN (gamma interferon), TNF (tumor necrosis factor) and LT (IV-3 - IV-9) and several others that are less well defined (IV-10 - IV- 13).
  • factor genes may be subject to a specific control mechanism in T cells, since the immunosuppressive drug cyclosporin A appears to abrogate their activation. As noted earlier, this drug acts during the early activation phase of T cells and interferes the production of IL-2, IL-3, and -IF (IV-14 - IV-17). The inhibitory effect is specific, since many other inducible genes such as c-fos and HSP 70 are unaffected (IV-15).
  • the present invention relates to DNA segments which encode amino acid sequences with similarities to members of a- newly emerging family of small, secreted proteins (see Figure IV-6 in Experimental Section IV) .
  • This family of structurally related factors includes platelet_ factor 4 (PF-4) (IV-18), platelet basic protein (PBP) and its processed forms ⁇ -thromboglobulin ( ⁇ ?-TG) and connective tissue activating peptide III (CTAP.
  • PF-4 platelet_ factor 4
  • PBP platelet basic protein
  • CTAP connective tissue activating peptide III
  • Ill Ill
  • IB- 10 gamma interferon inducible ⁇ protein
  • MIP macrophage inflammatory protein
  • NAF neurotrophil-activating factor
  • fibroblast ⁇ human growth related protein
  • H-gro human growth related protein
  • Inflammation and wound healing/tissue repair are very complex processes which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells.
  • This family of genes which may have been derived from one common ancestral gene, may well serve to establish such a net- work.
  • the individual factors may possess multiple functions, as shown for several of the members of this family (see below. Experi ⁇ mental Section IV, subsection Discussion) .
  • the conserved similarities between these proteins may indicate an interaction with conserved receptor elements. As enumerated above, the members of this family of proteins display a remarkable variety of tissue specificities. The distinctions between the factors may determine unique functions required during an immune reaction depending on the cell type activated.
  • This invention contemplates the application of methods of recombinant DNA technology to fulfill such needs and to develop means for producing certain protein factors which appear to be related to inflammatory or cell; growth processes, and which could not be produced otherwise.
  • This invention also contemplates the appli ⁇ cation of the molecular mechanisms of these factors related to inflammation and healing processes.
  • the present .invention relates to a development of recombinant DNA technology, which includes production of novel lymphokine-like or cytokine-like proteins, free of other peptide factors. Novel DNA segments, RNAs, and bioassay methods are also included.
  • the present invention in particular relates, in part, to DNA segments which encode mRNAs and/or proteins having structural and/or functional characteristics of such molecules related to lymphokines/cytokines that are inducible in T cells.
  • Members of one particular group of these proteins feature hydrophobic N-terminal leader or signal peptides characteristic of secreted proteins, have predicted sizes of about 8 kD (kilodaltons) upon cleavage of the putative leader peptide, and also share some critical amino acid sequence similarity with a newly emerging family of secreted factors which have been shown to display functions associated with an inflammatory response and/or to have mitogenic activities.
  • these DNA segments are capable of being expressed in suitable host cells, thereby producing lymphokine-like or cytokine-like proteins.
  • the invention also relates to mRNAs produced as the result of trans-, cription of the sense strands of the DNA segments of. this invention.
  • This invention furthe ' r comprises novel bioassay methods for determining effects of various mitogenic agents on expression in human cells of, the mRNAs and proteins produced from the genes related to DNA segments of the invention. - - _
  • the present invention comprises DNA segments encoding lymphokine-like or cytokine-like proteins, selected from the group con ⁇ sisting of: Act-2 [i.e., an approximately full length (-0.7 kb) cDNA clone of activated human peripheral blood mononuclear cells (PBMC) mRNA, number 2, hereinafter referred to as "full-length Act-2" DNA]; human cDNA clones of activated T cell mRNAs, pAT 744 and PAT 464; and related DNA segments which can be detected by hybridization to any of the above human DNA segments, which related segments encode related lymphokine-like or cytokine-like proteins.
  • Act-2 i.e., an approximately full length (-0.7 kb) cDNA clone of activated human peripheral blood mononuclear cells (PBMC) mRNA, number 2, hereinafter referred to as "full-length Act-2" DNA
  • PBMC peripheral blood mononuclear cells
  • this invention relates to an RNA transcript of a DNA of the invention.
  • these RNA transcripts are capable of being trans ⁇ lated to produce complete molecules of the encoded lymphokine-like or cytokine-like proteins.
  • the invention in another embodiment, relates to a recombinant DNA molecule comprising a vector and a DNA of the present invention.
  • recombinant molecules include molecules comprising full-length DNA and any of the following vector DNAs: a baculovirus vector (such as derivatives of Ac-Nuclear Polyhedro ⁇ is Virus; AcNPV); a bacteriophage lambda vector (gtlO); an Ml3 bacteriophage vector; or the RNA transcription vector pGEM-1.
  • Recombinant DNAs of this invneiton also include recombinant molecules comprising clone pAT 744 DNA or clone pAT 464 DNA and any of the following vector DNAs: lambda (GtlO); and Ml3 vector; and a mammalian expression vector (such as CDM-8 or PMT2-2) capable of expressing inserted DNAs in mammalian (e.g., COS) cells.
  • GtlO lambda
  • Ml3 vector a mammalian expression vector capable of expressing inserted DNAs in mammalian (e.g., COS) cells.
  • the invention comprises a cell, preferably a-mammalian or insect cell, transformed with a DNA -of the invention.
  • the invention comprises cells, including yeast cells and bacterial cells such as those of E_. coli and _B. subtilis, transformed with DNAs of the invention.
  • the transforming DNA is capable of being expressed in the cell, thereby increasing the amount of lymphokine/cytokine-like protein encoded by this DNA, in the cell.
  • the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells.
  • the invention comprises novel lymphokine/cytokine-like proteins made by expression of a DNA of the invention, or by translation of an RNA of the invention.
  • these proteins will be of a secreted form (i.e., lacking an apparent signal sequence) .
  • These protein factors can be used for f nc ⁇ tional studies, and can be purified for additional bio ⁇ chemical and functional analyses, such as qualitative and quantitative receptor binding assays.
  • the novel lymphokine/cytokine-like proteins will be pro ⁇ tein products of "unmodified” DNAs and mRNAs of the invention, or will be modified or genetically engineered protein products. As a result of engineered mutations in the DNA sequences, modified lymphokine/cytokine-like proteins will have one or more differences in amino acid sequence from the corresponding naturally occurring "wild-type" proteins.
  • This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention. In 'this embodiment of the invention, the antibodies will specifically bind to a lymphokine/cytokine-like protein which includes ⁇ the sequence of such peptide, preferably when that protein is in its native (biologically active) conforma ion. These antibodies can be used for detection .or purification of the protein factors. " • * • - ⁇ . -
  • This invention further comprises novel .bioas ⁇ ay methods for detecting the expression-of genes related -to
  • DNAs of the invention may be used as probes to determine steady state levels of or kinetics of induction of related mRNAs.
  • Such bioassays may be useful, for example, for identification of various classes of tumor cells or genetic defects in the inflammatory response.
  • the bioassays of this invention may also be useful for detecting activation of the immune system in vivo. Genes related to DNAs of this invention are expressed very early upon immune activation, in T cells (and also some other hematopoietic cells), and, there- fore, serve as extremely early markers for immune system activation.
  • various body fluids may be tested routinely with antibodies to protein factors of the invention, for example, to monitor whether tissues are rejected upon transplantation.
  • Fig. 1-1 shows Northern blots using a ⁇ ---F- labeled full-length CDNA probe and RNA derived from a variety of human cell types, which illustrate the speci ⁇ ficity of full-length gene expression, mainly in certain mitogenically stimulated immune cells.
  • Fig. 1-2 characterizes the temporal expression of mRNA in activation of normal T and B lymphocytes and monocytes (by means of Northern Blots), illustrating that Act-2 gene expression is both rapidly induced and termi ⁇ nated in response to mitogen.
  • Fig. 1-3 depicts the sequencing strategy, DNA sequence, and deduced .amino acid sequence of -0.7 kb
  • Fig. 1-4 presents a Kyte-Doolittle hydro- phobicity plot generated from the deduced amino acid sequence, which reveals an extremely hydrophobic N- terminus that appears to be a so-called leader or signal peptide.
  • Fig. 1-5 demonstrates translation of RNA species produced from full-length Act-2 cDNA in- cell-free systems, showing by gel electrophoresis a product with an apparent M r of approximately 11,000 similar to the calcu- lated molecular weight of 10,199 daltons, which apparent ⁇ ly translocates into microsomal membranes, as do secreted proteins.
  • Fig. 1-6 portrays results of using the full- length Act-2 cDNA to perform a genomic Southern blot of human DNA, a comparatively simple pattern of digestion most consistent with Act-2 representing a single copy or low copy number gene.
  • Fig. 1-7 directly compares the time course of expression of Act-2 with two other genes known to play roles in cell proliferation, c-fos, and c-myc, in stimu ⁇ lated lymphocytes, showing that Act-2 mRNA is induced coordinately with c-fos and its peak expression coincides with that of c-myc.
  • Table 1-1 presents results of N-terminal sequencing of Act-2 protein radiolabeled with * ⁇ 5 S- methionine or cysteine.
  • EXPERIMENTAL SECTION II Fig. II-l outlines the procedure used to iso ⁇ late genes which are induced immediately upon mitogenic stimulation of human periopheral blood T lymphocytes.
  • Fig. II-2 illustrates kinetics of mRNA induc ⁇ tion (with Northern blots and cDNA probes) of four selected inducible genes upon stimulation of • resting human blood T cells.
  • Fig. II-3 portrays diverse expression and regu ⁇ lation of selected cDNA clones in the human helper T -cell line Jurkat. _- ._ __ _. --. • __>
  • Fig. II-4 shows (Northern blot) analyses of expression of selected cDNA clones in human fibrobla ⁇ t ⁇ .
  • Table II-2 presents expres ⁇ ion analysis of T cell induced genes in the helper T cell line Jurka -and in human fibroblast ⁇ .
  • Fig. III-2 illustrate ⁇ (Northern blot) analysis of mitogen induced genes in CD28 T cells that _were stimulated with either anti-CD28 monoclonal antibody or ionomycin in the absence or pre ⁇ ence of phyto- hemagglutinin.
  • Fig. III-3 depict ⁇ (Northern blot) analy ⁇ i ⁇ of mitogen induced gene ⁇ .
  • T cell line Jurkat that were ⁇ timulated with either dioctanoyl-glycerol or ionomycin or both agent ⁇ together in the ab ⁇ ence or pre ⁇ ence of cycloheximide.
  • Table III-l summarizes mRNA analyses (Northern blot data) for mitogen-induced genes in response to agents used in Figs. III-1-3.
  • FIG. IV-1 portrays Northern Analy ⁇ i ⁇ of cDNA clone ⁇ pAT 464 and pAT 744 RNA in human peripheral blood T cell ⁇ , either un ⁇ timulated (control) cell ⁇ or cell ⁇ treated with cycloheximide or cyclo ⁇ porin.
  • Fig. IV-2 demon ⁇ trate ⁇ Northern analy ⁇ i ⁇ of pAT 464 and pAT 744 RNA in Jurkat cell ⁇ , either un ⁇ timulated (control) or cells stimulated with the agents indicated.
  • Fig. IV-3 illustrates the sequencing strategy, the nucleotide sequence, and the predicted ' amino acid sequence for pAT 464, including the open reading frame of 276 base pairs, corresonding to._92 amino acids. -• ⁇ *
  • Fig. IV-4 shows the sequencing strategy, the nucleotide sequence, and the predicted amino acid sequence for pAT 744, including the open reading frame corresponding to 92 amino acids.
  • Fig. IV-5 documents primer extension analysi ⁇ of the DNA ⁇ equence for pAT 744, using an oligonucleotide complementary to. positions 135 to 152 of the pAT 744 sequence annealed to RNA from human peripheral T cells.
  • Fig. IV-6 shows comparisons among the amino acid sequence ⁇ of proteins encoded by pAT 464, pAT 744 and a family of structurally related proteins.
  • Fig. IV-7 presents Southern blot analysi ⁇ of pAT 464 and pAT 744 gene ⁇ in human placental DNA digested with ⁇ everal restriction enzyme ⁇ .
  • Fig. IV-8 illu ⁇ trate ⁇ expre ⁇ ion of pAT 464 and pAT 744 mRNAs in promyelocytic (HL60) cell ⁇ and human tonsillar B Cells (by Northern blot methods).
  • Fig. IV-9 demonstrate ⁇ expression of appropriate secreted proteins in mammalian (COS) cells transformed with pAT 464 and pAT 744 DNAs in various expression vectors (by 35 s-cysteine labeling of proteins) .
  • Fig. IV-10 demonstrate ⁇ expre ⁇ ion of appro ⁇ priate secreted proteins in mammalian (COS) cells trans- formed with pAT 464 DNAs in variou ⁇ expre ⁇ ion vector ⁇ (by ⁇ S-cysteine labeling of proteins).
  • Fig. IV-11 shows the ues of rabbit antibody
  • Fig. IV-12 illustrates the activity of rabbit antibody (number 722) raised against the C-terminal 12 amino acids of the pAT 744 peptide to detect secreted peptide in supernatant ⁇ of COS cell culture ⁇ tran ⁇ formed with pAT 744 DNA (by Western blotting) .
  • Fig. IV-13 shows the use of rabbit antibody (number 720) raised against the C-terminal 12 amino acids of pAT 464 peptide to detect secreted peptide in super ⁇ natants of dbs " cell cultures transformed with pAT ⁇ 464 DNA.
  • Q Fig. IV-14 presents results with a second rabbit antibody (number 719) raised against the C- terminal 12 amino acid ⁇ of the pAT 464 peptide in reaction ⁇ with secreted peptide in supernatants of ' COS cell cultures transformed with pAT 464 DNA.
  • Fig. IV-15 demonstrates that mitogenically activated human peripheral blood T cells secrete pAT 464 protein (by Western blotting with anti-peptide antibody) .
  • DNA ⁇ of thi ⁇ invention include the following cDNA clone ⁇ : pAT 120; pAT 125; pAT 127; pAT
  • DNA ⁇ of thi ⁇ invention al ⁇ o include recombinant
  • a mammalian expression vector (exemplified by CDM-8 or PMT2-2) capable -of expressing inserted DNAs in CO
  • the sense strand DNA nucleotide sequence, and the predicted primary protein sequence encoded, are shown for full-length Act-2 DNA in Fig. 1-3.
  • four clones with identically sized inserts of approximately -0.7 kb were identified, all of which hybridized to the same 0.9 kb mRNA.
  • the ⁇ equencing ⁇ trategy u ⁇ ed, the re ⁇ ulting nucleotide ⁇ equence and the predicted amino acid sequence for the encoded protein are shown in figure IV-4 for' pAT 744 and Figure IV-3 for pAT 434.
  • the full-length Act-2 cDNA and pAT 744 cDNAs appear to be derived from es ⁇ entially the same gene.
  • base 131 is a T and 135 is A.
  • These differences cause predicted amino acid number 20 to be Pro and Leu, in full-length Act-2 and pAT 744, respectively, while the other base change does not alter the amino acid sequence. It is possible that such differences are due to inherited variations (i.e., polymorphism ⁇ ) in the gene ⁇ of the two different individuals from which the clones are derived.
  • pLD ⁇ 78 extends 7 nucleotide ⁇ further 5' than clone pAT 464 and sequence compari ⁇ on ⁇ how ⁇ a- difference of 5 nucleotides between both clones, none of which affect ⁇ the predicted amino acid ⁇ equence. These .differences in the nucleotide sequence may reflect polymorphisms between the genes of different donors. A* comparison of the sequences for pAT 744 and
  • the ⁇ e two gene ⁇ may have been derived from a common ance ⁇ tral gene and their encoded proteins may play functionally related roles.
  • Recombinant DNA molecules which comprise a vector and other DNAs of the invention are also within the scope of the invention.
  • Preferred recombinant molecules include molecules comprising full-length Act-2 DNA and any of the following DNAs: a bacteriophage lambda vector (gtlO); an M13 bacteriophage vector; or the RNA transcription vector pGEM-1.
  • Preferred recombinant DNAs of this invention also include recombinant molecules comprising clone pAT 744 or pAT 464 DNA and any of the following vector DNAs: lambda vector (e.g., gtlO); an M13 vector.
  • Most preferred recombinant molecules of tvhis invention include: molecules comprising full-length Act- 2 DNA and any of the following vector DNAs: a i>aculo- viru ⁇ vector (including pAc-Act'2, a plasmid derived from polyhedrin pla ⁇ mid pAc373; and a recombined baculovirus, vAc-Act2, derived from pAc-Act2 and a wild-type AcNPV strain E 2 DNA) .
  • a i>aculo- viru ⁇ vector including pAc-Act'2, a plasmid derived from polyhedrin pla ⁇ mid pAc373; and a recombined baculovirus, vAc-Act2, derived from pAc-Act2 and a wild-type AcNPV strain E 2 DNA
  • recombinant molecules are those comprising pAT 744 or pAT 464 DNA and a mammalian expres ⁇ ion vector (CDM-8 or PMT2-2) capable of expre ⁇ sing inserted DNAs in mammalian (e.g., COS) cells.
  • CDM-8 or PMT2-2 mammalian expres ⁇ ion vector
  • the full-length Act-2 human cDNA was synthe- ⁇ ized, clone and isolated as described in Experimental Section I.
  • 10° phage plaques from a HUT-102B2 cDNA library, constructed in lambda gtlO (1-14) were screened with a partial length (-0.4 kb) Act-2 cDNA insert, labeled using the random priming method (1-15).
  • the full-length (-0.7 kb) DNA sequence contains an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilizes the most 5' AUG, which generally is the one utilized in eucaryotic translation initiation.
  • the region of the first AUG has good homology with the consensus sequence determined for eucaryotic start sites (1-17).
  • the cDNA appears to extend to the 3' end of the mRNA since it contains classic AAUAAA poly- adenylation signals (1-18) and a start of a poly A tail.
  • the human cDNAs of this invention were synthe ⁇ sized, cloned and isolated as described in Experimental Section II by a process of "subtraction" cloning and hybridization using mRNA from activated T cells.
  • This strategy i ⁇ summarized in Figure II-l.
  • Human peripheral blood T cells were polyclonally activated for 4.5 hours in the presence of the mitogens phytohemagglutinin (PHA) and phorbol 12-mytri ⁇ tate 13-acetate (PMA) a ⁇ well as the protein synthe ⁇ i ⁇ inhibitor cycloheximide.
  • PHA phytohemagglutinin
  • PMA phorbol 12-mytri ⁇ tate 13-acetate
  • the ⁇ e clone ⁇ which exemplify DNA ⁇ of thi ⁇ invention, include: pAT 120; pAT 125; pAT 127; pAT 129; pAT 133; pAT 140S; pAT 140L; pAT 154; pAT 158; pAT 189; pAT 201; pAT 204; pAT 225; pAT 229; pAT 232S; pAT 232L; pAT 237; pAT 239; pAT 243; pAT 270; pAT 276; pAT 281; pAT 383; pAT 402; pAT 407; pAT 416; pAT 428; pAT 464; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496; pAT 516; pAT 542; pAT 563; pAT 591; pAT 594;
  • Clones pAT 464 and pAT 744 which were originally derived from a subtracted cDNA library (see Experimental Section II), have been characterized in more detail. Near full-length cDNA clones were isolated from a cDNA library which was derived from RNA extracted from human peripheral blood T cells stimulated for 4 ⁇ 5 hr with PHA-P and PMA in the presence of cycloheximide. This cDNA library was constructed with oligo ⁇ _T priming as described (IV-29) and was subsequently cloned into- lambda gt 10 (IV-30).
  • Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotide ⁇ long (including the Eco RI linker at 5' end).
  • Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5). It can.be concluded that about 10 nucleotides of the 5' end of the true mRNA sequence are mis ⁇ ing in thi ⁇ cDNA clone.
  • the first ATGs for pAT 464 and pAT 744 are at po ⁇ itions 84 and 74, respectively. In each case an open reading frame of 92 amino acids follows. DNA templates for transcription of full-length
  • Act-2 RNA in the pGEM-1 vector were prepared as follows (see also Experimental Section I): The -0.7 kb full- length Act-2 cDNA was digested with Ava I, filled in with Klenow and dNTPs, and then dige ⁇ ted with Eco RI to liberate a nearly full length fragment (ba ⁇ e 27 through the 3' end of the in ⁇ ert) from which the artificial 5' G- C tail had been removed. This fragment was subcloned into PGEM-1 (Promega Biotec) which had been digested with Sma I and Eco RI.
  • Baculovirus vector constructs containing full- length Act-2 cDNA were prepared from the -0.7 kb Act-2 cDNA by cloning into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing the full-length Act-2 cDNA under control of the polyhedrin promoter (see also Experimental Section I) .
  • SF9 cells were cotransfected with pAc-Act2 and wild- type AcNPV strain E2 DNA.
  • a recombined baculovirus vAc- Act2 was isolated and purified by a procedure ; -.of succe ⁇ ive round ⁇ of plaque hybridization (I-16a). ⁇
  • sub ⁇ tantially pre lymphokine/cytokine-like protein ⁇ thu ⁇ produced can be employed, using well-known techniques, in diagnostic as ⁇ ay ⁇ to determine the pre ⁇ ence of receptor ⁇ for the ⁇ e protein ⁇ in variou ⁇ body fluid ⁇ and ti ⁇ ue ⁇ ample ⁇ .
  • the invention also comprise ⁇ a cell, preferably a mammalian or in ⁇ ect cell, tran ⁇ formed with a DNA of the invention, wherein the transforming DNA is capable of being expressed.
  • the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells.
  • the invention also comprises novel lymphokine/cytokine-like proteins made by expres ⁇ ion of a DNA of the invention, or by tran ⁇ lation of an RNA of the invention.
  • the ⁇ e protein ⁇ will be of a secreted form (i.e. , lacking an apparent ⁇ ignal sequence) . These protein factors can be used for functional studies, and can be purified for additional biochemical and functional analyses, such as qualitative and quantitative receptor binding assays.
  • Insect cells transformed with a recombinant molecule containing full-length Act-2 DNA were prepared as described in Experimental Section I. , Briefly, the plaque purified, recombinant baculovirus, vAc-Act2, was used to infect SF9 cell ⁇ , according to standard -methods. The recombinant full-length Act-2/baculovirus vector was expressed in the SF9 cells to produce abundant Act-2 protein in the supernatant. Moreover, N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as predicted.
  • Mammalian cell ⁇ tran ⁇ formed with pAT 744 or pAT 464 DNA and secreting pAT 744 or pAT 464 protein are described in Experimental Section IV. Briefly, COS cells were transfected using standard DEAE-dextran methodology, with cDNA clones inserted into expres ⁇ ion sites of either CDM-8 or PMT2-T. The novel proteins were visualized by ⁇ S-cy ⁇ teine labelling of the cell ⁇ after the trans- faction of the DNA constructs using standard, well known methods. The size of the protein product ⁇ is appropriate as predicted from the cDNA sequence after the leader peptide is cleaved off.
  • This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention.
  • This embodiment of the invention is exempli ⁇ fied by rabbit antisera containing antibodie ⁇ which spe ⁇ cifically bind to pAT 744 or pAT 464 lymphokine/cytokine- like protein which includes the sequence of such peptide, were raised to synthetic peptides representing the C- terminal 12 amino acids of both pAT 744 and pAT 464 pro ⁇ teins, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier plus peptides into rabbits, according to standard methods of peptide immunology.
  • KLH carrier
  • Figs. IV-11 and IV- 12 show the use in Western blot experiments of two cLif- ferent rabbit antibodies (721 and 722) rai ⁇ ed again ⁇ t the pAT 744 peptide. Similar studie ⁇ with antibodie ⁇ to pAT 464 ⁇ -peptides are presented in Figs. IV-13 and IV-14. *a_fi_s is "evident from the figures, the appropriate secreted factors are detected -by antisera from rabbits immunized with synthetic"peptides. ⁇ - - -- ⁇ "• 5"
  • This invention further comprise ⁇ novel bioassay methods for detecting the expression of genes related to DNAs of the invention.
  • DNAs of this invention were used as probes to determine steady state levels of or kinetics of induction T Of related mRNAs.
  • Method ⁇ for the ⁇ e bioa ⁇ ays of the invention, using full-length Act-2, or using pAT 744 -or pAT 464 DNAs, and standard Northern blotting techniques, are described in detail in Experimental Sections I, -or II-IV, respectively.
  • pAT 744 -or pAT 464 DNAs are described in detail in Experimental Sections I, -or II-IV, respectively.
  • Such method ⁇ may be readily applied to analy ⁇ is of gene expression for lymphokine/cytokine-like proteins, either in isolated cell ⁇ or variou ⁇ ti ⁇ ue ⁇ .
  • Such bioa ⁇ ay ⁇ may be useful, for example, for identification of various classe ⁇ of tumor cell ⁇ or genetic defect ⁇ in the inflam- matory re ⁇ ponse.
  • a novel immune activation gene denoted Act-2, has been identified by differential hybridization screen ⁇ ing of an activated T Cell library. The gene is included rapidly following T cell --activation with phytohemagglu- tinin, B cell activation with Staphylococcus aureus Cowan I, and monocyte activation With lipopolysaccharide. ' cDNA containing the full length coding region has been isolated. The deduced amino acid sequence predicts ran open reading frame of 92 amino acids, including a very hydrophobic N-terminus, which by weight matrix score is predicted to be a signal peptide.
  • INTRODUCTION T cells play a pivotal role in the regulatory and effector functions of the immune response.
  • a ⁇ erie ⁇ of biochemical event ⁇ occurs, including an increase in intracellular calcium, pho ⁇ phorylation of protein ⁇ , and increa ⁇ ed phosphoinositol turnover (see references 1-1, 1-2).
  • These event ⁇ generate signals which lead to the activation of cellular genes and the production of cellular proteins that are not expressed or are very weakly expressed by resting cells.
  • These induced pro- teins are e ⁇ sential for the proliferation and differenti ⁇ ation of T lymphocytes into effector T cells mediating . helper, suppre ⁇ sor, or cytotoxic T cell functions.
  • Thi ⁇ section de ⁇ cribe ⁇ the cDNA cloning, ⁇ equencing, and characterization of a novel gene, denoted Act-2, which was identified by differential screening of an activated normal T cell cDNA library.
  • Circulating human peripheral-blood mononuclear cells were obtained from healthy volunteers and isolated by Ficoll Hypaque (LSM, Litton Bionetics) gradient centrifugation. Cells were generally cultured at 1-2 x 10 6 cell ⁇ /ml overnight in RPMI 1640 medium containing 10% fetal bovine ⁇ erum (FBS), L- glutamine, and antibiotic ⁇ .
  • FBS fetal bovine ⁇ erum
  • PMA phorbol myri ⁇ tate acetate
  • B lymphocytes were purified by incubating PBMC .(4% non-specific e ⁇ tera ⁇ e po ⁇ itive cells) with 2-aminoethyliso-thiouronium bromide-treated sheep erythrocytes and removing the rosette-focusing cells. In some experiments monocytes were depleted by plastic adherence. Such cells were 80% surface Ig posi ⁇ tive, latex non-ingesting cells, and were cultured for 30 min to 72 h in the presence of a 1:10,000 dilution of Staphylococcus aureus Cowan I (SAC, Calbiochem-Behring Corp. ) .
  • Monocytes were prepared by elutriation of PBMCs and were 95% pure by Giem ⁇ a ⁇ tain and flow cytometry. Cell ⁇ were cultured for 8 h in the presence of 10 ug/mg lipopolysaccharide (LPS, Sigma).
  • T cell line Jurkat, HUT-102B2, Molt-4, CEM, and Hut-78
  • B cell lines Raji, S3, Nall-1, 8392, GM4672, U266, and SUDHL-6
  • myeloid line ⁇ 562 and U937
  • RPMI 1640 medium containing 10% FBS
  • the osteosarcoma cell line 5887 J.-9 were maintained in 5 DMEM containing 15% FBS.
  • Human fetal -lung fibroblast HFL-1 cells were passaged to a " density of approximately * 1 x 10° cells per 175 cm 2 tissue culture flask in 25 ml of DMEM containing 10% FBS and allowed to grow to confluence over a period of 7 days at 37°C. The cells were stimu- 0 lated by removing the depleted medium and replacing it with fresh DMEM containing 20% FBS. Early pas ⁇ age HFL-1 cells were used for all experiments. "•
  • Nitro ⁇ cellulose fibers were incubated for 3 h at 65°C in 0.1 M 25 NaH 2 P0 4 pH 6.8, 0.85 M NaCl, 1 mM EDTA, 10 x Denhardt' ⁇ solution, 0.1% SDS, 100 ug/ml salmon sperm DNA and 10 ug/ml poly(rA) (prehybridization solution).
  • the filters were then hybridized at 65°C for 48 h using fresh pre ⁇ hybridization solution containing 10% dextran sulfate and 30 the radioactive probe.
  • the filters were washed 4. times for 20 min at 65°C in 2 x SSC, 0.5% SDS and twice for 30 min at 65°C with 0.1 x SSC, 0.1% SDS at 65°C and then autoradio-graphed.
  • HUT-102B2 cDNA library constructed in lambda gtlO (I-
  • DNA sequencing.- Sequencing was performed via the dideoxy chain termination method using Ml3 phage DNA and Sequenase (U.S. Biochemical Corporation) according to the manufacturer's recommendations.
  • Genomic Southern blot 10 ug of DNA from either U937 or 428 cells was digested with indicated enzymes, analyzed on a 1% agarose gel, and transferred to Gene Screen Plus nylon membrane ⁇ (duPont) and hybridized according to the manufacturer ⁇ suggested protocol.
  • the -0.7 kB Act- 2 cDNA was cloned into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing teh Act-2 cDNA under control of the poly ⁇ hedrin promoter.
  • SF9 cells were cotransfected with pAc- Act2 and wild-type AcNPV strain E2 "DNA.
  • Act-2 mRNA expressed at low or undetectable level ⁇ in re ⁇ ting PBMC, increased rapidly and dramatically in re ⁇ pon ⁇ e to mitogen ⁇ timulation. Peak level ⁇ occurred at approxi ⁇ mately 4 h, and then rapidly declined to negligible level ⁇ by 24 h after stimulation. Thus, Act-2 expression is both rapidly induced and terminated in respon ⁇ e to PHA.
  • Act-2 represents a novel gene. Comparison of the DNA sequence and the predicted amino acid sequence using DNA and protein homology search programs did not reveal any homology with published sequences in the most recent GenBank (version 55, March 31, 1988) and NBRF (versions 16 and 34, released March 31, 1988) databases.
  • the DNA ⁇ equence contain ⁇ an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilize ⁇ the rao ⁇ t 5' AUG, which generally i ⁇ the one utilized in eucaryotic translation initiation. Further, the region of the first AUG has good homology with the consen ⁇ u ⁇ sequence determined for eucaryotic start sites (1-17).
  • the cDNA wa ⁇ utilized to perform a genomic Southern blot (Fig. 1-6). A comparatively simple pattern of digestion was obtained. The same blot, was ; hybridized with an interleukin-2 receptor cDNA (the IL-2 receptor is encoded -by a single copy gene) , and bands of similar intensity were identified (not shown). These data are most consistent with A ⁇ ct-2 representing a sihgle copy or low copy number gene.
  • Act-2 activated -PBMC cDNA, number 2
  • Act-2 encodes an open reading frame.of 92 amino acids, It contains,a very.hydrophobic N-terminus that by the von Heijne weight matrix analysi ⁇ i ⁇ ⁇ trongly predicted to be a signal peptide.
  • in vitro translation analyses could not demonstrate cleavage of the signal peptide, a recombinant Act- 2/baculovirus vector has been expressed in insect cells and found that Act-r2 protein is abundant in the super- natant.
  • N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as pre ⁇ dicted. It remain ⁇ an interesting possibility that Act-2 may also exist in a membrane as ⁇ ociated form.
  • antibodie ⁇ are being produced in order - to be able to directly identify the distribution of Act-2 protein pro ⁇ scored in normal activated human lymphoid cells.
  • the 3' untranslated region is also of signifi ⁇ cant interest in that it is A-T rich and contains the consensus sequence ⁇ ATTTA (1-23) and TTATTTAT (1-24) that have been identified as common sequences in a variety of proto-oncogenes and ⁇ ecreted factors, including tumor necrosis factor, lymphotoxin, IL-1 (both alpha and beta), multiple interferons, and GM-CSF.
  • the TTATTTAT con ⁇ ensus is not commonly found in mammalian mRNAs in general but is particularly prevalent in mRNAs encoding proteins related to the inflammatory response (1-24); thus its presence in Act-2 is supportive of the idea that this gene may represent a new cytokine.
  • TTATTTAT con ⁇ ensus is not commonly found in mammalian mRNAs in general but is particularly prevalent in mRNAs encoding proteins related to the inflammatory response (1-24); thus its presence in Act-2 is supportive of the idea that this gene may represent a new cyto
  • Act-2 wa ⁇ minimally or not expressed in re ⁇ ting PBMC. However, it wa ⁇ rapidly induced following activa ⁇ tion of T cell ⁇ with PHA or PMA, B cell ⁇ with SAC, or monocyte ⁇ with LPS. However, ct-2 i ⁇ not expre ⁇ ed in every actively growing cell, a ⁇ evidenced by it ⁇ non- expression in HeLa an K562 cells, and its failure to be induced in re ⁇ ponse to serum stimulation of quiescent human fibroblasts.
  • the activatable genes can be further differentiated on the basis of their kinetics of induc ⁇ tion, their response to cycloheximide as well as their sensitivity to the immunosuppressive drug cyclosporin A. It is of note that this latter drug inhibits the expression of more than ten inducible genes, suggesting a broad genetic mechanism for the action of this agent.
  • MATERIALS -AND METHODS Cell Culture.
  • Human peripheral blood T cells were obtained from healthy volunteers and, were isolated, over a Ficoll-Hypaque gradient and nylon wool columns. The resulting cell preparations were consitently more than 90% T cells, as judged by anti-CD3 staining.
  • PB T cells were cultured at a concentration of 2 x 106 cells/ ml in RPMI 1640 containing 10% fetal calf serum (FCS).
  • FCS fetal calf serum
  • PB T cells were stimulated for various periods of time with PHAP (1 yUg/ml Burroughs-Welcome Co.) and PMA (20 ng/ml) either with or without cycloheximide (10 uq/ ⁇ al) .
  • the Jurkat cell line was provided by K. Hardy.
  • Jurkat cells were maintained in RPMI 1640 supplemented with 10% FCS and 25 q/ ⁇ al gentamicin.
  • Jurkat cells were stimu ⁇ lated for various periods of time at a concentration of 5 x 105 with PHA-P (1 Uq/ l ) and PMA (25 ng/ml) with or without cyclo ⁇ porin A (1 yg/ml; Sandoz).
  • Human fibro- bla ⁇ t lines, CCD-11LU and WI38 were obtained from the American Type Culture Collection. Fibrobla ⁇ t ⁇ were grown to confluence in MEM containing 10% FCS and then main ⁇ tained in MEM with 0.25% FCS for 3 to 5 Day ⁇ . In order to reinitiate growth, the ⁇ pent medium wa ⁇ replaced by MEM supplemented with 20% FCS either with or without cycloheximide (10 ⁇ q/ l) .
  • PB * - ⁇ cells were isolated and cultured as. escribed above.
  • RNA was isolated as described (II-8) from unstimulated cell ⁇ or after stimulating the cells -for 4.5 hours with PHA-P arid PHA in the pre ⁇ ence of cycloheximide.
  • Poly A+ ?RNA wa ⁇ purified by one pas ⁇ age over an oligo-dT column (II- 3).
  • this cDNA wa ⁇ hybridized to a.cot value to 2000 mole ⁇ x ⁇ /1 with a 10 fold exce ⁇ s of poly A+- RNA from unstimulated cells.
  • the single stranded molecule ⁇ then were ⁇ eparated from the double stranded cDNA:mRNA hybrids by chromatography using a hydroxy- apatite column (11-14, 11-20). After the fir ⁇ t round of ⁇ ubtraction, 15% of the molecule ⁇ appeared in the ⁇ ingle ⁇ tranded fraction a ⁇ judged by the di ⁇ tribution of count ⁇ .
  • Probes were ⁇ upplied by the following individuals or institutions: GM-CSF, Genetics In ⁇ titute; -"/ ' -Interferon, Meloy Laboratories; c-fos. Dr. T. Curran; IL-2 receptor. Dr. W. Greene; IL-3, Genetics Institute; Met- and Leu-preproenkephalin, Dr. S. Sabol; human IL-4, ATCC; p53, Dr. D. Givol; lymphotoxin, Genentech; IL-5, Dr. K. Aria (DNAX); ornithine-decarboxy- la ⁇ e. Dr. D. Nathans; bcl-2. Dr. A. Bakhshi; IL-6, Dr. H.
  • the latter agent is known to superinduce a number of growth related genes (II-l, 11-27, 11-30, 11-34).
  • cyclo ⁇ heximide prevents mRNA induction which follows IL-2 and IL-2 receptor ⁇ ynthe ⁇ i ⁇ and interaction.
  • Thi ⁇ focuses the analysi ⁇ on the primary re ⁇ pon ⁇ e of activated ceils, defined by those genes which are inducible independent of new protein synthesis'. To date, approximately 40% of the subtracted lambda gtlO cDNA ? library has eeh screened with subtracted probes.
  • phage that hybridized to subtracted probes were subjected further to a differential serein in which ' cDNA probes synthesized from activated and resting T cell mRNA were used on duplicate filters of the phage. Finally, 528 phage clones were selected which harbored induced cDNAs, a ⁇ judged by both the subtractive and the differential screening methodolo ⁇ gies.
  • the next step was to cross- hybridize subcloned cDNA insert ⁇ with the 528 phage ⁇ .
  • 66 unique cDNA clone ⁇ were identified, the majority of which appear to repre ⁇ ent di ⁇ tinct gene ⁇ .
  • a limited number of group ⁇ may derive from different segments of the same mRNA, thus leading to an overestima- tion of individual gene ⁇ .
  • the number of unique inducible gene ⁇ will exceed 66, as 120 of the 528 phages have not hybridized to the selected cDNA insert ⁇ te ⁇ ted to date.
  • the number of isolated phage clones belonging to a given group via cros ⁇ -hybridization varied con ⁇ siderably and ranged from 1 to as many as 86 phage clones (see Table II-l legend) .
  • Forty-four of the novel inducible gene clones have been studied, further (see below). All hybridized to an inducible and, with few exceptions, single-sized message by Northern blot analyses (Table II-l) .
  • none of the cDNA inserts contained repetitive sequences, but a few appeared to be members of small multi-gene families, as determined by Southern blot analyses (Table II-l).
  • composition of the subtracted library was shown to repre ⁇ sent a typical activated T cell phenotype as determined by the enrichment for clones encoding IL-2, GM-CSF, gamma-IFN, c-myc and c-fos, the latter of which i ⁇ induced in T cells at 4.5 hours in the presence of cyclo ⁇ heximide, but to a much les ⁇ er extent than c-myc (II- 36).
  • ⁇ elected 528 phage were detected several isolates of c-myc.
  • the 528 phage include cDNAs homologous to the IL-2 receptor, the IL3 and IL-4 growth factors and Met- and Leu-preproenkephalin (11-46, 11-47, 11-49) (Table II-l).
  • IL-2 in the library that are not pre ⁇ ent in the 528 phage selected with subtracted cDNA probes resulted in part from the much stronger signal generated by nick-translated versus heterogenous cDNA probes.
  • the length of the induction period (4.5 hours) was optimal for a great number of genes, it was not optimal for those genes which are expressed with relatively delayed kinetics. Nonethe ⁇ less, the hybridization data indicate that the 528 selected phage encompass many but not all of the genes expected, and the subtracted library contains the known induced genes which have been assayed.
  • FIG. 1 In order to assess the heterogeneity of expres ⁇ ion for ' the i ⁇ olated inducible gene ⁇ , kinetic analy ⁇ es of mRNA levels for many of the genes were per ⁇ formed.
  • Figure II-2 displays typical Northern analyses for four novel inducible genes.
  • One pattern of expression exemplified by pAT 249, displays a very rapid appearance after activation of T cells by PHA and PMS, i.e., by 30 min. or less; another common pattern shown for .
  • pAT 464 is characterized by mRNA appearance only after 2-4 hours. Additional mRNA species (e.g.
  • pAT 129 and pAT 139, Figure 11-2) are induced at intermediate times.
  • the human CD4+ helper cell line Jurkat i ⁇ known to have retained the inducibility of ⁇ everal gene ⁇ , including the lymphokine ⁇ IL-2 and gamma-interferon as well as the 11-2 receptor (II-II-19, 11-37, 11-45).
  • CsA immunosuppre ⁇ ive drug cyclo ⁇ sporin A
  • Many lymphokines elaborated by activated T cells are known to be suppressed by this drug which appears to inhibit transcriptional induction (11-16, II- 37, 11-39).
  • Figure II-3 and summarized in Table II-2 four different patterns of expres ⁇ ion could be distinguished.
  • the remaining 13 genes tested were not induced in Jurkat T cells. Ten of these (e.g. pAT 133, Figure II-3) failed to hybridize to any message in Jurkat cells ⁇ as determined at a number of time points following induction (data not shown) . Possibly, these message ⁇ exist in a cell type distinct from Jurkat, such as CD8+ T cells. Alternatively, Jurkat cells are transformed and may have lost or modified these genes or the signalling machinery nece ⁇ sary to induce their mRNAs. The members of the last group of genes (3 out of 35) were con ⁇ titutively expresed in Jurkat cells (e.g., pAT 129, Figure II-3). These genes may contribute to the uncontrolled growth of this tumor line (see Discussion).
  • a Frequency grouping indicates the number of cros ⁇ - hybridizing phage clones, among the 528 clones isolated, which are detected by the subcloned insert listed: 1, one cros ⁇ -hybridizing phage clone; 2, two to five cro ⁇ - hybridizing phage clones; 3, six to 10 cross-hybridizing phage clones; 4, eleven to twenty-five cross-hybridizing phage clone ⁇ .
  • mRNA size (in nucleotides) was determined relative to the migration of the 28S and 18S rRNA on formaldehyde-agarose gels. time ( inute ⁇ ) at which the iduced mRNA species was first detected following PHA (1 /g/ml) and PMA (20 ng/ml) stimulation of peripheral blood (PB) T cells; the second number denotes the time (minutes) at which maximal steady state mRNA levels were noted.
  • a 542 al ⁇ o detected a mRNA species of 4600 nucleotides which wa ⁇ coordinately expre ⁇ ed with the predominant species of 6800 nucleotides.
  • h pAT 563 also detected mRNA species of 3600, 4100 and 8500 nucleotides,. coordinately expressed with the predominant 2400 nucleotide species.
  • Table II-2 Expression analysis of T cell induced genes in the helper T cell line Jurkat and in human fibroblasts. Experimental details are as described in figure II-3 and II-4. Y - induction upon stimulation of quiescent human fibroblasts (HF) with serum N - no expression in quiescent or serum stimulated human fibroblast ⁇ ND - not determined a - " denote ⁇ expre ⁇ sion data for the 900 nucleotide constitutively expres ⁇ ed mRNA species (see Table II-l) denote ⁇ expre ⁇ sion data for the 2000 nucleotide induced mRNA specie ⁇ ( ⁇ ee Table II-l)
  • these genes may encode other functions such as modulation of the immune system.
  • those genes that exhibit limited tis ⁇ ue di ⁇ tribution of expre ⁇ ion (Table II-2) are to be expected to play such roles in the differentiated function of activated T cell ⁇ .
  • two of the ⁇ e gene ⁇ , pAT 464 and 744 have been ⁇ equenced and di ⁇ play predicted hydrophobic leader peptide ⁇ and structural homology with known secreted proteins, sugge ⁇ ting their identity a ⁇ potential lymphokine ⁇ (Experimental Section IV) .
  • lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony- stimulating actor activities:- Relationship to interleukin 5. Proc. Natl. Acad. Sci. USA 84:7388-7392.
  • T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with PHA induced all nine genes.
  • stimulation by fully mitogenic agents regardles ⁇ of cell ⁇ urface binding specificity, corre ⁇ lates with the expression of all those genes studied here.
  • heterogenous patterns of gene expression encompassing five regulatory classes, were revealed, by the use of PMA, calcium ionophore and anti-CD28 mono ⁇ clonal antibody, agents which mediate only a sub ⁇ et of intracellular event ⁇ and thus an incomplete mitogenic signal.
  • IL-2 and two novel lymphokines represent one regulatory clas ⁇ that appear ⁇ to require unique tran ⁇ criptional activation signals relative to the other mitogen, induced genes.
  • the acti ⁇ vating agents studied here include monoclonal antibodies (mAbs) directed against CD2, CD3 or CD28 which are dis ⁇ tinct, physically non-associated T cell surface molecules, the lectin phytohemagglutinin (PHA), calcium ionophore, phorbol 12, myristate 13-acetate (PMA), and dioctanoylglycerol (DiC3).
  • mAbs monoclonal antibodies directed against CD2, CD3 or CD28 which are dis ⁇ tinct, physically non-associated T cell surface molecules, the lectin phytohemagglutinin (PHA), calcium ionophore, phorbol 12, myristate 13-acetate (PMA), and dioctanoylglycerol (DiC3).
  • T cell receptor or the associated CD3 complex of T cells initiates the activation of T cells (III-2, 111-18, III- 23).
  • PHA-mediated stimulation appears to require the presence of the antigen receptor complex or a coordinate ⁇ ly expressed molecule, as determined by ⁇ tudies with selective surface loss mutants (111-20).
  • An antigen- independent pathway of T cell activation involving the CD2 complex (E rosette receptor) has al ⁇ o been described (111-19).
  • Binding of the above mi ogens to cell surface receptors on T cells results in the production of inositol 1,4,5-tripho ⁇ phate , an increased intracellular calcium concentration ([Ca 2+ ]i), membrane translocation of protein kinase C (PKC) and sub ⁇ equent proliferation (III-6, III-9, 111-13, 111-15, 111-22, 111-25).
  • PKC protein kinase C
  • the signal delivered by CD28 seems to be funda ⁇ mentally distinct from that of CD2 or CD3 because stimu ⁇ lation by anti-CD28 leads to an elevation of cytoplasmic cyclic GMP concentration and does not cause increase ⁇ in [Ca2+]; or activation of PKC (111-12, 111-14, 111-22, 111-26).
  • IL-2 production initiated by CD28 i ⁇ re ⁇ i ⁇ tant to the effect ⁇ of cyclo ⁇ porin A in contra ⁇ t to that induced by CD3 or calcium ionophore and PMA (III-8).
  • anti-CD28 - alone doe ⁇ not ⁇ timulate proliferation of T cell ⁇ but require ⁇ the ⁇ imultaneous presence of a_comitogen, such as PJJA ( 11-7,, III-8).
  • a_comitogen such as PJJA ( 11-7,, III-8).
  • T cells also may be activated, bypassing celi surface interactions, following treatment with calcium ionophore in addition to PMA or DiC8; the latter two agents act, at least in part, by stimulating protein kinase C (111-16, 111-21).
  • Ionophore-mediated increa ⁇ e ⁇ in [Ca 2+ ] act synergistically with DiC3 or PMA to ⁇ timulate prolifera ⁇ tion and induced gene expre ⁇ ion in purified PB T cell ⁇ and ⁇ ome induced gene expre ⁇ ion, including lymphokine ⁇ , in the T cell line Jurkat (111-16).
  • PB mononuclear cell ⁇ were obtained from normal healthy donor ⁇ by leukophore ⁇ i ⁇ and PB lymphocyte ⁇ i ⁇ olated by den ⁇ ity gradient centrifuga ⁇ tion through Lymphocyte Separation Medium (Litton Bionetic ⁇ ) , followed by the removal of adherent cell ⁇ (monocyte ⁇ and B cells) by passage over a nylon wool column.
  • the resulting cell ⁇ had approximately 1% B cells and ⁇ 1% monocytes a ⁇ determined by FACS.
  • the T cell ⁇ were cultured at 2 X 10 /ml in RPMI-1640 medium contain ⁇ ing 10% heat-inactivated fetal calf serum and stimulated with one of the following agents: anti-CD3 (mAb OKT3, Ortho Diagnostics), used at 10 ng/ml; PHA-P (Burroughs- Wellcome), used at 3 ⁇ q/ ⁇ al; anti-CD2 (ref. 111-19: anti- T112 and anti-T113 ascites, obtained from E. Reinherz), each ascites was used at a 1:200 dilution. All reagents were titrated to determine the optimal concentration as a ⁇ ayed by the ability to stimulate peak proliferation of PB T cells or IL-2 production by Jurkat cells.
  • cycloheximide (Sigma Chemical Co.) was used at 10 Uq/ml .
  • the ability of each agent to stimulate pro- liferation was determined by 3 H-thymidine incorporation assayed after 4 days.
  • a representative experiment showed 1,003 cpm for culture medium alone, 184 / 567 cpm for PHA, 107,908 cpm for anti-CD2 and 1,248 cpm for anti-CD3.
  • the lacksof response to anti-CD3 in PB T cells appeared due to monocyte depletion, since linfractionated PBL from the same donor gave 127,760 cpm.
  • CD28+ cell ⁇ were purified a ⁇ previously described (III-8). The cells were cultured as outlined above and stimulated for 4 h with anti-CD28 mAb 9.3 (obtained from J. A. Ledbetter) at 100 ng/ml, iono ⁇ mycin (Calbiochem; dissolved in DMSO) at 133 nM, and PMA (Sigma Chemical Co.; dissolved in DMSO) at 0.3 ng/ml.
  • a preliminary experiment showed all the gene ⁇ to be maxi ⁇ mally induced at 4 h.
  • the PMA concentration wa ⁇ determined by ⁇ H-thymidine incorporation to maximally ⁇ ynergize with either anti-CD28 or ionomycin yet not to be it ⁇ elf mitogenic.
  • the mitogenic activity of each agent wa ⁇ assayed by 3 H-thymidine incorporation after 3 days of stimulation.
  • a representative experiment showed 121 cpm for medium alone, 488 cpm for PMA, 236 cpm for mAb 9.3 alone, 58,590 cpm for mAb 9.3 and PMA. 186 cpm for ionomycin alone, 44,760 cpm for ionomycin and PMA and 69,360 cpm for immobilized anti-CD3.
  • Jurkat cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum at a den ⁇ ity of 2 X 10-Yml to 8 X 10°/ml. Fre ⁇ h aliquots of cells were thawed at approximately 6 week intervals. Cells were stimulated at 4 X 10 /ml with one or more of the follow- ing agents: DiC3 (Molecular Probes. Inc., Eugene, OR; dissolved in absolute ethanol) at 100 /M, ionomycin at 500 nM, cycloheximide at lOjU/ml. The DiC3 and ionomycin - 62 -
  • RNA (10 pg per lane) was separated by electrophoresi ⁇ in formaldehyde-agarose gels, ' blotted onto Genescreen membrane filter ⁇ (NEN Re ⁇ earch Product ⁇ ) and hybridized to 32 p-labelled purified cDNA inserts prepared by nick- translation.
  • Fig. Ill-i show ⁇ that for each gene, PHA, anti- CD2 and anti-CD3 stimulation resulted in ⁇ imilar re ⁇ pon ⁇ es.
  • a hierarchy in which the relative levels of mRNA expression can be graded from a low level of induci- bility (pAT 229) to strong inducibility (pAT 464) can be seen in response to any of the three mitogen ⁇ .
  • pAT 464, Fig. III-l pAT 744, data not shown
  • two were induced to a moderate level pAT 563, Fig. III-l; pAT 120, data not shown
  • two were induced to a low level pAT 416, Fig.
  • III-l pAT 237, data not shown) and three were weakly induced (pAT 229, Fig. III-l; pAT 154 and pAT 225, data not shown) by either PHA, or anti-CD2 or anti-CD3 mAbs. Stimulation with any one of the above mitogen ⁇ in the presence of PMA elicited an elevated response, relative to PHA. anti-CD2 or anti-CD3 mAbs alone, for all of the genes (data not shown) . In addition, although the kinetics of mRNA accumulation are unique for each gene, the ⁇ e kinetics are relatively uniform for each gene irrespective of the stimulus used. "
  • Table III-l Summary of Northern blot data for mitogen- induced genes in respon ⁇ e to agents used in Figs. III-l- 3. Symbols: +, mRNA hybridization to labelled probe; -, no hybridization to labelled probe; -, no hybridization; +/-, weak hybridization; Ab, abrogation of DiC8- and ionomycinr-mediated expression; Incr, increased levels of DiC8- and ionomycin-mediated expre ⁇ ion; ND, experiment not performed.
  • the ⁇ e agent ⁇ include calcium ionophore, PKC activators or anti- CD28 mAb, none of which stimulates DNA synthe ⁇ i ⁇ in puri ⁇ fied T cell ⁇ (III-8, 111-16, 111-24 and reviewed in III- 26).
  • purified T cell ⁇ proliferated after treatment with the combination of calcium ionophore or anti-CD28 mAb and PMA (see Materials and Methods).
  • Fig. III-2 Steady state mRNA levels resulting from stimu ⁇ lation of CD28+ T cells with PMA, anti-CD28 mAb 9.3, ionomycin, PMA plus anti-CD28 mAb and PMA plus ionomycin are shown in Fig. III-2. Unlike stimulation via CD2 or CD3, which elicited graded responses from all nine mitogen induced genes, activation through CD28 clearly divided the nine novel genes into distinct groups (Fig. III-2 and Table III-l). One group, exemplified by pAT 416 (Fig. III-2), and including pAT 120, pAT 154, pAT 225 and pAT 229 (data not shown), showed no respon ⁇ e to anti- CD28 mAb.
  • III-2 pAT 563 and pAT 744, data not ⁇ hown) and for IL-2R (III-8 and data not ⁇ hown) .
  • PMA alone induced varying level ⁇ of expre ⁇ ion, gene induc ⁇ tion with ⁇ oluble or cro ⁇ -linked mAb 9.3 alone was not seen in this group.
  • IL-2 mRNA in contrast, was not induced by PMA alone, but was induced in respon ⁇ e to a synergistic combination of PMA and mAb 9.3.
  • DiC8 is a cell-permeant synthetic diacylglycerol (DG) that mimics the effect of endogenous DG by reversibly acti ⁇ vating PKC (III-5, 111-16, 111-17) .
  • DG cell-permeant synthetic diacylglycerol
  • the fir ⁇ t cla ⁇ detected i ⁇ represented by pAT 237 in Fig. III-3 and includes genes which are PKC inducible but display either varible and minimal or no synergy with a [Ca 2+ ]i signal (pAT 237, Fig. III-3: pAT 120, pAT 154, pAT 225 and pAT 416, data not shown). No response to ionomycin alone was detected for this cla ⁇ of gene ⁇ .
  • the final cla ⁇ ob ⁇ erved in these cells includes pAT 464 (data not shown) and . AT 744 as well a ⁇ IL-2 (Fig. III-3), whose induction exhibited a require-** ment for both signals as ⁇ ayed in thi ⁇ experiment.
  • pAT 464 data not shown
  • AT 744 as well a ⁇ IL-2 (Fig. III-3)
  • Fig. III-3 a ⁇ IL-2
  • cycloheximide added ⁇ imultaneou ⁇ l with the activating agent ⁇ entirely prevented the accumu ⁇ lation Of IL-2, pAT 744 (Fig. III-3) and pAT 464 mRNA (data not shown) , implying a mechanism of gene induction requiring newly ⁇ ynthesized protein.
  • cyclo ⁇ heximide treatment .enhanced mRNA levels in the other two groups (Fig. III-3 and data not shown) .
  • the apparent homogeneity of expre ⁇ ion pattern ⁇ for each gene in re ⁇ pon ⁇ e to PHA, anti-CD3 or anti-CD2 mAb ⁇ may reflect that qualitatively, a similar series of intracellular biochemical events re ⁇ ult ⁇ from activating T cells with the ⁇ e agents.
  • This common pattern of gene expres ⁇ ion in re ⁇ ponse to a variety of inducing agents can potentially be mediated at a number of levels in the signalling pathway.
  • PB T cell ⁇ are heterogeneou ⁇ with regard to population subtype ⁇ or from differences in the basal state of activation between quiescent, nontrans- formed cell ⁇ and cycling, tumor cell ⁇ .
  • Jurkat cell ⁇ may abnormally over-expre ⁇ s or under-expres ⁇ ⁇ pecific regulatory component ⁇ required for ⁇ ome gene induction event ⁇ .
  • Thi ⁇ regulatory cla ⁇ of gene ⁇ responds to signal ⁇ mediated through CD28, in addition to CD2 and CD3 in PB T cell ⁇ , exhibit ⁇ a requirement for two ⁇ ignal ⁇ in Jurkat cell ⁇ , and their induction i ⁇ completely abrogated by cycloheximide treat ⁇ ment in Jurkat cell ⁇ .
  • a requirement for protein ⁇ ynthe ⁇ i ⁇ preceding mRNA induction in Jurkat appear ⁇ to be unique to thi ⁇ cla ⁇ of gene ⁇ , relative to many other mitogen induced gene ⁇ ⁇ tudied here and elsewhere, suggesting a novel and conserved regulatory mechanism for these and most likely other lymphokines.
  • T cell activation II A new activation pathway used by a major T cell population via a disul- fide.bonded dimer of a 44 kilodalton polypeptide
  • T.Cell pro ⁇ liferation involving the CD28 pathway is as ⁇ oci- ated with cyclo ⁇ porin.re ⁇ i ⁇ tant interleukin 2 gene expression. Mol. Cell. Biol. 7:4472-4481.
  • T. cell vari ⁇ ants lacking molecules involved in T. cell acti- vation T3 T. cell receptor, T44. and Til:
  • the two gene ⁇ can be expre ⁇ ed in T cell ⁇ , B cells and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expres ⁇ ion i ⁇ re ⁇ tricted to hematopoietic lineages.
  • the predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders -character ⁇ istic of secreted proteins.
  • the predicted size of both proteins is about 8 kD upon cleavage of the putative leader, peptide.
  • pAT 464 and pAT 744 are similar to each other and also share some critical amino acid sequence similar- ity with a newly emerging family of ⁇ ecreted factors including connective tissue activating factor III (CTAP III), platelet factor 4 (PF4), an interferon-gamma induced factor (IP-10), macrophage inflammatory protein (MIP) and a factor chemotactic to neutrophils (3-10C, MDNCF, NAF) .
  • CTAP III connective tissue activating factor III
  • PF4 platelet factor 4
  • IP-10 interferon-gamma induced factor
  • MIP macrophage inflammatory protein
  • MDNCF macrophage inflammatory protein
  • Human peripheral blood T cell ⁇ were obtained from healthy human donors by lympho- phore ⁇ is on a enwall CS 3000 or an_ IBM Cebe 2997 -appa- ratu ⁇ . Blood enriched for lymphocyte ⁇ wa ⁇ subsequently purified over Ficoll Hypaque gradients and nylon wool columns. As judged by anti CD3 staining, the ⁇ e cell preparation ⁇ con ⁇ i ⁇ tently were compo ⁇ ed of greater than 90% T cell ⁇ .
  • Cell Stimulation Human peripheral blood T cell ⁇ , Jurkat cell ⁇ and HL60 cell ⁇ were maintained in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum.
  • Penicillin, Streptomycin and Gentamycin at a cell density of 2 x 10° cells/ml for human T cell ⁇ , or 5 x 10 5 cell ⁇ /ml for Jurkat cell ⁇ , re ⁇ pectively.
  • the cell ⁇ were treated with either one or a combination of the following agent ⁇ : Phytohemagglutinin (PHA-P; Burirough ⁇ Wellcome) at a final concentration of 1 Afg/ml, Phorbol 12-myri ⁇ tate 13-acetate (PMA) at 20 ng/ml, cycloheximide at 10 jq/ml and cyclo ⁇ porin A at 1 juq/ml .
  • PHA-P Phytohemagglutinin
  • PMA Phorbol 12-myri ⁇ tate 13-acetate
  • cycloheximide at 10 jq/ml
  • cyclo ⁇ porin A at 1 juq/ml .
  • RNA Isolation and Northern Analysi ⁇ Total cellular RNA wa ⁇ isolated by the quanidinium isothiocy- nate method as described (IV-27) and either 8 ⁇ q (periph ⁇ eral blood T cells) or 10 ⁇ q (Jurkat cells) of total cellular RNA were separated on a formaldehyde agarose gel and subsequently transferred to nitrocellulose (Schleicher and Schuell) with 10 x SSC. ,
  • cDNA insert ⁇ were nick-translated (IV-28) and used as probe ⁇ for hybridization at 42°C in 40% Formamide, 4 x SSC, 10% dextran sulfate and lx Denhardt's ⁇ olution. After hybridization for 10-18 hr, the filters were washed at a final stringency of 0.1 x SSC at 60°C
  • Clones pAT 464 and pAT 744 originally were derived from a sub- ⁇ tracted cDNA library (Experimental Section II, above). Near full-length cDNA clone ⁇ were i ⁇ olated from a cDNA library which wa ⁇ derived from RNA extracted from human peripheral blood T cell ⁇ ⁇ timulated for 4.5-hr with. PHA-P and PMA in the pre ⁇ ence of cycloheximide. Thi ⁇ cDNA library was constructed with oligo dT priming as described (IV-29) and was sub ⁇ equently cloned into lambda gt 10 (IV-30).
  • Primers for the sequencing reactions were syn- the ⁇ ized a ⁇ 17mer ⁇ on an oligonucleotide ⁇ ynthe ⁇ izer model 380A (Applied Biosystems) following the protocol of the supplier. All oligonucleotides were purified on a 20% acrylamide gel prior to use. Both the cDNA sequences- and the predicted protein ⁇ equence ⁇ were compared with- a- computer data bank (Bionet, current release).
  • oligonucleotide comple-. mentary. to -nucleotide 135-152 of the pAT 744 sequence (Fig. IV-4B) was annealed to 20 q of RNA f_eom human peripheral blood T cells which were stimulated for 4.5 ⁇ ir with Pi_A and PMA in the presence of cycloheximide. Annealing was performed in 100 mM KC1, and the reverse transcription reaction was a ⁇ described (IV-29), using reverse transcripta ⁇ e (Seikageiku) to extend the annealed primer (100 (g/ml) .
  • Hybrid ⁇ were extracted'asrith an-equal volume of phenol/chloroform and then-were precipitated in ethanol.
  • the final reaction product wa ⁇ analyzed on a 8% denaturing polyacrylamide gel.
  • a DNA ⁇ equence ladder ⁇ erved a ⁇ size markers.
  • COS cell ⁇ were transfected msing ⁇ tandard.DEAE- dextran methodology, with cDNA clones inserted-- into expression sites of either of the following vector DNAs: CDM-8 [obtained from Brian Seed at Massachusett ⁇ . General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)]; or PMT2-T [obtained from Genetics In ⁇ titute, Cambridge, Ma ⁇ achu ⁇ ett ⁇ ; a related earlier ver ⁇ ion of thi ⁇ vector ha ⁇ been de ⁇ cribed in Yu-Chung Yang, et al.. Cell _47_, 3-10 (1986)].
  • CDM-8 obtained from Brian Seed at Massachusett ⁇ . General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)
  • PMT2-T obtained from Genetics In ⁇ titute, Cambridge, Ma ⁇ achu ⁇ ett ⁇ ; a related earlier ver ⁇ ion of thi ⁇ vector
  • pAT 464 and pAT 744 Two genes from this collection of cDNAs, named pAT 464 and pAT 744, were noted to ⁇ hare regulatory char- acteri ⁇ tic ⁇ with the IL-2 lymphokine gene (Experimental Section III). Like IL-2, the ⁇ e genes required absolutely the combined action of signal ⁇ initiated by ionomycin and PMA for optimal expression in highly purified T cells. In addition, mRNAs for pAT 464 and pAT 744 were induced by 2 hours and maintained at high levels for at least 24 hours, kinetics different from those seen with the major ⁇ ity of the isolated genes (Experimental Section II).
  • Nucleotide Sequence of pAT 464 and pAT 744 The nucleotide sequence of near full-length cDNA clone ⁇ for pAT 464 and pAT 744 wa ⁇ determined (see Materials and Methods). The sequencing strategies used, the resulting nucleotide sequences and the predicted amino acid ⁇ for the encoded protein ⁇ are ⁇ hown in Figure ⁇ rv-3 and IV-4 for pAT 464 and pAT 744, re ⁇ pectively.
  • Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotides long (including the Eco RI linker at the 5' end). Both cDNA sizes are in good agreement with the estimated mRNA sizes of 850 nucleotides (pAT 464) and 750 nucleotides (pAT 744), since the mRNAs are likely to include a much longer poly A tail.
  • Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5).
  • U ⁇ ing an oligonucleotide complementary to nucleotide ⁇ 135-152 (Fig. IV-4) of the pAT 744 ⁇ equence a ⁇ a primer for reverse transcriptase resulted in a fragment of about 155 nucleotides in size (138 nucleotides of synthesized cDNA and 17 nucleotides of primer) .
  • the first ATGs for pAT 464 and pAT 7.44 are at positions 84 and 74, respectively.
  • the ⁇ e open read- ing frame ⁇ are the longes to be found : for the two clone ⁇ .
  • the nucleotide sequences surrounding the predicted start sites for translation for both clones display a good match with the consensus sequences derived from known translation initiation site ⁇ (IV-36).
  • the 3' untranslated region ⁇ of the two cloned gene ⁇ contain several elements which may serve regulatory functions.
  • the ATTTA motif (underlined in »igure ⁇ : IV-3 and IV-4) that Jaa ⁇ been a ⁇ ociated with the instability of lymphokine mes ⁇ age ⁇ (IV-39) appear ⁇ four time ⁇ in clone pAT 464 and twice in clone pAT 744.
  • pAT J44. and 464 contain the octamer motif L TTATTTAT (a ⁇ ub ⁇ et of the ATTTA motif ⁇ ), which i ⁇ present in the 3' untran ⁇ lated region ⁇ of a number of distinct mRNAs induced during an inflammatory response ' (IV-40 ⁇ .
  • the ⁇ e two gene ⁇ may have been derived from a common ancestral gene and. their encoded proteins " may play functionally related roles•; The higher amino acid similarity sugge ⁇ t ⁇ a con ⁇ ervation of function and further ⁇ trengthen ⁇ the encoded protein ⁇ tructure ⁇ predicted from the open reading frame ⁇ in the cDNA clone ⁇ (see also comparison with other protein ⁇ below) .
  • the pre ⁇ umed ⁇ ecreted part ⁇ of the two pro- tein ⁇ exhibit a high degree of ⁇ imilarity, wherea ⁇ their N-terminal leader peptide ⁇ have little in common except their hydrophobic nature.
  • the hydrophilicity plot ⁇ of the two protein ⁇ are virtually superimpo ⁇ able which affirms further their structural relatedness (data not shown).
  • the primary structures of the proteins encoded by pAT 464 and 744 exhibit a striking similarity to a number of other proteins, ⁇ o e of which have been detected a ⁇ ⁇ ecreted proteins.
  • ⁇ o e of proteins have been detected a ⁇ ⁇ ecreted proteins.
  • Several members of this family of proteins have been shown to exhibit functions assiciated with an inflammatory response and with mitogenesis (see Discus ⁇ ion).
  • thi ⁇ similarity is based on four almost identically spaced cysteine residues and one proline residue, present in all proteins of this family (Fig. IV-6B).
  • These highly conserved amino acids are likely to play an important role in the structure of the proteins (see Discussion). In addition to this conservation, many other amino acids are shared between various members of this family.
  • genomic DNA wa ⁇ probed with the ⁇ e two gene ⁇ Human placenta DNA was digested with various restriction enzymes and subjected to a Southern analysi ⁇ with radioactive probe ⁇ derived from the cDNA in ⁇ ert ⁇ for the two gene ⁇ .
  • pAT 464 and 744 do not readily cros ⁇ -hybridize with each other, in ⁇ pite of their remarkable ⁇ imilarity.
  • pAT 464 and 744 are not expres ⁇ ed in quiescent or serum stimulated normal human fibroblast ⁇ . Here hematopoietic cell ⁇ other than T cell ⁇ were analyzed for expression.
  • Promyelocytic HL60 cells can be differentiated terminally into macrophages or into granulocyte ⁇ . Neither pAT 464 nor pAT 744 was expres ⁇ ed in undifferentiated HL60 cells but both were induced upon induction of differentiation into macrophages with PMA (Fig. IV-8A) . In contrast, using DMSO to differentiate HL60 cells into granulocytes did not result in any indue- tion of their mRNA ⁇ .
  • pAT 464 and pAT 744 may be expre ⁇ ed by a number of dif ⁇ ferent hematopoietic cell ⁇ upon being activated in ⁇ ome manner, including viral transformations.
  • Antibodies to predicted amino acid sequences Rabbit antisera were raised to synthetic peptide ⁇ repre- ⁇ enting the C-terminal 12 amino acids of both pAT 464 and pAT 744, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier plu ⁇ peptide ⁇ into rabbit ⁇ , according to ⁇ tandard method ⁇ of peptide immunology.
  • Figs. IV-11A and B show the use in We ⁇ tern blot experiment ⁇ of two different rabbit anti- bodie ⁇ (721 and 722) rai ⁇ ed again ⁇ t the pAT 744 peptide; Figs.
  • IV-12A and IV-B similarly illustrate two antibodies (719 and 720) to the pAT 464 peptide.
  • supernatants of COS cells transfected with various expres ⁇ ion vector con ⁇ truct ⁇ were analyzed with the anti- ⁇ era.
  • ⁇ the appropriate secreted factors are detected by antisera from rabbits immunized with synthetic peptides, but not by serum sam- ple ⁇ taken from the ⁇ ame rabbit ⁇ before immunization with peptide ⁇ .
  • Fig. IV-13 shows a Western blot of supernatant from human T cells activated in culture with PHA/PMA u ⁇ ing anti-pAT 464 antibody. The antibody detects a secreted product of appropriate size. Similar results were obtained with anti-pAT 744 antibody (data not shown). Therefore, human T cells produce not only an abundant amount of pAT 464 and pAT 744 mRNA upon mitogenic stimulation (see above), but also the secreted forms of the protein factors predicted from the cDNA sequences of these clones.
  • pAT 464 and ' 744 are very homologou ⁇ to each other, e ⁇ pecially in their pre ⁇ umed ⁇ ecreted port__6f ⁇ s-_, Thi ⁇ ⁇ ugge ⁇ t ⁇ con ⁇ ervation of function. Both gene pro-*- ducts also share important amino acid residues with a family of proteins, -whose functions are not oicL only-par ⁇ tially understood; some of these protein ⁇ have been detected a ⁇ secreted factors (see below). Although pAT 464 and pAT 744 do not possess significant amino acid homology with several well-defined immunoregulatory fac- tors like IL-2 or gamma-IFN, they are likely to ⁇ hare regulatory element ⁇ with the ⁇ e gene ⁇ .
  • pAT 464 and pAT 744 require two inducing signals for optimal expres ⁇ ion (e.g., PHA and PMA), they appear after 2 hour ⁇ of ⁇ timulation of T cell ⁇ , they are very sensitive to the inhibitory effects of the immuno ⁇ uppre ⁇ - ⁇ ive drug cyclo ⁇ porin A and their activation i ⁇ sup- pre ⁇ ed completely in Jurkat T Cell ⁇ in the pre ⁇ ence of cycloheximide.
  • the primary amino acid ⁇ tructure ⁇ of the-'vpro- tein ⁇ encoded by pAT 464 and 744 exhibit ⁇ ignificant homology with a newly emerging family of ⁇ ecreted fac ⁇ tors.
  • the homology is ba ⁇ ed primarily on the positions of four cysteine re ⁇ idue ⁇ ( ⁇ ee al ⁇ o IV-23, IV-42, IV-43) and one proline re ⁇ idue (boxed in Fig. IV-6B).
  • the ⁇ e amino acid ⁇ are found in all the protein ⁇ ⁇ hown here and the peptides were aligned accordingly in Figure IV-6B.
  • the cysteines are likely to provide a common structure.
  • cysteine ⁇ For one of the members of this family, ?-thromboglobulin (a processed form of PBP, see below), these cysteine ⁇ have been shown to form di ⁇ ulfide bonds (IV-44). Start ⁇ ing from the amino terminus, cysteine ⁇ 1 and 3 and cy ⁇ - teines 2 and 4 form ⁇ uch bonds.
  • Trp Val Gin WVQ
  • WV WV
  • Fig. IV-6B the amino acids Trp Val Gin (WVQ) (or WV) (boxed in Fig. IV-6B) are found in eight of the factors, ⁇ ix re ⁇ idue ⁇ down ⁇ tream from the la ⁇ t of the four conserved cysteines.
  • the genes listed in Figure IV- 6B can be subdivided into two principal groups: The first two cysteines are positioned in one of two way ⁇ , either next to each other as Cys (C) or separated by one amino acid as C X C.
  • IV-23) or monocyte-derived neutrophil chemotactic factor (MDNCF) (IV-43) or neutrophil activating factor (NAF) (IV-25) is a human factor which has chemotactic activity for granulocytes in vitro (IV-24, IV-45, IV-46),- induce ⁇ rapid granulocyto ⁇ is jua vitro or with systemic injections in vivo and causes skin reactions upon local injection (IV-46, IV-47).
  • IP-10 (IV-21) is an interferon induced -protein factor which is expre ⁇ ed during delayed-type hyper ⁇ en ⁇ i- tivity reactions incited by a form of tuberculin or by - lFN (IV-49). It can " be expressed in several cell types, including endothelial cells, monocytes, fibroblast ⁇ and keratinocyte ⁇ (IV-49, IV-50).
  • Platelet factor 4 i ⁇ relea ⁇ ed from the o-s-granule ⁇ in platelet ⁇ during injury and ha ⁇ chemotactic activity for monocyte ⁇ , neutrophils and fibroblasts (IV- 51, IV-52). Additional activities are the inhibition of collagenase (IV-53) and an immunoregulatory role (IV-54). Platelets al ⁇ o release the platelet basic pro ⁇ tein (PBP) ⁇ -thromboglobulin (A-TG) and connective tis ⁇ ue activating peptide III (CTAP III) (also known as low- affinity platelet factor-4).
  • PBP platelet basic pro ⁇ tein
  • A-TG ⁇ -thromboglobulin
  • CTAP III connective tis ⁇ ue activating peptide III
  • CTAP III and _>-TG represent succe ⁇ ive N-terminally proce ⁇ ed form ⁇ of PBB, a ⁇ indicated in Figure IV-6B.
  • _?-TG appear ⁇ to be chemotactic for fibroblasts (IV- 51). The remaining factors li ⁇ ted were all cloned on the ba ⁇ i ⁇ of being inducible or differentially expre ⁇ ed.
  • RANTES (IV-42) and TCA-3 (IV-57) are T cell derived gene ⁇ for which no function i ⁇ known yet.
  • the human H-Gro gene, the mou ⁇ e JE gene and the chicken 9E3 gene are all expressed by fibroblasts.
  • the JE gene is induced by various agents in 3T3 cell ⁇ , including serum and IL-1 (IV-58).
  • the 9E3 gene is acti ⁇ vated by transformation with the Rous Sarcoma Viru ⁇ and i ⁇ suppressed in growth-restricted cells (IV-59).
  • the H- Gro gene is the human counterpart of the hamster Gro gene, whose constitutive expres ⁇ ion correlate ⁇ with tumorigenic variants of the nontumorigenic Chinese ham ⁇ ster fibroblastic cell ⁇ (CHEF) (IV-26). Gro is tran- siently induced by ⁇ erum in the nontumorigenic CHEF cells. The expre ⁇ ion of thi ⁇ gene i ⁇ apparently not sufficient for induction of the phenotypes associated with the tumorigenic state.
  • the expression data for the genes discussed above is consi ⁇ tent with a role during inflammation and/or mitogene ⁇ i ⁇ a ⁇ ociated with tissue repair. This is deduced from the various functions demonstrated, from the agents inducing these genes and from the cell types involved. Therefore, pAT 464 and pAT 744 are expected to exercise similar functions, in particular because the pAT 464 gene appears to represent the human homolog of the mouse MIP gene. Inflammation and wound healing/tissue repair are very complex proce ⁇ es which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells. This family of genes, which may have been derived from one common ancestral gene, may well serve to establish such a network.
  • the individual factors may possess multiple functions, as shown for several of the members of this family (see above) .
  • the conserved similarities between these pro ⁇ teins may indicate an interaction with conserved receptor elements.
  • the members of this family of protein ⁇ di ⁇ play a remarkable variety of ti ⁇ ue specificities.
  • pAT 464 and pAT 744 are expres ⁇ ed in T cell ⁇ and other hematopoietic cell ⁇ , but not in fibro- blasts.
  • the distinction ⁇ between the factor ⁇ may deter-: mine unique function ⁇ required during an immune reaction depending on the cell type activated.
  • CDM-8 or PMT2-2 mammalian expres ⁇ ion vector
  • this section has illu ⁇ trated novel antibodie ⁇ of thi ⁇ invention, i ⁇ ade again ⁇ t a peptide encoded by a * DNA segment of the invention and able to specifically bind to pAT 744 or pAT 464 lymphokine/cytokine-like protein which includes the sequence of such peptide.
  • MDNCF monocyte-derived neutrophil chemotactic factor
  • the carboxyi- terminal tridecapeptide of platelet factor 4 is a potent chemotactic agent for monocytes. Bio- chem. Biophys. Res. Comm. 107:130 IV-53. Hit-Rarper, J., H. Wohl, and E. Harper. 1978. Platelet Factor 4: An Inhibitor of Collagen- ase. Science 199:991

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Abstract

L'invention concerne des découvertes présentant des aspects particuliers de technologies d'ADN de recombinaison que l'on peut utiliser avec succès afin de produire des protéines lymphokinoïdes/cytokinoïdes humaines inconnues jusqu'à aujourd'hui, exemptes d'autres lymphokines ou cytokines. On peut produire ces protéines à partir de segments d'ADN dans des cellules ou des systèmes exempts de cellules sous diverses formes fonctionnelles. Ces formes permettent de procéder de manières diverses à des études biochimiques et fonctionnelles de ces nouvelles protéines, ainsi qu'à la production d'anticorps. L'invention concerne également des moyens permettant de déterminer le niveau d'activation de gènes contre les protéines lymphokinoïdes/cytokinoïdes, par exemple par mesure de l'ARNm pendant l'activation mitogénique de cellules T isolées, ou par mesure des antigènes sécrétés dans les liquides extracellulaires ou biologiques.
PCT/US1989/005603 1988-12-16 1989-12-15 Nouveaux genes de lymphokine/cytokine WO1990007009A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005198A1 (fr) * 1990-09-14 1992-04-02 Chiron Corporation Expression de proteines inductibles par macrophages (mip) dans des cellules de levure
EP0506574A1 (fr) * 1991-03-29 1992-09-30 Sanofi Protéine à activité de type cytokine, ADN recombinant codant pour cette protéine, cellules et microorganismes transformés
EP0544743A1 (fr) * 1990-08-13 1993-06-09 THE UNITED STATES OF AMERICA as represented by the Secretary UNITED STATES DEPARTMENT OF COMMERCE Lymphokine 154
FR2685919A1 (fr) * 1992-01-08 1993-07-09 Sanofi Elf Proteine a activite de type cytokine, adn recombinant codant pour cette proteine, cellules animales transformees.
WO1996039521A1 (fr) * 1995-06-06 1996-12-12 Human Genome Sciences, Inc. Chemokine beta-13 humaine
WO1997029125A1 (fr) * 1996-02-09 1997-08-14 Schering Corporation Reactifs constitues de chemokine de cellules dendritiques de mammiferes
US6015883A (en) * 1996-04-17 2000-01-18 Incyte Pharmaceuticals, Inc. Rantes homolog from prostate
US6495129B1 (en) 1994-03-08 2002-12-17 Human Genome Sciences, Inc. Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3)
US6623942B2 (en) 1994-03-08 2003-09-23 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polynucleotides
CN115028739A (zh) * 2017-08-03 2022-09-09 泰加生物工艺学公司 用于治疗黑素瘤的方法和组合物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5001230A (en) * 1988-02-18 1991-03-19 Schering Corporation T cell activation markers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Journal of Immunology, Volume 142 Number 2 issued January 1989; BROWN et al. "A family of small inducible proteins secreted by leukocytes are members of a small superfamily that includes leukocyte and fibrablast-derived inflamitory agents, growth factors, and indicators of various activation process" pages 679-687 see sequence on page 681. *
Journal of Immunology; Volume 142, Number 5 issued March 1989; ZIPFEL et al. "Mitogenicativation of Human T Cells induces two closely related genes share structural similarities with a new family of secreted factors" pages 1582-1590 see sequences on page 1585. *
Procedings National Acadamy Sciences; Volume 85 issued December 1988; LIPES et al. "Identification, cloning, and charaterization of an immune activation gene" pages 9704-9708 see sequence page 9706. *
See also references of EP0449956A4 *

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EP0544743A1 (fr) * 1990-08-13 1993-06-09 THE UNITED STATES OF AMERICA as represented by the Secretary UNITED STATES DEPARTMENT OF COMMERCE Lymphokine 154
EP0544743A4 (en) * 1990-08-13 1994-05-18 Us Health Lymphokine 154
US5814484A (en) * 1990-09-14 1998-09-29 Tekamp-Olson; Patricia Expression of macrophage inducible proteins (MIPS) in yeast cells
WO1992005198A1 (fr) * 1990-09-14 1992-04-02 Chiron Corporation Expression de proteines inductibles par macrophages (mip) dans des cellules de levure
WO1992017586A1 (fr) * 1991-03-29 1992-10-15 Elf Sanofi Proteine a activite de type cytokine, adn recombinant codant pour cette proteine, cellules et micro-organismes transformes
EP0506574A1 (fr) * 1991-03-29 1992-09-30 Sanofi Protéine à activité de type cytokine, ADN recombinant codant pour cette protéine, cellules et microorganismes transformés
US5652123A (en) * 1991-03-29 1997-07-29 Elf Sanofi Protein having interleukin 13 activity, recombinant DNA coding for this protein, transformed cells and microorganisms
FR2685919A1 (fr) * 1992-01-08 1993-07-09 Sanofi Elf Proteine a activite de type cytokine, adn recombinant codant pour cette proteine, cellules animales transformees.
US6495129B1 (en) 1994-03-08 2002-12-17 Human Genome Sciences, Inc. Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3)
US6623942B2 (en) 1994-03-08 2003-09-23 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polynucleotides
WO1996039521A1 (fr) * 1995-06-06 1996-12-12 Human Genome Sciences, Inc. Chemokine beta-13 humaine
AU713267B2 (en) * 1995-06-06 1999-11-25 Human Genome Sciences, Inc. Human chemokine beta-13
WO1997029125A1 (fr) * 1996-02-09 1997-08-14 Schering Corporation Reactifs constitues de chemokine de cellules dendritiques de mammiferes
US6015883A (en) * 1996-04-17 2000-01-18 Incyte Pharmaceuticals, Inc. Rantes homolog from prostate
CN115028739A (zh) * 2017-08-03 2022-09-09 泰加生物工艺学公司 用于治疗黑素瘤的方法和组合物

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EP0449956A4 (en) 1992-08-19
DK113491A (da) 1991-08-16
CA2005639A1 (fr) 1990-06-16
AU626369B2 (en) 1992-07-30
AU4816090A (en) 1990-07-10
DK113491D0 (da) 1991-06-13
JPH03504331A (ja) 1991-09-26
EP0449956A1 (fr) 1991-10-09
IL92685A0 (en) 1990-09-17

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