EP0631624A1 - Receptor complexes with b29 (ig-beta or ig-gamma) and their uses - Google Patents

Receptor complexes with b29 (ig-beta or ig-gamma) and their uses

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Publication number
EP0631624A1
EP0631624A1 EP93907136A EP93907136A EP0631624A1 EP 0631624 A1 EP0631624 A1 EP 0631624A1 EP 93907136 A EP93907136 A EP 93907136A EP 93907136 A EP93907136 A EP 93907136A EP 0631624 A1 EP0631624 A1 EP 0631624A1
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Prior art keywords
accordance
igm
cells
expression
lymphocytes
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German (de)
English (en)
French (fr)
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Michel C. Nussenzweig
Thais E. Costa
Roland R. Franke
Mercedes Sanchez
Ziva Misulovin
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Rockefeller University
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Rockefeller University
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    • 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
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus

Definitions

  • the invention described herein relates generally to cellular immunity and the recognition of antigen by cellular immune system components.
  • cellular components contain antigen-recognizing receptors which fall generally within the Ig supergene family.
  • the differences between such cellular components, e.g., T- and B-lymphocytes can be utilized in accordance with the teachings contained herein.
  • Humoral immune responses are generally initiated by the binding of antigen to the immunoglobulins present on the membranes of cells.
  • the antigen and immunoglobulin proteins may react in the presence of one or more non-immunoglobulin proteins, e.g., MB1, and B29, which are implicated in receptor assembly.
  • the antigen receptors of B- and T-lymphocytes are members of the Ig supergene family whose specificity is determined by a series of genomic rearrangements.
  • the ligand binding domains of the receptors present on these cells are defined by the combination of independently encoded polypeptides that are linked together by disulfide bonds.
  • both receptors are associated with a number of other proteins on the cell surface.
  • T-lymphocytes are reactive to compounds which are complexed or presented by Antigen Presenting Cells (APC) and/or Major Histocompatibility Complex (MHC) .
  • APC Antigen Presenting Cells
  • MHC Major Histocompatibility Complex
  • MHC may trigger antigen recognition by the T-lymphocytes.
  • MHC is comprised of numerous molecules which tag and "activate" the antigen, causing recognition by the T-cell receptors, binding and ultimately internalization of the antigen by the cell.
  • the T cell receptor is comprised of at least seven different polypeptides that include the antigen specific alpha and beta chains, the zeta chain, which is a signaling molecule, and the gamma, delta and epsilon chains, which form a second signaling component. All are required for efficient assembly and surface transport (Weiss and Stobo, 1984; Ohashi et al., 1985; Berkhout et al. , 1988; Suss an et al. , 1988).
  • Comparable proteins to the T-cell receptor associated proteins in B-cells include MB1 and B29, also known as IgMa and IgMb respectively (Hermanson et al., 1988; Hombach et al., 1988; Sakaguchi et al., 1988; Hombach et al. , 1990; Campbell et al., 1991).
  • MB1 and B29 form a disulfide linked heterodimer that is associated with IgM (Hombach et al., 1990), and both proteins share a negatively charged intracytoplasmic sequence motif with CD3 g, d and z (Reth, 1989) .
  • MB1 and B29 appear to be required for transport of IgM to the plasma membrane of transfected fibroblasts and B-cells (Hombach et al. 1990; Venkitaraman et al. 1991) .
  • Crosslinking of the T-cell receptor leads to CD45-dependent activation of protein kinases, increased phosphoinositol turnover and calcium mobilization.
  • a very similar set of events is induced by the crosslinking of IgM on the surface of B-cells; however, the functional role of the IgM associated proteins has to date been inadequately defined.
  • T-lymphocyte receptor reactivity it has long been desired to modify T-lymphocyte receptor reactivity to eliminate the need for MHC and/or APCs.
  • one object of the present invention is therefore to define the role of the IgM-associated non- immunoglobulin proteins.
  • Another object of the present invention is to provide a method of modifying T-lymphocyte immunity to render such immunity essentially MHC non-dependent.
  • Yet another object of the present invention is to provide methods of assessing T- and B-lymphocyte and immune system activity, including, e.g., antigen recognition, MHC complexation or reactivity, B-cell surface IgM activity and other factors.
  • a method of modifying cellular immunity comprising transfecting a T-lymphocyte with B-cell derived immunoglobulin and B29.
  • transfected T-lymphocytes are included herein, as are various methods of use. Also included are mast cells and macrophage cells transfected with the B-cell derived immunoglobulin and B29.
  • DNA and RNA constructs described herein as well as antibody constructs which recognize such molecules, the immunoglobulins present on the surface of the T- lymphocytes and non-Ig proteins.
  • FIGURE 1 DNA constructs, and cell surface expression of human immunoglobulin
  • A Maps of immunoglobulin, B29 and MB1 expression vectors.
  • the Spleen Focus Forming Virus-Long Term Repeat (SFFV LTR) promoter was used in all constructs for T cell expression.
  • Human growth hormone (hGH) introns and polyadenylation signals were added to both B29 and MB1 cDNAs, and a histidinol resistance gene was included in the MB1 plasmid.
  • a phosphorylcholine (PC) specific mouse variable region from S107 was combined with a human heavy chain constant region engineered to be synthesized only in the membrane bound form.
  • the light chain was composed of an S107 kappa variable region and the human kappa constant region.
  • the heavy chain and light chain were on the same plasmid as a neomycin resistance gene.
  • (B) Flow cytometric analysis of surface expression of human IgM on transfected Jurkat cells. The relative cell number is plotted against fluorescence intensity on a logarithmic scale. The constructs transfected, cell line and the number of times the cell line was enriched by sorting (SX_) are indicated at the top of each panel. "Control” indicates unstained cells, and GAHIGM indicates staining with FITC conjugated goat anti-human IgM.
  • SFFV Spleen focus forming virus long terminal repeat
  • B29 B29 cDNA
  • MB1 MB1 cDNA
  • hGH human growth hormone splice and polyadenylation signals
  • His histidinol resistance gene
  • mlgM phosphorylcholine specific, membrane bound form of human IgM heavy chain
  • kappa phosphorylcholine specific kappa light chain
  • Neo neomycin resistance gene
  • # cell line number
  • SX_ the number of times a cell line was enriched for surface IgM by sorting
  • p467 B29 expression vector
  • p474 MB1 expression vector
  • p468 IgM expression vector.
  • FIGURE 2 Expression of IgM protein, B29 and MB1 mRNAs
  • FIGURE 3 Affinity purification of surface iodinated proteins from transfected cell lines
  • Phosphorylcholine binding immunoglobulins and associated polypeptides were affinity purified from 1% digitonin lysates of surface iodinated cells.
  • the cell types. constructs transfected and cell line number are indicated at the top of each lane, the same as in FIGURE 2A; m and k indicate the position of the heavy and light chains determined from purified standards; Kd: kilodaltons.
  • FIGURE 4 Calcium flux assays of transfected Jurkat cells
  • Cells loaded with Fura-2 were assayed fluorimetrically for calcium mobilization in response to 1.5 ug/ml anti-CD3, 10 ug/ml anti-human IgM, 10 ug/ml isotype control monoclonal antibody or 500 ng/ml of phosphorylcholine coupled to bovine serum albumin (PC- BSA) .
  • the cell types and constructs transfected are indicated at the left and the reagents added are indicated at the top.
  • IgGl isotype control monoclonal antibody
  • a-CD3 anti-CD3 antibody
  • a-IgM anti-IgM antibody
  • PC-BSA phosphorylcholine coupled to bovine serum albumin
  • Jurkat untransfected Jurkat cells
  • IgM Jurkat cells transfected with IgM #3 (FIGURE IB);
  • IgM+B29 IgGl+B29:
  • IgM+B29+MBl Jurkat cells transfected with IgM+B29+MBl #9 (FIGURE IB) ; time and [Ca +2 ] are indicated at the bottom right.
  • FIGURE 5 Generation of inositol triphosphates (IP3) by transfected Jurkat cells
  • IP3 inositol triphosphate
  • FIGURE 6 IL-2 secretion in response to anti-IgM by transfected Jurkat cells
  • PMA phorbol-myristate acetate
  • IgM+B29 Jurkat cells transfected with IgM+B29 #5 (FIGURE IB); other symbols as in FIGURE 4.
  • FIGURE 7 IL-2 secretion in response to phosphorylcholine by transfected Jurkat cells
  • BSA bovine serum albumin
  • IgM+B29 Jurkat cells transfected with IgM+B29 #5 (FIGURE IB); other symbols as in FIGURE 4.
  • FIGURE 8 Dose response to PC-BSA and blocking by monomeric phosphorylcholine in transfected Jurkat cells
  • FIGURE 9 Assays of alternative cell lines transfected with IgM, B29 and MB1.
  • FIGURE 10 Assays of Mast cells transfected with Igm, B29 and MB1.
  • A Stimulation of Mast cells P815 stimulated with anti-IgM.
  • B Stimulation of Mast cells P815 transfected with IgM, B29 and MB1, and stimulated with phosphorylcholine- BSA.
  • This invention relates to the receptors and related molecules on the surface of humoral immune system cellular components, e.g., T- and B-lymphocytes, DNA molecules and derivatives thereof, e.g., mRNA, which code on expression for these receptor compounds, sense and anti-sense RNA molecules which likewise code for such molecules or for other molecules, antibodies which recognize such proteins and nucleic acid derivatives, antibodies that recognize B-cell receptors and receptor related molecules individually and in combinations such as complexes, as well as anti-idiotype antibodies which recognize both these antibodies and ligands, including agonists, antagonists and the like to these B-cell receptor and receptor related molecules. Numerous methods of production and use are also included.
  • humoral immune system cellular components e.g., T- and B-lymphocytes
  • DNA molecules and derivatives thereof e.g., mRNA, which code on expression for these receptor compounds, sense and anti-sense RNA molecules which likewise code for such molecules or for other molecules
  • antibodies which recognize such proteins and nucleic
  • a preferred aspect of the invention is drawn to a method of modifying T-lymphocytes to render said cells substantially non-dependent upon the presence or activity of MHC.
  • Another preferred aspect of the invention is drawn to a method of modifying mast cells or macrophage cells to render said cells active in recognizing antigen in the presence or absence of MHC.
  • Another preferred embodiment of the invention relates to the DNA constructs described herein and in the plasmids incorporating such constructs.
  • Another preferred embodiment of the invention relates to a unicellular host transfected with the plasmids described herein.
  • Another preferred embodiment of the invention relates to the antibodies which are described herein, including polyclonal, monoclonal and chimeric antibodies.
  • One preferred chimeric antibody contains an immunoglobulin protein or fragment in combination with a B29 protein or fragment of B29.
  • Another preferred chimeric antibody contains an immunoglobulin molecule or fragment and a protein molecule or fragment other than B29, such as those described in NATURE, Vol. 338:383 (March 30, 1989). Many of these chimeric antibodies will contain the consensus amino acid sequence noted therein.
  • Expression control sequence a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • a DNA sequence is operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) upstream (in front of) the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • start signal e.g., ATG
  • Antibody an immunoglobulin molecule or functional fragment thereof, such as FAb, F(ab / ) 2 or dAb.
  • An antibody preparation is reactive for a particular antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen.
  • An antibody preparation is non-reactive for an antigen when binding of the individual immunoglobulin molecules in the preparation to the antigen is not detectable by commonly used methods.
  • the fragments of the antibodies described herein include the phosphorylcholine specific mouse variable region from S107, the human heavy chain constant region, a derivative of the human heavy chain constant region in membrane bound form, and the light chain S107 kappa variable region, alone and operatively linked to a human kappa constant region.
  • chimeric bodies are likewise included.
  • Standard hybridization conditions salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash.
  • DNA sequence polynucleotide sequences prepared or isolated using recombinant DNA techniques. These include cDNA sequences, DNA sequences isolated from their native genome and synthetic DNA sequences. The term as used in the claims is not intended to include naturally occurring DNA sequences as the exist in nature.
  • Receptor and receptor complex includes both the singular and plural, and contemplates the existence of one or more structures comprised of the protein(s) which make up the ligand recognition site.
  • one or more proteins may be inv- ved, as well as one or more compounds not directly it Ived in ligand recognition, e.g., MB1, B29 and other .
  • Expression of recombinant molecules as used herein may involve the post-translational modification of a resultant polypeptide coded by the sequence present in the host cell.
  • expression might include, among other things, the production of an mRNA molecule or a polypeptide, glycosylation, lipidation or phosphorylation of the polypeptide, or cleavage of a signal sequence to produce a "mature" protein.
  • polypeptide encompasses full-length polypeptides, fragments of mature proteins and modifications or derivatives thereof, such as glycosylated versions of such polypeptides, polypeptides retaining a signal peptide, truncated polypeptides having comparable biological activity and the like.
  • expression of a "derivative" of the sequence may involve the production of an intermediate molecule, which is generated in during the expression of the protein. Typically, this involves the expression of mRNA which likewise codes for the particular polypeptide to be ultimately expressed. In this instance, the mRNA molecule is deemed to be a derivative of the DNA coding sequence contained in the particular expression vector.
  • Signal and signal transduction refer to changes which occur in the cell in response to crosslinking (binding) of the receptor by reaction with a ligand. Examples of such cellular changes include initiation of a cascade of enzyme reactions, rapid increases in calcium flux, increase in phosphoinositol (IP3) turnover and secretion of lymphokines from lymphocytes.
  • IP3 phosphoinositol
  • the immunoglobulin antigen receptor in T- lymphocytes was reconstituted by transfection of cloned components derived from B-lymphocytes.
  • the transport of IgM to the surface of T-cells required the co-expression of the immunoglobulin heavy and light chains with B29.
  • the transfected receptor was fully active in the presence of B29.
  • MB1 a second IgM associated polypeptide, was not required for either transport or signal transduction.
  • Immunoglobulin receptor function was "reconstituted” by transfection of the B-cell cloned receptor components into human T-lymphocytes, and it was noted that the transport of IgM to the surface of the T-cells ("host") required the co-expression of B29. Furthermore, the expression of IgM and B29 in combination was sufficient to reconstitute antigen specific signal transduction by the expressed immunoglobulin in the transfected T-cells. This established a functional requirement for B29, and suggests that the signaling apparatus of T- and B-cells is structurally homologous.
  • the light chain was composed of the corresponding kappa variable region (Kwan et al., 1981) , coupled with a human kappa constant region gene (Hieter et al., 1982).
  • hGH Human growth hormone
  • splice consensus sequences (Saito et al., 1987) were added to mouse MB1 and B29 cDNAs, and a histidinol resistance gene was included as a second drug resistance marker in the MB1 expression vector (Hartman and Mulligan, 1988) .
  • IgM expression was readily detected on the surface of Jurkat cells transfected with IgM and B29, or a combination of IgM, MB1 and B29. In contrast, it was difficult to detect IgM on Jurkat cells transfected with the heavy and light chain alone, or a combination of IgM and MBl. In all cases we enriched for surface IgM positive cells by selection with a fluorescence activated cell sorter (FIGURE IB) . High levels of surface IgM expression were achieved after 1-2 rounds of selection of cell lines transfected with either IgM and B29, or IgM, B29 and MBl. However, eight rounds of sorting were required for cell lines transfected with IgM alone, and the combination IgM and MBl was always negative (FIGURE IB) .
  • FOGURE IB fluorescence activated cell sorter
  • Transfected IgM is associated with B29 and MBl
  • B29 and MBl were associated with IgM
  • a polypeptide with the appropriate electrophoretic mobility for B29 (44 Kd) was co-purified with immunoglobulin heavy and light chains from Jurkat cells transfected with IgM and B29.
  • the same polypeptide and three additional species were co-purified from the IgM, B29 and MBl transfectants (Fig 3).
  • the 32Kd band was consistent with MBl.
  • the additional bands at 52Kd and 39Kd may be alternate forms of B29 and MBl, or T-cell encoded proteins.
  • the function of the transfected immunoglobulin was initially assessed by measurement of calcium flux in response to receptor crosslinking.
  • Fura-2 loaded cell lines were treated with a monoclonal anti-human IgM antibody, a monoclonal anti-CD3, or an isotype matched monoclonal antibody control.
  • Jurkat cells transfected with either IgM and B29, or IgM, B29 and MBl responded to anti-IgM crosslinking with a rapid increase in free intracellular calcium (FIGURE 4) .
  • Untransfected Jurkat cells, and Jurkat cells that expressed high levels of IgM alone did not respond to anti-IgM crosslinking, but were fully competent to respond to anti-CD3 (FIGURE 4) .
  • the isotype control IgGl antibody had no effect (FIGURE 4)
  • polyclonal goat anti-IgM antibodies had the same ef ect as monoclonal anti-IgM (not shown) .
  • Inositol phosphate turnover is another measure of signal transduction by immunoglobulin.
  • inositol turnover was measured in response to anti-IgM antibody (FIGURE 5) .
  • IgM, B29 and MBl resulted in an increase in cellular inositol-3- phosphate (IP3) .
  • anti-IgM antibody had no effect on untransfected Jurkat cells (FIGURE 5) .
  • the combination of immunoglobulin and B29 expressed on the surface of T-cells was fully functional, and activated intracellular calcium mobilization and phosphoinositol turnover.
  • IL-2 production was measured in response to anti-IgM (FIGURE 6) .
  • Jurkat cells that expressed either IgM and B29, or IgM, B29 and MBl secreted IL-2 in response to anti-IgM treatment. This response was specific; untransfected Jurkat cells did not respond to anti-IgM.
  • the same control cell line was competent to produce IL-2 in response to anti-CD3 stimulation. None of the lines were induced with the isotype control monoclonal antibody (FIGURE 6) .
  • T-lymphocytes respond to processed peptides associated with MHC on the surface of other cells, whereas B-cells respond to native antigens.
  • transfected Jurkat cells were challenged with phosphorylcholine coupled to bovine serum albumin (PC-albumin) (Figs. 4, and 7).
  • PC-albumin bovine serum albumin
  • the challenge with PC-albumin induced calcium flux and IL-2 secretion in the T-cell lines that expressed B29 in addition to IgM, whereas controls with albumin alone were negative.
  • T-cells that expressed surface IgM alone failed to respond (FIGURE 4) .
  • MBl and B29 are important for the transport of immunoglobulin to the surface of B-cells and fibroblasts.
  • B-cell lines that lack MBl did not express surface IgM, and this phenotype can be restored by transfection of cloned MBl (Hombach et al., 1990).
  • MBl is required for receptor assembly in B-cells.
  • both MBl and B29 are required for surface expression of IgM.
  • other immunoglobulin isotypes can be expressed on the surface of fibroblasts with B29 even in the absence of MBl (Venkitaraman et al., 1991).
  • IgM receptor structure proposes that IgM interacts with a pair of MBl and B29 heterodimers.
  • MBl and B29 are disulfide linked and interact with IgM in part via polar amino acids in the transmembrane domain.
  • MBl was not required for either surface expression or. function of IgM antigen receptors on transfected T-cells.
  • the B-cell derived IgM receptor can interact with the T- cell signal transduction apparatus. There are many similarities between the signaling pathways of these two receptors as well as several significant differences. For example, much experimental evidence points to activation of tyrosine kinases as one of the first steps in the signal transduction pathways of the TCR (reviewed by (Klausner and Samelson, 1991)), and (Campbell and
  • IgM and B29 could overcome the differences between the signal transduction apparatus of T- and B-cells.
  • these molecules could interact directly with and activate one of the many T-cell protein tyrosine kinases by forming a complex with structural similarity to CD3 z or e.
  • Crosslinking of the isolated z or e chains is sufficient to induce signaling in transfected T-cells (Irving and Weiss, 1991; Romeo and Seed, 1991; Letourneur and
  • a second mechanism which explains signal transduction by IgM and B29 in T-cells involves the direct association of IgM with T-cell encoded proteins that in turn make the appropriate cellular connections.
  • the CD3 components are particularly appealing candidates for this function since they are both structurally and functionally related to B29 and MBl (Reth, 1989) .
  • T-cell receptor Another major difference between the T-cell receptor and immunoglobulin antigen receptors is the nature of the antigen recognized by the two receptors. Immunoglobulins recognize antigens directly, whereas recognition of antigen by the TCR requires the presence of MHC. The requirement for MHC in conjunction with or prior to T- cell recognition places severe limitations on the targets recognized by T-cells, and makes transfer of cellular immunity MHC restricted. For this reason, there has been considerable interest in modifying T-cell recognition to abrogate the MHC requirement.
  • T-cells that express functional immunoglobulin antigen receptors on the cell surface have the potential for recognizing any antigen recognized by antibodies in an MHC independent fashion.
  • immunoglobulin IgM and the B cell derived receptor molecules, MBl and B29 were transfected into cloned mast cells and macrophages. These constructs were then stimulated with antigen, phosphorylcholine-BSA (PC-BSA) or anti-human IgM antibodies. The results are shown in Figures 9 and 10. The ability to produce a functional receptor by transfection in these other cells indicates that there is structural and functional similarity in signal transduction between these various cell types. Hence, these other cell lines can be used to functionally reconstitute the T-cell receptor from cloned components.
  • PC-BSA phosphorylcholine-BSA
  • DNA sequences and constructs can be made as described in the Example which code for the B-cell derived immunoglobulin and related proteins. Such constructs can be inserted into host organisms to induce expression of these proteins. From the DNA sequences, one can readily ascertain the amino acid sequences of these B-cell derived proteins. IgM, B29, MBl and other related proteins, such as the isolated antibody polypeptide chains. This has been accomplished, and it also may be possible to make substantial alterations in the polypeptide sequence, including insertions as well as deletions, to obtain a variety of molecules which have substantially the same biological activity or immunological activity profile.
  • This approach may utilize antisense nucleic acids and ribozymes to block translation of specific mRNA, either by masking mRNA with an antisense molecule or cleaving it with a ribozy e. In this manner, coding and expression of these proteins can be inhibited.
  • Antisense DNA and RNA molecules are complementary to at least a portion of specific mRNA molecules. They may hybridize to form double stranded mRNA. The cell does not translate mRNA in this double stranded form, thus interfering with expression of the mRNA coded protein. Such anti-sense methods have been used to inhibit gene expression in vitro.
  • Ribozymes are RNA molecules which have the ability to cleave other single stranded RNA molecules, roughly analogous to DNA restriction endonuclease. Ribozymes were discovered in RNA molecules which excise their own introns. By modifying these RNA molecules, it is possible to produce specific nucleotide sequences which are recognized and cleaved. Because they are sequence specific, only particular mRNAs such as those which code for the B-cell derived proteins would be inactivated. Another feature of the invention involves the expression of the DNA sequences for B-cell derived IgM, MBl and B29. DNA sequences may be expressed by operatively linking them to an expression control sequence, inserting into an appropriate expression vector and employing that expression vector to transform an appropriate host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, e.g., ATG, in the correct reading frame upstream of the coding DNA sequence.
  • expression vector/host combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAs e.g., the numerous derivatives of phage ⁇ , e.g., NM989 and other phage DNA, e.g., M13 and Filamentous single stranded
  • hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli , Pseudomonas, Bacillus, Streptomyces , fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, BSC1, BSC30 and BMT10) , insect cells (e.g., Sf9) , and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli , Pseudomonas, Bacillus, Streptomyces , fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, BSC1, BSC30 and BMT10) , insect cells (e.g., Sf9) , and human cells and plant cells in tissue culture.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, the toxicity to the host of the product encoded by the DNA sequence(s) to be expressed, and the ease of purification of the expression products.
  • the cDNA can be inserted into the appropriate vector, such as the eukaryotic expression vector pCDM ⁇ .
  • This plasmid has certain advantages, including a high copy number in E.coli, a eukaryotic promoter and a high level of expression.
  • other vector expression systems can be used.
  • clones can be isolated which contain cDNA sequences coding for B-cell derived IgM, B29, MBl and other molecules.
  • cDNA sequences coding for B-cell derived IgM, B29, MBl and other molecules.
  • a variety of expression techniques can likewise be used. For example, antibody screening of proteins encoded by cDNA cloned into the vector, activity assays of conditioned media after injection of RNA from cloned cDNA or plasmid/phage carrying promoter or other indicator sequences may be evaluated.
  • Transfection can also be accomplished by a variety of methods. For example, spheroblast fusion, DEAE dextran and electroporation can be used, primarily for transient expression. Stable expression typically utilizes calcium phosphate, spheroblast fusion and electroporation.
  • Partial DNA sequences corresponding to the partial amino acid sequences of the proteins of the present invention or a portion thereof, or a degenerate variant of such partial DNA sequences may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species, such as humans.
  • the present invention thus extends to the probes so prepared that may be provided for screening cDNA and genomic libraries for clones that may correspond to genes expressing the respective proteins.
  • the probes may be prepared with a variety of known vectors.
  • the present invention also includes the preparation of the plasmids including such vectors.
  • the receptor proteins are ideally prepared by the recombinant techniques described herein, isolation and purification from cells known to bear or produce the receptor proteins, such as B-lymphocytes.
  • the cells or active fragments likely to participate in receptor protein synthesis or to have receptor protein associated therewith may be subjected to a series of isolation techniques, such as for example elution of detergent- solubilized proteins from an affinity matrix, whereupon the present receptor proteins may be recovered.
  • isolation techniques such as for example elution of detergent- solubilized proteins from an affinity matrix
  • the present invention also extends to antibodies including polyclonal, monoclonal and chimeric, which are comprised of or recognize the IgM receptor and the non- immunoglobulin proteins described herein.
  • these antibodies may find use in a variety of research, diagnostic and therapeutic applications.
  • the antibodies can be used to screen expression libraries to obtain the gene that encodes either the receptor complex or non-Ig proteins.
  • those antibodies that neutralize receptor component activity can be employed in intact animals to better elucidate the biological role that the receptor and the related proteins play.
  • Such antibodies can also participate in drug screening assays to identify drugs or other agents that may exhibit the same or a contrary activity as the proteins.
  • Polyclonal, monoclonal and chimeric antibodies to the receptor and components thereof are thus contemplated.- These molecules are capable of preparation by known techniques such as the hybridoma technique, utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Naturally, these antibodies are merely illustrative of antibody preparations that may be made in accordance with the present invention.
  • the chimeric molecules may contain an immunoglobulin protein or a fragment of an immunoglobulin protein in combination with the polypeptide molecule B29.
  • the chimeric molecules may also include immunoglobulins and immunoglobulin fragments as mentioned above, combined with other polypeptides and polypeptide fragments which are active as described herein.
  • such other polypeptides may include the following:
  • IgE type receptor proteins (Fcl Rl) on mast cells CD3- gamma- ⁇ " and zeta component proteins of T- cells: other polypeptides where two amino acids are negatively charged aspartic or glutamic acid groups; the third and fifth amino acids are tyrosine, and the fourth and sixth are leucine or isoleucine.
  • chimeric molecules contained herein therefore include molecules and molecular fragments selected from the following compounds: hCD3-gamma, mCD3- S, hCD3- ⁇ , mCD3-rS, mCD3-zeta, BLVGP 30, hMBl, MBl, mB29, pFC j I-f-T and rFC.RI-B.
  • the B-cell derived receptor and related proteins may be prepared alone on in operative association with other molecules or pharmaceutical agents in a form suitable for administration for either diagnostic or therapeutic purposes.
  • the invention thus includes both diagnostic and pharmaceutical compositions including the receptor complex and/or the component proteins, individually and in combination with other diagnostic reagents in the former instance, and in combination with pharmaceutically acceptable carriers, and possibly, other therapeutic agents where coadministration is deemed appropriate or desirable.
  • the receptor Ig protein and related proteins possess significant diagnostic and therapeutic capabilities in connection with conditions involving the activity of T- and B-lymphocytes and other humoral immune disease, e.g., autoimmune disease, viral, e.g., HIV and the like, mycobacterial infection, etc.
  • diagnostic and therapeutic utilities are predicated on the structures and activities of the receptor and the related proteins. Diagnostic utilities include assays such as immunoassays with labeled quantities of the receptor, non-immunoglobulin proteins, antibodies, ligand and binding partners thereto, receptor assays, and drug screening assays to evaluate new drugs for the ability to promote or inhibit receptor or non- receptor protein production or activity.
  • the above assays can also be used to detect the presence or activity of the receptor or non-receptor proteins vis-a ⁇ vis invasive stimuli, pathology or injury, the presence or absence of which affect such receptor or non-receptor protein production or activity.
  • Therapeutic methods and pharmaceutical compositions are based upon the receptor and related proteins, and the vectors described above, which contain sequences coding for materials having the same or antagonistic activity thereto. Therapeutic methods are generally based on the promotion or inhibition of the activities of the receptor and non-receptor proteins, and thus extends to the treatment of disease or dysfunction attributable to the excess activity or the absence of such activity.
  • compositions described herein include effective amounts of the receptor molecules and non- immunoglobulin proteins, their agonists, antagonists, antibodies or like drugs, in combination with pharmaceutically acceptable carriers.
  • Carriers include the pharmacologically inactive ingredients or components used in dosage formulations, e.g., water for injection, tablet adjuvants, capsule fillers and the like.
  • Such compositions can be prepared for a variety of protocols, including where appropriate, oral and/or parenteral administration. Exact dosages and dosing schedules would be determined by the skilled physician. Diagnostic and research applications extend generally to the proteins, immunoglobulin as well as non- immunoglobulin, involved in receptor reactivity, and the difference in such reactivity between B- and T-lymphocyte receptors.
  • One preferred diagnostic application generally extends to a method for the assessment of immune function.
  • Assays of immune function in animals including body fluids such as blood, plasma and urine, tissue samples, and biomolecules such as DNA, will assist in the detection and evaluation of pathology or other systemic dysfunction.
  • the diagnostic and research applications include the performance of several competitive assay protocols, involving the analyte, a ligand and one or more binding partners of interest, where the binding partners are typically selected from the present receptor immunoglobulin and/or non-immunoglobulin proteins.
  • the binding partners may be generally selected from the group consisting of cells and cellular components having the present receptor, the non-immunoglobulin proteins and other cell proteins.
  • the ligands useful in these applications are generally molecules which are recognized by B-cell receptor IgM proteins. These ligands may be detected either alone or in combination with a second detecting partner such as avidin.
  • Standard assays based on the cell components or proteins themselves which employ extract formats may be used. Each assay is capable of being based on enzyme linked and/or radiolabeled ligands and their binding partners, including the Igm-type receptors and non-receptor proteins disclosed herein.
  • the broad format of the assay protocols which are possible within the present invention extends to assays wherein no label is needed for detection.
  • one of the formats contemplates the use of a bound protein-specific receptor. In such instance, the analyte would need only to be added to the receptor, and the bound analyte could then be easily detected by a change in the property of the binding partner, such as by a change in the receptor.
  • assays contemplated herein include assays such as those set forth in the Example, where a specific indicator sequence is contained in the DNA construct. Expression of the protein is thus detected.
  • the assays of the invention may follow formats wherein either the ligand or the binding partner, be it a receptor or an antibody, are bound.
  • the assays include the use of labels which may be selected from radioactive elements, enzymes and chemicals that fluoresce.
  • the diagnostic applications also include methods for the assessment of immune and other disorders in animals, including humans.
  • the methods comprise assays involving in addition to the analyte, one or more binding partners of the advanced glycosylation endproducts, and one or more ligands.
  • These diagnostic assay methods broadly comprise the steps of:
  • Suitable analytes are typically selected from blood, plasma, urine, cerebrospinal fluid, lymphatic fluid and tissue. Also, the analyte is typically assessed for immune system function.
  • the present receptor immunoglobulin protein, a non-Ig protein or a cellular material bearing such proteins is combined with the analyte and the ligand, and the binding activity of either or both of ligand and analyte to the receptor may then be measured to determine the extent of binding. In this way, the differences in affinity between the components in the assay serve to characterize reactivity.
  • a control quantity of a binding partner to the Igm on non-Igm proteins may be prepared and optionally labeled, such as with an enzyme, a compound that fluoresces and/or a radioactive element, and may then be introduced into a tissue or fluid sample of a mammal. After the labeled material or its binding partner(s) has had an opportunity to react with the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label.
  • immunological assays which utilize either a binding partner to the IgM or ligand thereto, optionally labeled with a detectable label, and further optionally including an antibody Ab, labeled with a detectable label, an antibody Abj labeled with a detectable label, or a chemical conjugate with a binding partner to the protein labeled with a detectable label.
  • the procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled. "BP" in this instance stands for all binding partners of the compound under examination:
  • a further alternate diagnostic procedure employs multiple labeled compounds in a single solution for simultaneous radioimmuno assay.
  • a composition may be prepared with two or more analytes in a coordinated compound having the formula radioisotope-chelator- analyte.
  • IgM or another protein can be used to form complexes with one or more binding partners, and one member of the complex may be labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by the known applicable detection methods.
  • One assay format contemplates a bound receptor to which are added the ligand and the analyte.
  • the receptor can be a compound which binds to the analyte, such as a capture antibody.
  • the resulting substrate is then washed after which detection proceeds by the measurement of the amount of ligand bound to the receptor.
  • a second format employs bound ligand to which the receptor and the analyte are added. Both of the first two formats are based on a competitive reaction with the analyte, while a third format comprises a direct binding reaction between the analyte and bound receptors.
  • bound receptor-specific carrier or substrate is used. The analyte is first added after which the receptor is added, the substrate washed, and the amount of receptor bound to the substrate is measured.
  • the present invention includes the following protocol within its scope:
  • a method for determining immune system function comprising:
  • Abj a characteristic property of Abj is that it will react with Ab,.
  • Ab raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens.
  • Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab is referred to as the primary antibody and Abj is referred to as a secondary or anti-Ab, antibody.
  • the labels most commonly employed the various procedures described herein are radioactive elements, enzymes, chemicals with fluoresce when exposed to ultraviolet light, and other such labels.
  • Suitable radioactive elements may be selected from the group consisting of 3 H, 14 C, 32P, 35 S, 36 C1, 51 Cr, "Co, 58 Co, 59 Fe, 90 Y, I25 I, I31 I, and 186 Re.
  • a radioactive label such is prepared with one of the above isotopes is used, known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro- photometric, thermometric, amperometric or gasometric techniques known in the art.
  • the enzyme may be conjugated to a protein of interest, binding partners or carrier molecules by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • indicator enzymes which can be used in these procedures are known and can be utilized.
  • the preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D- galactosidase, urease, glucose, oxidase plus peroxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkaline phosphatase, NAD oxidoreductase plus luciferase, phosphofructokinase plus phosphoenol pyruvate carboxylase, aspartate aminotransferase plus phosphoenol pyruvate decarboxylase, and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternative labeling material and methods.
  • fluorescent materials are also known and an be utilized as labels. These include, for example, fluorescein, rhodamine and auramine.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the assays described above may be prepared or used in the form of test kits.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to a binding partner, such as the IgM protein, a receptor or ligand as listed herein; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of- binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .
  • a binding partner such as the IgM protein, a receptor or ligand as listed herein
  • additional immunochemical reagents at least one of which is a free or immobilized ligand, capable either of- binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .
  • kits suitable for use by a medical specialist may also be prepared.
  • one class of such kits contains at least a labeled binding partner as state above, and directions, of course, depending upon the method selected, e.g. "competitive”, “sandwich”, “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • the corresponding diagnostic test kit may comprise:
  • test kit comprises; (a) a labeled component which has been obtained by coupling the above binding partner to a detectable label;
  • a ligand capable of binding with at least one of the binding partners of at least one of the components to be determined; and (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the compound presently assessed and the binding partner.
  • T-lymphocytes taken from a patient can be modified as described herein and reinfused into the patient to restore T-cell function in an immunocompromised patient.
  • a therapy can be designed to improve T- cell function in the patient in need of such treatment.
  • T-lymphocytes are the effectors of a large number of cellular immune responses, including but not limited to tumor rejection and protection from infection (viral, parasitic and mycobacterial) such diseases can be treated in accordance with the teachings contained herein.
  • the nature of the antigens normally recognized by these cells is tailored to the particular individual, because T- lymphocytes recognize a combination of antigen plus cell surface MHC molecule(s) .
  • MHC is highly polymorphic in humans, naturally occurring T-cells that recognize any of the above mentioned challenges are not useful if transferred between individuals; the T-cells from one individual are unable to recognize antigen in other individuals because the endogenous MHC is not perceived by the T-lymphocytes which are infused.
  • T- cells from individual Y only recognize tumor cells in individual Y. When transferred to individual X, the T- " cells do not recognize tumor cells because of the differences in MHC between individuals X and Y. This is the case even between like tumor types.
  • T-cells in accordance herewith can be transfected with monoclonal antibodies which recognize a given antigen.
  • two individuals with the same tumor or tumor type can be treated by administering to each an effective amount of the T-cells described above, which recognize the particular tumor or tumor type.
  • This overcomes or circumvents the differences in MHC between individuals.
  • specific antibody genes to target the T-cells to specific antigens in different individuals the same composition can be used in all patients with a given disease.
  • T-lymphocytes as described herein with B-cell derived IgM and B29, and a monoclonal antibody which recognizes and binds to a specific antigen can impart recognition of the antigen to T-cells without the presence or activity of MHC.
  • Specific monoclonal antibodies now used to target radiolabeled compounds or toxins to tumors can be included as donors of the antibody specificity to be transferred into T-cells.
  • T-cells described above, and the infusion thereof into patients can also used to produce a T-cell line having the desired non-MHC specificity for purposes of secreting selected lymphokines and delivering such lymphokines to specific sites of activity within the mammal.
  • T-cells with this specificity can then be used to deliver normal T- cell lymphokines, such as IL-2, IL-4 and interferons to the specific areas of involvement.
  • IL-2, IL-4 and interferons normal T- cell lymphokines, such as IL-2, IL-4 and interferons to the specific areas of involvement.
  • T-cells themselves are cytotoxic and kill tumors directly.
  • T-cells play a role in combating infection.
  • One potential target is HIV.
  • T-cells can be engineered to be specific for the virus by transferring antibodies that are specific for the portion of gpl20 that binds to CD4.
  • These recipient T-cells would preferably have the CD4 protein component thereof deleted, such as by a gene knockout technique so that these modified cells would not in and of themselves be susceptible to infection.
  • mycobacterial organisms which are at least in part resistant to chemotherapeutic agents.
  • mycobacterial organisms which are at least in part resistant to chemotherapeutic agents. Examples include the tuberculosis and leprosy mycobacteria. Monoclonal antibodies specific for these mycobacteria could be transfected into T-cells, which are the key mediators for successful immune responses in these diseases. The T-cells would then be introduced into the patient, to in turn deliver the required lymphokines and the T-cell cytotoxicity at the site of infection.
  • the Spleen Focus Forming Virus, Long Term Repeat (SFFV LTR) promoter from pFNeo (Saito et al. , 1987) was combined with the BamHl to EcoRl fragment of the human growth hormone (hGH) to produce an SFFV cDNA expression vector (p463) .
  • the hGH sequences and polyadenylation signals were added to increase mRNA stability for cDNA expression.
  • MBl and B29 cDNAs obtained by polymerase chain reaction (PCR) were sequenced and cloned into the polylinker of p463 to obtain the p466-B29 and the p467-MBl expression vectors.
  • the EcoRl fragment of the MBl expression clone was then transferred to the EcoRl site of pSV2His (Hartman and Mulligan, 1988) to produce a plasmid that carries both an MBl expression vector and His resistance, p474-MBl.
  • the IgM heavy chain minigene was composed of the V region of S107 (Crews et al. , 1981) ligated to the Hind3 to BamHl fragment of a human IgM constant region gene that has been modified to produce only the membrane bound form of IgM protein (Danner and Leder, 1985) .
  • the IgM minigene was cloned just 3' of the SFFV LTR in pFneo in the Sail to BamHl site to make plasmid p459.
  • the kappa minigene was composed of the S107 light chain V region (Kwan et al., 1981) ligated to the EcoRl to BamHl fragment of human kappa light chain constant region (Hieter et al., 1982) and the promoter was the EcoRl to BamHl fragment of the SFFV LTR.
  • the heavy and light chains were combined in a single plasmid by cloning the light chain minigene into the unique BamHl site of p459 to produce p468, a plasmid that carries the PC specific heavy and light chains, and neomycin resistance genes.
  • Jurkat cells were grown in RPMI 1640 supplemented with
  • Goat anti-human IgM either labeled with fluorescein or unlabeled, IgGl monoclonal isotype control antibody, and goat anti-mouse IgG were from Southern Biotechnology.
  • RNA was prepared as described (Chirgwin et al., 1979). Following electrophoresis, and transfer blots were hybridized with either B29 cDNA or MBl anti-sense RNA as described (Sambrook et al. , 1989) .
  • 3-6X10 7 viable cells were labeled with 3-4 mCi of Na[ 125 I] and lactoperoxidase/glucose oxidase as described (Hubard and Cohn, 1972) and solubilized in 1% digitonin, 100 mM NaCl, 50 mM Tris HC1 (pH 6.8) and 1 mM phenylmethylsulfonyl fluoride at 4 ° C for 30 minutes. Insoluble material was separated by centrifugation and the supernatant was incubated with phosphorylcholine beads for 4-5 hours. The beads were washed three times with 0.1% digitonin, 100 mM NaCl and 50 mM Tris HCl (pH 6.8) for five minutes.
  • IgM Elution of IgM was carried out by incubating the beads for 1 hour with wash buffer supplemented with 20 mM phosphorylcholine. Protein samples were analyzed on 8-12% SDS-PAGE on gradient gels under reducing conditions. The protein bands were visualized by silver staining. A Phosphor Imager (Molecular Dynamics) was used to visualize 125 I labeled proteins.
  • PC-albumin and PC-sepharose Para-diazoniumphenylphosphorylcholine were synthesized (Chesebro and Metzger, 1972) and BSA dissolved in PBS was added and incubated overnight. Modified BSA (PC- albumin) was separated from unreacted DPPC by column chromatography on Sephadex G25 in PBS.
  • the dipeptide alanyltyrosine was coupled to CNBr-activated Sepharose (Pharmacia) .
  • the beads were washed in PBS and freshly synthesized DPPC was added, incubated overnight, washed with water and stored at 4 ° C.
  • Cells to be tested for IL-2 production were resuspended at 1.2X10 6 /ml in tissue culture media and used directly or pretreated with either 10 ug/ml anti-CD3, 20 ug/ml of
  • PMA Phobol myristate acetate
  • the cells were sequentially stimulated with 1 ug/ml of either anti-CD3, or DA4.4 monoclonal anti-human IgM or the IgGl isotype control followed after 10 seconds by 10 ug/ml of goat anti-mouse IgG crosslinking reagent.
  • the incubation was terminated after 3 min by the addition of 8 ml of 2:1 methanol:chloroform, and the extracted material was subjected to chromatography on 1ml columns of Dowex AG-1-8 (Bio-Rad) .
  • IP Inositol phosphates
  • T-cell antigen receptor zeta chain is tyrosine phosphorylated upon activation. J. Biol. Chem. 263, 18225-30.
  • IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes. Proc. Nat. Acad. Sci. U.S.A. 88, 3982-6.
  • the z chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70 a 70kDa tyrosine phosphoprotein. Proc. Natl. Acad. Sci. U.S.A. 88, 9166-9170.
  • VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of the antibody.
  • T-cell growth factor Parameters for production and a quantitative microassay for activity. J. Immunol. 120, 2027-2032.
  • Chimeric immunoglobulin-T-cell receptor proteins form functional receptors: Implications for T-cell receptor complex formation and activation. Cell 60, 929-939.
  • B29 a member of the immunoglobulin gene superfamily exclusively expressed on B-lineage cells. Proc. Nat. Acad. Sci. U.S.A. 85, 6890-4.
  • Ig-beta components of the IgM antigen receptor complex by amino-ter inal sequencing. Eur. J. Immunol. 20, 2795-9.
  • T-cell antigen receptor activation pathways the tyrosine kinase connection. Cell 64, 875-8.

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