William C. Raschke

Salk Institute, لا هویا, California, United States

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Publications (38)284.37 Total impact

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    ABSTRACT: The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal "ignorance."
    No preview · Article · Nov 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor signaling by dephosphorylating the inhibitory tyrosine of the src family kinases. CD45-deficient mice fail to fully unmask the role of CD45 in B cells because of the expression of a partially redundant tyrosine phosphatase, CD148. However, mice that are doubly deficient in CD45 and CD148 exhibit a very early block in B-cell development, thereby obscuring later roles for CD45. To overcome these limitations, here we take advantage of an allelic series of mice in which CD45 expression is titrated broadly (0-180%). Although high expression of CD45 inhibits T-cell receptor (TCR) signaling, we show that CD45 plays a purely positive regulatory role during B-cell receptor (BCR) signaling. In concert with exaggerated BCR signaling, increasing CD45 expression drives enhanced receptor editing in the bone marrow and profound loss of follicular and marginal zone B cells in the spleen. In the context of the IgHEL/sHEL model of B-cell tolerance, such high CD45 expression transforms anergy into deletion. Unexpectedly, elimination of the autoantigen sHEL in this model system in order to block clonal deletion fails to rescue survival of mature B cells. Rather, high CD45 expression reduces B-cell activating factor receptor (BAFFR) expression and inhibits B-cell activating factor (BAFF)-induced B-cell survival in a cell-intrinsic manner. Taken together, our findings reveal how CD45 function diverges in T cells and B cells, as well as how autoreactive B cells are censored as they transit development.
    Full-text · Article · Nov 2011 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The kinase-phosphatase pair Csk and CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn. T cell receptor (TCR) signaling and thymic development require CD45 expression but proceed constitutively in the absence of Csk. Here, we show that relative titration of CD45 and Csk expression reveals distinct regulation of basal and inducible TCR signaling during thymic development. Low CD45 expression is sufficient to rescue inducible TCR signaling and positive selection, whereas high expression is required to reconstitute basal TCR signaling and beta selection. CD45 has a dual positive and negative regulatory role during inducible but not basal TCR signaling. By contrast, Csk titration regulates basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons-to downmodulate inducible TCR signaling during positive selection and to counteract Csk during basal TCR signaling.
    Full-text · Article · Mar 2010 · Immunity
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    ABSTRACT: Ebola virus (EBOV) infection of humans is a lethal but accidental dead-end event. Understanding resistance to EBOV in other species may help establish the basis of susceptibility differences among its hosts. Although rodents are resistant to EBOV, a murine-adapted variant is lethal when injected intraperitoneally into mice. We find that mice expressing reduced levels of the tyrosine phosphatase CD45 are protected against EBOV, whereas wild-type, CD45-deficient, or enzymatically inactive CD45-expressing mice succumbed to infection. Protection was dependent on CD8(+) T cells and interferon gamma. Reduced CD45-expressing mice retained greater control of gene expression and immune cell proliferation following EBOV infection, which contributed to reduced apoptosis, enhanced viral clearance, and increased protection against the virus. Together, these findings suggest that host susceptibility to EBOV is dependent on the delicate balance of immune homeostasis, which, as demonstrated here, can be determined by the levels of a single regulator.
    Full-text · Article · Sep 2009 · Cell host & microbe
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    ABSTRACT: The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.
    Full-text · Article · Apr 2009 · Journal of Biological Chemistry
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    Elizabeth L Virts · Oscar Diago · William C Raschke
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    ABSTRACT: Transgenic mice have been generated that carry a CD45 minigene under control of the human leukocyte function-associated antigen (LFA-1, CD11a) promoter. CD45-null mice carrying the transgene exhibit the lymphocyte lineage-specific isoform expression patterns of wild-type mice. Furthermore, these mice have normal thymocyte development and peripheral T-cell numbers. The proliferative ability of T cells in response to mitogens and antigen also is regained, as is B-cell responsiveness to anti-IgM. The antibody response to antigen is also restored and is similar to that of normal mice. Therefore, introduction of a functional CD45 minigene is sufficient to overcome the principal severe combined immunodeficiency (SCID)-associated defects and represents a potential route to a gene therapy for human CD45-deficent SCID.
    Full-text · Article · Mar 2003 · Blood
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    E L Virts · W C Raschke
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    ABSTRACT: Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5′, 3′, and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a β-galactosidase reporter construct. Furthermore, the CD45 3′-untranslated region and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed that contain either the cytomegalovirus promoter, the leukocyte function-associated antigen (LFA-1; CD11a) promoter, or various CD45 5′ regions. Neither the cytomegalovirus nor the LFA-1 promoter was capable of generating detectable levels of expression in constructs with CD45 cDNA. However, when CD45 intron sequences between exons 3 and 9 were inserted in the cDNA construct to generate a CD45 minigene, the LFA-1 promoter was able to drive reproducible, significant expression of CD45. CD45 minigenes using the CD45 5′ sequences up to 19 kilobases upstream of the transcriptional start produced very little protein. The LFA-1 CD45 minigene construct produced correct cell type-specific isoforms when expressed in T and B lymphocyte lines. Therefore, we conclude that the regulation of CD45 expression and cell type-specific splicing requires elements within the intron sequences.
    Preview · Article · Jul 2001 · Journal of Biological Chemistry
  • E Virts · D Barritt · W C Raschke
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    ABSTRACT: The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous. All Th lines produced mRNA encoding this isoform, but in only three of the Tc lines was the 89 isoform detectable by RT/PCR. RNase protection assays with RNA isolated from normal CD8+ splenic cells demonstrated the 89 splice product was present in low abundance. The relative abundance of the different isoforms in the thymic lymphoma, BW5147, was determined through RNase protection analysis. The 789 isoform predominates, representing approximately 75% of the CD45 mRNA whereas the 89 form constitutes about 24%. In addition, an isoform lacking exons 4-8 (isoform 9) also was detected and comprises approximately 1% of the total CD45 mRNA in this cell line. Finally, these studies demonstrate that exon 10 is also used as an alternatively spliced exon.
    No preview · Article · Mar 1998 · Molecular Immunology
  • Elizabeth Virts · Diana Barritt · Edward Siden · William C. Raschke
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    ABSTRACT: CD45 isoform expression patterns were examined in various mast and monocyte cell populations. The reverse transcription-polymerase chain reaction (RT/PCR) and Southern analysis showed these myeloid cells express characteristic sets of CD45 isoforms. Mast cells produce mRNA for two splice variants, one containing exons 5, 7 and 8 of the alternatively expressed exons (therefore lacking exons 4 and 6) and another containing variable exons 7 and 8. Monocytes express three prominent CD45 mRNA species, one which includes exons 5, 7 and 8, another with exons 7 and 8 and the third containing only exon 8 of the variable exons. These results show that there are clear differences within the myeloid lineage sub-populations with respect to CD45 exon usage which appear to delineate mast cell and monocyte specific patterns.
    No preview · Article · Nov 1997 · Molecular Immunology
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    ABSTRACT: Pichia pastoris (Pp) and Hansenula polymorpha (Hp) are methylotrophic yeasts commonly used for industrial purposes. Growth of either of these yeasts in the presence of methanol as the carbon source results in high-level induction of alcohol oxidase expression. The respective alcohol oxidase genes, AOX1 in Pp and MOX in Hp, have similar regulatory characteristics. Our studies show that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp. Furthermore, the size of an AOX1p-heterologous gene-AOX1 terminator cassette transcript synthesized in Hp is indistinguishable from that synthesized in Pp suggesting that transcription both initiates and terminates at the same sites in both yeast species. Induction of AOX1p in Hp demonstrates that the methanol-inducible regulatory mechanism in Hp is able to recognize and activate the Pp promoter in spite of extensive sequence variations between AOX1p and MOXp.
    Preview · Article · Nov 1996 · Gene
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    ABSTRACT: The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.
    No preview · Article · Oct 1995 · Journal of Biological Chemistry
  • William C. Raschke · Michelle Hendricks · C M Chen

    No preview · Article · Feb 1995 · Immunogenetics
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    ABSTRACT: In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.
    Full-text · Article · Jan 1995 · Biochimica et Biophysica Acta
  • James M. Cregg · Thomas S. Vedvick · William C. Raschke
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    ABSTRACT: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.
    No preview · Article · Sep 1993 · Bio/Technolgy
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    ABSTRACT: The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.
    No preview · Article · Aug 1992 · Biochemical and Biophysical Research Communications
  • S.L. Zebedee · D Barritt · R Epstein · W.C. Raschke
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    ABSTRACT: Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and BALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele. Both assays, DNA-PCR and protein-immunofluorescence, yielded identical results for each strain examined. Placement of the Ly5 gene with respect to other characterized markers of mouse chromosome 1 for these recombinant inbred mouse strains shows a gene order of Idh-1:Ity:Pep3:[Ly5, Cfh].
    No preview · Article · Jul 1991 · European Journal of Immunogenetics
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    Suzanne L. Zebedee · Diana S. Barritt · William C. Raschke
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    ABSTRACT: The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced. A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5' end of the Ly5a LCA gene, generated using polymerase chain-reaction techniques on genomic DNA, reveals eight additional nucleotide differences between the Ly5a and Ly5b alleles.
    Preview · Article · Feb 1991 · Developmental Immunology
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    W C Raschke
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    ABSTRACT: The murine T200 family of cell-surface glycoproteins is expressed on hematopoietic lineage cell types in a developmentally regulated manner with different lymphocyte subpopulations expressing cross-reactive but structurally distinct forms. To investigate the differences between these forms and the regulation of these differences, murine T200 cDNA clones were isolated by using a probe obtained by subtractive hybridization. This procedure made use of a T200+ L-cell transfectant and the parent Ltk- cell line. The 1.9-kilobase (kb) cDNA clone was sequenced and found to contain the coding region of the COOH-terminal 331 amino acids and 0.9 kb of 3' untranslated region. Where the sequence overlapped with the rat sequence, 80-90% homology was observed. RNA blot analysis revealed that B-lineage cell lines express either a 5.6-kb or a 6.5-kb mRNA correlating to the size difference of the T200 glycoprotein synthesized. Similarly, in the T-cell lineage a helper T-cell clone and a cytotoxic T-cell clone express T200 transcripts of 5.6 kb and 5.9 kb, respectively, which correlate with the distinct sizes of their T200 glycoproteins. A T200-negative mutant of a T-cell line was found to express full-length T200 mRNA, although at a diminished level.
    Preview · Article · Feb 1987 · Proceedings of the National Academy of Sciences
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    M Hagiya · D D Davis · T Takahashi · K Okuda · W C Raschke · H Sakano
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    ABSTRACT: Both alleles of immunoglobulin (Ig) heavy-chain joining region (JH) genes in three Ig-negative Abelson murine leukemia virus (Ab-MuLV)-transformed cell lines were characterized by DNA cloning and nucleotide sequence determination. These studies unambiguously identified two distinct types of Ig-negative B-lineage cells. The first type of cell (e.g., R8) is an "immature pre-B cell," and it contains at least one intermediate recombinant structure containing heavy-chain diversity (DH) and JH sequences but no variable region (VH) sequence. This type of cell, which has also been characterized by other investigators, generates mu-positive sublines during subsequent culturing of cells and represents a precursor stage to pre-B cells. The second type of cell (e.g., RAW253) is an "abortive pre-B cell," in that both JH alleles contain nonfunctional VH-DH-JH structures. The nucleotide sequence determinations in this study demonstrated that these nonfunctional V-D-J structures were generated by nonproductive somatic recombinations, involving either out-of-phase joining events, or the formation of termination codons in the DH coding sequences. The identification of abortive pre-B cells suggests that the recombinational joining of Ig VH, DH, and JH segments is not actively regulated by a putative recombinase to preserve the translational reading frame. This in turn implies that a large portion of precursor cells at the early stage of B-cell differentiation are abortive and possibly blocked to further differentiation.
    Preview · Article · Feb 1986 · Proceedings of the National Academy of Sciences
  • William C. Raschke · Robert Hyman
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    ABSTRACT: L-cells, which normally do not express the mouse lymphocyte T200 antigen, were transfected with DNA from the mouse T-cell lymphoma, BW5147, and a T200+ L-cell line isolated. The detection and enrichment of T200+ L-cells from a pool of transfectants was accomplished using monoclonal anti-T200 antibody and fluorescence-activated cell sorting. A subclone has been selected that is stable for expression of T200. The T200 molecules expressed by BW5147 and the T200+ L-cell are similar in size, around 190 Kd, compared to the 220-Kd B-cell form of T200. The BW5147 T200 molecule is 3000 daltons larger than the molecule expressed by the transfected L-cell, a size difference due to glycosylation moieties, since treatment of the molecules with Endoglycosidase F or treatment of cells with tunicamycin yields T200 molecules of the same size from the 2 cell sources. In comparison, the B-cell form of T200 retains a size difference of 25,000 daltons from the T-cell form after these treatments. Monoclonal antibodies specific for the B-cell form of T200 do not recognize the T200+ L-cell, providing further evidence that the T-cell form of T200 is expressed by the transfected L-cell.
    No preview · Article · Nov 1985 · Molecular Immunology

Publication Stats

2k Citations
284.37 Total Impact Points


  • 1974-1995
    • Salk Institute
      • • Molecular Biology and Virology Laboratory
      • • Developmental Biology Laboratory
      لا هویا, California, United States
  • 1979-1986
    • University of California, Berkeley
      Berkeley, California, United States
  • 1985
    • Fred Hutchinson Cancer Research Center
      Seattle, Washington, United States
  • 1977
    • Stanford Medicine
      Stanford, California, United States