The Journal of Experimental Medicine

Dohi, Taeko; Fujihashi, Kohtaro; Rennert, Paul D.; Iwatani, Koichi; Kiyono, Hiroshi; McGhee, Jerry R.

doi: 10.1084/jem.189.8.1169pmid: 10209035

To investigate the potential involvement of T helper (Th)2-type responses in murine models of intestinal inflammation, we used trinitrobenzene sulfonic acid (TNBS)–hapten to induce inflammatory bowel disease in situations where Th1-type responses with interferon (IFN)-γ synthesis are either diminished or do not occur. Intracolonic administration of TNBS to either normal (IFN-γ +/+ ) or Th1-deficient IFN-γ knockout (IFN-γ −/− ) BALB/c mice resulted in significant colitis. In IFN-γ −/− mice, crypt inflammation was more severe than in IFN-γ +/+ mice and was accompanied by hypertrophy of colonic patches with a lymphoepithelium containing M cells and distinct B and T cell zones resembling Peyer's patches. Hapten-specific, colonic patch T cells from both mouse groups exhibited a Th2 phenotype with interleukin (IL)-4 and IL-5 production. TNBS colitis in normal mice treated with anti–IL-4 antibodies or in IL-4 −/− mice was less severe than in either IFN-γ +/+ or IFN-γ −/− mice. Our findings now show that the Th2-type responses in TNBS colitis are associated with colonic patch enlargement and inflammation of the mucosal layer and may represent a model for ulcerative colitis. inflammatory bowel diseases mouse T cells cytokines hapten-induced colitis Footnotes Abbreviations used in this paper: GALT gut-associated lymphoreticular tissues IBD inflammatory bowel diseases PNA peanut agglutinin SLN sacral lymph nodes TNBS 2,4,6-trinitrobenzene sulfonic acid Submitted: 2 March 1998 Revision received 25 February 1999

Marie-Cardine, Anne; Kirchgessner, Henning; Bruyns, Eddy; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Ratnofsky, Sheldon; Meuer, Stefan; Schraven, Burkhart

doi: 10.1084/jem.189.8.1181pmid: 10209036

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2. T lymphocytes T cell receptor transmembrane adaptor proteins signal transduction Footnotes Anne Marie-Cardine is a recipient of a fellowship from the Training and Mobility of Researchers program of the European Community (ERBFMBICT950472). This work was supported in part by Deutsche Forschungsgemeinschaft grants SCHR/533/1-1 and SCHR/533/4-1 and by Sonderforschungsbereich (SFB) 405, projects A5 (to B. Schraven) and B9 (to F. Autschbach). The work in M. Mann's laboratory was partially supported by a grant from the German Technology Ministry (BMBF). M. Mann's present address is Center of Experimental Bioinformatics, Odense University, Campusveij 55, DK-5230 Odense M, Denmark. Anne Marie-Cardine and Henning Kirchgessner contributed equally to this work. Abbreviations used in this paper: aa amino acid EST expressed sequence tag Grb2 growth factor receptor binding protein 2 IEF isoelectric focusing ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine based inhibition motif LAT linker for activation of T cells NF-AT nuclear factor of activated T cells PI3-K phosphatidylinositol 3-kinase PLC phospholipase C PTK protein tyrosine kinase PTYR phosphotyrosine SH src homology SHIP SH2 containing inositol phosphatase SHP SH2 containing protein tyrosine phosphatase SIT SHP2-interacting transmembrane adaptor protein SLP SH2 domain containing leukocyte phosphoprotein TRIM T cell receptor–interacting molecule Submitted: 8 December 1998 Revision received 8 February 1999

Sabelko-Downes, Kimberly A.; Cross, Anne H.; Russell, John H.

doi: 10.1084/jem.189.8.1195pmid: 10209037

We have previously demonstrated a role for Fas and Fas ligand (FasL) in the pathogenesis of experimental allergic encephalomyelitis (EAE). However, using an active induction paradigm we could not distinguish between FasL expressed on activated CD4 + T cells from that expressed on other inflammatory or resident central nervous system (CNS) cells. To address this issue, we have conducted reciprocal adoptive transfer experiments of nontransgenic or myelin basic protein–specific T cell receptor transgenic wild-type, lpr , or gld lymphocytes into congenic wild-type, lpr , and gld hosts. We found that FasL expressed on donor cells is important for the development of EAE, as FasL-deficient lymphocytes transfer attenuated disease. Furthermore, Fas expressed in the recipient animals is important for the progression of EAE, as clinical signs of disease in lpr recipients were dramatically attenuated after transfer of either wild-type or lpr T cells. Surprisingly, these experiments also identified CNS cells as a source of functional FasL. Host-derived FasL appears to be especially important in the recovery from EAE, as many gld recipients of wild-type lymphocytes develop prolonged clinical signs of disease. Thus it appears that FasL plays distinct roles in EAE during the initiation of and recovery from disease. autoimmunity inflammation T cell regulation demyelinating disease apoptosis Footnotes Abbreviations used in this paper: B6 C57BL/6 CNS central nervous system EAE experimental allergic encephalomyelitis FasL Fas ligand GVHD graft-versus-host disease MBP myelin basic protein MS multiple sclerosis TUNEL TdT-mediated dUTP nick-end labeling Submitted: 21 December 1998 Revision received 12 February 1999

Tristan Brandhorst, T.; Wüthrich, Marcel; Warner, Thomas; Klein, Bruce

doi: 10.1084/jem.189.8.1207pmid: 10209038

Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood. In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products. In Blastomyces dermatitidis , the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor. DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer. These findings establish the pivotal role of WI-1 in adherence and virulence of B . dermatitidis yeasts. To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms. dimorphic fungi gene targeting virulence factor pathogenic mechanism adhesin Footnotes Abbreviation used in this paper: HMM Histoplasma macrophage medium Submitted: 24 December 1998 Revision received 26 February 1999

Léonetti, Michel; Galon, Jérome; Thai, Robert; Sautès-Fridman, Catherine; Moine, Gervaise; Ménez, André

doi: 10.1084/jem.189.8.1217pmid: 10209039

Using a snake toxin as a proteic antigen (Ag), two murine toxin–specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag–specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20–100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP–Ab–Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS ® analyses showed that an Ag–Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag–Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor–containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response. Ag presentation protein A B cell superantigen Footnotes J. Galon was a recipient of fellowships from Ministère de la Recherche and of Association de Recherche contre le Cancer. Abbreviations used in this paper: CTLL cytotoxic T cell line FCC fluorescein-5(6)-carboxamidocaproic hIg human immunoglobulin IBP immunoglobulin binding protein SAPE streptavidin-PE SpA staphylococcal protein A ssp. subspecies Submitted: 29 June 1998 Revision received 30 December 1998

Nieborowska-Skorska, Malgorzata; Wasik, Mariusz A.; Slupianek, Artur; Salomoni, Paolo; Kitamura, Toshio; Calabretta, Bruno; Skorski, Tomasz

doi: 10.1084/jem.189.8.1229pmid: 10209040

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor–dependent myeloid precursor cells, STAT5 activation–deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor–independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor–independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation–defective BCR/ABL SH3+SH2 mutants to induce growth factor–independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor–independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis. oncoprotein domains cooperation transformation leukemia Footnotes Abbreviations used in this paper: aa amino acid(s) BrdU 5-bromo-2′-deoxyuridine CML chronic myelogenous leukemia CNS central nervous system DAM dominant-active mutant DNM dominant-negative mutant EMSA electrophoretic mobility shift assay PI-3k phosphatidylinositol-3 kinase P.Tyr phosphotyrosine RT reverse transcription SH src homology STAT signal transducer and activator of transcription WT wild-type Submitted: 15 October 1998 Revision received 13 January 1999

Law, Che-Leung; Ewings, Maria K.; Chaudhary, Preet M.; Solow, Sasha A.; Yun, Theodore J.; Marshall, Aaron J.; Hood, Leroy; Clark, Edward A.

doi: 10.1084/jem.189.8.1243pmid: 10209041

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain–containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL–SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells. GrpL SLP-76 Grb2 nuclear factor of activated cells cell activation Footnotes C.-L. Law's present address is Xcyte Therapies Inc., 2203 Airport Way South, Suite 300, Seattle, WA 98134. C.-L. Law, M.K. Ewings, and P.M. Chaudhary all contributed equally to this study. Abbreviations used in this paper: GEF guanine nucleotide exchange factor GrpL Grb2-related protein of the lymphoid system IP immunoprecipitate MAPK mitogen-activated protein kinase NF-AT nuclear factor of activated T cells PTK protein tyrosine kinase SH src homology SHP SH2 domain–containing protein tyrosine phosphatase SLP-76 SH2 domain–containing leukocyte protein of 76 kD Submitted: 27 October 1998 Revision received 18 February 1999

Massberg, Steffen; Sausbier, Matthias; Klatt, Peter; Bauer, Markus; Pfeifer, Alexander; Siess, Wolfgang; Fässler, Reinhard; Ruth, Peter; Krombach, Fritz; Hofmann, Franz

doi: 10.1084/jem.189.8.1255pmid: 10209042

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia–reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3′,5′-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet–endothelial cell and platelet–platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI −/− ) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation. fluorescence microscopy endothelial cell microcirculation nitric oxide cyclic guanosine 3′,5′-monophosphate–dependent protein kinase Footnotes S. Massberg and M. Sausbier contributed equally to this work. Abbreviations used in this paper: cAK cAMP-dependent protein kinase cGKI cGMP-dependent protein kinase I cGMP cyclic guanosine 3′,5′-monophosphate I/R ischemia/reperfusion NO nitric oxide PRP platelet-rich plasma VASP vasodilator-stimulated phosphoprotein Submitted: 3 November 1998 Revision received 25 January 1999

Davis, Daniel M.; Mandelboim, Ofer; Luque, Isabel; Baba, Eishi; Boyson, Jonathan; Strominger, Jack L.

doi: 10.1084/jem.189.8.1265pmid: 10209043

Molecular interactions with the extracellular domains of class I major histocompatibility complex proteins are major determinants of immune recognition that have been extensively studied both physically and biochemically. However, no immunological function has yet been placed on the transmembrane or cytoplasmic amino acid sequences of these proteins despite strict conservation of unique features within each class I major histocompatibility complex locus. Here we report that lysis by a subset of natural killer (NK) cells inhibited by target cell expression of human histocompatibility leukocyte antigen (HLA)-Cw6 or -Cw7 was not inhibited by expression of chimeric proteins consisting of the extracellular domains of HLA-C and the COOH-terminal portion of HLA-G. Assays using transfectants expressing a variety of HLA-Cw6 mutants identified the transmembrane sequence and, in particular, cysteine at position 309 as necessary for inhibition of 68% (25/37) of NK cell lines and 23% (33/145) of NK clones tested. Moreover, these NK clones inhibited by target cell expression of HLA-Cw6 and dependent upon the transmembrane sequence were found not to express or to only dimly express NK inhibitory receptors (NKIR1) that are EB6/HP3E4-positive. Furthermore, assays using monoclonal antibody blocking suggest that an NK receptor other than NKIR1 or CD94 is responsible for recognition dependent upon the transmembrane sequence of HLA-Cw6. NK cells class I MHC protein transmembrane sequence NK receptors cysteine Footnotes Presented in abstract form at The British Society for Immunology 6th Annual Congress, Harrogate, England. 1–4 December 1998. Abbreviations used in this paper: β 2 m β 2 -microglobulin GPI glycosylphosphatidylinositol NKIR natural killer cell inhibitory receptor Submitted: 18 November 1998 Revision received 24 February 1999

Ruiz, Pedro J.; Garren, Hideki; Hirschberg, David L.; Langer-Gould, Annette M.; Levite, Mia; Karpuj, Marcela V.; Southwood, Scott; Sette, Alessandro; Conlon, Paul; Steinman, Lawrence

doi: 10.1084/jem.189.8.1275pmid: 10209044

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87–99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87–99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease. experimental autoimmune encephalomyelitis mimicry altered peptide ligand autoimmunity multiple sclerosis Footnotes Abbreviations used in this paper: APL altered peptide ligand CNS central nervous system EAE experimental autoimmune encephalomyelitis gpSCH guinea pig spinal cord homogenate HPV human papilloma virus HSV herpes simplex virus HVS herpes virus Saimiri MBP myelin basic protein MOG myelin oligodendroglial glycoprotein MS multiple sclerosis Submitted: 18 November 1998 Revision received 2 February 1999