The Journal of Experimental Medicine

Phung, Quy H.; Winter, David B.; Cranston, Aaron; Tarone, Robert E.; Bohr, Vilhelm A.; Fishel, Richard; Gearhart, Patricia J.

doi: 10.1084/jem.187.11.1745pmid: 9607916

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2 −/− mice were immunized with oxazolone, and B cells were analyzed for mutation in their V κ Ox1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2 +/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G·C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C. biological sciences genetics genes, immunoglobulin mutation DNA repair Footnotes We gratefully thank Francis Chrest for assistance in flow cytometry, Michael Neuberger for antigen, Andrew Wakeham for advice in genotyping splenic DNA, Heinz Jacobs for sharing data before publication, and Richard Wood and Dennis Taub for many insightful comments on the manuscript. 1 Abbreviations used in this paper: MLH, Mut L homologue; MSH, Mut S homologue; Pfu , Pyrococcus furiosus; PMS, post-meiotic segregation; VκOx1, V gene for the κ chain that binds to oxazolone. Submitted: 6 March 1998 Revision received 7 April 1998

Wei, Sheng; Gamero, Ana M.; Liu, Jin Hong; Daulton, Angela A.; Valkov, Nichola I.; Trapani, Joseph A.; Larner, Andrew C.; Weber, Michael J.; Djeu, Julie Y.

doi: 10.1084/jem.187.11.1753pmid: 9607917

The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal–regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h 51 Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation. natural killer cell mitogen-activated protein kinase perforin granzyme B signal transduction Footnotes Abbreviations used in this paper: AMAPK active MAPK ERK extracellular signal–regulatory kinase KD kinase deficient MAPK mitogen-activated protein kinase MBP myelin basic protein MEK MAPK kinase PI phosphatidylinositol PLC phospholipase C TRITC tetramethyl rhodamine isothiocyanate Submitted: 30 July 1997 Revision received 27 March 1998

Wykrzykowska, Joanna J.; Rosenzweig, Michael; Veazey, Ronald S.; Simon, Meredith A.; Halvorsen, Katherine; Desrosiers, Ronald C.; Johnson, R. Paul; Lackner, Andrew A.

doi: 10.1084/jem.187.11.1767pmid: 9607918

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7–14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4 + T cell counts. AIDS T cell homeostasis apoptosis animal model pathogenesis Footnotes Abbreviations used in this paper: dpi days after infection SIV simian immunodeficiency virus Submitted: 10 December 1997 Revision received 11 March 1998

Ishii, Satoshi; Kuwaki, Tomoyuki; Nagase, Takahide; Maki, Kazushige; Tashiro, Fumi; Sunaga, Shinji; Cao, Wei-Hua; Kume, Kazuhiko; Fukuchi, Yoshinosuke; Ikuta, Koichi; Miyazaki, Jun-ichi; Kumada, Mamoru; Shimizu, Takao

doi: 10.1084/jem.187.11.1779pmid: 9607919

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor–deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock. platelet-activating factor platelet-activating factor receptor anaphylaxis endotoxic shock gene targeting Footnotes Abbreviations used in this paper: ANOVA analysis of variance ; ES, embryonic stem HR heart rate LTB 4 leukotriene B 4 MAP mean arterial pressure NO nitric oxide PAF platelet-activating factor R L total lung resistance Submitted: 20 January 1998 Revision received 10 March 1998

Cutler, Antony J.; Botto, Marina; van Essen, Dominic; Rivi, Roberta; Davies, Kevin A.; Gray, David; Walport, Mark J.

doi: 10.1084/jem.187.11.1789pmid: 9607920

The role of the classical complement pathway in humoral immune responses was investigated in gene-targeted C1q-deficient mice ( C1qA − /− ). Production of antigen-specific immunoglobulin (Ig)G2a and IgG3 in primary and secondary responses to T cell–dependent antigen was significantly reduced, whereas IgM, IgG1, and IgG2b responses were similar in control and C1qA − /− mice. Despite abnormal humoral responses, B cells from C1qA − /− mice proliferated normally to a number of stimuli in vitro. Immune complex localization to follicular dendritic cells within splenic follicles was lacking in C1qA − /− mice. The precursor frequency of antigen-specific T cells was similar in C1qA − /− and wild-type mice. However, analysis of cytokine production by primed T cells in response to keyhole limpet hemocyanin revealed a significant reduction in interferon-γ production in C1qA − /− mice compared with control mice, whereas interleukin 4 secretion was equivalent. These data suggest that the classical pathway of complement may influence the cytokine profile of antigen-specific T lymphocytes and the subsequent immune response. complement deficiency immune response interferon γ gene targeting Footnotes Abbreviations used in this paper: FDC follicular dendritic cell GCDC dendritic cell identified within murine germinal center HAGG heat-aggregated human γ-globulin IDC interdigitating dendritic cell PNA peanut agglutinin TD T cell–dependent Submitted: 18 February 1998

Antalis, Toni M.; La Linn, May; Donnan, Karen; Mateo, Luis; Gardner, Joy; Dickinson, Joanne L.; Buttigieg, Kathy; Suhrbier, Andreas

doi: 10.1084/jem.187.11.1799pmid: 9607921

The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2–mediated induction of constitutive low-level interferon (IFN)-α/β production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2–mediated inhibition of tumor necrosis factor-α induced apoptosis, ( a ) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and ( b ) demonstrate a novel activity for a eukaryotic serpin. plasminogen activator inhibitor serpin interferon alphavirus Ross River virus Footnotes Abbreviations used in this paper: CAT chloramphenicol acetyl transferase CPE cytopathic effect IRF interferon response factor ISGF3 interferon-stimulated gene factor 3 ISRE interferon-stimulated response element MOI multiplicity of infection OAS oligoadenylate synthetase PAI-2 plasminogen activator inhibitor poly IC polyinosinic-polycytidylic acid RRV Ross River virus serpin serine proteinase inhibitor TCID 50 50% tissue culture infectivity dose uPA urokinase-type plasminogen activator Submitted: 17 July 1997 Revision received 27 February 1998

García-Ojeda, Marcos E.; Dejbakhsh-Jones, Sussan; Weissman, Irving L.; Strober, Samuel

doi: 10.1084/jem.187.11.1813pmid: 9607922

In the principal pathway of α/β T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4 + and CD8 + T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4 + and CD8 + T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4 + CD8 + intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function. extrathymic bone marrow T cell development thymus Footnotes M.E. García-Ojeda and S. Dejbakhsh-Jones contributed equally to the research described in this paper. Abbreviations used in this paper: FBS fetal bovine serum HSA heat-stable antigen Submitted: 9 September 1997 Revision received 26 March 1998

Davidson, Wendy F.; Giese, Thomas; Fredrickson, Torgny N.

doi: 10.1084/jem.187.11.1825pmid: 9607923

B cell malignancies arise with increased frequency in aging individuals and in patients with genetic or acquired immunodeficiency (e.g., AIDS) or autoimmune diseases. The mechanisms of lymphomagenesis in these individuals are poorly understood. In this report we investigated the possibility that mutations at the Fas ( lpr ) and Fasl ( gld ) loci, which prevent Fas-mediated apoptosis and cause an early onset benign lymphoid hyperplasia and autoimmunity, also predispose mice to malignant lymphomas later in life. Up to 6 mo of age, hyperplasia in lpr and gld mice results from the predominant accumulation of polyclonal T cell subsets and smaller numbers of polyclonal B cells and plasma cells. Here, we examined C3H- lpr , C3H- gld , and BALB- gld mice 6–15 mo of age for the emergence of clonal T and B cell populations and found that a significant proportion of aging mice exclusively developed B cell malignancies with many of the hallmarks of immunodeficiency-associated B lymphomas. By 1 yr of age, ∼60% of BALB- gld and 30% of C3H- gld mice had monoclonal B cell populations that grew and metastasized in scid recipients but in most cases were rejected by immunocompetent mice. The tumors developed in a milieu greatly enriched for plasma cells, CD23 − B cells and immunodeficient memory T cells and variably depleted of B220 + DN T cells. Growth factor–independent cell lines were established from five of the tumors. The majority of the tumors were CD23 − and IgH isotype switched and a high proportion was CD5 + and dull Mac-1 + . Considering their Ig secretion and morphology in vivo, most tumors were classified as malignant plasmacytoid lymphomas. The delayed development of the gld tumors indicated that genetic defects in addition to the Fas / Fasl mutations were necessary for malignant transformation. Interestingly, none of the tumors showed changes in the genomic organization of c-Myc but many had one or more somatically-acquired MuLV proviral integrations that were transmitted in scid passages and cell lines. Therefore, insertional mutagenesis may be a mechanism for transformation in gld B cells. Our panel of in vivo passaged and in vitro adapted gld lymphomas will be a valuable tool for the future identification of genetic abnormalities associated with B cell transformation in aging and autoimmune mice. lpr gld Fas Fas ligand lymphoma Footnotes 1 Abbreviations used in this paper: ALPS, autoimmune lymphoproliferative syndrome; DN, double negative; IAL, immunodeficiency-associated lymphomas; MAIDS, mouse AIDS; RT, room temperature. Submitted: 10 October 1997 Revision received 16 March 1998