The Journal of Experimental Medicine

Krause, Anja; Guo, Hong-Fen; Latouche, Jean-Baptiste; Tan, Cuiwen; Cheung, Nai-Kong V.; Sadelain, Michel

doi: 10.1084/jem.188.4.619pmid: 9705944

Most tumor cells function poorly as antigen-presenting cells in part because they do not express costimulatory molecules. To provide costimulation to T lymphocytes that recognize tumor cells, we constructed a CD28-like receptor specific for G D2 , a ganglioside overexpressed on the surface of neuroblastoma, small-cell lung carcinoma, melanoma, and other human tumors. Recognition of G D2 was provided by a single-chain antibody derived from the G D2 -specific monoclonal antibody 3G6. We demonstrate that the chimeric receptor 3G6-CD28 provides CD28 signaling upon specific recognition of the G D2 antigen on tumor cells. Human primary T lymphocytes retrovirally transduced with 3G6-CD28 secrete interleukin 2, survive proapoptotic culture conditions, and selectively undergo clonal expansion in the presence of an antiidiotypic antibody specific for 3G6-CD28. Polyclonal CD8 + lymphocytes expressing 3G6-CD28 are selectively expanded when cultured with cells expressing allogeneic major histocompatibility complex class I together with G D2 . Primary T cells given such an antigen-dependent survival advantage should be very useful to augment immune responses against tumor cells. adoptive cell therapy chimeric receptors costimulation ganglioside gene transfer Footnotes Abbreviations used in this paper: GAM goat anti–mouse GAR goat anti– rat LNGFR low-affinity nerve growth factor receptor NTP inactive mutant form of the human LNGFR scFv single chain variable region fragment Submitted: 5 November 1997 Revision received 14 May 1998

Nicolas, Nathalie; Moshous, Despina; Cavazzana-Calvo, Marina; Papadopoulo, Dora; de Chasseval, Régina; Le Deist, Françoise; Fischer, Alain; de Villartay, Jean-Pierre

doi: 10.1084/jem.188.4.627pmid: 9705945

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku–DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination. human immunodeficiency V(D)J recombination DNA repair severe combined immunodeficiency Footnotes Abbreviations used in this paper: CHO Chinese hamster ovary cs catalytic subunit D diversity dsb double-strand break J joining PK protein kinase RAG recombination activating gene RSS recombination-specific sequences T − B − absence of circulating T and B lymphocytes V variable XRCC x-ray cross-complementing Submitted: 9 December 1997 Revision received 12 June 1998

Wen, Tong-Chun; Tanaka, Junya; Peng, Hui; Desaki, Junzo; Matsuda, Seiji; Maeda, Nobuji; Fujita, Hiroko; Sato, Kohji; Sakanaka, Masahiro

doi: 10.1084/jem.188.4.635pmid: 9705946

In the central nervous system, interleukin (IL)-3 has been shown to exert a trophic action only on septal cholinergic neurons in vitro and in vivo, but a widespread distribution of IL-3 receptor (IL-3R) in the brain does not conform to such a selective central action of the ligand. Moreover, the mechanism(s) underlying the neurotrophic action of IL-3 has not been elucidated, although an erythroleukemic cell line is known to enter apoptosis after IL-3 starvation possibly due to a rapid decrease in Bcl-2 expression. This in vivo study focused on whether IL-3 rescued noncholinergic hippocampal neurons from lethal ischemic damage by modulating the expression of Bcl-x L , a Bcl-2 family protein produced in the mature brain. 7-d IL-3 infusion into the lateral ventricle of gerbils with transient forebrain ischemia prevented significantly hippocampal CA1 neuron death and ischemia-induced learning disability. TUNEL (terminal deoxynucleotidyltransferase–mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling) staining revealed that IL-3 infusion caused a significant reduction in the number of CA1 neurons exhibiting DNA fragmentation 7 d after ischemia. The neuroprotective action of IL-3 appeared to be mediated by a postischemic transient upregulation of the IL-3R α subunit in the hippocampal CA1 field where IL-3Rα was barely detectable under normal conditions. In situ hybridization histochemistry and immunoblot analysis demonstrated that Bcl-x L mRNA expression, even though upregulated transiently in CA1 pyramidal neurons after ischemia, did not lead to the production of Bcl-x L protein in ischemic gerbils infused with vehicle. However, IL-3 infusion prevented the decrease in Bcl-x L protein expression in the CA1 field of ischemic gerbils. Subsequent in vitro experiments showed that IL-3 induced the expression of Bcl-x L mRNA and protein in cultured neurons with IL-3Rα and attenuated neuronal damage caused by a free radical–producing agent FeSO 4 . These findings suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-x L protein, which is known to facilitate neuron survival. Since IL-3Rα in the hippocampal CA1 region, even though upregulated in response to ischemic insult, is much less intensely expressed than that in the CA3 region tolerant to ischemia, the paucity of IL-3R interacting with the ligand may account for the vulnerability of CA1 neurons to ischemia. interleukin 3 transient forebrain ischemia DNA fragmentation receptor Bcl-x L Footnotes This project was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare of Japan. Abbreviations used in this paper: MAP2 microtubule-associated protein 2 RT reverse transcription TBS Tris-buffered saline TUNEL terminal deoxynucleotidyltransferase–mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling Submitted: 30 December 1997 Revision received 3 June 1998

Rathmell, Jeffrey C.; Fournier, Sylvie; Weintraub, Bennett C.; Allison, James P.; Goodnow, Christopher C.

doi: 10.1084/jem.188.4.651pmid: 9705947

Peripheral tolerance mechanisms normally prevent delivery of T cell help to anergic self-reactive B cells that accumulate in the T zones of spleen and lymph nodes. Chronic exposure to self-antigens desensitizes B cell antigen receptor (BCR) signaling on anergic B cells so that they are not stimulated into clonal expansion by CD4 + T cells but instead are eliminated by Fas (CD95)-induced apoptosis. Because a range of BCR-induced signals and responses are repressed in anergic B cells, it is not known which of these are critical to regulate for Fas-mediated peripheral tolerance. Display of the costimulatory molecule, B7.2 (CD86), represents a potentially important early response to acute BCR engagement that is poorly induced by antigen on anergic B cells. We show here that restoring B7.2 expression on tolerant B cells using a constitutively expressed B7.2 transgene is sufficient to prevent Fas-mediated deletion and to trigger extensive T cell–dependent clonal expansion and autoantibody secretion in the presence of specific T cells. Dysregulated expression of B7.2 on tolerant B cells caused a more extreme reversal of peripheral tolerance than that caused by defects in Fas or Fas ligand, and resulted in T cell–dependent clonal expansion and antibody secretion comparable in magnitude to that made by foreign antigen-specific B cells. These findings demonstrate that repression of B7.2 is critical to eliminate autoreactive B cells by Fas in B cell–T cell interactions. The possible role of B7.2 dysregulation in systemic autoimmune diseases is discussed. B7.2 (CD86) Fas (CD95) B cell antigen receptor B cell autoimmunity Footnotes J.C. Rathmell's current address is Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E. 57th St., R402, Chicago, IL 60637. Abbreviations used in this paper: BCR B cell antigen receptor CTLA4 cytotoxic lymphocyte–associated antigen 4 HEL hen egg lysozyme HPRT hypoxanthine phosphoribosyltransferase L ligand PI propidium iodide RT reverse transcription tg transgenic Submitted: 2 February 1998 Revision received 26 May 1998

Lucius, Ralph; Gallinat, Stefan; Rosenstiel, Philip; Herdegen, Thomas; Sievers, Jobst; Unger, Thomas

doi: 10.1084/jem.188.4.661pmid: 9705948

The renin-angiotensin system (RAS) has been traditionally linked to blood pressure and volume regulation mediated through the angiotensin II (ANG II) type 1 (AT 1 ) receptor. Here we report that ANG II via its ANG II type 2 (AT 2 ) receptor promotes the axonal elongation of postnatal rat retinal explants (postnatal day 11) and dorsal root ganglia neurons in vitro, and, moreover, axonal regeneration of retinal ganglion cells after optic nerve crush in vivo. In retinal explants, ANG II (10 −7 –10 −5 M) induced neurite elongation via its AT 2 receptor, since the effects were mimicked by the AT 2 receptor agonist CGP 42112 (10 −5 M) and were entirely abolished by costimulation with the AT 2 receptor antagonist PD 123177 (10 −5 M), but not by the AT 1 receptor antagonist losartan (10 −5 M). To investigate whether ANG II is able to promote axonal regeneration in vivo, we performed optic nerve crush experiments in the adult rats. After ANG II treatment (0.6 nmol), an increased number of growth-associated protein (GAP)-43–positive fibers was detected and the regenerating fibers regularly crossed the lesion site (1.6 mm). Cotreatment with the AT 2 receptor antagonist PD 123177 (6 nmol), but not with the AT 1 receptor antagonist losartan (6 nmol), completely abolished the ANG II–induced axonal regeneration, providing for the first time direct evidence for receptor-specific neurotrophic action of ANG II in the central nervous system of adult mammals and revealing a hitherto unknown function of the RAS. axonal regeneration angiotensin receptor PD 123177 losartan apoptosis Footnotes R. Lucius and S. Gallinat contributed equally to this work. Abbreviations used in this paper: ANG angiotensin AT 1 ANG II type 1 AT 2 ANG II type 2 BDNF brain-derived neurotrophic factor CNS central nervous system DRG dorsal root ganglia GAP growth-associated protein GAPDH glyceralaldehyde-3-phosphate dehydrogenase GFAP glial fibrillary acidic protein RAS renin–angiotensin system RGC retinal ganglion cell RT-PCR reverse transcriptase PCR Submitted: 9 March 1998 Revision received 16 April 1998

Ram, Sanjay; McQuillen, Daniel P.; Gulati, Sunita; Elkins, Christopher; Pangburn, Michael K.; Rice, Peter A.

doi: 10.1084/jem.188.4.671pmid: 9705949

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N . gonorrhoeae . Neisseria gonorrhoeae factor H porin serum resistance Footnotes Abbreviations used in this paper: CMP-NANA 5′-cytidinemonophospho- N -acetyl neuraminic acid DGI gonococci that cause disseminated infection LOS lipooligosaccharide NHS normal human serum PID pelvic inflammatory disease Submitted: 13 March 1998 Revision received 2 June 1998

Li, Xiaomao; Sambhara, Suryaprakash; Li, Cindy Xin; Ewasyshyn, Mary; Parrington, Mark; Caterini, Judy; James, Olive; Cates, George; Du, Run-Pan; Klein, Michel

doi: 10.1084/jem.188.4.681pmid: 9705950

Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon γ after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit β-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy. respiratory syncytial virus DNA vaccine vector design F protein immune modulation Footnotes We wish to express our sincere appreciation to Nancy Scollard and Anjna Kurichh for their expertise in viral and CTL assays, and to Bill Bradley and Diane England for oligonucleotide synthesis and DNA sequencing. We also wish to thank Dr. Raymond Oomer and Shahneela Usman for the purification of the RSV F protein. We are grateful to Dr. David Brownstein (Yale University, New Haven, CT), who evaluated the lung histopathology. Abbreviations used in this paper: BC cells Balb/c fibroblasts DNA-F optimized plasmid DNA vector expressing the RSV F protein FI-RSV formalin-inactivated RSV F protein fusion protein RSV respiratory syncytial virus Submitted: 25 March 1998 Revision received 21 May 1998

Storb, Ursula; Klotz, Emily L.; Hackett, John; Kage, Karen; Bozek, Grazyna; Martin, Terence E.

doi: 10.1084/jem.188.4.689pmid: 9705951

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57–65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA. Ig genes somatic hypermutation RNA secondary structure hot spots RNA polymerase pausing Footnotes We are greatly indebted to Phil Schumm for the statistical analysis. We are grateful to Peter Engler, Nancy Michael, Larry Loeb, David Roth, and Kevin Struhl for critical reading of the manuscript. Dr. Klotz's current address is NCI, NIH, Bethesda, MD 20892. Dr. Hackett's current address is Abbott Laboratories, North Chicago, IL 60064. Abbreviations used in this paper: C constant D diversity EPS alternating EcoRV and PvuII restriction enzyme sites J joining MuF mutator factor pol RNA, polymerase II V variable Submitted: 9 April 1998 Revision received 10 June 1998

Kee, Barbara L.; Murre, Cornelis

doi: 10.1084/jem.188.4.699pmid: 9705952

The transcription factors encoded by the E2A and early B cell factor ( EBF ) genes are required for the proper development of B lymphocytes. However, the absence of B lineage cells in E2A - and EBF -deficient mice has made it difficult to determine the function or relationship between these proteins. We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied. We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF. Moreover, we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes, including EBF, IL7Rα, λ5, and Rag-1, and the ability to induce κ light chain in response to mitogen. Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12-inducible traits. Our data demonstrate that, in the context of this macrophage line, E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage–associated genes. B cell development transcription lineage determination gene expression Footnotes B.L. Kee was supported by a fellowship from the Medical Research Council of Canada and is a Special Fellow of the Leukemia Society of America. This work was funded by the National Institutes of Health (NIH), the Council for Tobacco Research and the Edward Mallinckrodt Jr. Foundation. Abbreviations used in this paper: bHLH basic helix-loop-helix c-fms macrophage colony-stimulating factor EBF early B cell factor EMSA electrophoretic mobility shift assay RT reverse transcriptase Submitted: 17 April 1998 Revision received 28 May 1998

Miyazaki, Toru; Lemonnier, François A.

doi: 10.1084/jem.188.4.715pmid: 9705953

The potential involvement of early growth response (Egr)-1, a zinc-finger transcription factor belonging to the immediate-early genes, in positive/negative selection of thymocytes has been implicated by its expression in the population of CD4 + CD8 + double positive (DP) cells undergoing selection. To further investigate this possibility, transgenic mice overexpressing Egr-1 in thymocytes were bred with a transgenic mouse line expressing a T cell receptor (TCR) recognizing the H-Y male antigen in the context of H-2 b class I major histocompatibility complex (MHC) molecules. In Egr-1/TCR H-Y double-transgenic mice, efficient positive selection of H-Y CD8 + T cells occurred, even in mice on either a nonselecting H-2 d background or a β2-microglobulin (β2m)-deficient background in which the expression of class I MHC heavy chains is extremely low; no positive selection was observed on a K b−/− D b−/− β2m −/− background where class I MHC expression is entirely absent. Similarly, when the Egr-1 transgene was introduced into a class II MHC–restricted TCR transgenic mouse line, Egr-1/TCR double-transgenic mice revealed increased numbers of CD4 + T cells selected by class II MHC, as well as significant numbers of CD8 + T cells selected by class I MHC (for which the transgenic TCR might have weak affinity). Thus, Egr-1 overexpression allows positive selection of thymocytes via TCR–MHC interactions of unusually low avidity, possibly by lowering the threshold of avidity required for positive selection. Supporting this possibility, increased numbers of alloreactive T cells were positively selected in Egr-1 transgenic mice, resulting in a strikingly enhanced response against allo-MHC. These results suggest that expression of Egr-1 and/or its target gene(s) may directly influence the thresholds required for thymocyte selection. Egr-1 positive selection T cell avidity transgenic mouse Footnotes F.A. Lemonnier is supported by the Association pour la Recherche sur le Cancer. Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche Ltd. (Basel, Switzerland). Abbreviations used in this paper: DP double positive Egr-1 early growth response 1 Egr/H-Y Egr-1 transgene–positive H-Y transgenic I 0 class I MHC–deficient II 0 class II MHC–deficient ISP immature SP NL/H-Y Egr-1 transgene–negative H-Y transgenic PNAr peanut agglutinin receptor RAG recombination-activating gene SP single positive TG transgenic Submitted: 21 April 1998 Revision received 3 June 1998