The Journal of Experimental Medicine

Farr, Andrew G.; Rudensky, Alexander

doi: 10.1084/jem.188.1.1pmid: 9653078

Klein, Ludger; Klein, Thomas; Rüther, Ulrich; Kyewski, Bruno

doi: 10.1084/jem.188.1.5pmid: 9653079

Inducible serum proteins whose concentrations oscillate between nontolerogenic and tolerogenic levels pose a particular challenge to the maintenance of self-tolerance. Temporal restrictions of intrathymic antigen supply should prevent continuous central tolerization of T cells, in analogy to the spatial limitation imposed by tissue-restricted antigen expression. Major acute-phase proteins such as human C-reactive protein (hCRP) are typical examples for such inducible self-antigens. The circulating concentration of hCRP, which is secreted by hepatocytes, is induced up to 1,000-fold during an acute-phase reaction. We have analyzed tolerance to hCRP expressed in transgenic mice under its autologous regulatory regions. Physiological regulation of basal levels (<10 −9 M) and inducibility (>500-fold) are preserved in female transgenics, whereas male transgenics constitutively display induced levels. Surprisingly, crossing of hCRP transgenic mice to two lines of T cell receptor transgenic mice (specific for either a dominant or a subdominant epitope) showed that tolerance is mediated by intrathymic deletion of immature thymocytes, irrespective of widely differing serum levels. In the absence of induction, hCRP expressed by thymic medullary epithelial cells rather than liver-derived hCRP is necessary and sufficient to induce tolerance. Importantly, medullary epithelial cells also express two homologous mouse acute-phase proteins. These results support a physiological role of “ectopic” thymic expression in tolerance induction to acute-phase proteins and possibly other inducible self-antigens and have implications for delineating the relative contributions of central versus peripheral tolerance. self-tolerance inducible self-antigens acute-phase proteins thymic medullary epithelium deletion Footnotes This study has been supported by the DFG (Ky 7/7-1), the German Cancer Research Center (B. Kyewski) and the Volkswagenstiftung (U. Rüther). Abbreviations used in this paper: APP acute-phase protein DC dendritic cell Dep dominant epitope DN double negative DP double positive hCRP human C-reactive protein RIP rat insulin promoter mSAP mouse serum amyloid P component Sep subdominant epitope SP single positive Tag large T antigen Submitted: 3 March 1998 Revision received 1 April 1998

Smith, David J.; Salmi, Marko; Bono, Petri; Hellman, Jukka; Leu, Taina; Jalkanen, Sirpa

doi: 10.1084/jem.188.1.17pmid: 9653080

Vascular adhesion protein 1 (VAP-1) is a human endothelial sialoglycoprotein whose cell surface expression is induced under inflammatory conditions. It has been shown previously to participate in lymphocyte recirculation by mediating the binding of lymphocytes to peripheral lymph node vascular endothelial cells in an L-selectin–independent fashion. We report here that the VAP-1 cDNA encodes a type II transmembrane protein of 84.6 kD with a single transmembrane domain located at the NH 2 -terminal end of the molecule and six potential N -glycosylation sites in the extracellular domain. In vivo, the protein exists predominantly as a homodimer of 170–180 kD. Ax endothelial cells transfected with a VAP-1 cDNA express VAP-1 on their cell surface and bind lymphocytes, and the binding can be partially inhibited with anti–VAP-1 mAbs. VAP-1 has no similarity to any currently known adhesion molecules, but has significant identity to the copper-containing amine oxidase family and has a monoamine oxidase activity. We propose that VAP-1 is a novel type of adhesion molecule with dual function. With the appropriate glycosylation and in the correct inflammatory setting, its expression on the lumenal endothelial cell surface allows it to mediate lymphocyte adhesion and to function as an adhesion receptor involved in lymphocyte recirculation. Its primary function in other locations where it is expressed, such as smooth muscle, may depend on its inherent monoamine oxidase activity. vascular adhesion protein 1 adhesion molecule monoamine oxidase sialoglycoprotein endothelial Footnotes D. Smith was a postdoctoral fellow of the European Molecular Biology Organisation and subsequently in receipt of a European Community Human Capital and Mobility Fellowship during the course of this work. T. Leu's current address is Department of Medical Microbiology, University of Turku, FIN-20520 Turku, Finland. Abbreviations used in this paper: BSAO bovine serum amine oxidase CHO Chinese hamster ovary DAO diamine oxidase GCG Genetics Computer Group HEV high endothelial venule(s) MAO monoamine oxidase PLN peripheral lymph node(s) PNAd PLN addressin SSAO semicarbazide-sensitive amine oxidase VAP-1 vascular adhesion protein 1 Submitted: 18 September 1997 Revision received 5 March 1998

Putterman, Chaim; Diamond, Betty

doi: 10.1084/jem.188.1.29pmid: 9653081

Anti–double-stranded DNA (dsDNA) antibodies are the serologic abnormality characteristically associated with systemic lupus erythematosus (SLE) and may play an important role in disease pathogenesis. Although the anti-dsDNA antibodies present in SLE are indicative of an antigen-driven response, the antigen has not been conclusively identified. By screening a phage peptide display library, we demonstrated previously that the decapeptide DWEYSVWLSN is specifically bound by the pathogenic murine IgG2b anti-dsDNA antibody R4A. To investigate the possibility that a protein antigen might trigger lupus-like autoimmunity, we immunized BALB/c mice with DWEYSVWLSN in adjuvant. Mice developed significant titers of IgG anti-dsDNA antibodies 2–3 wk after the initial immunization. Immunized mice also developed antibodies against some other lupus autoantigens, and immunoglobulin deposition was present in renal glomeruli at 49 d. Although an immune response to peptide and dsDNA was evident in BALB/c mice, there was little response in other inbred strains. This study demonstrates that lupus-like anti-dsDNA reactivity can be generated in nonautoimmune mice by immunization with a peptide antigen. Peptide-induced autoimmunity may prove useful in understanding the spreading of antigenic specificities targeted in SLE. However, most importantly, the demonstration that a peptide antigen can initiate a SLE-like immune response opens a new chapter on the potential antigenic stimuli that might trigger SLE. systemic lupus erythematosus anti-DNA peptide library autoantibodies inbred strains Footnotes Abbreviations used in this paper: ANA antinuclear antibody dsDNA double-stranded DNA GXM glucoronoxylomannan MAP multiple antigenic peptide RNP ribonucleoprotein Submitted: 31 October 1997 Revision received 17 April 1998

Omer, Fakhereldin M.; Riley, Eleanor M.

doi: 10.1084/jem.188.1.39pmid: 9653082

We have examined the role of the immunomodulatory cytokine transforming growth factor (TGF)-β in the resolution and pathology of malaria in BALB/c mice. Circulating levels of TGF-β, and production of bioactive TGF-β by splenocytes, were found to be low in lethal infections with Plasmodium berghei . In contrast, resolving infections with P. chabaudi chabaudi or P. yoelii were accompanied by significant TGF-β production. A causal association between the failure to produce TGF-β and the severity of malaria infection was demonstrated by treatment of infected mice with neutralizing antibody to TGF-β, which exacerbated the virulence of P. berghei and transformed a resolving P. chabaudi chabaudi infection into a lethal infection, but had little effect on the course of P. yoelii infection. Parasitemia increased more rapidly in anti–TGF-β–treated mice but this did not seem to be the explanation for the increased pathology of infection as peak parasitemias were unchanged. Treatment of P. berghei –infected mice with recombinant TGF-β (rTGF-β) slowed the rate of parasite proliferation and prolonged their survival from 15 to up to 35 d. rTGF-β treatment was accompanied by a significant decrease in serum tumor necrosis factor α and an increase in interleukin 10. Finally, we present evidence that differences in TGF-β responses in different malaria infections are due to intrinsic differences between species of malaria parasites in their ability to induce production of TGF-β. Thus, TGF-β seems to induce protective immune responses, leading to slower parasite growth, early in infection, and, subsequently, appears to downregulate pathogenic responses late in infection. This duality of effect makes TGF-β a prime candidate for a major immunomodulatory cytokine associated with successful control of malaria infection. transforming growth factor β malaria tumor necrosis factor α interleukin 10 immunomodulation Footnotes Submitted: 15 November 1997 Revision received 18 February 1998

Lüneberg, Edeltraud; Zähringer, Ulrich; Knirel, Yuriy A.; Steinmann, Dorothee; Hartmann, Maike; Steinmetz, Ivo; Rohde, Manfred; Köhl, Jörg; Frosch, Matthias

doi: 10.1084/jem.188.1.49pmid: 9653083

With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625–positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625–positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila . This is the first description of phase-variable expression of Legionella LPS. Legionella pneumophila LPS phase-variation serum resistance virulence Footnotes This work is dedicated to Dieter Bitter-Suermann on the occasion of his 60 th birthday. Abbreviations used in this paper: BYCE buffered CYE CI chemical ionization CYE charcoal yeast extract EI electron impact GLC gas–liquid chromatography MAC membrane attack complex MOMP major outer membrane protein NHS normal human serum NMR nuclear magnetic resonance SG serogroup Submitted: 5 December 1997 Revision received 8 April 1998

Busch, Dirk H.; Pilip, Ingrid; Pamer, Eric G.

doi: 10.1084/jem.188.1.61pmid: 9653084

The mechanisms underlying the genesis and maintenance of T cell memory remain unclear. In this study, we examined the evolution of a complex, antigen-specific T cell population during the transition from primary effector to memory T cells after Listeria monocytogenes infection. T cell populations specific for listeriolysin O (LLO) 91–99 , the immunodominant epitope recognized by H2-K d –restricted T lymphocytes, were directly identified in immune spleens using tetrameric H2-K d –epitope complexes. The T cell receptor (TCR) Vβ repertoire of specific T cells was determined by direct, ex vivo staining with a panel of mAbs. We demonstrate that LLO 91–99 -specific, primary effector T cell populations have a diverse TCR Vβ repertoire. Analyses of memory T cell populations demonstrated similar TCR diversity. Furthermore, experiments with individual mice demonstrated that primary effector and memory T cells have indistinguishable TCR repertoires. Remarkably, after reinfection with L. monocytogenes , LLO 91–99 -specific T cells have a narrower TCR repertoire than do primary effector or memory T cells. Thus, our studies show that the TCR repertoire of primary effector T lymphocytes is uniformly transmitted to memory T cells, whereas expansion of memory T cells is selective. T cell receptor repertoire cytotoxic T lymphocytes Listeria monocytogenes effector/memory T cells recall Footnotes Abbreviations used in this paper: β 2 m β 2 microglobulin BirA biotin operon repressor protein A LLO listeriolysin O SB staining buffer Submitted: 17 February 1998 Revision received 8 April 1998

Sourdive, David J.D.; Murali-Krishna, Kaja; Altman, John D.; Zajac, Allan J.; Whitmire, Jason K.; Pannetier, Christophe; Kourilsky, Philippe; Evavold, Brian; Sette, Alessandro; Ahmed, Rafi

doi: 10.1084/jem.188.1.71pmid: 9653085

Viral infections often induce potent CD8 T cell responses that play a key role in antiviral immunity. After viral clearance, the vast majority of the expanded CD8 T cells undergo apoptosis, leaving behind a stable number of memory cells. The relationship between the CD8 T cells that clear the acute viral infection and the long-lived CD8 memory pool remaining in the individual is not fully understood. To address this issue, we examined the T cell receptor (TCR) repertoire of virus-specific CD8 T cells in the mouse model of infection with lymphocytic choriomeningitis virus (LCMV) using three approaches: ( a ) in vivo quantitative TCR β chain V segment and complementarity determining region 3 (CDR3) length repertoire analysis by spectratyping (immunoscope); ( b ) identification of LCMV-specific CD8 T cells with MHC class I tetramers containing viral peptide and costaining with TCR Vβ–specific antibodies; and ( c ) functional TCR fingerprinting based on recognition of variant peptides. We compared the repertoire of CD8 T cells responding to acute primary and secondary LCMV infections, together with that of virus-specific memory T cells in immune mice. Our analysis showed that CD8 T cells from several Vβ families participated in the anti-LCMV response directed to the dominant cytotoxic T lymphocyte (CTL) epitope (NP118–126). However, the bulk (∼70%) of this CTL response was due to three privileged T cell populations systematically expanding during LCMV infection. Approximately 30% of the response consisted of Vβ10 + CD8 T cells with a β chain CDR3 length of nine amino acids, and 40% consisted of Vβ8.1 + (β CDR3 = eight amino acids) and Vβ8.2 + cells (β CDR3 = six amino acids). Finally, we showed that the TCR repertoire of the primary antiviral CD8 T cell response was similar both structurally and functionally to that of the memory pool and the secondary CD8 T cell effectors. These results suggest a stochastic selection of memory cells from the pool of CD8 T cells activated during primary infection. immunological memory CD8 T cells viral immunity T cell receptor lymphocytic choriomeningitis virus Footnotes C. Pannetier's current address is Laboratory of Immunology, National Institute of Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Abbreviations used in this paper: C constant D diversity ELISPOT enzyme-linked immunospot J joining LCMV lymphocytic choriomeningitis virus LDA limiting dilution assay PCC pigeon cytochrome C V variable Submitted: 18 February 1998 Revision received 9 April 1998

Chun, Tae-Wook; Engel, Delphine; Mizell, Stephanie B.; Ehler, Linda A.; Fauci, Anthony S.

doi: 10.1084/jem.188.1.83pmid: 9653086

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1–infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4 + T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4 + T cells derived from HIV-infected individuals who are antiretroviral therapy–naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro . Given that an array of cytokines, including IL-6, TNF-α, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART. HIV-1 latency resting CD4 + T cells cytokines antiretroviral therapy Footnotes Abbreviation used in this paper: HAART highly active antiretroviral therapy Submitted: 26 February 1998 Revision received 4 May 1998

Chan, Vivien W.F.; Mecklenbräuker, Ingrid; Su, I-hsin; Texido, Gemma; Leitges, Michael; Carsetti, Rita; Lowell, Clifford A.; Rajewsky, Klaus; Miyake, Kensuke; Tarakhovsky, Alexander

doi: 10.1084/jem.188.1.93pmid: 9653087

The B cell–specific transmembrane protein RP-105 belongs to the family of Drosophila toll -like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C β I/II (PKCβI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105–mediated signaling cascade in B cells. We also find that negative regulation of RP-105–mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor–mediated arrest of RP-105–mediated B cell proliferation. RP-105 B lymphocytes signal transduction mice Footnotes Abbreviations used in this paper: BCR B cell antigen receptor CsA cyclosporin A dnMEK double negative mutant of MEK FcγR Fcγ receptors MAP mitogen-activated protein MEK MAP kinase kinase PAMPS pathogen-associated molecular pattern PKCβI/II protein kinase C β I/II PRR pattern recognition receptor PTK protein tyrosine kinase sIgM surface IgM Submitted: 26 March 1998 Revision received 17 April 1998