The Journal of Experimental Medicine

Toes, René E.M.; Ossendorp, Ferry; Offringa, Rienk; Melief, Cornelis J.M.

doi: 10.1084/jem.189.5.753pmid: 10049938

Pieper, Rembert; Christian, Robert E.; Gonzales, Monica I.; Nishimura, Michael I.; Gupta, Gaorav; Settlage, Robert E.; Shabanowitz, Jeffrey; Rosenberg, Steven A.; Hunt, Donald F.; Topalian, Suzanne L.

doi: 10.1084/jem.189.5.757pmid: 10049939

CD4 + T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II–restricted human CD4 + T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4 + tumor-infiltrating lymphocyte (TIL) line. The HLA-DRβ1*0101–restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer. melanoma antigen CD4 + T cells HLA-DR1 triosephosphate isomerase Footnotes 1 Abbreviations used in this paper: ID, inner diameter; MART, melanoma antigen recognized by T cells; μESI, microelectrospray ionization; MS, mass spectrometry; OD, outer diameter; TIL, tumor-infiltrating lymphocyte(s); TPI, triosephosphate isomerase. Submitted: 11 November 1998 Revision received 9 December 1998

Chaux, Pascal; Vantomme, Valérie; Stroobant, Vincent; Thielemans, Kris; Corthals, Jurgen; Luiten, Rosalie; Eggermont, Alexander M.M.; Boon, Thierry; van der Bruggen, Pierre

doi: 10.1084/jem.189.5.767pmid: 10049940

MAGE -type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE -type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE -encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4 + T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4 + T cells. We isolated CD4 + T cell clones that recognized two different MAGE-3 epitopes, MAGE-3 114–127 and MAGE-3 121–134 , both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1 , -2 , and - 6 . Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination. human invariant chain peptide tumor histocompatibility leukocyte antigen class II Footnotes P. Chaux and R. Luiten were supported by a postdoctoral fellowship from the Training and Mobility of Researchers Program of the European Commission. V. Vantomme was partially supported by the Fonds National de la Recherche Scientifique (TELEVIE grants), Brussels, Belgium. This work was partially supported by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming, and by grants from the Association contre le Cancer, Brussels, Belgium, from the BIOMED 2 programme of the European Community, from the Fonds J. Maisin, Belgium, and from the Caisse Générale d'Epargne et de Retraite (CGER)-Assurances and VIVA, Brussels, Belgium. Abbreviations used in this paper: aa amino acid(s) Ii invariant chain(s) Submitted: 11 December 1998

Kehren, Jeanne; Desvignes, Cyril; Krasteva, Maya; Ducluzeau, Marie-Thérèse; Assossou, Olga; Horand, Françoise; Hahne, Michael; Kägi, David; Kaiserlian, Dominique; Nicolas, Jean-François

doi: 10.1084/jem.189.5.779pmid: 10049941

Contact hypersensitivity (CHS) is a T cell–mediated skin inflammation induced by epicutaneous exposure to haptens in sensitized individuals. We have previously reported that CHS to dinitrofluorobenzene in mice is mediated by major histocompatibility complex (MHC) class I–restricted CD8 + T cells. In this study, we show that CD8 + T cells mediate the skin inflammation through their cytotoxic activity. The contribution of specific cytotoxic T lymphocytes (CTLs) to the CHS reaction was examined both in vivo and in vitro, using mice deficient in perforin and/or Fas/Fas ligand (FasL) pathways involved in cytotoxicity. Mice double deficient in perforin and FasL were able to develop hapten-specific CD8 + T cells in the lymphoid organs but did not show CHS reaction. However, they did not generate hapten-specific CTLs, demonstrating that the CHS reaction is dependent on cytotoxic activity. In contrast, Fas-deficient lpr mice, FasL-deficient gld mice, and perforin-deficient mice developed a normal CHS reaction and were able to generate hapten-specific CTLs, suggesting that CHS requires either the Fas/FasL or the perforin pathway. This was confirmed by in vitro studies showing that the hapten-specific CTL activity was exclusively mediated by MHC class I–restricted CD8 + T cells which could use either the perforin or the Fas/FasL pathway for their lytic activity. Thus, cytotoxic CD8 + T cells, commonly implicated in the host defence against tumors and viral infections, could also mediate harmful delayed-type hypersensitivity reactions. cytotoxic T lymphocyte contact hypersensitivity contact dermatitis hapten dinitrofluorobenzene CD8 + T cells Footnotes Abbreviations used in this paper: CHS contact hypersensitivity DNBS dinitrobenzene sulfonic acid DNFB dinitrofluorobenzene DTH delayed-type hypersensitivity ELISPOT enzyme-linked immunospot HPRT hypoxanthine phosphoribosyltransferase I 0/0 MHC class I–deficient II 0/0 MHC class II–deficient P 0/0 perforin-deficient LC Langerhans cell SFC spot-forming cell Submitted: 5 June 1998 Revision received 11 January 1999

Cantoni, Claudia; Bottino, Cristina; Vitale, Massimo; Pessino, Anna; Augugliaro, Raffaella; Malaspina, Angela; Parolini, Silvia; Moretta, Lorenzo; Moretta, Alessandro; Biassoni, Roberto

doi: 10.1084/jem.189.5.787pmid: 10049942

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2– activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor–associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-γ/δ + T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6. natural killer cells activating receptor natural cytotoxicity immunoglobulin superfamily cDNA Footnotes 1 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activating motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; KARAP, killer activating receptor–associated polypeptide; KIR, killer inhibitory receptor; ORF, open reading frame; RT-PCR, reverse transcriptase-polymerase chain reaction; SF, superfamily. Submitted: 14 August 1998 Revision received 5 November 1998

Basu, Sreyashi; Srivastava, Pramod K.

doi: 10.1084/jem.189.5.797pmid: 10049943

Calreticulin (CRT), a peptide-binding heat shock protein (HSP) of the endoplasmic reticulum (ER), has been shown previously to associate with peptides transported into the ER by transporter associated with antigen processing (Spee, P., and J. Neefjes. 1997. Eur. J. Immunol. 27: 2441–2449). Our studies show that CRT preparations purified from tumors elicit specific immunity to the tumor used as the source of CRT but not to an antigenically distinct tumor. The immunogenicity is attributed to the peptides associated with the CRT molecule and not to the CRT molecule per se. It is further shown that CRT molecules can be complexed in vitro to unglycosylated peptides and used to elicit peptide-specific CD8 + T cell response in spite of exogenous administration. These characteristics of CRT closely resemble those of HSPs gp96, hsp90, and hsp70, although CRT has no apparent structural homologies to them. heat shock protein stress protein antigen presentation cytotoxic T lymphocyte response cancer immunity Footnotes Note added in proof. During the course of these studies, we have learned that C. Nicchita, E. Gilboa, and colleagues have independently discovered that calreticulin can elicit, in a cell- and tumor-specific manner, CTL responses (Nair, S., P. Wearsch, D.A. Mitchell, J.J. Wassenberg, E. Gilboa, and C.V. Nicchita. Calreticulin displays in vivo peptide binding activity and can elicit cytotoxic T lymphocyte responses against bound peptides. J. Immunol. In press.). Abbreviations used in this paper: CRT calreticulin ER endoplasmic reticulum HSP heat shock protein PDI protein disulfide isomerase TAP transporter associated with antigen processing VSV vesicular stomatitis virus Submitted: 17 August 1998 Revision received 15 December 1998

Schüler, Thomas; Qin, Zhihai; Ibe, Sabrina; Noben-Trauth, Nancy; Blankenstein, Thomas

doi: 10.1084/jem.189.5.803pmid: 10049944

It is widely accepted that cellular immune responses are induced by CD4 + T helper 1 (Th1) cells secreting interleukin (IL)-2 and interferon (IFN)-γ. Tumor immunity is often mediated by cytotoxic T lymphocytes (CTLs) whose activation is supported by Th1 cytokines. Since IL-4 directs Th2 development and has been shown to inhibit Th1-dominated responses, we assumed that IL-4–deficient (IL-4 −/− ) mice would develop vigorous CTL-mediated tumor immunity compared with IL-4–competent (IL-4 +/+ ) mice. Surprisingly, IL-4 −/− mice were severely impaired to develop tumor immunity to both a mammary adenocarcinoma line and a colon carcinoma line. The lack of tumor immunity in IL-4 −/− mice was associated with reduced IFN-γ production, diminished levels of tumor-reactive serum IgG2a, and undetectable CTL activity, indicating a defective Th1 response in the absence of endogenous IL-4. Anti–IL-4 monoclonal antibody blocked tumor immunity in IL-4 +/+ mice when administered at the time of immunization but not at the time of challenge. Additionally, tumor immunity could be induced in IL-4 −/− mice, if IL-4 was provided by gene-modified cells together with immunizing tumor cells. These results demonstrate that tumor immunity requires IL-4 in the priming phase for the generation of effector cells rather than for their maintenance and exclude secondary, developmental defects in the “knockout” strain. Together, our results demonstrate a novel and previously unanticipated role of IL-4 for the generation of Th1-associated, CTL-mediated tumor immunity. tumor vaccination interleukin 4 T cell immunity interleukin 4–deficient mice Footnotes Submitted: 5 October 1998 Revision received 29 December 1998

Sarma, Supria; Guo, Yong; Guilloux, Yannik; Lee, Cheng; Bai, Xue-Feng; Liu, Yang

doi: 10.1084/jem.189.5.811pmid: 10049945

Unmutated tumor antigens are chosen as primary candidates for tumor vaccine because of their expression on multiple lineages of tumors. A critical issue is whether unmutated tumor antigens are expressed in normal cells, and if so, whether such expression imposes special restrictions on cytotoxic T lymphocyte (CTL) responses. In this study, we use a transgenic approach to study the development and effector function of T cells specific for P1A, a prototypical unmutated tumor antigen. We report here that although P1A is expressed at low levels in normal tissues, including lymphoid tissues, the P1A-specific transgenic T cells develop normally and remain highly responsive to the P1A antigen. The fact that transgenic expression of P1A antigen in the thymus induces T cell clonal deletion demonstrates that normal hematopoietic cells can process and present the P1A antigen and that P1A-specific T cells are susceptible to clonal deletion. By inference, P1A-specific T cells must have escaped clonal deletion due to low expression of P1A in the thymus. Interestingly, despite the fact that an overwhelming majority of T cells in the T cell receptor for antigen (TCR)–transgenic mice are specific for P1A, these mice are no more resistant to a P1A-expressing plasmocytoma than nontransgenic littermates. Moreover, when the same TCR-transgenic mice were challenged simultaneously with B7-1 + and B7-1 − tumors, only B7-1 + tumors were rejected. Therefore, even though P1A can be a tumor rejection antigen, the effector function of P1A-specific CTL is restrained in vivo. These results have important implications for the strategy of tumor immunotherapy. cytotoxic T lymphocytes unmutated tumor antigen T cell development antitumor immunity T cell receptor–transgenic mice Footnotes This study is supported by grants from the National Institutes of Health (NIH) (CA58033 and CA69091), Kaplan Comprehensive Center of New York University Medical Center, and Ohio State University Comprehensive Cancer Center. Part of the study was carried out when Supria Sarma was supported by NIH training grant CA09161. Present addresses are as follows: S. Sarma, Howard Hughes Medical Institute, Rockefeller University, 1230 York Ave., New York, NY 10021; Y. Guo, Hoechst Marion Rousse, Inc., CNS-Molecular Biology, Bridgewater, NJ 08807; C. Lee, 3161 Broadway, Apt. 6C, New York, NY 10027; X.-F. Bai, Department of Pathology, Ohio State University Medical Center, 146 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210; and Y. Guilloux, Institut de Biologie, INSERM U463, 9 Quai, Moncousu, 44093, Nantes cedex 1, France. S. Sarma, Y. Guo, Y. Guilloux, and X.-F. Bai contributed equally to this study. Abbreviations used in this paper: E μ IgM heavy chain enhancer GAPDH glyceraldehyde 3-phosphate dehydrogenase RT reverse transcriptase SW Swiss Webster TG transgenic mouse Submitted: 20 October 1998 Revision received 7 January 1999

Cella, Marina; Salio, Mariolina; Sakakibara, Yoichi; Langen, Hanno; Julkunen, Ilkka; Lanzavecchia, Antonio

doi: 10.1084/jem.189.5.821pmid: 10049946

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses. dendritic cell maturation and activation influenza virus double-stranded RNA type I interferon MxA Footnotes M. Salio holds an EC fellowship (EUNIDI). The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche, Basel, Switzerland. Abbreviations used in this paper: CD40L CD40 ligand DC dendritic cell ds double-stranded MOI multiplicity of infection Submitted: 10 November 1998 Revision received 7 January 1999

Gruenheid, Samantha; Canonne-Hergaux, François; Gauthier, Susan; Hackam, David J.; Grinstein, Sergio; Gros, Philippe

doi: 10.1084/jem.189.5.831pmid: 10049947

The natural resistance associated macrophage protein ( Nramp ) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90–100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe 2+ across the endosomal membrane and into the cytoplasm. iron anemia transport infection macrophage Footnotes 1 Abbreviations used in this paper: CHO, Chinese hamster ovary; CHON, Nramp-transfected CHO cells; Endo H, Endo-β-acetylglucosaminidase H; GST, glutathione S-transferase; MEL, erythroleukemia; Nramp, natural resistance associated macrophage protein; TM, transmembrane. Submitted: 29 September 1998 Revision received 11 December 1998