The Journal of Experimental Medicine

Pende, Daniela; Parolini, Silvia; Pessino, Anna; Sivori, Simona; Augugliaro, Raffaella; Morelli, Luigia; Marcenaro, Emanuela; Accame, Laura; Malaspina, Angela; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro

doi: N/Apmid: 10562324

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3ζ chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies. natural killer cells triggering receptor natural cytotoxicity Footnotes 1used in this paper: GAM, goat anti–mouse antiserum; HRPO, horseradish peroxidase; Ig-SF, Ig superfamily; ITAM, immunoreceptor tyrosine-based activating motif; KIR, killer inhibitory receptor; NCR, natural cytotoxicity receptor; ORF, open reading frame; RT, reverse transcriptase Submitted: 12 July 1999 Revision requested 13 September 1999 Accepted: 14 September 1999

Richter, Anne; Löhning, Max; Radbruch, Andreas

doi: N/Apmid: 10562319

T helper (Th) lymphocytes, when reactivated, recall expression of those cytokines they had been instructed to express in earlier activations, even in the absence of specific cytokine-inducing factors. In cells that memorize their expression, the cytokine genes are modified by chromatin rearrangement and demethylation, suggesting that they have been somatically imprinted. Here we show, by using inhibitors blocking the cell cycle in various stages, that for the instruction of a Th cell to express interleukin (IL)-4 or IL-10 upon restimulation, entry of the cell into the S phase of the first cell cycle after initial activation is required. Separation of the IL-4 receptor (IL-4R) and T cell antigen receptor (TCR) signals in time, demonstrates that this instruction is dependent on concomitant signaling from both receptors. In Th cells, inhibited to progress into the first S phase after activation, the IL-4R and TCR signals can be memorized for at least 1 d, priming the T cell to become instructed for expression of IL-4 upon restimulation, when entering the S phase after release of the cell cycle block. The requirement of the initial S phase of T cell activation, for instruction of Th cells to express IL-4 or IL-10 upon restimulation points to the decisive role of epigenetic modification of cytokine genes as a molecular correlate of the memory to express particular cytokines. cytokine memory T cell differentiation interleukin 4 interleukin 10 CFSE Footnotes 1used in this paper: CFSE, 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester; Dig, digoxigenin; MACS, high-gradient magnetic cell separation; STAT, signal transducer and activator of transcription; tg, transgenic Submitted: 22 April 1999 Revision requested 24 August 1999 Accepted: 7 September 1999

Rebel, Vivienne I.; Hartnett, Sheila; Hill, Geoffrey R.; Lazo-Kallanian, Suzan B.; Ferrara, James L.M.; Sieff, Colin A.

doi: N/Apmid: 10562323

Hematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-α as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55 −/− mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75 −/− or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55 −/− mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55 −/− marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level. hematopoietic stem cell cell differentiation cytokine receptors Footnotes G.R. Hill's present address is Mater Medical Research Institute, South Brisbane QLD 4101, Australia; J.L.M. Ferrara's present address is Departments of Internal Medicine and Pediatrics, Division of Hematology and Oncology, University of Michigan Cancer Center, Ann Arbor, MI 48109. Abbreviations used in this paper: BFU-E, erythroid burst-forming unit; BM, bone marrow; CFC, colony-forming cell; CFU-GEMM, multilineage CFU; CFU-G/M, granulocyte/macrophage CFU; CFU-S, CFU in the spleen; CRU, competitive repopulating unit(s); HSC, hematopoietic stem cell; Lin, lineage; PB, peripheral blood; PI, propidium iodide; SSC, side scatter; WBC, white blood cell; WT, wild-type. J.L.M. Ferrara and C.A. Sieff contributed equally to this paper. Submitted: 7 July 1999 Revision requested 3 September 1999 Accepted: 7 September 1999

Cao, Ming Yu; Davidson, Dominique; Yu, Jie; Latour, Sylvain; Veillette, André

doi: N/Apmid: 10562326

We have identified a novel Src homology 2 domain–containing leukocyte protein of 76 kD (SLP-76)–related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling. adaptor SLP-76 Blnk signaling lymphocytes Footnotes 1used in this paper: BCR, B cell antigen receptor; Blnk, B cell linker protein; BMMC, bone marrow–derived mast cell; Clnk, cytokine-dependent hemopoietic cell linker; Gads, Grb2-related adaptor downstream of Shc; NFAT, nuclear factor of activated T cells; PECAM, platelet-endothelial cell adhesion molecule; PLC, phospholipase C; RAH, rabbit anti–hamster; SAM, sheep anti–mouse; SH2, Src homology 2; SLP, SH2 domain–containing leukocyte protein of 76 kD M.Y. Cao and D. Davidson contributed work of equal importance to this paper and both should be viewed as first author. Submitted: 29 July 1999 Revision requested 9 September 1999 Accepted: 10 September 1999

Bell, Diana; Chomarat, Pascale; Broyles, Denise; Netto, George; Harb, Ghada Moumneh; Lebecque, Serge; Valladeau, Jenny; Davoust, Jean; Palucka, Karolina A.; Banchereau, Jacques

doi: N/Apmid: 10562317

We have analyzed the presence of immature and mature dendritic cells (DCs) within adenocarcinoma of the breast using immunohistochemistry. Immature DCs were defined by expression of CD1a-, Langerin-, and intracellular major histocompatibility complex class II–rich vesicles. Mature DCs were defined by expression of CD83 and DC-Lamp. Breast carcinoma cells were defined by morphology and/or cytokeratin expression. We demonstrate two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature CD1a + DCs, mostly of the Langerhans cell type (Langerin + ), were retained within the tumor bed in 32/32 samples and (b) mature DCs, CD83 + DC-Lamp + , present in 20/32 samples, are confined to peritumoral areas. The high numbers of immature DCs found in the tumor may be best explained by high levels of macrophage inflammatory protein 3α expression by virtually all tumor cells. Confirming the immature/mature DC compartmentalization pattern, in vitro–generated immature DCs adhere to the tumor cells, whereas mature DCs adhere selectively to peritumoral areas. In some cases, T cells are clustering around the mature DCs in peritumoral areas, thus resembling the DC–T cell clusters of secondary lymphoid organs, which are characteristic of ongoing immune reactions. breast cancer dendritic cells tumor immunity MHC class II chemokines Footnotes 1used in this paper: DCs, dendritic cells; HPCs, hematopoietic progenitors; int, interstitial; LCs, Langerhans cells; MIP, macrophage inflammatory protein; TAAs, tumor-associated antigens Presented partly at the 5th International Symposium on Dendritic Cells in Fundamental and Clinical Immunology, Pittsburgh, Pennsylvania, September 23–28, 1998. Submitted: 20 July 1999 Revision requested 7 September 1999 Accepted: 10 September 1999

Cordon-Cardo, Carlos; Prives, Carol

doi: N/Apmid: 10562311

Hudson, James D.; Shoaibi, Mahmood A.; Maestro, Roberta; Carnero, Amancio; Hannon, Gregory J.; Beach, David H.

doi: N/Apmid: 10562313

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis. macrophage migration inhibitory factor p53 inflammation and cancer growth arrest apoptosis Footnotes 1used in this paper: FBS, fetal bovine serum; GFP, green fluorescent protein; GSNO, S-nitrosoglutathione; MBP, maltose binding protein; MEF, mouse embryonic fibroblast; MIF, macrophage migration inhibitory factor; NO, nitric oxide; SNP, sodium nitroprusside Submitted: 26 July 1999 Accepted: 5 August 1999

Fernandez, Victor; Hommel, Marcel; Chen, Qijun; Hagblom, Per; Wahlgren, Mats

doi: N/Apmid: 10562315

Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30–45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P . falciparum . Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5–6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P . falciparum . Prominent polypeptides of 20, 22, 76–80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro–propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P . falciparum on the surface of the infected erythrocyte. P . falciparum surface antigen rif gene family antibodies strain-specific Footnotes 1used in this paper: CHO, chinese hamster ovary; HUVECs, human umbilical vein endothelial cells; PECAM1, platelet/endothelial cell adhesion molecule 1; PfEMP1, P. falciparum erythrocyte membrane protein 1 Submitted: 26 February 1999 Revision requested 3 August 1999 Accepted: 3 September 1999

Bosselut, Rémy; Zhang, Weiguo; Ashe, Jennifer M.; Kopacz, Jeffrey L.; Samelson, Lawrence E.; Singer, Alfred

doi: N/Apmid: 10562325

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4 + CD8 + thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex–engaged TCR complexes. T cell receptor development signaling thymus phosphorylation Footnotes 1used in this paper: GEM, glycolipid-enriched membrane microdomain; ITAM, immunoreceptor tyrosine-activated motif; LAT, linker for activation of T cells; PI-3, phosphatidylinositol-3; PLC, phospholipase C; PTK, protein tyrosine kinase; SH2, src homology 2 Submitted: 22 July 1999 Revision requested 10 September 1999 Accepted: 16 September 1999

Adamko, Darryl J.; Yost, Bethany L.; Gleich, Gerald J.; Fryer, Allison D.; Jacoby, David B.

doi: N/Apmid: 10562321

Asthma exacerbations, many of which are virus induced, are associated with airway eosinophilia. This may reflect altered inflammatory response to viruses in atopic individuals. Inhibitory M 2 muscarinic receptors (M 2 Rs) on the airway parasympathetic nerves limit acetylcholine release. Both viral infection and inhalational antigen challenge cause M 2 R dysfunction, leading to airway hyperresponsiveness. In antigen-challenged, but not virus-infected guinea pigs, M 2 R dysfunction is due to blockade of the receptors by the endogenous antagonist eosinophil major basic protein (MBP). We hypothesized that sensitization to a nonviral antigen before viral infection alters the inflammatory response to viral infection, so that M 2 R dysfunction and hyperreactivity are eosinophil mediated. Guinea pigs were sensitized to ovalbumin intraperitoneally, and 3 wk later were infected with parainfluenza. In sensitized, but not in nonsensitized animals, virus-induced hyperresponsiveness and M 2 R dysfunction were blocked by depletion of eosinophils with antibody to interleukin (IL)-5 or treatment with antibody to MBP. An additional and unexpected finding was that sensitization to ovalbumin caused a marked (80%) reduction in the viral content of the lungs. This was reversed by the antibody to IL-5, implicating a role for eosinophils in viral immunity. eosinophils muscarinic receptors parainfluenza virus antigen challenge viral immunity Footnotes 1used in this paper: AbIL5, antibody to IL-5; AbMBP, antibody to eosinophil MBP; ECP, eosinophil cationic protein; MBP, major basic protein; M2R, M2 muscarinic receptor; NO, nitric oxide; Ppi, pulmonary inflation pressure; RSV, respiratory syncytial virus; TCID50, tissue culture ID50 Some of the data in this manuscript were published previously in abstract form (Adamko, D.J., B.L. Yost, A.D. Fryer, and D.B. Jacoby. 1999. Am. J. Respir. Crit. Care Med. 159:229). Submitted: 9 June 1999 Revision requested 12 August 1999 Accepted: 10 September 1999