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Wesley, Umadevi V.; Albino, Anthony P.; Tiwari, Shakuntala; Houghton, Alan N.
doi: 10.1084/jem.190.3.311pmid: 10430620
Dipeptidyl peptidase IV (DPPIV) is a cell surface peptidase expressed by normal melanocytes, epithelial cells, and other cells. Malignant cells, including melanomas and carcinomas, frequently lose or alter DPPIV cell surface expression. Loss of DPPIV expression occurs during melanoma progression at a stage where transformed melanocytes become independent of exogenous growth factors for survival. Tetracycline-inducible expression vectors were constructed to express DPPIV in human melanoma cells. Reexpressing DPPIV in melanoma cells at or below levels expressed by normal melanocytes induced a profound change in phenotype that was characteristic of normal melanocytes. DPPIV expression led to a loss of tumorigenicity, anchorage-independent growth, a reversal in a block in differentiation, and an acquired dependence on exogenous growth factors for cell survival. Suppression of tumorigenicity and reversal of a block in differentiation were dependent on serine protease activity, assessed using mutant DPPIV molecules containing serine→alanine substitutions. Surprisingly, dependence on exogenous growth factors was not dependent on serine protease activity. Reexpression of either wild-type or mutant DPPIV rescued expression of a second putative cell surface serine peptidase, fibroblast activation protein α, which can form a heterodimer with DPPIV. This observation suggests that rescue of fibroblast activation protein α may play a role in regulating growth of melanocytic cells. These results support the view that downregulation of DPPIV is an important early event in the pathogenesis of melanoma. melanoma fibroblast activating protein α serine protease tumorigenicity differentiation Footnotes 1used in this paper: dox, doxycycline; DPPIV, dipeptidyl peptidase IV; FAPα, fibroblast activating protein α; TRP, tyrosinase-related protein; TUNEL, TdT-mediated dUTP-biotin nick-end labeling A.P. Albino's present address is American Health Foundation, 1 Dana Rd., Valhalla, NY 10595. Submitted: 5 March 1999 Revision requested 21 May 1999 Accepted: 7 June 1999
Schrader, Carol E.; Edelmann, Winfried; Kucherlapati, Raju; Stavnezer, Janet
doi: 10.1084/jem.190.3.323pmid: 10430621
Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti–δ-dextran, IL-4, IL-5, and transforming growth factor (TGF)-β1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS ® . B cells from MMR-deficient mice show a 35–75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM + IgD + B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by 3 Hthymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented. class switch recombination msh2 mlh1 pms2 Footnotes 1used in this paper: AchR, acetylcholine receptor; CSR, class switch recombination; DC-PCR, digestion-circularization PCR; DSB, double strand break; GC, germinal center; MMR, mismatch repair; nt, nucleotide(s); PALS, periarteriolar lymphatic sheath; S, switch; wt, wild-type Note added in proof. Another group has recently reported that B cells from Msh2-deficient mice show reduced IgG switching in vitro and also in vivo (Ehrenstein, M.R., and M.S. Neuberger. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:3484–3490). Submitted: 26 March 1999 Revision requested 2 June 1999 Accepted: 9 June 1999
Bos, Martine P.; Hogan, Daniel; Belland, Robert J.
doi: 10.1084/jem.190.3.331pmid: 10430622
The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae . N . gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66–Opa binding is mediated by the NH 2 -terminal domain of the receptor and occurs through protein–protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa–CD66 interaction. Neisseria gonorrhoeae carcinoembryonic antigen bacterial adhesion opacity protein mutagenesis Footnotes 1used in this paper: CAM, cell adhesion molecule; CHO, Chinese hamster ovary; CEA, carcinoembryonic antigen; GAM, goat anti–mouse; GAR, goat anti–rabbit; ICAM, intercellular adhesion molecule; IgSF, immunoglobulin superfamily; mut, mutant; N-domain, NH2-terminal Ig variable–like domain; Ngo, Neisseria gonorrhoeae; Nme, Neisseria meningitidis; Opa, opacity-related Submitted: 25 February 1999 Revision requested 20 May 1999 Accepted: 8 June 1999
Schwarz, Margaret A.; Kandel, Jessica; Brett, Jerald; Li, Jun; Hayward, Joanne; Schwarz, Roderich E.; Chappey, Olivier; Wautier, Jean-Luc; Chabot, John; Gerfo, Paul Lo; Stern, David
doi: 10.1084/jem.190.3.341pmid: 10430623
Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% ( P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II ( P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls ( P < 0.003). Tumors from human breast carcinoma–derived MDA-MB 468 cells were suppressed by >80% in EMAP II–treated animals ( P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, ≈80% were inhibited with maximum diameter <2 mm ( P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature. tumor capillary endothelium programmed cell death cell growth inhibitors blood vessels Footnotes 1used in this paper: bFGF, basic fibroblast growth factor; BrdU, 5-bromodeoxyuridine; DAP-1, 6-diamidino-2-phenylindoledilactate; EC, endothelial cell; EMAP, endothelial-monocyte activating polypeptide; LLC, Lewis lung carcinoma; meth A, methylcholanthrene A–induced fibrosarcoma; RT, reverse transcription; SMC, smooth muscle cell; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling; VEGF, vascular endothelial growth factor R. Schwarz's current address is Department of General Oncologic Surgery, City of Hope National Medical Center, 1500 East Duarte Rd., Duarte, CA 91010. Submitted: 2 March 1999 Revision requested 24 May 1999 Accepted: 2 June 1999
van Elsas, Andrea; Hurwitz, Arthur A.; Allison, James P.
doi: 10.1084/jem.190.3.355pmid: 10430624
We examined the effectiveness of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)–expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8 + and NK1.1 + cells but occurred irrespective of the presence of CD4 + T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells. CTLA-4 immunotherapy autoimmunity vitiligo melanoma Footnotes Andrea van Elsas' present address is Department of Immunohematology and Bloodbank, Tumor Immunology lab. E3-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Submitted: 26 March 1999 Revision requested 1 June 1999 Accepted: 8 June 1999
Bender, Jeremy; Mitchell, Tom; Kappler, John; Marrack, Philippa
doi: 10.1084/jem.190.3.367pmid: 10430625
We investigated the mechanism by which α/β T cells expand upon transfer to T cell–deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester–labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4 + T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4 + T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4 + T cells in irradiated mice. These data establish that class II–bound peptides control the expansion of CD4 + T cells transferred to T cell–deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice. T cell homeostasis peptides peripheral selection T cell receptor–major histocompatibility complex interaction T cell–deficiency Footnotes 1used in this paper: CFSE, carboxyfluorescein diacetate succinimidyl ester; CyC, cychrome; Ii, invariant chain; KO, knockout; SEB, staphylococcal enterotoxin B Submitted: 25 February 1999 Revision requested 19 May 1999 Accepted: 15 June 1999
Holdorf, Amy D.; Green, Jonathan M.; Levin, Steven D.; Denny, Michael F.; Straus, David B.; Link, Vinzenz; Changelian, Paul S.; Allen, Paul M.; Shaw, Andrey S.
doi: 10.1084/jem.190.3.375pmid: 10430626
The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation. Lck CD28 costimulation tyrosine kinase SH3 domain Footnotes 1used in this paper: GST, glutathione S transferase; ITAM, immunoreceptor tyrosine-based activation motif; MAP, mitogen-activated protein; SH, Src homology Submitted: 26 February 1999 Revision requested 4 June 1999 Accepted: 15 June 1999
Rabinovich, Gabriel A.; Daly, Gordon; Dreja, Hanna; Tailor, Hitakshi; Riera, Clelia M.; Hirabayashi, Jun; Chernajovsky, Yuti
doi: 10.1084/jem.190.3.385pmid: 10430627
Galectin-1 (GAL-1), a member of a family of conserved β-galactoside–binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2–polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1–expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A q -restricted, collagen type 2–specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1–mediated autoimmune disorders. galectin-1 gene therapy apoptosis collagen-induced arthritis T cells Footnotes 1used in this paper: CIA, collagen-induced arthritis; CII, collagen type II; GAL-1, galectin-1; mGAL-1, mouse GAL-1; PI, propidium iodide; RA, rheumatoid arthritis; rGAL-1, human recombinant GAL-1; TDG, thiodigalactoside Gordon Daly and Hanna Dreja contributed equally to this work. Submitted: 6 March 1999 Revision requested 6 May 1999 Accepted: 1 June 1999
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