Lymphocyte Activation and Differentiation: Fundamental and Clinical Aspects. Proceedings of the 18th International Leucocyte Culture Conference. La Grande Motte, France, June 19–24, 1987 9783110850253, 9783110107609

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Lymphocyte Activation and Differentiation Fundamental and Clinical Aspects

Lymphocyte Activation and Differentiation Fundamental and Clinical Aspects Proceedings of the 18th International Leucocyte Culture Conference La Grande Motte, France, June 19-24,1987 Editors J. C. Mani • J. Dornand

W DE G Walter de Gruyter • Berlin • New York 1988

Editors

Dr. Jean Claude Mani Dr. Jacques Dornand Laboratoire de Biochimie des Membranes 8, Rue de l'Ecole Normale F-34075 Montpellier France

Library of Congress Cataloging in Publication Data International Leucocyte Culture Conference (18th : 1987 : La Grande Motte, France) Lymphocyte activation and differentiation : fundamental and clinical aspects : proceedings of the 18th International Leucocyte Culture Conference, La Grande Motte, France, June 19-24,1987 / editors J. C. Mani, J. Dornand. p. cm. Includes bibliographies and indexes. ISBN 0-89925-446-2 (U.S.) 1. Lymphocyte transformation-Congresses. 2. Cell differentiationCongresses. 3. Immunogenetics-Congresses. I. Mani, J. C. (Jean Claude), 1939- . II. Dornand, J. (Jacques), 1945- . III. Title. [DNLM: 1. Cell Differentiation-congresses. 2. Lymphocyte Transformation-congresses. 3. Lymphocytes-immunology-congresses. W3 IN725 18th 1987L / WH 200 1576 1987L] QR185.8.L9I555 1987 599'.087612~ dcl9 DNLM/DLC

CIP-Kurztitelaufnahme der Deutschen Bibliothek Lymphocyte activation and differentiation : fundamental and clin, aspects ; proceedings of the 18 th Internat. Leucocyte Culture Conference, La Grande Motte, France, June 19-24,1987 / ed. J. C. Mani ; J. Dornand. Berlin ; New York : de Gruyter, 1988. ISBN 3-11-010760-0 NE: Mani, Jean C. [Hrsg.]; International Leucocyte Culture Conference

Copyright © 1988 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Lüderitz & Bauer Buchgewerbe GmbH, Berlin. - Printed in Germany -

PREFACE This volume contains the Proceedings of the 18th International Leucocyte Culture

Conference. This Conference was held

under the auspices of the French Society

of Immunology

and

the Institut de la Santé et de la Recherche Médicale, in la Grande-Motte (France) on June 19-24 1987. About 500 participants

and

65

speakers

and

workshop

chairmen

attended

the

lecture on oncogenes

and

Conference. The Conference included a plenary

five Symposia on: biochemical and cellular

aspects of

lym-

phocyte activation, differentiation of T and B lymphocytes, molecular

biology

of

leucocytes,

virus-induced

lymphocyte

dysfunctions, accessory cells. 26 papers correspond to these lectures. 380 posters have been selected and displayed

all along

the

Conference; their abstracts were published in a special issue of Immunobiology

(supplement vol 3, 1987). They were in-

cluded within 16 workshops and 100 of them have been selected for oral presentation. The papers corresponding to these presentations are published in this volume together with the lectures of the chairmen of the workshop sessions. 15 workshops were held in the Convention Center of La Grande Motte: growth and differentiation

factors

mais, M. Dy), mechanisms of autoimmunity genes in lymphocyte

activation

(P. Jeanteur, D.

experimental models of immunomodulation afico), immunodeficiencies

(chairmen C. Da(J.F. Bach), oncoStehelin),

(M. Parant, F. Spre-

(F. Rosen, M. Seligmann), natural

killer cells (R.B. Herberman, E. Klein), differentiation and activation of T lymphocytes

(L. Boumsell, C. Mawas), diffe-

rentiation and activation of B lymphocytes E.

Lipsky),

lipidic

mediators

of

the

(A. Coutinho, P.

immune

response

(J.

Benveniste, J.S. Goodwin), neural modulation of immunity (H.

VI

Besedovsky, M. Dardenne), biochemistry of leucocyte stimulation and effector mechanisms (P. Golstein, K. Resch), accessory

cells-lymphocyte

O'Brien),

idiotypic

interactions

regulation

(A.

Novogrodsky,

(M. Fougereau,

J.

R.L.

Urbain),

complement receptors (M.P. Dierich, M.D. Kazatchkine ) , major histocompatibility complex (D. Charron, E.O. Long). The 16th workshop was held in the SANOFI Research Center of Montpellier and concerned the clinical applications of antileucocyte monoclonal antibodies. The papers corresponding to the lectures and the selected

short communications

are

in-

Most of the papers are in the form of original studies

re-

cluded in this volume (workshop P, pp. 827-942).

porting

important

new data; a few ones are overview

arti-

cles. Credit for the quality of the volume goes, of course, to the individual authors.

Jean-Claude MANI Jacques DORNAND

CONTENTS

INTRODUCTION Fundamental and Clinical Aspects of Lymphocyte Activation and Differentiation J.C. Mani, J. Dornand PLENARY

1

LECTURE

Oncogenes and Multistep Carcinogenesis D. Stehelin

5

SYMPOSIA BIOCHEMICAL

AND CELLULAR

ASPECTS

OF

LYMPHOCYTE

ACTIVATION Biochemical and Molecular Events Controlled by Lymphoid and Myeloid Growth Factors W.L. Farrar, D.K. Ferris, A. Harel-Bellan

17

PGE2 Inhibition of Human T Lymphocytes Proliferation: Regulatory Effect of PGE2 on the Intra-Cellular Signaling Pathways S. Chouaib, J. Bertoglio, D. Fradelizi

27

Structure-Function Relationships for the Interleukin 2 Receptor R.J. Robb

35

Monovalent Cation Fluxes as Regulators of Critical Nuclear Events in Lymphocyte Activation: Mirror Image Processes in Differentiation and Proliferation J.G. Kaplan, K.V.S. Prasad, B. Sahai, A. Severini

45

Diacylglycerol, Calci um, cAMP and cGMP Mediate Qualitatively Distinct Protein Kinase C Translocation Events in Normal B Lymphocytes J.C. Cambier, 1 . 1 . Chen

57

Phosphorylation of Prosolin and Leucocyte Growth Regulation H.L. Cooper, R. Braverman

67

VIII DIFFERENTIATION OF T AND B LYMPHOCYTES Regulation of Human B Cell

Function

J.L. Ambrus Jr., H. Goldstein, K.R. Young, P. McFarland, T. Nakagawa, N. Nakagawa, E. Brown, F. Uckun, A. Tenner, M. Peters, N. Witzel, H. Mostowski, A.S. Fauci

73

The Role of Lymphokines in the Immunoglobulin Class Switch C.M. Snapper, W.E. Paul

83

Mouse and Human B Cell Activating

Factors

J. Theze, C. Roth, A. Diu, D. Jankovic, M. Körner, 0. Abehsira-Amar, J.L. Moreau, M. Fevrier, M.L. Gougeon Molecular Cloning of the Murine T11

97

(CD2) cDNA

P.H. Sayre, L.K. Clayton, E.L. Reinherz, J. Novotny

111

Antigen Expression and Proliferative Activity during B and T Cell Development D. Campana, G. Janossy

123

MOLECULAR BIOLOGY OF LEUCOCYTES (ANTIGENS, RECEPTORS,

INTERLEUKINS)

The Molecular Biology of the Multisubunit Interleukin-2

Receptor

T.A. Waldmann, C. Goldman, M. Tsudo

133

Expression and Rearrangement of T Cell Receptor Genes in Human Leukemic T Cel1 Lines N. Kimura, R.-P. Du, T.W. Mak The Human T-Cell Rearranging y Genes

143 (TRGy)

M.-P. Lefranc, A. Forster, T.H. Rabbitts Immunoglobulin Gene

153

Expression

M.S. Neuberger, G.P. Cook, J.O. Mason, C. Milstein, S. Pettersson, W. Reik, M.J. Sharpe, R. Sitia, M.A. Surani, K. Weston, G.T. Williams

159

Structure and Expression of Mouse Fc-y Receptor II (FcyRII) J. Even, N. Varin, M. Daeron, C. Bonnerot, W.H. Fridman, P.M. Hogarth, E. Witort, L. Bonadonna 165

IX

VIRUS-INDUCED LYMPHOCYTE

DYSFUNCTIONS

The Impact of the Presence of EBV on the Interaction of B Cell Lines with T Lymphocytes E. Klein, G. Klein

175

Induction of B Cell Activation Markers by Epstein-Barr Virus A. Calender, M. Billaud, G. Lenoir Production of Interleukin-1

183

(IL1) by EBV-Infected Cells

T. Tursz, C. Bensimon, P. Busson, Y. Mahe, H. Wakasugi

191

DNA-Mediated Gene Transfer of Epstein-Barr Virus Receptor onto Murine Fibroblasts J.F. Cantaloube, P. Poncelet, A. Calender, G. Lenoir, M. Piechaczyk

.. 201

ACCESSORY CELL FUNCTIONS Modulation of IL1 Receptor Expression by IL1, Prostaglandins and Glucocorticoids J.J. Oppenheim, T. Akahoshi, K. Matsushima, G. Tosato

209

Tumor Necrosis Factors: Modulation of Synthesis and Biological Activities G.H.W. Wong, D.V. Goeddel

217

Analysis of the Structure and Function of Immunectin J. Schwartz, G. Freeman, R. Patarca, R. Singh, S. Zolnay, G. Orfanos, V. Anderson, S.J. Galli, T. Durfee, F. Blattner, H. Cantor Natural Effector Cells of the Intestinal Epithelium: Function, and Regulation

227

Ontogeny,

J.R. Klein, M.D. Howell

235

Analysis of Ia-Transfected Fibroblasts: Antibody and T Cell Recognition Sites Segregate with the 31 Domain of the E3 Polypeptide Chain N. Rebai, F. Bekkhoucha, D. Emilie, M. McMillan, M. Pierres, B. Mai is sen

245

WORKSHOPS WORKSHOP A. GROWTH AND DIFFERENTIATION

FACTORS

Inhibition by Cyclosporin A of the IL2 Dependent Autocrine Growth in a Human Tumor T Cell Line and Fate of IL2 Receptors after Endocytosis A. Dautry-Varsat, V. Duprez, A. Hemar, M. Ferrer

259

X Interleukin B, a B Cell-Derived

Lymphokine

P. Del Guercio

263

Induction and Enhancement of Lymphokine Gene Expression in Human T Cell Clones Transfected with HTLV-I pX cDNA R. De Waal Malefijt, H. Yssel, H. Spits, J.E. De Vries, S. Miyatake, T. Yokota, K.I. Arai, M. Seiki, M. Yoshida

267

Evidence for a Human B Cell Activating Factor Acting on Resting B Lymphocytes A. Diu, M. Fevrier, J.L. Moreau, M.L. Gougeon, J. Theze, E.L. Reinherz

271

Splicing Intermediates of the IL-2 Receptor (p55 Subunit) P. Froussard, P. Chastagner, J. Theze, S. Longacre The Role of Plasminogen Activator in Regulating T Cell and y Interferon Production

275 Proliferation

P. Gladstone, M. Leahy, C.A. Spooner

279

Membrane and Soluble IL2-R Produced by Human T Cell Clones: A Comparative Study Y. Jacques, B. Le Mauff, F. Boeffard, A. Godard, J.P. Soulillou Phenotype and Cytokines Production by Human Myeloma Cell

287

Lines

B. Klein, M. Jourdan, C. Dupperay, R. Bataille B + Null Cell-Derived Prothymocyte Differentiating Activity Biological Properties and Physicochemical Characterization

291 (PTDA):

J.C. Lecron, M.D. Mossalayi, L. Sutton, J. Gombert, J. Tanzer, P. Goube De Laforest

297

TDM-Activated Macrophages Produce a Cytostatic Factor Distinct from TNFa, IFNa,6, and IL-1 M. Lepoivre, G. Lemaire, J.F. Petit

301

Characterization of a Factor Produced by Human Alloreactive T Cell Clones J.F. Moreau, H. Gascan, A. Godard, M. Bonneville, J.P. Soulillou

W O R K S H O P B. M E C H A N I S M S OF

AUTOIMMUNITY

Effector Mechanisms of Autoimmune J.F. Bach

305

Diseases 309

XI

T Cell Response to Myelin Basic Protein in Healthy Subjects and Multiple Sclerosis Patients M.A. Bach, E. Tournier-Lasserve, G. Hashim

323

Active T Cell Regulation of Autoimmune Responses in Autoimmune and "Nonautoimmune" Spleen Cell Populations C.E. Calkins, R.D. Miller, S.A. Cochran, A.L. Rothermel, J.S. Moore, M.J. Caulfield

327

Interleukin-2 Receptor Targeted Immunotherapy of Autoimmune Diseases: Treatment Strategies for Type I Diabetes T. Diamantstein, A. Dunger, I. Kloting, B. Kuttler, H.D. Volk, H.J. Hahn, R. v. Baehr

331

Protection of Non-Obese Diabetic (NOD) Mice from Insulitis by Neonatal Injection of F^ Spleen Cells A.R. Hayward, P. Chantler, E. Simpson

335

WORKSHOP C. ONCOGENES AND LEUCOCYTE

TRANSFORMATION

Complex Transcriptional Regulation of Nuclear Proto Oncogenes: of a Block to Elongation in C-Myc and C-Fos Genes

Existence

J.M. Blanchard, M. Piechaczyk, P. Fort, N. Mechti, H.G. Suarez, B. Lebleu, P. Jeanteur Determinants of C-Fos and C-Myc mRNAs

339

Instability

A. Bonnieu, M. Piechaczyk, P. Fort, J. Rech, J.M. Blanchard, P. Jeanteur

345

Cloning and Functional Analysis of the Human C-fms Gene P. Dubreuil, M. Courcoul, D. Birnbaum, J. Planche, M.J. Pebusque, P. Mannoni, F. Birg

351

Effects of Proliferative (Interleukin, Colony Stimulating Factors) and Antiproliferative (Cyclic AMP, Interferon) Signals on Gene Expression in Myeloid and Lymphoid Cell Lines A. Harel-Bellan, W.L. Farrar

355

The Progession to Tumourigenes is Following Transformation of Murine Myelomonocytic Cell by V-Myc G. Symonds, P.B. Rowe, Y. Takayama, P. Stapleton, A. Bruskin, J.M. Bishop

359

XII HTLV-I Infection of a Human Antigen Specific Proliferating/Cytotoxic T Cell Clone Results in a Loss of Cytotoxicity but does not Affect the T Cel1 Receptor H. Yssel, J.E. De Vries, H. Spits, M. Due Dodon, L. Gazzolo

WORKSHOP D. EXPERIMENTAL MODELS OF

363

IMMUNOMODULATION

Roles of Monokines in the Nonspecific Host Defence Stimulated by Exogenous Agents M. Parant

367

Enhancement of the Immune Response Using a Synthetic B-Lymphocyte Mitogen Covalently Linked to Antigens T. Böltz, W.G. Bessler, G. Jung, K.H. Wiesmüller J. Metzger

379

Effects of in vivo Treatment of Mice with Anti-Murine T3 Antibody: Suppression of Transplantation Responses R. Hirsch, 0. Leo, D.H. Sachs, J.A. Bluestone

383

Gamma-Interferon Protects Listeria-Susceptible BALB/c Mice from Lethal Listeriosis without Affecting the Development of Protective Immunity A.F. Kiderlen, M.L. Lohmann-Matthes

387

Effect of a Lipophilic Muramylitri peptide on Gamma-Interferon Induced Major Histocompatibility Antigen (MHC) and Leu-M3 Antigen Changes in Human Monocytes R. Landmann, M. Wesp, P. Dukor

393

Effect of Cyclosporin A (CsA) on the Induction of Memory T Lymphocytes I. Motta, B. Shidani, P. Truffa-Bachi

399

Modulation by Cyclosporin A (CsA) of the Immune System of Mice Infected with Lymphocytic Choriomeningitis Virus (LCMV) B. Shidani, M.-F. Saron, J.-C. Guillon, P. Truffa-Bachi

403

Modulation of Lymphocyte Activation in vitro by a Soluble Placental Factor A. Skibin, S. Segal, M.R. Quastel

407

The Role of Interleukins and the Interleukin-2 Receptor in the Pathogenesis of Murine Cutaneous Leishmaniasis W. Sol bach, M. Lohoff, C. Gunther, H. Streck, M. Röl1inghoff, T. Diamantstein

413

XIII In vitro Studies of Synthetic Glycolipids: A New Class of Compounds with Immunomodulating Activity K.G. Stlinkel, 0. Lockhoff, G. Streissle, V. Klimetzek, A. Paessens, H.D. Schlumberger Imm'unotoxicity Studies of PCB (Aroclor 1254) in the Adult Rhesus mulatta) Monkey

(Macaca

H. Tryphonas, S. Hayward, L. O'Grady, J.C.K. Loo, D.L. Arnold, F. Bryce, Z.Z. Zawidzka Human Cytomegalovirus

Induces Alterations in Mixed Lymphocyte

427 Reactions

C.L. Wright, S.J. Forman, J.A. Zaia, K.G. Blume

WORKSHOP E.

421

433

IMMUNODEFICIENCIES

Immunological Aspects of AIDS and HIV

Infection

M. Seligmann

437

Studies Regarding the Interaction of the T4 (CD4) Molecule with the Envelope Protein gp120 of the Human Immunodeficiency Virus (HIV) 445

D.K. Ferris, D. Littmann, W.L. Farrar Low Level Re-Expression of the Silent MHC Class-II Genes in the Lymphoblasts from Patients with the HLA-Class II Deficient SCID A. Lisowska-Grospierre, A. Guyot, M.T. Dimanche, B. Mach, C. Griseel 1 i

449

Adenylate Cyclase and 5'Nucleotidase Activities of Peripheral Mononuclear Cells is Psoriasis

Blood

J.C. Mani, A. Chocquet, J.C. Bonnafous, J. Dornand, M. Andary, J. Clot, J.J. Gui 1 hou

453

WORKSHOP F. NATURAL KILLER CELLS Natural Killer Cells E. Klein Characterization and Population Kinetics of Liver-Associàted Granular Lymphocytes (Natural Killer Cells)

465 Large

L. Bouwens, E. Wisse

469

Modulation of Lymphonkine Activated Killer Activity of Lymphocytes Isolated from Human Peripheral Blood and Bone Marrow by Recombinant IL4 C.G. Figdor, E. Wierenga, J.E. De Vries, H. Spits

473

XIV Inhibition of Human Natural

Killer Cells by Resistant Target Cells

M.K. Heiskala

479

Changes in Gene Expression Associated with Activation or of Natural Killer Function

Inhibition

J. Kornbluth, R. Hoover

483

Fibronectin Expression by Endogenous and Activated NK Cells S. Morrone, S. Scarpa, R. Testi, G. D'Orazi, A. Gismondi, A. M. Piccoli, L. Frati, A. Modesti, A. Santoni Cytolytic Potential of C D 3 + 4 ~ 8 ~ T Cells with Differently T Cell Receptor Y Chains

Punturieri, 489

Organized

R.J.' Van De Griend, R.L.H. Bolhuis, J. Borst, S.L. Ang, D. Hafler, J.G. Seidman

493

WORKSHOP G, DIFFERENTIATION AND ACTIVATION OF T LYMPHOCYTES CD1a Molecules Form Intermolecular Complexes with HLA Class I Molecules or Other CD1 Molecules on Normal Human Thymus Cells M. A m i o t , H. Dastot, A. Bernard, L. Boumsell

499

The CD2 Pathway of Activation. Epitope Requirements, Linkage to Other Surface Molecules and Early Biochemical Events D. Olive, A. Pierres, C. Cerdan, M. Bagnasco, C. Mawas

511

A T Cell Surface Molecule Different from CD2 is Involved in Spontaneous Rosette Formation with Erythrocytes F. Aubrit, B. Raynal, A. Bernard, L. Boumsell

521

A Dual Function of the T4 Molecule in the Cytolytic Activity of Human T Cell Clones D. Blanchard, J.E. De Vries, H. Spits

525

Role of Antigen-Specific T Cell Receptor and Associated Proteins in the Activation of a Cytolytic T Cell Clone Analysed by Protein Phosphorylation and by T Cell Receptor Negative Variants C. Boyer, P. Kaldy, A. Guimezanes, C. Langlet, M. Buferne, A.M. Schmitt-Verhulst Y-Chain T Cell Receptors on Dendritic Epidermal

529

Cells

J.E. Coligan, F. Koning, A.M. Lew, W.L. Maloy, W.M. Yokoyama, G. Stingl, E.M. Shevach

533

XV Stimulation of T Cells via LY-6C: A Phssphatidylinositol-Anchored Cell Surface Molecule which Generates Intracellular Calcium Mobilization upon Cross-Linking F.J. Dumont, R.R. Palfree, R.C. Boltz

537

New Subgroups in the Human T Cell Rearranging VY Gene Locus A. Forster, S. Huch, N. Ghanem, M.P. Lefranc, T.H. Rabbits

541

Genomic Organisation of the Constant Regions of the Human T-Cell Rearranging Gene Y M.P. Lefranc, A. Forster, T.H. Rabbits

545

Second Signals for T Cell Mitogenesis Provided by a mAbs CD45 and CD5 (T1)

(T200)

J. Martorel1, R. Vilella, L. Borche, I. Rojo, J. Vives

549

WORKSHOP H. DIFFERENTIATION AND ACTIVATION OF B

LYMPHOCYTES

Immunobiology of B Lymphocytes A. Coutinho

553

Regulation of B Lymphocyte Responsiveness by Multiple Cytokines D.F. Jelinek, P.E. Lipsky

557

Influence of IL4 on the Induction of Immunoglobulin Secretion by BCAF-Containing Supernatant 0. Abehsira-Amar, D.L. Jankovic, M. Körner, J. Theze

565

Early B Cells Immortalized by Epstein-Barr Virus (EBV). A Phenotypic Study C.D. Gregory, C. Kirschgens, L.S. Young, M. Rowe, A.B. A. Forster, T. Rabbits

Rickinson, 569

The Human B Cell Repertoire: Multispecificity as a Major Characteristic of Antibodies Secreted by Human EBV-Transformable Cord Blood B Cells T. Logtenberg, A. Kroon, F. Gmelig-Meyling, R.E. Ballieux Analysis of Excessive B Cell Growth in Lymph Nodes of L. major Mice: The Effect of L. major Specific T-Cell Clones

573 Infected

M. Lohoff, M. Röl 1 inghoff Antibody Response in vitro of Human Peripheral Application of a Limiting Dilution Method A.L. Luzzati, E. Giacomini, P. Frugoni

577 Blood Lymphocytes: 581

XVI Frequency Analysis of Functional LPS-Reactive Murine B Cells

Immunoglobulin C^. Gene Expression in

H.F.J. Savelkoul, P.C.L.M. Linssen, J. Termeulen, A.A. Van der Linde-Preesman, R. Benner

585

Changes in Gene Expression in Differentiating B Lymphocytes S. Sharma, L.B. King. R.B. Corley Regulation of Lipopolysaccharide by Ly-5 and Uyb-2 Molecules

591 (LPS)-Induced B Cell

Differentiation

H. Yakura, G. Tate, H. Sakata, I. Kawabata, T. Ashida, M. Katagiri

WORKSHOP

... 595

I. LIPIDIC MEDIATORS OF THE IMMUNE RESPONSE

Phospholipid-Derived Mediators and the Immune Response Y. Thomas, J. Benveniste, J.S. Goodwin

599

Anti-Oxidants and IL2 Synthesis J. Dornand, M. Gerber, C. Sekkat, J.C. Mani

609

Role of Lipoxygenase Pathway Products in IL2 Synthesis M. Gerber, B. Longhi, F. Michel, A. Crastes De Paulet

615

Platelet Activating Factor (PAF) Induces Human Suppressor Cell

Activity

M. Rola-Pleszczynski, C. Pouliot, L. Bouvrette, B. Pignol, P. Braquet PAF-Acether Induces an Enhanced Release of Interleukin-1 Human Monocytes

621

(IL-1) from

P. Salem, S. Deryckx, J. Benveniste, Y. Thomas

625

Down-Regulation of CD2 and CD3 Antigen Expression by PAF-Acether E. Vivier, A. Dulioust, J. Benveniste, Y. Thomas

WORKSHOP J. NEURAL MODULATION OF

629

IMMUNITY

Neuroendocrine Modulation of Immunity H. Besedovsky, A. Del Rey

633

Cyclic AMP Response to Dopamine in the Human Monocyte-Like Cell Line U-937 E. Kouassi, Y.S. Li, R. Rouet, J.P. Revillard

645

ß-Endorphin Inhibits Thy-1 Antigen Expression on Murine Thymocytes and Antagonizes Thymosin Fraction V Action M. Rouabhia, P.A. Deschaux

649

XVII

WORKSHOP K. BIOCHEMISTRY OF LEUCOCYTE STIMULATION EFFECTOR

AND

MECHANISMS

Genes Preferentially Expressed in Cytolytic T Cells P. Golstein, J.F. Brunet, K. Harper, F. Denizot

653

Signal Transduction in Lymphocyte Activation K. Resch, M. Szamel

659

IL-2 Mediated Induction of Lytic Granules, Perforin, and BLT-Esterase in Potent, Granule-Free Cytolytic T Lymphocytes Indicates an Alternative Function of Lytic Granules G. Berke, D. Rosen

669

Extracel1ular ATP Initiates the Proliferative Response of PMA-Activated Medullary Thymocytes by Increasing their Cytosolic Free Calcium Concentration J. Dornand, C. El Moatassim, J.C. Mani

673

Evidence for a Kinase Cascade Stimulated by Interleukin 2 in Cytotoxic T Cell Clones W.L. Farrar, S.W. Evans

679

The Regulation of Cytoskeletal

Protein Gene Expression in Lymphocytes

E. McCairns, D. Fahey, R. Tayler, D. Sauer, P.B. Rowe Protein Phosphorylation During T Cell

683

Activation

J.F. Peyron, M. Fehlmann

687

Clues to Mechanisms of Activation for Killing from the Observations of Changes in Intracellular ( C a + + ) in Individual Cytolytic T Cells and Targets A.M. Schmitt-Verhulst, M. Poenie, R.Y. Tsien

691

Induction of Activation Signals in Human T Lymphocytes M. Szamel, B. Krebs, M. Kracht, K. Resch

697

Both L y t 2 + and L3T4 + Cell Subsets Secrete the Serine Esterase Granzyme A in Response to a Specific Antigenic Stimulus F. Velotti, M. Nabholz, H.R. MacDonald

WORKSHOP L. ACCESSORY CELLS-LYMPHOCYTE Accessory Cells-Lymphocyte

701

INTERACTIONS

Interactions

R.L. O'Brien, A. Novogrodsky

705

XVIII

A Role for HLA-Class I Molecules from Accessory C e l l s in T-Cell Activation S. Huet, B. Raynal, A. Bernard, L. Degos, J. Dausset, L. Boumsell

709

Adhesion Molecules of Monocytes M. Patarroyo, J. P r i e t o , P.G. Beatty, E.A. C l a r k , C.G. Gahmberg

713

C e l l u l a r and Molecular Basis of Self-Induced P r o l i f e r a t i o n of T Lymphocytes ( S I P ofT) B.M. Sahai, J.G. Kaplan

717

Human T Cell A c t i v a t i o n : P a r t i c i p a t i o n of T Cell D i f f e r e n t i a t i o n Antigen Cluster 2 (CD2) in the Generation and/or Transduction of Accessory Cell S i g n a l s M. Suthanthiran, J. H i l l J r . , V.K. Murthi, R. Schwartz, A. Subramaniam, A. Novogrodsky, K.H. Stenzel WORKSHOP

PI.

IDIOTYPIC

723

REGULATION

The Influence of Vk Gene Polymorphism on the Induction of S i l e n t Idiotypes in the Arsonate System M. B r a i t , J. Marvel, J. Tassignon, J.D. Capra, J. Urbain

727

Allogeneic Manipulation of the "GAT" I d i o t y p i c Cascade does not Influence the VH Gene Usage S. Corbet, M. Hirn, C. S c h i f f , M. Fougereau, C. Roth

731

S p e c i f i c Suppression of the Primary Antibody Response with Immune Complexes Formed in Antibody Excess Depends on the Idiotype and the Isotype of the Antibody Forming the Complex M.J. C a u l f i e l d

739

The HLA-Restricted Presentation of Idiotype to Human A n t i - I d i o t y p i c T Cell Clones does not Require Processing by Antigen Presenting C e l l s I . Claassen, G. Ten Hoor, H. Bunschoten, A. Osterhaus, F. Uytdehaag .. 743 I d i o t y p i c Predominance A l t e r a t i o n in Anti-Arsonate Response by Priming with A n t i - I d i o t y p i c Antibodies A. Gaya, E. A l s i n e t , M. F r e i x a s , J. Vives

749

A n t i - I d i o t y p e Induction of Humoral Immunity in Cattle R.A. Goldsby, A. Arulanandan

753

Anti MHC-Receptor Cytotoxic T Lymphocytes: Their Role in the S p e c i f i c Resistance to Graft-Versus-Host Reaction K. Kosmatopoulos, D. Scott-Agara, 0. Halle-Pannenko, S. Orbach-Arbouys

757

XIX Altered Anti-Idiotype Response to a(1-3)Dextran-Associated in Mice Bearing an X-Linked Immune Defect

Idiotypes

H.C. Seiinka, R. Bösing-Schneider

761

WORKSHOP N, COMPLEMENT RECEPTORS Human CD3 Complement

Receptors

M.D. Kazatchkine, M.P. Dierich

765

Influence of Antigen-Bound Complement Components C3b and C4b in Activation of Human T-Cell Clones J. Arvieux, M.G. Colomb

781

Human C3 and C3dg Stimulate Proliferation of a Pre-B Lymphoblastoid Cell Line via CR2 Complement Receptor A. Hatzfeld, E. Fischer, J.P. Levesque, J. Hatzfeld, M.D. Kazatchkine

785

Activation of B Lymphocytes by Zymosan-Bound C3 Fragments C. Kai, R. Szigeti, P. Aman, E. Klein

789

Contribution of C3 Fragments to Lymphocyte Mediated Lysis of CR2 Carrying Cells E. Klein, 0. Ramos, G. Sarmay, C. Kai, E. Yefenov, J. Gergely "In vivo" and "in vitro" Modulation of CR1 (C3b Receptor) by its Ligand C3b

Expression

F. Porteu, A. Mir, L. Halbwachs-Mecarelli

WORKSHOP 0. MAJOR HISTOCOMPATIBILITY

793

797

COMPLEX

MHC in Leucocyte Biology D. Charron HLA Class I a1, a2, and ß2-Microglobulin Domain

801 Interactions

B. Kahn-Perles, A. Gil let, R. Hakem. B.R. Jordan, C. Layet, B. Perarnau, P. Pontarotti, J. Sire, R. Sodoyer, A. Toubert, F.A. Lemonnier 805 Diversity and Function of Human Class II MHC Antigens E.O. Long, S. Rosen-Bronson, S. Jacobson, H.F. McFarland, R.P. Sekaly

809

DRa-DQß Mismatched Isotype Pairing Occurs in EBV Transformed B Cell Lines V. Lotteau, L. Teyton, D. Burroughs, D. Charron

813

XX Distinct Epitopes for K d - R e s t r i c t e d Cytolytic T Cells Specific for HLA-CW3 or HLA-A24 Map to the same Region of HLA J.L. Maryanski, P. Pala, J.C. Cerottini, G. Corradin, B. Jordan, T. Strachan, R. Sodoyer

819

Resistance or Susceptibility of Human Target Cells to NK Lysis: Correlation with the Surface Expression of HLA Class I Molecules and Induction of Susceptibility by Monoclonal Anti Class I Antibodies A. Quillet, A. Harel-Bellan, C. Marchiol, D. Fradelizi

823

WORKSHOP P (SANOFI), CLINICAL APPLICATIONS OF A N T I LEUCOCYTE MONOCLONAL

ANTIBODIES

Anti-Leucocyte Monoclonal Antibodies: From Laboratory Reagents to Therapeutic Agents P. Poncelet

827

Treatment of Donor Bone Marrow with FabT101 Ricin A-Chain Immunotoxin for the Prevention of Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation H.E. Blythman, G. Laurent, J.M. Derocq, E. Gluckman, D. Maraninchi, J.P. Vernant, B. Rio, M. Michallet, J. Reiffers, M. Boasson, F. Dreyfus, P. Schneider, F.K. Jansen

831

Monoclonal Antibodies for Bone Marrow Purging before Transplantation: A Brief Review D. Campana, G. Janossy, E. Coustan-Smith, C. Bouloux, P. Poncelet, L. Foroni

835

Comparative Efficiency of an Immunomagnetic Purging Procedure and a Rabbit Complement Lysis to Eliminate BL Cells from the Bone Marrow V. Combaret, M.C. Favrot, I. Philip, P. Poncelet In vivo Antigenic Modulation of CD5+ Cells in Renal Patients Receiving Anti-CD5 Antibody Infusions

845 Transplanted

G. Cordier, J.P. Revillard, J. Brochier, M. Blanc, J. Traeger, E. Carosella, M. Latour, J. Armand

849

In vivo Therapy by CD8 Monoclonal Antibody for Delays to Hematological Recovery after Autologous Bone Marrow Transplantation M. Favrot, T. Philip, P. Biron, V. Combaret, J.M. Zucker, A. Bernard . 855 Autologous Bone Marrow Transplantation for Acute Lymphoblastic Leukemia in Remission. In vitro Purging of Inoculum with Monoclonal Antibodies and Complement P. Herve, E. Racadot, E. Plouvier, M. Flesch, J.Y. Cahn, A. Noir

859

XXI

Immunosuppression by Murine Monoclonal Antibodies: The Rhesus Monkey Used for P r e c l i n i c a l Testing M. Jonker

865

Therapeutic Applications of Monoclonal Antibodies for Lymphoma and Leukemia M.S. Kaminski, K.A. Foon BMA 031 - The TCR S p e c i f i c Monoclonal Antibody for C l i n i c a l

871 Applications

R. Kurrle, R. Zerban, F.R. S e i l e r , B. Nashan, K. Wonigeit

885

Immunoassays for the Monitoring of Kidney G r a f t ' s Transplanted Patients under 33B3.1 MOAB Therapy B. Le Mauff, F. Boeffard, J.P. S o u l i l l o u , Y. Jacques

891

Prospective Randomized Studies of T Cell Depletion in HLA Matched Bone Marrow Transplantation (BMT) for Leukemia: 2 Studies of the GEGMO in 92 Patients D. Maraninchi, E. Gluckman, D. Guyotat, B. Rio, D. B l a i s e , J.L. Pico, V. Leblond, M. M i c h a l l e t , G. Laurent, M.H. Gaspard, F. Dreyfus, N. I f r a h , J. R e i f f e r s , A. Bordigoni, A. Bernard, M. Delage, F. Jansen

895

A Monoclonal Anti Interleukin 2 Receptor Prevents Human Kidney Graft Rejection J.P. S o u l i l l o u , B. Le Mauff, Y. Jacques, P. Peyronnet, M. Hourmant, D. Cantarovich, P. Dubigeon, D. O l i v e , C. Mawas, M. Delage, M. Hirn .. 901 C l i n i c a l Pharmacology of 0KT3 L. Chatenoud, J.F. Bach

905

Introduction to the C l i n i c a l Use of Monoclonal Antibodies as Immunosuppressive Agents C. Mawas

915

General Considerations on the Quality Control of Monoclonal Antibodies before Therapeutic Use in Man R. Thorpe, G.C. S c h i l d

927

Immunotoxins with Therapeutic Use in Man F.K. Jansen, H.E. Blythman, C. Bouloux, B. Bourrie, P. Carayon, P. C a s e l l a s , J.M. Derocq, D. Dussossoy, 0. Gros, P. Gros, G. Laurent, J.C. Laurent, G. Richer, P. Schneider, P. Vandest, H. Vi d a l , G.A. Voisin

935

AUTHOR

943

SUBJECT

INDEX INDEX

949

FUNDAMENTAL AND

AND

CLINICAL

ASPECTS

OF

LYMPHOCYTE

ACTIVATION

DIFFERENTIATION

J.C. Mani, J. Dornand CNRS ER 228. ENSCM, Montpellier

(France)

Chairmen 18th LCC

The International

Leucocyte

Culture Conferences

are the ma-

jor world meetings for the study of white blood cells at the cellular

level

under

in

vitro

conditions.

This

series

of

Conferences, which originated in the United States, has been held alternatively in North America and the European and this is the first time that one was held first

meetings

discovered

were

mainly

phenomenon

of

concerned

in France.

with

lymphocyte

region,

the

The

then

newly

transformation

under

conditions of cell culture. Since then, the Conferences have become multidisciplinary, covering subjects ranging from basic aspects of membrane

structure

and

function

to

applications of leucocyte phenomena. For the last the

room

let

to clinical

problems

and this was the case for the Meeting was

"Fundamental

into account major of the

advances

The

Meetings,

predominant, theme of

of

occurred

and

of

triphosphate,

in

recent

intracellular role

of

this

Lymphocyte

It has been chosen

that

interleukins

gers, such as inositol

become

LCC.

and Clinical Aspects

Activation and Differentiation". discovery

has

18th

clinical

the

to take years: messen-

oncogenes

in lymphocyte transformation, tremendous advances of the applications of molecular use of monoclonal

biology

antibodies

as

to the immunological diagnostic

agents, evidence of a neural modulation new immunomodulators

and

field,

therapeutic

of immunity,

use of

(efficient immunosuppressors for trans-

plantation, synthetic vaccines), intensive research on inhe-

Lymphocyte Activation and Differentiation © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

2 rited and acquired immunodysfunctions. Studies of leucocyte function

are

currently

advancing

rapidly

along

several

fronts and their applicability to a wide variety of medical problems is being demonstrated. One of the goal of the 18th LCC was to link these developments to parallel

advances

in

biochemistry and cell biology. The discovery of the T Cell Growth Factor

(TCGF), interleu-

kin-2, has been a crucial step in the understanding of lymphocyte activation mechanisms. The regulation of

lymphocyte

proliferation, either by natural effectors or added

drugs,

may involve one of the three main steps of this

mechanism:

IL-2 production and release, expression

receptors,

IL-2-induced

cell proliferation.

The

of

IL-2

role

of

ionic

trans-

port, cell aggregation, the involvement of leukotrienes and inositol

triphosphate,

these different

steps

the are

effects under

of

immunomodulators

current

investigation

on and

are presented in a Symposium, while the role of lipidic mediators of the immune response is the subject of a special Workshop. The importance of pharmacological applications of these fundamental advances was emphasized during these sessions . The differentiation of T and B lymphocytes plays a critical role both in the mechanism of normal immune response and in immunopathology. Monoclonal antibodies have allowed the easy identification of differentiation markers of the lymphocyte plasma membranes and the characterization tiation

stages

of

lymphocyte

of the

subpopulations.

differen-

The

genetic

control of the immune response is reflected by genetic factors controlling the differentiation of the various cell types. Impairment of cell differentiation leads to severe diseases, such as inherited

immunodeficiencies

or

leukemia. A

Symposium and two workshops were devoted to fundamental and clinical aspects of lymphocyte differentiation and a special Workshop concerned

immunodeficiencies.

Advances in molecular

biology

and genetic

been spectacular during the last few years

engineering

have

and their appli-

3 cations to immunology highly fruitful: immunoglobulins, interleukins, T cell receptor, oncogenes. Here again advances of

fundamental

and

clinical

problems

are

parallel.

Symposium en Molecular Biology of Leucocytes was

The

supplemen-

ted by a plenary lecture and a Workshop on oncogenes. One of the major advances in the understanding of the immune response has been the evidence of a necessary

cooperation

between different cell types: various lymphocyte

subpopula-

tions and different leucocytes. Accessory cell functions include the production

of soluble

factors, antigen

tion, cell-to-cell contact, and are

involved

presenta-

in normal

and

pathological immune responses. A Symposium and a Workshop on accessory

cells,

a Workshop

on

complement

receptors

have

presented recent advances on this growing field. The viral origin of several

leukemia

and

lymphosarcoma

more recently that of acquired immunodeficiencies well

documented

and extensive

research

and

(AIDS) is

are carried

out

on

this subject. For the first time in the LCC history, a full Symposium was "devoted to this problem. Other

clinical

as-

pects of leucocyte culture were concerned by specific Workshops: autoimmunity,

natural

killer

cells,

immunodeficien-

cies, experimental models of immunomodulation. The relationship between immune response and nervous system has become more than a simple hypothesis. research

teams work

Interdisciplinary

on the neural modulation

of

immunity,

both in University laboratories and in pharmaceutical firms, evidencing the real importance of this problem. A Workshop was devoted to these researches. The developments that the Conference might have include: the applications of recent

advances

stimulated

in the mecha-

nisms of membrane recognition, transmembrane signalling, intracellular

messengers,

to

the

cell

biology

of

leucocyte

function; the applications of such developments to improvement of clinical diagnostic and therapeutic techniques. This type of development requires no special emphasis, since the tremendous advances in clinical immunology which have occur-

4 red

in

recent

years,

to

a

great

extent

dependent

on

the

findings and applications of leucocyte culture, are evident in the wide variety

of

services

now

supplied

to

clinical

medicine in this field. The great interest in this field is demonstrated by the future

plans

for

subsequent

conferences

of

this

series

Canada and Belgium, as well as by many publications area.

The

latter

show

increased

application

to

in

in the

practical

clinical problems as well as enhanced understanding of basic mechanisms.

ONCOGENES AND MULTISTEP

CARCINOGENESIS

D. Stehelin Laboratory of Molecular Oncology, INSERM U186 Institut Pasteur, Lille (France)

My talk will deal with

the cooperation

of oncogenes

defined cell systems, where we try to reconstitute by which one oncogene is able to cooperate with

in well

processes

another

on-

cogene, to transform a given tissue. Most or all proto-oncogenes

serve normal

functions

probably

implicated in the mitogenic pathways of normal cells

(Figure

1). Some encode growth factors, some encode cell surface receptors,

membrane

signals

teins), encode cytoplasmic sent nuclear

signals.

(tyrosine

kinases,

G-like

protein kinases, and some

Spontaneous

or

induced

pro-

repreacci-

genetic

dents can pervert the nature or regulation of these signals: proto-oncogenes may turn into oncogenes, leading

susceptible

cells to become modified in their growth properties or vated.

But

we

also

know

that

a single

oncogene

is

acti-

usually

not enough to generate malignancy. Cancers are multistep processes. It is classical to say that these

steps

include

initiation,

promotion, progression

gure 2). So far, these terms bear analyzed in molecular terms.

little

significance

In other words,

(Fiwhen

it is probably

quite abusive to say that a given oncogene is always an initiator and another always a progressor. Let us illustrate the purpose with the oncogene a-myo : myc may be an initiator, by a viral LTR

for example

through

its

Burkitt's lymphoma, myc could well be something moter, being

linked

immunoglobulin

activation

(Long Terminal Repeat) in avian lymphomas. In through chromosomal

activator

signal.

And

like a pro-

rearrangement in

some

Lymphocyte Activation and Differentiation © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

SCLC

to an (Small

6

ONCOGENES

CLASSIFICATION

Products Localisation External

Growth factor homology

sis

PDGF (3 chain Growth factor Receptor homology

EGF receptor

Cytopl. Mbr.

erbBi fins

CSF-1 receptor

(trans.)

ros

Homology with Insulin receptor

Cytopl. Mbr.

src

tyrosine kinase activity

(inner face )

yes fes/fps

GTPase activity (G proteins homology)

ras

serine/threonine kinase activity

mil/raf

Cytopl.

mos

genes activators ?

myc

Nucleus

myb fos

erbA

T 3 , T 4 Hormones

Receptor

Not yet classified ets rei

Figure 1 Cell

Lung

Carcinoma)(or

progressor

gene

through

sion, respectively). We may participate in tumor

cervical gene

carcinomas),

amplification

conclude

that

formation

(or

this

it may

be

a

overexpres-

single

oncogene

in three different

types

of tumors at three different steps, with three different and probably

characteristic

types

of

activation

processes.

All

these cancers most certainly harbour other activated oncogenes, that so far have remained undetected very difficult

to uncover since

and appear

we possess no hint

to

be

as to

7

NORMAL

INITIATED

TUMOR

MALIGNANT

CELL

CELL

CELL

CELL

\ 1/ ONCOGENE ACTIVATION (S)

Figure 2 what we should search A major which

oncogens

which

mechanisms

Possibilities

are

cooperate

target

of

say

tissues out

fortunately

able

task

is now

to

cooperate

activation,

are apparently

cogenes able to be that

for.

and challenging

activated by of

in

with

which

discouraging by

pairs the

to attempt

to

200

three

which kind

decipher others

of

cancers.

different

means

each,

specifically

our body are

able

one

contains!)

there are good reasons to believe that only

specific associations of oncogenes

by

(say: 50 known on-

transform

that

to

to exhibit

but very coo-

peration . We recently

decided

to

study in-vitro

models, where

one

can

modulate the events imposed onto the cells. Our approach

us-

ed some retroviruses that we showed in the early eighties contain

not only

(Figure

3).

one

One example I will tosis Virus steps

oncogene,

rapidly

but

two

go through

distincts

is Avian

(AEV) that has captured probably

the erbA

and erbB

oncogenes

Erythroblas-

in two

proto-oncogenes.

distinct

These

two

distinct

RNA

species yielding two distinct proteins. By using mutants

af-

genes are now activated

cellular

to

and expressed

from

two

8

AEV-DNA

LTR A gag I

erb A ~

Aenv

erb B

LTR

AUG

Q-*— P75

m RNAs •

"V*

gag-erb A

(nucí.)

AUG

STOP

T

O"*--. SD

" V

*

„„erb B cf.x.TjoQo.xixjooco gpoo

(trans membr.)

LTR ^ g a g

myb

A env

ets

LTR

STOP

m

T

RNA

gag-myb-ets

rcaxxToxooooooooooexxxio p 135

(nucl.)

LTR

^gag

mil

LTR

mvc

MH2-DNA STOP

AUG

m

T

-\r

„-««gag-mil a OOOOOOGOOOOOOOOOOOO P100

RNAs

(cyto.)

O-»-, so *.

STOP

T

SA

OODOQQDOOOO p62/63 (nucl.)

myc

Figure 3 fected

in one

including

or

our

the

other

own have

of

shown

these

that

genes,

these

tinct functions. And it's probably

several

genes

not by

chance

that

two genes are found together within the virus: they te to transform specifically dangerous for

chickens.

It

groups

perform

disthese

coopera-

given cells. This virus is very

induces

an

acute

erythroleukemia

that kills most of them within four weeks. Let erbA

us

examine

what

we

know

about

the

viral erbB

and

genes as compared to their cellular progenitors.

has been shown to be the receptor

for

the

epidermal

the erbB

growth

9 factor,

a

transmembrane

extra-cellular ne-kinase cellular

cellular

protein

domain, a transmembrane

domain. And progenitor,

if you compare you

find

out

that

contains

an

stretch and a tyrosithe viral

at

least

gene

two

to

its

interesting

things. The first is that putative been

lost

in

the

viral

growth

gene.

factor

It

is

binding

supposed

domain

that

this binding domain is lacking, the viral gene products become

constitutively

active

(without

molecule of growth factor to come

by

having

to

and bind

has

because

wait

have for

to the

a

recep-

tor ) . A second modification

is that the C-terminus is truncated

the virus as compared

to the cellular

progenitor.

This

tributes to the activation of the gene leading to the formation of erythoblasts, move that piece, you

do not

you

can

transform

interesting because

but

also

still

fibroblasts it

indicates

target cell specificity

for

fibroblasts.

transform

con-

trans-

If you

re-

erythroblasts,

but

any more. that

I think

truncations

this

may

alter

the-

re are also some mutations which may bear some importance the transforming process yet to be Other alterations of this erbB human

glyoblastomas.

gene may be involved in other

lated So

gene, erbB2

if I went

Finally,

a

mutation

within

(neu

in the rat),

through

and

in 3T3 mouse

that,

to an active oncogene.

It's

to

shown

show

In

some cases

by

again

these

perate to enhance the potency of

that

of

cases

transformation, also

the

alter

but

the type

in of

other

Bob

re-

Wein-

cells.

proto-oncogene may undergo several modifications

may

in so-

domain has been observed in a very closely

berg to be transforming

in

evaluated.

types of tumors: its amplification has been described transmembrane

is

transformation.

Beside these two main differences present in the virus,

me

in

gene

a

given

to turn

in-

alterations

coo-

for

type

distinct

transformation

that

a given

alterations

that will

be

in-

duced . I said that there was another gene present in Avian

Erythro-

10 blastosis Virus: the erbA This

gene

has

been

gene.

shown

to

be

a

hormone

receptor

gene.

1

Bjorn Vennstrom and Hartraut B e u g s people in Heidelberg, Jacques

Ghysdael

Lille, found

with

that

Yves

Goldberg

the cellular

in

our

counterpart

laboratory

of

the erbA

product is probably the receptor of thyroid hormones thyronine and thyroxin. The erbA

and in gene

triiodo

gene product contains a DNA

binding domain that is common to glucocorticoid receptors. A small

region

bears

relatedness

function of which is not yet bly,

the

hormone

binding

to

carbonic

known,

domain,

anhydrase,

if any. And most

or

at

least

an

about

compared

nine to

amino

the

acids

cellular

of

the

C-terminus

progenitor.

And

in the vi-

are

when

proba-

important

part of it, is located at the C-terminus, because rus

the

lost

as

try

to

you

bind hormone to the viral protein, the hormone does not bind any more. Thus the v-erbA

protein might modifiy cells

se it is now constitutively In-vitro

as well

active without the hormone.

as in-vivo

, avian

erythroblasts

are

rently barely affected by the viral erbA

gene product

They

gene

are

transformed

which interferes with

by

becau-

the

their

viral erbB proper

appaalone.

product

differentiation

alone

into

ma-

ture erythroblasts. But when both gene products are expressed in erythroblasts throblasts become

infected with wild-type

fully

level, their maturation grow wildly

blocked

is prevented

in a self-renewal

AEV,

at the colony

mode

and

the

these

ery-

forming

unit

immature

cells

highly

leuke-

and become

mic . In the limited time that I have left, I will not examine cooperation of the two oncogenes myc

and ets contained

in the avian

directly

retrovirus

tention to a third 2

E26.

I will

retrovirus,

call

namely Mil-Hill

(MH2). This virus is highly oncogenic

your

virus

the

withat-

number

in chickens that

die

3-6 weeks post-infection with multi clonal tumors spread

all

over the body, probably mainly of macrophage origin. MH2 virus has transduced the mil Again,

the

two

gene

and the myc

products

are

oncogenes

made

from

(Figure

two

3).

distinct

11

,

-»««lllllii^

Biolog.

M H 2

N.R

CEF

Proliferation

Transformation

S y s te m s

Virus

+ +

mil+mjc+

MUTANTS n i l * MH2 U-200 MH2 PA-200 (In vitro) («pontan«.) LUI* Paris MUTANTS mye+ MH2 OB MH2 CI2S (In vitro) («pontan«.) un» p«n.

Figure 4

i — •

+

= Figure 5

mRNAs, leading to a P60 protein

+

^^

for myc

fusion protein for mil and a

. Again we have now

these genes can cooperate

good

evidence

for transformation.

that

But we wanted

to test that in a straighforward manner; and this turned out to be sort of tedious work, but

led to some interesting

re-

sults that I would like to present. We

introduced

deletions

of this virus, press

in order

correctly

be mil-plus

in

only

mutants),

would be myc-plus

the mil gene

to prepare

or

only

in

mutants

the mil gene

mutants).

or

the myc

that

product

the myc gene

gene

would

(these product

ex-

would (those

Having at hand such mutants, we

showed that infection of cells with these mutants led to the formation of the proper proteins. This was pressing these proteins to

the mil or

through

the myc gene

assessed

a vector, making

products,

transformed or the modified cells

and

by

ex-

antibodies

testing

now

for the expression

the

of

the

given viral oncogene. Cells infected by the mil-plus mutant exhibit

the

ristic cytoplasmic

to

product.

When

they exhibited

fluorescence

cells

were

corresponding

infected

the characteristic

by

the

nuclear

charactethe

myc-plus

v-mil mutant

fluorescence

cor-

responding to the v-myc

product. So having at hand both

tants that were active

in their way of

and

producing

the

right

protein,

we

infecting asked

the

the

mu-

cells

following

questions. First, we asked if we could define biological tests for each

12

of

these

oncogene

products.

neuroretina cells because

We

used

our

mutants

to

it was known previously

by George Calothy's group in Orsay

(France) that

infect

and

shown

the

wild-

type MH2 virus could induce these cells to grow in culture. We asked whether these cells were growing because of the gene product mil or the gene product myc Using

our mutants,

number

of

culture very

it became

neuroretina

cells

clear

that

versus

(Figure 4) the wild-type

quickly.

The mil expressor

, or both. if

the

virus

you

induces

mutants

score of

number

days

cell

(mil-plus

the in

growth

mutants)

do almost as well. And the myc expressors don't have any detectable

effect

in

those

conditions.

In

other

mil gene product is necessary and sufficient cell

growth

of

these

neuroretinal

cells.

words,

the

to promote

the

This

is

shown

in

Figure 5 in another form. We wanted to have a similar test

for

the myc expressor

tants. We showed that these mutants were another

type

of

cells,

chick

embryo

able

to

mu-

transform

fibroblasts.

Although

these cells are readily transformed by the myc-plus mutants, mil-plus mutants do not apparently modify these cells in our conditions

(Figure 5).

In conclusion, what is useful and convenient

in

is that for both genes, we have at disposal two

this

sytem

independent

tests where each one is positive for one of the genes, whereas it's negative for the other gene. The second question was to see if we could show

cooperation

of these two oncogenes in the transformation of given cells. Again, the answer was yes; If you test now not only the proliferation of neuroretina cells, but also the transformation of those cells, as tested by soft agar cloning, you will see (Figure 5) that the wild-type virus that genes mation

is

able

to

induce

proliferation

expresses and

also

the

two

transfor-

(colonies in solf agar); mil-plus mutants are able to

induce proliferation, but do not induce colonies, so they do not transform these neuroretina cells. And the myc mutants, since they don't

induce

proliferation

in

those

conditions.

13

In 7d-old Ck

InCk

emb. N.R cells

macrophages /^TTN

Quiescent NR cells

Ï

^ ¿ J

v - m l l -^J

v-myc

Transformed L ^ J I O Ì Mildly tumorig. — N o n metastatic l^OA c-MGF dep

Mitogenized Non-transf.

v-myc

Normal macrophages

1

Y «XU

v-mil Very tumorig. Metastatic c-MGF indep

Transformed

(autocrine)

(Coll. with

(Coll. with

C A L O T H Y ' s group)

GRAF's

group)

Figure 6 could hardly, of course, effects

of

wild-type

induce colonies.

MH2

and

mil-plus

simple possible explanation, which

But comparing

viruses

for

their

to

is that cells induced

proliferate by the mil gene product now become the mya product

leads

the

transformation.

And

sensitive that

a to to

turned

out to be the case. We could show by reconstitution

experiments

(Figure

6) that

neuroretina cells infected with the mil-plus mutant proliferate and will grow for weeks or months, but they rapidly become transformed. We conclude

that

the

two

genes

cooperate

for the transformation of chicken neuroretina cell. We asked whether we could demonstrate cooperation in another cell type than neuroretina cells. We used chicken macrophages, instead of taking embryo neuroretina

cells,

and

observed

that

normal

macrophages

can

be

transformed with the mya expressor mutants. But these transformants depend

for their growth

on a growth

factor,

the

14

myelomonocytic growth factor in the medium. If you don't ges

do

not

grow.

If

you

(c-MGF), which has to be add the factor, these

now

superinfect

these

added

macropha-

macrophages

with the mil-plus mutant, you can see that they rapidly come

independent

of the growth

is true in-vitro duced

by

these

two

bird and are very ments

with

factor

as well as in-vivo. genes

together

(Figure

6). And

Graf's

group

cells have now become autocrine

are

found

all

in

over

from

Heidelberg

that

for this

(or a very themselves.

So the conclusion

is that we can clearly cooperate

in

the

experi-

related) factor. They produce the factor oncogenes, my a and mil can

this

The tumors that are in-

aggressive. And it is clear

Thomas

be-

these closely

show that the the

two

transformation

of two distinct types of tissues, neuroretina cells and macrophages.

And

we

know

already

that

this

recombination

is

also able to work in again other tissues. Is then the myc oncogene

always bound to cooperate with

the

mil oncogene? Again this idea is too simple-minded. Many authors have shown that, for example, myc can cooperate with the ras gene to transform rat embryo fibroblasts. So in that case, we have another perate to transform

selection

another

of oncogenes

type of cells.

Of

that

coo-

course,

this

has to be explored much further before we try to uncover proper rules concerning Undoubtedly

the

the

cooperation.

availability

of

simple in-vitro

tests that allow to observe within

a few days

of given oncogenes or the cooperation nes should

accelerate

the accumulation

concerning

transformation

and

biological

the

activity

of couples of

oncoge-

of pertinent

results

tumorigenesis.

They

allow

to

proto-oncogenes can turn

and to test

into active oncogenes,

test

to

define the active domains of a given oncogene,

how

rapidly multiple combinations of potentially cooperative oncogenes . They may

shed

light on the complex ways by which probes

reagents should be designed

to detect

specifically

the

or

dif-

ferent perversions by which proto-oncogenes turn into active

15

oncogenes in specific target tissues.

References

Review: Graf, T, D. Stehelin. 651, 245.

1982. Biochim. Biophys. Acta

AEV: Sap, J., A. Munoz, K. Damm, Y. Goldberg, J. Ghysdael, A. Leutz, H. Beug, B. Vennstrom. 1986. Nature 324, 635. Debuire, B., C. Henry, M. Benaissa, G. Biserte, J.M. Claverie, S. Saule, P. Martin, D. Stehelin. 1984. Science 224, 1456. MH2: Coll, J., M. Righi, C. De Taisne, C. Dissous, A. Gegonne, D. Stehelin. 1983. EMBO J. 2, 2189. Galibert, F., S. Dupont de Dinechin, M. Righi, D. Stehelin. 1984. EMBO J. 3, 1333. Bechade, C., G. Calothy, B. Pessac, P. Martin, J. Coll, F. Denhez, S. Saule, J. Ghysdael, D. Stehelin. 1985. Nature 316_, 559 . Martin, P., C. Henry, F. Ferre, C. Bechade, A. Begue, G. Calhoty, B Debuire, D. Stehelin, S. Saule. 1985. J. Virol. 5J7, 1191. Martin, P., C. Henry, F. Denhez, P. Amouyel, C. Bechade, G. Calothy, B. Debuire, D. Stehelin, S. Saule. 1986. Virology 152, 2 7 2 Graf, T., F. Von Weizsaecker, S. Grieser, J. Coll, D. Stehelin, T. Patschinsky, K. Bister, C. Bechade, G. Calothy, A. Leutz. 1986. Cell 45, 357.

BIOCHEMICAL MYELOID W.L.

AND MOLECULAR

GROWTH

EVENTS

CONTROLLED

BY L Y M P H O I D

AND

FACTORS.

FARRAR,D.K.

FERRIS,

and

A.HAREL-BELLAN

L a b o r a t o r y of M o l e c u l a r I m m u n o r e g u l a t i o n , B i o l o g i c a l R e s p o n s e Modifier Program, National Cancer Inst.-Frederick Cancer Res. F a c i l i t y , F r e d e r i c k , MD 21701 USA Introduction We

have

examined

stimulated leukin

by

3(IL

GM-CSF.

cytokines,

3),

Here,

a number and

the

we have

three principal

of b i o c h e m i c a l including

clonly

areas;

1) t h e

activation

pathways

2)

regulation

of g e n e

the

and anti-growth cleotides)

and

lar a c t i v a t i o n

interleukin

stimulating

d i v i d e d the

kinase

topics

evidence

from ancient

AMP,

2),inter-

factors,

G-CSF

suggests

interkinase

transcription

the potential

2(IL

events

to be d i s c u s s e d

which

and perhaps

signals(i.e.cyclic 3)

and molecular

multiple

regulation,

by the g r o w t h "anti-sense"

evolution

of g r o w t h

stress-response

and into

factors oligonu-

or

cell-

mechanism(s).

Results The

results

from numerous

are summarized many

of

cell

lines

the b i o c h e m i c a l

also been IL 3.

in F i g u r e

but also

response

unrelated studied,

studies

includes

seen with

It is c l e a r

erative

studies

from many

unique

activation

ically

regulated

of e i t h e r of p r o t e i n

stable kinase

One mechanism

growth

m a y be

factors. kinase

(PK-C),

of s t u d y i n g

that

Among

phorbol

esters

intracellular

have

including

cellular

prolifagents

those

best

, are stimulants C activation.

independently addition

others

2-dependent

factors,

Both

or,

cultures

in t h e

(PMA,TPA,

events

of

pharmacolog-

to c e l l

cylic AMP analog(8-Br-cAMP) C

IL

features

thatthe

of a c t i o n

the e x o g e n o u s

with

and

encompasses

by p h a r m a c o l o g i c a l

or p r o t e i n

pathways by

stimulating

studies

mechanisms

cyclic AMP accumulation kinase

performed

be m o d u l a t e d

to t h e p r i n c i p a l with

laboratory

presented

those genral

the c o l o n y

may

by o u r

1. T h e m o d e l

would

Lymphocyte Activation and Differentiation © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

case

etc.). be

to

18 compare the effects of direct activators of specific

kinases

with the desired growth factors for the biochemical or molecular

observation. We have used this strategy for the exam-

ination of growth factors modulation of cellular protein phosphorylation

and gene

regulation.

IL 2 (1) and IL 3 (2) have both been shown to induce the phosphorylation of a 68Kd protein on threonine-serine Synthetic diacylglycerol(OAG),

residues.

the endogenous activator of

PK-C also was able to stimulated phosphorylation of the p68 molecule on the same residues. Moreover, purified PK-C could phosphorylate the p68 protein in vitro. In addition to the finding that direct activators of PK-C and IL 2 or IL3 stimulated the common phosphorylation of

certain proteins, the

physiological ligands also induced the phosphorylation of additional sets of proteins possibly not substrates of PK-C.

W? cAMP-Dependent Kinase

J L \

/8Br

VCAMP

1.

Inhibition ol protein s y n t h e s i s

2.

Stimulation of c - f o s , c - m y b

G r o w t h Factor K i n a s e a n d or Protein K i n a s e C

S6 Kinase

s S

'mrivh^

1.

Stimulation of transcription a n d translation

Ca2+

?

Fig. 1. Biochemcial and Molecular Events Stimulated by IL 2.

19 One

such kinase

somal ated

protein in C T 6

1 hr. The

substrate

S6

(3). T h e

cells with

a similar

phorylate kinase

was

ated cells which t h e S6 p r o t e i n situ when The data

ligand

these

osphorylations

were

of

ase activity

PK-C

also

This observation

suggested

system which

would

ivity by the

action

attractive

since

olism and ponse not

observed

to G - C S F ,

induce

however,

G - C S F all

induced

deoendent

cell

raises

ation may exist potential ility

lines,

that

This

many

a cell

idea

line

the

t h e S6

is

direct

multiple

protein.

may be a b l e

of p 6 8

to

res-

to G M - C S F IL 2,

IL3,

of k i n a s e

interdigitate have

"shunt"to

the

did and

factor

observation

pathways

metab-

Finally, in

in r e s p e c t i v e

would also

act-

activa-

of c e l l u l a r

responsive

receptors

cascade

kinase

synthesis.

This

kin-

particularly

(4). A l t h o u g h

GM-CSF did not.

Such a system

receptors

activat-

of a n o t h e r

how m u l t i p l e processes

of p 6 8

and specific

schemes.

action

of a k i n a s e

phosphorylation

that

to t h e

of k i n a s e Second,

ph-

Additional

of o n e k i n a s e

p68 phosphorylation

the p o s s i b l i t y

PK-C.

unrelated

of m a c r o m o l e c u l a r

phosphorylation

First/

some protein of

system

in

stimulants.

following:

phosporylated

explain

the

as o b s e r v e d

the p o s s i b l i t y

to c o n t r o l

regulation

also

soluble

stimul-

the m o d u l a t i o n

of a n o t h e r .

it c o u l d

tion could arise we have

involve

did

phosphorylated

activators

for by PK-C.

in-

phos-

another

u s e d as

the

extr-

of S6 a s

IL 2 o r O A G

pattern were

protein

with

could not

in t h e a c t i v a t i o n

specifically

S6

within

of P K - C

pattern PK-C

stimulated

observed

resulted

which

activator

In f a c t ,

suggested

ligands

to d i r e c t

of t h e

conditions

ligands

ions than c o u l d be a c c o u n t e d activation

levels.

a more-elaborate

suggesting

status

from either

peptidic

studies

growth

common

phosphorylations PK-C

same

phosphoryl-

chain elongation

2, p u r i f i e d

the p h y s i o l o g i c a l from

is m a x i m a l l y

ribo-

2 or d i a c y l g l y c e r o l

a direct

u n d e r _in v i t r o

the p h y s i o l o g i c a l

of

IL

isolated

in t h e

to b e t h e 4 0 S

phosphorylation

S6 to p h y s i o l o g i c a l

activity

IL

protein

Although

peptidic

the physiological

by

phosphorylation

increased

acted polyribosomes. duced

identified

S6 p r o t e i n

stimulated

increased

correlated

was

clearly activ-

among

the

flexib-

alternative

20 pathways native

in t h e e v e n t

systems

the cell.

Our

ine k i n a s e s . dimensional kinase

would

data does

gels does seen

remains

intrinsic factor CSF-1 have

to the

will

also posess identified

case

teins

function,

inturn generates

function which

synthesises

the known

IL

intrinsic

kinase

protein. been

2

identified,

ptors which molecule

its

contain

to p r o b a b l y

One possible domains

might

kinases

may

couple

be

through

the

Evans,

could

stimulate

activty

olated were

cell

in e r r o r

subsequent was

of

regarding

experiments

stimulate

addition

the

membranes.

by

IL

which

the

relative

size

would

participation et.al.(5)

devoid

revealed

that

IL 2 to t h e m e m b r a n e s

to o t h e r the

activity in f a c t rather

increased

rece-

p70

activity. of

kinase to

of G T P - b i n d i n g

recently

the o r i g i n a l

levels

recently

or d i r e c t l y

of a G T P - b i n d i n g specfic

any

(Tac)

has

suggest

such enzymatic systems

horm-

regarding

the p55 (p70)

the

enzyme

to e s t a b l i s h of

protein

receptors

system

adrenergic

Information

domain

activty

2 at pmol

none

here.

regulates

cyclase

been able

small

Although

for

so far

mentioned

In t h e c a s e of

to a m p l i f i e r

(G-proteins).

will

growth

to a n a m p l i f i e r

messenger

IL 2 b i n d i n g

teins

itself

follow

pro-

receptor

by w h i c h

not

In

not contain

mechanism

pos-

the m e m b r a n e ?

has not

rather

The

a GTP-binding

is t h e a d e n y l a t e

tyrosine

tyrosine

kinases

activity,

two

traverse

to the s t r u c t u r a l

another

tyrosby

such as that

factors

from ATP(fig.l).

receptor

While

the

system cAMP

kinase

of

for

stimulants,

a second kinase.

of a p r o t e i n

the a m p l i f i e r

receptors

signal

hormone

to c o u p l e

lines.

tyrosine

of t h e r e c e p t o r

the g r o w t h

factor

of a d r e n e r g i c

which ones

intrinsic

with

cell

of

alter-

kinases

the minute

cascades which

some

Such

advantages

the a c t i v a t i o n

detect

domain

in t h e k i n a s e Clearly,

survival

serine-thronine

a family

structural

How does the g r o w t h the

of

in n o n t r a n s f o r m e d

stimulation.

been

not exclude

that

blockade.

obvious

not readily

high

also participate

possible

have

The evaluation

activty

sibility

of s o m e also

showed protein

other pro-

that in

IL2

is-

calculations of G T P

bound,

GTP-binding than nmol.

The

the a p p a r e n t

af-

21

f i n i t y of a G - p r o t e i n possible protein

interaction activity.

receptor

complex

c, or d i r e c t l y Lymphokine We have wth

Whether

recently

evaluated

accumulation

of For

been shown

to s t i m u l a t e

interferon

of

mRNA

colony lear

Bellan

stimulating

imulated

that

the

by

comparison

IL 2 a n d

those

essentlially

(10)

temporal

events

cells will

stimulated

revealed

by

in t h e

seen

lymphoid

the

to s t u d y i n g

anti-growth The addition

we

signal of

of c l o n e d

cell

nuc-

clearly

fibroblasts

Early

such

genes

line and not

fibroblastic.

of a

on

potent

proliferation.

or s t a b l e

derivat-

the p r o l i f e r a t i v e

IL 2 (11)

as

the

stimulants

and myeloid

inhibit

c-fos

observed

lines and not

the effects

to e i t h e r

same.

stimulated

c-myb was

(PGE2)

among

of

of g r o w t h

lymphoid

AMP agonist

lines

the

st-

stimulation

cell

and

at

all

be the

oncogenes

in t h e

effects

(8-BR-cAMP)

cell

of

s e r u m w i t h irouse

examined

to b o t h

cyclic

i v e s of c y c l i c A M P ponse

have

IL 2

three

and GM-CSF

probably

early

Likewise,

In a d d i t i o n

gamma

(9).

IL 2. T h e d a t a

fibroblastic

lineages.

lineage

expression,

found that

shown

lineages.

in l y m p h o i d a n d m y e l o i d

gene

most

a consensus

KC w e r e

IL 2

of

decarboxylase

expresión

by

the

associated with growth

those

the disinct

only

have

as s t i m u l a t e d

and c-myc between m y e l o i d or

we have

, IL 3, G - C S F ,

between

of

the synthesis

and

have

c-myc, and c-myb(6,7).

of o r n i t h i n e

gro-

state

esters

that

nuclear

the

, secretory

& Farrar

lymphoid and myeloid A direct

of

transcription

stimulated

the same

relatively

several

and steady

levels

factors

IL 2

unkown.

and post-transcriptional

proto-oncogenes

suggests

c-fos,

also

G-

phospholipase

is

2 and phorbol

sequential

activation

both transcritional Furthermore,

IL

(8). M o r e r e c e n t l y , the

of

proto-oncogenes

example,

PK-C

the a

Expression

transcription

the

with

the

such as

kinases

the ability

the

proto-oncogenes,

thus detected complex

couples

system,

associated

several

genes.

IL 3 s t i m u l a t e

G-protein

of G e n e

to m o d u l a t e

and activators

the

membrane

structural nuclear

This assay

IL 2 r e c e p t o r

to an a m p l i f i e r

to

Regulation

factors

mRNA

for G T P . of t h e

or

colony

res-

22 stimulating elevation are

factors

of

largely

(10). The m o l e c u l a r

cyclic AMP unknown.

When

CT6 cells,

stimulation

cumulation

was

when

inhibit c-myc

factor

response

the a b i l i t y

mRNA.

cAMP

ation

(9).

c-fos

expresión

In m y e l o i d and

cells/

The effects

of

these mRNAs

Client t h e e l e v a t i o n macromolecular in r e s p o n s e

of

same

gene

inhibited genrally cAMP

again

were

This of

"Anti-Sense"

of

the data a v a i l a b l e

remains genes

sources

to h o w

on the

proach

this

cell

cycle

Two

to the

of c - m y c . O l i g o m e r s respective The

coding

15-mers

and remained Both IL

the

factor

were

2 stimulation

the

suggested levels

e x o n of

started with

of

to r a p i d l y

stimulus

for

of p r e a c t i v a t e d

The

3-6

resting T cells

of

these to

of t h e

were

ap-

specific

synthesized

the s e c o n d

initiation

for

question

oligonucleotides

codon

15 n u c l e o t i d e s penetrate

undegraded

multiple

attempted

sequence

c-fos and

the

and were

from

function

We have

to the c o d i n g

first

shown

syn-

proto-oncogenes

such oligonucleotides

relatively

activation

of

u s e of c o m p l e m e n t a r y

regions

the

proliferation

stimulation.

progression.

orientation

in q u e s t i o n .

complementary

the e x p r e s s i o n

growth

by the

at

Proto-Oncogenes.

t o selectively d e t e r m i n e

issue

in a n t i - s e n s e gene

of

that

accompanies

accumul-

factors.

from much

biology

stimul-

stimulation

at m u l t i p l e

It w o u l d a p p e a r cell

2

the p r o t e i n

inhibition

of

expres-

stimulated

Oligonucleotide

gnerally

IL

the

inhibited.

s y n t h e s i s "to e f f e c t

however/

not solely

during

had effects

IL 2

did/

c-myc mRNA

was

severely

inhibit

seen upon

induced

because

deprived

ac-

increased

of c A M P ,

to t h e g r o w t h

2

the a c c u m u l a t i o n

the

analog

CSF

which

growth

observed

cAMP analog

by e l e v a t i o n

of c A M P

to IL

expression

cAMP did not

genes.

cAMP analog

expression

c-fos and Tac mRNA

is a d d e d

IL 2 t o s t i m u l a t e also

by

regulated

c-myb, and Tac mRNA

used.

inhibited

of m R N A

analog

these

accumulation

regulation thesis

was

of

ation(lO). of

The

of

analog

s i o n of O D C m R N A

cAMP

mechanism(s)

lymphokine

of c - f o s ,

seen(12).

the g r o w t h

induced mRNA

inhibits

the

in

cells

of

exon the

length. at

37°C

hrs. T cells

results

(PHA)

and

in t h e

ac-

23 cumulation of c-fos and c-myc mRNA. We allowed

anti-fos

or anti-myc oligomers to penetrate either resting(unact.) T cells or the preactivated stimulated

IL 2-dependent counterparts and then

the cells with either PHA (resting T cells) or

IL 2/ respectively. The sense myc 15-mer was used as a control. From the data in figure 2 it can be seen that anti-myc

oligo-

mer inhbited the proliferative response of resting T cells to PHA

(3 day assay)panel A, and the proliferative response of

activated T cells to IL 2, panel B. Sense myc-oligomer

had

moderate inhibition only at very high dosages of oligomer.

A: Resting T Cells/PHA

B: IL2 Dependent, Growth Arrested T Cells/IL2

Oligodeoxynucleotides (/¿M) Fig. 2. Oligonucleotide O-O anti-myc 1 5 m e r ; A - Z \

"Anti-Sense" of C-Fos and C-Myc. anti-fos 15mer; • - • sense myc

15mer.

Panel A: Human T cells + PHA; Panel B: Act.T cells + IL 2.

24 Anti-fos

oligomer

liferation entially

delete

consequences sequent

greatest

and not

specifc

on the

events

was on PHA

technique

cellular

cell

intracellular

and other

effect

IL 2. T h i s

proteins

biology,but

events

stimulated

now allows

and observe

more

important/

such as late gene

that m a y be a c o n s e q u e n c e

pro-

u s to

pot-

the on

sub-

expression

of s p e c i f i c

protein

expression. Do G r o w t h F a c t o r s or C e l l u l a r Response Mechanisms? From what mechanisms qualitatively speculated

respond

from

isms governing may

study.

We

almost

all

to h e a t heat

some the

have developed

An example

of

shock by

response

the

Since

mechanism

havior

which

HSP 90 a n d

of

HSP

phorbol

ester.

controlling

HSP

This

human

that

suggested

that

activation

a family

of g e n e s

lymphokine

mediated

responses

conserved

70

of p r o t e i n s

mechanisms.

Figure at 37°C

by

Resting

in r e s p o n s e

T

cells

to P H A

the b i o c h e m i c a l

participating

are highly

and

conserved

Since

physiological

w e m a y be a b l e

to a p p r o a c h

their

anti-sense

primitive

suggesting

function.

relative

or

mechanisms

in a n t i g e n

to n o r m a l same

shows

stimulated

than proto-oncogenes,

the

3

family

in c e l l u l a r

by

be-

protein

in r e s p o n s e

activation

under

shock

role

cellular

cells

stress-

are extraordinarily

that

called

to a l l

stimulation

appear

rapidly

like

any biochemical

the heat are

stimuli

following

conserved

ligand

T lymphocytes.

andsuggests

important

of

have mechan-

respond

response

a highly evoked

will

to

signals.

lymphocytes

a family

ancient

90 a n d HSP

cellular

in f a c t m o r e

of

such

these

70 p r o t e i n s

synthesized

on that

to e u b a c t e r i a

two m e m b e r s

IL 2 w i t h a c t i v a t e d also

from the

temperature

utilized

induction

the

can be seen

we asked whether

physiological

come? We that

to e x t r a c e l l u l a r

is a u b i q u i t o u s

this was

cells

biochemical

synthesis This

research

Stress

conserved

early

from mammal

proteins.

of m a m m a l i a n

of a c e l l

from highly

Stimulate

information

earlier

response

normal the

to e x t e r n a l of o u r

relatively

cells

and tissues.

the ability

this hypothesis

found

shock

has

Activators

these

and are an

proteins stimulants

participation oligomer

in

process.

25 A

B

C

• HSP90

HSP70

0

3

6

Hour« ol IL2 Treatment Fig. 3. Induction of Heat Shock Protein Synthesis by IL2. Panel A; IL2-deprived activated T cells;Panel B. 3 hrs post IL 2 treatment; Panel C; 6 hrs post IL 2 treatment. References 1 -Evans, SW and WL Farrar. 1987. J. Cell.

Biochem.34,47.

2 .Evans SW, D. Rennick, W.L. Farrar.1986. Blood. 68,906. 3 Evans, S.W. and W.L. Farrar.1987. J. Biol. Chem.

262,4624.

4 Evans, S.W., D. Rennick,W.L. Farrar.1987. Biochem. J. 244,683• 5.Evans,S.W., S. Beckner,W.L. Farrar. 1987. Nature.

325,166.

6.Farrar, W.L.,S.W., Evans,F. Ruscetti,E. Bonvini, H. Young, M. Sparks.1986.In. Role of Leukocytes in Host Defense (JJ Oppenheim and D. Jacobs, eds.) Alan R. Liss, NY p.75. 7.Cleveland, J.L.,U.R.Rapp, W.L. Farrar.1987. J.

Immunol.138,

3495. 8.Farrar, W.L.,M. Sparks,H. Young.1986. J.

Immunol.137,3836•

9.Farrar,W.L.,J.L. Cleveland,A.Harel-Bellan,M.

Vinocour.1987.

(Submitted) 10.Bellan, A. and W.L.

Farrar.1987.(Submitted)

11.Beckner, S.K. and W.L. Farrar.1986. J. Biol. Chem. 12.Farrar, W.L.,S.W. Evans, U.R. Rapp, J.L. J. Immunol, in press.

261,3043

Cleveland.1987.

PGE2 INHIBITION OF HUMAN T LYMPHOCYTES PROLIFERATION : REGULATORY EFFECT OF PGE2 ON THE INTRA CELLULAR SIGNALING PATHWAYS

S. CHOUAIB , J. BERTOGLIO Laboratoire d'Immunologie, France

et D. FRADELIZI UA

1156

CNRS/IGR,

Villejuif,

INTRODUCTION PGE2, at physiological concentrations, induces a profound inhibition of T Lymphocytes proliferation after in vitro stimulation either with PHA or with monoclonal anti T3 antibodies(1). We have previously demonstrated that PGE2 inhibits the production and release of IL2(2). We have observed however, that supplementing these inhibited culures with exogenous IL2, did not fully restore the T cell response. Further experiments have indicated that the PGE2 mediated inhibition is associated with a parallel increase in intra cellular levels of cyclic adenosine monophosphate (cAMP)(3). This observation led us to investigate the effect of PGE2 on the molecular events involved in the cellular activation process at the level of transmembrane signaling and induction of cytosolic second messengers. The phosphoinositol cascade is known to play a crucial role in lymphocyte activation(4). Elevation of cytosolic free calcium (Ca2+)i and protein kinase C activation are reactions which appear to mediate the response to extracellular ligands in lymphocytes(5). We have examined in the present study, the regulatory effect of PGE2 on T cell activation with respect to calcium mobilization and activation of PKC. Our results indicate that PGE2 immununosupressive effect involves predominantly inhibition of PKC. MATERIALS AND METHODS Peripheral blood mononuclear leukocytes(PBL) were prepared from heparinized venous blood from normal healthy donors. The PBL were isolated by Ficoll-Hypaque density gradient centrifugation. Enriched T cell preparations were obtained by filtration of PBL through a nylon wool column as described earlier(3). Purity was about 95%.

Lymphocyte Activation and Differentiation © 1988 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

28

T lymphocyte proliferation assays were with PHA (10ug/ml) or anti T3 antibodies (25 ng/ml). The tests were performed in microcultures in RPMI 1640 with 10% Human serum as previously described(3). Proliferation was evaluated by 3HTdr incorporation. IL2 activity in supernatants was evaluated using the murin IL2-dependent cytotoxic T cell line CTL-L2(6). IL2 receptor quantification was performed using anti Tac binding or indirect immunofluorescence for the low affinity sj£gs(7). I)IL2 binding was performed for determinatio^gf the ( number of tjigh affinity sites (specific affinity of I-IL2 was 7.5x10 dpm/pM and initial concentration in the assay was 200pM)(8) IL2 used in the experiments was highly purified human recombinant or natural IL2. Calcium mobilisation was studied by the 45 Ca2+ uptake method using CaC12 in the culture medium (luC/ML) and incubation of lymphocytes for 60mn at 37°. Intra cellular free Calcium (Ca2+)i was measured using the fluorescent indicator Quin2 as previously described (9). PHA was purchased from Wellcome or Difco (PHA-P). PGE2 was purchased from Upjohn Co, dissolved in ethanol at 10 mg/ml and kept at -70°. Dibutiryl cAMP (dBcAMP), Cholera toxin (CT), TPA and Isoproterenol (Iso) were obtained from Sigma. A23187 was purchased from Boehringer-Mannheim and Quin2 AM from Calbiochem.

RESULTS

PGE2 INHIBITION OF T LYMPHOCYTE PROLIFERATION AFTER IN VITRO STIMULATION Resting human T Lymphocytes were cultured in vitro in the presence of PHA or monoclonal antj^ T3 antibodies and increasing concentrations of PGE2 (10 to 10 M). In both system T cell proliferation was inhibited in a dose dependent manner (table 1). Addition of exogenous IL2 did not fully restore the lymphocyte proliferation in PGE2 treated cultures compared with untreated cultures.

29

Addition to the culture of TPA, at a concentration which by itself does not stimulate resting T cells, overcome the PGE2 induced inhibitory effect on T lymphocyte proliferation, suggesting a probable interference of PGE2 with PKC activation. ANTI T3 ng/ml 0 25 25 25

Exp2

CULTURES SUPPLEMENTED NONE I L 2 TPA

PGE2 M 0

10

PHA Jig/ml

150 12 7 0 0 32 7 2 0 0 ( 4 3 % ) 19 12 200( 4 % ) 36

8

0

0

1 000

0 , 10.1Q 10

1 0

!0

100 200 700 146 4 0 0 200(41%) 142 000 900( 0 % ) 148 500

CULTURES SUPPLEMENTED NONE

PGE2 M

75 000 19 000(74%) 76 000( 0 % )

WITH

WITH TPA

1 500 110 0 0 0 112 0 0 0 ( 0 % ) 113 0 0 0 ( 0 % )

EFFECT OF PGE2 ON INTRA CELLULAR CALCIUM MOBILIZATION The effects of PGE2 on Ca 2+ flux and intra cellular Ca 2+ mobilization in PHA stimulated lymphocytes were examined. As shown on table 2, PHA stimulation of T lymphocytes resulted in a marked calcium influx measured by 45 Ca 2+ uptake. Addition of PGE2 (10 M) resulted in a decreased 45 Ca2+ uptake. Control experiments indicate that PGE2 does not affect the calcium influx mediated by calcium ionophore A23187. EFFECT

OF

PGE2

4 5

on

AFTER

Ca

2 +

PHA

UPTAKE

IN T

cpn

Cell Treatment

Exp.

Medium

830

LYMPHOCYTES

[Ca 2 + ]|

STIMULATION

I

Exp.

SEM

Exp.

II

Ill

1350

1065

PHA PHA

3930

(100) •

4320

(100)

4130

(100)

PGE2+PHA

2290

(42)

2110

(52)

2080

(50)

A23187

6210

(100)

8025

(100)

7435

(100)

PGE2+A231B7

5630

(91)

7490

(93)

7100

(90)

Jr PGE2

PHA ^-M*.

Table

2

172 n M

30

Intra cellular free calcium was studied using the fluorescent indicator Quin2. As shown on figure 1 the addition of PHA on T lymphocyte increase the intra cellular free calcium to 172 nM within 2 minutes. In contrast, preincubation of T lymphocytes with PGE2 prior to PHA stimulation, resulted in a lower level in free Ca2+ (126 nM). PGE2 EFFECT ON EXPRESSION OF HIGH AFFINITY IL2 RECEPTOR EXPRESSION Previous experiments (not shoyn) have indicated that PGE2 do not affect expression of Tac antigen. However, IL2 receptors with markedly different affinities for IL2 have been described and there are a number of evidence indicating that only the high affinity receptors are involved in the proliferation process. We examined high affinity binding sites on anti T3 stimulated cells (range 3500-5500 sites per cell on 3 experiments) (figure 2). When PGE2 was added to the culture, this number of receptors was on average, reduced by 80 % . In the presence of TPA, however, the density of IL2 high affinity sites was not diminished by PGE2.

FREE 1L2 I M I

• Aliti —T 3

(0-0)

a n t i T3 and PGE2 ( • - • ) a n t i T3 PGE2 and

PHA ( A - Û )

Figure

EFFECT OF PGE2 ON TRANSFERRIN STIMULATED LYMPHOCYTES

2

RECEPTOR EXPRESSION BY PHA

As shown on figure 3 using 0KT9 antibody for detection of the transferrin receptor, addition of PGE2 (10 M) to PHA stimulated cultures inhibits transferrin receptor expression (28 % vs 67 % 0KT9 positive cells). When TPA was added to

31

the culture (but n o t A 2 3 1 8 7 ) a c o m p l e t e r e s t o r a t i o n of t h e expression of transferrin receptor was achieved (78% v s 84%). Transferin receptor expression induced by costimulation w i t h A23187 and TPA was not affected by PGE2. P H A + A231B7

L L L. L. C7H»

72H*

M H