Immunobiology Zeitschrift für Immunitätsforschung

Editors E . D . ALBERT, München • D. BlTTER-SUERMANN, Mainz • M. P. DlERICH, Innsbruck • M. FELDMANN, London • K. HAVEMANN, Marburg • S. H . E . KAUFMANN, Freiburg • H . KIRCHNER, Heidelberg • E . KLEIN, Stockholm • W. KÖHLER, Jena • M. LOOS, Mainz • T . L U G E R , Wien • H . H . PETER, Freiburg • K. RESCH, Hannover • G. RIETHMÜLLER, Mün­chen • M. RÖLLINGHOFF, Erlangen • D . SCHENDEL, München • V. SCHIRRMACHER, Heidel­berg • C . SORG, Münster • R. VAN FURTH, Leiden • H . W AGNER, Ulm • G . WlCK, Innsbruck • R. ZlNKERNAGEL, Zürich

Editor-in-Chief

D. G E M S A , Marburg

Editorial Advisory Board

R. A V E R D U N K , Berlin • J . F . B A C H , Paris • H . B A L N E R , Rijswijk • R. B E N N E R , Rotterdam • H . v. B O E H M E R , Basel • G. B O N N A R D , Bern • D. G. B R A U N , Basel • V. B R A U N , Tübingen • J . BROSTOFF, London • A. C O U T I N H O , Paris • T . D I A M A N T S T E I N , Berlin • W. D R Ö G E , Heidelberg • P. Ö U K O R , Basel • P. ERB, Basel • H . - D . F L A D , Borstel • O. G Ö T Z E , Göttingen • E . G Ü N T H E R , Freiburg • U . H A D D I N G , Mainz • H . H A H N , Berlin • K. H Ä L A , Innsbruck • G . J . H Ä M M E R L I N G , Heidelberg • K. U . H A R T M A N N , Marburg • H . ZUR H A U S E N , Heidelberg • M. HESS, Bern • J . K A L D E N , Erlangen • B. K I N D R E D , Tübingen • T . J . K I N D T , New York • U . K O S Z I N O W S K I , Tübingen • E . K O W N A T Z K I , Freiburg • P. K R A M M E R , Heidelberg • W. L E I -B O L D , Hannover • K. LENNERT, Kiel • F . L I L L Y , New York • J . LlNDEMANN, Zürich • E . M A C H E R , Münster • H . M E T Z G E R , Bethesda • V. TER M E U L E N , Würzburg • H . J . M Ü L L E R -E B E R H A R D , La Jolla • W. M Ü L L E R - R U C H H O L T Z , Kiel • H . PETERS, Göttingen • E . PlCK, Tel Aviv • O. P R O K O P , Berlin • M. Q U A S T E L , Beer Sheva • J . P. R E V I L L A R D , Lyon • E . P. RlEBER, München • E . R Ü D E , Mainz • E . SCHÖPF, Freiburg • H . G. S C H W I C K , Marburg • K. S E T H I , Heidelberg • G . S U N S H I N E , London • N . T A L A L , San Francisco • G. T I L L , Ann Arbor • G. U H L E N B R U C K , Köln • M. W A G N E R , Jena • H . W E K E R L E , Würzburg • P. W E R N E T , Tübingen

Volume 174

Gustav Fischer Verlag • Stuttgart • New York • 1987

IMiversitäts-ßibüothek München

ISSN Immunobiology • Zeitschrift für Immunitätsforschung • 0171-2985 © Gustav Fischer Verlag • Stuttgart • New York • 1987 Alle Rechte vorbehalten Printed by Druckerei Ungeheuer + Ulmer KG GmbH + Co, Ludwigsburg Printed in Germany

Contents Volume 174 • 1987

B E T T M A N N , R., J . D E G W E R T , J. H E U E R , and E. K Ö L S C H : An Evaluation of the Existence of Soluble Suppressor Factors in Cloned Antigen-Specific T Suppressor Lymphocytes 339

B R O C K E , S., L. T A K A C S , J. G E R D E S , H . O S A W A , and T. D I A M A N T S T E I N : The Ontogeny of the Interleukin 2 Receptor Expression and of the Interleukin 2 Responsiveness in the Rat Thymus 266

C H A V A L I , S. R., and J. B. C A M P B E L L : Adjuvant Effects of Orally Administered Saponins on Humoral and Cellular Immune Responses in Mice 347

C H E N - B E T T E C K E N , U., E. W E C K E R , and A. SCHlMPL: Transcriptional Control of u,- and x-Gene Expression in Resting and Bacterial Lipopolysaccharide-Activated Normal B Cells 162

C H O U , Y . K., W . S T A N L E Y , G. V I R E L L A , J. M. K I L P A T R I C K , and H . H . F U D E N B E R G :

Generation of Phenotypically Distinct Macrophage-Hepatoma Hybrid Clones . . . . 444 C S E R M E L Y , P., and J. SOMOGYl: The Possible Pitfalls of the Measurement of Intracellular

Calcium Concentration of Lymphocytes with the Fluorescent Indicator Quin2 . . . . 380 D O R N A N D , J., C. E L M O A T A S S I M , and J.-C. M A N I : Amiloride-Induced Suppression of

Lymphocyte Proliferation Inhibition of IL 2 Receptor Expression after Blockade of Early Sodium Influx 365

E L R I D I , R., A. F. W A H B Y , A . -H . S A A D , and M. A . -W. S O L I M A N : Concanavalin A Responsiveness and Interleukin 2 Production in the Snake Spalerosophis diadema . . . 177

G O O D E L L , E. M. , J. K. S T O L T E N B O R G , and W . E. B O W E R S : Accessory Cell Dependent T Lymphocyte Proliferation: Potent Activity of Dendritic Cells 30

I R A H A R A , M. , M . K A M A D A , T. M O R I , T. S U D O , and T. M O R I : Inhibitory Effects of Tunicamycin on the Mixed Lymphocyte Reaction and Mitogen-Induced Lymphocyte Blastogenesis 190

K A M P E R D I J K , E. W . A., C. D. DiJKSTRA, and E. A. D ö P P : Transport of Immune Complexes from the Subcapsular Sinus into the Lymph Node Follicles of the Rat . . . 395

K I M U R A , S., H . K l Y O N O , S. M. M I C H A L E K , S. H A M A D A , and J. R. M C G H E E : Haptenated Streptococcal Antigens Elicit Either T Cell-Dependent Type 1 or T Cell-Independent Type 2 Immune Responses 146

K R A A L , G., R. V E R D A M , and A. T w i S K : The Influence of Oxazolone on the Recirculation Capacity of B and T Cells 326

K R I S H N A M U R T I , C , P. A H R E N S , and H . A N K E L : Differential Effects of ß- and y-Interferons on Natural Killer Cell-Mediated Lysis of Lung Carcinoma Cells 221

L A N G I I O R N E , J., and R. ASOI ' SKY: Influence of Plasmodium Chabaudi Adami on the Isotypic Distribution of the Antibody Response of Mice to Sheep Erythrocytes . . . . 432

M Ä N D I , Y . , R. P u s z T A i , K. B A R A N J I , G. S E P R E N Y I , B. T A R Ö D I , M. B A K A Y , and I . B E L Ä D I :

The Role of Interferon in the Adenovirus-Induced Augmentation of Granulocyte-Mediated Cytotoxicity in Chicken 210

M Ä N D I , Y . , T. V E R O M A A , K. B A R A N J I , A. M J C Z Ä K , I . B E L Ä D I , and P. T O I V A N E N :

Granulocyte-Specific Monoclonal Antibody Inhibiting Cytotoxicity Reactions in the Chicken 292

M A Y U M I , H . , K. H i M E N O , N . T O K U D A , J.-L. F A N , and K. N O M O T O : Drug-Induced Tolerance to Allografts in Mice. X. Augmentation of Split Tolerance in Murine Combinations Disparate at Both H-2 and Non-H-2 Antigens by the Use of Spleen Cells from Donors Preimmunized with Recipient Antigens 274

M R O W I E T Z , U. , E. C H R I S T O P H E R S , and J.-M. S C H R Ö D E R : C5a-Specific Modulation of Phagocyte Functions in Patients with Localized Bacterial Infections 460

N i C K E R S O N , S. C , and L. M. S O R D I L L O : Origin, Fate, and Properties of Multinucleated Giant Cells and their Association with Milk-Synthesizing Tissues of the Bovine Mammary Gland i . . . 200

484 • Contents

O C K L I N D , G.: Stimulation of Human Lymphocytes by Oxidized Red Blood tells (RBC) in the Presence of Polyethylene Glycol (PEG) 20

P A W E L E C , G., U . S C H W U L E R A , M. B L A U R O C K , F. W . B U S C H , A. R E H B E I N , I . B A L K O , and

P. W E R N E T : Relative Cloning Efficiencies and Long-Term Propagation Capacity for T Cell Clones of Highly Purified Natural Interleukin 2 Compared to Recombinant Interleukin 2 in Man 67

P E R E I R A , C. A., A. M. S T E F F A N , F. K O E H R E N , C. R. D O U G L A S , and A. K I R N : Increased Susceptibility of Mice to MHV 3 Infection Induced by Hypercholesterolemic Diet: Impairment of Kupffer Cell Function 253

P O P E , B. L . : Secretion of a Suppressor Cell-Inducing Factor by an Interleukin 3-Dependent Cell Line with Natural Cytotoxic Activity. I I . Range, Potency, and Kinetics of Suppressive Activity 107

P R O H A S K A , R., and G. R. A D O L F : Characterization of the Human Erythrocyte Comple­ment Receptor CR1 (C3b Receptor) by Epitope Mapping 93

R E A R D O N , C. L., and D. O. L U C A S : Heavy-Metal Mitogenesis: Thymocyte Activation by Zn + + Requires 2-Mercaptoethanol and Lipopolysaccharide as Cofactors 233

R E D D E H A S E , M. J . , R. Z A W A T Z K Y , F. W E I L A N D , H.-J . B Ü H R I N G , W . M U T T E R , and U . H .

K O S Z I N O W S K I : Stable Expression of Clonal Specificity in Murine Cytomegalovirus-Specific Large Granular Lymphoblast Lines Propagated Long-Term in Recombinant Interleukin 2 420

R O B I N S O N , J . H . , and R. K. J O R D A N : Delayed Type Hypersensitivity Responses to the Qa-Tla Region of the Mouse Major Histocompatibility Complex 1

S C H I L T , U . : Murine Cytomegalovirus 10 S C H M I D T , H . , T. F O R S T H U B E R , H.-J . B Ü H R I N G , and C. A. M Ü L L E R : Differential Expres­

sion of the HLA-B7 and the HLA-A2 Gene in Transfected Mouse L(tk~) Cells after Stimulation by Mouse Interferon 51

S C H M I T T , E., B. F A S S B E N D E R , K. B E Y R E U T H E R , E. S P A E T H , R. S C H W A R Z K O P F , und E.

R Ü D E : Characterization of a T Cell-Derived Lymphokine that Acts Synergistically with IL 3 on the Growth of Murine Mast Cells and is Identical with IL 4 406

S E L I G E R , B., G. K R U P P A , and K. P F I Z E N M A I E R : Stable Expression of a Selectable Myeloproliferative Sarcoma Virus in Murine T Lymphocyte and Monocyte Cell Lines 313

S M I N I A , T., C. J . A. D E G R O O T , C. D. D I J K S T R A , J . C. K O E T S I E R , and C. H . P O L M A N :

Macrophages in the Central Nervous System of the Rat 43 S W O B O D A , R., E. W E C K E R , and A. SCHlMPL: Regulation of IL 2 Expression in Mitogen-

activated Murine T lymphocytes 300 S Z A M E L , M. , V. K A E V E R , and K. R E S C H : Functional Domains of the T Lymphocyte

Plasma Membrane: Characterization of the Polypeptide Composition 76 W A N G , K. C , C. R. V E R R E T , R. K. Y U , R. K. G E R S H O N , and S. L E E : An Immunosup­

pressive Factor Interacts with Membrane Glycolipid Asialo-GM 1 Structure 139

Short Communications

A K I Y O S H I , T., F. K O B A , T. K I K U C H I , S. A R I N A G A , and H . T s u j i : Production of Interleukin 2 by Human Lymph Nodes 360

D R Ö G E , W . , H.-P. E C K , M. B E T Z L E R , and H . N Ä H E R : Elevated Plasma Glutamate Levels in Colorectal Carcinoma Patients and in Patients with Acquired Immunodeficiency Syndrom (AIDS) 473

W A L T E R , M. , L. G I S S M A N N , H . Z E N T G R A F , and H . K I R C H N E R : Measurement of Cell-Mediated Immunity Against Bovine Papilloma Virus by Lymphoproliferative Reac­tions 244

Letter to the Editor

W A T T R E , P., A. D E W I L D E , C. H A C O T , and P. R A M O N : Herpes Virus IgM Antibodies in Broncho-Alveolar Lavage of Immunocompromised Patients with Interstitial Pneu­monia 480

Authors' Index

A D O L F , G . R . 93

A H R E N S , P. 221

A K I Y O S H I , T . 360

A N K E L , H . 221

A R I N A G A , S. 360

A S O F S K Y , R. 432

B A K A Y , M . 210

B A L K O , I . 67

B A R A N J I , K . 210,292

B E L Ä D I , 1 .210,292

B E T T M A N N , R. 339

B E T Z L E R , M . 473

B E Y R E U T H E R , K . 406

B L A U R O C K , M . 67

B O W E R S , W . E . 30

B R O C K E , S. 266

B Ü H R I N G , H . - J . 51 ,420

B U S C H , F . W . 67

C A M P B E L L , J . B . 347

C H A V A L I , S. R. 347

C H E N - B E T T E C K E N , U . 162

C H O U , Y. K. 444

C H R I S T O P H E R S , E . 460

C S E R M E L Y , P. 380

D E G W E R T , J . 339

D E W I L D E , A . 480

D I A M A N T S T E I N , T . 266

D I J K S T R A , C . D . 43, 395

D Ö P P , E . A . 395

DORNAND, J . 365

D O U G L A S , C. R. 253

D R Ö G E , W . 473

E C K , H . - P . 473

E L M O A T A S S I M , C. 365

E L R I D I , R. 177

F A N J . - L . 2 7 4

F A S S B E N D E R , B . 406

F O R S T H U B E R , T . 51

F U D E N B E R G , H . H . 444

G E R D E S J . 266

G E R S H O N , R. K. 139

GlSSMANN, L . 244

G O O D E L L , E . M . 30

G R O O T , C J . A . D E 43

H A C O T , C . 480

H A M A D A , S. 146

H E U E R J . 339

H I M E N O , K . 274

I R A H A R A , M . 190

J O R D A N , R. K. 1

K A E V E R , V . 76

K A M A D A , M . 190

K A M P E R D I J K , E . W . A . 395

K I K U C H I , T . 360

K I L P A T R I C K J . M . 444

KlMURA,S. 146 K I R C H N E R , H . 244

K I R N , A . 253

K I Y O N O , H . 146

K O B A , F . 360

K O E H R E N , F . 253

K Ö L S C H , E . 339

K O E T S I E R J . C. 43

K O S Z I N O W S K I , U . H . 420

K R A A L , G . 326

K R I S H N A M U R T I , C. 221

K R U P P A , G . 3 1 3

L A N G H O R N E J . 432

L E E , S . 139

L U C A S , D . O . 233

M Ä N D I , Y. 210,292

M A N I J . - C . 3 6 5

M A Y U M I , H . 274

M C G H E E J . R . 146

M l C H A L E K , S. M . 146

MiCZÄK, A . 292

M O R I , T . 190

M R O W I E T Z , U . 460

MÜLLER, C . A . 51

M U T T E R , W . 420

N Ä H E R , H . 473

N l C K E R S O N , S. C. 200

N O M O T O , K . 274

O C K L I N D , G . 20

O S A W A , H . 266

P A W E L E C , G . 67

P E R E I R A , C . A . 253

P F I Z E N M A I E R , K . 313

P O L M A N , C . H . 43

P O P E , B . L . 107

P R O H A S K A , R. 93

P U S Z T A I , R . 2 1 0

R A M O N , P. 480

R E A R D O N , C. L . 233

R E D D E H A S E , M J . 420

R E H B E I N , A . 67

R E S C H , K . 76

R O B I N S O N J . H . 1

R Ü D E , E . 406

S A A D , A . - H . 177

S C H I L T , U . 10

SCHlMPL, A . 162, 300

S C H M I D T , H . 51

S C H M I T T , E . 406

S C H R Ö D E R J . - M . 460

S C H W A R Z K O P F , R. 406

S C H W U L E R A , U . 67

S E L I G E R , B . 313

S E P R E N Y I , G . 210

S M I N I A , T . 43

S O L I M A N , M . A . - W . 177

S O M O G Y I J . 380

S O R D I L L O , L . M . 200

S P A E T H , E . 406

S T A N L E Y , W . 444

S T E F F A N , A . M . 253

S T O L T E N B O R G J . K . 30

S U D O , T . 190

S W O B O D A , R. 300

S Z A M E L , M . 76

T A K A C S , L . 266

T A R Ö D I , B . 2 1 0

T O I V A N E N , P. 292

T O K U D A , N . 274

486 • Authors' Index

TSUJI, H . 360

TwiSK, A. 326

V E R D A M , R. 326

V E R O M A A , T . 292

V E R R E T , C . R . 139

V l R E L L A , G . 444

W A H B Y , A. F. 177

W A L T E R , M . 244

W A N G , K . C . 139

W A T T R E , P. 480

W E C K E R , E . 162,300

W E I L A N D , F . 420

W E R N E T , P. 67

Yu,R.K. 139

Z A W A T Z K Y , R. 420

Z E N T G R A F , H . 244

Subject Index

Accessory cell dependent lymphocyte proliferation 30

Accessory cells, dendritic cells 30 Acquired immunodeficiency syndrome

(AIDS), glutamate levels 473 Adjuvant effects of saponins 347 Allografts, drug-induced tolerance . . . . 274 Amiloride-induced suppression of lym­

phocyte proliferation 365 Antibody response, plasmodium chabau-

di adami 432 Antigen-specific T suppressor lympho­

cytes, factors 339

B cells, lipopolysaccharide-activated . . . 162 B and T cells, recirculation 326 Bacterial infections phagocyte functions 460 Blastogenesis, tunicamycin 190 Broncho-alveolar lavage, herpes virus

IgM antibodies 480

Calcium concentration, intracellular . . . 380 Calcium concentration, measurement . . 380 Cell-mediated immunity, papilloma vi­

rus 244 Chicken, cytotoxicity reactions 292 Cloning efficiencies, interleukin 2 . . . . 67 Colorectal carcinoma, glutamate levels . 473 Complement receptor CRl, characteriza­

tion 93 Concanavalin A responsiveness, Snake

Spalerosophis diadema 177 Cytomegalovirus 10 Cytomegalovirus-specific large granular

lymphoblast lines 420 Cytotoxicity, granulocyte-mediated . . . 210 Cytotoxicity, the role of interferon . . . 210 Cytotoxicity reactions, chicken 292 C5a-specific modulation, phagocyte

functions 460

Delayed type hypersensitivity 1 Dendritic cells, lymphocyte prolifera­

tion 30 Drug-induced tolerance, allografts . . . . 274

Epitope mapping, complement receptor CRl 93

u- and x. u,cnc expression, B cells 162 Glutamate levels, colorectal carcinoma . 473

Glycolipid asialo-GM! structure, immu­nosuppressive factor 139

Granulocyte-mediated cytotoxicity . . . 210 Granulocyte-specific monoclonal antibo­

dy, cytotoxicity 292

Haptenated streptococcal antigens . . . . 146 Heavy-metal mitogenesis 233 HLA-B7 and HLA-A2 gene expression 51 Hybrid clones, macrophage-hepatoma . 444 Hypercholesterolemic diet, MHV 3 in­

fection 253 H-2 and non-H-2 antigens, split tole­

rance 274

IL 2 expression, T lymphocytes 300 IL 2 receptor expression, amiloride . . . 365 IL 4 acts synergistically with IL 3 . . . . 406 IL 4, growth of murine mast cells . . . . 406 Immune complexes, lymph node follic­

les ' 395 Immune complexes, transport 395 Immune responses, haptenated strepto­

coccal antigens 146 Immune responses, saponins 347 Immune responses, T cell dependent . . . 146 Immunocompromised patients, herpes

virus IgM antibodies 480 Immunosuppressive factor, glycolipid

asialo-GM1 structure 139 Interferon, gene expression 51 Interferon, granulocyte-mediated cyto­

toxicity 210 Interferons, differential effects 221 ß- and y-interferons, natural killer lysis . 221 Interleukin 2, cytomegalovirus-specific

lymphoblast lines 420 Interleukin 2, human lymph nodes . . . . 360 Interleukin 2, natural and recombinant . 67 Interleukin 2 production, snake Spalero­

sophis diadema 177 Interleukin 2 receptor and responsive­

ness 266 Interleukin 2 receptor expression . . . . 266 Interleukin 2 responsiveness, rat thymus 266 Interleukin 3-dependent cell line 107 Interstitial pneumonia, immunocompro­

mised patients 480

Kupffer cell function, MHV 3 infection 253

488 • Subject Index

Lipopolysaccharide-activated B cells . . . 162 Long-term propagation of T cell clones,

interleukin 2 67 Lung carcinoma cells, natural killer lysis 221 Lymph node follicles, immune com­

plexes 395 Lymph nodes, production of interleukin

2 360 Lymphoblast lines, cytomegalovirus-spe­

cific 420 Lymphocyte plasma membrane, poly­

peptide composition 76 Lymphocytes, intracellular calcium con­

centration 380 Lymphocytes, stimulation by oxidized

red blood cells 20 Lymph ©proliferative reactions, papillo­

mavirus 244

Macrophage-hepatoma hybrid clones . . 444 Macrophages, central nervous system . . 43 Major histocompatibility complex,

mouse 1 Mammary gland, multinucleated giant

cells \ . . 200 Mast cells, IL 4 406 Membrane, glycolipid asialo-GMi struc­

ture 139 MHV 3 infection, susceptibility of mice 253 Milk-synthesizing tissue 200 Mixed lymphocyte reaction, tunicamy-

cin 190 Monocyte cell lines, Sarcoma virus . . . . 313 Multinucleated giant cells 200 Murine cytomegalovirus 10

Natural cytotoxic activity, suppressor cell-inducing factor 107

Natural killer cell-mediated lysis 221 Nervous system, macrophages 43

Oxazolone, recirculation capacity . . . . 326 Oxidized red blood cells, stimulation of

lymphocytes 20

Papilloma virus, cell-mediated immunity 244 Phagocyte functions, C5a-specific modu­

lation 460 Plasma membrane, T lymphocytes . . . . 76

Plasmodium chabaudi adami, antibody response 432

Pneumonia, herpes virus IgM antibodies 480

Qa-Tla region 1 Quin 2, measurement of intracellular cal­

cium concentration 380

Recirculation capacity, B and T cells . . . 326

Saponins, immune responses 347 Sarcoma virus, expression in lymphocy­

tes and monocyte cell lines 313 Sarcoma virus, T lymphocyte and mono­

cyte cell lines 313 Snake Spalerosophis diadema, concana-

valin A responsiveness 177 Snake Spalerosophis diadema, interleukin

2 production 177 Sodium influx, amiloride 365 Soluble suppressor factors, cloned T sup­

pressor lymphocytes 339 Split tolerance, allografts 274 Subcapsular sinus, immune complexes . . 395 Suppression of lymphocyte proliferation,

amiloride-induced 365 Suppressor cell-inducing factor 107

T cells, recirculation 326 T lymphocyte proliferation, dendritic

cells 30 T lymphocyte, sarcoma virus 313 T lymphocytes, IL 2 expression 300 T lymphocytes, plasma membrane . . . . 76 T suppressor lymphocytes, soluble sup­

pressor factors 339 Thymocyte activtion, heavy-metal mito-

genesis 233 Thymus, interleukin 2 receptor expres­

sion 266 Transfected mouse L (tk~) cells, HLA-B7

expression 51 Tunicamycin, mitogen-induced lympho­

cyte blastogenesis 190 Tunicamycin, mixed lymphocyte reac­

tion 190

Zn4" + , mitogenesis 233

Immunobiol., vol. 174, pp. 420-431 (1987)

1 Federal Research Center for Virus Diseases of Animals, Tübingen, 2 Institute for Virus Research at the German Cancer Research Center, Heidelberg, and 3 Medical University Clinic, Tübingen, Federal Republic of Germany

Stable Expression of Clonal Specificity in Murine Cytomegalovirus-Specific Large Granular Lymphoblast Lines Propagated Long-Term in Recombinant Interleukin 2

M . J . R E D D E H A S E 1 , R . Z A W A T Z K Y 2 , F . W E I L A N D 1 , H . - J . B Ü H R I N G 3 , W . M U T T E R 1 , and U . H . K O S Z I N O W S K I 1

Received March 25,1987 • Accepted April 13,1987

Abstract

The somatic stability of cloned long-term cytolytic T lymphocyte lines (CTLL) specific for antigens encoded by murine cytomegalovirus (MCMV) was tracked for more than two years of continuous in vitro propagation. Clone Si retained its original specificity for a structural (S) antigen of MCMV for about eight months in the presence of antigen and interleukin 2 (IL 2), but not in IL 2 alone. In the following months, however, in spite of the continued presence of antigen, clonal variants developed that displayed distinct patterns of target cell recognition, including loss of the original specificity and acquisition of exclusive specificity for the natural killer target cell YAC-1. On the other hand, large granular lymphoblast (LGL) lines, line I E l -IL and a series of sublines thereof, could be established that stably expressed Ld-restricted specificity for a viral nonstructural immediate-early (IE) antigen more than two years after withdrawal of antigen and feeder cells when propagated in the presence of pure recombinant human IL 2. The finding that the presence of antigen was not essential for the stability of clone IEl-derived CTLL indicates that maintenance of specificity in LGL lines is not a result of antigen-mediated selection, but reflects an intrinsic property.

Introduction

Based on different in vitro propagation requirements, three types of C T L L have been distinguished (for review, see ref. 1). Type 1 C T L L do not require external supply of I L 2, but depend on the presence of antigen. Such a state lasts only for a few weeks. Type 2 C T L L depend on both, antigen and I L 2, and can then be maintained for several months. Most long-term virus-specific C T L L reported so far belong to this group (2-5). Finally, C T L L growing in lymphokine-supplemented medium in the absence of feeder cells and antigen (6-8) are referred to as type 3 C T L L . Since virus-

Abbreviations: Con A = concanavalin A; CTL = cytolytic T lymphocyte(s); CTLL = CTL line(s); IE = immediate-early; IFN = interferon; IL2 = interleukin 2; LGL = large granular lymphoblast(s); LPS = lipopolysaccharide; \sIL2 = lymphokine supernatant IL 2; MCMV = murine cytomegalovirus; MHC = major histocompatibility complex; NK = natural killer; PFU = plaque-forming units; rhIL2 = recombinant (DNA) human IL2 ; U = units.

Stability of Cytolytic T Lymphocyte Lines • 421

specific type 3 C T L L can be propagated in any laboratory without the need to establish the technology for viral infection, it should be an aim to select such C T L L as reference lines for the definition of virus-encoded epitopes.

When compared with type 2 C T L L , however, the selection of influenza virus-specific type 3 C T L L proved to be a rare event (9), and type 3 clones in general were found to display profound changes in the karyotype (10), frequently associated with expression of nonspecific cytolytic activity (for review, see ref. 11). Degeneration of specificity to an NK-like (12), an aged killer (13) or an indiscriminate, promiscuous target cell recognition pattern was found to be accompanied by the acquisition of L G L morphology and an N K cell surface marker profile (for review, see ref. 14). It has been proposed recently that during adaptation to antigen-independent long-term growth in the only presence of lymphokines, C T L L invariably and rapidly lose the original specificity, supposedly due to overgrowing specificity loss variants (15).

In this communication, we report on virus-specific type 3 C T L L with L G L morphology that could be established with relative ease and did not acquire the promiscuous reactivity predicted. Clone IE1, specific for the viral nonstructural I E membrane antigen of MCMV (16, 17), was selected in July 1984 (18), and deprived of antigen and feeder cells in November 1984. Since then, line I E 1 - I L and sublines thereof have been maintained continu­ously in rhIL2 till now (March 1987) without losing viral antigen specific­ity. The existence of such an L G L line contradicts published opinions. First, an L G L phenotype is not necessarily associated with nonspecific cytolytic activity, and second, antigen is not required for the maintenance of specificity in C T L L .

Materials and Methods

Propagation of CTLL

Technical details regarding the selection of MCMV-specific CTL clones were given in a previous report (18). Line Si-A was split twofold in 6-day intervals and restimulated in 2-ml culture with 1 x 106 30-Gy y-ray-irradiated splenocytes, derived from MCMV-infected BALB/c (MHC-d) mice, and 1 x 105 PFU of infectious MCMV (ATCC VR-194, Smith strain) in the presence of 50 U of rhIL2 per ml. Line IE l - IL and its sublines were split likewise and maintained in the presence of 50 U of rhIL2 per ml without restimulation. The number of restimulations as well as of passages with IL 2 alone is coded in the form AnJLm with full stops indicating recloning (e.g. code A22IL11.9 signifies that the line has been recloned after 22 restimulations followed by 11 passages in IL 2 alone, and was then maintained in IL 2 without antigen for further 9 passages). Lot 89050/84802 of rhIL2 (Sandoz Forschungsinstitut, Vienna, Austria) is > 99 % pure IL 2 (1.04 ± 0.35 ng of LPS per mg of protein) with a specific activity of 7.3 x 106 U/mg of protein normalized to a reference standard (lot ISP-841) supplied by the Biological Resources Branch at the National Cancer Institute, Frederick, MD, U.S.A.

Recloning and statistical analyses

Recloning was routinely done by seeding 0.5 cells per round-bottomed 96-tray (0.2 ml) microtiter well. The clone probability was calculated from the Poisson equation (19). In split

422 • M. J. R E D D E H A S E , et al.

assays (see Table 1), the probability for an independent distribution of distinct monospecific clones (null hypothesis) was obtained by applying Fisher's exact probability test (20).

Target cells and cytolytic assay

The Moloney leukemia virus-induced T cell lymphoma of A/Sn (MHC-a, K k D d ) mice, YAC-1, was used to probe NK activity, and the methylcholanthrene-induced mastocytoma of DBA/2 (MHC-d) mice, P815, served to detect aged killer reactivity. Simian virus 40 (SV40)-transformed kidney cells of B10.D2 (MHC-d) origin, KD2SV (21), and bovine papilloma virus-transformed fibroblasts derived from DBA/2 mice, DBA/2-BPV (provided by Dr. H . Pfister, Erlangen, F.R.G.), were used as probes for MCMV-specific CTL after either MCMV infection (multiplicity of 20 PFU/cell) under conditions of selective and enhanced expression of IE genes (IE-KD2SV and IE-DBA/2-BPV) or presentation of MCMV structural proteins (S-KD2SV) (16, 17). Specific lysis was determined in a standard 3-h 3 l Cr release assay.

Cell surface phenotyping

For cytofluorometric analysis of asialo-GMl expression, the CTL were labeled with rabbit anti-asialo-GMl serum gammaglobulin (0.1 mg per 106 cells; code no. 014-09801, Wako Pure Chemical Industries, Osaka, Japan). Fluorescein-conjugated F(ab)2 fragments of goat anti-rabbit IgG (H and L chain-specific; Cat. No. 1312-0081, Cappel, Malvern, PA, U.S.A.) was used as second antibody. The relative fluorescence intensities of 104 viable cells measured with a fluorescence-activated cell sorter (FACS IV; Becton Dickinson FACS Systems, Sunnyvale, CA, U.S.A.) were expressed in a linear scale of 255 channels, and the fluorescence in the channel that separated the population in two halves is given in Table 5 to represent the median fluorescence of the population.

Assay for I FN

The supernatants of ten IE l - IL CTL cultures were pooled 4-6 days (about one population doubling) after seeding of 105 cells. At the time of harvesting, the differences in the average cell numbers between different lines did not exceed 40%. The IFN assay was performed essentially as described (22). IFN titers were expressed in international reference units (IU/ml) of murine IFN (NIH standard G-002-904-511). Sensitivity to pH 2 characterized the IFN as IFN-y. This result was confirmed by complete neutralization of the IFN activity with monoclonal anti murine IFN-y antibody (hybridoma R4-6A2 secreting IgGl ; kindly provided by Dr. Havell, Saranac Lake, NY, U.S.A.).

Results

Selection and attributes of the CTL clones SI and IE1

The viral antigen specificity of these two clones has been reported previously (18). Clones were derived from mesenteric lymph nodes of 10-month-old BALB/c mice which had been infected with M C M V as new­borns and had established a latent infection after a period of persistent viral replication. Already when clones became first visible in microcultures after only five in vitro restimulations could two discrete morphologic pheno-types be distinguished: clones of large lymphoblasts and clones of small, densely growing lymphocytes. After six months of periodic restimulation (stage A30), the cloned lines IE1-A (Fig. la) and Sl-A (Fig. lb) were inspected by electron microscopy. IE1 cells showed the characteristics of L G L with typical cytolytic granules (23), while Si cells were considerably

Stability of Cytolytic T Lymphocyte Lines • 423

Fig. 1. Morphologic phenotype of the MCMV-specific CTL clones IEl and SI. Electron microscopic analysis of clone morphology. Clone IEl cells (a) are large granular lymphoblasts with a diameter of 16-17 jim, whereas clone Si cells (b) are considerably smaller in size and display a pronounced protopode-uropode polarity. Both bars represent 2 jam. Ultrathin sections were stained with uranyl acetate and lead citrate.

smaller and displayed a pronounced polarity with a protopode occupied by the nucleus, a uropode containing the organelles, and one or more pseudopodia. The morphologic characteristics remained stable in clone Si for about eight months and apparently indefinitely in the L G L clone I E l . Both clones recognized a viral antigenic determinant specified by MCMV, the epitope SI of a structural antigen of the virus and the nonstructural I E epitope I E l , respectively, in conjunction with cellular (D)L d class I glyco­protein, and expressed the surface markers Thy-1.2 and Lyt-2.2, but not Lyt-1.2 and L3T4 (18). Clone I E l was recently found to recognize L fibroblasts (MHC-k) cotransfected with the L d gene and the major I E gene of MCMV, gene iel, that codes for the regulatory, intranuclear phospho-protein pp89 (24). Neither of these clones lysed ConA-induced lympho­blasts of the congenic mouse strains BIO, B10.D2, B10.S, B10.BR, B10.Q, B10.M, B10.P, and B10.RIII (b, d, s, k, q, f, p, and r M H C haplotypes, respectively).

Morphologic alteration and somatic variation of specificity within line SI-A

Line Sl -A proved to be dependent on both, I L 2 and antigen. In the presence of I L 2 alone, cytolytic activity was gradually lost within two months (not shown). Continuous selection by restimulation with infectious virus and irradiated, primed splenocytes resulted in retention of cytolytic

424 • M. J. R E D D E H A S E , et al.

activity, but not specificity. After 15 months of propagation (stage A78), the line displayed morphologic heterogeneity with a high proportion of L G L , and had acquired reactivity against the lymphoma YAC-1 . The synchronism of these alterations along with published experience concern­ing the reactivity of L G L clones (reviewed in ref. 14) suggested L G L to be the mediators of that NK-like activity. To test this prediction, L G L clones and uropodial lymphocyte clones were separated by recloning and probed for cytolytic activity against the specific target S-KD2SV and the prototype N K target YAC-1 (Table 1). It was found that the parental line at that stage comprised four types of clones: the original type consisting of S-specific uropodial lymphocytes and, in addition, uropodial lymphocytes as well as L G L with dual specificity and, against all prospects, S-specific L G L . This result demonstrated that either reactivity can be associated with either morphologic phenotype, and thus proved that both alterations are independ­ent events.

With the aim to rescue clone Si in its original form, long-term lines were established from the four S-specific, uropodial subclones (see Table 1) of line S l -A (U1-U4, Table 2). When the lines were tested 3.5 months after recloning (stage A78.18), line S1-A-U4 had already begun to switch to the L G L phenotype, while line S1-A-U3 retained the original morphology for at least three further months. The reactivity pattern observed at stage A78.34 typified line S l - A - U l as NK-like with dual specificity for the

Table 1. Independent line Sl-A

segregation of morphologic phenotype and NK-like reactivity within

Morphologic phenotype

Number of clones

Proportion of negative cultures (clone probability)

Median value (range) of % specific target cell lysis

Proportion of negative cultures (clone probability)

S-KD2SV YAC-1

Large granular lymphoblast

24 [2]'

12

348/384 (0.952) 29 (13-53)

26 (17-35)

20 (10-40)

< SLb

Uropodial lymphocyte

9 [Of

4

371/384 (0.983) 59 (46-65)

36 (21-60)

38 (11-52)

< SLb

Note: At stage A78, line Sl-A was recloned by seeding 0.5 cells in a 0.2 ml microculture along with feeder cells and infectious MCMV as antigen. After 3 weeks, microcultures screened positive for growth were split and quarter aliquots tested for cytolytic activity. a Data in brackets represent the statistically expected number of double-positive cultures as a consequence of non-clonality. Lysis of S-KD2SV and YAC-1 was mediated by the same clone, because the observed number of double-positive cultures could not be explained statistically (P < 10"9, Fisher's test).

b Significance limit (upper 99% tolerance limit of spontaneous lysis) that was 5.2%.

Stability of Cytolytic T Lymphocyte Lines • 425

Table 2. Somatic variation of specificity in Sl-A sublines

Sl-A subline Morphologic Protocol code Target cells (% specific lysis at E/T phenotype3 ratios of 10-2.5-0.6:l)b

S-KD2SV KD2SV YAC-1 P815

Sl -A-Ul UL A 78.18 32-17-9 < SLC 11-5-3 _d

UL A 78.26 25-15-8 < SL 11-8-4 < SL UL-LGL A 78.34 29-19-16 < SL 22-13-3 < SL

S1-A-U2 UL A 78.18 34-13-7 < SL 6-4-1 _ UL A 78.26 27-21-11 8-5-0 13-9-4 < SL UL-LGL A 78.34 35-33-25 23-10-4 23-21-15 < SL

S1-A-U3 UL A 78.18 26-17-7 < SL 34-18-6 _ UL A 78.26 27-15-11 < SL 22-12-8 < SL UL A 78.34 40-32-18 17-13-6 44-33-25 < SL

S1-A-U4 UL-LGL A 78.18 40-18-12 < SL 21-11-5 _ LGL A 78.26 < SL < SL 20-10-3 < SL LGL A 78.34 < SL < SL 31-20-12 < SL

a Uropodial lymphocyte (UL) or large granular lymphoblast (LGL), or intermediate forms (UL-LGL).

b Mean value of six replicate determinations for each effector-to-target-cell (E/T) ratio tested. c Specific lysis below the significance limit (upper 95 % confidence limit for the mean value of

spontaneous lysis) at the highest E/T ratio tested. d Not determined.

specific target and YAC-1 . Lines S1-A-U2 and U3 represent a novel type of C T L characterized by the potential to lyse non-infected fibroblasts (includ­ing non-transformed embryonal fibroblasts, not shown) which are resistant to YAC-type N K reactivity. The genesis of line S1-A-U4 proved that N K reactivity can be mediated by cells of the C T L lineage, as the original specificity for S antigen of MCMV converted into an exclusive specificity for YAC-1 . None of those variants lysed the NK-resistant aged killer target cell P815 (13).

In conclusion, attempts to recover the original form of clone Si failed and, instead, three distinct target cell recognition patterns were found to have developed in formerly antigen-specific Sl-A subclones.

Stability of specific cytolytic activity in long-term IE1 CTLL maintained in rhIL2

As with clone SI, clone IE1 was first propagated by repeated restimula-tions. At stage A22, line I E l - A was split, and a subline, designated I E l - I L , was maintained without virus or feeder cells in the presence of rhIL2. After withdrawal of I L 2 , I E l - A and I E l - I L cells inevitably died within 48 h, demonstrating that both lines were IL2-dependent. When assayed for

426 • M. J. R E D D E H A S E , et al.

Table 3. Long-term stability of lytic activity and specificity within IEl lines maintained in rhIL 2

Experi­ Effector Protocol Target cells (% specific lysis at E/T ment cell line code ratios of 10-2.5-0.6:!)3

IE-KD2SV S-KD2SV KD2SV YAC-1 P815

1 IE1-A A42 35-15-3 2 0 2 0 IE1-IL A22IL20 39-17-6 3 1 0 1

IE1.14-IL A22IL11.9 57-33-11 4 2 2 0 IE1.18-IL A22IL11.9 43-25-8 1 1 2 3 IE1.2ML A22IL11.9 56-35-14 4 2 4 2

2 IE1.14-IL A22IL11.39 49-29-12 3 1 2 2 IE1.18-IL A22IL11.39 41-24-11 2 0 0 1 IE1.21-IL A22IL11.39 36-30-22 0 2 1 0

IE1.18.15-IL A22IL11.21.18 63-46-35 0 1 2 0 IE1.18.20-IL A22IL11.21.18 70-61-32 3 3 1 2 IE1.18.34-IL A22IL11.21.18 51-44-31 1 4 2 2

3 Mean values of six replicate determinations. In case of non-significant lysis, only the value for an effector-to-target-cell (E/T) ratio of 10 is shown.

specificity after 20 passages in rhIL2 (4 mo, stage A22IL20), line I E l - I L displayed the same specificity pattern as did line I E l - A that had been further restimulated (Table 3, Expt. 1). In detail, both lines lysed KD2SV fibroblasts only when MCMV I E genes were expressed (target IE-KD2SV, but not KD2SV and S-KD2SV), and not YAC-1 or P815. After 11 passages in rhIL2, line I E l - I L was recloned (26 of 384 cultures positive for growth; clone probability 0.965). Non-lytic variants were not observed, and all subclones had retained the original specificity. Three representative sublines (IE1.14-IL, IE1.18-IL, and IE1.21-IL) were established by long-term pro­pagation in rhIL2, and assayed together with the parental lines at stage A22IL11.9 (Table 3, Expt. 1) as well as six months later at stage A22IL11.39 (Table 3, Expt. 2). At stage A22IL11.21, subline IE1.18-IL was again recloned (37 of 384 cultures positive for growth; clone probability 0.950), and sublines thereof (IE1.18.15-IL, IE1.18.20-IL, and IEl.18.34-IL) were assayed at stage A22IL11.21.18 after a total of 50 passages (10 months) in rhIL2 without antigen (Table 3, Expt. 2). All sublines displayed exactly the specificity pattern of the parental lines, and none had developed NK-like, aged-killer, or promiscuous reactivity. This demonstrated that I L 2 was necessary and sufficient for the maintenance of cytolytic activity and specificity in the L G L clone I E l and all its descendants.

Retention of specificity in presence of high doses of rhIL2

Recent findings have indicated the induction of NK-like and promiscu­ous reactivity in antigen-specific C T L clones by high doses of lymphokines

Stability of Cytolytic T Lymphocyte Lines • 427

(12, 14). We therefore tested the disposition of the established C T L L IE1.18-IL to alter the pattern of target cell recognition under the influence of high doses of rhIL2. Table 4 documents that the specificity remained stable when the line was grown for one month (6 passages) in 500 U of rhIL2 per ml (4.4 nM), tenfold the amount used routinely for propagation. After two weeks, part of the line was exposed for further two weeks to 5000 U of rhIL2 per ml, again without induction of significant NK-like or promiscuous reactivity. In detail, KD2SV as well as DBA/2-BPV fibro­blasts were lysed only after expression of MCMV I E genes, and YAC-1 or P815 were not lysed. The same specificity pattern was observed when rhIL2 was replaced for one month by 40 % supernatant of lectin (Con A)-induced rat lymphoblasts (lsIL 2) known to contain a variety of lympho-kines. These data imply that line IE1.18-IL was refractory to an I L 2-mediated change in specificity. It should be noted that prolonged propaga­tion in lsIL2 (50 U of IL2/ml) resulted in a reduced growth and ultimately in the loss of the subline, suggesting that the use of rhIL 2 was essential for the long-term maintenance of line I E l - I L .

Somatic variation of asialo-GMl expression and IFN-y secretion in line IEl-IL

The glycolipid asialo-GMl has been regarded as a surface marker for NK-cells (25). Previous studies have shown that long-term C T L L can also display this marker (14). The demonstration of constitutive asialo-GMl expression in highly specific C T L L disproves a causal linkage between the presence of this glycolipid and NK-like function (compare Tables 4 and 5). At stage A22IL64, line IE1.18-IL and all its sublines still expressed Thy-1.2 and Lyt-2.2 antigens (not shown), but varied in the degree of asialo-GMl

Table 4. Invariant antigen specificity of line IE1.18-IL during exposure to high doses of IL 2

Protocol code for line IE1.18-IL

Lymphokine (U of IL 2/ml)

Target cells (% speci ific lysis at E/T ratios of 10-1-0.1 : l ) a Protocol code for line IE1.18-IL

Lymphokine (U of IL 2/ml)

IE-KD2SV

KD2SV IE-DBA/ 2-BPV

DBA/ 2-BPV

YAC-1 P815

A22IL11.53 rhIL2(50) b 50-45-20 2 52-35-16 0 1 1 A22IL11.47(6)C rhIL2(500)b 46-34-14 1 48-31-11 3 6 2 A22IL11.47 (3/3) rhIL 2 (500/5000) 31-10-5 3 47-17-1 0 4 3 A22IL11.47(6) lsIL 2 (200)d 37-23-11 2 45-25-2 1 3 2

a Mean values of six replicate determinations. b 6.8 and 68 ng (0.44 and 4.4 pmol) of pure IL 2/ml, respectively. c Numbers in parentheses indicate the number of passages in the high dose of lymphokine noted in the second column.

d Lymphokine supernatant of Con A-stimulated rat lymphoblasts (lsIL 2), containing 500 U of I L 2/ml and 54 IU of IFN-y/ml besides other lymphokines that have not been quantitated. Residual Con A activity was blocked by adding a-methyl-mannoside (50 mM).

428 • M. J . R E D D E H A S E , et al.

Table 5. Expression of the NK-cell surface marker asialo-GMl and secretion of IFN-y by I E l - I L sublines

I E l - I L subline FA CS median channel fluorescence3 Titer of IFN-y b

(U of rhIL 2/ml) IU/ml Control anti asialo-GMl

1.18-IL (50) 20 91 54; 36 (<3 ; < 3) 1.18.15-IL (50) 25 163 54; 54 (<3 ; < 3) 1.18.20-IL (50) 15 26 18; 36 (<3 ; < 3) 1.18.34-IL (50) 30 38 12; 12 (<3 ; < 3) 1.18-IL (500) 22 231 162; 162 (<3 ; <3 ) c

a Results are expressed as relative fluorescence intensities on a linear scale with 104 cells analyzed. Staining with FITC-labeled second antibody alone served as control.

b Double determination. Values in parentheses represent the IFN titer after pH2 treatment. The detection limit was 3 IU/ml. Medium and rhIL 2 did not contain IFN.

c Control experiments with up to 1 u.g of LPS per mg of IL 2 ensured that LPS present in the rhIL2 preparation (1 ng per mg of IL 2) did not induce IFN.

expression. The fluorescence profile of line IE1.18-IL (not depicted) indi­cated that the population comprised individual cells with an asialo-GMl expression ranging from almost negative to very high. On the population level, sublines stably displayed a higher (IEl.18.15) or lower (IEl.18.20 and I E l . 18.34) median fluorescence intensity than did the parental line. Expres­sion of the glycolipid was elevated after exposure of the line for one month to 4.4 nM rhIL2 (500 U/ml).

The variation in the amount of asialo-GMl detected at the cell membrane corresponded with the secretion of IFN-y. When maintained in 0.44 nM rhIL2, all lines displayed a low basic level of IFN-y secretion that was enhanced by cultivation in higher doses of I L 2.

These findings indicated that somatic variation did occur in line I E l - I L , but, apparently, antigen recognition specificity was conserved.

Discussion

The selection of antigen-independent antiviral C T L L with constant specificity appears to be a rare event. TAYLOR and ASKONAS have pointed out in a recent review that they could keep only a single influenza-specific antigen-independent C T L L sufficiently long enough for detailed analysis, though a series of antigen-dependent clones were obtained with ease (9). Our long-term study on the cloning of C T L specific for antigens specified by MCMV, a member of the herpes virus group, has now documented long-term stability of antigen-independent C T L L in an essentially different virus system, thus confirming that selection of stable virus-specific type III C T L L is in principle reproducible. To our knowledge, the existence of antigen-independent C T L L has not been reported before for any other

Stability of Cytolytic T Lymphocyte Lines • 429

herpes virus. With respect to the continuity of MHC-restricted cytolytic specificity in the absence of antigen and feeder cells, line I E l - I L and descendants thereof are without precedent among virus-specific C T L L . It is an important aspect that previous attempts to maintain C T L L have employed lsIL 2 comprising a variety of lymphokines/monokines, while line I E l - I L was propagated in purified rhIL2. We have experienced that replacement of rhIL2 by lsIL2 did not induce rapid alterations in the specificity or cytolytic activity of line IE1.18-IL (Table 4), but caused reduced growth, and ultimately resulted in the extinction of the line. The use of rhIL2 may therefore be crucial for a successful maintenance of antigen-independent C T L L .

It has been proposed recently that stable expression of specificity in long-term C T L L is a matter of antigen-mediated selection against clonal specific­ity loss variants rather than the consequence of structural stability of the idiotypic T cell receptor (15). Consistent with that idea, line Sl-A retained specificity only when restimulated periodically with viral antigen. Later on, however, and without any apparent change in the propagation conditions, a spectrum of variants developed, including L G L that had lost the original specificity and had instead attained specificity for the NK-target YAC-1 . Thus, somatic variation in line Sl-A was finally able to override the counterselection by antigen. This proved to be a slow process, since recloning revealed a coexistence of ancestor-type C T L and variant C T L seven months after the first signs for specificity drift in that line.

It is evident from our data that selection by antigen can be excluded as an explanation for the stability of specificity displayed by line I E l - I L . The objection that this clone might be autostimulatory by constitutively expres­sing M C M V I E antigen can be refuted, because neither viral nucleic acid nor I E protein (pp89) was detectable (not shown). Transformation does not offer an alternative explanation either, since after withdrawal of I L 2 all I E l -I L sublines inevitably died. Such a strict IL2-dependence is a strong criterion for a nontransformed state.

It appears that uropodial type II C T L L have a programmed tendency to switch to the L G L phenotype that is characterized by a higher degree of cytoplasmic organization (Fig. 1), while a revert to uropodial morphology was not observed. This unidirectional development suggests that L G L represent the more mature, more specialized, and perhaps also the more stable type of C T L . The morphologic difference between antigen-depend­ent type II and antigen-independent type III C T L L has been recognized before (7, 9). In this context, it is important to point out that clone IE1 was isolated from lymph nodes of latently MCMV-infected mice already as an L G L . Latent herpes virus infection provides a unique condition for the in vivo development of C T L clones, as reactivation events can result in frequent in vivo restimulation, generating C T L L in vivo. It could be speculated that selective expression of I E antigens during reactivation from viral latency (26) selectively favors the in vivo maturation of IE-specific

430 • M . J . R E D D E H A S E , et al.

C T L clones, while S-specific C T L clones must be expected to outlast in a less developed state. Altogether, the genesis of clone I E l suggests that stable type III C T L clones exist in vivo and thus do not represent an artificial state attained only in vitro during long-term propagation.

We hypothesize that, in vivo and in vitro, C T L clones undergo frequent somatic variation until reaching a more stable functional phenotype. This state appears to be signified by L G L morphology, regardless of the final recognition specificity. Cloning of those L G L whose terminal functional phenotype is the expression of MHC-restricted antigen-specificity should result in stable, IL2-dependent C T L L which can then serve as certifiable probes for the definition and identification of antigens. Work to test this prediction is in progress.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 120 Leukemia Research and Immunogenetics, and Grant Ko 571/8 Persistent Virus Infections: Molecular Mechanisms and Pathogenesis.

We thank Dr. D. A R M E R D I N G (Sandoz Forschungsinstitut, Vienna, Austria) for the gener­ous supply of rhIL2, Dr. H . K I R C H N E R (German Cancer Research Center, Heidelberg, F.R.G.) for cooperation and advice, Ms. I . H U B E R for technical assistance, and Ms. S. G R A U

for preparing the manuscript.

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Dr. U. H . K O S Z I N O W S K I , Federal Research Center for Virus Diseases of Animals, P.O. Box 1149, D-7400 Tübingen, Federal Republic of Germany