Kinetics of lymphocyte stimulation in vitro by non-specific mitogens

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form resevoed

Experimental

Cell Research 8.5 (1974) 351-361

INETICS OF LYMPHOCYTE ST1 BY NON-SPECIFIC TH. B. WEBER,l V. T. SKOOG,2 ANITA lMineraa

MATTSSON” and KERSTIN

~I~~A~~-K~~~S~~~~~

institute for Medical Research, SF-00101 Helsinki IO, Finland, and “htitutejor Medical Genetics, S-752 20 Uppsala, Sweden

SUMMARY The role of mitogens during lymphocyte activation was studied with kidney bean leucoagglutinin, concanavalin A and kidney bean phytohemagglutinin. The mitogens were removed by treatment with appropriate antisera, which was demonstrated to remove also mitogens already attached to the cells. The process of lymphocyte activation in vitro can be divided into four distinct steps, three of which are mitogen-dependent and the fourth is mitogen-independent. The first step consists of attachment of the stimulatory molecules to the cell membrane. The second step consists of reaction between mitogen and an activating system. During these two phases the cells become preactivated. The establishment of a preactivated state involves at least -some synthesis of cvtoolasmic RNA. The nreactivated state is reversible and during the third step of lymnbocvte ac&ation the final result of preactivation is determined. Depending on the presence br absence of mitogen the cells may remain preactivated for over 60 h, they may return to the resting state or they may proceed through the final stages of the proliferation cycle and eventually divide. This fourth step is independent of the presence or absence of mitogen. A prolonged contact between cells and mitogen is required during the mitogen-dependent steps. The process of lymphocyte activation by mitogen is thus continuously being regulated by the stimulatory molecuies on the lymphocyte membrane, which may be of considerable significance also for in vivo immunologic& reactions at the cellular level.

Lymphocyte activation in vitro is initiated by the interaction of mitogens with receptors on the cell surface 17, 10, 14, 221.The role of the mitogen after the initial attachment to the cell membrane has been subject to controversy among scientists in this field. Several authors stress that the prolonged presence, i.e. over 20 h, of mitogen during stimulation is necessary [3, 16, 19, 20, 25, 261, while others claim that only a short contact, i.e. a few minutes up to some hours, is needed [4, 7, 1I]. However, the latter reports may be criticized on the ground that there was no control whether the mitogen attached to the cells in fact was removed at the times indicated. Recently it has been suggested that 23 - 741813

the mitogen is required during certain critical steps in the cell cycle IS, 9, 12]. The present investigation was ~~d~r~ake~ to clarify the kinetics of lymphocyte activation in vitro and the role of the mitogen during different stagesof the cell cycle. The mitogens chosen for study were kidney bean Ieacopurified agglutinin, ~hytohernagg~u~~~~~ prepared from kidney Reagents Lab. and concanavahn A _front jack beans. Antilym~hocyte globulin was used for evaluation of the function of mitogen-treated cells. The kinetics of the reactions between plant mitogens and cell receptors studied in a number of investigations [7, 15;

352 Weber et al. Lymphocytes were isolated from peripheral blood 17, 241.The reaction conforms to the laws of and cultured according to Lindahl-Kiessling [8], massaction and thermodynamics and appears with some minor modifications. Triplicate cultures to be completely reversible [17, 241. The of 2 x lo6 cells in 2 ml Eagle suspension medium were set up in polycarbonate tubes (16 x 125 mm, Daniel foIlowing reaction formula should then be Ltd, Finland). The medium did not contain methylcellulose. During incubation the gas phase consisted valid

Re + Mi Z ReMi

(1)

where Re is lymphocyte receptor, Mi, mitogen and ReMi, mitogen-receptor complex. Changing the concentrations of the reactants results in corresponding changes in the equilibrium. This means that mitogens can be removed from the cells by transferring the cells to mitogen-free medium. However, this method of removing mitogen is dependent on the establishment of an equilibrium between free and bound mitogen molecules and in caseswhere the cells contain a great number of receptors the complete removal of mitogen may require several changes of medium. The removal of mitogens from lymphocytes can be greatly enhanced by the use of hapten inhibitors or antisera to mitogens, which very rapidly cause an almost complete elimination of any free mitogens in the cell cultures, thereby promoting the detachment of cell bound mitogen molecules. a-Methyl-D-mannoside is a very efficient hapten inhibitor for the jack bean mitogen ConA, but there are no efficient inhibitors known for the kidney bean mitogens. Therefore antisera to mitogens were used to remove mitogens from cells in this investigation. MATERIALS AND METHODS The mitogens used were: purified phytohemagglutinin, Wellcome Reagents Ltd, UK (PHA-W) 1 ,ug/ml; crystallized leucoagglutinin, Medix Laboratories Ltd., Finland (LA) 5 Pg/ml; concanavalin A, Calbiochem, (ConA) 62 pg/ml, and antilymphocyte globulin produced in rabbits (ALG) 160 ,ug/ml. These substances have been characterized in detail previously [13]. Antisera to mitogens were prepared as described 1131. All radiochemicals were obtained from the Radiochemical Centre, Amersham, UK. Exptl Cell Res 85 (1974)

of 5 % CO, in air. DNA and RNA synthesis were measured by the incorporation of 0.1 ,&i 14C-thymidine (suet. act. 60 mCi/mM) and 2 &i 14C-uridine (spec.‘ -act. 58 mCi/mM), into the acid-insoluble fraction of the cultured lymphocytes [6]. The results are given as the mean cpm for each triplicate culture. Tritium-labelled mitoaens were nreuared bv acetylation with 3H-acetic-anhydrideA (ipec. aci 5.8 CiimM) accordine to Weber 1231. Autoradiographs’of lymphocytes<ured with iabelled mitogens were prepared using AR 10 stripping film (Eastman Kodak Co.). Newly synthesized cytoplasmic RNA was studied according to Cooper & Kay [2]. 70-80 x lo6 lymphocytes were incubated in 50 ml medium. 250 pCi $Huridine (spec. act. 5 Ci/mM) was added at 5 or 18 h and the incubation then continued for 3 h. The cultures were terminated by addition of ice-cold medium and then washed twice with ice-cold buffered saline. The cytoplasmic RNA was isolated and the sedimentation profile studied in sucrose gradients. The gradients were monitored for EzeD nm and the radioactivity was subsequently determined by liquid scintillation counting.

RESULTS Effect of mitogen antisera on DNA synthesis in lymphocytes and on mitogen binding When appropriate antiserum to the different mitogens as added within 1 h of establishment of the cultures, almost complete inhibition of DNA synthesis was obtained at 42-48 h. Later addition of antiserum caused less inhibition and addition after 6 h was essentially without effect, which is in agreement with previous reports [4, 261. The results of the experiments where antiserum was added after 1 h are shown in table 2. The inhibitory effect can be explained in two different ways; either the mitogen is removed from the cell, or its mode of attachment is changed to a form not compatible with stimulation. In order to clarify this point, mitogens were labelled with tritium and autoradiographs prepared of lymphocytes cultured with the labelled mitogens in the

Fig. 1. Autoradiographs of lymphocykx cultured in vitro in presence of tritium-labelkd &ph {c’o~), PHA-W {mik’&) and ConA (60tfom) for 24 h. (Left) Cells from cultures wjvilhout antiserum LO mhtogens: (r@f) C‘ellls from cultures to which antiserum was added after 1 h. Expptl Cd? Wes 85 (ls7$j

354 Weber et al. Table 1. Effect of anti-mitogen serum on the binding of mitogen Number of labelled lymphocytes after 24 h in vitro culture with tritiated LA and PHA-W. Anti-mitogen serum was added after 1 h. Cells containing 4 grains or more were considered labelled

Labelled cells Unlabelled cells Total

3H-PHA-W

3H-PHA-W -t antiserum

415 25 500

459 500

41

3H-LA + antiserum

3H-LA

18

499 1

482 500

500

presence and absence of antiserum. Fig. 1 seems to detach the cell-bound mitogen shows autoradiographs obtained in some molecules. The autoradiographs with ConA typical experiments with 3H-labelled LA, were not quantitatively evaluated. Grain PHA-W and ConA. In the absence of counts were performed in autoradiographs antiserum almost all cells are labelled. with labelled LA and PHA-W and the After the addition of antiserum the label is results are summarized in table 1. It can be concluded that addition of antifound as loose aggregates in the culture medium, while the cells are almost devoid of mitogen serum to lymphocyte cultures relabel. moves the mitogen molecules from the cells The interpretation of the autoradiographs i.e. the mitogen receptor complex is broken with ConA is difficult, becauseof the marked up. The antisera probably react predomitendency to form aggregates in the cultures nantly with free mitogen molecules, which also in the absence of antiserum. However, changes the equilibrium in eq. (1) to the left. also in this case the addition of antiserum Table 2. DNA- and RNA-synthesis

after activation mitogen serum and activation with second mitogen

with first mitogen, inhibition

with anti-

Incorporation of 14C-thymidine at 42-48 h and 14C-uridine at 5-8 and 18-21 h into the acid-insoluble fraction of 2 x lo6 lymphocytes stimulated with ConA (62 ,ug/ml), LA (5 pug/ml) and PHA-W (1 pg/ml). Appropriate antisera were added at 1 h and the second mitogen, ALG (160 pg/ml), at 24 h. The resuhs are expressed as net cpm I S.E.M. Hours

14C-Thymidine (cpm f S.E.M.) 42-48 h

0

1

24

ConA ConA ConA LA LA LA PHA-W PHA-W PHA-W

-

-

8 597fl 1 179,

ALG -

6210&1656

antiConA antiConA antiPHA antiPHA antiPHA antiPHA -

Exptl Cell Res 85 (1974)

983 479

11770f

777 297 6096&l 164 16899+_ 992 757+_

;LG ikG i!LG

271+

63

13441 129+ ill+

139 23 18

14C-uridine (cpm i S.E.M.) 5-8 hours

18-21 hours

1237+159 1 538 + 261 -

-

1 521 k160 857i 89 -

11789F2046 1 530+ 417 -

1370+_167 676+ 80

20066+2431 1034+ 146 -

-

560+

48

-

606121337 3148+ 922

862+

126

Lymphocyte stimulation by non-spec$c rn~to~e~~ 355 ~t~,~ul~tio~ of preactivated lymphocytes by a second mitogen The results of these experiments are summarized in table 2. The lymphocytes were stimulated with ConA, PHA-W and LA and antisera to the mitogens were added after I h. At 24 h ALG was added together with FUdR as described previously [S, 91

36

44

56

66

time of harvest (hours); afuliizate: cpm/l x IO6 cells. Stability of the preactivated state. C&ures mcubated with LA and inhibited by addition of antiPHA at 1 h were given ALG as second mitogen. This was added at intervals, 24 h before harvest. The added 12 h before incorporation of l%thymidine harvest was measured. For explanations see fig. 2.

Fig. 3. Abscissa:

!

36

48

60

Fig. 2. Abscissa: time of harvest (hours); ordinate: cpm/l x lo6 cells. Kinetics of response to second mitogen. Cultures incubated with PHA-W (top), LA (middle) and ConA (bottom) were inhibited by anti-PHA and anti-ConA respectively at 1 h. At 24 h ALG was added as second stimulator. The cells were harvested at 36, 48 and 60 h. At 72 h before harvest 14C-thymidine was added and the incorporation measured. @, first mitogen + antiserum; q , second mitogen given to preactivated cells; B , second mitogen only.

in order to synchroaize the cells and facilitate NA synthesis at 42arallel control cultures without mitogens or with only ALG added at 24 R were established. The incorporation of ihymidine in these cultures did not differ significantly from each other. Cells treated with Con A and LA and appropriate antiserum could be stimulated to start DNA synthesis 12 h after the addition of ALG (fig. 2). On the other hand cells initially stimulated with PNA-W could not be activated in the same way by a second mitogen. Thus cells stimulated with ConA and LA during a short period become preactivated, whereas cells treated with PHA-W are not. owever, &be cells treated Exptl Cd [email protected] (1974)

285

w.s

4s

28s

18s

4s

Fig. 4. Abscissa: sedimentation rate; ordinate: AzGO. Sedimentation profiles of cytoplasmic RNA extracted from lymphocytes cultured with LA or PHA-W (-); lymphocytes cultured with LA or PHA-W with antiserum added after 1 h (-----) and from control cultures (-*-.-.-). (Left) Cultures with LA; (right) with PHA-W. The cells were cultured for 8 h (top) and 21 h (bottom).

with PHA-W and anti-PHA were perfectly viable and could be stimulated by ALG, but the kinetics of the stimulatory process was then similar to that of previously untreated cells, i.e. DNA synthesis started 24 h after the addition of ALG (fig. 2). ALG as well as Exptl Cell Res 85 (1974)

LA or PHA-W could be used as second mitogen on lymphocytes primarily stimulated with ConA. LA and PHA-W could not be tested in the same system as the antiserum used cross-reactedwith these mitogens. RNA synthesis measured as uridine in-

250

i

,,I

/’ I f-t I \ ’ \I / \ ’ \\ /I : \\, I I\ f \ --. \.-./ \ IlfJ

/> !

250 i

I /’

I

I

I-., / \ 1

I

! ‘\ i

II

\ ./“\ ,’ I/ v 1.1

28s

IBS

i

\

\

45

2.3s

cpm. Sedimentation profiles of radiolabelled cytoplasmic RNA A-W (---); lymphocytes cultured with LA or PHA-W control cultures (-.-.-.-). (Left) Cultures with LA; (right) ,&i/ml TI-uridine added 3 h before harvest at 8 h (top line)

as

4s

Fig. 5. .Abscissa: sedimentation rate; ordinate:

corporation into preactivated lymphocytes was also investigated. As can be seen in table 2 the incorporation into cells treated with PHA-W and antiserum was virtually abolished. Also in the LA anti-PHA system it was greatly diminished, while in the Con A and anti-ConA system it was not inhibited at all during 5-8 h and only to 50 “/b at 18-21 h.

extracted from lymphocytes cultured with LA or with antiserum added after 1 h (-----j and from with PHA-W. The cells were cultured with 250 and 21 h (bottom line).

Stability of preactivated state The stability of the preactivated state was studied by preserving preactivated lymphocytes for different time periods at 37°C before adding the second mitogen. Two experiments are shown in fig. 3. In general the preactivated state was preserved for 3256 h. owever, the stability varied considerably and in some instances cells remained Exptl

Cell Res 85 (1974)

358

Weber et al.

unstimulated cultures the RNA synthesis is very low, as judged by the incorporation of uridine into acid-insoluble material. When Lymphocytes treated with ConA and subsequently inhibited by the addition of antiCon A at 1 h received antiserum is added to stimulated cells the PHA-W as second mitogen after 24 h. By adding antiPHA at different times the response to the second RNA synthesis at 5-8 h is not at all influenced mitogen was followed by 14C-thymidine incorporation. when LA is used as mitogen, whereas the Figures represent cpm/106 cells RNA synthesis in cells stimulated with PHA-W is depressed to the level of control Time of addition of antiPHA (hours) cultures. At 18-21 h the RNA synthesis is invariably greatly depressed in cells treated 24 25 30 42 with PHA-W if antiserum is added at 1 h. 3 026 ConA The same held usually true also for LA 523 Con A + antiCon A treated cells, although the results were not as Con A + antiCon A + 2 419 PHA-W consistent as for PHA-W treated cells, and in Con A + antiCon A + some experiments the RNA synthesis at 423 731 2200 3 217 PHA-W + antiPHA 18-21 h remained fairly high. The reason for this variability is not known, but it may be preactivated for over 60 h. The disappearance due to the complexes formed between LA of the preactivation was not associated and anti-PHA serum. The difference bewith loss of cell viability, as the preserved tween PHA-W and LA seemsto be that the cells could be stimulated to DNA synthesis, RNA synthesis is turned off rapidly when but the stimulation kinetics became similar antiserum is added to cells stimulated with PHA-W, while in cells stimulated with LA to that of previously untreated cells. If the loss of the preactivated state is it proceeds for at least some hours. Although caused by breakdown of certain cellular the incorporation of uridine is significantly constituents, for instance RNA or proteins depressed at 5-8 h in cells treated with LA this process would be slowed down by and antiserum (table 2) synthesis of cytokeeping the cells at a low temperature. plasmic RNA is taking place (fig. 5). The However, keeping the cells at t4”C did not incorporation of uridine is related to both nuclear and cytoplasmic RNA and it seems increase the stability of preactivation. logical to assumethat the labelling of nuclear RNA synthesis in preactivated lymphocytes RNA becomes depressed upon treatment The different classesof cytoplasmic RNA in with antiserum prior to any changes in the activated and preactivated lymphocytes were labelling of cytoplasmic RNA. studied by ultracentrifugation in sucrose gradients. The results are shown in figs 4 and Effect of antiserum towards the second mitogen 5. As can be seen from fig. 4 the sedimenta- The response to the second mitogen after tion patterns of the cytoplasmic RNA ex- treatment with anti-mitogen serum was tracted from cells treated with mitogens and studied, using antiserum added at different with mitogens plus antisera do not differ times after the second mitogen. As can be significantly from each other or from control seen from table 3 the response is inhibited. cultures. However, when newly synthesized The inhibition follows a time schedule radiolabelled RNA was studied a different similar to that of lymphocytes treated with pattern was obtained as seen in fig. 5. In antiserum after the first mitogen. It seems Table 3. Effect second mitogen

of antiserum

Exptl Cell Res 85 (1974)

towards the

quite clear that activation by the first mitogen, different stages of Pymphocyte activation or by the second, is not a ‘hit and run’ process, in vitro. In previous reports it was s but that the mitogens, in order to activate the lymphocytes or push them forward in lymphocytes during a short c the cycle, have to remain in prolonged Con A in vitro in someway becamestimulated although not sufficiently to enter the DNA contact with the cells, i.e. for about 6 h. synthetic S phase of the cell cycle [8,9]. Such Kinetics of secondary stimulation cells were termed preactivated lymphocytes. The entering of cells into the DNA synthe- The kinetics of the activation and preactivasizing period (S phase) after the addition of tion of lymphocytes has now been studied the second stimulant was studied in cultures using various mitogens, i.e. IA, A-W and without added FUdR. Fig. 2 shows that it ConA together with ALG. The mitogens takes about 12 h before DNA synthesis were removed from the cells by treatment starts, which means that the preactivation with appropriate antisera. was found that does not push the cells through the entire cells pretreated briefly wi LA or con A 61 period. The second mitogen is thus could later be rapidly activated by ALG, necessary for completion of the Gl period i.e. LA and ConA caused the cells to become while the S period is mitogen-independent. preactivated. PHA- on the other hand did not cause the cells to become preactivated under similar conditions. The reason for ISCUSSION this difference between mitogens is nor It has previously been shown that antisera easily explained. It has been shown in a to mitogens inhibit their effect on lympho- previous report that BHA-W also differs in cytes in vitro, if added at the beginning of the other respects from Con A and LA [13]. cell culture [1, 4, 5, 261. On the other hand, 1PMA-Wseemsto be more strongly attached antiserum added after 6 h in culture is to the cell membrane. ne explanation for essentially without effect on the DNA syn- the differences with respect to preactivation thesis for at least 72 h [4, 261. In earlier of lymphocytes could be that antiserum in t experiments [9] it was considered likely that case of IV&A-W, in addition to reacting the antibody-mitogen complex stayed on with free mitogen also reacts with cell-bound the cell surface. Wowever, using radiolabelled mitogen, and that somehow this reaction mitogens we have now been able to demon- immediately stops the stimulator-y process. strate that antisera to mitogenic lectins However, the cells are not irreversibly and in fact remove those from the cells. As it has damaged by treatment with WAey can be stimulated later been shown that the reaction between the cellular receptors and the mitogens is rever- with AIG. Nevertheless it seems clear that sible it is probable that antiserum changes the reaction of P A-W with the activating the equilibrium in the cultures by reacting receptor system f lymphocytes is very predominantly with free mitogen, thereby different from the reaction of LA and Con A. The mechanism of preactivatlon was causing increasing amount of already bound mitogen to become detached and subse- studied by measuring e ir,corporatioi2 of quently precipitated by the antiserum. Thus radioactive uridin the use of mitogen antisera provides a tool certain degree of for the study of the role of mitogens during for the cells to reach a preactivated state. Exptl Cell

Res 85 (1,974)

360

Weber et al.

However, it is by no means clear whether the new synthesis of RNA is the limiting step or if this occurs in some other metabolic reactions. The RNA extracted from preactivated cells did not differ qualitatively from cells not treated with antiserum, but the quantitative differences, especially with respect to labelling, were highly significant. Investigations of the kinetics and the mechanism of preactivation shed considerable light upon the controversial problem concerning the role of mitogens during different stages of the cell cycle. Several investigators have claimed that only a short contact between mitogen and cell is required for stimulation [4, 7, 1I], while others claim that the mitogen is required to push the cell from the resting state, GO, into Gl and also during a considerable part of, or during the entire Gl phase [3, 16, 19, 20, 25, 261, and even during the DNA synthetic S phase [19]. These discrepancies have probably been created by the use of different mitogens with highly divergent binding and stimulatory characteristics. This and previous studies clearly show that different mitogens may react with the lymphocytes in fundamentally different ways [13]. The present investigation has established that a short contact between the lymphocytes and the mitogen in the beginning of the culture period is not enough to push the cells into the DNA synthetic phase and to undergo mitotic division. Apparently the mitogens are required at the beginning of the cell cycle in order to make the cells enter the 6-l phase from the resting phase, GO. This event is designated preactivation. The time required for the cells to reach the S period is very variable according to previous investigations [3, 18, 261. For the moment it is not known whether this heterogeneity is due to differences in the length of Gl or differences in the time the lymphocytes require to go from GO to G 1. Exptl Cell Res 85 (1974)

Table 4. Activation of lymphocytes StepI

Attachment of mitogen to outer cell membrane of lymphocytes in the resting phase, GO. Step II Activation of resting lymphocytes by reaction with activating receptor system. The lymphocytes graduaIly enter the Gl phase. Step III Determination of lymphocyte response. Depending on time to contact and amount of mitogen the activation may proceed further, or the cells may remain in a preactivated state, or they may return to a resting state. This occurs in the Gl phase. StepIV Point of no return is reachedat the end of the Gl uhase.Then the cells oroceedto the S phase and eventually they may divide, regardless of the presenceor absenceof mitogen.

However, a considerable fraction of the cell population seems to require contact with mitogens only during 1 or 2 h in order to go into Gl as judged from the studies on preactivation of cells with LA and ConA. Once the cells are preactivated they proceed for some time in the Gl phase, but they are not able to complete it and go into S. Thus, in order to go into S the cells require a new contact with mitogen, which again has to remain established for some hours before the Gl is completed. Once the cells have reached a certain point in the G 1 they proceed into the S phase, which probably is mitogen-independent. The activation of lymphocytes in vitro seems to be a very complex process and results obtained with different mitogens are not necessarily comparable, even if the mitogens come from the same plant species, as is the case for LA and PHA-W. The first step in the stimulatory process is attachment of the mitogen to the cell membrane, which step can be clearly distinguished from the following one [9, 211. The attachment is followed by a second step leading to activation of the cells. This step requires the pres-

ence of mitogen on the cell for at least 1 h. Such a short contact with mitogen leads to preactivation of the cells, i.e. removal of the mitogens LA and ConA after 1 h allows the cells to continue in the G 1 phase for a certain time before the cells are turned off. The cells then remain in a so-called preactivated state for up to 3 days before they return to the resting state. Treatment of the cells with a second mitogen during the preactivated state causesthem to proceed further through the Gl and eventually into S. Again, in order to start, the reaction requires prolonged contact with the mitogen. However, presence of mitogen throughout the Gl period and In the S phase is apparently not required. The process of lymphocyte activation according to our present concept is summarized in table 4. A short contact with a stimulator leads to preactivation. Depending on the fate of the stimulator after this initial event the lymphocytes may continue to proliferate, they may remain in a preactivated or primed state for a considerable period or they may return to a resting state. It may thus be concluded that lymphocyte stimulation is not a simple hit and run process, but a complex series of events continuously regulated by the stimulatory molecules. The in vitro activation of lymphocytes with mitogens probably reflects many of the processestaking place during immunological stimulation of lymphocytes in vivo. The results of the present investigation may be of considerable significance for our understanding of immunological reactions. Skiiful technical assistance was provided by Miss lnger Karlberg and Mrs Lucia Killat. This work was aided by grants from the Finnish Research Council for Medical Sciences, the Sigrid Jusehus Foundation, Finska Lakarsaiilskapet, the

SwedishCancer Society (no. 84 AhEn Foundation and the Swe MedicalResearch.

WXA),

the

Society for

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21. Weber, T, Scandj clin lab invest, sup& ill (E969) p. 66. 22. Weber, T, Lindahl-Kiessling, K, Mattson, A & Aim, G V, Life sci I1 (1972) 343. 23. Weber, T, J iabelled camp 8 (1972) 449. 24. - Experientia 29 (1973) 863. 25. Yamamoto, H, Nature 212 (1966) 997. 26. Younkin, L R, Exptl cell res 75 (1972) 1. Received November 30; 1973