lymphocyte mediated cytotoxicity in vitro. induction and inhibition by humoral antibody and nature...

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Transplant. Rev. (1972), Vol. 13 91-114 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the authorfs) Lymphocyte Mediated Cytotoxicity m Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells PETER PERLMANN\ HEDVIG PERLMANN^ & HANS WIGZELL- During the past decade, experimental evidence has been brought forward in many laboratories that lymphocytes are capable of destroying appropriate target cells in vitro. These cytotoxic effects of lymphocytes have been assumed to reflect their effector role in tissue damaging immune responses such as occur in allograft rejection, tumor surveillance and certain auto- immune diseases (Perlmann & Holm 1969). Lymphocyte mediated cytotoxicity in vitro is complex and may involve a number of different pathways. The contention that a cytotoxic reaction is lymphocyte induced does not necessarily imply that the lymphocytes which start the reaction also are the cytotoxic effector cells, nor that the latter are lymphocytes at all. In addition, even in those instances in which there is evidence that both inducer- and effector cells are lymphocytic, lymphocytes of different origin and function may participate in the cytotoxic reaction. Work with mice in alloimmune situations, involving the H2 system, has provided evidence that thymus derived lymphocytes (T-cells) are required for cytotoxicity (Blomgren et at. 1970, Cerottini et al. 1970a,b, Golstein et al. 1972a). In a few instances available data suggest that T-cells may be both inducer- and effector cells (Golstein et al. I972a,b). Recent evidence also indicates that humoral antibodies may induce cytotoxicity of thymus-independent lymphoid cells (Harding et al. 1971, Van Boxel ct al. ^ Department of Immunology, Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, Sweden. - Department of Tumor Biology, Karolinska Institute, 104 01 Stockholm 60, Sweden.

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Page 1: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

Transplant. Rev. (1972), Vol. 13 91-114

Published by Munksgaard, Copenhagen, DenmarkNo part may be reproduced by any process without written permission from the authorfs)

Lymphocyte Mediated Cytotoxicity m Vitro.

Induction and Inhibition

by Humoral Antibody and Nature

of Effector Cells

PETER PERLMANN\ HEDVIG PERLMANN^ & HANS WIGZELL-

During the past decade, experimental evidence has been brought forwardin many laboratories that lymphocytes are capable of destroying appropriatetarget cells in vitro. These cytotoxic effects of lymphocytes have beenassumed to reflect their effector role in tissue damaging immune responsessuch as occur in allograft rejection, tumor surveillance and certain auto-immune diseases (Perlmann & Holm 1969).

Lymphocyte mediated cytotoxicity in vitro is complex and may involvea number of different pathways. The contention that a cytotoxic reactionis lymphocyte induced does not necessarily imply that the lymphocyteswhich start the reaction also are the cytotoxic effector cells, nor that thelatter are lymphocytes at all. In addition, even in those instances in whichthere is evidence that both inducer- and effector cells are lymphocytic,lymphocytes of different origin and function may participate in the cytotoxicreaction. Work with mice in alloimmune situations, involving the H2 system,has provided evidence that thymus derived lymphocytes (T-cells) arerequired for cytotoxicity (Blomgren et at. 1970, Cerottini et al. 1970a,b,Golstein et al. 1972a). In a few instances available data suggest that T-cellsmay be both inducer- and effector cells (Golstein et al. I972a,b). Recentevidence also indicates that humoral antibodies may induce cytotoxicity ofthymus-independent lymphoid cells (Harding et al. 1971, Van Boxel ct al.

^ Department of Immunology, Wenner-Gren Institute, University of Stockholm,Norrtullsgatan 16, S-113 45 Stockholm, Sweden.

- Department of Tumor Biology, Karolinska Institute, 104 01 Stockholm 60,Sweden.

Page 2: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

92 P- PERLMANN, H. PERLMANN & H. WIGZELL

1972, Britton et al. 1973). This antibody induced cytotoxicity has beenobserved in xeno- or autoimmune, or in tumour situations Perlmann &Holm 1969, Bubenik et al. 1970, MacLennan et al. 1970a, Ringertz et al.1971, Skurzak et al. 1972, Pollack 1972). It seems that these two pathwaysof target cell destruction, the one brought about by sensitized T-lymphocytesand the other by thymus independent lymphocytes, are not mutually ex-clusive. Rather, they may either potentiate or inhibit each other and theirinteractions may vary during different phases of an immune response (Lamonet at. 1972, Perlmann et al. 1972a).

In this article we will describe the results of some recent experimentsintended to throw light on 1) the mode of induction of this antibodydependent cytotoxicity, and 2) the nature of the effector cells involved.

1. THE EXPERIMENTAL MODEL

In the present model, the target cells are chicken erythrocytes (Crbc) labeledwith ^^Cr-chromate and the effector cells are highly purified lymphocytesfrom human peripheral blood. Cytotoxicity, assessed as ^^Cr-release fromthe target cells, is induced by minute amounts of anti-Crbc antibodies fromhyperimmunized rabbits, in the absence of added complement. In a fewinstances, results of Crbc lysis by lymphocytes activated with phytohem-agglutinin (PHA) have been included, since this cytotoxic reaction reflectsa T-cell involvement not noted in the antibody induced reactions (Brittonet al. 1973). "Cr-release reported in Tables and Figures in this paper isin all cases based on measurements of independent duplicate incubations(SE < 2 per cent within experiments). Complete lysis corresponds to 85-90I>er cent of ^^Cr-released from cells to medium. Experimental details havebeen described (Perlmann & Perlmann 1970, Perlmann et d. 1972b) andwill also be given in text and figure legends below.

a. Target cells and effector cells. Chicken erythrocytes are suitable targetcells since they are highly susceptible to cell-mediated lysis. At the sametime, they are not lysed when exposed for 3-4 days to lymphocytes in theabsence of any inducing agents, or when kept under culture conditionswithout lymphocytes (Perlmann & Perlmann 1970). This distinguishes themfrom most tissue culture cells which under similar conditions release ^ Crspontaneously at a rate of 1-1.5 per cent/hr already during the first 24 hoursof culture (Perlmann & Holm 1969). Erythrocytes are metabolically moreinert than tissue culture cells and would therefore seem to be less capableto repair surface lesions. Moreover, antigens which may be well expressedon tissue culture cells may be sparse or undetectable on erythrocytes. While

Page 3: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 93

these differences are of minor importance in the present context, a moreserious source of error is the high susceptibility of Crbc to phagocytosisand ensuing lysis by monocytes, macrophages or polymorphonuclear leuco-cytes. When antibody induced cytotoxicity of lymphocytes is studied withthese target cells it is therefore mandatory to remove non-lymphoid cellsfrom the effector cell preparations as completely as possible.

Previous results have indicated that > 5 per cent of monocytes andpolymorphonuclear leucocytes in a lymphocyte preparation increased theinitial rate of lysis without, however, affecting its final extent (Perlmann& Perlmann 1970). In the present experiments, the lymphocytes wereextensively purified. Thus, lymphocytes were prepared by gelatin sedimenta-tion from defibrinated blood without heparin, subsequent removal ofadherent cells by nylon-fiber filtration (Perlmann & Perlmann 1970),followed by removal of remaining red blood cells by centrifugation througha dense layer of ficoll-isopaque (Boyum 1968). When indicated, the lympho-cytes were incubated in tissue culture flasks in medium containing 15 percent foetal calf serum at 37°C for an additional 15-20 hr, in order toremove remaining adherent cells. In some experiments, nylon-filtration wasreplaced by addition of colloidal iron and centrifugation in order to removephagocytic cells (Lundgren & Moller 1969).

100

50

= = = = Q -10 20 30

Figure I. Time course of cytotoxic reaction.Lymphocytes were purified by nylon-fiber filtration (dots) or colloidal iron uptake

(circles), followed by ficoll-isopaque centrifugation. (Nylon lymphocytes contained0.9 per cent polymorphonuclear leucocytes, 0.2 per cent monocytes; iron lymphocytes0.7 per cent polymorphonuclear leucocytes, 0.1 per cent basophils, 0.1 per centmonocytes). 2.5 x 10" lymphocytes were then incubated with 1 x 10" "Cr-Crbc in thepresence of either anti-Crbc serum (solid lines) or normal rabbit serum (dashed lines),final dilutions 10^. Total volumes 1.5 ml Eagle's medium + 5 per cent foetal calfserum. All sera heat inactivated.

Abscissa: hr of incubation.Ordinale: per cent ""Cr-release, corrected by subtracting per cent "Cr-release in

lymphocyte free controls (1.4-3.0 per cent).

Page 4: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

94 p. PERLMANN, H. PERLMANN & H. WIGZELL

b. The cytotoxic reaction. Figure 1 illustrates the typical reaction ratesobtained with lymphocytes added in a 25:1 ratio over the target cells,corresponding to an excess of lymphocytes. The antiserum induced reactionis fully significant after only 1-2 hours of incubation when it actuallyproceeds at its highest rate; during the first 5 hr approximately 8,000 Crbc/hr were lysed by iron-purified lymphocytes, versus 5,500/hr by nylon-purified lymphocytes. The reason for the differences seen between the twopreparations is unknown but is not correlated with any differences in con-taminating non-lymphoid white cells (see legend to Figure 1). When lympho-cytes are freed from contaminating human red cells and incubated withCrbc in round-bottomed tubes as in this experiment, the reaction startswithout time lag and reaches completion within 20-40 hr of incubation.Incubating the cells at the same concentrations but in smaller volumes (e.g.,0.3 or 0.75 ml) and in conical tubes usually results in complete lysis within10-20 hr.

Further information on this cytotoxic reaction is provided by experimentsin which varying numbers of antibody treated Crbc are exposed to a constantnumber of lymphocytes, purified by nylon filtration and ficoU isopaquecenthfugation. The results of one experiment are shown in Figure 2. Verysimilar results were obtained in 7 additional experiments of this kind. Inthis experiment, lymphocytes and Crbc were incubated under stationary

20.10-4

15

10

20 100

Figure 2. Influence of target cell concentration.1 X 10* lymphocytes were incubated with varying numbers of "Cr-Crbc, pretreated

with either anti-Crbc serum or normal rabbit serum, final dilutions 10^. Total volume0.6 ml. Eagle's medium + 5 per cent foetal calf serum. All sera heat inactivated.

Abscissa, number of "Cr-Crbc in incubation mixture.Ordinate, number of killed Crbc after 20 hr (dots) or 40 hr (circles') of incubation,

corrected by subtracting numbers of killed Crbc in normal rabbit serum controls (3-5per cent of numbers killed in antiserum samples. 85 per cent "Cr-release = 100 percent lysed Crbc in this experiment).

Page 5: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 95

TABLE IInhibition of cytotoxicity by cytoclialasin B'

Effector cells -

Crude

Purified

Inhibitor*

5 / i g2.5 .

_

5 /Jg

2.5 .

% "Cr released '

64.16.4

12.3

43.14.9

20.1

% Inhibition"

-9081

_

8953

' 2.5 X 10" leucocytes or lymphocytes, 4 x 10 "Cr-Crbc, total volume 1.5 ml. Timeof incubation, 18 hr.

" Crude: leukocytes obtained by gelatin sedimentation. Purified: lymphocytes furtherpurified by nylon fiber filtration and ficoll-isopaque centrifugation.

' Final concentration of cytochalasin B in incubation mixture.' Induced by heat inactivated anti-Crbc serum added to incubation mixture, final

dilution 10'", and corrected by subtracting per cent release in incubation mixturescontaining normal rabbit ssrum + 5 /ig inhibitor (crude: 1.3 per cent; purified: 0.9per cent). Release in lymphocyte free controls 1.2-1.3 per cent.

' Inhibition as per cent of "'Cr-release in absence of inhibitor.

conditions in conical tubes in a total volume of 0.6 ml. The number ofCrbc lysed increases steadily with the number of Crbc added, withoutreaching saturation. Saturation is obtained, however, when the number ofadded Crbc exceeds 5 x 1 0 ^ (unpublished). At high target cell concentrations,increasing the number of Crbc/tube from 2 x lOMo 1 x 10* (5x) resulted inan increment of 70,000 Crbc lysed during both the first and the secondday of incubation. This speaks against lysis being due to the < 0.5 per centpolymorphonuclear leucocytes present but dying during the first day ofincubation (Perlmann & Perlmann 1970). At lymphocyte:Crbc ratios of1:1, statistically 5-7 lymphocytes were required to lyse one Crbc. Thisargues against the notion that Crbc lysis is due to a phagocytic mechanismby the < 0.1 per cent mature monocytes present in this lymphocyte prepara-tion, since it would imply that each such cell would have to lyse 200-400Crbc under the indicated conditions. Other evidence against monocyticinvolvement is given in the next paragraph (see below). The kinetics ofthe lytic reaction also speak against lysis by soluble factors released fromeffector cells to the medium. This latter alternative has been ruled out inprevious experiments which showed that antibody free bystander cells ina target cell mixture (Crbc - duck erythrocytes) were not destroyed, thusindicating that ^^Cr-release requires close contact between effector cells and

Page 6: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

96 P PERLMANN, H. PERLMANN & H. WIGZELL

antibody coated target cells (Perlmann & Perlmann 1970). Previous light-and electron microscopic observations have also demonstrated that antibodyinduced cytotoxicity is non-phagocytic and that the effector cells haveiymphocytic appearance. When incubated with Crbc or other target cellsin the presence of antibody, lymphocytes show typical signs of surfaceactivation, reflected by uropod formation and increased motility apparentwithin a few hours (Biberfeld et al. 1970, 1972). The cytotoxic reaction cantherefore be inhibited by anti-metabolites inhibiting energy supply, motilityand other surface bound activities of the effector cells (Perlmann & Holm1969, Wasserman et cd. 1971). A typical example, showing inhibition, bycytochalasin B, is given in Table I. This drug is known to affect cellularmotility, phagocytosis and adhesion (Carter 1967, Malawista el al. 1971,Wessels et al. 1971, Sanger & Holtzer 1972).

2. INDUCTION

a. Nature and quantities of antibody. Cytotoxicity of lymphocytes is inducedby antibodies of the IgG-class while antibodies of IgM-class are inactive(MacLennan et al. 1970a, Perlmann & Perlmann 1970). Induction requiresthe presence of an intact Fc-piece. F(ab')2 or Fab-fragments of anti-CrbcIgG, while inhibiting the reaction provoked by intact antibody, lack inductivecapacity (Moller &Svehag 1972, Larsson & Perlmann 1972). The cytotoxicinteraction between human lymphocytes and Crbc, coated with rabbit anti-Crbc, is inhibited by human myeloma proteins belonging to subclasses IgGl,IgG2 and IgG3. The inhibitory capacity of IgG4 is dubious (Larsson et al.1972). In similar experiments, others have recently observed inhibitions byall four subclasses (MacLennan, personal communication). The resultsindicate that Fc-receptors on the effector cells (MacLennan 1972, Lareson& Perlmann 1972, Moller & Svehag 1972), presumably intrumental duringcytotoxicity induction, react with IgG from different species^ and do notdistinguish between the subclasses, with the possible exception of IgG4.It should be pointed out, however, that the interaction between rabbitIgG-Fc and human lymphocytes may differ from that between humanlymphocytes and human IgG-Fc. In any event, different results wereobtained in parallel experiments with human blood monocytes, phago-cytosing Crbc coated with the same rabbit anti-Crbc antibodies. As wasto be expected (Huber & Fudenberg 1968, Huber et al. 1971, Hay et al.1972), in these experiments inhibition was only obtained with IgGl andIgG3 (Larsson et al. 1972).

The number of antibody molecules needed for induction of Crbc lysis

Page 7: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 97

TABLE IIAdsorption of antigen-antibody complexes to lymphocytes

1. Lymphocytes purified by nylon-fiber filtration and ficoll-isopaque centrifugation.2. Incubated for 15-20 hr with

2:1, "^I-IgG from anti-Crbc serum2:2, "'MgG from anti-Crbc serum + CrbcZ:3, '^I-IgG from normal rabbit serum + Crbc.

3. Lymphocytes separated from Crbc by ficoll-isopaque centrifugation. Lymphocytesfrom interphase washed 4 x.

4. Determination of adsorbed antibody by ^"I-counting of aliquots of lymphocytesfrom 2:1, 2:2 and 2:3, respectively.

5. Cytotoxicity of lymphocytes from 2:1, 2:2 and 2:3, respectively. Lymphocytesincubated with5:1, normal rabbit serum + ""Cr-Crbc5:2, anti-Crbc serum + "Cr-Crbc5:3, PHA + ^Cr-Crbc"^Cr-release measured after 20 hr.

is small. Utider the standard conditiotis of the present tests (2.5 x 10^lymphocytes, 10^ ^^Cr-Crbc in 1.5 ml, 20 hr) 50 per cent lysis is obtainedwith 0.1-1.0 ng of IgG-antibody, corresponding to antiserum dilutions oflO'^-IO" (Perlmann et al. 1972b). Some sera are active at dilutions of10"*-10"'^ Thus, otily a few hundreds of antibody molecules/Crbc aresufficient for induction, provided that antibodies are added to the Crbcin advance or to the incubation mixture.

b. Uptake of antigen-antibody complexes. Pretreatment of lymphocytesfollowed by washing requires about 10,000 x more antibody for equivalentcytotoxicity than adding it to target cell-lymphocyte mixtures (Perlmann& Perlmatin 1970). This is probably due to the fact that lymphocytes areknown to firmly bind antibody complexed with antigen, or otherwise aggre-gated (Basten et al. 1972a,b, Dickler & Kunkel 1972) whereas free IgGin only loosely bound. Since the interaction between lymphocytes and anti-body bound to target cells is believed to constitute the triggering step forthe cytolytic reaction, experiments were performed in order to 1) establishif there was a difference in the uptake by lymphocytes of antibody boundto surface antigens of the Crbc, as compared to uptake of antibody alone,and 2) to investigate the cytotoxic potential of lymphocytes pretreated inthis way. The experimental protocol is outlined in Table II. Experimentaldetails have been published elsewhere (Perlmann et al. 1972b).

The restilts of 5 representative experiments are sutnmarized in Table III.Although the recovery of antibody adsorbed to lymphocytes was low (1-2

Transplant. Rev. (1972), Vol. 13 -

Page 8: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

98 P. PERLMANN, H. PERLMANN & H. WIGZELL

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Page 9: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 99

per cent of input), there was a highly significant difference between theamounts adsorbed in the presence or absence of Crbc, respectively; theamounts adsorbed in the former case were, on an average, 40 x greater,corresponding to 2 -6x10* antibody molecules/lymphocyte. The amountsof IgG adsorbed in the absence of Crbc corresponded to those adsorbedfrom control mixtures consisting of Crbc + IgG from normal rabbit serum,or IgG from unrelated antiserum (Perlmann et al. 1972b).c. Acquisition of a specific cytotoxic potential. Pretreatment of the lymph-ocytes with antibody carrying Crbc significantly changed their performancein subsequent cytotoxicity tests. As seen from Table III, these lymphocyteshad acquired a significant cytotoxic potential when tested in cytotoxicitytests with ''^Cr-Crbc in the absence of added antibody. This cytotoxicityis strictly specific for Crbc. Lymphocytes incubated with anti-Crbc alone,with anti-Crbc and non-crossreacting duck erythrocytes, or with IgG fromnormal rabbit serum + Crbc, did not acquire this type of cytotoxicity(Perlmann et al. 1972b). See also Figure 3.

It may be concluded that in these tests cytotoxicity is mediated by anti-body, picked up by lymphocytes from the surface of antibody coated Crbc,and reutiUzed when new antigen in the form of fresh "Cr-Crbc is introducedinto the system. In terms of amounts of antibody actually adsorbed, thisacquired cytotoxicity was, however, smaller than expected. This may be

- 1 5 25

Figure 3. Cytotoxicity of lymphocytes preincubated with Crbc and either anti-Crbcserum or normal rabbit serum.

25 X 10" lymphocytes were preincubated for 18 hr witb varying numbers of Crbcin presence of either anti-Crbc serum (solid lines) or normal rabbit serum (dashedlines), final dilutions 10' .

2.5x10" lymphocytes (see text and Table II) were then incubated with 1x10""Cr-Crbc in the presence of either anti-Crbc serum (dots) or normal rabbit serum(circles), final dilutions 10" . Total volumes 1.5 ml Eagle's medium + 5 per centfoetal calf serum. Time of incubation 18 hr. All sera heat inactivated.

Abscissa: lymphocyte:Crbc ratios during preincubation.Ordinate: per cent "Cr-release, corrected by subtracting per cent "Cr-release in

lymphocyte free controls («- 1 per cent).

Page 10: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

100 p. PERLMANN, H. PERLMANN & H. WIGZELL

TABLE IVCytotoxicity of lymphocytes pretreated with plasma membrane

fractions from Crbc and anti-Crbc serum

Cytotoxicity- of lymphocytes inPretreatment oflymphocytes'

Normal rabbit serum PHA

Fraction I + anti-Crbc serum 1 26Fraction I + normal rabbit serum 1 31Fraction II + anti-Crbc serum 19 33Fraction II + normal rabbit serum 1 32

' 8 X 10* purified lymphocytes in 1 ml Eagle's medium + 5 per cent foetal calf serum;0.5 ml heat inactivated normal rabbit serum or anti-Crbc serum, final dilutions10"*; 0.5 ml of Crbc-plasma membrane fraction I or II, respectively (150 /ig protein/ml). See text. Lymphocytes were incubated for 1 hr at 4°C, separated by centrifuga-tion, washed 4 x.

= Per cent ='Cr-release from 0.5 x 10" Crbc after 20 hr of incubation with 1.25 x 10"preincubated lymphocytes, corrected by subtracting per cent "Cr-release in lympho-cyte free controls. Incubation mixtures contained heat inactivated normal rabbitserum, final dilution lO""", or PHA (100 /^g/ml), in Eagle's medium + 5 per centfoetal calf serum, total volume 0.75 ml.

due partly to the presence of Crbc-antigens transferred to the lymphocytesurface, and partly due to internalization of complexes by endocytosis.Direct evidence for the presence of antigen on the lymphocyte surface isobtaitied by measuring adsorption of ^-^I-anti-Crbc added after isolationof the lymphocytes preincubated with unlabeled anti-Crbc -t- Crbc. Suchexperiments and various autoradiographic procedures indicate that most ofthe bound material occurs as relatively big antigen-antibody aggregates onthe surface of a minority of the lymphocytes (Perlmann, Perlmann & Biber-feld, in preparation).

Available evidence would thus suggest a transfer from Crbc to lympho-cytes of antigen-antibody complexes, perhaps set free in the process oflymphocyte induced Crhc lysis during preincabation. In order to establishthat uptake of antigen-antibody complexes from solution would equip thelymphocytes with similar reactivities, and also to rule out that transfer ofwhole Crbc or debris during lymphocyte separation (step 3, Table II)would be responsible for the results, experiments with proteins from isolatedCrbc-plasma membranes were performed. Crbc, washed 3 x in saline, werelysed in 10 vol. aq. dest., homogenized and centrifuged at 3000 g for 30min. Membranes were then separated from nuclei on a discontinous sucrose

Page 11: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CVTOTOXICITY 10!

gradient, washed and extracted with 1 per cent Na-deoxycho!ate. Proteinfractions were separated on Sepharose 6B and dialysed. Details will bepublished elsewhere (Berzins, Blomberg, Carlsson & Perlmann, in prepara-tion). Table IV shows the results of a typical experiment in which twodifferent protein fractions from these membranes were mixed with anti-Crbc serum or normal rabbit serum. The Table shows the cytotoxic activityof lymphocytes, pretreated with these mixtures and subsequently incubatedwith ^^Cr-labeled Crbc in normal serum. When complexed with antibody,one of the fractions conferred a significant cytotoxicity to the lymphocytes.The reasons why the other fraction was inactive are not known. The cyto-toxic potential of the lymphocytes mediating PHA induced Crbc lysis wasnot significantly affected.

d. Inhibition of antibody induced cytotoxicity after uptake of the sameantibody complexed with antigen. The last two columns in Table III indicatea second important change in the cytotoxic behaviour of lymphocytes, pre-treated with antibody carrying Crbc. When added to ^^Cr-labeled Crbcin the presence of fresh anti-Crbc, the normal antibody induced cytotoxicityis considerably reduced. Lymphocytes pretreated with antibody alone, orwith IgG from normal rabbit serum + Crbc, are not inhibited. The PHA-induced cytotoxicity of lymphocytes preincubated with antibody -i- Crbcis unchanged or slightly increased, thus indicating that this reduction isnot due to a general reduction of their effector function because of treatment(Perlmann et al. 1972b).

An experiment illustrating the above points is shown in Figure 3.Maximal inhibition of cytotoxicity was obtained when antibody treated Crbcand lymphocytes were incubated at ratios of 1:1. At ratios between 1:1 and5:1 the present system reached saturation. Increase of the ratio to 25:1 didnot change inhibition or aquired cytolytic potential.

Inhibition of cytolysis by these preincubation procedures is open to dif-ferent interpretations. Thus, when lymphocytes and Crbc are separated byficoll-isopaque centrifugation after preincubation (Table II), more effectorlymphocytes may be trapped in the Crbc sediment when anti-Crbc antibodiesare present. Microscopic cell counts of the sediments did not support thissince the numbers of lymphocytes trapped in the Crbc sediments were notdifferent. Nevertheless, it was considered important to compare the cyto-toxic potential of lymphocytes separated from Crbc by ficoll-isopaquecentrifugation (step 3, Table II) with that of those isolated from thesediments by a second ficoll-isopaque centrifugation. The results of twotypical experiments are shown in Table V. When incubated with ^^Cr-Crbcand normal rabbit serum, both lymphocyte preparations were equally

Page 12: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

102 P. PERLMANN, H. PERLMANN & H. WIGZELL

cytotoxic. Thus, there is no evidence for a selective removal of a subpopula-tion of antibody reactive lymphocytes accounting for the inhibition pheno-menon. Blocking of lymphocytic Fc-receptors by antigen-antibody complexeswould be one way of interfering with the cytolytic interaction. It is knownthat antigen-antibody complexes, involving antibodies against third partyantigens, or elevated concentrations of aggregated IgG, can block antibodyinduced cytolysis (MacLennan 1972). In the present case, however, inducingand blocking antibodies seem to be directed against the same antigens andto be physico-chemically identical. The lymphocyte associated antibodiesare cytolyticaJly less efficient than the antibodies added directly to lympho-cyte-target cell mixtures, probably because of partial masking of antibodysites by bound antigen. Blocking is presumably ascribable to competitionfor Fc-receptors on the lymphocytes and for antigen on the target cells.

3. SURFACE ASSOCIATED IMMUNOGLOBULINON EFFECTOR CELLS

a. Fractionation on anti-immunoglobuUn columns. The experiments de-scribed above provide direct evidence for the importance of Fc-receptorson the effector cells in antibody induced cytotoxicity. While thymus derivedlymphocytes in the resting stage lack these receptors (Basten et al. 1972a,b),they are found on macrophages-monocytes and polymorphonuclear leuco-cytes (Berken & Benacerraf 1966, Lo Buglio et al. 1967, Lay & Nussen-zweig 1968, Henson 1969, Huber et al. 1969, Phillips-Ouagliata et al. 1971)and on bone-marrow derived lymphocytes (B-cells) (Basten et al. 1972a,b).An additional marker of the latter cells is their high concentration of surfacebound immunoglobulin (Pemis et al. 1970, Raff 1971, Unanue et al. 1971,Wilson & Nossal 1971, Wigzell et al. 1972). We therefore investigated howfractionation of the effector cells through columns charged with rabbitanti-human immunoglobulin (Ig) would affect their cytotoxic performances.

Columns were prepared as described earlier (Wigzell & Andersson 1969,Wigzell et al. 1972). In brief, acid treated glass beads were first incubatedfor 30 min at 45 °C and at 4''C overnight, with 0.5 per cent human IgG in0.15 M phosphate btiffered saline, pH 7.4. They were then added to columns(1.5 X 30 cm, 30—40 ml glass beads) and treated with low dilutions of rabbitanti-human Ig-sera. The anti-sera used were either polyvalent or directedagainst Fab-determinants. The antiserum dilutions were chosen to ascertainan excess of anti-Ig on the beads, thus providing the necessary density offree antibody sites to retain Ig-bearing lymphocytes. Before addition ofthe cells, excess antiserum was washed off and the columns were filled with

Page 13: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 103

TABLE VAcquired cytotoxicity' of pretreated lymphocytes

Expt.no.

1

2

Lymphocytesfrom

Interphase *Sediment *

Interphase *Sediment'

Lymphocytes pretreated'

Normal rabbit serum

11

I< 1

with Crbc and

Anti-Crbc serum

1926

1312

' Per cent "Cr-release from 1 x 10" *'Cr-Crbc after 20 hr of incubation with 2x 10"lymphocytes, corrected by subtracting per cent "Cr-release in lymphocyte freecontrols « 1.0 per cent in both experiments). Incubation mixtures contained heatinactivated normal rabbit serum, diluted 10', 5 per cent foetal calf serum andEagle's medium, total volume 1.5 ml.

" 20x10" lymphocytes, purified by nylon fiber filtration and ficoll-isopaque cen-trifugation; 8x 10* Crbc; heat inactivated normal rabbit serum or anti-Crbc serum,final dilutions lO"*. 15 ml Eagle's medium + 15 per cent foetal calf serum. Incuba-tion at 37° for 18 hr.

' Lymphocytes prepared from interphase obtained at ficoU-isopaque centrifugation,step 3, Table II.

* Lymphocytes isolated by second ficoll-isopaque centrifugation of tbe cells sedimentsdat step 3, Table II. Washed 4 x.

tissue culture medium containing 5 per cent inactivated foetal calf serum.In these experiments, nylon-fiber purification of the lymphocytes wasomitted. The lymphocytes were centrifuged through ficoll-isopaque, eitherdirectly after gelatin-sedimentation or after treatment with colloidal iron.Finally, they were incubated overnight in tissue culture flasks. From 10 to40 X 10 * cells were added to each column, at a concentration of 5 x 10^/ml.The cells were immediately eluted with tissue culture medium at a flow rateof 2-3 ml/min. Fractionation was performed under sterile tissue cultureconditions. The yield of lymphocytes recovered from the anti-Ig columnswas from 50-80 per cent, while that from control columns, charged eitherwith human-lg or with foetal calf serum, was 80-100 per cent. The anti-Igcolumns efficiently remove B-cells, as judged from the results of indirectimmunofluorescence assay of surface bound Ig. At the same time, theproportion of T-lymphocytes increases, as assessed by their capacity to formrosettes with sheep erythrocytes (Jondal et al. 1972).

Figure 4 shows the results of a typical experiment in which lymphocytesafter column fractionation were tested at three different lymphocyte:Crbcratios. The cytotoxic potential of the cells passed through the anti-Ig column

Page 14: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

104 P- PERLMANN, H. PERLMANN & H. WIGZELL

was almost completely abolished in comparison both with unpassaged cellsand with cells passaged through a control column. The inhibition of cyto-toxicity corresponded to a reduction of Ig positive cells assayed by meansof a polyvalent anti H-chain serum in the immunofluorescence test from12 per cent to 0 per cent and to an increase in T-cells {sheep erythrocyterosettes) from 50 per cent to 92 per cent.

The reduction of cytotoxicity by column passage is specific for antibodyinduced cytotoxicity and irreversible. This is illustrated by the experimentof Figure 5. Tlie antibody induced cytotoxicity of the lymphocytes, passagedthrough the anti-Ig column, was as low after 40 hr as after 20 hr incubation.In contrast the PHA-induced cytotoxicity was only slightly reduced at 20 hrbut not at 40 hr. Five other experiments gave similar results. These experi-ments would thus also suggest that the major effector cells in the two cyto-toxic systems are different.

The cell-separating principle in these experiments is assumed to be theanti-Ig activity of the columns. Similar results were obtained with columnscharged with antibodies specific for Fab-determinants. In experiments withmouse lymphocytes, the specificity of retention of lymphocytes carrying therelevant Ig determinants has been demonstrated (Wigzell et al. 1972).However, since these columns at the same time are charged with antigen-

100 r

50

- o -

625 125 25

Figure 4. Cytotoxicity of lymphocytes after passage through column charged withanti-human immunoglobulin.

Lymphocytes were passaged either through anti-Ig column {dots), or through columncharged with human Ig {circles), or were untreated {square).

1.25 X 10° lymphocytes were then incubated with varying numbers of ^'Cr-Crbc inpresence of either anti-Crbc serum {sotid lines) or normal rabbit serum {dashed line),final dilutions 10^, total volumes 0.75 ml Eagle's medium - 5 per cent foetal calfserum. Time of incubation 20 hr. All sera heat inactivated.

Abscissa: lymphocyte: Crbc ratios in incubation tubes.Ordinate: per cent "Cr-release, corrected by subtracting per cent "Cr-release in

lymphocyte free controls (2.ft~3.3 per cent).

Page 15: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 105

100

50

20 20

Figure 5. Time course of cytotoxicity exhibited by lymphocytes passaged throughcolumn charged witb anti-human immunoglobulin.

Lymphocytes were passaged either through anti-Ig column (dots) or column chargedwith human Ig (circles), or were untreated (squares).

1.25 X 10" lymphocytes were then incubated with 2x 10* "Cr-Crbc in presence ofeither anti-Crbc serum (Figure 5a, solid lines) or normal rabbit serum {dashed lines),final dilutions lO"*, or PHA-P (Difco, Detroit, 111.) (Figure 5b, solid lines), 25 fA% inincubation mixture. Total volumes 0.75 mi Eagle's medium + 5 per cent foetal calfserum. All sera heat inactivated.

Abscissa: hr of incubation.Ordirmte: per cent °Cr-release, corrected by subtracting per cent "Cr-release in

lymphocyte free controls {Figure 5a: 1.1-1.7 per cent, 5b: 1.6-3.4 per cent).

TABLE VICytotoxicity of lymphocytes treated with antibody to human immunoglobulin

NoneRabbit IgG, 30 / g

60 /ig120 ^g240 / g

Anti-Fab 1.5 fig6 fig

20 i g

Cytotoxicity induced

% "Cr-release

6469644849

31217

by anti-Crbc serum '

% Inhibition

000

2523

526789

"'Cr-Crbc pretreated with heat inactivated antiserum to Crbc, diluted 10" . "Cr-release measured after 20 hr of incubation, corrected by subtracting "Cr-re!easefrom Crbc treated with normal rabbit serum (< 2 per cent). Per cent inhibition

= (1 -Per cent release with inhibitor

) X 100.Per cent release without inhibitor

- Final concentration (^glm\) in test tube of IgG from normal rabbit serum, or im-munosorbent purified anti-Fab IgG (see text). All incubation mixtures contained5 per cent foetal bovine serum.

Page 16: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

106 P. PERLMANN, H. PERLMANN & H. WIGZELL

antibody complexes (human Ig-rabbit anti-human Ig) it is possible that partof the fractionation may be due to column binding of cells via their Fc-receptor. Final answers on the principles involved should be obtained bymeans of columns coated with pepsin-fragmented F(ab')2 fragments of theanti-Ig antibody.

b. Effect of treatment of the lymphocytes with rabbit anti-human Fab orits pepsin-fragment. Previous experiments have shown that rabbit antisera tohuman-Ig light chains efficiently inhibit the cell-mediated cytotoxic reactioninduced by rabbit antibody to target cell antigens (Holm & Perlmann 1971,Perlmann & Perlmann 1971). Antisera to various heavy chains were lessefficient. An example of this inhibition is shown in Table VI. The antiserumused herein was prepared by injecting rabbits with human IgM. Antibodieswere isolated by adsorption to unsolubilized human IgG and elution at pH2.8. They were tested for specificity by immunodiffusion and for concen-tration by the ELISA procedure (Engvall & Perlmann 1972). Although notstrictly monospecific, this preparation, here designated 'anti-Fab', containedantibodies to human Ig-Fab-determinants in high concentrations.

In this and similar experiments, 0.2 ml of lymphocytes (1 x 10*) were firstincubated for 1 hr at 37°C with anti-Fab-IgG (0.2 ml) or with an appro-priate concentration of normal rabbit IgG. 0.2 x 10 ^^Cr-Crbc and mediumwere then added to a final volume of 0.6 ml. The inhibitor concentrationsgiven are final concentrations present during the cytotoxic test. The lympho-cyte:Crbc ratios were kept sub-optimal (5:1) in order to achieve maximalsensitivity of inhibition. As will be seen, normal rabbit IgG also had a slightinhibitory effect at elevated concentrations. However, the inhibitory effectsof the anti-Fab antibodies were of a different order of magnitude.

These results raise the question whether or not surface bound immuno-globulin on effector lymphocytes has any function in the cytotoxic reaction.(Table VII). In order to investigate this, the inhibitory capacity of theanti-Fab preparation was compared with that of its pepsin fragment F(ab')2-Pepsin fragmentation, purification on Sephadex G-200, and analysis ofantibody activity were done as described elsewhere (Larsson & Perlmann1972). Good inhibition of the cytolytic reaction was obtained regularly withanti-Fab IgG. In contrast, inhibition with its pepsin fragments was in mostexperiments (5/6) weak or absent, in spite of good antigen binding and theexpected bivalency (precipitating in immunodiffusion) of these fragments.This speaks against any direct participation of lymphocytic immunoglobulinin the cytolytic reaction. Therefore, inhibition of cytotoxicity by anti-FabIgG may reflect a blocking of Fc-receptors, known to be entities differentfrom lymphocytic immunoglobulin (Basten et al. 1972b). Inhibition via

Page 17: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

LYMPHOCYTE MEDIATED CYTOTOXICITY 107

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Page 18: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

108 P PERLMANN, H. PERLMANN & H. WIGZELL

Fc-receptors on effector cells may be caused either by the Fc-part of therabbit anti-Fab IgG, or by human IgG, complexed with the rabbit antibodyand released from cells during incubation (Cone et ai 1971). A similar kindof self-inhibition may also be responsible for the weak inhibitory effectsobtained with pepsin fragmented anti-Fab. However, cross-Unking of surfaceareas by these fragments (Taylor et al. 1971) or lymphocyte agglutinationmay have caused these reductions.

4. CONCLUSIONS

The lymphoid cells exhibiting cytotoxicity in the present model are charac-terized by high affinity for antigen-antibody complexes (Fc-receptors). Theyhave high concentrations of surface bound immunoglobulin, as indicated bytheir selective removal on anti-Ig columns. They also have complementreceptors, since passage through columns charged with C3b strongly reducescytotoxicity in this model (Perlmann, Perlmann & MuUer-Eberhard, inpreparation). This distinguishes them from the thymus derived lympho-cytes known to be instrumental as initiator- and perhaps also as effectorcells in other cytotoxic reactions (Blomgren et al. 1970, Cerottini et al.1970a,b, Golstein et al. 1972a,b). Evidence for the indej>endence of T-cellparticipation of the antibody induced cytotoxicity of lymphocytes hasalso been obtained in other models (Harding et al. 1971, Von Boxel et al.1972, Britton et al. 1973). The data presented here and previously leavelittle doubt that these cells are lymphocytic cells. However, although theyhave the surface markers characteristic for B-lymphocytes, there is no directevidence that they are B-cells in a strict sense, i.e. that t h ^ also are precursorcells of antibody producing cells. Their lymphocytic appearance and surfacemarkers do not rule out that we are dealing with circulating precursor cellsof a thus far undefined type (A. C. Allison, personal communication).

The most important factor for the initiation of this cytotoxic reactionwould seem to be the Fc-receptor. Such receptors are present on manydifferent cell-types (Lo Buglio et al. 1967, Lay & Nussenzweig 1968,Henson 1969, Huber et al. 1969, Warner & Ovary 1972) and it is thereforenot surprising that antibodies bound to target cells may induce a cytotoxicresponse in different cells, including immature precursor cells from foetalliver (Britton et al. 1973). In addition, evidence presented here and else-where indicates that cytotoxicity reflects an energy dependent activation ofsurface bound processes in the effector cells (Perlmann & Holm 1969).These processes may well be the same, regardless of whether they are setin motion by reaction of the Fc-receptors present on bone-marrow derived

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LYMPHOCYTE MEDIATED CYTOTOXICITY 109

cells, with target cell bound IgG or by the reaction of the antigen receptor onT-cells with target cell antigens. It should be stressed, however, that merecontact with the target cell is not sufficient to induce cytotoxicity. Thus,although complement receptor lymphocytes (Lay & Nussenzweig 1968,Bianco et al. 1970) are taking part in killing reactions induced by igG-anti-body, they do not lyse target cells coated with C3b, in spite of the formationof stable rosettes with such cells (Perlmann, Perlmann & Muller-Eberhard,in preparation).

The present results provide no evidence for any primary function of thelymphocytic immunoglobulin in the cytotoxic reaction. However, they do notrule out that such immunoglobulin may become imjxjrtant during laterphases of interaction with the target cells. Although blast transformationis not essential for the lytic reaction (Perlmann & Holm 1969), lymphocytesincubated for longer time with antibody coated target cells do transformand proliferate. Such activation has earlier been achieved by means ofantigen-antibody complexes (Bloch-Shtacher et al. 1968, Moller 1969). Itis possible that activation by antigen-antibody complexes in the presence oftarget cells may lead to selective proliferation of cells producing their owntarget cell specific antigen receptors ( = antibodies). The existence of suchan amplifying mechanism is presently under study.

It is of particular interest to note that lymphocytes may become spe-cifically cytotoxic after having picked up antibody in the form of complexes,either from the medium, or from target cells with which they have interacted.Thus, lymphocytes participating in specific cell mediated lysis might be oftwo major types in regard to their antigen-binding surface receptors; thereceptors may either be actively synthesized by the cells which carry them,or may be passively adsorbed to them in the form of antigen-antibodycomplexes. No evidence yet exists, as to whether or not non-thymus derivedB-lymphocytes carrying antigen-binding receptors of own manufacture haveany direct cytolytic function in the system described herein.

The present findings further underline the complexity of lymphocytemediated effector pathways in cytolytic immune systems. Antibody and anti-body producing B-cells are present in most immune responses together withlymphocytes of thymus derived type. Thus, for example, when removal froman immune system of thymus-derived lymphocytes causes increased survivalof a graft, this may be explainable in at least two ways: 1) cytolytic effectorcells of thymus-derived type and necessary for rejection have been removed,or 2) necessary 'helper' cells for 7S antibody production have been removed,thereby interfering with the production of the antibodies needed for cellmediated reactions, involving no T-cell participation at all. Production ofIgG antibodies is frequently a highly thymus dependent phenomenon (Taylor

Page 20: Lymphocyte Mediated Cytotoxicity in Vitro. Induction and Inhibition by Humoral Antibody and Nature of Effector Cells

p. PERLMANN, H. PERLMANN & H. WIGZELL

et al. 1967). Cell fractionation experiments would here be necessary forfinal determination of the effector cell type.

Present knowledge of major effector pathways in different immunesystems, subjected to in vitro analysis, is conflicting. If one limits thediscussion to lymphocyte mediated cytolytic systems, it would seem clear thatthymus derived lymphocytes play a predominant role when major histo-compatibility differences within the species are involved (Cerottini et al.1970a,b, Golstein et al. 1972a). However, in other systems, involvingcytolysis of heterologous cells (Harding et al. 1971, Van Boxel et al. 1972,Britton et al. 1973) or of tumour cells in syngeneic animal models (Lamonet al. 1972, Pollack 1972) or cancer patients (Skurzak et al. 1972, Perlmannet al. 1972), cell niediated effector pathways may be T-cell independent.In the tumour systems referred to, the relative role of thymus derived versusnon-thymus derived lymphocytes in the in vitro cytolytic test seems to varyimder different phases of the disease. The relevance of the different cellulareffector pathways seen in vitro, for tissue damage occurring in vivo in dif-ferent immune situations remains largely to be estabhshed.

ACKNOWLEDGMENTS

This work was supported by grants no. 2032-135 from the Swedish NaturalScience Research Council and no. B73-16X-159 from the Medical ResearchCouncil (P.P., H.P.); grants no. 72:161 from the Swedish Cancer Society andfrom Karolinska Institute Medical School (H.W.). We thank M. Jondal forI>erforming rosette tests and immunofluorescence staining, and E. Engvalland A. Larsson for anti-human Fab preparation. The skilful technicalassistance of G. Halld6n, U. Kallstrom and B. Olsson is gratefully ac-knowledged.

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