Effect of Human Immunoglobulin Preparations on Fc Rosette Formation between Anti-D-Coated Erythrocytes and Lymphocytes

Original Papers Z. Immun.-Forsch. vol. 154, pp. 387-398 (1978) Laboratory of Blood Products, Department of General Biologics Control, National Institute of Health, Tokyo, Japan

Effect of Human Immtllioglobulin Preparations on Fc Rosette Formation between Anti-D-Coated Erythrocytes and Lymphocytes H. ISIDYAMA, K. OKUYAMA, K. MASUDA, and J. YASUDA With B Figures Received September 12, 1977 . Accepted in Revised Form February 28, 1978

Abstract Human immunoglobulin (Ig) preparations were tested for their inhibit,ory effect on Fc rosette formation between anti-D-coated hmnan erythrocytes and lymphocytes, as compared to their complement activating capacity. Both of the two biological activities ascribed to the sites in the Fc portion of the IgG molecules were found to be reduced in the pepsin-treated, as well as in the S-sulfonated Ig preparations, as compared to the activities of the normal hmnan Ig preparation. In the plasmin-treated Ig preparation, which was found to be cmnposed of three major components: plasmin-Fab, plasmin-Fc and plasminresistant IgG, the activity of inhibiting the Fc rosette formation was well retained, in contrast to its low complement activating capacity.

Introduction

One of the most important prerequisites for an intravenous immunoglobulin preparation to be used for prophylaxis and therapy of infections is that it should be free of risk of side-effects upon its administration. Such reactions are said to be caused by aggregated IgG molecules capable of activating the complement system at the specific site in their Fc portion (1). Consequently, various attempts have been made to prepare Ig preparations for intravenous use by removing the Fc fragment, by modifying IgG molecules to block the complement site or by employing manufacture processes which are expected to yield aggregate-free Ig (2). All these attempts were aiming at (or at least attributing their effectiveness to) reduction of the complement-activating capacity. On the other hand, the Fc receptor on the human lymphocyte surface is known to fix IgG molecules through the Fc portion, in particular when they are complexed with their corresponding antigens or nonspecifically aggregated (4). The presence of the Fc receptor can be

388 . H. ISHIYAMA, K.

OKUYAl\L~,

K. MASUDA, and.J. YASUDA

detected by rosette formation by sensitized ery'thl'Ocytes around the lymphocytes, which, in turn, is inhibited by addition of aggregated Ig to the lymphocyte suspension prior to its incubation with sensitized red cells. In the following report., the inhibitory effect of various human Ig preparations on Fc rosette formation will be studied in relation to their complement activating capacity in order to see whether both of the biological activities ascribed to the sites in the Fc portion behave in parallel. Materials and Methods H uma,n immunoglob'Ldins (I g) Three batches of human normal immlilloglobulin (NG; Green Cross Corporation, Osaka, .Japan), 2 batches of pepsin-treated human immlilloglobulin (PG; Behringwerke, Marburg/Lahn, Germany), 2 batches of plasmin-digested immunoglobulin (PLG; Green Cross Corporation) and 2 batches of S-sulfonated immunoglobulin (SG; supplied from Teijin Institute for Biomedical Research, Tokyo, .Japan) (12) were used either in the untreated state or after being treated by heating (vide infra). The NG was prepared by cold ethanol fractionation of pooled human plasma separated from venous blood. It was available in the form of about 15% solution containing aminoacetic acid (0.3M) as the stabilizer. On free-boundary electrophoretic analysis, more than 95% of the total protein was found to have the Inobility of IgG. The molecular components contained in the PG are as stated by BARANDUN et al. (2) and those in the SG were as stated by MASUHO et al. (12). According to the manufacturer, PLG has been prepared by the following method: the pseudoglobulin fraction prepared by the method of SLOTTA (17), which contains plasminogen was treated with human urokinase (Green Cross Corporation, Osaka, .Japan) at 25° C and pH 7.4 as described by SGOURIS et al. (15). The resulting plasmin-containing material was added to the human IgG of 99% or higher purity in the ratio of 30 caseinolytic units per 1 gram of IgG and the mixture was incubated for 96 hours at 20° C. Thereafter the plasmin and urokinase were removed by adsorption on asbestos. The purified PLG was available in a lyophilized form which was reconstituted to a 5% solution in distilled water. PLG was further fractionated to its eomponents by the following proeedures: PLG was applied to a Sephadex G-200 (Pharmacia, Uppsala, Sweden) eolumn (2 X 90 em) and eluted in 0.05M aeetate buffer eontaining 0.9% NaCI (pH 6.6). The eluate at the first protein peak was pooled: it was found to be eomposed of <.whole» IgG molecules resistant to plasmin digestion. The eluate at the second protein peak was pooled and further fractionated by twicerepeated colunill ehromatography (column size 5 X 90 cm) in CM-Cellulose (Pharmacia, Uppsala, Sweden). The elution was first carried out with 0.01 M phosphate buffer (pH 7.6) and then 'with the same buffer containing 0.3M NaCl. The first protein fraction was found to contain plasmin-Fc while the later fraction elut€.d by the addition of 0.3M NaCl eontained plasmin-Fab, respectively. The purity of the Fab and Fc fractions were confirmed immunologically by the double immunodiffusion technique against antisera to human IgG fragments (Miles Laboratories Inc., Kankakee, Ill.).

Effect of human Ig on Fe rosette formation . 389 From these results, the molecular components present in our PLG seem to be similar, if not identical, to those observed by BARANDUN et al. (2) and dif· ferent from t h e ones reported by MACLENNAN et al. (Il).

Anti·D serum Anti·D(Rho) serum of individual ongm from a hyperimmlmized male was kindly supplied from the Nihon Seiyaku Co., Tokyo, Japan. Its a ntibody titer was 1: 512 in the indirect antiglobulin test as well as in the bromelin m eth od. H eat·treatment of If! Ig preparations diluted 1: 10 with the physiological saline solution were h ea ted at 63 ° C for 20 minutes with continuous shaking. Immediately b efore use, the samples w ere centrifuged at 1000 g for 30 minutes to remove deposits. Sensitization of er·yth1"Ocytes Group 0, D(Rho)·positive human erythrocytes adjusted to the concentrat ion of 10' cells/ml were sensitized with equal volumes of appropriately diluted anti·D serum. Sensitization was carried out at 37° C for 1 h; the cells w er e washed three times with physiological saline solution and suspended to the concentration of 1 X 10' cells/m!. Lymphocyte preparation Human mononuclear cells w ere isolated from peripheral blood of a h ealthy individual by Ficoll.Paque (Pharmacia, Uppsala, Sweden) gradients and sus· p ended in Hanks balanced salt solution to the concentration of 2 X 10' cells/ml after being wash ed three times with the said salt solution.

Inhibition of Fc r·osette formation One hundred ,ul of lymphoeyte sllspension were mixed with an equal volume of appropriately diluted Ig preparation and the mixture was incubated at 37 0 C for 5 min. Thereafter, 100/11 of sensitized hmnan red cell suspension were added to the above mixture and subsequently incubated at 37 ° C for 20 min. As a control, t h e lymphocytes were treated with sensitized r ed cells alone . At the end of t h e second incubation p eriod, test mixtl.ll·es were centrifuged at 400 g for 5 min in order to enhance the reaction. A drop of the test mixtur·e was mounted on an eosinophil counter (Kayagaki Irikakogyo Co., Japan, 0.2 mm depth) and examined microscopically. Rosettes w ere counted, among 400 ~ 600 lymphocytes in each of the samples. L y mphocytes surrounded by four 01' more erythrocytes w er e cmmted as «rosettes ». Triplicate t ests were performed on each of the samples a nd the inhibition ratio was calculated as follows: Number of rosettes in control - Number of rosettes in t est sample x 100 Number of rosettes in control = Inhibition ratio ( % )

T est for complement activating capacity Unless otherwise specified, 100 CRoo of guinea pig complem ent in 1 ml of gelatin-Veronal buffer solution (pH 7.5) and 3 ml of the same buffer solution were added to 1 ml of the Ig preparation. After incubation of the mixture at 37 ° C for 1 h , the residual eomplem ent activity was m easured with sheep red cells sensitized with rabbit anti-sh eep erythrocyte antibody, from which the consum ed CHoo dose was calculated . For th e details of the technique, see KABAT and MEYER (8).

390 . H.

ISHIYANL~,

K. OKUYAMA, K. MASUDA, and J. YASUDA

Test for complement.mediated 1:mmune hemolysis Half ml of the PLO or various fractions of it (in protein concentrgtion of 10 mg/ml) were mixed with equal volumes of sheep erythrocyte suspension. 0.1 ml of guinea pig complement (200 CH5o/ml) was then added and the mix· tures were incubated at 37° C for 30 minutes and thereafter at 4-6° C overnight. If necessary, the supernatant fluid was centrifuged prior to determination of hemolysis at 541 nm. Percent hemolysis was calculated against the reading obtained with the control tube which received 0.5 ml of the same erythrocyte suspension which was hemolyzed by the addition of 0.6 ml of distilled water. vVhen the hemolysis in the supernatant fluid was negligible, the residual com· plement activity therein was measured with sensitized sheep red cells as specified above.

Results When the lymphocyte suspension was incubated with the red cells sensitized with the anti-D serum of 2-fold dilution series ranging from the undiluted serum to 1: 16,16.5%,14.6%,11.8%,10.2% and 9.6% of the lymphocytes were found to be rosette forming cells, respectively. 0----0 Normal limrunoglobulin (NG)

y..---x

Pepsin-treated

10 I:i.--6 plasmin-digested cr---~S-sulfonated

•

" " It

(pc)

(PLG) (SG)

• Bovine serum albumi n (BSA)

50

o 0.01

0.1

1.0

Ig concentration (mg/ml) Fig. 1. Inhibition of Fc rosette formation by human immunoglobulin preparations.

392 . H.

ISHIYAMA ,

K.

OKUYA.l"WA,

K.

MASUDA,

and J.

YASUDA

From the results, a 1 : 2 dilution of the anti-D serum was chosen for sensitization of the red cells in subsequent experiments. The inhibitory effect of the untreated Ig preparations on the Fc rosette formation is given in Table 1, in whichPLGwasfound to be inhibitory in all of the concentrations used. NG was found to be inhibitory in concentrations of 0.05 mgjml or higher. The addition of 0.1 mgjml of NG or PLG resulted in the reduction of rosette formation to 8.8% or 3.9%, respectively. When the Ig concentration was raised to 0.5 mgjml, the rosette forming ratio was 8.3% for the NG and 3.1 % for the PLG, respectively. In Figure 1, the inhibition ratios are shown with the Ig preparations in concentrations ranging from 0.01 mgjml to 1.0 mgjml. At a concentration of 0.05 mgjml and of 0.1 mgjml, for example, the inhibition ratios with the NG were 24 % and 40 %, whereas those by the PLG were 47% and 72 %, respectively. Thus, the PLG was found more inhibitory than the NG even in its untreated state. On the other hand, no inhibitory effect was observed with the PG, nor with the SG, nor with bovine serum albumin (BSA) which was used as a control.

100

,....

c ....J.J0 11)

H

~

....0 .....D J.J

.... ....

.c

SO 0 -··· -0

~

NG

J.J

Y- - - Y

PLG PRG

I/)

'I1"---- it

Fc

)( - -'/.

Fab,.,


J.J
0 H

o 0 . 01

0.1

1.0

protein concentration (mg/m1) Fig. 2. Inhibition of Fc rosette formation by the components of PLG. * The rosette formation of higher than the control is shown as «0 inhibition» in this figure.

+ 1.2 -1.0 +1.3 -1.6 +3.6 -0.5

14.5

U.8

14.6

Pepsin-treated (PO)

Plasmin-digested (PLO)

S-sulfonated (SO)

15.1 +0.6 -1.2

+2.3 -2.7

+2.3 -0.2

14.2

7.9

+1.4 -1.0

11.2

0.05

0/0

14.5

3.9

15.0

8.8

+1.1 -1.0

+1.5 -2.2

+ 1.0 -0.5

+1.1 -1.6

0.1

of Fc rosette formation

14.2

3.1

14.2

8.3

+0.6 -1.1

+1.1 -2.4

+0.7 -1.2

+0.5 -1.6

0.5

13.6

1.0

14.0

7.2

+0.5 -1.3

+1.6 -1.0

+0.5 -1.7

+0.1 -0.7

1.0

Mean of the Fc rosette forming cells in the control was 14.6%. Triplicate tests were carried out on each sample. * As the interlot variation was negligible, mean and variations were collectively calculated with products belonging to each of the categories of the Ig preparations.

+0.3 -1.3

14.2

Concentration (mg/ml) 0.01

Normal (NO)

Immunoglobulin preparations *

Table 1. Effects of immunoglobulin preparations on Fc rosette formation

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<0

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ro

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Effect of human Ig on Fe rosette formation . 393

For the analysis of the potent inhibitory effect observed with the PLG, each of the components obtained from it were separately studjed for their inhibitory effects. As is shown in Figure 2, the strongest inhibitory effect was observed in all the concentrations with the plasminFc which exhlbited 61 % inhibition, twice as great an inhibitory effect as that of the PLG, at the concentration of 0.01 mg/m!. With the plasmin-Fab, no inhibitory effect was observed at all. Rather, the plasminFab seemed to have enhanced the rosette formation , since 130% to 153% as many rosettes as in the control were observed with various concentrations of the plasmin-Fab. The inhlbitory effect of the plasminresistant I gG (PRG) was somewhat weaker than that of NG in all the concentrations used.

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.....0

... ell

I

~

~

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.....0 .....

...

.... .J:! ....I:::

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Ig concentration (mg/ml)

0-.---0

Normal Inununoglobulin (NG)

:(--_·x

Pepsin-treated

"

(PG)

~--A

plasmin-digested "

(PLG)

O----{J

5- sulfonated

(SG)

•

Bovine serum albumin

(BSA)

•

Fig . 3. Inhibition of Fe rosette forma tion by heat-aggregated human immunog lobulins .

394 . R.

ISHTYAMA,

K.

OKUYAMA,

K.

MASUDA,

and J.

YASUDA

Thus, the highest inhibitory effect of the PLG among the Ig preparations can be attributed to the potent binding capacity of its free Fc component to the Fc receptors of the lymphocytes. Thereafter, the inhibitory effect of the heat-treated Ig preparations was studied. As shown in Figure 3, about twice as great an inhibitory effect was observed with the heat-treated NG and PLG as with the corresponding untreated samples. With 0.05 mg/ml of the heat-treated NG, 44% of rosettes were inhibited, whereas 78 % inhibition was observed with the same amount ofthe heat-treat ed PLG. At a concentration of 0.1 mg/ml, approximately 60% was inhibited by the NG and 90% by the PLG. Finally, no rosette could be seen when 0.5 mg/ml of the NG or PLG was used . The heat-treated PG and SG had shown some inhibitory effect on the Fc-rosette formation only when such high concentrations as 1.0 mg/ml was used, which could rather be attributed to the intact IgG molecules contained in these preparations. None of t he intravenous Ig preparations were found to have consumed more than 20 CHao while more than 100 CH 50 were consumed by the NG, when being tested at the concentration of 50 mg/ml (Tab. 2). The PLG and its components were then separately studied for their complement-activating capacity (Tab. 3). In the presence of guinea pig complement, sheep erythrocytes were hemolyzed by PLG and PRG. Thi~ must have been caused by natural, heterologous erythrocyte antibodies, since the hemolyt ic activity was no longer observed when the PLG was absorbed with packed sheep red cells. The supernatant fluids recovered from the sheep red cells which had been treated with either plasmin-Fab or plasmin-Fc in the presence of complement showed negligible coloring (if any) due to released hemoglobin. Table 2. Complement activating capacity of human Ig preparations Immunoglobulin preparations P epsin.treated No.1 No.2 P lasmin-digested No. 1 No.2 S·sulfonated No.1 No.2 Normal No .1 No .2 No.3

CR;o consumed

7

6 4 9

9 15

;;;; 100 ;;;; 100 ;;;; 100

Effect of human Ig on Fe rosette formation . 395 Table 3. Complement-rnediated immune hemolysis and non-specific complement act.ivation by the PLG and its fractions

PLG Plasmin-resistant IgG (PRG) Plasm.in-Fab Plasmin-Fe Controls Complement activation Hemolysis

Immune Hemolysis (%)

Activated Complement (CH;.)

25.1 100 negligible negligible

0.3 2.9

100

o

However, when the consumption of the added complement was tested in these supernatants, 2.9 CH50 of the complement were found to have been activated by the plasmin-Fc. Thus, most, if not all, of the complement-activating capacity of the PLG must have been borne by its free Fc component which is capable of activating the complement system non -specifically. Even so, the complement activating capacity of the PLG and its Fc component was found less active than their binding activity to the Fc receptor. In other words, the latter activity of the Fc portion was found much better retained than the former in the plasmin-digested Ig preparation and also in the free Fc component thereof. Discussion Membrane receptors recognizing the Fc portion of the Ig molecules are found on various cells related to the immune system such as Blymphocytes, T-Iymphocytes, so-called «Null cells» (5) etc. and can be clearly differentiated from surface Ig which bind antigens or from complement receptors (10, 18, 19). Consequently, rosette formation using antibody-coated human erythrocytes has been tried for detecting the Fc receptors on viable lymphoid cells (5). Aggregated Ig is thought to have much greater binding affinity for the lymphocyte Fc receptor than native Ig due to the availability of greater number of Fc pieces on the aggregates (5). Moreover, the free Fc fragments contained in the PLG were found even more avid in binding to the Fc receptors than the Fc portions made available on the whole Ig molecules. This fact must have contributed to our finding that the PLG inhibited the formation of rosettes more efficiently than the NG even without being subjected to the heat aggregation. The exact site of the Ig molecule that is recognized by lymphocytes is not yet definitely established. Based on the fact that reduction and

396 . H.

ISHIYAlI'IA,

K.

OKUYAMA,

K.

MAS UDA,

and J.

YASU DA

alky lation markedly affect the ability of Ig to bind to the ly mphocytes, the site is assumed to be localized in the CH 2 domain (20) . On the other hand, a murine myeloma protein having the intact Cll 2 but lacking the Cll 3 region was found unable to bind to B lymphocytes (14). Our observation that the SG, unlike the PLG, could not prevent the sensitized red cells from binding to the Fc receptors seems to favour the former assumption. Nevertheless, it may be also likely that reduction and alkylation could bring about the structural change affecting not only the Cll 2 but also the CH 3 region. A complement-binding site is known to be localized in the Cll 2 region (9). There are known at least two different pathways of breakdown of the I gG molecules by plasmin digestion. The first one requires a treatment in an acid pH prior to the proteolytic process by which an IgG molecule is said to yield a Facb fragm ent by cleaving the C-terminal parts of the F c portions from each of the two y-chains (3). The biological activities of the Facb fraction thus obtained have been studied in detail by MAC LENNAN et al. (11). Another pathway of plasmin digestion is t he one studied by SGOURIS et al. (15). Here, the resulting components are thought to be similar, if not identical, to the ones obtained by the papain digestion, namely two Fab and one Fc fragments (7, 16). Since our PLG was prepared according to the latter method, it is no wonder that the same major components as those observed by SGOURIS et al. and later characterized by BARANDUN et al. (2) were also present in it: the plasmin-resistant IgG (PRG) , plasmin-Fab and plasmin-Fc fractions. Among the three components, the whole IgG molecules (PRG), after having combined to the corresponding antigens, were able to activate complement as is shown by the occurrence of immune hemolysis (Tab. 3). The PRG was also capable of binding to the Fc-receptors. The Fab fraction, unlike the Facb , was unable to activate complement although the antigen-binding sites must be present in it. The free Fc fraction , containing both of the CH 2 and the Cll 3 domains, was shown to activate the complement system non-specifically in addition to its ability to inhibit rosette formation. Although the intact inter chain disulfide bonds in t he F c portion are thought to be essential for the integrity of the sites for the activation of complement and for the binding to the Fc-receptor, these two act.ivities did not behave in parallel in the cases of our PLG and of its Fc fraction , in which the latter activity was better retained than the former. The binding of introduced I g to the Fc receptors on the various lymphoid cells and the specific activation of complement, as can be expected with the administration ofPLG, might well be advantageous to the defence mechanism of the individnals, in particular in those

Effect of human Ig on Fc rosett fJrmation . 397

equipped with undisturbed immune function whose Ig concentration is not below the normal level. Nevertheless, in an extremely immunodeficient patient, who should have normally developed Fc receptors on the lymphocytes without the surface Ig of one's mvn, as in the case of the X-linked agammaglobulinaemia (6), all the Ig introduced via the intravenous route must suddenly be fixed to the receptors available. In this respect, it should be remembered that anaphylactic reactions in guinea pigs to equine albumin could be inhibited by the administration of antibody to the Fc fragment of autologous IgG (13). Therefore, caution must better be taken in administering to immunodeficient patients those intravenous IgG preparations which contain whole IgG molecules and/or intact Fc fragments to avoid too rapid exposure of the Fc receptors to the newly introduced IgG, even though there is as yet no proven case of any clinical side reaction that can be explained by the above mentioned assumption. References 1. BARANDUN, S., P. KISTLER, F. JEUNET, and H. ISLIKER. 1962. Intravenous administration of human l'-globulin. Vox Sang. (Basel), 7: 157-174. 2. BARANDUN, S., F. SKVARIL, and A. MORELL. 1975. Prophylaxis and treatment of diseases by means of imnl.lilloglobulins. In: Inderbitzin, T. M. (Ed.) : Prophylaxis of infectious and other diseases. Monogr. Allergy, 9: 39-60, Karger, Basel. 3. CONNELL, G. E., and R. R. PORTER. 1971. A new enzymic fragment (Facb) of rabbit immunoglobulin G. Biochem. J., 124: 53 p. 4. DICKLER, H. B., and H. G. KUNKEL. 1972. Interaction of aggregated l'-globulin with B lymphocytes. J. expo Med. 136: 191-196. 5. DICKLER, H. B. 1976. Lymphocyte receptors for immunoglobulin. Adv. Immunol., 24: 167-214. 6. FR0LAND, S. S., and J. B. NATVIG. 1973. Identification of three different human lymphocyte populations by surface markers. Transplant. Rev., 16: 114-162. 7. HINMAN, J., J. L. TULLIS, C. A. SARAVIS, and R. B. PENNELL. 1967. Intravenoususe of plasmin treated immunoglobulin G. Vox Sang. (Basel), 13: 85-89. 8. KABAT, E. A., and M. M. MAYER. 1961. Experimental Immunochemistry, 2nd Ed., P. 214, Charles & Thonms, Springfield, Ill. 9. KEBOE, J. M., and M. FOUGEREAU. 1969. Immunoglobulin peptide with complement fixing activity. Nature, 224: 1212-1213. 10. LAY, W. H., and V. NusSENZWEIG. 1968. Receptor for complement on Leukocytes. J. expo Med., 128: 991-1007. 11. MAC LENNAN, 1. C. M., G. E. CONNELL, and F. M. GOTCH. 1974. Effector activating determinants on IgG. II. Differentiation of the combining sites for C 1q from those for cytotoxic K cells and neutrophils by plasmin digestion of rabbit IgG. Immunology 26: 303-310. 12. MASUHO, Y., K. TOMIBE, K. MATSUZAWA, and A. OHTSU. 1977. Development of ::m intravenous l'-globulin with Fc activities. 1. Preparation and charac.terization of S-sulfonated human l'-globulin. Vox. Sang. (Basel), 32: 175-181.

398 . H. ISIDYAMA, K. OKUYAMA, K. MASUDA, and J. YASUDA 13. MATRE, R., and A. GROV. 1976. Inhibition of anaphylactic reaction in guinea pig by antibodies to fragments of Fc from autologous IgG. Int. Arch. Allergy, 52: 422-424. 14. RAMASAMY, R., D. S. SECHER, and K. ADETUGBO. 1975. CH3 domain of IgG as binding site to Fc receptor on mouse lymphocytes. Nature, 253: 656. 15. SGOURIS, J. T. 1967. The preparation of plasmin treated immune serum globulin for intravenous use. Vox Sang. (Basel), 13: 71-84. 16. SGOURIS, J. T., R. W. STOREY, K. B. MAcCALL, and H. D. ANDERSON. 1962. The purification, assay, sterilization, and removal of pyrogenicity of hmnan urokinase. Vox Sang. (Basel), 7: 739-749. 17. SLOTTA, K. H., and .J. D. GONZALEZ. 1964. Native plasminogen. Biochem., 3: 285-291. 18. THEOFILOPOULOS, A. N., F. J. DIXON, and V. A. BOKISCH. 1974. Binding of soluble immune complexes to human lymphoblastoid cells. 1. characterization of receptors for IgG Fc and complement and description of the binding mechanism. J. expo Med. 140: 877-894. 19. VVARNER, N. L. 1974. Membrane irrul1unoglobulins and antigen receptors on Band T lymphocytes. Advanc. Immlllol. 19: 67-216. 20. WISL0FF, F., T. E. MICHAELSEN, and S. S. FR0LAND. 1974. Inhibition of antibody-dependent human lymphocyte-mediated cytotoxicity by immtlloglobulin classes, IgG subclasses and IgG fragments. Scand. J. Immunol. 3: 29-38. H. ISHIYAlVIA, Ludwig Institute for Cancer Research, Lausanne Unit of Human Cancer Immunology, Ch. des Boveresses, CH-I066 Epalinges SjLausanne, Switzerland