Research in

ExperimentalMedicine

Res. Exp. Med. (Berl.) 173, 131--144 (1978)

@ Springer-Verlag 1978

Isolation of a Receptor Protein for Lymphocyte Specific Cytotoxic Antibodies from Human Lymphocyte Membranes H. Kr6mker 1, H. Warnatz 1, and H. J. Aschhoff 2 1Institute of Clinical Immunology, University Erlangen-Ntimberg, Krankenhausstr. 12, D-8520 Erlangen, Federal Republic of Germany 2Institute of Physiological Chemistry, University Erlangen-N~rnberg

Summary. Membrane proteins from lymphocytes prepared from human spleen or thymus were solubilized by extraction with hypermolar potassium chloride solution. The extracted protein mixture consisted of more than 20 protein bands in the disc polyacrylamid gel electrophoresis. Single proteins could be isolated by separation on Sephadex G200 column and preparative polyacrylamid gel electrophoresis. For demonstration of lymphocyte antigens, antisera to human lymphocytes of different origin and specificity were used. In the immunoprecipitation test according to Ouchterlony, it was shown that membrane protein preparations of spleen cells contained seven proteins which precipitated with antilymphocyte sera. Three or four precipitation lines were formed with an antiserum to thoracic duct lymphocytes and with an antiserum to cultured Bcells. Two of these precipitation lines crossreacted with each other. Using fractions of the preparative polyacrylamid gel electrophoresis, it was demonstrated that the precipitating antigens of the spleen cell extract had molecular weights of 500 000 respectively 220 000 daltons. Another precipitating protein had a molecular weight of about 60000 daltons. The thymus cell extract formed one single precipitation line with an antithymus cell serum; this precipitating protein of the thymus cell extract was not present in spleen cell extracts. The cytotoxicity of antilymphocyte sera was inhibited by the spleen cell extract as well as by the thymus cell extract. Using fractions of the preparative polyacrylamid gel electrophoresis the inhibitory protein could be detected in the same fraction as the precipitating proteins of high molecular weight. The precipitating lower molecular weight protein (about 60 000 d) was not inhibitory for cytotoxicity of the antilymphocyte sera. The inhibition pattern of the spleen cell extract or the thymocyte extract showed differences depending on Offprint requests to: Prof. Dr. H. Warnatz (address see above)

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Since the p u b l i c a t i o n o f M e d a w a r [1] on solubilized cell m e m b r a n e antigens in 1963 m a n y a p p r o a c h e s were u n d e r t a k e n to s e p a r a t e a n d characterize surface antigen o f cell m e m b r a n e s a n d to study these antigens for their p h y s i o l o g i c a l f u n c t i o n a n d their role in i m m u n o l o g i c a l reactions. F r e q u e n t l y used m e t h o d s for s o l u b i l i z a t i o n o f m e m b r a n e p r o t e i n s are u l t r a s o n i c a t i o n [ 2 - - 8 ] , autolytic destruction o f cells [9] as well as t r e a t m e n t with p r o t e o l y t i c enzymes [10--13] o r detergents [14--19]. H o w e v e r , these p r o c e d u r e s for p r e p a r a t i o n o f m e m b r a n e p r o t e i n s are often u n s a t i s f a c t o r y because o f the low yield o f native m e m b r a n e p r o t e i n s o r b e c a u s e the m a t e r i a l s have lost their f u n c t i o n a l a n d i m m u n o l o g i c a l properties. Reisfeld a n d c o w o r k e r s [20--23] described an e x t r a c t i o n m e t h o d o f cells with 3 M p o t a s s i u m chloride as a highly effective m e t h o d for s o l u b i l i z a t i o n o f cell surface antigens as H L A o r H 2 antigens f r o m h u m a n or m u r i n e l y m p h o c y t e s . P a r t i c u l a r l y , h e t e r o p o l a r b o u n d p r o t e i n s which are t h o u g h t to be r e c e p t o r s in i m m u n o l o g i c a l reactions are e x t r a c t e d f r o m the cell m e m b r a n e s with this m e t h o d whereas the s t r u c t u r a l p r o t e i n s are n o t touched. The i n t e n t i o n o f the present s t u d y was to isolate proteins f r o m l y m p h o c y t e m e m b r a n e s which react specifically with a n t i b o d i e s to l y m p h o c y t e s in the i m m u n o p r e c i p i t a t i o n test a n d which inhibit the c y t o t o x i c activity o f these antibodies.

Materials and Methods 1.1 Cell Extraction Membrane proteins of lymphocytes were prepared from normal human spleens or the thymus which were removed either during laparotomy or during cardiac surgery of children. After removal of the capsule, the organs were minced into small pieces and the lymphocytes were isolated by a loose fitting glass homogenizer. The cells were suspended in Hanks solution containing 5 percent serum of AB-blood group donors and the lymph0cytes were separated from erythrocytes by centrifugation on a ficoll-hypaque gradient (d= 1.077) 40min at 250g. Microscopic examination of the spleen cell suspension showed that the spleen cells contained between 20 and 50 percent erythrocytes. The lymphocytes were small or medium sized cells. 95 percent of the cells were viable according to trypan blue exclusion test. The extraction of membrane proteins was performed according to Meltzer et al. [25]. 10s cells were washed repeatedly with Hanks solution and phosphate buffered saline. Afterwards they were extracted by 10ml 3M KC1 in 5mM potassium phosphate buffer pH 7.4 under stirring for 18h at 4°C. The mixture was centrifugated at 40000g for 60min at 4°C in a Beckman Spinco L2 centrifuge. The supernatant was dialysed twice against the 200-fold volume of distilled water for one hour. The precipitate was centrifuged at 40 000 g for 15 min and the supernatant was concentrated to a protein content of about 170 mg per ml by ultrafiltration in a Minicon filter (Amicon). In order to remove insoluble particles the material was centrifuged a second time at 110 000 g for 1 h and was subsequently filtered through a Millipore

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Fig. 1. Elution pattern o f m e m b r a n e proteins from spleen cells (--), thymus cells ( . . . . . . ) and erythrocytes ( - - - - - - ) after separation on Sephadex G200 (flow rate 15 ml/h). ~ indicates the m a x i m u m o f the elution o f dextranblue (MW 2000000, void volume) filter (pore size 0.45 p). The protein content was estimated photometrically at 215/225 n m with bovine serum albumin as standard [24]. F o r preparation o f erythrocyte m e m b r a n e proteins we used ghosts o f h u m a n red blood cells (blood group 0, rhesus negative) which were p r o d u c e d after hemolysis in distilled water. M e m b r a n e proteins o f colon carcinoma cells were prepared from the cell line C373 which grows as established line in culture. The procedure o f m e m b r a n e protein extraction was the same as described above for h u m a n lymphocytes.

1.2 Gel Filtration of the Crude Extract The proteins of the 3 M I~CI extract were separated on a Sephadex G200 column (2.5 diameter, 50cm height) in 0.1 M p h o s p h a t e buffered saline p H 7.4 at a flow rate o f 15ml per hour and fractionated with a Ultra-Rac fraction collector (LKB, Uppsala, Sweden) using an Uvicor II for optical control o f the effluent proteins. Corresponding to the elution profile the eluted material was divided into three main fractions F1, F2 and F3 (Fig. 1).

1,3 Analytic Polyacrylamidgel-Disc Electrophoresis F o r further fractionation we used a modified disc electrophoresis gel system la according to Maurer [26]. The polyacrylamid gel (PAG) consisted o f 7.5 percent acrylamide with 0.2 percent N-Nmethylen-bis-acrylamid in a 0.3 M Tris/HC1 buffer p H 8.9. The gel was polymerized in the presence o f a m m o n i u m sulfate and T E M E D . 100--150 lag o f the m e m b r a n e extracts mixed with 100lal o f 2.5% P A G p H 6.7 were put in the top o f the gel (sample gel). Electrophoresis was p e r f o r m e d in a Tris/glycin buffer (5 m M / 4 0 m M ) p H 8.3. At the beginning the current was 1 m A / g e l ; it was increased to 4 m A / g e l at 300 V after 15 rain. Electrophoresis was finished after 1 h. The buffer front marked by b r o m p h e n o l red was at that time at a distance of 75 m m from the sample gel. The proteins were fixed and stained with a 1 percent amidoblack solution in 7 percent acetic acid for 90 rain and subsequently destained in 7 percent acetic acid for 1--2 days. Besides amidoblack staining, carbohydrate containing proteins were d e m o n s t r a t e d by treatment with periodic acid and Schiff's reagent according to Caldwell and Pigman [27]. The benzidine reaction according to Clarke [28] was used for staining o f iron containing proteins.

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1.4 Preparative PA G Disc-Electrophoresis of Crude Membrane Extract For preparation of single protein bands from analytical disc electrophoresis standardized series of gels (8.5 cm length, running distance 7.5 cm, protein 150 gg) were cut into 5 mm pieces with a razor blade set. The corresponding gel pieces were collected and homogenized in a glass homogenizer. Elution of proteins from the gels was performed by repeated addition of 5 ml phosphate buffered saline p H 7.4. Identical eluates were collected after centrifugation at 40 000 g for 30 rain and the supernatants were concentrated by ultrafiltration in Minicon-filters (Amicon) to a protein content of about 10--20 mg per ml. The fractions eluted from the PAGs were enumerated as E l - - E 1 6 beginning with E1 at the sample gel.

1.5 Immunological Analysis As antigenic materials for immunological analysis of the membrane proteins we used 1. the unfractionated 3 M KC1 extract of spleen cells or thymocytes 2. the fractions F 1 - - F 3 of spleen cells separated on Sephadex G200 columns 3. the fractions E l - - E 1 6 of spleen cells prepared from the PAG disc electrophoresis. These antigenic preparations were examined for their capacity to precipitate with antisera to lymphocytes and for their capacity to inhibit the cytotoxic reactions of antisera to lymphocytes after absorption. Antisera to lymphocytes were kindly supplied by the Behring Werke, Marburg. The following preparations were used: 1. Antisera to spleen cells and to lymphnode cells (cytotoxic titer 1:4000 or 1:8000, respectively, using peripheral blood lymphocytes as target cells). 2. Antisera to thoracic duct lymphocytes (cytotoxic titer 1 : 64 000 using peripheral blood lymphocytes as target cells). 3. Antisera to thymus cells, absorbed with cultured B lymphoblasts (cytotoxic titer 1 : 32000 using T-lymphocytes as target cells and 1 : 128 000 using thymus cells as target cells). 4. Antisera to cultured B-lymphoblasts, absorbed with thymus cells (cytotoxic titer 1 : 64 000 using peripheral blood lymphocytes as target cells). 5. Antiserum to h u m a n immunoglobulin (anti-Ig-serum) or to h u m a n serum proteins. The antilymphocyte sera were prepared in horses by long term immunization with mixed h u m a n spleen cells, lymphnode cells, thoracic duct cells, thymus cells or with cultured B-cells from an established cell line, respectively. All antisera were absorbed with human erythrocytes of the blood groups A and B and with insolubilized human serum protein. The anti-immunoglobulin sera were produced in rabbits or goats. In order to absorb lymphocyte specific antibodies in the antisera 0.1ml antiserum was incubated with 10 gg protein of the antigen preparations for 18 h at 4 ° C. The formed precipitate was centrifuged for 20 min at 20 000 g. In control series the antisera were incubated with equal concentrations of membrane antigens from erythrocytes or colon carcininoma cell extracts.

1.5.1 Immunoprecipitation. Immunoprecipitation was performed in agar gel according to the method of Ouchterlony [29]. The antigenic materials prepared according to 2.4 and the antisera were filled into radially arranged wells of 2 m m diameter in the agar gel (conditions: 1.5% agar in sodium barbital HC1 buffer pH 8.6 on slides). The results were read after incubation for 48 h at room temperature either natively or after removal of non precipitated proteins and staining with amidoblack 108 (Merck). 1.5.2 Cytotoxicity Inhibition Test. As target cells in the cytotoxicity inhibition test we used 1. lymphocytes of the peripheral blood, separated on a ficoll-hypaque gradient ( d = 1.077), 2. T-cells of the peripheral blood enriched according to the method of Julius et al. [30] by passage through a nylon wool column of 10 cm length in PRMI 1640 medium with 2.5 percent AB-serum. The passed cells contained 93--95 percent E-rosette forming cells [31] and 3. cells from normal h u m a n thymus.

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100000 viable target cells suspended in 10 pl Hanks solution with 2 percent bovine serum albumin were incubated with 0.1 ml of a geometric dilution series of different heat inactivated (56° C for 30 min) absorbed and not absorbed antisera for 20 min at room temperature. 0.1 ml rabbit serum as complement was added for 1 h at 37°C and the cytotoxic titer was determined by the trypan blue exclusion test. In control experiments the target cells were incubated with horse gamma globulin and complement; less than 5 percent dead cells were found in the controls. The cytotoxic reaction was considered as positive when 20 percent dead cells over the controls were counted with not absorbed antisera.

Results

2.1 Results of Separation on Sephadex G200 Figure 1 shows the elution profiles o f m e m b r a n e proteins f r o m spleen cells, t h y m u s cells and erythrocytes after gel filtration on Sephadex G200. Near the void volume proteins o f high molecular weight were eluted in one peak (fraction F1) which consisted o f about 10 protein bands in the disc electrophoresis (less than 10 percent o f total proteins). The second fraction F2 included a b o u t 60 percent o f the total protein content o f the m e m b r a n e extract; here besides other proteins hemoglobin f r o m the erythrocytes mixed to the spleen cells was identified by disc electrophoresis and staining with the benzidene reagent according to Clarke. Fraction F3 contained proteins of lower molecular weight. F r a c t i o n a t i o n o f thymus cells resulted in a peak F1 which consisted o f about 25 percent o f the total protein content.

2. 2 Results of PA G-Disc-Electrophoresis In PAG-disc-electrophoresis o f the spleen cell m e m b r a n e extract more than 25 protein bands were differentiated. Special staining procedures showed that several bands were iron containing proteins. The b r o a d b a n d in the midth o f the electrophoresis o f spleen cell membranes and of fraction F2 o£ the P A G

Fig. 2. PAG-disc-electrophoresis of 3 M KC1 extract prepared from erythrocyte membranes (EE) and of 3 M KC1 extract from spleen cells (SE) and the fractions F1, F2 and F3 of the spleen cell extract after separation on Sephadex G200

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Fig. 4a and b. Immunoprecipitation of spIeen cell extract with antisera to spleen cells (1), to thymus cells (2), to thoracic duct cells (3,6), to lymph node cells (4) and to cultured B-lymphoblasts (5). The photo of the original plate (a) and a schematic graph (b) of the precipitation pattern are given

electrophoresis of erythrocyte membranes corresponded to hemoglobin (Fig. 2). Most of the PAS-positive proteins did not enter the PAG, a part of PAS positive proteins were found in the region of fast moving peptides. Disc electrophoresis runs of membrane proteins from lymphocytes and of proteins of erythrocytes membranes or serum led to different band patterns except hemoglobin. The proteins E l - - E 6 prepared by disc electrophoresis in P A G were eluted from the gel cuts. Reelectrophoresis on PAG-gels showed that these fractions consisted of 2 - - 3 bands of similar mobility. In Figure 3 the electrophoretic patterns of E l - - E l 3 are shown. The molecular weight of these proteins was determined using a polyacrylamid gel gradient from 3--35 percent polyacrylamid and 21 standard proteins of distinct molecular weights for calibrating [34]. In such calibrated PAG-gradient the proteins of fraction E3 exhibited molecular weights of about 500000 daltons or 220000 daltons, respectively. They were liable to aggregation when they were repeatedly frozen and thawed.

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Fig. 5 a and b. I mmunoprecipitation of thymus cell extract (TE) with antisera to spleen cells (1), thoracic duct lymphocytes (3, 6),thymus cells (2), lymph node cells (4) and cultured B-lymphoblasts (5). The original photo (a) and a schematic graph (b) of the precipitation pattern are given

Fig. 6. Immunoprecipitation of antiserum to spleen cells with unfractionated spleen cell extract (SE), and the fractions F1, F2 and F3 separated on the Sephadex G200 column

2.3 Immunoprecipitation The Figures 4 to 7 represent the results of immunoprecipitation of protein preparations from spleen cells and thymus cells. Unfractionated spleen cell proteins produced seven precipitation lines with anti-spleen cell serum absorbed with h u m a n erythrocyte and serum (Fig. 4). Three of these lines were crossreacting with the precipitation lines of the antithymocyte serum and two antisera to thoracic duct lymphocytes. The other antigens which precipitated with the antispleen serum were not demonstrated with the antithymocyte serum. Five of the seven precipitation lines produced with the spleen cell proteins were removed by absorption of the anti-spleen cell serum with thymus cells or with peripheral blood lymphocytes. Precipitation lines were also observed between the spleen cell extract and an antiserum to cultured B-cells. Unfractionated extract of thymus cells produced one broad and distinct band with the antithymocyte serum which was not present in the combination with antispleen cell serum or antisera to peripheral blood lymphocytes (Fig. 5). It was absorbed by incubation of antithymocyte serum with thymus cells, but not completely with peripheral blood lymphocytes. Besides this line one other weak line was observed between antithymuscell serum and thymus cell extract. With antispleen cell serum and antisera to thoracic duct lymphocytes, the thymus cell

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Fig. 7a--d. Immunoprecipitation of fractions E3, E6, El0, and El3, eluted from PAG-sections with antisera to spleen cells (1), to thymus cells (2), to thoracic duct cells (3), to peripheral blood lymphocytes (4), to cultured B-lymphocytes (6) and with anti-immunoglobulin serum (5) extract formed two minor precipitation lines which showed cross reactions with the weak precipitatidn lines produced by antithymus cell serum. Fraction F1 of the spleen cell extract separated on Sephadex G200 column precipitated with antisera to spleen cells, to thoracic duct cells and to cultured Bcells; one or two precipitation lines were formed (Fig. 6). Also fraction F2 reacted with these antisera; here three precipitation lines appeared. Fraction F3 was not reactive with antiserum to thoracic duct lymphocytes. Antigens separated by disc-PAG electrophoresis showed the following precipitation pattern with the antilymphocyte sera (Fig. Ta--d). The fractions E 2 - - E 4 precipitated with antispleen cell serum. Fraction E3 (Fig. 7a) produced two precipitation lines with the antiserum to spleen cells. They were not identical with the precipitation lines formed by the anti thoracic duct lymphocyte serum. Fraction E3 from spleen cells formed one precipitation line with antithymus cell serum. In the fractions E6 to E8 another precipitating protein was found. Fraction E6 (Fig. 7b) contained a protein crossreacting with antispleen cell serum, antithymocyte serum and antiserum to thoracic duct lymphocytes (reaction o f identity), but not with antiimmunoglobulin serum. A third precipitation protein was demonstrated in the fractions Et0 to El2 (Fig. 7c); they formed one precipitation line with antispleen cell serum which cross reacted with antiserum to thoracic duct lymphocytes. Fraction El3 (Fig. 7d) finally contained one protein which produced an identical precipitation line with antispleen cell serum, antiserum to thoracic duct lymphocytes and antithymocyte serum.

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2.4 Results of the Cytotoxicity Inhibition Test Figure 8 shows the inhibition curve of the cytotoxic activity of the antispleen cell serum after absorption with increasing concentrations of spleen cell protein. Inhibition was observed beginning from the protein concentration of 0.5 lag to 20 lag. Addition of proteins prepared from ghosts of human erythrocytes or from h u m a n colon carcinoma cells in the same protein concentrations was ineffective in inhibiting the cytotoxicity of the antiserum. Inhibition of the cytotoxicity of antisera after incubation with membrane protein fractions is given in Figure 9. Antispleen cell serum had a cytotoxic titer of 1:4000, using peripheral blood lymphocytes as target cells. After absorption with unfractionated spleen cell extract, the titer was reduced to 1:128. The inhibitory activity was located in the fraction F1 of the spleen cell extract fractionated on Sephadex G200 column. Fraction F2 was less inhibitory. Absorption of the antisera with erythrocyte or colon carcinoma cell membrane extracts in the same protein concentrations did not reduce the cytotoxic titer of the antiserum. Thymocyte extract was less inhibitory than spleen cell extract in

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Inhibition of cytotoxicity of anti-spLeen-ceLt-serum by fractions of spleen celt membrane antigens e[uted from disc-elektrophoresis . ~ precipitating fraction //~, cytotoxic titer Fig. 10. Inhibition of cytotoxicity of antispleen cell sera after incubation with fractions of spleen cell extracts eluted from PAG-sections. The fractions E2--E4 contained two proteins of 500 000 and 220 000 daltons which precipitated with antilymphocyte serum and inhibited the cytotoxicity of antilymphocyte serum. In fraction El3 (68 000 dalton) a protein was localized which precipitated with antilymphocyte serum

In conclusion, the proteins present in the fractions E 2 - - E 4 were inhibitory for the lymphocytotoxicity of antisera and they precipitated with the antilymphocyte sera (Fig. 7a). The precipitating proteins of lower moleCular weight (Fig. 7 b - - d ) , however, did not show inhibitory activity on the lymphocytotoxicity of the antisera. Discussion Membrane receptors of lymphocytes are usually characterized by immunological techniques. Particularly, immunofluorescent methods are available for the demonstration of antigens on lymphocyte surface membranes such as immunoglobulins or blood group substances. Other serological techniques for instance cytotoxic assays are used for the detection of histocompatibility antigens or the

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receptor of antilymphocyte globulin. The presence or absence of lymphocyte antigens are the basis for the differentiation of several lymphocyte populations. To-day, only a few antigens of biological membranes are analysed biochemically as for instance the blood group antigens or the immunoglobulin receptors of lymphocytes. The analysis of these antigens is easy because they are secreted from cells in large amounts. Many methods for the isolation or characterization of cell associated antigens have been developed; but the separated products often did not correspond to the original properties and formations of the membrane associated material. Attempts to isolate histocompatibility antigens have been successfully performed by Reisfeld and coworkers [20]. They solubilized membrane associated antigens by extraction of cells in hypertonic salt solutions. The material was inhibitory for the cytotoxicity of antisera to histocompatibility antigens and was stimulatory for lymphocyte in shortterm cultures. In the present paper, another surface membrane antigen of lymphocytes has been separated, namely the receptor protein for antilymphocyte gammaglobulin. The antigen was prepared by 3 M KC1 extraction of lymphocytes and was further purified by column chromatography on a Sephadex G200 column and preparative disc PAG-electrophoresis. This purified antigen precipitates with antilymphocyte sera and inhibits the cytotoxic activity of these antisera. It is a soluble protein. It is not sedimented by centrifugation at 110 000 g for 60 min and can be filtrated through a filter of 0.45 g pore size. After reelectrophoresis it forms a strong protein band accompanied by a faint other one. The purified proteins have molecular weights of about 500 000 daltons, respectively 220 000 daltons; they are liable to aggregation. The antigen is immunologically active. The precipitation pattern of the unfractionated spleen cell extract shows seven different precipitation lines with an anti-spleen cell serum. Antisera to isolated or enriched B-lymphocytes or T-lymphocytes form two or three partially crossreacting precipitation lines with this antigenic material. This finding indicates that B-cells and T-cells carry common as well as different antigens. Thymus cell extract shows a precipitation line after diffusion against anti-thymus cell serum which did not appear when antisera to thoracic duct lymphocytes or antisera to B-lymphocytes reacted with the thymus cell extract. Except that antigen, the thymus cell extract also contains some other proteins which cross react with the antisera to peripheral blood lymphocytes. These findings are in agreement with the results of the cytotoxicity inhibition test. Antigen prepared from thymus cells had the strongest inhibitory effect on the cytotoxicity of anti-thymus cell serum to thymus cells, and a weak effect on the cytotoxicity of this antiserum to purified peripheral T-lymphocytes. The spleen cell extract, on the other hand, is more inhibitory for the antisera to lymphocytes of the peripheral blood, particularly for antisera to B-lymphocytes. From these results it seems possible that different receptor proteins for the cytotoxic antibodies of antilymphocyte sera exist on the membranes of human thymus cells and of peripheral blood lymphocytes. The presented data suggest that the antigenic membrane substrate which reacts with antilymphocyte sera is a pure protein, for it does not produce a positive staining in the PAS procedure. However the results do not exclude that the receptor protein contains carbohydrate.

Lymphocyte Receptor Protein for Antilymphocyte Antibody

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O t h e r r e p o r t e d d a t a have s h o w n a g l y c o p r o t e i c n a t u r e o f the r e c e p t o r for antil y m p h o c y t e serum. In these studies it has been d e m o n s t r a t e d that a n t i l y m p h o cyte s e r u m reacts with m a n n o s e a n d sialic acid c o n t a i n i n g g l y c o p r o t e i n s o f the l y m p h o c y t e surface a n d t h a t the reactive sites o f the l y m p h o c y t e m e m b r a n e s are m a s k e d b y glucidic sub-units ( B o n a et al., 1972). Einstein et al. have shown t h a t a n t i s e r a to g l y c o p r o t e i n s o f the l y m p h o c y t e m e m b r a n e s are able to s t i m u l a t e the DNA-synthesis. F u r t h e r studies are necessary to allow a definite s t a t e m e n t a b o u t the n a t u r e a n d structure o f the r e c e p t o r protein.

Acknowledgement. We are indebted to Dr. F. Seiler, Behringwerke Marburg, for kindly supplying us with anti-lymphocyte sera. We wish to thank Dr. K. Felgenhauer, Univ.-Nervenklinik K6ln, for determining the molecular weights of the membrane proteins. This work was supported by research grant Wa 234/6 of the Deutsche Forschungsgemeinschaft.

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Received January 31, 1978 / Accepted May 10, 1978

Isolation of a receptor protein for lymphocyte specific cytotoxic antibodies from human lymphocyte membranes.

Research in ExperimentalMedicine Res. Exp. Med. (Berl.) 173, 131--144 (1978) @ Springer-Verlag 1978 Isolation of a Receptor Protein for Lymphocyte...
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