Immunobiol. , vol. 159, pp. 1-216 (1981)

14th International Leucocyte Culture Conference, Heidelberg

Structure and Function of the Lymphocyte Plasma Membrane

Ludwig Institute for Cancer Research . Lausanne Branch, Epalinges, Switzerland, and Institute of Biochemistry, Vi:lle Benedetto XV, 1 Genova, Italy

Structural variations of human Ia subsets

R. S. ACCOLLA, N. GROSS, S. CARREL, and G. CORTE

Human Ia molecular pool is composed of dist inct subsets of molecule.~ \\ hich can be identified by specific monoclonal antibodies. Recently, we have been able to positively define twO la subsets. NG 1 and NG2, present in all individuals irrespective of their DR phenotype by their reaction with 2 distinct monoclonal ant ibodies, 01-12 and 04-22, respecti vely (I ). It has also been shown thal such antibodies react with th('ir specific la subset via the p-chain (2). We have therefore analY7.ed at the biochemicallevt'l the degree of structu ral differences between NG t and NG2 subsets by using a very sensitive! 2-dimcnsional peptide mapping tcchnique. Results show that. in the la molecu lar poo l of a si ngle individual, ~-chai n s of NG t and NG2 subsets differ from each other . Moreover, when different DR specificities were compared, important structural variations were observed among fl-chains of either subset. In addition , analysis of a-chains associated to the two la subsets revealed important differenct'~ between NGta and NG2a Ia molecules of the same individual. However, within the same subset. no differences were observed among a-chains of differem individuals. Thtst rtsuIts show that both NGI and NG2 subscts have distinct forms of (3- and a-subunits; both subsets can carry polymorphic spec ificities which are however confined only at level of small fl -subunit.

I. ACCOLlA et aI. , Vllith Int. Histocomp. Wo rkshop Newletter 20, 46, 1980 2. CAR.R.El et al.. Molecular 101m. (in press) 1981

2 14th Internationa l Leucocyte Culture Conference, Heidelberg

I Abteilung fur Immunologie, J. Med. Universitatsklinik, H ambu rg, D-2000 Hamburg, 2 KJinik fi.ir Abdominal- und Transplantationschirurgie, Mcdi7.inisehe Hochschule Hannover, D-3000 H annover, Federal Republic of Germany

Partial molecular characterization of the rat-T-Iymphocyte antigen RT-Ly 2

Recently the cell surface antigen (RT-Ly 2) was detected on peripheral T-cells by alloanti ­sera (W ONIGEIT, K.: Transplant. Proc. 11 : 1631 [1979]). After enzymatic labeling of the cell membrane of ig (-) rat lymphnode cells with I23J, solubilisation in Triton X- IOD immunoad­sorption on Protein-A Sepharose anti-RT-Ly 2 and subsequent 50S-PAGE two protein bands slightly differing in their apparent molecular weights (21 and 25 k) could he detected. From crosslinking experiments and ultracentrifugation analysis it can be concluded that the antigen is on one chain molecule appearing in two different molecu lar forms. The fact that under reducing cond itions in 50S-PAGE the molecular weight of the rwo bands shifts from 21 to 25 and 25 to 29 k, respectively, suggests that the peptide chain of these proteiDS have intramolecu­lar disulfide bridge(s). Metabol ic as well as cell su rface labeling of glycoconiugates from Iymp hnode lymphocytes by the periodatc NaB3H4 method revealed that the antigen is not or, if at all, faintly glycosylated. By sequential binding experiments it could be shown that the 21 and 25 k proteins are not only detectable via their alloantigenic but also via xenogenic determinantS.

VA Medical Center, Washington, D.C., USA

Glucagon and insulin receptors on Iymphoblasts and leukemic cells

S. BHATHENA, A. F. GAZDAR, G. P. SCHECHTER, L. RECA!\'T

In fresh ly isolated mononuclear leukocytes (MNL) we have shown that glucagon receptors, similar to insulin receptors, arc found primarily on monocytes, with little specific binding by glucagon occurring on normal lymphocytes (BH ATHENA et aI. , Di:Jbeces, in press, February 1981). Using hormones radiolabellcd with m I, we have now studied receptors for glucagon and insulin on ( I) normal MNL activated by PHA or TCGF, (2) MNL isolated from patients with B cell malignancy (chro ni c lymphocytic leukemia, 2 patients) and T cell malignancy (Seury Syndrome, 3 patients) and (3) cells from Band T long term Iymphoblastoid lines. Insulin and glucagon binding rose 30-fold in normal MNL cultured with PHA for 5 days. Glucagon receptor number increased without a c hange in receptor affi nity. The insulin receptor number also increased but with an associated decrea.~e in affinity. MNL cultured with TCGF showed an early plateau rise in ins ulin binding (8-fold, with increased receptor numbers and no change in affinity) and a progressive rise up to 50-fold in glucagon binding due to both increased receptor number and affinity, suggesting that a T lymphoblast subset with very high glucagon binding had been selected out, or that glucagon receptors were being induced by TCGF. Monocyte-poor leukemic cells from 4 of the 5 patients with lymphoid malignancy (both Band T cell types) showed increased giuc:lgon binding (7 to 36-fold that of normal lymphocytes), Two patients (IB, IT) showed increased in~ulin binding (4- and 6-fo ld i). In contrast to the fresh ly isolated leu kemic cells, the T and B lymphoblastoid cci llines

14th International Leucocyte Culture Conference, Heidelberg 3

showed distinct patterns of insulin and glucagon binding. Six B cell lines and 5 T cell lines were studied. Glucagon binding by T lymphoblastOid cells was seven fold higher than that of B lymphoblast cells (p < .01), and insulin binding was correspondingly lower (p < .01 ). T cell lines had twice the number of glucagon receptors but the affinity was similar, whereas B lines had 4-fold the insulin receptors, with much greater affinity. Induction of both insulin and glucagon receptors on activated Iymphoblasrs suggests they may playa role in cell function. The reasons for alteration of distribution of the receptOrs on T and B lymphoblasts are unknown. The large increase in insulin receptors in B lymphoblastOid cells relative to T lymphoblastoid cells may be consistent with the involvement of B cells in immunoglobulin synthesis for export.

Dept. of Biochemistry. University of Lausanne!, ISREC1 and LICR" 1066 Epalinges. Switzer­land

Membrane proteins of murine T cell clones

Functionally differenr T cell subsets can be distinguished on the basis of antigenic determi­nants associated with cell surface molecules. The relationship of these differentiation antigens with the specialised function of the T cells that bear them is not well defined. The recent availability of doned murine l' lymphocytes provides homogeneous populations of funct­ionally distinct cells necessary for such biochemical studies. Thus we have compared the membrane protein composition of the following T cell clones: Balbl c helper T cells specific for beef apocytochrome sand pep tides thereof; B6 cytotoxic T cells specific for H-2Dd; B6 cytotoxic T cells specific for mouse sarcoma virus associated antigens; B6 proliferating l' cell specific for I-A k; B6 helper T cells specific for MIs' . Non-ionic detergent extracts of enzymati­cally surface labeled cells using !lSI or 3H-borohydride were analysed on 2-dimensional polyacrylamide gels by non~equilibrium pH gradient electrophoresis and by electrophoresis on I-dimensional gels in sodium dodecyl-sulfate. In addition, certain T cell specific proteins obtained by immunoprecipitation from different dones were analysed by peptide mapping. Comparison of the cell surface polypeptide profiles revealed significant differences between cytolytic and helper T cell clones. Moreover, structural differences were detected among homologous proteins expressed by the cytolytic and helper clones, thus suggesting a different processing of these surface components in functionally different lymphocyte subsets.

Laboratory of tumor immunology inserm U 152 Hopital Cochin and department of hemato­logy c.h.u. Pitie-Salpetrihe, Paris, France

Human Ia antigens on T cells. Structure, synthesis and molecular transfer

D . .J. CHARRON, M. HOA NG-XUAN, and D. LEVY

Initial description of the human Ia system indicatcd that the fa antigens are primarly expressed on B lymphocytes and macrophages. In contrast to resting T ceils, activated T cells were recently shown to be la+. Using a monoclonal anti HLA DR antibody and a 2-

4 14th International Leucocyte Culture Conference. Heidelberg

dimensional (2D) gel electrophoresis genotyping method (1) we have previously demonstrated that alloreactive T cell synthesize Ia antigens of the responder type (2). The 2 D gel Ia patterns of activated T cells and B cells are similar. Moreover tryptic peptide mapping of HLA DR subunit chains isolated from B cells and activated T cells are identica1. Comparison of Ia fingerprints from 1251 lactoperoxidase surface labeled and 355 methionine internally labeled alloreactive T cells shows that both Ia molecules of the responder and of the stimulator phenotype are present on the T cells at day 4 of a one way MLR. No Ia antigens were detected at day 11. Purified HLA DR molecules were isolated on a monoclonal anti Ia immunoabsor­bant and labeled with 1251. The binding capacity of allogeneic and autologous HLA DR molecules on differents Human T subpopulations was investigated. This may provide a molecular model to study the T cell receptor (s) for syngeneic andlor allogeneic Ia antigens.

1. CHARRON, D., and Ho McDEV1-rr. ]. Exp. Med. 152,2: 18 (1980). 2. CHARRON, D., E. G. ENGLEMAN, C. BEN1KE, and H o McD.EVITI. J. Exp. Med. 152,2: 127

(1980).

Inst. of Med. Microbiology, Vnlv. of Mainz, Hochhau.~ Augustusplatz, D-6500 Mainz, FRG.

Monoclonal antibodies directed against human C3b-receptors

M. P. DJERICH, H. H. MUSSEL, O. SCHEINU, M. SCHMIT!', T. EHLEN, and R. BURGER

A C3b-binding glycoprotein was purified by solubilization of human erythrocytes and subsequent affinity chromatography on C3-coated Sepharose 4B (MUSSEL et a1.. 1980, lmmunobioJ. 157,257). This material-exhibiting reactivity for C3b as tested by inhibition of immune adherence and of C3b-dependent rosette formation - served as antigen for immuniza­tion of BALB/c mice. Spleen cells of these mice were fused with mouse myeloma NSl cells according to the standard technique. Hybridomas were cloned by the limited dilution method. The supernatants of these clones were screened by incubation with Raji-lymphoblastoid cells and tonsil lymphocytes, respectively, which then were tested for the remaining capacity to

form rosettes with EAC14"'Y23bhu",. A ~eries of monoclonal antibodies was obtained. Three of these antibodies (A, B, C) were characterized in more detaiL They showed different reactivity patterns as determined by inhibition of C3b-dependent rosette formation. The antibody of clone A inhibited the binding of EAC14oxY23bhum to Raji cells up to a dilution of 1 X 105

whereas the reactivity against tonsil lymphocytes was markedly lower. The antibodies of clone B exhibited the reverse pattern. Antibodies of clone C inhibited the C3b-receptor activity of both target cells up to a dilution of 1 X 105• Neither antibody showed reactivity against human granulocytes or monocytes. This inhibition was C3b-receptor-specific since the adherence of EAC14°"Y23bihum and EACI4"x)'23dhum to complement receptor cells was not influenced. In support of a specific reaction with C3b-receptors we found that only binding of EAC140X)'23bhum to human renal glomeruli was inhibited by clone C but not of EACl4oxY23bihum. Further characterization regarding physicochemical and immunochemical properties of these antibodies is in pr'ogrcss .

Supported by the DFG, SFB 107, AS.

14th International Leucocyte Culture Conference, Heidelberg 5

Department of Pathology, University of Southern California School of Medicine, University of Southern California, Cancer Center, Los Angeles, USA

A subset of lymphocyte surface glycoproteins that are Con-A receptors and oxidized by galactose oxidase

JEAN J. FAVERO *, IAN L. GORDON, JOHI\ W. PARKER, and RICHARD L. O 'BRIEN

We have pr~viously r~ported that concanavalin A inhibits galactose oxidase induced mitogenesis. We therefore undertook to determine which, if any, lymphocyte surface protein!> are both oxidized by galactose oxidase and bind ConA. We labeled lymphocyte proteins by incubating with }H-tyrosine and surfac~ glycoproteins by oxidation with galactme oxidase or period ate followed by reduction of the aldehydes produced with borohydride. Gel elec­trophoresis of lysates of oxidation reduction labeled lymphocytes demonstrated that more than 20 lymphocyte surface glycoproteins were oxidized . Labeled lymphocytes were also incubated with ConA and the ConA receptors were isolated by immunoprecipitation with anti-ConA serum and fixed Staph A. Electrophoretic analysis of the immunoprecipitates demonstrated that incubating cells with tyrosine labeled more than 12 ConA receptor pro­teins. Immunoprecipitation of ConA receptors from oxidation reduction labeled cells revealed only 4 high molecular weight glycoproteins that w~re hath oxidi7.ed by galactose oxidase and bound ConA. These .~amc high molecular weight glycoproteins are also oxidized by periodare. Because ConA inhibits galactose oxidase mitogenesis, one or more of these four high molecular weight glycoproteins probably represent rhc necessary sites of oxidative mitogenic action and are also good candidates for the targets of eonA mitogenesis.

*) Current address: Laboratoire de Biochimie des Membranes, Ecole Nationale Superieure de Chimie, 8, rue de l'Ecole Normale, 34075 Montpdlier Cedex, France

Laboratory of Histology and Cell Biology, University of Amsterdam, Amsterdam, The Netherlands

Surface immunoglobulin-associated structures in the plasma membrane of B lymphocytes as revealed by freeze-etching

C. DE GROOT, and W. LUNI:

It has recently been shown that surface immunoglobulin (sIg), at least the IgM isotype, present on the surface of B lymphocytes protrudes through the lipid bilayer of the plasma membrane via hydrophobic peptide chains, not present in secreted IgM (1). In the present study the morphological ba.~is of such transmembrane character was invesrjgared. sig on rabbit and mouse spleen B lymphocytes was redistributed into patches, and glutaraldehyde-fixed cells frozen in 35 % glycerol were freeze-etched following a newly developed procedure (2), allowing the direct visualization of antibodies - not conjugated to electron microscopic markers - on the cell surface and intramemhraneous particles (IMP's) in the adjacent fracture face. It appeared in hath species, that no correlation could be found between sIg and IMP's of 8-10 nm diameter. However, careful examination of replicas at low Pt shadow angle revealed that very small IMP's (3--6 nm diameter), hardly protruding through the lipid bilayer, were found on the external fracture face associated with sig-anti Ig patches. This finding may r~present a morphological basis for the concept that sIg is a membrane-spanning protein and is

6 14th International Leucocyte Culture Conference, Heidelberg

in line with the hypothesis of VERKLEIJ and VERVERGAERT (3) that non-complementary intramembraneous particles of very small diameters are candidates for membrane-spanning proteins.

1. ROGERS, J., P. EARLY, C. CARTER, K. CAlAMJl., M. BOND, 1. HOOD, and R. WALL. 1980. Two mRNAs with different 3' ends encode membrane-bound and secreted forms of immunoglobulin f.i.-chain by alternative RNA processing pathways. Cell 20: 303.

2. KAPSENBERC, M. 1., T. HOGENES, and W. LEENE. Mechanism of lymphocyte activation. Tntramembraneous particles are not involved in patch and cap formation of PHA reception. (Submitted for publication.)

3. VERKLF.IJ, A. J., and P. H. J. TH. VFRVERCAERT. 1978. Freeze fracture morphology of biological membranes. Biochim. Biophys. Acta 515: 303.

MRC Clinical & Population Cytogenetics Unit, Western General Hospital, Edinburgh, Scotland

Analysis of HLA D-related and other human B ceIl surface determinants by means of monoclonal antibodies

VERONICA VAN H EYNINGEN, K. GUY, C. M. STEEl, B. B. COHEN, D. L. DEANE, D . CRJCH­TON, and SANDRA LAWRIE

Five monoclonal anti-B cell antibodies have been generated following immunisation of a mouse with the human B lymphoma line DAUDI. The specificities of the set of reagents have been studied by radio-immunobinding and immunofluorescence on a panel of human cell types including peripheral blood Band T cells and monocytes, bone marrow, leukaemic and lymphoma cells, Band T lymphoid lines, myeloma lines and cultured non-lymphoid cells. The antibodies have also been examined for their ability to inhibit mixed lymphocyte reactions and the nature of their respective target antigens has been investigatc=d by biochemical analysis of solubilised cell membranes. These studies shed light on the structure and cellular distribution of HLA D-related gene products and on the role of these antigens in mixcd lymphocyte reactions.

Dept. of Medicine, U.S.U.H.S., Bethesda, Maryland, and Dept. of Immunology , Merck Inst. for Therap. Research, Rahway, New Jersey, USA

Biochemical properties of the Ia antigen(s) of the I-A mutant B6.C-H-2bmt2 mouse

S. W. KESSLER and T. H. H ANSEN

The B6.C_H_2bmll mouse has a mutation in the I-A region of the major histocompatibility complex that results in skin graft rejections, MLR stimulation, and failure ro respond to certain protein antigens. In order to define the nature of the mutation in molecular terms, fa and H-2K molecules from B6.C_H_2hmI2 and parc=ntal C57BL/6 spleen cells were radiolabeled either biosynthetically or by surface membrane, lactoperoxidase-catalyzed iodination , were immunoprecipitated from detergent celllysates with (AI] X BIO.A) anti-B10.A(SR) serum,

14th Inwrnational Leucocyte Culture Conference, Heidelberg 7

and were then compared by 2-dimensional polyacrylamide gel electrophoresis. No discernible difference was noted in the locations of biosynthetically or cell surface-radiolabeled alpha, beta, or invariant Ia component polypeptides, either in intermediate stages of glycosylation or in unglycosylated form. Also, there was no difference in the amount of Ia synthesized relative to H-2K molecules. In contrast, Ia antigens radiolabeled on the cell surface were expressed in mutant mice at 3D--40 % of the level .~een in C57BL/6. A similar difference was noted when cells were stained with a f1uoresceinated anti-Ia monodonal antibody and analyzed with a fluorescence-activated cell sorter. However, 3 additional anti-lab monoclonal antibodies failed to stain B6.C_H_2bmI2 cells at all. In order to determine if the lesion in the mmant .~pecifically involved transport of Ia to the surface membrane, upward modulation of Ia antigens was induced by in vivo injection of anti-IgD antibodies. 1a expression, detected with all 4 fluorescent anti-Ia reagents, increased approximately 3-fold on C57BLl6 cells. A proportional increase was observed in the mutant with one of the reagents, but the 3 others remained nonreactive. A simple interpretation of our findings might implicate an alteration in the B6.C_H_2bmll mouse of one or more conformationally important uncharged (or compensati ng acidic and basic) amino acids, or even an abberant site of glycosylation, on one of the component Ia polypeptides. However, the possibility also exi.m that the mutation affects another molecu le that forms part of the functional 1a complex on the cell surface, but is too weakly associated to be recovered after detergent solubilintion and immunoprecipitation.

Centre d'lmmunologie INSERM-CNRS de Mar.~eille-Luminy, Case 906, 13288 Marseille Cedex 9, France

Topology of epitopes of the l' gene products analyzed by 35 monoclonal anti-la' alloantibodies

M. PIERRES, C. D EVAUX, M. DOSSETO, A. PI ERRES, J. P. R EBOUA H, and F. M. K OU RILSKY

In order to estimate the diversity of the IS anti-Ik B cell reperroire, a series of 35 monoclonal antibodies (mAb) have been constructed by fusion between NSI-Ag 4.1 or X63-Ag 8.653 myeloma and A.TH (KsfsDd) mouse .~pleen cells immune to A.TL (KslkDd) lymphoid cells. Amongst the 18 mAb reacting with the I_Ak molec\!les, six defined private determinants analogous to the conventional la.2 specificity, other mAb identified five epitopes having a strain distribution idelHical to that of the la.l specificity (i.e. present on H k -2\ H-21, and H_2r haplotypes); the other 7 mAb identified determinants of the I-A molecule that did not correlate with known Ia specificities: two mAb reacted only with I-A k. ant! J_Af moleC\lles, and five other mAb defined public epitopes present on various standard haplotypes.

Amongst 16 mAb which reacted with the I-E/Ck. molecule(s), five mAb detected epitopes with a strain distribution identical to that of the la.7 specificity of the Eo.-chain, one mAb non specific fo r a private determinant of the £j3k chain , and the remaining 10 mAb detected I-F.lC determinants which did not correlate with previously described Ia specificities. Finally, one mAb apparently identifies a new epirope shared by the I_Ak and l-E/C k molecules also expressed on H_2b, H_2'I, H-2' and H-2J haplotypes.

This collection of monoclonal ami-Iak antibodies is currently being used to investigate various aspects of the serology, the function, and the structure of la antigens, and results will be presented concerning: 1) analysis of the spatial urganization of the epitopes of the l_Ak and I-EI C k molecules using competitive inhibition of radiolabeled anti-lal< mAb ; 2) analysis of interspecies crossreactions between la-like molecules, in particular the croos-reaction of anti­I-A k and anti-I-E/Ck mAb with human HLA-D/Dr; 3) functional studie.~ of the la amlgens by means of inhibition of T cell responses by various mAb; 4) induction of-Ycllogcncic (rabbit) or syng~ncic (A.Tl.) anti-idiorypic r~agents against various anti_Iak mAb and their use in the study of Band T cells anti-I region products alloreactivity.

8 14th International Leucocyte Culture Conference, Heidelberg

Institut fur Virusforschung, Deutsches Krebsforschungszentrum, 6900 Heidelberg, FRG

Modulation of enzyme activities in isolated lymphocyte plasma membranes by lysolecithin acyltransferase-dependent modification of phospholipid fatty acid composition

KLAUS RESCH and MARTA SZAMEL

Shortly upon activation of lymphocytes with mitogens the activity of several plasma membrane bound enzymes are modulated. We have shown recently that in concanavalin A stimulated calf thymocytes (K + + Na +)ATPase and lysolecithin acyltransferase were acti­vated while other enzymes were suppressed in their specific activity. Due to a preferential activation of acyltransferase for polyunsaturated fatty acids mitogen-activated lymphocytes accumulate these fatty acids in the plasma membrane. We have suggested that the increase in the degree of fatty acid unsaturation underlies the observed changes of plasma membrane enzymes in activated lymphocytes. To test this plasma membranes were purified from calf thymocytes as described. With the exception that instead of a dextran gradient 35 % sucrose (w/w) was used. Isolated plasma membranes were incubated with increasing concentrations of the coenzyme A derivatives of long chain fatty acids (acylcoA's) in the presence of lysolecithin. Control experiments showed that under these conditions added acyl coA's were converted nearly quantitatively into membrane associated lecithin. Lysolecithin alone or lysolecithin acyltransferase mediated incorporation of palmitic acid (16 : 0) or oleic acid (18 : 1) at concentrations ranging from 10 to 400 nmol/mg membrane protein had no effects on the specific activities of any of the plasma membrane em~ymes tested. In contrast, incorpora­tion of linoleic aeid (18 : 2) modulated dose dependently these enzymes: The activity of (K + + Na+)ATPase was increased. maximally when 150 to 250 nmollinoleic acid were incorpo­rated per mg membrane protein. At higher uptake enzyme activity reverted to control values. Specific activity of lysolecithin acyltransferase (measured with 14C-oleoyl coA) was also increased with a similar dose response for incorporated linoleic acid. Concomitantly the activities of Mg+ + ATPase and y-glutamyl transferase were decreased, depending also on the degree of fatty acid substitution. The specific activity of alkaline p-nitrophenylphosphatase was marginally decreased, too. Identical results as in membranes modified with linoleic acid were obtained in plasma membranes into which arachidonic acid (20 : 4) was incorporated. Inhibition of lysolecithin acyltransferase with 5 X 1O-5M ethylmercurithiosolicylate during incubation of membranes with the polyunsaturated acyl coA's left all enzyme activities enchanged indicating that transfer of these fatty acids to phospholipid is required for modulating the activity of plasma membrane associated enzymes. The changes observed in the specific activities of plasma membrane enzymes after increasing the content of polyunsaturated fatty acids experimentally strikingly resembles those changes observed in the membranes of mitogen stimulated lymphocytes. The data thus fit a hypothesis that lysolecithin acyltransfe­rase is crucially involved in the molecular mechanism initiating lymphocyte activation. The autocatalytical activation of acyltransferase furthermore provides a basis for the limited time requirement of mitogen binding to allow progression through cell division.

Supported by grants of the DFG and the DAAD.

14th international Leucocyte Culture Conference, Heidelberg 9

Instirut fur Immunologie der Universitat Maim'., 6500 Mainz, FRG

Biochemical studies of murine I_A' subregion associated antigens

K. R ESKE

NP40-solubilized I_Ak products were isolated by immunoprecipitation with various mono­elonal anti I_Ak antibodies from biosynthetically and surface labeled C3H spleen cells. To determine physicochemical parameters of single I-A associated polypeptide chains, anti I_Ak antigens were analyzed by !ieveral electrophoretic techniques. In the absence of reducing agents they separate on one-dimemional SDS polyacrylamide gradient gels (lD PAGE) into four monomeric components, designated II 35,000 d, Y 32,000 d, II I 27,000 d, III 25,000 d. In addition, the following dimer-(oligomer) bands were observed: d l 52,000 d, d2 57,000 d, dJ

63,000 d, d~ 74,000 d. After reduction of anti I_Ak immunoprecipitates with dithiotreitol (DIT) only three bands remain detectable in the monomer region of the gel: a 37,000 d, y 32,000 d, ~ 31,000 d. Both light chains Il l' 112 coincide after disulfide bond cleavage and shift to fl. Additional internal radiolabeling studies with JH Man,)H CluN, and 3-,S Cys indicated that all four I-A polypeptide chains were glycoproteins. Each chain incorporated 3;cysteine. When spleen cells were surface labeled by lactoperoxidase cataly~ed iodination, 131 and 112 were labeled in addition to all light chain containing dimers, suggesting thal both light chains are easily accessible at the cell surface. Papain digestion studies of whole spleen cells, as well as shedding experiments indicated that all four polypeptide chains are present on the cell surface. 2-dimcnsional (2D) O'Farrell analysis ( td, IEF 2D , SDS PAGE) of unreduced anti-I-A k

antigens revealed a rather complex separation pattern. All monomer and dimer components were separated and showed their expected molecular weight positions. After reduction with DTT the established three chain pattern (a, 'I, ~) was obtained. a and P displayed small arrays of spots with p13.9-4.6 and 8.8-9.5, respectively, whereas 'I focuses at pI 8.3, indicating the similarity or even identity of y to the invariant chain recently reported by P. P. JONES. Dimer analysis was performed by running anti-I-A~ immunoprecipitates on 20, SDS PAGE (10 without OTT, 2D with OTT). All dimers (oligomers) except d\ contained either one light chain. Dimer d1 was a heterodimer composed of one heavy and one light chain, whereas dimer dJ , usually the most prominent dimer band of anti I_Ak immunoprecipitatcs, proved to be a homodimer of y. Treatment of the I-A antigenic complex with 0.1°/" SDS in tris buffer released the excess of the y-chain selectively with a concomitant increase of its homodimer dJ•

All four I-A associated polypeptide chains were purified to homogeneity by preparative SDS

PACE. The physicochemical parameters obtained by examining isolated polypeptide chains corroborated data from the analysis of whole anti I-A immunoprecipitates.

We conclude that I_Ak subregion associated antigens are composed of four hydrophobic polypeptide chains which arc present in the plasma membrane arranged in an oligo chain complex with surface exposed light chains. Experiments in progress are aimed at defining their structural relationship and to assess their individual immunofunction.

NIH, Bethesda; University of Wisconsin, Madison; University of Pennsylvania, Philadelphia, USA

HLA-SB: a new gene centromeric to HLA-DR which encodes B cell antigens similar to DR antigens

STEPHEN SHAW, PAULA K AVATHAS, ROBERT DEMARS, and LOIS LAMI'SON

We have identified five "new" HLA-linked antigens (1 ) (designated SB or Secondary B cell antigens) by twO different cell-mediated typing assays: secondary lymphocyte proliferative

10 14th International Leucocyte Culture Conference, Heidelberg

responses and secondary cell-mediated cytotoxicity. Each antigen can be identified by two independently derived primed lymphocyte typing reagents with excellent concordance between reagents (r > 0.87). Population studies of 200 unrelated donors ind icate that these SB­antigens are distributed in accordance with the hypothesis of Hardy-Weinberg equilibrium of alleles at a single locus, and these five antigens can account for the alleles of about 65 % of Caucasian haplotypes. The SB antigens are similar to DR antigens in genetics, tissue distribu­tion, function and probably structure. Both SB and DR antigens are encoded in the region of HLA between HLA-B and GLO. Both DR and SB are expressed strongly on B cells and macrophages but weak ly if at all on T cells. Both stimulate strong secondary proliferative and cytotoxic responses. Certain monoclonal antibodies to l a-like B cell antigens can inhibit both DR-specific cell-mediated cytotoxicity and some SB-specific cell-mediated cytotoxicity. How­ever, three lines of evidence demonstrate that SB and DR antigens are products of distinct genes. First, one family (out of 25 studied) has been identified in which recombination has occurred between SB and DR. One sibling has inherited A2-B5-DRt from one maternal haplotype but has lost the SB3 from that haplotype and acquired the SB2 gene from the maternal A I-B8-DR3 haplotype. Second, y-ray induced HLA variant cell lines which had been characteri7.cd previously with respect to loss of HLA-DR antigens have now been charac­terized for expression of SB antigens (2). Of five cell lines which had lost expression of DR 1, BS, and A2 from one haplotype, two had lost expression of the cis-linked SB2 antigen while three had retained the SB2 antigen. The thi rd, less direct, supporting evidence is that SB antigens Occur independently of the DR antigens in the population and the linkage disequilib­rium between DR and SB alleles is roughly comparable to that between HLA-DR and HLA­B. These data indicate that SB and DR have many functional similarities but that they are coded by distinct genetic loci.

1. SHAW, S., A. H. JOHNSON, and G. M. SHEARER. 1980. Evidence for a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogeneic proliferative and cytotoxic responses. J. Exp. Med. 152: 565-580.

2. KAVATIiAS, P., F. H. BACH, and R. DEMARS. 1980. Gamma ray-induced loss of expression of HLA and glyoxalase T alleles in Iymphoblastoid cells. Proc. Nat!' Acad. Sci. USA 77: 4251-4255.

The Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, University of London, Mortimer Street, London W1P 7PN, U.K.

The physical properties of lymphocyte plasma membranes and effects of fatty acid supplementation

C. D. STUBBS

Plasma membranes were isolated from mouse spleen lymphocytes and the effect of varying the temperature (10--45 °C) on membrane fluidity wa.~ examined on control membranes and membranes prepared from cells which had been supplemented with linoleate. Membrane fluid ity was assessed as the steady state fluorescence anisotropy of the hydrophobic fluores­cent dye 1,6-diphenyl-l,3,5-hcxatriene, inserted into the membrane lipids. Arrhenius type plots of fluorescence anisotropy with temperature were linear but with a marked discontinuity at 30°C where the slope changed. Below 30 °C there were possibly other discontinuities but these were less easily identifiable. The effect of membrane proteins was assessed by repeating the experiment on multilayer liposomes made from the extracted lipids. 'fhe discontinuity was ~t ill present at 30 ~C showing it to be a property of the membrane lipids. 'fhe temperatu re of the discontinuity was not affected by linoleate supplementation. inspite of a marked change in the fatty acid profile of the phospholipids, although the slope of the 30--45 "C portion of the

14th Internationa l Leucocyte Culture Conference, Heidelberg 11

curve was less. The change in slope may indicate a difference in the physical state of the lipids wllich is likely to be reflected in a modulation of membrane funct ion5. In previou.~ studies (Biochim. Biophys. Acta, 496, 155-156, 1977) supplementation of lymphocytes with linoleate produced a marked inhib ition of transformation by mitogens and the results of the present study indicate that a lipid modulation of membrane protein conformation, important in the process of transformation, may be involved in this effect.

Institute fo r General and Experimental Pathology, University of Innsbruck, Medical School, Innsbruck, Austria

Immunochemical characterization of chicken bursa and thymus cell antigens

H UGO W OLF and GEORG WICK

This contribulion deals with the immunochemical characterization of ch icken bursa and thymus lymphocyte membrane determinants by specific anti-bursa cell sera (ABS) and anti­thymus cell ~era (A TS). These antisera, raised in turkeys and absorbed as described in detail previously, can also be applied for in Yj·vodepletion of B- and T -cells. In this study living bursa and thymus cells were radiolabelled either by lactoperoxidase catalized 12'-1-iodination (for the protein moiety) or by galactose-oxidase/NaB3H 4-technique (for carbohydrates and glycopro­teins). After solubilization by Nonidet N P-40 and immunoprecipitat ion the relevant antigens were analyzed in SDS-polyacrylamide-gcl-clectrophores is (SDS-PAGE). The bursa cell sur­face proteins, precipitated by ABS and reduced with mercaptoethanol, give pcaks correspond­ing to molecular weights (MW) of 151,000, 108,000, 75,000 and 43,000 Daltons. Determinant5 recogni7.ed on thymus cells by A T5 show MW -peaks of t 72,000, 98,000, 62,000 and 45,000 Daltons. Further applications of this techn ique are thus two-fold: a) add itional studies on the modificat ion of these differenriation antigens during ontogeny, and b) production of antisera for the identification of functionally different lymphocyte subpopulations. So far monoclonal antibodies of avian origin cannot yet be prepared due to the lack of appropriate avian myeloma lines.

Supported by the Austrian Cancer Research Fund.