Workshop B-3 Role of Cell Surface Molecules in T Cell Triggering

T cell activation markers

I.C.R.F. Human Tumour Immunology Unit and Department of Clinical Haematology, School of Medicine, University College London, WClE, London, U.K.

124. Tumour promoter phorbol esters induce IL-2 receptor in T and B cells and antigen specific unresponsiveness in T lymphocytes

I. ANDO, D. H. CRAWFORD, G. HARIRI, D. WALLACE, and P. C. L. BEVERLEY

When exposed to tumour promoter phorbol esters, T and B cells express receptor~ to T-cell growth factor. This expression of growth factor receptor is paralleled by a decrease in expression of cell surface molecules associated with the T3-receptor complex. These pheno­typic changes are accompanied by a high sensitivity to IL-2 and antigen specific unresponsive­ness in T-cell clones. The metabolic requirements and changes associated with these pheno­typic and functional alterations are being investigated.

Medizinische Klinik, Hugstetterstr. 55, 7800 Freiburg, and Deutsches Primatenzentrum, Kell­nerweg 4, 3400 Gottingen, FRG

125. Expression of a Hodgkin cell associated antigen on subsets of activated T - and B-cells

R. ANDREESEN, J. OSTERHOLZ, K.]. BROSS, T. CHOSA, and G. HUNSMANN

The origin and identity of the neoplastic cell in Hodgkin's Lymphoma are still under debate. Recently, a monoclonal antibody (Ki-1) has been described which reacted specifically with Hodgkin and Sternberg-Reed cells but not with any other cells in the biopsy material. Here we report that this antibody binds to a surface structure expressed on T -responder cells reactive to autologous and allogeneic stimulator lymphocyte and soluble antigens, as well as on EBV transformed B-lymphocyte. In contrast, in lymphocytes cultures with ConA and PHA only 2-6 % of the transformed blasts react with the Ki-1 antibody and less than 1 % of the T-helper lymphoblasts generated by oxidative mitogenesis (sodium periodate) express the Ki-1 antigen. Ki-1 positive cells appear in mixed lymphocyte cultures first on day 3, reach a maximum on day 6-7 and decline rapidly from day 10 on. These cells can be identified as activated T-helper lymphocytes which express the T3, T4, T9, the la-like (HLA-DR) antigen and the IL-2 receptor as detected with the immunoperoxidase technique on poly lysine coated slides. About 45-60 % of all T -lymphoblasts generated by stimulation with autologous and 35-50 % lymphocytes stimulated with allogeneic non T-cells express the Ki-1 antigen. About 6 days

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after stimulation with EBV 35 % of B-lymphoblasts react with the Ki-l antibody. Established B-lymphoblastoid cell lines constantly express the Ki-l antigen up to 95 %. The Ki-l antigen was also found on the MTI T-cell line infected with Human T-cell Leukemia Virus. Interestingly, this cell line appears to have lost some T-cell surface markers and thus closely resemble the phenotype of Hodgkin's cells. Cells of the permanent cell lines MOLT-4 (60-80% positive cells), K-562 (50-60%, weak) and HL-60 (3-7%) react with the Ki-l antibody, the U 937, Raji, Daudi and Reh cell lines do not react. In summary, the Ki-l antibody binds to a surface structure expressed on T- and B-lymphocytes after stimulation with viruses or specific cellular or soluble antigens, but is not expressed after polyclonal T-cell stimulation by mitogens. If auto reactive cells are the first to be activated in the generation of an allospecific immune response the Ki-l antibody could aid to further characterize the role of autostimulation in immunity. On the other hand the result may indicate a relationship between the autoreactive subset of T-lymphocytes and the pathogenesis of Hodgkin's lymphoma and help to explain the many immunopathological abnormalities seen in patients with this disease. It may be hypothesized that Hodgkin's disease is a malignancy of auto reactive T-cells which have lost their T-cell markers during malignant transformation.

The Ki-l antibody was provided by Dr. H. STEIN, Pathologisches Institut, Hospitalstr., D-2300 Kiel.

Departement d'Immunologie, Institut Pasteur, 25 rue du Docteur Roux, Paris, France

126. Regulation of Interleukin 2 receptor expression on a murine GAT­specific T helper cell clone

G. BISMUTH, A. DAUTRY, M. DUPHOT, and J. THEZE

The level of Interleukin 2 receptor (IL 2 R) expression on a GAT-specific T helper cell clone has been studied. T cell clone 52.3 which is cultured with GAT and irradiated spleen cells as antigen presenting cells (APC) presents two distinct levels of IL 2 R expression. High levels are obtained three to four days after stimulation with antigen and APC. From this time the amount of IL 2 R slowly decreased to reach 12 to 15 days later a very low value. This has been measured with two different rat monoclonal antibodies against the murine IL 2 R and with labelled human IL 2. In the presence of cloned human IL 2, the recently activated cells divide actively while the non recently stimulated cells do not, but survive very well with a very slow decrease of IL 2 bioactivity in the medium. These results are consistent with the fact that IL 2 R expression is probably the main parameter by which the antigen specific signal controls the T cell proliferation. Furthermore, we show here clearly that IL 2 is not only mitogenic but also a survival factor for helper T cells depending on the level of IL 2 R expression on these cells.

Rotterdam Radio-Therapeutic Institute, The Netherlands

127. Reversible and non-reversible loss of IL-2 receptors and proliferative capacity in long term cultured human cytotoxic T cell clones

R. L. H. BOLHUIS, B. A. VAN KRIMPEN, and R.]. VAN DE GRIEND

Induction of IL-2 receptor expression by antigens or lectins preceeds the in vitro prolifera­tion of human T-lymphocytes as can be determined with the anti-Tac monoclonal antibody.

16th International Leucocyte Culture Conference, Cambridge . 83

IL-2 receptor densities on activated T cells can be reduced by «resting» the cells in mediu~ without lectins (1). In peripheral blood lymphocytes (PBL) the loss of IL-2 receptors is reversible not only by a renewed exposure of cultured lymphocytes to Phytoheamagglutin (PHA) but also by addition of monoclonal antibodies directed against the T3 receptor after incubation of the cells for 18 h. In contrast long term cultured cytotoxic T cell clones, which always express IL-2 receptors and loose these receptors when rested for a couple of days, but do not reexpress IL-2 receptors upon restimulation with PHA. Although IL-2 expression on cloned T cells is almost completely abolished after four days of «resting», the cytotoxic potential against the original stimulation cell is completely retained. Reexposure of these cells to optimal culture conditions that include PHA, feeder cells as well as IL-2 shows a 100 fold reduction in plating efficiency for the «rested» cells as compared to «non-rested» cells. Because cloned cells that maintained their proliferative capacity did express normal levels of IL-2 receptors, these finding suggest that the loss of IL-2 receptors in most of these long term cultured cloned effector cells is irreversible. The loss of IL-2 receptors is accompanied by loss of proliferative capacity as expected but not by a loss of cytotoxic potential. Expression of IL-2 receptors can be reinduced by lectins and anti-T3 in short term cultured lymphocytes but not in long term cultured cytotoxic T-cell clones.

1. CANTRELL, D. A., and K. A. SMITH, 1983 J.E.M. 158: 1895.

The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, U.S.A.

128. Distinct patterns of endogenous retroviral antigen expression during lymphocyte maturation determined by individual ev loci

D. L. EWERT and M. HALPERN

Inbred strains of chickens that carry endogenous retroviral genes (ev loci) in their germ line that encode the Chf phenotype were found to express retroviral envelope glycoprotein (VEG) on cells of the lymphocyte lineage. The relationship between lymphocyte maturation and VEG expression controlled by three ev loci (ev 3, ev 6, and ev 9) was analyzed. Immature or resting peripheral T or B cells were negative for VEG expression when analyzed by immunofluo­rescence techniques. However, immunochemical analysis of radiolabeled cell proteins revealed VEG in purified populations of both T cells (thymocytes) and B cells (bursacytes), suggesting that the antigens are constitutively expressed in lymphocytes. To determine the effect that activation of resting lymphocytes would have on the expression of VEG, a culture system was developed that would support maturation of both peripheral T and B lymphocytes following pokeweed mitogen (PWM) activation. Both immunoglobulin (Ig)-positive and -negative lymphoblasts present in PWM-stimulated cultures expressed levels of VEG detectable by immunofluorescence analysis. Similarly, Ig-lymphoblasts in concanavalin A-stimulated cul­tures were also positive for VEG. These results indicate that the level of VEG in mitogen­activated lymphoblasts of either the T or B cell lineage exceeds the level in immature and resting cells from strains of chickens carrying any of the three ev loci. To determine the relative level of VEG expression associated with terminally differentiated cells of the lymphocyte lineage, plasma cells obtained from either spleen or gland of Harder were examined. Plasma cells from lines of chickens possessing the ev 6 locus expressed higher levels of VEG detectable by immunofluorescence analysis than did lymphoblasts of the same genotype. By contrast, little or no VEG expression was detected on plasma cells of chickens possessing either the ev 3 or ev 9 retroviral locus in the absence of ev 6. These data suggest that the pattern of endogenous retroviral antigen expression during lymphocyte maturation is a function of the

84 . 16th International Leucocyte Culture Conference, Cambridge

particular ev gene. Furthermore, the observation that the indocability of antibody to VEG determinants, following infection within exogenous virus (RA V -1), is inversely related to the presence of ev 3 genes suggests that the expression of VEG tolerizes the immune system to cross-reactive antigens of an exogenous retrovirus.

lLudwig Institute for Cancer Research and 2Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland

129. Regulation of TCGF-receptor expression in a CTL clone by antigen

J. W. LOWENTHAL!, C. TOUNGE2, H. R. MACDoNALD!, and M. NABHOLZ2

We have isolated and characterized a H-2Kb-specific CTL clone, CRxCD.B.1.8 (B1.8), whose growth is strictly dependent on TCGF and periodic exposure to stimulator cells. A 3 h exposure to specific antigen induces B1.8 cells to become responsive to affinity-pure TCGF and proliferation occurs without the need for further antigenic stimulation. To demonstrate this, we made use for the fact that monoclonal antibodies directed against either the Lyt-2 antigen on the responder cells or against the stimulating H-2Kb antigen, block the acquisition of TCGF-responsiveness by resting B1.8 cells, but only when the antibodies are added less than 3 h after the addition of antigenic stimulators. B 1.8 cells, once activated, will grow exponentially for about 5 days, during which time they undergo 6 or 7 cell doublings. After this time, TCGF-responsiveness declines and cell proliferation stops, until by addition of fresh stimulator cells. Using a radiolabelled-TCGF binding assay, we found that exponentially­growing cells, 3 days after transfer with stimulator cells and TCGF, express an average of 7000 receptors per cell. From this time on, the level of receptor expression declines and is 10-fold lower on day 7. This decrease, which occurs even in saturating concentrations of TCGF, is not due to dilution by cell division, because the total number of receptors per culture increases about IO-fold during the 7 days. Loss of TCGF-receptors preceeds a decline in cell growth as measured by viable cell number, DNA synthesis and rate of progression through the cell cycle. In contrast to the decline in TCGF-receptor expression and arrest of cell growth, there is no concomitant decrease in cytolytic activity per cell.

Institute of Medical Microbiology, Freie Universitat, Berlin, and lImmunology Research Unit, Klinikum Steglitz, Freie Universitat, Berlin, West-Germany

130. Dynamics of IL-2 receptor expression of Listeria monocytogenes specific T helper cell clones

R. STOLPMANN, H. NAHER, H. OSAWA!, T. DIAMANTSTEIN!, and H. HAHN

It is generally thought, that interaction of antigen with the antigen receptor induces IL-2 receptor expression and interaction of IL-2 with the IL-2 receptor results in cell proliferation. T helper cell clones require the presence of IL-2 and antigen for continous growth. As a working hypothesis we postulated (1) that the antigen induced IL-2 receptor expression is a transient event and that the presence of antigen is required for maintenance of IL-2 receptor expression. To test this hypothesis L. monocytogenes-specific W3/25 + rat helper cell clones were cultivated; expansion of cells critically depended on heat-killed Listeriae presented by irradiated syngeneic spleen cells and addition of conditioned medium (CM) containing IL-2.

16th International Leucocyte Culture Conference, Cambridge . 85

At various days after antigenic stimulation expression of IL-2 receptors was determined by an indirect radioactive antibody binding assay using a monoclonal anti-rat IL-2 receptor antibody ART 18 (2). In parallel, cultures were divided and T cells subcultured either with addition of CM and antigen or addition of CM alone. T cells cultured with CM and restimulated with antigen expressed high levels of IL-2 receptors and maintained a high degree of proliferation. Proliferative responses of T cells subcultivated with CM alone without antigen gradually declined reaching background levels by d 10 after antigenic stimulation. IL-2 receptor expres­sion paralleled the decline of the proliferative response. In order to study the influence of antigenic restimulation on proliferation and expression of the IL-2 receptor, cultures of cloned T cells 14 d after antigenic stimulation (proliferation had reached background levels) were split and subcultures initiated with heat-killed Listeriae presented by syngeneic irradiated spleen cells either with or without addition of CM. Determination of 3HTdR uptake various days later revealed a marked increase of proliferation but only in those restimulated cultures where CM was present. IL-2 receptor expression paralleled the degree of proliferation. The data show that the antigen-induced expression of the IL-2 receptor on T cells is a transient event. Repetitive stimulation by antigen is required for maintenance of IL-2 receptors at the cell surface, the prerequisite for IL-2 dependent continous growth of T cells.

Part of this work was supported by DFG grant Di/153/8.

1. DIAMANTSTEIN, T., and H. OSAWA. 1984. Molecular Immunol. in press. 2. OSAWA, H., and T. DIAMANTSTEIN. 1983. J. Immunol. 30: 51.

T cell activation signals (other than activation complex)

Biochemistry department, Universite de Sherbrooke, Sherbrooke, Quebec, Canada

131. Purification and partial characterization of phytohemagglutinin­reactive glycoproteins from porcine lymphocytes

G. DUPUIS, G. LAGACE, M. LETELLIER, B. BASTIN, and J. CARDIN

Phaseolus vulgaris phytohemagglutinin (PHA) is a lectin which is well recognized as a potent activator of lymphocytes in vitro. It is generally accepted that PHA-induced lympho­cyte activation results from a specific recognition of the oligosaccharidic portion of plasma membrane glycoproteins (GP). The nature of these PHA-reactive GP remains a subject of experimental investigation. We have addressed the question of the nature of PHA-reactive GP in porcine splenic lymphocytes. We have shown that NP-40 is efficient to solubilize porcine plasma membrane GP and that the electrophoretic pattern compares to the one obtained from purified porcine plasma membranes. Binding of 125I_PHA to the electrophoretic ally separated plasma membrane constituents revealed the presence of major PHA-reactive GP of molecular weight 50-55, 75, 95, 110, 130 and 155 K and of minor bands. We have devised two complementary approaches to better characterize these PHA-reactive GP. The first approach makes use of affinity chromatography for selective purification of porcine PHA-reactive glycoproteins. Selective elution from a PHA-Affi Gel 10 column gives a heterogeneous GP fraction that shows by SDS-PAGE analysis an identical protein profile to the one observed previously by 125I-PHA binding to SDS-PAGE patterns. We have termed the affinity chromatography fraction «PHA-reactive porcine lymphocyte glycoproteins» (PPGP). We

86 . 16th International Leucocyte Culture Conference, Cambridge

have studied the biological properties of PPGP and shown that the fraction inhibits, in a dose­dependent manner, PHA-induced lymphocyte activation, the binding of labeled PHA to porcine lymphocytes and PHA-induced IL-2 production in a IL-2 dependent murine cell line. Our second approach is an attempt to probe the nature of the PHA receptor(s) by in situ labeling of the lectin receptor(s) with the use of photoreactive heterobifunctional reagents. Two reagents have been synthesized in our laboratory and used to modify PHA. The modified lectin retains its ability to activate lymphocytes. It has been used to tag PHA receptor(s) in porcine splenic lymphocyte plasma membranes. Electrophoretic analyses of the PHA-GP complexes show a good correlation, in terms of molecular weights, with the major proteins purified by affinity chromatography. Taken together these results suggest that the PPGP fraction contains lymphocyte plasma membrane GP involved in the biological activity of PHA. The components of the PPGP fraction have been incorporated in artificial vesicles with the aim of using this preparation in reconstitution experiments. Our preliminary results show that approx. 60 % of the proteins are incorporated with the oligosaccharidic portions oriented «right side» out as determined by agglutination experiments and proteolytic digestion. In addition, we are using a photoreactive bifunctional reagent to analyze the topological distribution of the components of the PPGP fraction in the artificial vesicles.

Supported by the Medical Research Council of Canada (Grant MT-6343).

Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel­Aviv 69978, Israel

132. Antibodies to nonpolymorphic determinants of the Thy-t molecule inhibit T cell proliferation

N. HOLLANDER

Thy-1 is an abundant cell surface glycoprotein of murine thymus derived lymphocytes. Although the Thy-1 molecule has been extensively studied with regard to its biochemistry, genetics, and selective expression on distinct cells, its functional role is not known. One approach to identification of «functiona]" T cell surface molecules is to determine effects of antibodies on T cell activity in the absence of complement. Using such an approach, we demonstrated that monoclonal rat antibodies against nonpolymorphic determinants of the murine Thy-1 antigen inhibited cell proliferation induced by alloantigens or by concanavalin A. In contrast, antibodies against allotypic determinants of Thy -1 had no effect on T cell activation. Inhibition of T cell proliferation was not dependent on the isotype of the blocking antibody, since both IgG and IgM anti Thy-1 antibodies were inhibitory. In addition, the blocking activity could not be attributed to the xenogeneic (rat) origin of antibodies to monomorphic Thy-1 determinants, since rat anti Thy-I.2 antibodies as well as mouse anti Thy-I.2 and mouse anti Thy-I.1 antibodies were not inhibitory, whereas rat anti monomor­phic Thy-1 determinants suppressed murine T cell proliferation. Thus, the efficacy of anti Thy-l antibodies as T cell inhibitors was determined by the antibody specificity. The suppressive mechanism of anti Thy-1 antibodies was effective throughout the entire course of allogeneic mixed lymphocyte reactions. Addition of antibodies at any time point during the first 90 hours of a 120 hour mixed lymphocyte culture resulted in significant suppression of the proliferative response. The inhibitory effect on antigen-induced responses did not result from a nonspecific mitogenic effect of the antibodies on T lymphocytes since no effects were observed when antibodies were added to responder cells alone. These results suggest that the Thy-l molecule, or a molecule which is located on the cell membrane in close proximity to the Thy-1 antigen, is involved in the activation of T cells.

16th International Leucocyte Culture Conference, Cambridge· 87

Dept. Medicin I, Erasmus University Rotterdam, P.O. Box 1738, The Netherlands

133. New markers on T cell subpopulations in the rat defined by monoclonal antibodies

P. JOLING, F. J. TIELEN, L. M. B. VAESSEN, and J. ROZING

In view of our interest in the role of various subpopulations of T cells in relation to allogeneic renal transplantation in a rat model, we have started to produce mouse monoclonal antibodies directed against cell types at various stages along the T cell lineage in rats. Various purified cell populations, for instance thymocytes and lymphoid tumour cells, were used for immunization. Approximately 50 clones producing monoclonal antibodies directed against surface determinants specifically carried by rat T cells and not by rat B cells have been selected so far. The cellular distribution of these markers in various lymphoid organs has been determined using FACS flow-cytometry as well as an immunoperoxidase technique on cryostat sections. From these data a differential recognition pattern mainly within the T cell compartment was observed. In spleen and lymph nodes the reactivity ranged from all T cells to a very small subpopulation of T cells. Several of these monoclonal antibodies are most likely candidates for use in further distinction of the T cell population in smaller subsets. Data obtained by two-colour fluorescence analysis of these antibodies in combination with existing markers for T suppressor and T helper subpopulations in the rat indicated a further specifica­tion within these two major functional categories. Data of immunoprecipitation studies confirmed that some antibodies are directed against existing T cell markers, while several other antibodies recognise new markers on cells in the early as well as the late phase of T cell differentiation. Also these antibodies have been tested on large granular lymphocytes (LGL), which are supposed to playa major role in the natural killer (NK) activity in rats, in normal and nude rat strains, resulting in a rather discriminating pattern on such cells. Experiments with the renal transplantation model in which several of these antibodies are used both for monitoring using specific histology and flow-cytometry as well as treatment of rejection episodes are in progress. Furthermore, we are now also using this panel for the characteriza­tion of the phenotypes of both cytotoxic T cells and their precursors against class I as well as class II molecules in the rat.

Centre de Biophysique Moleculaire, CNRS, 45045 Orleans, France

134. Specific membrane lectin of human T suppressor cells

CLAUDINE KIEDA and M. MONSIGNY

Previous studies had shown that lymphocytes possess membrane lectins (1). The presence of such specific sugar receptors could be evidenced by fluorescent neoglycoproteins (2). Neogly­coproteins are synthetic glycosylated derivatives of bovine serum albumin which differ in their saccharidic residues. Certain of these neoglycoproteins specifically bind to human peripheral blood leucocytes endogenous lectins. Among circulating T cells a subpopulation (30 % of the whole T cell population) was specifically labelled by fluorescein - substituted rhamnosylated bovine serum albumin, in agreement with previous results obtained by using a pokeweed polysaccharide (3). This «Rha( +) T cells» population can be separated from other T cells' «Rha( -) T cells'» using a three steps procedure: a) incubation of cells in the presence of

88 . 16th International Leucocyte Culture Conference, Cambridge

rhamnosylated bovine serum albumin (RhaBSA), b) immunoadsorption of RhaBSA-coated cells onto anti-BSA antibody coated Petri dishes, c) specific elution by BSA containing buffer. T 8 monoclonal antibodies which bind specific cell surface antigens of suppressor T cells labelled 95-99 % of Rha( +) T cells, while T 4 monoclonal antibodies specific for Helper! Inducer cells labeled 1-2 % Rha( +) T cells. In contrast, Rha( -) T cells were not labeled at all by T 8 monoclonal antibodies. The Rha( +) T cells population was found to act as functional suppressor cells: Rha( +) T cells were kept in culture medium for 48 h; culture supernatants were added to pokeweed mitogen-stimulated B cells. While Rha( + ) T cells supernatants did not inhibit B cell mitosis, it drastically inhibited protein synthesis and suppressed immunog­lobulin production as shown by radioimmunoassay and plaque forming cells assay.

1. KIEDA, C. M., D.]. BOWLES, A. RAVID, and N. SHARON 1978. FEBS Letters 94: 391-395. 2. KIEDA, C. M., A. C. ROCHE, F. DELMOTIE, and M. MONSIGNY. 1979. FEBS Letters 99:

329-332. 3. KIEDA, c., M. MONSIGNY, and M. ]. WAXDAL. 1982. Endogenous lectins on human

peripheral mononuclear leukocytes, in Lectins: Biology, Biochemistry, Clinical Biochemis­try. T. C. BOG-HANSEN and G. A. SPENGLER, eds. W. de Gruyter, Berlin, 3: 427-433.

Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland

135. Biosynthesis of mouse Thy-t antigen

B. LUESCHER and C. BRON

Thy-l antigen is a glycoprotein found on the surface of different cell types in a number of mammalian species. In the mouse, it is a specific marker for T lymphocytes. Recent studies have indicated that anti-Thy-l antibodies can activate functional subsets of T lymphocytes to

secrete lymphokine thus suggesting that Thy-l may be a T cell function-associated molecule. Here, we investigated the biosynthesis and the maturation of this protein in thymocytes and in a cell-free translation system using reticulocyte lysate and polyadenylated RNA from a cloned cytotoxic T cell line. The protein was detected intracellularly in pulse labeled cells with a xenogeneic rabbit anti-mouse Thy-l antibody. The earliest form of Thy-l detected after 10 min. pulse with 35S-methionine and 35S-cysteine had an apparent molecular weight of 26,500. During chase, this band converted to a polypeptide of 25,000 apparent molecular weight probably derived from the latter by trimming of the glucose residues of the «high mannose» oligosaccharide units. Mature Thy-l molecules were detected at the cell surface after 15 min. chase. At least one of the three Asn-linked oligosaccharide units was shown to be in the «high mannose» form as indicated by its susceptibility to endo-~-N-acetyl­glucosaminidase H (endo-H) digestion. Treatment of the «high mannose» forms or of the mature glycoprotein with endo-~-N-acetylglucosaminidase F (endo-F) generated a single precursor polypeptide of 13,500 molecular weight. The same result was obtained when cells were pulsed in the presence of tunicamycin. Finally, recent in vitro translation experiments suggested that the primary product has a molecular weight of 15,500 corresponding probably to the precursor of Thy-l molecule with its leader sequence. So far, we could not demonstrate the presence of any Thy-l molecule containing the 30 additional amino acids predicted by the sequence of the genomic clone recently isolated 0. SILVER; communicated at the 21st Harden Conf. on Structure and Biology of Lymphocyte membranes, Sept. 1983).

16th International Leucocyte Culture Conference, Cambridge· 89

Ludwig Institute for Cancer Research, Lausanne Branch, Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland

136. Production and characterization of monoclonal anti-Thy-t antibodies which stimulate lymphokine production by cytolytic T cell clones

H. R. MACDONALD, C. BRON, and J.-c. CEROTIINI

Recent studies have indicated that cloned T lymphocytes (in man) or T-T hybridomas (in the mouse) can be activated to secrete lymphokines in the presence of antibodies directed against either a putative antigen receptor structure or a nonpolymorphic molecular complex (T3) which is assumed to be associated with the receptor. In an effort to derive a panel of monoclonal antibodies with such lymphocyte-activating activities, we have immunized rats with a cloned murine cytolytic T lymphocyte (CTL) line (clone 11), and assayed the sera of individual rats for their ability to stimulate the release of macrophage activating-factor (MAF) by the immunizing clone. Spleen cells from rats with postive activity were then fused with the mouse myeloma AG-8653 and supernatants from growing hybrids were assayed for their ability to stimulate MAF production by the CTL clone. From one such fusion, 4 of 420 hybrids were initially identified as positive, of which 3 were subcloned and retained for further study. These three hybrid supernatants (designated 1-22, 3-5 and 5-8) all stimulated MAF production in a dose-dependent fashion; however, none of the supernatants significantly inhibited cytolytic activity. Flow microfluorometric analysis revealed that the antigen recog­nized by the three monoclonal antibodies was expressed not only on the immunizing clone but also on other CTL clones as well as on thymocytes and peripheral T lymphocytes. This distribution, as well as the pattern of staining in the thymus, prompted a detailed analysis of the relationship of this antigen to Thy-l. Competition binding studies on thymocytes or on clone 11 using 3H-leucine-labeled antibodies revealed that 1-22, 3-5, and 5-8 antibodies competed for binding with each other and with a rat monoclonal anti-Thyl.2 antibody (A T1 5, provided by Dr F. Fitch). Further analysis using con genic Thy 1.1 and Thy 1.2 mice revealed that 2 of the antibodies (1-22 and 3-5) reacted with thymocytes from both strains, whereas 5-8 reacted only with Thy 1.2 cells. Preliminary immunoprecipitation analysis of lysates from surface-iodinated clone 11 cells indicated that each of the antibodies precipitated a 25-28KD structure which could not be distinguished from Thy-I, in addition to some higher molecular weight material. Taken together, these data demonstrate that some monoclonal rat antibodies which react with polymorphic or non-polymorphic determinants on the Thy-l molecule can trigger lymphokine production by cloned CTL. In view of the functional analogy between these results and those obtained for the T3 antigen in man, it will be of considerable interest to characterize other surface proteins associated with Thy-l which may be detected by these reagents.

Immunology Branch, Centers for Disease Control, Atlanta, GA 30333, U.S.A.

137. Production of Interleukin 2 (112) by LeuS+ and LeuS- subsets of the T4 helper cell population in peripheral blood lymphocytes (PBL) isolated from normal individuals

A. C. MAWLE, J. K. A. NICHOLSON, G. D. CROSS, and J. S. McDOUGAL

The major T cell subset responsible for IL2 production is the helper population defined by the T4 monoclonal antibody (1-3). This population can be subdivided by a second monoclonal

90 . 16th International Leucocyte Culture Conference, Cambridge

antibody detecting the T cell antigen Leu8. Leu8- cells comprise about 20 % of the T 4 + population and are primarily involved in providing helper function for B cell stimulation and differentiation into immunoglobulin-producing cells (4). We have studied the capacity of T4+Leu8+ and T4+Leu8- populations to produce IL2 in response to the mitogens con­canavalin A (Con A) and phytohemagglutinin (PHA). IL2 was assayed on a continuously growing, IL2-dependent mouse T cell line (5). Our results show that the T4+Leu8- popula­tion is the primary IL2 producer on a per cell basis, regardless of whether Con A or PHA is used as an inducer. No differences in mitogen stimulation as measured by tritiated thymidine incorporation were found between the two populations.

1. MEUER, S. c., R. E. HUSSEY, A. C. PENTA, K. A. FITZGERALD, B. M. STADLER, S. F. SCHOSSMAN, and E. L. REINHERZ. 1982. Cellular origin of interleukin 2 (IL2) in man: evidence for stimulus-restricted IL2 production by T4+ and T8+ T lymphocytes. J. Immunol. 129: 1076.

2. SOLBACH, W., S. BARTH, M. ROLLINGHOFF, and H. WAGNER. 1982. Interactions of human T cell subsets during the induction of cytotoxic lymphocytes: the role of interleukins. Clin. Exp. Immunol. 49: 167.

3. FISHBEIN, E., J. ALCOCER-VARELA, and D. ALARCON-SEGOVIA. 1983. Cellular bases of the production and response to interleukin-2 in man: role of autologous rosette-forming T-cell subsets defined with monoclonal antibodies. Immunol. 50: 223.

4. GATENBY, P. A., G. S. KANSAS, C. Y. XIAN, R. L. EVANS, and E. G. ENGELMAN. 1982. Dissection of immunoregulatory subpopulations of T lymphocytes within the helper and suppressor sublineages in man. J. Immunol. 129: 1997.

5. GILLIS, S., M. M. FERM, W. Ou, and K. A. SMITH. 1978. T cell growth factor: parameters of production and quantitative microassay activity. J. Immunol. 120: 2027.

Dept. of Dermatology, Karolinska sjukhuset, Stockholm, Sweden

138. Mercuric chloride-A polyclonal activator with direct action on thymocytes and peripheral T lymphocytes

K. NORDLIND

Mercury is a lymphocyte mitogen and a strong allergic sensitizer (1). In this study, human thymocytes and peripheral blood lymphocytes were separated on the basis of buoyant density on Percoll discontinuous gradients and the different cell populations tested as regards mitogenic response to mercuric chloride. Thymocytes with a density of 1.065-1.067 glml and peripheral blood lymphocytes with a density of 1.063-1.065 glml were enriched in cells responsive to mercuric chloride. These populations were also reactive to mitogenic lectins, which is in agreement with results of others for thymocytes (2) and peripheral blood lymphocytes (3). Thus, mercuric chloride is a polyclonal activator of human thymocytes (virgin T cells) and peripheral blood T lymphocytes. When macrophageslmonocytes were depleted by incubation on a plastic surface followed by removal of iron phagocytic nonadhe­rent cells with a magnet, the stimulation remained at about the same level in the responding thymocyte and peripheral blood lymphocyte population. A direct binding of labelled mercury to the cell membrane of both thymocytes and peripheral blood lymphocytes was demonstrated by autoradiography. This labelling was not found if the cells were washed 30 min after addition of the isotope. However, after incubation for three days it was not possible to remove labelled mercury from the cell surface. Taken together, these findings indicate that mercury

16th International Leucocyte Culture Conference, Cambridge . 91

might act dire~t!y on the responsive lymphocytes and not via interaction with antigen presenting cells.

1. NORDLIND, K., and A. HENZE. 1984. Int. Archs Allergy appl. Immun. 73: 162. 2. GOUST, J. M., and L. R. PERRY jr. 1981. Thymus 3: 25. 3. ULMER, A. J., and H. D. FLAD. 1979. J. immunol. Methods 30: 1.

Biophysics Department, Weizmann Institute of Science, Rehovot, and lLife Sciences Depart­ment, Bar-Ilan University, Ramat Gan, Israel

139. Aldehyde groups formed in oxidative mitogenesis are essential for Interleukin-2 synthesis but not for expression of Interleukin-2 receptors

E. ROFFMAN, B. SREDNIl, and M. WILCHEK

Neuraminidase plus galactose oxidase (NAGO) treatment does not cause stimulation of human thymocytes. However, stimulation can be achieved upon addition of exogenously prepared Interleukin-2 (IL-2). The stimulation induced by IL-2 was inhibited by anti-Tac antibody, a putative antibody against the receptor of IL-2, indicating that NAGO-oxidized cells can serve as inducers of functional IL-2 receptors on IL-2-responding T cells. The induction of IL-2 receptors by the oxidized cells was not inhibited by immediate reduction with borohydride, since the cells could still be stimulated with IL-2. The same results were also observed when oxidized cells were treated with dinitrophenylhydrazide prior to reduction. The presence of IL-2 receptors was also confirmed by flow cytometry using anti-Tac antibodies followed by fluoresceinated goat anti-mouse IgG antibodies. Peripheral blood lymphocytes can be stimulated by NAGO-treatment, and the NAGO-conditioned medium can support the growth of an IL-2-dependent line. This stimulation can be inhibited with borohydride and restored with IL-2. The conditioned medium derived from the borohydride­reduced cells cannot support the growth of the IL-2-dependent line, indicating that borohy­dride inhibited the oxidation-induced IL-2 production. The results suggest that NAGO­oxidized sites can be modified chemically, and the modified cells do not lose their potential to express IL-2 receptors. Preliminary observations indicate that the cells which undergo successive treatments with NAGO and borohydride accumulate at the G2 phase of the cell cycle, whereas cells treated with NAGO alone complete the mitotic cycle.

Karolinska Institute and Danderyds Hospital, Stockholm, Sweden

140. Simple electronic counting of rosettes formed by human peripheral blood lymphocytes

G. SANDBERG and M. BJORKHOLM

A simple procedure earlier used for the counting of guinea pig thymocyte rosettes with a standard electronic laboratory cell counter (Cellcounter 134) was here adapted for use with human rosettes. The numer of rosettes (i.e. particles above a preselected size) was counted in ordinary preparations used in clinical work and, after lysis of the erythrocytes, the concentra-

92 . 16th International Leucocyte Culture Conference, Cambridge

tion of white cells was determined in the same samples. Since there is some overlap in the electronic detection of the smallest rosettes and the largest lymphocytes, the calculation of rosette frequency requires consideration of a «background», which is measured in parallel lymphocyte samples to which no sheep erythrocytes had been added. We have also introduced a correction for some «double rosettes» which may occur, by counting the largest particles in the samples. These double rosettes were «mathematically disintegrated» into single rosettes. The fixed settings of discriminator and sensitivity levels were slightly different than when counting thymocyte rosettes from guinea pigs. The formula which we have developed is

500(r+d-b) R = 100 w- 5 b

where R is the percentage of rosettes, r is the displayed number of rosettes, d the displayed number of double rosettes, b the displayed background and w the displayed number of lymphocytes. Starting from this formula we also developed a graphical method for determina­tion of rosette frequency. The counting of rosettes from healthy individuals, patients with chronic lymphatic leukemia and lymphoma resulted in excellent agreement with conventional rosette counting in a microscope.

!Institute of Biochemical Sciences, II Medical School, Naples, Italy; zMemorial Sloan­Kettering Cancer Center, New York, N.Y., U.S.A.; JDepartment of Microbiology and Immunology, New York Medical College, Valhalla, N.Y., U.S.A.

141. Anti-HLA class I monoclonal antibodies inhibit T cell proliferation

M. C. TURCO!, L. CORBO!, G. MORRONE!, K. WELTEZ, C. Y. WANd, R. MERTELSMANNz, S. FERRONEJ, and S. VENUTA!

We have studied the role of HLA class I products in early steps of T cell stimulation by testing the effect of anti-HLA-A, B (a-HLA-A, B) and a-~zmicroglobulin (a-~zm) mono­clonal antibodies (MoAbs) on Pan Tr and OKT3-stimulated lymphocytes. Phytohemaggluti­nin (PHA)-stimulated lymphocytes were used as control. The a-HLA-A, B MoAbs were directed to monomorphic determinants of the molecule. In order to avoid effects due to the Fc fragments of the used MoAbs (antibody-dependent cytotoxicity, interactions of Fc fragments with macrophage Fc receptors), F (ab')z fragments of the a-HLA-A, B were also prepared and used. We found that two of three a-HLA-A, B and the a-~2m tested and F(ab')z fragments strongly inhibited PHA-, Pan Tr and OKT 3-induced T cell proliferation. This inhibition was effective at concentrations of purified MoAbs as low as 100 ng/ml. It was not due to physical hindrance of Pan Tz and OKT 3 by the a-HLA, since a-HLA-A, B MoAbs were effective even if added to cells preincubated with OKT 3 or Pan Tz (or PHA). Suppressor cdlls were not induced by the inhibiting MoAbs, since we could not transfer the inhibition with cells or supernatants from an inhibited culture to a stimulated one. We found that the inhibition could not be reversed by addition of exogenous IL-1 or pure IL-2 and that IL-2 production was normal or increased in the inhibited cultures. These data suggested that the a-HLA-A, B action affected the expression or the function of the IL-2 receptor. In order to distinguish between these two alternatives, we studied the effect of a-HLA-A, B on T cells expressing the IL-2 receptor. Peripheral blood lymphocytes (PBL) were stimulated for 72 hours with PHA, then washed and incubated in presence or absence of pure IL-2. We show that a-HLA-A, B is not able to inhibit the IL-2 induced proliferation of activated T cells. We conclude that a­HLA-A, B does not block the activity of a receptor present on T cell membrane and propose that the inhibition is exerted at the level of the IL-2 receptor expression.

16th International Leucocyte Culture Conference, Cambridge· 93

Forschungsinstitut Borstel, D-2061 Borstel, FRG

142. Stimulation of highly purified (hp) T -lymphocytes by phytohemagglutinin (PHA) in presence or absence of monocytes

A. ]. ULMER, W. SCHOLZ, and H.-D. FLAD

To answer the question whether PHA can stimulate human peripheral blood T lymphocytes in absence of monocytes we developed an ultra micro culture in glass capillaries at a volume of 1 III or 2 III containing 1000 cells/culture. Hp T cells were isolated from human peripheral blood mononuclear cells (MNC) by subsequent removing of carbonyl-iron phagocytic cells, removing of low density cells by density gradient centrifugation, and isolation of E-rosette forming cells (E-RFC). These hp T lymphocytes were found to contain 0.1 % or less of monocytes as determined cytochemically by alpha-naphtyl acetate-esterase (ANAE) activity. When we stimulated hp T cells by PHA in the ultra micro culture the following results were obtained: 1. Hp T cells can be stimulated by PHA in absence of any ANAE positive monocyte. 2. We did not find ANAE-positive monocytes after culture of hp T cells in presence or

absence of mitogens (PHA, LPS). 3. We never found ANAE positive monocytes within the clusters formed after stimulation of

hp T lymphocytes by PHA. 4. A monoclonal antibody against Ia did not inhibit the response of the cells to PHA. s. A rabbit anti-serum against human leukocytic pyrogen (containing also anti IL-1 activity)

only reduced but did not abrogate the stimulation of the cells by PHA. 6. Depletion of HLA-DR positve cells from hp T cells or isolation of OKT 11 + /HLA-DR­

cells by fluorescence gated cell sorting only reduced but did not abrogate the response of the T lymphocytes to PHA.

7. Addition of adherent cells (> 9S % monocytes) resulted in an enhancement or in an inhibition of the response of hp T lymphocytes to PHA, depending on cell concentration and culture time. From these results we conclude that a distinct T cell population from human peripheral

blood can be stimulated by PHA in absence of ANAE positive monocytes. Nevertheless monocytes may have positive but also negative regulatory activity on T lymphocyte response to PHA. However, our results raise the question whether there is a distinct T cell population which may be stimulated by PHA without the absolute requirement for accessory cells.

Supported by DFG, FL 104/4-1.

Transplantation Laboratory and Fourth Department of Medicine, University of Helsinki, Helsinki, Finland

143. Effects of a human urinary mitogen (UM) on subpopulations of peripheral blood mononuclear leucocytes (PBML)

A. WANGEL, K. KAYHKO, and S. REITAMO

We have earlier isolated, to apparent homogeneity, a 27 000 d human basic protein (UM) from the urine of a patient with myelomonocytic leukaemia. UM is a mitogen for resting human peripheral blood lymphocytes (both OKT 4+ and OKT 8+) at lO-IlM concentration. We have now further defined the effect of UM on human PBML and their subpopulations in

94 . 16th International Leucocyte Culture Conference, Cambridge

six-day cultures. Cell proliferation was measured by lH thymidine uptake and Ig production by the plaque-forming cell (PFC) response using a staphylococcal protein A reverse haemoly­tic plaque assay. Whole PBML responded to UM with proliferation and a 40-fold increase in PFC. The increase in PFC was equal to that produced by pokeweed mitogen at a final concentration of 1 :800 and occurred in the three major Ig classes. To test the effect of UM on subpopulations of PBML, adherent cells (AC) were isolated by plastic adherence and T and B enriched populations by rosetting with sheep red blood cells. The proliferative response of T cells needed the presence af AC whilst the effect on Ig production by B cells required both T cell help and the presence of AC. The results show that, in addition to being a T lymphocyte mitogen, UM is also a T cell dependent polyclonal B cell activator.

University of Stockholm, Department of Immunology, S-106 91 Stockholm, Sweden

144. Helper function of human T cells with different affinity for Helix pomatia A hemagglutinin (HP) in a tetanus toxoid induced B cell differentiation system

MARGARETA WlKEN, ULLA HELLSTROM, and P. PERLMANN

T cells from human peripheral blood were enriched in 14+ cells by lysis of T8+ cells with the monoclonal antibody OKT 8+ complement. The T4 + subset was separated into 4 fractions differing in avidity for the lectin Helix pomaria A hemagglutinin (HP). The fractions were studied for their capacity to help autologous B cells to differentiate and mature into immuno­globulin synthesis and secretion after activation with tetanus toxoid (IT) in vitro. The T4+ cells with low avidity for HP were poor mediators of help for B cell differentiation. Removal of these cells enhanced the helper function of the remaining T 4 + cells, indicating that suppressor cells were contained within these low avidity T4 + cells. In contrast, efficient B cell help was provided by the two subsets of 14+ cells with high avidity for HP. However they differed in the quality of help exerted. Thus, while one subset mainly induced IgG secretion that mainly comprised IT-specific antibody, the second subset mainly induced a polyclonal IgM production. These findings indicate, that T 4 + cells vary in their stage of differentiation as seen by differences in expression of the HP-marker and more, differences in HP-marker expression appear to be associated with differences in cellular functions.