Immunobiol., vol. 178, pp. 1-166 (1988)

XX. Tagung der Gesellschaft fur Immunologie Society of Immunology· L' Association d'Immunologie October 6-8, 1988. Dusseldorf, Federal Republic of Germany

Abstracts

Workshop A T Cell Repertoire, T Cell Activation

lnstitut fur lmmunologie und Genetik, Deutsches Krebsforschungszentrum, 6900 Heidelberg, lm Neuenheimer Feld 280, FRG

A.1 A carbohydrate epitope shared by mouse CD2 and FcR proteins: involvement in CD2-LFA3 interaction

P. ALTEYOGT, G. HECKL-OESTREICHER, M. MICHAELIS, P. YON HOEGEN, and V. SCHIRR· MACHER

We studied a monoclonal antibody (mab 12-15) that reacted to murine Fc receptor proteins. On ESb-MP tumor cells, the antibody bound to beta 1, beta2 and alpha proteins and undefined molecule of 37 kd (beta3). The beta3 chain is also expressed on mouse thymocytes and was identified as the mouse CD2 antigen. Sequential immunoprecipitation experiments indicated that in the thymus not all CD2 molecules carry the 12-15 epitope.

The antigenic determinant recognized by mab 12-15 seemed to be composed of carbohy­drate. Treatment of ESb-MP cells with sodium m-periodate abrogated binding of the anti­body. Sialic acid was essential for epitope recognition, since neuraminidase treatment greatly decreased binding. Endoglycosidase F treatment had no effect, however, glycopeptidase F and alkaline borohydrate treatment were effective. The antibody did not react with several macrophage-like cell lines and not with melanoma cells transfected with a cloned Fc receptor beta2 gene, suggesting that the expression of the epitope was cell type dependent.

CTL inhibition experiments using anti tumor specific cytotoxic T lymphocytes (CTL) and tumor target cells indicated that mab 12-15 could specifically inhibit CTL-mediated lysis at the effector cell level, presumably by interfering with CD2-LFA-3 interaction. The possible role of carbohydrates in the interaction of adhesion molecules with immunoglobulin-like domains is discussed.

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Division of Biology, California Institute of Technology, Pasadena, CA 91125, U.S.A.

A.2 Diversity of T cell receptor 6 chain expressed at different stages of T cell differentiation

B. ARDEN, D. ZALLER, K. WANG, and L. HOOD

A repertoire study of the T cell receptor (TCR) a-chain variable (V) region genes expressed in thymocytes from adult C57BI/Ka mice revealed that the Va genes could be divided into 10 different subfamilies (1). V gene segments from each subfamily were subcloned and used as probes to screen the cDNA library from adult C57Bl/Ka thymocytes. The organization of the immunoglobulin heavy chain locus in multiple isotypes led us to test the possibility that the a-chain variable region gene pool might be utilized by novel constant (C) region genes other than Ca. Therefore, only those Va-hybridizing clones that did not hybridize to a Ca probe were isolated. Meanwhile, a frequent rearrangement within the a locus was cloned and identified as the TCR b chain (2). We used an oligonucleotide probe specific for Cb to screen our Va+' Ca - cDNA clones. Two out of seven clones contained Cb combined with V gene segments from the Va7 and Va10 subfamilies. The remaining clones were transcripts of germline Va or of Va to Ja rearrangements. With a Cb-specific cDNA probe another member of the Va7 subfamily and two Vb'S representing new single and two member subfamilies, respectively, were found. These new Vb-gene segments are linked to the Cb locus as well as to a Va-gene segment, as we showed by pulse field gel electrophoresis. cDNA libraries have been constructed from mRNA of day 16 and day 18 fetal thymocytes to investigate Vb-gene segment usage in fetal development in comparison to the adult Vb repertoire.

1. ARDEN, B., J. L. KLOTZ, G. SIU and L. HOOD, 1985. Nature 316: 783. 2. CHIEN, Y., M. IWASHIMA, K. B. KAPLAN,]. F. ELLIOTT and M. M. DAVIS, 1987. Nature327:

677.

Department of Tumor Immunology, Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02115, U.S.A., and! Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School

A.3 Crosslinking CD3 with CD2 employing Sepharose-linked antibodies enhances T lymphocyte proliferation: study of lymphokine synthesis by Northern analysis

K. DEUSCH, P.]. ANDERSON!, and S. F. SCHLOSSMAN

T lymphocyte proliferation can be induced through either triggering the CD3 : Ti-complex, the target of antigen specific activation, or CD2, the antigen-independent activation pathway. In this work, we employed Sepharose beads coupled with a submitogenic dose of CD3 (0.5 ng/ bead) and rabbit-anti-mouse antibodies in the study of proliferative responses of T lympho­cytes that were preincubated with antibodies directed towards several functionally relevant T cell surface molecules. Thus, crosslinking of CD3 with the latter gave the following stimula­tion indices: CD2 (60-fold), CD4 (4-fold), CDS, 2H4 and 4B4 (2-3-fold), class I and class II major histocompatibility antigens (less than twofold). The observed enhancement of prolifera­tion by CD3/ -accessory molecule-crosslinking was abolished in the presence of soluble antibody against the respective accessory structure. Interestingly, when comparing the effects of crosslin king of the above-listed surface structures on T4+2H4- and T4+2H4+ T lympho-

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cyte subpopulations, we found that the former was preferentially stimulated by the CD3 : CD4 combination. Finally, we will present data that reveal the pattern of lymphokine synthesis in T lymphocytes triggered through crosslinking of CD3 with the accessory structures cited, as obtained by Northern analysis. In conclusion, our results suggest that specific interactions between T cell surface molecules may play a role in the regulation of lymphocyte activation.

Abt. Angewandte Immunologie, Inst. fur Radiologie und Pathophysiologie, Deutsches Krebs­forschungszentrum, Heidelberg, FRG

A.4 T lymphocyte/monocyte interaction in the generation of antigen­specific primary immune responses in man

B. ENDLER-JOBST and S. C. MEUER

One function of monocytes (MO) in antigen (A g) specific immune responses relates to processing and presentation of Ag in association with MHC class II molecules. A second contribution by MO, independent of Ag presentation, is related to providing help for the induction of interleukin (IL) 2 synthesis and secretion by T cells, a process which is not understood at present. The availability of monoclonal antibodies (mAbs) which directly trigger human T cells via their Ti-T3 Ag receptor complex allows to circumvent the MO requirement for Ag presentation, but not for induction of IL 2 synthesis. Therefore, such mAbs can be used to investigate selectively MO function in IL 2 production by T cells. Experiments employing vigorously purified small resting human T lymphocytes, MO and Sepharose-linked anti CD3 mAbs (S-T3) yielded the following results: Triggering of T cells via their Ti-T3 Ag receptor complex induces the production of a variety of mediators from MO including IL 1 and tumor necrosis factor (TNF). This T cell/MO interaction requires cell/cell contact and is accompanied by the release of 51Cr from labelled MO. Unlike classical cytotoxic T cell function, induction of mediator release is radiosensitive and does not lead to MO death. Moreover, this T cell activity appears to be specifically directed towards cells of the MO lineage. More important, cell free supernatants, containing no detectible concentrations of IL 2, generated during incubation of small resting human T cells, MO and S-T3 for a culture period of 12 h, can replace MO to support IL 2 production by T cells. Neither IL 1 nor TNF alone is able to produce such an effect. These data suggest that an unknown mediator participates in primary antigen specific immune responses, whose function is involved in the induction of IL 2 synthesis of Ag receptor-triggered T lymphocytes.

Institut fUr Immunologie, Universitat Munster, Domagkstr. 3, D-4400 Munster, FRG

A.5 Selective in vitro activation of tumor specific T suppressor (Ts) lymphocytes::-

H.-D. HAUBECK, I. MINKENBERG, and E. KOLSCH

Despite its immunogenicity, the plasmacytoma ADJ-PC-5 grows progressively in the syngeneic BALB/c host. In an experimental model, simulating very early phases of tumor growth, we were able to show that the apparently first reaction of the immune system towards

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the growing tumor is the activation of tumor specific Ts cells. These Ts cells, which suppress the induction of a cytotoxic T cell response against the tumor, have been characterized in detail. There is evidence that the Ts cell-inducing antigen (Ts-Ag) on ADJ-PC-5 cells is expressed to some extent on normal BALB/c spleen cells and is therefore a «self» antigen rather than a tumor specific neoantigen (1). To characterize Ts-Ag in more detail, we have developed an in vitro system for the selective induction of tumor specific Ts cells, because Ts cells function would be masked in the in vitro Ts assay in the presence of activated cytotoxic T cells (CTL) which are no longer susceptible to suppression. Activation of CTL is prevented by fixation of ADJ-PC-5 stimulator cells with glutaraldehyde. In contrast, specific Ts cells are activated by this approach. They suppress activation of CTL in a primary mixed lymphocyte anti-tumor culture (MLTC) of BALB/c spleen cells against ADJ-PC-5 but not against the syngeneic control tumors ULMC (lymphoma) and METH A (fibrosarcoma). Even in lectin­kill assays, these Ts cells have neither cytolytic nor NK-like activity, thus excluding a veto­effect. These Ts cells could be eliminated by treatment with cytotoxic monoclonal antibodies against Thy-1.2, Lyt-2.2, UT4, I-Ad and I-Ed plus complement. Thus, they seem to belong to the same Ts cell category recently described (2). The in vitro system will allow a more detailed study on the induction of Ts cells and the Ts cell effector mechanisms and will be helpful in isolating and characterizing Ts-Ag.

1. KOKE, 0., H.-D. HAUBECK, and E. KOLSCH. 1986. Eur. J. Immunol. 16: 659. 2. HEUER, J., J. DEGWERT, and E. KOLSCH. 1986. Immunogenetics 24: 316.

". Supported by the Deutsche Forschungsgemeinschaft through SFB 310 and the Minister fur Wissenschaft und Forschung des Landes NRW - Az. IV B 5 - 50000286.

Medizinisch-Naturwissenschaftliches Forschungszentrum, Ob dem Himmelreich 7, D-7400 Tubingen, FRG; lDept. Rheumatology, Hospital of Joint Diseases, New York, NY; 2Genetics Institute, Cambridge, MA, U.S.A.

A.6 Bi-linear differentiation pattern of mono-myeloid or mature T cell phenotypes from an undifferentiated acute leukemia by recombinant growth factors

S. LEIPPOLD, I. LORENZ, S. GOYERTl, S. c. CLARK2, H. ZISCHLER, P. WERNET, and E. M. SCHNEIDER

Bone marrow-derived homogeneous malignant blasts from a patient with acute leukemia expressed the early hematopoietic progenitor marker CD34 as well as HLA class II antigens and CD7, however lacked lineage specific differentiation markers. The karyotype revealed aneuploidy with 45, xx and a characteristically elongated chromosome 2 (chr 2q +). These cells exhibited strong proliferative responses when cultured in the presence of highly purified or recombinant growth factors. Using a T cell-conditioned medium (TCM) or a mixture of highly purified interleukin 2 (IL2) plus recombinant interleukin 3 (rIL3) and recombinant granulo­cyte-macrophage colony stimulating factor (rGM-CSF), the cells lost CD34 expression within 14 days of in vitro culture and gained strong expression of mature T cell antigens (CD3, CD2, CD8) as well as the T cell receptor (TCR) a/~ chains detected by WT31 binding. In cultures with IL 2 or rIL 3, the generation of a homogeneous and mature T cell population was incomplete (IL2) or did not occur at all (IL3). GM-CSF alone, however, induced differentia­tion along the mono-myeloid pathway. Mono-myeloid cells were entirely positive for CD14 but negative for CD2, 3 and 8. These results were confirmed by molecular genetic analyses. TCR a/~ chain message and rearrangements of the TCR ~ and y chain genes were detectable by cells cultured with TCM or the mixture of IL 2, IL 3 and GM -CSF, whereas fresh leukemic

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cells as well as IL 3 or GM-CSF cultured cells lacked TCR a/~ mRNA and maintained the germline configuration of TCR ~ and y chain genes. GM-CSF induced mono-myeloid cells expressed CD14-specific mRNA. These results demonstrate the successful induction of T cell as well as mono-myeloid differentiation from a leukemic homologue of a hypothetical multipotent progenitor by recombinant growth factors alone.

Institute of Microbiology and Immunology, University of Ulm, D-7900 Ulm, FRG

A.7 Interleukin 4 and interleukin 2 synergize during primary activation of murine CD S T lymphocytes: evidence that interleukin 4 acts as cytotoxic differentiation factor

T. MIElliKE, R. SCHMIDBERC;ER, K. HEEG, and H. WAGNER

In order to analyze interleukin 4 (IL 4) and interleukin 2 (IL 2) for synergistic interactions during primary activation of resting murine CD 8 T cells, a model system was used which bypasses antigen-presenting cells by utilizing anti-T3 monoclonal antibodies immobilized on Sepharose beads. In this system, IL 4 and IL 2 were shown to promote growth via different growth pathways. When IL 2 and IL 4 are combined in concentrations which by themselves sustain growth, both proliferative as well as cytolytic responses are amplified. At low concentrations however, IL4 synergizes with IL2. This synergistic interaction endows resistance to the suppressive effect of Cyclosporin A (CsA) in the sense that cytolytic responses become induced efficiently, even though proliferative responses are largely sup­pressed. Accordingly, cell proliferation and maturation in terms of acquisition of cytolytic potential can be dissociated from each other. These results imply a physiologic role of IL 4 as «cytotoxic T cell differentiation facton> and thus extend the spectrum of functions of this pleiotropic Iymphokine. They also imply that transition of cytotoxic T cell precursors (CTL-p) into CTL (maturation) is not necessarily linked with extensive cell proliferation.

Institute of Immunology and Genetics and Institute of Nuclear Medicine, German Cancer Research Center, D-6900 Heidelberg, FRG

A.S Regulation of interleukin 2 production by ornithine decarboxylase and its product putrescine

S. MIHM, A. Risso, F. OBERDORFER, and W. DROGE

Treatment of EL-4lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin 2 (IL2) and the expression of IL2-specific mRNA within 4-8 h. This system is a model for the in~uction of a differentiation function in a homogeneous lymphoid cell population by a well­defined signal. In addition, TP A induces an increase in ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of the intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culrure medium. However, putrescine

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cannot induce the production of T cell growth factor (TCGF) in the absence of TPA and cannot reconstitute the TCGF production in cultures with cyclosporin A, which is a well­known immunosuppressive substance and known to inhibit the ODC activity. Putrescine has rather a counterregulatory (suppressive) effect on the TCGF production, as demonstrated by the fact that the ODC inhibitor a-difluoromethylornithine (DFMO) augments TCGF pro­duction and IL 2-specific mRNA expression. The administration of putrescine or polyamines to DFMO-treated cultures was found to suppress TCGF production and IL 2-specific mRNA expression. However, TCGF production is not induced by DFMO in the absence of TPA. Moreover, DFMO does not augment total protein synthesis eH-leucine incorporation), indicating that the effect of DFMO on the TPA-induced TCGF production is selective.

Children's Hospital, University of Wiirzburg, FRG

A.9 Unusual response pattern to PMA in a patient with common variable immunodeficiency

C. R. POLKE and H. W. KRETH

Common variable immunodeficiency (CVI) is a heterogenous disease that affects homeo­stasis, activation, differentiation, and maturation of the immune system. In the study of immunological defects in our CVI patients, one patient showed an unusual response to phorbol myristate acetate (PMA), an activator of protein kinase C (PKC). The EBV-cellline of this patient was unresponsive to PMA treatment with regard tei modulation of CD 25 (low affinity receptor for IL2) in FACS analysis and immunoglobulin secretion analyzed by ELISA. Peripheral blood mononuclear cells (MNC) and T cells lacked responsiveness to an optimal dose of PMA (10 ng/ml) with respect to CD 25 induction (FACS analysis) and proliferation, alone and in combination with an optimal dose of ionomycin, a calcium ionophore. Surprisingly, the costimulation assay including a suboptimal dose of PMA (0.5 ng/ ml) and ionomycin was normal with respect to CD 25 induction and proliferation. In addition, all mitogen proliferation assays of T cells and MNC were markedly reduced and could not be restored to normal levels by the addition of either IL 2 or indomethacin. The induction of CD 25 by PHA was also impaired. These data show that, in this patient, activation of lymphocytes is impaired in response to an optimal but not to a suboptimal dose of PMA. We are presently investigating whether this abnormal response is due to an altered PKC molecule or whether a different activation pathway is involved.

'Abt. Angewandte Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, FRG, 2 Ciba-Geigy, Basel, Switzerland

A.tO Selective phosphorylation of (A) 22-24 KO protein(s) in both TCR/CD3- and C02-mediated activation of resting human T lymphocytes

Y. SAMSTAG', T. STAEHELIN2, and S. C. MEUER'

Recent work on T cell receptor/CD3 (TcR/CD3)-mediated activation of resting human T cells by stimulating with crosslinked anti-CD3 antibodies led to the discovery of the selective,

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strong phosphorylation of one (or two) 22-24 KD protein(s) distinct from the TcR/CD3-complex (SAMSTAG et aI., in press). The kinetics of its phosphorylation were quite similar to those of IL 2 receptor (IL 2 R) expression, interferon gamma and interleukin 2 (IL 2) produc­tion. We now show that this specific protein phosphorylation is not restricted to T cell activation via the classical T cell receptor pathway, but also a highly characteristic and therefore probably essential phenomenon of T cell activation via the alternative CD2-mediated pathway. Activation of resting purified T cells by either immobilized anti-CD3 or by the three synergistic anti-CD2 monoclonal antibodies (anti-Tt1 b -T112' -Ttl)) in solution, resulting both in a proliferative response, induced the strong phosphorylation of the 22-24 KD component(s) with similar kinetics and to the same extent. The pattern of total protein phosphorylation was also identical. Since both pathways of T cell activation are known to be dependent on the IL2/IL2R autocrine loop, it is possible that the observed activation­associated phosphorylation of the 22-24 KD protein(s) is related to signals via the IL 2 R rather than the antigen receptor or CD2. These possibilities will be addressed by blocking IL 2 binding, employing anti-IL2 R antibodies or by means of suboptimal T cell stimulation where proliferation is dependent on exogenous lymphokines (IL2, IL4).

Klinik fiir Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, FRG

A.ll High and low responsiveness to the stimulatory effect of TCRI CD3 antibodies of mouse IgG2b isotype

H. J. SCHLITT, R. SCHWINZER, and K. WONIGEIT

T cell stimulation by soluble monoclonal antibodies to the T cell receptor/CD3 complex usually requires the presence of monocytes in the culture. For mouse IgG 1 antibodies, it could be demonstrated that the stimulatory effect for PBMNC is determined by a polymorphism of macrophage Fc-receptors which defines responder and nonresponder individuals. It was the aim of our study to characterize interindividual differences in the stimulatory potency of the mouse IgG2b antibodies BMA031 (a/~-TCR) and UCHTt (CD3) and to analyze the relevance of monocytes for proliferation induced by these antibodies.

Results: 1) Of PBMNC from 35 individuals tested only two showed significant proliferative response to 15 ng/ml BMA031 and UCHTt (<<high responders»), whereas all responded to OKT3 (lgG2a, CD3). 2) Analysis of 13 family members of a high responder showed a frequency of high responsiveness of about 50 % with a pattern suggesting dominant inheri­tance. 3) High responsiveness for the IgG2b antibodies could be transferred to low responders by an irradiated monocyte-enriched fraction of high responder PMBNC. 4) 1.5 Itg/ml BMA031, but not UCHTt, could stimulate PBMNC also from other individuals (SI: 2-25) (<<low responders») and its effects were not abolished by monocyte depletion below 0.1 % (SI: 3-10).

Conclusions: High responsiveness for the IgG2b antibodies BMA031 and UCHT1 is very rare in the general population. The response pattern is genetically determined and seems to be controlled by monocytes. Most likely, it reflects a genetic polymorphism of the Fc-receptor. In contrast, the general responsiveness to high concentrations of BMA031 (but not UCHTt) and its relative independence of monocytes seems to indicate different activation requirements and may be related to the specific target structure of BMA031.

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Abt. Angewandte Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, FRG

A.12 A novel cell surface molecule selectively involved in C02-mediated human T cell activation

B. SCHRAVEN, B. HUTMACHER, M. Roux, and S. C. MEUER

Resting T cells can be activated in vitro either through the T cell antigen receptor complex (CD3/Ti) or alternatively by monoclonal antibodies to particular epitopes of the CD2/T11-sheep-erythrocyte receptor (i.e. anti-Tll, plus T113). The recent identification of a natural ligand binding to CD2 (LFA3) and activating resting T cells in concert with submitogenic concentrations of anti-Tll, and anti-T113 has provided strong support for the notion that this «alternative pathway» of T cell activation has a functional significance in vivo. Here, we describe a novel cell surface molecule, defined by a monoclonal antibody T AI211 which appears to be selectively involved in T cell activation via CD2. TA1211 detects a monomer of 150 KD under both reducing and nonreducing conditions on resting and activated human lymphocytes, most hematopoietic cell types, but not on erythrocytes and platelets. When added to purified resting human T cells, anti-TAI211, which is itself not mitogenic, leads to a strong proliferative response in concert with submitogenic concentrations of anti-Tll, plus anti-T11 3. This amplification of alternative pathway activation is different from the effect produced by LFA3 or TllTS (the sheep form of LFA3), or additional anti-CD2 antibodies that mimick the action of LFA3 (anti-T1lTt.) for the following reasons: 1) Anti-TAI211 does not exert its function through binding to CD2. 2) In the case of optimal T cell triggering via CD2, anti-TAI211 produces an additional stimulatory effect. Interestingly, while amplifying CD2 activation, antigen receptor-mediated T cell responses are rather inhibited by anti-TAl 211. Given that immune responses to alloantigen and soluble antigens result from concomitant induction of several individual activation pathways, it is not surprising that anti-TAl211 exerts partial blocking effects in these systems as well. These data support the concept that antigen receptor mediated and alternative pathway of T cell activation represent distinct mechanisms which are controlled through selective signals. Moreover, the fact that T A/211 is not restricted in its expression to T lymphocytes might suggest that analogous activation mechanisms are functional in regulating other hematopoietic cell types.

Departments of 1 Medical Microbiology and Immunology, and' Anatomy and Cell Biology, University of Ulm, 7900 Ulm, FRG

A.13 A single T cell receptor-associated GTP-binding protein triggers granule exocytosis in human cytotoxic T lymphocytes

H. SCHREZENMEIERI, G. AHNERT-HILGER', and B. FLEISCHERl

In order to study the subcellular events occurring after T cell activation, we used cloned human CTL permeabilized with alpha~toxin of Staphylococcus aureus. This method of permeabilization leads to stable transmembi"ane channels that permit the introduction of small molecules into the cell but preserve the cellular structures and macromolecular contents of the

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CTL. We used the exocytosis of CTL-specific serine esterases as a marker of T cell activation. The T cell receptor-activated exocytosis functions in such permeabilized CTL.

Introduction of the G-protein-activating, membrane-impermeable, GTP-analogue, GTPyS into permeabilized CTL triggers exocytosis in the presence of Ca++. For optimal exocytosis, ATP is required. The G-protein inactivating GDP-analogue GDPpS inhibited exocytosis triggered via the T cell receptor/CD3 complex but not that triggered by activating the protein kinase C. If protein kinase C was depleted in CTL, the response to GTPyS was strongly inhibited. After antibody-induced modulation of the T ceH receptor/CD3 complex exocytosis could no longer be triggered by G-protein activation. These experiments demonstrate the presence of a G-protein involved in T cell receptor-mediated CTL triggering. In the sequence of signalling steps, this G-protein is localized after T cell receptor triggering but before the formation of the protein kinase C-activating phosphoinositol breakdown product diacylglyc­erol. The finding that this G-protein is functionally inactivated after CD3-modulation provides a molecular basis for the general unresponsiveness observed after modulation of the T cell receptor.

Institute for Hygiene, Innsbruck, Austria

A.14 Involvement of an 85 kd adherence molecule in the initiation of T cell responses

T. F. SCHULZ, H. NEUMAYER, and M. P. DIERICH

We have recently described the identification and characterization, by means of the monoclonal antibody-7F7, of an 85 kd activation antigen found on activated T and B cells, cultured monocytes, endothelial cells, follicular dendritic cells and fibroblasts stimulated with interferon-gamma (T. F. SCHULZ, 1988, Eur. J. Immunol. 18: 7-11, and 1988, Mol. Immunol. in press). This membrane molecule contains a protein core of 55 kd and N-linked as well as O-linked carbohydrates. It is differently glycosylated, and individual epitopes are variably expressed on cells of different lineage. Because of it structural features, the 7F7 antigen is related to the recently described adherence molecule ICAM-I. 7F7 inhibits the T cell proliferation induced by anti-CD3 antibody, PHA, Con A but not the proliferation of IL2-dependent T cells. The T cell response to allogenic stimulator cells is weakly inhibited. An inhibition of these T cell responses was only seen if the antibody was added within the first 8 h (24 h for Con A) of culture. 7F7 also inhibits the formation of cellular aggregates seen in stimulated cultures. Preincubation of purified monocytes cultured in the presence of serum with 7F7, followed by washes to remove unbound antibody, inhibits the capacity of these monocytes to act as accessory cells for T cells stimulated with anti-CD3 antibody. We conclude that the 85 kd 7F7 antigen, an adherence molecule related to ICAM-l, plays a role in the initiation of T cell responses, probably by contributing to the contact between T cells and accessory cells.

This work was supported by: FWF P-6054.

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Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Pittsburgh, PA, U.S.A.

A.IS A laminin B-2-chain-like crossreactive surface structure (human P48 equivalent) is involved in target cell recognition by human non-MHC-restricted interleukin 2-activated T lymphocytes

R. E. SCHWARZ, T. L. WHITESIDE, and J. C. HISERODT

Whereas human non-MHC-restricted lymphokine-activated killer (LAK) cell activity is predominantly mediated by Leu19+ ICDr LGLlNK cells, it is now well established that Leu19+/CD3+ non-MHC-restricted T cells can also contribute to this cytolytic activity. In order to investigate the molecular mechanisms employed in this cytolytic process, highly purified Leu19+ ICD3+ populations (> 98 % pure) of LAK cells were obtained by cell sorting of human peripheral blood lymphocytes (PBL) after 7 days of culture with human recombi­nant interleukin 2 (rIL 2). Two-color flow cytometry could demonstrate that up to 30 % of the activated Leu19+/CD3+ T cells expressed a surface structure which was immunochemically crossreactive with the B2 subunit of laminin, as defined by affinity purified F(ab/)2 fragments of anti-laminin antibody and sequence homology with a eDNA probe reacting with B2 mRNA. This laminin B2-like structure was also expressed on Leu19+/CD3- LAK and PBLINK cells, but not on resting T cells. The surface structure recognized by anti-laminin F(ab/)2 was a 48 KD protein (p48) on both reduced and non-reduced SDS-PAGE gels. Anti-laminin F(ab/h inhibited cytolytic activity of sorted Leu19+/CD3+ cells in a dose dependent manner. Single cell cytotoxicity assays demonstrated that p48 is involved in target cell recognition (activation of cytotoxicity) and not in primary target cell adhesion. Similar findings were made using Leu19+ I CD3- LAK cells. It appears, therefore, that the same surface recognition structure is used by both non-MHC-restricted LAK populations of either NK or T cell phenotype.

Max-Planck-Arbeitsgruppe fUr Rheumatologie/Immunologie, D-8S20 Erlangen, and 1 EMBL, D-6900 Heidelberg, FRG

A.16 Involvement of the protooncogene jun in T cell activation and proliferation

E. SIEBERT, R. BRAVO!, F. EMMRICH, and R. A. KROCZEK

The cellular homo logs of viral oncogenes, the protooncogenes, act in the control of cell growth and differentiation or mediate intracellular signalling systems. The products of oncogenes can be subdivided according to their cellular location into secreted, surface, cytoplasmic, and nuclear oncoproteins. The proteins coded by the oncogenes myc, myb, ios, and ski are targeted to the cell nucleus, where at least some are thought to act directly as specific transactivators and regulators for RNA and DNA synthesis. Jun, a recently identified protooncogene, also codes for a protein central to RNA and DNA synthesis. The product of jun is closely related to or identical with AP-l, a nuclear oncoprotein mediating the effects of phorbol esters on gene expression. The aim of our study is to characterize the involvement of jun in various stages of T cell activation and proliferation. To this end, we are analyzing the kinetics of jun expression after signals either non-mitogenic or mitogenic for T cells. We will also present results defining the interrelationship of jun transcription to the expression of other genes intimately involved in T cell activation such as myc, ios, myb, IL2, IL2R, and IFN-y.

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Department of Molecular Pharmacology, Medical School Hannover, FRG

A.17 Incorporation of polyunsaturated fatty acids into plasma membrane phospholipids is a required early signal for interleukin-2 production

M. SZAMEL, B. REHERMANN, B. KREBS, M. KRACHT, M. GOLOMBEK, M. SCHMIDT, and K. RESCH

Anti-T-cell receptor and anti-CD3 monoclonal antibodies (BMA 031 and BMA 030, respectively), as well as the combination of the phorbol ester TP A with a calcium ionophore induce interleukin 2 (IL2) production and proliferation in human peripheral blood lympho­cytes. In contrast, the synthetic diacylglycerol (DiC8) and ionomycin induce no IL 2 secretion; however, expression of high affinity receptors for IL2 is detectable. The incorporation of linoleic acid (cis-18 :2) into plasma membrane phospholipids of DiC8+ionomycin cells restores their proliferative capacity and their ability to produce IL2. Neither palmitoic acid (16:0) nor linelaidic acid (trans-18:2) can substitute for cis-18:2. TPA induces a long-term activation of protein kinase C. Both BMA 030 und DiC8 cause a transient activation of protein kinase C. Both BMA 030 and DiC8 cause a transient activation of the enzyme. Cis-18:2 prolongs the time of activation of protein kinase C. BMA 030, BMA 031 and TP A activate the incorporation of polyunsaturated fatty acids into plasma membrane phospholipids. The key enzyme of plasma membrane phospholipid metabolism, lysophosphatid acyltransferase, which specifically incor­porates polyunsaturated fatty acids into plasma membrane phospholipids, is activated by anti­TCR-CD3 antibodies and by TPA within 4 h of exposure. Thus, our data suggest that elevated incorporation of polyunsaturated cis-fatty acids into plasma membrane phospholipids repre­sents a necessary early event for IL 2 production and proliferation by prolongation of activation signals in human lymphocytes.

Genzentrum der Universitat Miinchen, Am Klopferspitz, D-8033 Martinsried, FRG

A.IS The role of the CD2/LFA-3 system in antigen- and mitogen­induced T cell activation

G. TIEFENTHALER and T. HONIG

A new monoclonal antibody (mAb) to the LFA-3 molecule, termed G26, was isolated. The mAb appears to recognize the same epitope as the original TS2/9 mAb described by KRENSKY et al. With the help of mAb G26, the function of the CD2/LFA-3 systems was investigated in further detail in various T cell activation systems. MAb G26 inhibits the activation of peripheral blood T lymphocytes following stimulation with the lectins Con A or PHA, neuraminidase­glucose oxidase treatment, with mAbs to CD3 or Ti, and with allogeneic cells. This inhibition (about 50 %) matches the value that has been reported for the anti-LFA-3 mAb TS2/9. Only the PHA-driven T cell activation could be reconstituted with either purified TIlTS (the sheep form of LFA-3 that binds to human CD2) or with SRBC presenting TllTS in a membrane bound form. This shows that an accessory cell function sufficient to activate T cells with PHA is the provision ofLFA-3. Next, anti CD3 mAbs were coupled to SRBC to present ligands both to the T cell receptor complex (anti-CD3 mAb) and to the CD2 molecule (TllTS) on the same cell surface. These CD3 mAb coupled SRBC were able to stimulate T cells. This stimulation could be blocked by either mAbs to TIl TS or by soluble anti-CD3 mAbs. The mAb coupled SRBC could, however, form rosettes with human T cells in the presence of mAb L180/1 to sheep

12 . XXth Meeting of the Society of Immunology

Ttl TS via the anti-CD3 mAbs. This shows that crosslinking of the T cell receptor complex by anti-CD3 mAbs coupled to the SRBC surface was, by itself, insufficient for T cell activation but was rendered stimulatory by the additional interaction of CD2 with its natural ligand, TIl TS. Accordingly, the role of the CD2/LF A -3 system exceeds that of a pure adhesion molecule and is active in T cell activation via the T cell receptor complex.

Institut fiir Immunologie und Genetik, Deutsches Krebsforschungszentrum, D-6900 Heidel­berg, 1m Neuenheimer Feld 280, FRG

A.19 Functional and phenotypical analyses of in situ activated anti­tumor immune cells using a sponge matrix tumor model

U. ZANGEMEISTER, K. THIEDE, B. KYEWSKI, and V. SCHIRRMACHER

The analysis of the cellular microenvironment at sites of tumor rejection or tumor vaccine application has been hampered by methodological limitations. Here, we use a subcutaneously implanted cell-free sponge matrix as an experimentally confined in situ environment to study MHC-restricted local interactions of presensitized host immune T cells against a highly metastatic murine lymphoma (ESb). The compartmentalization of the tumor cells allows us to recover the infiltrating host cells by excising and gently squeezing the sponge tissue. When ESb immune mice were injected with inactivated ESb tumor cells as vaccine into the sponge, an accumulation and activation of lymphocytes within the sponge were observed. The infiltrating CD 8+ T cells exhibited highly specific cytotoxicity against the transplanted tumor cells in a 4-h 51Cr release assay. In contrast, no cytolytic activity was detectable in the regional lymph nodes. Analysis of the CD 4 + ICD 8+ ratio of T lymphocytes within the sponge at various times after challenge with the autologous tumor vaccine showed a gradual relative and absolute increase of the CD 8+ subset. Thus, at the time of maximal cytolytic activity of sponge-derived T cells, the CD 4+ ICD 8+ ratio was 1 :3, in contrast to the ratio 2:1 in regional lymph nodes. Interestingly, significant IL 2 Rec expression (> 15 %) was detectable only in the CD 4 + subset. When transplanted into naive recipients, the activated sponge cells could recruit other circulating host cells to the tumor recognition site and initiate a tumor protective response. Thus, the sponge matrix model enables a more precise definition of the contribution of distinct cell types and interactions to recognition and rejection of a metastatic syngeneic tumor.

Abt. f. Padiatrische Hamatologie und Onkologie, Medizinische Einrichtungen der Universitat Diisseldorf, Diisseldorf, FRG; Hematology Division, Stanford University Medical Center, Stanford, CA, U.S.A.

A.20 Control of erythropoiesis by the T cell CD2 determinant is mediated at a pretranslationallevel

N. ZESSACK, K. BACHTENKIRCH, M. SHATSKY, L. LEVITT, and S. BURDACH

We have previously demonstrated that the T cell determinant CD2 is involved in control of erythropoiesis by the lymphokine cascade: blockade of the CD2 T cell determinant induces down modulation of 1) T cell p55 IL2 receptor expression, 2) interleukin 2 (IL2)-induced inhibition of early erythroid progenitors (BFU-E) and 3) IL2-induced marrow T cell

XXth Meeting of the Society of Immunology . 13

interferon-y (IFy) release (BURDACH et al. Blood, 1988). We have now attempted to further clarify the molecular mechanism of CD2 mediated control of IFy release and inhibition of erythropoiesis. We asked whether decrease of T cell IFy protein release is preceded by decrease ofT cell IFy mRNA. T cell p55 mRNA and p55 membrane receptor expression were induced via triggering of the antigen receptor associated CD3 epitope. Before CD3 triggering T cells were preincubated with a CD2 blocking (Leu 5b) or isotope control CDS antibody. T cell pellets were employed during incubation to facilitate physical contact between T cells and interaction between CD2 and its ligand, lymphocyte function antigen-3, which is present on various cell types including T cells. CD2 blockade caused a > 60 % inhibition of p55 expression after 72 h of culture with IL2. CD3 triggered T cell supernatants caused a > 70 % inhibition of BFU-E. Blockade of CD2 caused a > 90 % abrogation ofT cell-mediated BFU-E inhibition. In addition, CD2 blockade reduced IFy release from CD3 triggered T cells by > 80 %. Addition of IFy neutralizing monoclonal antibody to CD3 triggered T cell supernatant caused> 95 % abroga­tion of IL2 inhibition of BFU-E. T cell RNA was recovered following 16 h of incubation utilizing vanadylribosides for inactivation of RNAses and cesiumchloride gradients for RNA separation. Kinetic studies revealed maximum IFy mRNA accumulation at 16 h of culture. RNA recovery was about 10 f,lg/107 cells. A full length cDNA for IFy (G. RICCA) served as template to generate a molecular probe employing random priming and Klenow polymerase. The specific activity of the probe was 2-3 X 107 cpm/f,lg DNA. No IFy mRNA was detectable after 16 h of incubation of CDS-triggered control cells. In contrast, a strong IFy signal was detectable in RNA derived from CD3-triggered T cells. Blockade of p55 resulted in a decrease in IFy mRNA concentration, indicating receptor specificity of IL 2-induced IFy mRNA increase. Blockade of CD2 also caused a distinct decrease in IFy mRNA concentration. Thus, decrease in IFy protein release was preceded by a decrease in IFy mRNA concentration, induced by blockade of CD2. This data suggests that regulation of IFy production and control of erythropoiesis by CD2 is exerted at a pretranslationallevel.

Institut fUr Immunologie, * Institut fur Medizinische Mikrobiologie der Joh.-Gutenberg­Universitat, 6500 Mainz, FRG

A.21 T cell activation in serum-free medium

F. ZIMMERMANN, W. DAUBENER*, T. HORN", E. RODE, and U. HADDING'f

We have developed a defined serum-free medium based on Iscove's (I)-medium supplemented with albumin, transferrin and linoleic acid, further referred to as Iscove's serum-free (ISF)­medium, which allows long-time culturing of mouse T cells.

It was now investigated whether human peripheral blood T cells can also be activated in ISF­medium. First, we stimulated peripheral blood mononuclear cells (PBMC) with the T cell mitogen PHA. This type of activation was possible in I-medium and in ISF-medium. In contrast, stimulation of PBMC by HLA differences (alloantigens) was possible only in ISF­medium. Next, we tested whether ISF-medium allows the triggering of T cells by nominal antigen (tetanus toxoid). No significant proliferation of PBMC after stimulation with tetanus toxid was observed either in 1- or ISF-medium. Furthermore, increasing the concentrations of supplements (transferrin, albumin, linoleic acid), of antigen, of the responder cell density or addition of rh-IL 1~ or rh-IL2 did not allow a tetanus toxoid-specific proliferation ofT cells in ISF-medium.

We conclude that the unresponsiveness of PBMC to nominal antigen in ISF-medium cannot be due to a lack of nutrients because the T cells were able to proliferate in ISF-medium after mitogenic or alloantigeneic stimulation. Since processing and presentation of antigen is not required in either PHA or in alloantigen-driven systems, this process could be deficient in the absence of serum, explaining the lack of tetanus toxoid-induced proliferation.