Workshop D Lymphocyte Activation and Apoptosis

IClinical Research Unit for Rheumatology, University of Freiburg, Freiburg, Germany, 2Burnham Institute, La Jolla, CA, USA

O. 1 C04+ cells from patients with Wegener's granulomatosis express high levels of C095

The CD4+ cells of peripheral blood lymphocytes (PBL) from healthy donors express CD95 (Fasl APO-l) in low and high levels. In contrast, CD4+ cells of PBL from patients suffering from Wegener's granulomatosis express high levels of CD95. CD95 triggering induces apoptosis and alteration in the CD95/CD95L system is associated with autoimmune disorders. Therefore we analyzed if the different expression pattern correlates with a different susceptibility to CD95mediated apoptosis. There is no difference in the apoptotic response of lymphocytes from patients or healthy donors after incubation with CD95 antibodies or activation with CD3 antibodies. Tyrosine phosphorylization after CD95 stimulation shows differences in CD95 sensitive and resistent cell lines. By Western blot analysis using an antiserum specific for the phosphatase FAP-l we found FAP-l expressed in cell lines resistent against CD95-mediated apoptosis as well as in CD4+ and CD8+ lymphocytes from patients whereas FAP-l was not detectable in cell lines sensitive for CD95-mediated apoptosis. In conclusion, FAP-l expression correlates with a decreased susceptibility to CD95-mediated apoptosis. The expression of FAP-l might be one mechanism to protect activated cells from CD95-mediated apoptosis and to maintain an autoimmune response.

Clinical Research Unit for Rheumatology, Albert-Ludwig-University, Freiburg, Germany

O. 2 Determination of the human C8 protein domains binding to the C095L 1. BARTH, C. SCHATZLEIN, H.-H. PETER, H. Eibel The C8 protein is a subunit of the human 20S-proteasome. Proteasomes are protein degrading machineries of cells. They are involved in diverse cellular processes including the degrading of ubiquitinated and false folded proteins, cell cycle control and in processing of MHC I class presented peptides. As an a-type subunit the C8 protein is located in the outer heptameric ring of the proteasome complex. It displays no proteolytic activity rather it is thought to playa structural role. C8 was shown to bind together cytoplasmatic domain of CD95L by using interaction trap cloning with yeast as well as in higher eukaryotic cells. To determine the domains of the C8 protein which interacts with the CD95L cytoplasmatic domain, we constructed several C8 mutants and tested their ability to interact with CD95L. As expected, deletions of large portions of the C8 protein resulted in the distruction of protein structure and subsequently no binding was observed. Furthermore depending on the crystallo-

168 . 28th Annual Meeting 1997 graphic 3-D-structure of the C8 protein, several short sequences (up to four amino acids) were deleted or amino acids with prominent side chains were substituted by amino acids with small side chains. These panel of mutated C8 protein variants are currently tested on their binding capacity to the CD95L, revealing the structural backbone of C8-CD95L interaction.

'Bernhard-Nocht-Institut fur Tropenmedizin, Hamburg, and 2Institut fur Klinische und Molekulare Virologie, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Germany

O. 3 C02 hyperreactivity in primate T cells transformed by Herpesv;rus sa;m;r; B. M. BROKER', C. STEEG', U. KLAUENBERG!, B. FLECKENSTEIN2, B. FLEISCHER!, and H. FICKENSCHER2

Herpes~irus saimiri transforms T cells of various primate species to continuous growth. Whereas

New World primate T cells can be transformed by virus strains of any subgroup, human T cells are transformed by subgroup C strains only. The subgroups differ mainly in the transformationassociated genomic region. In human T cells, research has been focussed on the transformationassociated proteins StpC and Tip and the pronounced hyperreactivity to CD2 ligation. In order to investigate the correlation of virus strain subgroup and level of CD2 reactivity, we compared transformed T cell lines from marmoset monkeys with transformed human T cell lines. Interleukin-2 secretion, after contact with cells bearing the CD2 ligand CD58/LFA3 was observed in T cells from any species but only if they had b€en transformed by virus strain C488. In contrast to human cells the C488 transformed marmoset cells were not activated by cross-linked antiCD2.1 monoclonal antibodies. Marmoset cell lines transformed with virus strains of other subgroups did not secrete interleukin-2 following CD2.1 ligation. However, cytokine production could be induced by mitogen stimulation. Thus, both virus subgroup and host species determine the degree of CD2 hyperreactivity in primate T cells transformed by Herpesvirus Sarmlrl.

Institut fur Klinische und Molekulare Virologie der Universitat Erlangen-Nurnberg, Germany

0.4 Inhibition of a~ptosis in lymphocytes by the Herpesv;rus so;m;r; encoded Bel-2 homolog T. DERFUSS, H. FICKENSCHER, G. HENNING, B. FLECKENSTEIN, E. MEINL Infection with Herpesvirus (H.) saimiri transforms human T cells to stable growth. These transformed T cells do not release infectious virus and only a minority of the viral genes is actually expressed in transformed human T cells. H. saimiri codes for a gene with homology to conserved domains of Bcl-2, named HVS-Bcl-2. This study aims in getting insight into the function and expression pattern of HVS-Bcl-2. HVS-Bcl-2 was not expressed in transformed human T cells, but abundantly in virus-producing cultures and seen by Northern and Poly-A Northern analysis. To analyse its function, Jurkat cells and murine T cell hybridomas were stably transfected with HVS-Bcl-2. HVS-Bcl-2 inhibited dexamethason induced apoptosis in murine T cell hybridomas and irradiation induced apoptosis in Jurkat cells, while apoptosis induced by recombinant CD95 ligand in murine T cell hybridomas was not affected. In conclusion, HVS-

28th Annual Meeting 1997 . 169 BcI-2 is not needed for stable growth of transformed human T cells, but might favour viral replication by prolonging the life of infected cells. The differential inhibition of apoptosis we observed in murine T cell hybridomas indicates that HVS-BcI-2 might be complementary to the H. saimiri encoded FUCE inhibitory protein that inhibits apoptosis by death receptors like CD95.

Institute for Virology and Immunobiology, Julius-Maximilians-University, Wurzburg, Germany

O. 5 The role of C095-mediated apoptosis for human HIV-l-specific C08+ cytotoxic Tlymphocyte-{CTLI-meCIiated cytolysis R. EHRET and C. JASSOY Objective: To examine the contribution of perforin/granzyme and Fas (CD95/APO-1)/FAS ligand-mediated cytotoxicity to cell lysis mediated by human HIV-1- specific CD8+ CTL. Methods: HIV-specific CTL clones were used as effector cells in 51 chromium release assays. Target cells were either B-lymphoblasts, T-cell lines or autologous primary T cells either preincubated with peptides or infected with recombinant vaccinia viruses expressing the relevant protein or with HIV-l. Assays were performed in the absence and presence of EGTA, an inhibiting anti-CD95 antibody (ZB-4) and peptide inhibitors of CD95-mediated apoptosis (Z-VAD, YVAD). Results: HIV-specific CD8+ CTL lysed antigen-presenting and HIV-infected transformed cell lines by both the perforin and the Fas/Fas ligand mechanisms. The CD95-mediated fraction constituted up to 10-20% of the total cytolytic activity in these cells and was completely blocked by an inhibiting anti-CD95 monoclonal antibody and inhibitors of caspases. Preliminary data indicate that cytolysis of primary HIV-infected cells is almost completely executed by perforin/granzyme release. Conclusions: Cytolysis of HIV-infected cell lines and primary T helper cells by antigenspecific CD8+ CTL is mediated predominantly by perforin/granzyme release. This observation indicates that CD95-mediated cytotoxicity by CD8+ CTL plays a minor role in HIV-1 infection.

Institut fur Klinische und Molekulare Virologie, Erlangen, Germany

O. 6 A herpesviral superantigen in transformed Tcells? H. FICKENSCHER, A. KNAPPE, C. HILLER, M. THURAU, and B. FLECKENSTEIN

Herpesvirus saimiri C488 transforms human T lymphocytes to stable growth in culture. The transformed human T cells harbor the viral genome in non-integrated episomal form without production of virus particles. In order to analyze virus gene expression in more detail, we applied a subtractive hybridization technique and compared stimulated virus-transformed cells with uninfected parental T cells of the same donor. A number of known T cell activation genes were isolated. Viral cDNAs from the transforming gene stpC/tip were enriched after subtraction. In addition, the viral immediate-early and superantigen-homologous gene ie14/vsag was represented by numerous eDNA clones that comprised the entire spliced transcript. Whereas a weak basal expression of ie14/vsag was detected by RT-PCR only, the phorbol ester induced transcripts were readily shown by Northern blotting, ie14/sag, which before had been classified

170 . 28th Annual Meeting 1997 as a major immediate-early gene of herpesvirus saimiri, is localized within a highly conserved region with extensive homologies to the cellular genome. Mutant viruses without gene ie14/vsag are replication competent and fully capable of transforming human and marmoset T cells. Since ie14/vsag is transiently expressed after stimulation, it may increase T-cell proliferation in an activation dependent, superantigen-like, but apparently Vp-independent way.

German Cancer Research Center (DKFZ), Department of Tumorprogression and Immune Defense, Heidelberg, Germany

o. 7 Co-induction of Tcell antigen receptor-mediated apoptosis by C044s N. FaGER, Z. ROZSNYAY and M. ZOLLER The CD44 standard isoform (CD44s) has long been known as one of the costimulatory molecules capable of enhancing T cell receptor (TCR)-mediated proliferation and cytokine production. Whether it plays a similar role in the modulation of TCR-induced programmed cell death has not yet been clarified. To investigate the possible influence of CD44s on TCR-dependent apoptosis, an influenza virus hemagglutinin-specific T cell clone (IP12-7) expressing high level of CD44 was used. Cross-linkage of CD44s, which by itself did not induce detectable apoptosis, synergistically increased and accelerated TCR-mediated programmed cell death. Comparison of a variety of monoclonal anti-CD44s antibodies revealed that their capacity to influence the apoptosis correlated strongly with their function as costimulator in T cell activation. These data demonstrate that co-ligation of CD44s with TCR (1) augments antigen receptor-mediated programmed cell death; (2) increases the susceptibility of cells for the induction of TCR-mediated functions without modifying the outcome of the response which would be elicited by stronger TCR triggering in the absence of CD44 co-stimulation.

Institute for Clinical Immunology, Friedrich-Alexander-University, Erlangen-Nurnberg, and lGSF Munich, Germany

O. 8 UV·B irradiated cell lines use different pathways to execute apoptotic cell death M. HAGENHOFER, H. GERMAIER 1, C. HOHENADL1, P. ROHWER,]. R. KALDEN, and M. HERRMANN In most cell lines induction of apoptosis by UV-B irradiation resulted in changes of cellular morphology, exposure of phosphatidylserine, oligonucleosomal DNA fragmentation, and generation of hypochrome nuclei. Most cell lines (e.g. HL60) showed oligonucleosomal and isolate high molecular weight (hmwt) DNA fragments, hypochrome nuclei, morphological changes, annexin-B vinding and positive TUNEL reaction. No oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. However, using a method developed for detection of oligonucleosomal DNA fragments we were able to isolate hmwt DNA fragments from cytoplasm of apoptizing HaCaT cells, which are typical for early apoptotic chromatin degradation. Therefore, this method is useful for preparation of hmwt apoptotic DNA fragments without need of inversed or pulse field gel electrophoresis. In contrast, Raji cells were TUNEL negative, formed low amounts of hmwt DNA, displayed minor morphological changes and showed an atypical hypochrome shift. Nevertheless, they excluded propidium iodide,

28th Annual Meeting 1997 . 171 bound annexin-V, and stopped proliferation. Therefore we conclude that Raji cells underwent programmed cell death without most characteristics of apoptosis. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation, as well as chromatin condensation is not suitable to exclude programmed cell death.

Department of Medicine, Institute of Immunology, University of Munster, Germany

D. 9 CD14 expression as an early effector mechanism of monocyte apoptosis S. HEIDENREICH, M. SCHMIDT, A. RADEMAEKERS, H.-G. PAVELS The present study was undertaken to investigate the role of CD14 for human monocyte apoptosis. For that the effects of LPS and IL-4 on CD14 expression and apoptosis, indicated by annexin V binding or PI staining, were studied simultaneously and in a kinetic fashion by flow cytometry. Flow cytometric detection of monocyte apoptosis was confirmed by DNA electrophoresis, DNA fragmentation assay and morphologically by electron microscopy. Our data show, that LPS-induced increase of CD14 expression rescued monocytes from apoptosis, whereas IL-4 at first suppressed CD14 expression transcriptionally and consecutively provoked apoptosis. Other antigens such as HLA class 1 molecules or CD33 were not down-regulated in association with apoptosis suggesting the specific role of the CD14 antigen for cell survival. Enzymatic removal of membrane-linked CD14 by PI-PLC similarly induced apoptosis as IL-4 did. Our findings suggest that regulation of CD14 receptor expression is an early effector mechanism determining the lifespan of human monocytes.

lInstitut fur Klinische und Molekulare Virologie, Universitat Erlangen-Niirnberg, Germany, and 2Novartis Forschungsinstitut, Wien, Austria

D. 10 Expression and function of signaling lymphocytic activation molecule (SLAM) on Herpesvirus soimir; transformed T cells G. HENNING!, G. AVERSA 2, B. FLECKENSTEIN!, and E. MEINL 1 T cell activation is mediated by the T cell-receptorlMHC complex as well as other costimulatory receptors. The recently described signaling lymphocytic activation molecule (SLAM) is a costimulatory molecule expressed on activated T cells, memory T cells and activated B cells. SLAM is thought to interact with soluble or membrane-bound SLAM. Engagement of SLAM induces proliferation and IFN-y-production of T cells as well as proliferation and Ig synthesis of B cells. We analyzed SLAM expression and function on human T cells transformed to stable growth by Herpesvirus (H) saimiri. SLAM was abundantly expressed on H saimiri transformed T cells, while CD28, another costimulatory molecule, was hardly detectable. After engagement of SLAM by an agonistic antibody H saimiri transformed T cells are activated and produce IFN-y and IL-2. SLAM/SLAM interaction may be involved in autocrine activation of H saimiri transformed T cells.

172 . 28th Annual Meeting 1997 Department of Medicine III, Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nuremberg, Germany

D. 11 Inhibition of apc?ptosis in short term activated human lymphocytes by "fe-chain cytokines is independent of activation of PI-J kinase T. HIERONYMUS, H.-M. LORENZ, M. GRUNKE, B. MANGER and J. R. KALDEN Activated peripheral blood lymphocytes undergo apoptosis when deprived of growth factors, but the intracellular signaling pathways responsible for mediating cell survival have not been described. In this study, we compared different cytokines in their ability to rescue short term activated lymphocytes from apoptotic cell death and the role of PI-3 kinase and pp 70S6K . We found that only IL-2, IL-4, IL-7 and IL-15 can inhibit induction of apoptosis in this experimental model with similar dose kinetics. These cytokines utilize the common y-chain of the IL-2 receptor in their specific receptor complex and all have the ability to activate PI-3 kinase. We detected no difference in the expression of bcl-2 in IL-2 treated or untreated cells. Including the fact that IL-4 is unable to activate p21 ras or MAP kinase, we considered a potential role for PL-3 kinase and the following downstream activation of pp 70S6K in the signal transduction leading to inhibition of apoptosis in this system. To examine this we used wortmannin which inhibits PI-3 kinase and rapamycin which prevents activity of pp 70S6K . However, both agents failed to induce apoptosis in cells treated with the above noted Yc-chain utilizing cytokines, but showed strong inhibition on proliferation. Therefore, this study suggests an important role for the activation of PI-3 kinase and pp 70 S6K for cellular growth in this system, but not for cell survival. IL-2, IL-4, IL-7 and IL-15 thus appear to utilize signaling pathway(s) independent of PI-3 kinase for cell survival in short term activated lymphocytes. This work was supported by DFG grant Lo 437/3 and SFB 263.

Molecular Immunology, Robert Koch-Institute, Berlin, Germany

D. 12 Identification and initial characterization of a novel Tcell-specific cell surface activation antigen A. HUTLOFF, K. BEIER, A.-M. DITfRICH, R. A. KROCZEK By generating the mAb 8F4 we have recently identified an antigen expressed on activated but not resting human T cells. No signal was detected on activated B cells, monocytes, or NK cells. The Mr of the antigen immunoprecipitated by mAb 8F4 (27 and 29 kDa) and the comparison of the expression pattern on T cells with known activation antigens strongly indicates that 8F4 is a novel cell surface molecule. Immunohistology revealed 8F4+ cells in germinal centers of tonsillar tissue. Analysis of ex vivo tonsillar T cells determined that 50-80% of the cells bear the 8F4 antigen. These T cells are 15% CD25+, 93% CD69+ and 18% CD71+. Further analysis showed that 8F4+ tonsillar T cells predominantly belong to the mature T cell compartment, with a clear correlation between the degree of 8F4 expression and CD45RO expression. In functional terms, the 8F4 antigen showed strong co-stimulatory capacity (80-fold) on T cell proliferation, when resting peripheral blood T cells were suboptimally stimulated by CD3 crosslinking (for comparison: CD28: 90-fold). A similar co-stimulatory effect was seen on the upregulation of several activation antigens and Iymphokines (TRAP, CD69, ATAC). Furthermore, the 8F4 antigen exhibited potent co-stimulatory effects on IgM and IgG synthesis on co-culture of CD3-activated T cells with tonsillar B cells. Taken together, our data suggest that the 8F4 antigen plays a significant role in the T cell help for B cells in lymphoid tissue.

28th Annual Meeting 1997 . 173 Institute of Veterinary Virology, University of Berne, Berne, Switzerland

O. 13 Activation-induced apoptosis in bovine macrophages: Does interferon-a playa role in eliminating infected cells? 1. W. lUNGI, M. BRCIC, B. ADLER, H. ADLER and E. PETERHANS The number of immunological cells is controlled by two major mechanisms: cell division and programmed cell death. The withdrawal of growth factors causes both a stop of proliferation and onset of cell death by apoptosis. Similar activating stimuli may induce either proliferation or apoptosis, depending on the stage of differentiation and/or corollary stimuli. This ambiguity of cell stimulation is widely recognized for lymphocytes, although the underlying mechanisms are not yet fully understood. Here we show that activation of macrophages (M, alveolar M and monocyte-derived M divide infrequently in vitro, do not depend on exogenous addition of colony stimulating factors, and are long-lived. When exposed to IFN-a for 2 days, then stimulated with LPS, M undergo apoptosis within 24 to 40 h. IFN-y primes M for LPS-induced apoptosis to a lesser degree. Apoptosis is neither dependent on NO production nor on TNF-a release. However, high TNF-a concentrations may also induce apoptosis, and IFN-a-primed M respond to lower concentrations of TNF-a by apoptosis. This and other evidence suggests that activationinduced apoptosis is a mechanism whereby M infected with intracellular pathogens (ineluding viruses) are eliminated to the benefit of the host, and that IFN-a not only protects uninfected cells from virus infection, but may also contribute to the elimination of infected cells.

Tumorimmunology Program, German Cancer Center, and IPediatric Oncology Department, University Children's Hospital, Heidelberg, Germany

O. 14 FLiCE is involved in regulation of activation-induced cell death (AICO) of peripheral T cells F. C. KISCHKEL, M. E. PETER,]. P. MEDEMA, C. SCAFFIDI, C. SCHEUERPFLUG 1, K.-M. DEBATIN 1, P. H. KRAMMER Resting peripheral T cells (dO T cells) express low amounts of CD95(APO-l/Fas) on the surface and are resistant to CD95-induced apoptosis. After stimulation with PHA for one day (d1 T cells) CD95 is upregulated but the T cells are still resistant. Subsequent culture in interleukin 2 for 5 days (d6 T cells) does not change CD95 surface expression. However, d6 T cells are sensitive to CD95-mediated apoptosis. To determine the mechanism of this apoptosis resistance d1 and d6 T cells were tested for formation of the CD95 death-inducing signaling complex (DISC). CAP1 and 2 (FADD) and CAP3 were recruited to the receptor after stimulation of T cells at both activation stages. However, CAP4/FLICE was only found in the DISC of the apoptosissensitive d6 T cells. A FLICE activation assay with in vitro-translated FLICE showed that only the DISC of d6 T cells was active in processing FLICE. Among the molecules tested only the expression of bel-xL correlated with CD95-induced apoptosis sensitivity as determined by both PCR and immunoblotting. However, in bel-xL transfected and CD95 resistant MCF8 cells neither recruitment of FLICE to the DISC nor FLICE processing activity of the DISC was altered indicating that bel-xL might act downstream of DISC-bound FLICE.

174 . 28th Annual Meeting 1997 Ben May Institute for Cancer Research, University of Chicago, Chicago IL 60637, USA

D. 15 Giant-1, a gene induced in anergic T lymphocytes, is a member of the integrin regulatory protein/cytohesin family U. KORTHAuER, E. O. MITCHELL, R. S. MENON, N. N.s. and A. BWESTRONE The goal of our project was to identify (novel) molecules that are crucial in maintaining anergy in murine T cell clones. Since anergy can persist for weeks we reasoned that it could be maintained through altered gene expression. Employing a PCR based screen for genes expressed differently in anergic versus responsive murine T cells «
lInstitut fur Klinische und Molekulare Virologie, Universitat Erlangen-Nurnberg, Germany 2Institute of Biochemistry, University of Lausanne, Switzerland

D. 16 Herpesvirus soimiri transforms human Tcell clones to stable growth witllout inducing resistance to apoptosis M. KRAFT!, H. FICKENSCHER', D. LENGENFELDER', MEINL'

J.

TSCHOPp2, B. FLECKENSTEIN' and E.

Infection with Herpesvirus (H) saimiri transforms human T cells to stable growth. This study aims in obtaining further insight into the mechanism of transformation and in assessing how broadly this system can be applied to study T cell physiology. Effects of H saimiri on the sensitivity to apoptosis, on the regulation of proliferation and the cell cycle progression were examined by comparing two native CD4+ T cell clones with their transformed derivatives. The expression levels of the apoptosis preventing molecules Bcl-2, Bcl-XL and of the apoptosis promoting protein Bax were not changed after transformation. Expression and function of TNF-RI, CD95, TRAMP, and DR4 (TRAIL receptor) on transformed and parental clones was analysed. CD95 and TNF-RI continued to be expressed after transformation. The transformed and parental T cell clones were sensitive to CD95-ligation, but resistant to TNF-a and TRAIL mediated apoptosis. DR4 was hardly detectable on both cell types. The H saimiri encoded FLIP, which inhibits apoptosis mediated by death receptors like CD95, was not expressed by H. saimiri transformed human T cells. Native and transformed T cell clones showed similar sensitivity to cell death induction or inhibition of T cell activation mediated by irradiation, oxygen radicals, dexamethasone, CsA, and prostaglandin E2. Altogether, this study strongly suggests that the growth transformation by H. saimiri is not based on a resistance to apoptosis, but rather on the utilization of normal cellular activation pathways.

28th Annual Meeting 1997 . 175 Institute of Hygiene, Department of Medical Virology, University of Heidelberg

O. 17 The interleukin-1 0 homologue of Epstein-Barr Virus modulates the expression of the B7-receptors C028 and CTLA-4 on T cells to induce anergy A. MULLER, L. SCHMITT, G. SCHONRICH The Epstein-Barr Virus (EBV) encodes a protein homologous to the human interleukin-l0 gene (IL-I0). IL-I0 is known to be a mediator of immunosuppression. For efficient T cell stimulation a T cell receptor (TCR)-dependent signal as well as a costimulatory signal delivered through the binding of B7 to its receptor CD28 are required. In previous studies we demonstrated that viral interleukin-l0 (vIL-I0) inhibits B7-mediated rejection of tumor cells by interfering with costimulation. Therefore we analysed the effect of vIL-I0 on the B7-receptors CD28 and CTLA-4 expressed on the surface of T cells. In our study we used splenocytes of T cell receptor transgenic mice (TCR mice) recognizing the major histocompatibility complex (MHC) class I molecule Kb. To analyse the effect of vIL-I0 on the expression of the B7-receptors we cotransfected the murine tumor cell line P81S (MHC-haplotype H2-d) with the MHC class I molecule Kb and the costimulatory molecule B7 (P81S-Kb-B7). In addition we established a triple-transfectant expressing the Kb-molecule, the B7-molecule and vIL-I0 (P81S-Kb-B7-vIL-I0). The splenocytes were incubated with the transfectants and the expression of CTLA-4 and CD28 was monitored over a period of 4 days. 48 hours post stimulation we could detect an upregulation of CTLA-4 on activated T cells incubated with P81S-Kb-B7. The CD28 expression was not altered. In contrast T cells incubated in the presence of vIL-I0 did not express CTLA-4 48 hours post incubation and the CD28-molecule was significantly downregulated. Therefore the B7-molecule can no longer bind its receptor on T cells stimulated in the presence of vIL-I0. This results in TCR-occupancy without sufficient costimulation (anergy). In conclusion downregulation of CD28 might be one mechanism by which vIL-l 0 inhibits an efficient antiviral T cell response.

Max-Planck-Institut fur Immunbiologie, Freiburg, Germany

O. 18 C022 is a negative regulator of Bcell receptor signalling L. NITSCHKE, R. CARSETTI, B. OCKER, G. KOHLER and M. LAMERS CD22 is a B cell receptor (BCR)-associated transmembrane protein, whose cytoplasmic tail contains 3 immunoreceptor tyrosine inhibitory motifs (ITIM's). They are phosphorylated upon BCR crosslinking, and can bind the tyrosine phosphatase SHP-l, a putative negative regulator of BCR-signalling. To assess the role of CD22 in vivo, we have generated CD22-deficient mice. CD22-deficient mice show a normal B cell development. They have normal numbers of peripheral B cells, but they have a more mature phenotype and recirculating B cells are absent from the bone marrow. After BCR-crosslinking in vitro, splenic CD22-I - B cells show an increased Ca 2+ response and a lower survival due to an increased induction of apoptosis. In contrast, responsiveness to the B-cell mitogen LPS is increased. In vivo, a shorter average lifespan in the B-cell compartment is found. T cell independent immune responses are impaired, whereas T cell dependent responses are normal. Thus the absence of CD22 lowers the signalling threshold for BCR-crosslinking. We propose that the low threshold leads to hyperresponsiveness of the B cells and a chronic basal activation. In this model, engagement of the receptor without T cell help leads to an increased induction of apoptosis, explaining the shorter lifespan of B cells and the low response to T cell independent antigens. The changed phenotype and the LPS-reactivity is explained by the chronic basal stimulation.

176 . 28th Annual Meeting 1997 Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany

O. 19 Activation-induced Tcell death (AICO): Susceptibility of individual Tcell clones to undergo AICO relates to FasL surface expression and potentially to TH2 phenotype H.-H. OBERG, D. KABELITZ, and O. JANSSEN':Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD). Activated T cells also undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. It has been shown that the two cell death pathways are causally related since AICD is in part depending on Fas/FasL interactions. Thus, stimulation of T cells leads to FasL gene transcription and surface expression. FasL binding to Fas molecules on the same cell or on a neighbor cell then triggers the Fas-dependent death signaling cascade. Comparing a set of different T cell clones, we found that resistance or susceptibility of individual clones to undergo AICD is regulated at the level of FasL surface expression. All tested clones were CD95-positive and died upon Fas-ligation. In AICD-resistant clones, TCR-CD3-induced tyrosine phosphorylation or FasL mRNA expression were not impaired. However, FasL surface expression as judged by immunofluorescence staining and flow cytometry was markedly reduced compared to AICD-sensitive clones. We also found that in response to CD3-stimulation, the majority of AICD-resistant clones produced less interferon gamma than AICD-sensitive clones. In addition, two of the resistant clones secreted significant amounts of IL-4. Although no direct correlation could be drawn in terms of IL-2-production, our data also point to a potential correlation of the TH2 phenotype to AICD-resistance. ':-0. J. is supported by a grant from the BMBF (AIDS-Stipendienprogramm).

Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany

O. 20 Activation-induced Tcell death (AICO): C04-ligation inhibits AICO on individual Tcell clones H.-H. OBERG, R. SANZENBACHER, O. JANSSEN" and D. KABELITZ Using the T cell panel of antibodies against leukocyte antigens provided by the 6th Leukocyte Typing Workshop, we have tested the effect of ligation of 'accessory' molecules prior to the induction of AICD in T cell clones. We found that anti-CD4 mAb inhibited AICD but not Fas antibody-induced apoptosis of established T cell clones. Similar effects were seen with F(ab')z but not with Fab fragments of anti-CD4 mAb and gp120 of HI\!. Aside from individual clones, CD4-inhibition was also demonstrated in a significant portion of short-term T cell clones. Thus, out of 137 CD4-positive clones tested, 92% of OKT3-sensitive and 21 % of SEA/SEB-sensitive clones were rescued from AICD by pretreatment with anti-CD4 mAb. Anti-CD4 mAb inhibited FasL surface expression and CD3- and/or SEA-induced FasL mRNA synthesis. In addition, pre-ligation of CD4 molecules inhibited anti-CD3- and SEA-induced but not phorbolester and calcium ionophore-triggered cytokine production (e.g. interferon gamma and interleukin2). In terms of signaling events, CD4-ligation resulted in a reduction of TCR-induced tyrosine phosphorylation of individual proteins. These results indicate that certain CD4-associated kinases or signaling molecules are uncoupled from the TCR/CD3-complex upon prestimulation with anti-CD4. Of note, opposite AICD-inhibitory or AICD-inducing effects of CD4-ligation in activated T cells versus resting T cells may also have implications for T cell survival and/or T cell responses in HIV-infected individuals. ':-0. J. is supported by a grant from the BMBF (AIDS-Stipendienprogramm).

28th Annual Meeting 1997 . 177 Institute for Virology and Immunobiology, Julius-Maximilians-University, Wurzburg, Germany

0.21 Contact of HIV·1 infected cells with C04+ T lymphocytes leads to a~ptosis which involves caspases but bypasses C095 (Fas/APO·1) and TNF·R1 H. OHNIMUS, M. HEINKELEIN, C. JASSOY

Objective: The mechanism of CD4+ T cell depletion during HIV-infection remains still unclear. We examined whether the cell death upon contact of CD4+ T lymphocytes with HIV glycoprotein-expressing cells is caused by apoptotic processes. Methods: CD4+ T lymphocytes were coincubated with either HIV-infected or HIV glycoprotein-expressing cells in the absence or presence of peptides (ZVAD-FMK; YVAD-CMK; DEVD-CHO) inhibiting caspases or antibodies (ZB-4; H398) directed against CD95 (Fas/APO-1) and tumor necrosis factor receptor 1 (TNF-R1). Lysis of the cells was analysed by standard chromium release assay. The cleavage of poly(ADP-ribose)-polymerase (PARP) was monitored by standard Westernblot technique. DNA fragmentation was assessed after preparation of intracellular low molecular weight DNA by agarose gel electrophoresis. Results: Contact of CD4+ T lymphocytes with HIV-infected or HIV glycoproteinexpressing cells leads to lysis of both populations, to PARP cleavage and DNA fragmentation. Cell death is blocked by addition of peptides inhibiting caspases but not by antibodies directed at CD95 and TNF-Rl. Inhibition of caspase activity has no effect on syncytium formation. Conclusions: These results indicate, that the contact of CD4+ T lymphocytes with HIVinfected or HIV glycoprotein-expressing cells leads to an apoptotic process in both populations. The apoptotic process can be blocked by inhibition of caspase activity. Neither the CD95- nor the TNF-R1-molecule is involved in the activation process. Apoptosis may contribute to CD4+ T lymphocyte cell death in vitro and possibly to CD4+ T cell depletion in vivo.

IChildren's University Hospital Tubingen, Dept. of Oncology/Hematology, Tubingen, Germany, 2Dept. of Microbiology & Immunology, Valhalla, New York, NY, USA, 3Bristol Meyer Squidd

0.22 The upregulation of MHC class I protects Tcells from antibody· mediated deletion T.

ORLIKOWSKY', ZHI-QIN WANG 2, A. DUDHANE 2, R. MITILER 3, G. E. DANNECKERl, D. NIETHAMMERl, M. K. HOFFMANN 2

Macrophages are known to stimulate the immune response of T cells when they engage the antigen receptor and costimulatory surface components of T cells with which they form cellular conjugates. Macrophages can also destroy T cells which they target for conjugate formation in a non stimulatory fashion. We show here that T cells can protect themselves against macrophagedependent antibody-mediated cellular cytotoxicity (ADCC) through the upregulation of MHC class I molecules. Resting or virgin CD45RO T cells express class I molecules in relatively low density and are easily killed by macrophages, whereas memory CD45RO T cells, which express class I at elevated levels, resist ADCC. Both T cell types are readily destroyed in the ADCC response when their class I molecules are masked.

178 . 28th Annual Meeting 1997

Klinische Forschergruppe fur Rheumatologie, Albert-Ludwigs-Universitiit Freiburg, Freiburg, Germany

D. 23 Identification of proteins interacting with the intracellular part of the human CD95 ligand

J. RADONS, C. SCHATZLEIN, H.-H. PETER, and H. EIBEL CD95 ligand (CD95L), a member of the TNF-a type II membrane protein family, is expressed on several cell types including activated T- and B-lymphocytes, NK cells as well as in lung, eye and kidney. Binding of CD95L to his corresponding receptor (CD95/Fas) induces a cascade of ICE-like protease reactions leading to apoptosis of CD95-bearing cells. Whereas much information is available on the structural features of CD95L, little is known about the mechanisms regulating CD95L expression. One mechanism might be the interaction of proteins with the prolinerich domain within the cytoplasmic part of CD95L. In order to identify possible interactors of CD95L, an interaction trap was performed using the yeast two-hybrid system. Therefore, the yeast strain EGY48 was transformed with pEG202/CD95L(1-80), an expression vector encoding only the cytoplasmic domain of CD95L fused to the bacterial repressor LexA. Afterwards, EGY48 was transformed with the reporter plasmid pJKI03 for LexA fusion proteins containing a single high affinity overlapping type colEJ operator, and pJG4-5 for making galactose-inducible yeast eDNA encoded proteins fused to the bacterial activation domain B52. The interaction trap cloning revealed 12 new clones encoding proteins that interact with the intracellular part of CD95L. The analysis of these interactions and the effects on CD95L expression and activity will be discussed.

Tumorimmunology program, German Cancer Research Center, Heidelberg, Germany

D.24 Two FLiCE isoforms (Caspase 8/a and Caspase 8/b) are both recruited and activated by the CD95 death inducing signaling complex (DISC) C. SCAFFIDI, J. P. MEDEMA, F. C. KISCHKEL, P. H. KRAMMER and M. E. PETER The induction of apoptosis by the cell surface receptor CD95 (APO-l/Fas) has been shown to involve activation of a family of cysteine proteases (caspases). Recently, we identified a new member of this family, designated FLICE (CAP4/caspase-8/MACH/Mch5) as part of the CD95 death inducing signaling complex (DISC). Therefore FLICE is the most upstream caspase in the CD95 apoptosic pathway. A total of 8 different isoforms of FLICE (caspase-8/a-h) have been described. Using a set of monoclonal anti-FLICE antibodies we could demonstrate, that only two of the FLICE isoforms (caspase-8/a and caspase-8/b) were predominantly expressed in a number of cells of different origin. We could further demonstrate that the proform of both FLICE isoforms binds to FADD in the DISC through their N-terminal DEDs. Binding results in activation by proteolytic cleavage leading to the release of the active subunits pl0 and p18 into the cytosol, while the prodomains of both isoforms remain in the DISC. The activation of FLICE is the first step in a cascade of caspases and occurs only at the DISC.

28th Annual Meeting 1997 . 179 Clinical Research Unit for Rheumatology, Albert-Ludwigs-University, Freiburg, Germany

D. 25 The intracellular domain of CD95L regulates protein expression by interaction with a proteasome protein C. SCHATZLEIN, P. FIEDLER, E. VON SEYDLITZ, H.-H. PETER and H. EIBEL The CD95L (FasL, APO-1L) is a type II membrane protein and belongs to the TNF-family. Binding of the CD95L to the CD95 (Fas, APO-1) receptor induced apoptosis in susceptible cells. CD95L expression in T cells is induced upon activation and the CD95: is involved in activation induced cell death of preactivated T cells as well as in lymphocyte homeostasis, peripheral tolerance, and the immune privilege of sites like testis or eye. The comparison of the amino acid sequences of CD95L and the other TNF-family members reveals a long polyproline stretch within the intracellular part of CD95L which is unique to CD95L. To analyze if this region is involved in the regulation of CD95L activity, we employed the yeast two hybrid system to identify proteins that may interact with the intracellular part of CD95L. The C8 subunit of the human 20 S proteasome was identified as an interacting partner of the CD95L. Deletion mutants revealed that the C8 protein interacts specifically with the proline-rich domain of the CD95L in the yeast system. Interaction of the isolated protein with the CD95L in mammalian cells was confirmed by co-immunoprecipitation. Increased levels of CD95L expression are achieved in 293T and COS-7 cell lines by deletion of the intracellular prolinerich domain. Reversely, overexpression of the C8 protein in CD95L expressing cells downregulates CD95L surface expression. Several cotransfection assays with CD95L constructs lacking different partS of the intracellular domain gave insights in the mechanisms involved in CD95L solubilization. In conclusion, expression of the human CD95L is posttranscriptionally regulated by interaction of the C8 subunit of the human 20 S proteasome with the intracellular domain of CD95L.

IThe Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, Australia and 2Institut fUr Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universitat Munchen, Munich, Germany

0.26 Requirements for proteolysis during apoptosis D. L. VAUX', S. WILHELM 2 and G. HAcKER 2 Apoptotic cell death is a major regulatory mechanism in the immune system. The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of the caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase-K were able to cleave the caspase substrate DEVD-AMC, while neither proteinase-K nor non-activated extracts were able to do so. Caspase-like activity could be inhibited by the specific caspase inhibitor DEVD-CHO or the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation. As DNA cleavage could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases and DNA cleavage.

180 . 28th Annual Meeting 1997 IInstitute for Medical Biochemistry, 2Institute for Immunology, JInstitute for Pathology, University of Rostock, Germany

D. 27 Induction of apoptosis in the human leukemic Tcell line Jurkat by placental galectin- 1 H. WALZEL 1, U. SCHULZ2, P. NEELSt, L.JONAS3,J. BROCK 1 Placental galectin-1, a member of the galeetin family of ~-galactoside binding proteins, inhibits the cellular proliferation of the human leukemic T cell line Jurkat, of HL-60 human promyelocytic cells and of THP-1 human monocytic leukemia cells. In Jurkat T cells galectin-1 induces a concentration dependent increase in intracellular calcium ([Ca2+];). However, cell calcium signaling induced by galectin-1 was not recorded in THP-1 cells and HL-60 cells. As detected by electron microscopy galectin-1 mediated proliferation inhibition of Jurkat T cells was associated with morphologic changes characteristic for apoptosis. Flow cytometric measurements revealed that the lectin induces a time and concentration dependent increase in phosphatidylserine expression as detected by annexin V FITC/PI staining. Galectin-1 mediated effects on phosphatidylserine expression were found to be reduced in the presence of lactose. In agarose gels a DNA ladder pattern appeared after incubation of Jurkat T cells with galectin-1 for 6 h. The results indicate that galectin-1 may induce apoptosis as a mechanism for terminating T cell-mediated responses by eliminating activator T cells.

Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany

D. 28 Monocyte-de~ndent cell death (MDCD) of peripheral blood Tcells by mitogen stimulation D. WESCH, S. MARX and D. KABELITZ Activated T cells undergo apoptosis when triggered upon CD3/TCR stimulation, involving in most cases the Fas/FasL interaction. In contrast, resting T cells are largely resistant to apoptosis mediated by anti-Fas (CD95) mAb, anti-CD3 mAb or phorbol12-myristate 13-acetate (PMA). Recent studies indicate a PMA-triggerd, monocyte-dependent apoptosis in freshly isolated «resting» T cells. We now demonstrate that in addition to PMA, the mitogens phytohaemagglutinin A (PHA) and, to a lesser degree, concanavalin A (Con A) induce MDCD of freshly isolated T cells, including both CD4+ and CD8+ subsets. Moreover, our experiments reveal that CD45RA+ cord blood T cells are as susceptible to PMA-stimulated induction of MDCD as peripheral blood T cells from adults. The mechanism whereby monocytes enable freshly isolated T cells to undergo cell death is still not clear. We show that MDCD triggered by optimal concentrations of PMA or PHA is not significantly inhibited by Fas-blocking reagents, i.e. neutralizing anti-Fas mAb M3 and Fas-Fc fusion protein. Similarly, inhibitors of ICE-like proteases did not inhibit MDCD. In striking contrast, the H 20 2 scavenger catalase completely prevented the PMA-stimulated MDCD, thereby revealing a strong T cell expansion after 4 days of culture. To a lesser extent, MDCD was inhibited by protein kinase inhibitor herbimycin A. We conclude that induction of MDCD of freshly isolated T cells is a triggered by PMA, as well as by mitogens, such as PHA and Con A. Established myelotic- or monocytic cell lines could not substitute for monocytes, suggesting that cell death-inducing and cell expansion-stimulating activities are different functions of accessory cells. The complete inhibition by catalase of PMA-triggered MDCD points to a pivotal role of reactive oxygen intermediates.

28th Annual Meeting 1997 . 181 Institute for Virology and Immunology, University of Wiirzburg, Wiirzburg, Germany

D.29 Defective activation induced cell death and clonal contraction in IL-2-deficient cells M. WOLF, B. KNEITZ, T. HERRMANN, T. HONIG AND A. SCHIMPL IL-2-deficient mice develop a disease syndrome characterised by lymphadenopathy, splenomegaly and an accumulation of T cells with a memory phenotype. To study T cell expansion and termination two approaches were taken. 1) SAg was injected into IL-2-/- mice and wt littermates. While in vivo expansion of SAg reactive cells was normal in IL-2-/- mice, late deletion of CD4 T cells was incomplete. Despite high Fas expression, CD4 T-cell-blasts from IL2-/- mice failed to undergo activation induced cell death (AICD) when cultured on plates coated with anti Fas mAb (1). When IL-2 was present during activation, blasts derived from lymph node cells of IL-2-/- mice became sensitive to FAS-mediated AICD while other modes of apoptosis were comparable between IL-2-/- and IL-2+ cells. 2) In the second system, IL2-/- mice were bred to tg mice expressing the D011.10 TCR, specific for Ova + I-Ad. CD4 T cells from IL-2-/- and IL-2 producing TCR transgenic mice were transferred into nu/nu recipients. Expansion and contraction of KJ1-16-Id+ T cells was measured after injection of the relevant Ova peptide. While TCR tg CD4 cells from IL-2 producing donors expanded and contracted as described by KEARNEY et al. (2), numbers of IL-2-/- cells stayed high even after 3 weeks unless WT T cells were cotransferred. When transferred into WT recipients, both IL-2 producing and defective cells only showed a transient increase after peptide injection, indicating that WT Cells, either through the provision of paracrine IL-2 or through regulatory cells, limit uncontrolled survival of IL-2-/- cells. Both sets of data suggest a defect in the termination of T cell immune responses in the absence of IL-2 and emphasize its role in T cell homeostasis.

1) B. KNEITZ, T. HERRMANN, S. YONEHARA and A. SCHIMPL, Eur. J. Immunol. 25: 2572-2577 (1995). 2) E. R. KEARNEY, K. A. PAPE, D. Y. LOH and M. K. JENKINS, Immunity 1: 323-339 (1994).