Workshop L: Infection and Immunity

WORKSHOP L

Infection and Immunity

Dept. of Immunology, Medical School of Hannover, Hannover, Germany

L.l Systemic viral infections induce significant amounts of MxA-protein in human PBL in vivo C. ARMBRUST, D. JAKSCHIES, K. U. NOLTE, M. WALTHER, H. DEICHER, and P. VON Wussow 251 HIV -negative patients with a fever episode were serially tested for the presence of type I Interferon inducible MxA-protein in the PBL. 50 fll of lysed blood were measured in an ELISA consisting of two monoclonal antibodies directed against the MxA-protein. Of the 251 patients 62 had a proven bacteriaemia (I4x staphylococcus, lSx streptococcus, lOx E.coli, 4x Klebsiella, 2x Borellia, Ix Yersinia, Ix Listeria). Only one of these patients possessed Mx-concentrations above 0.03 U/IO.OOO leukocytes. In contrast, of the 251 patients tested 44 had an acute viral disease (25x H. zoster, 13x Mononucleosis, 2x influenza A and 4x a CMV infection). 60 % of these patients were Mx-positive above 0.03 U/I0.000 leukocytes. We therefore conclude that even severe bacterial infections do not induce a measurable Mx-A response. In contrast, systemic viral infections induced this IFN-inducible protein. Thus, viral diseases may become recognizable already at the onset of clinical symptoms by the appearance of the MxA-protein.

Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, AG Infektabwehr, RuhrUniversitat Bochum, Bochum, Germany; lHoffmann-LaRoche AG, Basel, Switzerland; 2Institut fur Hygiene und Mikrobiologie, Abt. Virologie, Ruhr-Universitat Bochum, Bochum, Germany

L.2 Interleukin-8, IL-6, and sTNF-RI release from human epithelial cells (A549) infected with respiratory syncytial virus (RSV) R. ARNOLD, H. GALLAnl, H. WERCHAU 2, and W. KONIG We used the human epithelial cell line A549 in order to investigate the release of interleukin-S (IL-S), IL-6, tumor necrosis factor-a (TNFa), and the soluble forms of TNFa-receptor (sTNF-R) during infection with RSV. Our data showed that A549 cells secreted IL-6 and IL-S in a time- and RSV-dose dependent manner. Northern blot analysis revealed that the increased IL-S release was accompanied with an elevated cytoplasmic IL-S mRNA level. In contrast, TNFa was not detected in cell supernatants of infected A549 cells; but an increased release of the sTNF-RI was measured following infection. The sTNF-RII was not detected. In addition, we investigated the IL-S release

24th Meeting of the Society of Immunology . 167 of A549 cells in the presence of the cytokines IL-la/~, TNFa/~, IL-3, IL-6, IFN-y, and the colony-stimulating factors G-CSF and GM-CSF. With exception of IL-6, all investigated factors modulated the IL-S release of RSV -infected and noninfected A549 cells in a specific manner. These data suggest that the airway epithelium plays an important role during the onset of RSV-infection with regard to cytokine release. TGF~,

Dept. of Immunology, University ofUlm, Ulm, Germany

L.3 Immunopathological mechanisms in the liver of ~2-microglo­ bulin deficient mice during Listeria monocytogenes infection j. ARNOLDI, S. THOMA-USZYNSKI, C. LADEL,j. DIONYSIUS-BECKH, and S. H. E. KAUFMANN In the liver of normal mice, i.v. infection with Listeria monocytogenes (L.m.) induces a complex cellular immune response, histologically manifested by infiltrative granulomalike lesions, consisting of CD4+ and CDS+ T-cells. Using ~2m-l- mutant mice, deficient in the ~2-microglobulin as part of the MHC class I molecule, and consequently in functional CDS+ T-cells, we have characterized the impact of ~2m dependent mechanisms on tissue reactions during listeriosis. As revealed by CFU analysis, ~2m-l- mutant mice showed enhanced susceptibility to L.m. infection. Immunopathological tissue reactions, mainly represented by perivascular infiltrations persisted for a long time, indicating delayed and incomplete focussing of the specific immune response. Granuloma formation, used as indicator of cell-mediated immunity was not detected before day 21. Interestingly, in secondary infections, formation of small granuloma-like lesions developed similar to those in control mice. As demonstrated by immunohistochemical analysis, lesions as well as liver parenchyma contained some CDS+ T -cells; most of which expressed the y/o T-cell receptor. Our results indicate that ~2m deficiency leads to enhanced susceptibility to the intracellular pathogen L.m. The ability to develop granulomas was delayed but not abolished by this immune defect. Therefore, ~2m independent mechanisms, probably involving y/o T -cells may compensate for the lack of ~2m - and consequently for CDS+ a/~ T-cell deficiency.

Institut fur Hygiene und Mikrobiologie, Universitat Wurzburg, Wurzburg, Germany

L.4 Yersinia enterocolitica infection in resistant and susceptible strains of mice: a possible essential role of interferon gamma 1. B. AUTENRIETH, M. BEER, E. BOHN, and]. HEESEMANN Y. enterocolitica is an invasive enteropathogenic microorganism causing intestinal and extraintestinal diseases. We could demonstrate that T lymphocytes are involved in and required for overcoming primary Yersinia infection. In the present study we show that by day 7 and 10 postinfection (p.i.) splenic T cells from Yersinia-resistant C57BLl6 mice can transfer immunity into naive mice while the transfer of T cells from Yersiniasusceptible Balb/c mice does not mediate protection. While T cells on day 7 p.i. from C57BLl6 mice exhibit a marked proliferative response and do produce significant

168 . 24th Meeting of the Society of Immunology quantities of interferon gamma upon stimulation with heat-killed yersiniae in vitro, Yersinia-specific T cells from Balb/c exhibit a merely weak proliferative response and do not produce interferon gamma. In line with these results we could demonstrate that resistance to Y. enterocolitica could be abrogated by in vivo neutralization of interferon gamma in resistant C57BL/6 but not in susceptible Balb/c mice. On the other hand, administration of recombinant interferon gamma rendered Balb/c mice more resistant to Y. enterocolitica while this treatment did not affect the course of infection in C57BL/6 mice. These results indicate that the different regulation of the T cell response and interferon gamma production is related to susceptibility of mice to Y. enterocolitica.

First Dept. of Medicine and Dept. of Anatomy, University of Mainz, Mainz, Germany

L.S In situ analysis of superantigen-induced cytokine burst M. BETTE, M. SCHAFER, E. WEIHE, and B. FLEISCHER The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain bacterial infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal Balb/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for IL-2, y-IFN, TNF-a and TNF-~ were not expressed at detectable levels in tissues of unstimulated animals but became visible in the spleen already 30 min after i.p. application of 50 [!g staphylococcal enterotoxin B. All mRNAs levels showed peak expression around 3 h after injection and a slow decrease up to 24 h p.i. Expression of the mRNAs was restricted to the T-cell dependent area of the peri arteriolar lymphatic sheets of the spleen. Interestingly, TNF-a mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-a mRNA showed a broader distribution indicating a second cell population producing TNF-a. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage-depleted mice. Only the second phase of TNF-a mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of Cyclosporin A. These data show that non-phagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF-a is T cell derived.

Dept. of Pediatrics, University of Leipzig, Leipzig, Germany

L.6 Phagocytic activities of neutrophils from healthy and high risk neonates - influence of intrauterine growth retardation M. BORTE, G.-M. KRAUSE, C. VOGTMANN, and W. BRAUN The increased frequency of bacterial infections during the neonatal period is multifactorial but is principally due to developmental deficiencies of the neonatal immune system. One of the most important appears to be in the function of the polymorphonuclear

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leukocyte (PMN). Numerous studies have shown subtle functional abnormalities of neutrophils from term and preterm neonates, but only little information is available regarding the influence of intrauterine growth retardation (IUGR) on phagocytic activities of neutrophils from small-for-gestational-age (SGA) neonates. Using a microassay first we studied the uptake of non-living particles (cadmium-microcrystals, CM, and microspheric hydrophile particles, MSHP) by glass-adherent PMN from 37 SGA term neonates and 46 term neonates. Simultaneously we studied in 11 SGA respectively 10 term neonates the adherence and luminol-dependent chemiluminescence (CL) of separated neutrophils. In PMN from SGA neonates the phagocytosis of CM and MSHP was significantly decreased. In addition adherence of neutrophils to nylon fibre was decreased in cells from SGA when compared with term neonates. Phagocytosis-associated CL during uptake of opsonized zymosan was also reduced in neutrophils from SGA compared with term neonates. To evaluate the degree of IUGR, the differences between the infant's birthweight and that of the 10th percentile on the LUBCHENCO intrauterine growth curve were calculated. We found a strong correlation between the impaired phagocytosis of neutrophils from SGA neonates and the degree of IUGR (r= -0.6006, p < 0.01). We believe, that these abnormalities of neutrophils from SGA neonates could be a cause for their increased susceptibility to infections.

IDept. of Pediatrics and 2Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany

L.7 Lymphocyte surface markers in preterm neonates compared with term neonates M. BaRTEl, I. LEHMANN2, and S. ARNOLDI The neonatal host defense system is influenced by various perinatal risk factors. One of the most important is related with the problem of prematurity. Although numerous investigators reported of cellular immunologic parameters in term newborns, only little information is available regarding the preterm newborn. In previous studies we could demonstrate that various phagocytic parameters of neutrophils were significantly lower in preterm neonates as compared with term neonates. Now using flow cytometry we studied lymphocyte subpopulations (CD3, CD4, CDS, HLA-DR in CD3, CD19, CD16 + CD 56) in peripheral whole blood samples form preterm and term neonates. We found significant differences in the number of T lymphocytes between both study groups. In preterm neonates we found decreased numbers of activated T cells compared with term neonates. In addition, we observed enhanced T4/TS-ratio in prcterm neonates, caused by an increased share of T helper cells. No differences were found in the B- and NK-cell population. Striking was the very decreased number of non-MHC-restricted cytotoxic T cells (CD3+ICD56+ICD16+) in preterm neonates. Between small-forgestational-age (SGA) preterm neonates and appropriate-for-gestational-age preterm neonates we observed no differences regarding to lymphocyte subpopulations, while in a previous study we found a strong correlation between impaired phagocytosis of neutrophils from SGA neonates and the degree of intrauterine growth retardation.

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Dept. of Immunology, University of Ulm, Ulm, Germany

L.S Antigen responses of Listeria monocytogenes specific T lymphocytes during primary and secondary infection S. DAUGELAT, B. SCHOEL, and S. H. E. KAUFMANN Murine listeriosis represents an experimental model for intracellular bacterial infections. Cells of the natural immune system as well as specific CD4+ and CD8+ T lymphocytes contribute to host defense. Only little is known about the relative importance of the antigens of L. monocytogenes for specific protection. In order to develop new strategies of vaccine design against intracellular bacteria we used this system to decide whether distinct proteins are predominantly recognized by T cells and have protective character or whether multiple antigens are seen. Proteins of L. monocytogenes were separated by native 2D-electrophoresis and transferred into 480 liquid fractions by electroelution «< Tcell blot») (1). Antigen specific T cells from spleens of infected C57Bl!6 mice were cultured with these fractions in proliferation assays and a growth pattern was obtained by labelling cells with JH -thymidine. In parallel, culture supernatants were screened for IFN-y, IL-4 and IL-I0 in order to obtain further information about the effector functions of the cells. Comparison of proliferation and cytokine patterns lead to the following results: a multitude of antigens is recognized during primary as well as secondary infection rather than unique dominant antigens. The strength of the proliferative T cell response depended on the stage of infection and correlated with IFN-y secretion. IL-4 could not be demonstrated indicating a major role for TH1-type cells. The IL-10 secretion pattern is currently investigated. In addition, it is attempted to define protein ligands capable of stimulating nonspecific elements of early host resistance, i.e. NK cells and macrophages. 1. H. GULLE et al. (1990):

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Imm. Meth. 133, 253-261.

Institute of Immunology and Transfusion Medicine, University of Lubeck, Medical School, Lubeck, Germany

L.9 Stimulation with mitogens and superantigens is differently affected by zinc C. DRIESSEN, L. RINK, and H. KIRCHNER

Zinc is an important trace element for maintenance of the immune response. Lipopolysaccharide (LPS) has a direct role in the pathogenesis of gram-negative bacterial septicemia; Toxic Shock Syndrome Toxin 1 (TSST -1) is a superantigen produced by Staphylococcus aureus, Mycoplasma arthritidis-derived superantigen (MAS) causes synovitis in rats. We tested the influence of zinc on the cytokine production of PBMC after stimulation with LPS, TSST-l, MAS and Phytohaemagglutinin (PHA), which are potent inducers of cytokine release in vitro. Interestingly, we found a different effect of zinc on stimulation with superantigens compared to LPS and PHA. Addition of ZnS04 ranging between 0.05 mM and 0.25 mM led to an up to three-fold increased level of TNF-a, IL-l/3 and IFN-y when PBMC were stimulated with PHA or LPS, while CUS04 controls showed no stgnificant effect. In contrast, after stimulation with TSST -lor MAS, zinc led to a dose-dependent decrease of TNF-a, IL-1 ~ and IFN -y. Cytokine levels

24th Meeting of the Society of Immunology . 171 reached only 10 % of the control values without zinc. The different effect of zinc on stimulation with PHA or LPS and superantigens suggests that zinc is involved in different immunological pathways. Either surface binding of superantigens or the subsequent signal-transduction might be affected by zinc.

Max-Planck-Society, Research Units for Rheumatology, Erlangen; 1Institute of Hygiene and Microbiology, University Hospital, Wurzburg; 2Max-Planck-Institute for Biochemistry, Martinsried, Germany

L.10 Characterization of the yersinia outer membrane protein YadA mediated yersinialcollagen type IV interaction A. FLUGEL, H. SCHULZE-Koops,J. HEESEMANN 1, K. KUHN2 , and F. EMMRICH Yersinia enterocolitica is enteropathogenic for humans. It has been shown that a 70 kb plasmid is necessary for full virulence expression in Yersinia. The most prominent plasmid encoded outer membrane protein YadA mediates binding to different types of collagen including collagen type IV which is one of the major components of basal membranes. Interaction of microorganisms with basal membranes is very likely to be a crucial step in initiating infectious diseases. In order to elucidate YadA/collagen type IV interaction we used recombinant Yersinia strains expressing the cloned YadA gene. Collagen type IV was purified from human placenta. Digestion of type IV collagen with different proteases resulted in several fragments spanning the entire protein. In adhesion assays with radiolabeled collagen fragments YadA positive Yersinia were found to bind specifically to the 58 nm N-terminal fragment 7s1. In contrast, Yad negative Yersinia did not bind to any of the fragments. Binding of the 7s1 fragments could be inhibited by type II collagen indicating similar binding sites in both proteins. The C-terminal globular NC1 domain of collagen type IV and the 30 nm N-terminal 7s fragment lacking the Cterminal aminoacids of the 7s1 had only minor binding capacities and were unable to block 7s1!YadA interaction. Binding of 7s1 to YadA was rapid and almost complete after 30 minutes. The interaction was stable over a broad pH range and independent on bivalent cations. Based on these findings, further enzymatic fragmentation of the 7s1 fragment may allow to define peptide sequence(s) relevant for YadA binding and might enable us to answer the question whether YadA/collagen type IV interaction contributes to virulence of enteropathogenic Yersinia.

Abt. Virologie, Universitat Ulm, Ulm, Germany

L.ll Naturally processed peptides from cytomegalovirus infected organs G. GEGINAT, H. HENGEL, T. RUPPERT, and U. KOSZINOWSKI Host resistence to murine cytomegalovirus (MCMV) infection is mediated by virus specific CD8+, CD4- T-Iymphocytes (REDDEHAsE et al. J. Virol. 61, 3102). An immunodominant antigen recognized by protective CTL has been defined as the nonapeptide YPHFMPTNL from the MCMV iel protein which is presented by the

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MHC I Ld molecule and is extractable from in vitro infected cells (DEL VAL et aI., Cell 66, 1145). We analyzed the tissue distribution of the nonapeptide in MCMV -infected Balb/c mice. Mice either sublethally irradiated or not irradiated (immunocompetent) were infected with MCMV, and organs were collected from different times post infection. The peptide and the virus load were determined simultaneously. HPLC fractionation of acidic organ extracts from selected target tissues relevant for MCMV infection yielded positive fractions when tested with a nonamer-specific T -cell line. The major active fraction co eluted with the synthetic nonapeptide YPHFMPTNL. Immunogenic peptide was detectable already 24 h post infection and disappeared after virus clearance. The peptide yield from organs did not correlate with virus load. In salavary glands peptide was detectable in the early stages of infection but not later (10 days post infection) despite of an increasing virus load. The discrepancies between virus replication and antigen presentation are discussed.

Dept. of Medical Immunology, Medical Academy Magdeburg, Magdeburg, Germany

L.12 Expression and purification of the 34 kilodalton immunogen of Treponema pallidum A. GERBER, S. KRELL, H. STRUY, and]. MORENZ Integral membrane proteins of pathogenic treponemes are highly immunogenic proteins. One clone from a library of Treponema pallidum DNA, designated pSK1, was used for the amplification of the 34 kD immunogen. The PCR product was inserted into pUC1S and subcloned into the expression vector pTrc 99A. The inducible expression of the recombinant 34 kD antigen was detected by Western blotting. A distinct band with an apparent molecular weight of 34 kD corresponded to the precursor molecule. After cleavage of the leader peptide the acylated form migrated as a smear in the molecular weight range of 29-32 kD. Coomassie brillant blue stained SDS polyacrylamide gels showed two weak bands of corresponding molecular weight. After phase partitioning with the nonionic detergent Triton X-114 the 34 kD detergent phase protein was purified to near homogeneity by gel filtration, ion exchange chromatography, preparative electrophoresis, and electroelution. The purified recombinant 34 kD antigen might be useful in combination with other strong immunogens for the serodiagnosis of syphilis.

Forschungsinstitut Borstel, Dept. of Immunology and Cell Biology, Borstel, Germany

L.13 Modulation of T-cell costimulatory antigens on human monocytes following stimulation with Mycobacterium tuberculosis ]. GERCKEN, H.-D. FLAD, and M. ERNST Recently we have shown that Mycobacterium tuberculosis (M.tb.) is preferentially phagocytosed by a major subpopulation of monocytes exhibiting a lower HLA-DR antigen density, whereas cells belonging to a minor subpopulation with high HLA-DR expression usually do not take up bacteria. In the present study our interest was focussed on the phenotypic modulation of the T-cell costimulatory surface molecules LFA-3

24th Meeting of the Society of Immunology . 173 (CD58), ICAM-l (CD54) and B7/BBl on either monocyte subpopulation following stimulation with M.tb. Highly purified monocytes were derived by counterflow elutriation and stimulated with viable FITC-Iabelled M.tb. (H37Rv). By means of 3-color flowcytometry analysis we were able to determine simultaneously the expression of HLADR and of a costimulatory antigen on active phagocytes (FITC-M.tb. positive) and bystander (FITC-M.tb. negative) monocytes. We found that M.tb. induces a downregulation of the antigen density of LFA-3, ICAM-l and B7/BBl on active phagocytes, especially on those which had ingested many bacteria. On the other hand a downregulation of antigens was not observed for the small subpopulation of phagocytically inactive monocytes showing a high HLA-DR expression. Here, with regard to LFA-3 and B7/BBl the percentage of antigen-positive monocytes increased during mycobacterial stimulation, whereas it remained high in case of I CAM -1 expression. These results support our previous suggestion that monocytes can functionally by divided into a large fraction of «professional phagocytes» the antigen-presenting capacity of which is suppressed by M.tb., and a small fraction of monocytes more specialized for the presentation of antigenic pep tides and the activation of T-cells. This work was supported by BMFT, grant no. 01KI 8827.

Ilnstitut fur Klinische Mikrobiologie der U niversitat Erlangen- N umberg, Erlangen; 2Behringwerke AG, Marburg, Germany

L.14 Soluble IL-4 receptor acts as a physiological antagonist of IL-4 in murine leishmaniasis A. GESSNER I, K. SCHROPPEL I, A. WILLI, K. ENSSLE 2, L. LAUFfER2, and M. ROLLINGHOPpl The neutralization of IL-4 by monoclonal antibody has previously been shown to be one possible way for induction of protective immune responses towards infection with Leishmania major in otherwise susceptible BALB/c mice. Conversely, even high doses of recombinant IL-4 can not reverse the resistant phenotype of C57BL/6 mice. Here we show that not the expression of IL-4 alone but the balance between the endogenously produced soluble IL-4 receptor and its ligand determines the phenotype of immune response against L. major. While concentrations of IL-4 in sera of infected C57BLl6 and BALB/c rose to comparable levels during the first 2 weeks after infection only in sera of resistant C57BLl6 mice the soluble IL-4 receptor, a potential IL-4 antagonist, was detectable. In addition, it is demonstrated that the susceptible phenotype of BALB/c mice can be reverted to resistance by systemic application of recombinant soluble IL-4 receptor.

174 . 24th Meeting of the Society of Immunology Dept. of Medical Immunology, Medical Academy Magdeburg, Magdeburg; lRobert Koch Institute of the Federal Health Office Wernigerode, Wernigerode, Germany

L.15 Plasmid-dependent serum resistance of isogenic strains of Escherichia coli K12 and Salmonella fyphimurium S. GLOCKNER, H. STRUY, H. TSCHAPE 1, and]. MORENZ Virulence plasmids encode pathogenic factors of microorganisms. The influence of two virulence plasmids of S. typhimurium, a 90 Md plasmid pathogenic for man and two isolates (tra+ and tra-) of a 60 Md plasmid pathogenic for animals, were investigated for their influence on the serum resistance of bacteria. Bacterial survival rates were detected by incorporation of 75Se-methionin. Total complement activity was compared with activity of the classical and alternative pathway with and without antibodies. E. coli K12 showed a plasmid-dependent increase of serum resistance in both pathways alone but not against total complement. S. thyphimurium showed an increased resistance versus alternative pathway activation without absorption and against total complement. The increase of serum resistance was more pronounced with the 60 Md plasmid than with the 90 Md plasmid. With the tra+ isolate of the 60 Md plasmid a high increase of serum resistance was detected.

Institut fur Chemotherapie, Bayer AG, Wuppertal, Germany

L.16 Influence of muramyldipeptide or lipopolysaccharide on prostaglandin-E2 synthesis in SCID mice A. C. GRUBERT and H. BRUNNER Mice with severe combined immunodeficiency (C.B-17, SCI D) exhibit phagocyte mediated resistance towards infections but lack functionally competent T and B lymphocytes. The bacterial cell wall components, muramyldipeptide (MDP) and lipopolysaccharide (LPS) are effective activators of macrophages. Nonspecific resistance against infections with various bacteria could be enhanced in SCID-mice by prophylactic administration of LPS or MDP. The activation of peritoneal macrophages of SCID-mice by MDP or LPS was then examined in vitro by determination of the amount of released prostaglandin-E2 (PGE2) using a radioimmunoassay as compared to immunocompetent C.B-17-mice. MDP induced a significant increase in PGE2-release in vitro. Macrophages from C.B-17mice showed a similar response. Indomethacin completely inhibited PGE2-release. Incubation of cells with MDP together with interferon-gamma resulted in an increased PGE2-production. As expected, cells incubated with LPS secreted more PGE2 than those incubated with MDP. Thus, macrophages of SCID-mice showed a good in vitro response to cell wall components of bacteria. The data support the concept that the SCID-mouse represents a suitable model to study the effects of immunomodulators on nonspecific host defense.

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Dept. of Med. Immunol., Medical School (Charite), Humboldt-University, Berlin, Germany

L.17 Selection and characterization of a human anti-P24 19M hybridoma A. HANSEN, D. ROGGENBUCK, T. PORSTMANN, R. VON BAEHR, and L. FRANKE Human PBMC obtained from a HIV-I infected donor during seroconversion were immortalized by PEG-mediated fusion to the heteromyeloma cell line CB-F7. Regarding the protective action of murine monoclonal antibodies (mabs) to the major core protein p24 in previous in-vitro studies, cell culture supernatants were screened for anti-p24 reactivity by ELISA. The anti-p24 reactive hybridoma CB-6Al with stable Ig-production was further characterized after limiting dilution cloning and scaling up. Supernatant derived from CB-6Al (IgM/lambda) reacted with different auto- and foreign antigens (ssDNA, heparan sulfate, diphtheria toxin, keratin, TNF, staphylolysin, chondroitin sulfate) when tested against a broad panel of antigens by ELISA. Enzyme immunoassays (ELISA, Western-Blot) using purified 6Al-antibody are in progress to verify this polyreactive binding pattern. Analysis of the immunoglobulin variable region genes expressed in CB-6Al revealed nearly germline-identical VHIII (VH26)/VlambdaI genes. The characteristics of the 6Al-antibody are discussed in contribution to the known polyclonal B-cell activation caused by HIV -infection. It may be suggested that an activation of B-cells from the natural repertoire is involved in anti-viral first line defense mechanisms and/or acts as a basis for the selection of mono reactive anti-HIV antibodies after somatic mutation.

lFraunhofer Institute for Toxicology, Hannover; 2Drug Research Center, Bayer AG, Wuppertal, Germany

L.18 The therapeutic effect of acetylsalicylic acid on the Listeria monocytogenes infection S. HOCKERTZl, K. ROGALLA 2 , and T. SCHETTLER 2 The influence of acetylsalicylic acid (ASA, CAS 50-78-2) on the Listeria monocytogenes infection in balb/c mice was investigated. One day before lethal or sublethal infection, balb/c mice were treated intravenously with therapeutic concentrations of ASA alone or in combination with murine recombinant interferon gamma, a lymphokine from Thelper cells. Three days post infection, parasite burdens of spleen and liver were determined by the colony forming unit assay. It was shown that the prophylactic application of ASA in a concentration of 5 mg/kg body weight resulted in a more than lO-fold reduction of viable Listeria monocytogenes in spleen and liver of balb/c mice. In addition, the combination of a suboptimal dosage of interferon gamma with ASA resulted in a significant higher survival rate compared to the untreated controls. These results are discussed according to the current opinion, that ASA has the capability to enhance the production and secretion of interferon gamma in vitro and in vivo.

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Institute of Clinical Microbiology, Erlangen, Germany

L.19 A fraction of cytosolic 21 kDA proteins from Leishmania maior is a potent T cell stimulator A. HOERAUF, M. ROLLINGHOFF, and W. SOLBACH The aim of this study is to identify antigens that playa critical role in the experimental infection of mice with Leishmania major. Therefore, soluble leishmanial antigen, i.e. a mixture of cytosolic proteins obtained after ultracentrifugation of a Leishmania major lysate, was separated according to molecular weight using SDS-PAGE. A fraction containing proteins in the molecular weight range of 21 kDa (F21) was found to specifically stimulate splenic cells from L. major infected mice and a L. major specific T cell clone (LI/1). Further purification of the antigenic fraction using twodimensional electrophoresis revealed five protein spots in the range of pH 5.5-7, which are stimulatory for L1I1 cells. Thus, the stimulatory protein has several isoelectric points most likely due to posttranslational modification. Finally, preliminary data suggest that F21 induces protection in vivo: Genetically susceptible BALB/c mice immunized with 3 x 50 Ilg of F21 prior to infection with 1 x 106 Leishmania major parasites into the right footpad don't develop ulcerous lesions and survive infection for more than one year. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 263/A1).

IMax-Planck -Institut fur Immunbiologie, Freiburg; 2 Abteilung fur angewandte Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany

L.20 Structural and functional analyses of a Borrelia burgdorferi mitogen(s) N. HONARVAR I, U. E. SCHAIBLE\ C. GALANOS\ H. HOSCHOTZKY\ R. WALLICH 2 , and M. M. SIMONI The spirochete Borrelia burgdorferi (B. burgdorferi) is the causative agent of Lyme disease, a tick borne infection in human. Recent studies in a mouse model have shown that experimental inoculation with spirochetes leads to specific cellular and humoral immune responses to various antigens including the outer surface lipoproteins A and B (OspA and OspB) as well as the flagellum associated protein Flagellin (fla). Preliminary findings that lymphocytes from both infected and naive mice exhibited similar proliferative responses to B. burgdorferi in vitro indicated that spirochetes have potent mitogenic activity. Further analysis demonstrated that among lymphocytes, B-cells are the main target for this activity. Quantitative analyses revealed frequencies of B-cells responding to B. burgdorferi preparations with IgM and IgG secretion similar to those obtained with LPS. However, the finding that the B. burgdorferi mitogen activated B-cells from both LPS susceptible and -resistant strains of mice similarly suggested that the respective structure(s) is distinct from LPS. Recombinant OspA and OspB were shown to be unable to induce non-specific proliferation of B-cells. Pretreatment of preparations with proteases (pronase or proteinase K) did not eliminate the activity. B. burgdorferi

24th Meeting of the Society of Immunology . 177 structures enriched by Phenol-chloroform-petroleum ether (PCP) extraction were found to express mitogenic activity similar to that of the B. burgdorferi lysate. Further purification of the PCP-material by SDS-PAGE revealed the presence of two major mitogenic structures with molecular masses in the range of 14 KD and 3 KD, which are presently characterized.

Institute of Medical Microbiology and Infectious Diseases Immunology, Freie Universitat Berlin, Berlin, Germany

L.21 Mycobacterium bovis (BCG)-induced immunomodulation of OTH to SRBC results in C011 b-independent, C04+ T cellmediated myelomonocytic cell recruitment R. IGNATIUS, M. E. A. MIELKE, and H. HAHN BCG-infected mice develop delayed-type hypersensitivity (DTH) in response to doses of sheep red blood cells (SRBC) that cause suppression of DTH in non-infected mice. However, this BCG-modulated DTH differs qualitatively from that in non-modulated mice. Although both are exclusively mediated by CD4+ T cells, significant differences were shown in the mechanisms of myelomonocytic cell invasion: while the expression of non-modulated DTH to SRBC was markedly inhibited by anti-CDllb mAb, BCGmodulated DTH resisted to such treatment. It is concluded that one of the immunomodulating effects of BCG is based on the induction of a qualitatively different SRBC-specific CD4+ TDTH cell, able to induce myelomonocytic cell recruitment by a CDII b-independent mechanism.

Institute of Clinical Microbiology, Universitat Erlangen-Niirnberg, Erlangen, Germany

L.22 Expression of the IL-1-receptor-antagonist (IL-1 RA) in murine yersiniosis M. JORDAN, M. ROLLING HOFF, and H. U. BEUSCHER In patients with infectious diseases, plasma levels of IL-l are elevated. The IL-IRA is a naturally occurring inhibitor of IL-I which competes with IL-l for occupancy of IL-I receptors. Here we examined the expression of the IL-IRA in mice after oral infection with the enteropathogenic bacteria Yersinia enterocolitica 08. In Peyer's Patches (PP), the local site of infection, induction of the IL-IRA mRNA was temporarily delayed when compared to IL-I (a and ~) mRNA. Expression of IL-lmRNA was also detectable in other organs, e.g. spleen but not in the liver. When activated macrophages were subjected to immunofluorescent staining using antibodies to IL-Ia, IL-l~ and IL-IRA, about 1 % of the cells produced all three proteins, 5 % produced IL-Ia and IL-IRA, and no cells were found to produce IL-I RA in combination with IL-I ~ alone. The results indicate that expression of IL-I ~ and IL-IRA are regulated differentially and further suggest that IL-I a and IL-I RA are produced by the same cell type, i.e. aged macrophages.

178 . 24th Meeting of the Society of Immunology Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Hannover; 1Institut fur Medizinische Mikrobiologie, Universitat Dusseldorf, Dusseldorf, Germany

L.23 Characteristics of interferon induced tryptophan metabolism in human macrophages in vitro M. KIEKENBECK, F.-C. BANGE, U. VOGEL, W. DAUBNER 1, and E. C. BbTIGER Interferon gamma has potent growth-inhibitory effects on intracellular parasites, such as Toxoplasma gondii, Chlamydia psittaci and Chlamydia trachomatis. This effect has been attributed to the intracellular depletion of tryptophan as IFNy induces expression of indoleamine 2,3-dioxygenase (IDa). More recently, we have demonstrated that IFNy additionally affects tryptophan metabolism by mediating expression of tryptophanyltRNA synthetase (IFP 53) and suggested that induction of tryptophanyl-tRNA synthetase may represent a defense mechanism for rendering residuable tryptophan accessible for host cell protein synthesis to exclusion of the intracellular parasite. In this study, we have investigated the regulatory effects of IFNy on IDa and IFP 53 expression in human macrophage cell lines (U937, HL-60, KG-I, THP-I) both at the level of mRNA and at the level of functional enzyme activity. Our results demonstrate that these two genes although functionally linked - are differentially regulated as treatment of cells with IFNy readily induced IFP 53 expression, but did not affect IDa. Induction of IDa by IFNy required pretreatment with phorbol ester resulting in differentiation of the cells and was only seen in THP-I cells. These results point to a potential additional function of IFP 53 besides tryptophanylation of tRNA.

Med. Microbiology and Immunology, Ruhr-Universitat, Bochum, Germany

L.24 Immunosuppressive activity of Helicobacter pylori: effects on isolated lymphocytes and monocytes U. KNIPP, S. BIRKHOLZ, W. KAUP, and W. OPI'ERKUCH

Helicobacter pylori (H.p.) colonization of the human gastric mucosa causes a longterm, not self-limiting inflammation, suggesting that the microbe has properties to protect itself against the host immune defense. Recently we were able to demonstrate that H.p. suppresses the in vitro proliferative response of human peripheral blood mononuclear cells (PBMC) activated by mitogens as well as by antigens. The aim of this study was to clarify which cell subsets of PBMC were influenced by the immunosuppressive activity of H.p. The use of monocytes which had been pretreated with a soluble cytoplasmic fraction of H.p. led to a suppressed proliferation of T cells after PHA-activation. This effect could be neutralized by the addition of untreated monocytes, suggesting that monocyte accessory functions are altered by the suppressive activity. Activation of PBMC or of isolated T cells with PHA in combination with PMA revealed that the proliferative response of lymphocytes could be also inhibited by H.p. independent of monocytes. Furthermore, FACS~analysis of PHA/PMA-activated lymphocytes demonstrated a significant reduction of IL-2 receptor expression as well as an inhibition of blastogenesis of these cells in the presence of H.p. FACS-analysis of HLA-DR, CDl4 and ICAM-I expression on the surface of monocytes revealed an influence of H.p. on

24th Meeting of the Society of Immunology . 179 CD14 expression, while the expression of HLA-DR and ICAM-l antigens was not altered in the presence of the bacterium. The effect of H.p. on the CD14 expression was heat-labile, suggesting that it is not lipopolysaccharide mediated. Although the immunologic mechanisms involved in H.p.-associated gastritis are not yet clearly defined, it is reasonable to presume that alteration of MNC functions may contribute to the pathogenesis of this disease.

1Dept. of Rheumatology and 2Dept. of Molecular Pharmacology, Medical School, Hannover, Germany

L.25 U937 cells as a model for infection of monocytic cells with

Chlamydia trachomatis

L. KClHLER 1, E. NETTELNBRECKER I, H. HOLTMANN2, and H. ZEIDLER 1 Infection with Chlamydia trachomatis (Chl.tr.) is the most common sexually transmitted disease in the Western world. Acute urethritis and cervicitis might result in chronic inflammation such as Chlamydia-induced arthritis or tubal obstruction with infertility in women. U937 cells were used as a model to investigate a persistent infection of Chl.tr. in monocytic cells. Undifferentiated U937 cells and U937 cells differentiated with the phorbol ester TPA were inoculated with Chl.tr. serovar K at doses which induced formation of inclusion bodies in virtually all cells of the Hep-2 culture. In TPAdifferentiated U937 cells inclusion bodies were observed starting at day 2 of the 14 days cultivation period and reaching its maximum at day 7 to 10. At that time approximately 0.5 to 2 % of the cells contained inclusion bodies. In contrast in undifferentiated U937 cells only at day 2 and 3 of the cultivation period inclusion bodies were observed in 0.005 % of the cells by immuno-fluorescence. The data were consistent with the detection of chlamydial RNA in the culture and infectiousness on Hep-2 cells. In summary like in human peripheral blood monocytes most of the chlamydia show a low level of infection. Thus U937 cells might serve as an interesting model for investigating the interaction of Chl.tr. with monocytic cells especially in view of a replicative and non replicative infection as well as the state of persistence.

Med. Mikrobiologie Immunologie, AG Infektabwehr, Ruhr-Universitat Bochum, Bochum, Germany

L.26 Induction of suppression of inflammatory mediator release two strategies of Pseudomonas aeruginosa B. KONIG, P. FRIEDL, and W. KONIG Pseudomonas aeruginosa is known as an opportunistic pathogen in immunosuppressed patients. We focussed on P. aeruginosa isolated from Cystic Fibrosis (CF) as well as from burn patients. While P. aeruginosa isolated from burn patients induced chemiluminescence response, leukotriene B4 , histamine release from human inflammatory cells, CF strains, expressing a pronounced mucoid phenotype, induced no release of the abovementioned inflammatory mediators. Moreover, CF patients impaired the host response

180 . 24th Meeting of the Society of Immunology towards external stimuli. In further experiments interleukin-8 (IL-8) release, a cytokine with chemotactic properties, was monitored. In human neutrophils as well as in a lymphocyte/monocyte/basophil cell suspension performed IL-8 was present in the cytoplasm; IL-8 mRNA was constitutively expressed. P. aeruginosa strains from CF patients as well as from other sources induced IL-8 release (up to 30ng/l0 7 cells) in a time- and dose-dependent manner. IL-8 release was observed after an incubation time of 60 min and increased over time (4 h). An increase in IL-8 specific mRNA was observed after an incubation time of 60 min and prolonged up to an incubation time of 18 hours. Our results clearly indicte a distinct regulation of the diverse inflammatory mediators. In dependence of the mediator release pattern the outcome of infection may differ as is well documented by two severe types of P. aeruginosa infection, the chronic cystic fibrosis and the acute wound sepsis.

Institut fur Med. Mikrobiologie und Immunologie, Ruhr-Universitat Bochum, Bochum, Germany

L.27 Influence of staphylococcal enterotoxins (A, B) on the in vitro immune response of patients with atopic dermatitis and healthy donors C. KOERNER, u. STEPHAN, and W. KONIG

Patients suffering from atopic dermatitis (AD) frequently show a skin colonization with Staphylococcus aureus. S. aureus strains isolated from the skin of AD patients have been shown to secrete superantigenic exotoxins, e.g. Staphylococcal enterotoxin A (SEA), B (SEB) and toxic shock syndrome toxin 1 (TSST -1 ). We investigated the potential role of SEB and SEA in the inflammatory skin reaction and elevated IgE-level of AD patients. The influence of SEB/SEA on proliferation, Ig-synthesis, expression of the low-affinity receptor for IgE CD23 and secretion of its soluble IgE-binding factor sCD23 of the lymphocyte-monocyte-basophil (LMB) fraction from AD patients and healthy blood donors was studied. Both enterotoxins markedly enhanced proliferation and suppressed IgG,A and M-synthesis of LMBs from atopic and healthy donors. SEA and SEB modulated the spontaneous CD23-expression donor-specific (varying between upregulation and suppression) in both donor groups. The IL4-induced CD23-expression of LMBs from both atopic and healthy donors was suppressed by the enterotoxins. SEB/ SEA increased the release of sCD23 of LMBs prestimulated with IL4 and also of unstimulated LMBs from AD patients, but not of unstimulated LMBs from healthy donors. IgE-synthesis was modulated donor-specific. Our data suggest that the preactivation of patients' lymphocytes determines the different response towards staphylococcal superantigens, which may be a pathogenetic mechanism in atopic dermatitis.

24th Meeting of the Society of Immunology . 181 Dept. of Medical Immunology and 1Dept. of Medical Microbiology, Medical Academy Magdeburg, Magdeburg, Germany

L.28 Molecular characterization of the recombinant clone PSKl expressing the «34 KD immunogen» and a 63 KD antigen of

T. pallie/urn

S. KRELL, A. GERBER, E. GRABERT!, H.-L. BORKHARDT!, andJ. MORENZ The clone pSKI was isolated by colony immunoblot from a genomic DNA library of Treponema pallidum in pUCI8. No homologous DNA to the cloned fragment was detectable in T. phagedenis by Southern blotting. The expression of T. pallidum antigens of 37 kD and 63 kD was shown by Western blotting and by in vitro transcription translation. The antigen coding regions of the insert were determined by deletion cloning. DNA sequencing revealed the identity of the 37 kD antigen of pSKI to the 34 kD immunogen (NORGARD et al.) and to TpD (SCHOULS et al.). Using affinity purified antibodies against the recombinant antigens the corresponding proteins of T. pallidum were identified. According to the designation of T. pallidum proteins by NORRIS et al. the recombinant 37 kD-antigen corresponds to protein m and the recombinant 63 kD antigen to protein e. Protein e is believed to be a member of a group of homologous heat shock proteins detected in various bacteria species (e.g. GroEl of E. coli). The 37 kD antigen is one of the highly immunogenic detergent phase proteins specific for pathogenic Treponema species. The demonstrated reactivity of patient sera from all stages of syphilis shows possible application of the 37 kD antigen in serodiagnosis of syphilis.

Institut fur Biochemie der Medizinischen Fakultat (Charite) an der Humboldt-Universitat zu Berlin, Berlin, Germany

L.29 Generation of recombinant human monoclonal antibodyfragments against HBV using a phage display library in

E. coli

G. KUTTNER, E. GIESSMANN, and G. SCHMIDTKE Human antibodies against neutralizing epitopes of the HBV envelope protein are important for preventing the reinfection of patients after liver transplantation. Some epitopes of the HBV env-protein (pre SI-region comprising the amino acids 21-47 and pre S2-region comprising the amino acids 120-145) are known to be inducers of virus neutralizing and protective antibodies. We generated an immune response including these epitopes by vaccination of healthy volunteers using HEVAC-B Paster (Institut Merieux GmH). Lymphocytes from day 7 after the third immunization were used for the generation of a single chain Fv library in E. coli. Using the phage display (MCCAFFERTY et al. 1990, Nature 348, 552-554) the library was enriched and screened with synthetic peptides of the pre SI- and pre S2region. The properties of the resulting soluble antibody-fragments (affinity constants, cross reactivity and sequences of the VH and VL) expressed in E. coli are presented.

182 . 24th Meeting of the Society of Immunology Dept. of Immunology, University of Ulm, Ulm, Germany

L.30 Listeria monocytogenes infection in MHC deletion mutants C. LADEL, C. BLUM, J. ARNOLDI, and S. H. E. KAUFMANN We investigated the role of MHC expression in immunity to intracellular bacteria by using MHC deficient mice strains. Deletion mutants deficient in MHC class I (kindly provided by R. JAENISCH, MIT) or II (kindly provided by D. MATHIS, INSERM) lack functional CD8+ and CD4+ T cells, respectively. These mice were infected with sublethal doses of Listeria monocytogenes and the course of infection assessed by determination of CFU in spleens and livers. Both strains were more susceptible to listeriosis compared with their normal controllittermates. Furthermore liver lesions in both mutants markedly differed from those in normal control littermates. There seem to be a strict correlation between the expression of the MHC molecules, and, therefore, maturation of the different T-cell subsets, and formation of characteristic lesions in the infected organs. Our findings demonstrated the importance of MHC class I or II in listeriosis. Because these molecules represent the necessary restriction elements for CD8+ and CD4+ T-cells, respectively, we conclude that both CD8+ and CD4+ T-cells are required for optimum protection.

Institut fur Klinische Mikrobiologie, Universitat Erlangen, Erlangen, Germany

L.31 Natural killer cells participate in the early defence against Leishmania major infection in mice T. LASKAY, M. ROLLINGHOFF, and W. SOLBACH In this study the role of natural killer (NK) cells in the course of experimental L. major infection was investigated. NK cells in genetically resistant C57BLl6 mice were depleted by in vivo administration of anti asialo-GMl or anti NKl.l antibody. A marked exacerbation of the infection was found in the NK-depleted mice within the first two weeks of infection. Both the local tissue swelling and the number of parasites in the lesions were significantly higher than in animals not depleted for NK cells. Lymph node cells taken from NK-depleted mice released less IFN-y than those taken from nondepleted animals, when cultured in vitro. As an alternative approach we have used poly I:C treatment in order to activate in vivo the NK cell activity of BALB/c mice, which are genetically susceptible to L. major infection. Poly I:C treatment led to milder symptoms and to a significantly lower parasite burden during the early course of infection. Also, lymph node cells from parasite-infected and poly I:C treated BALB/c mice released higher amounts of IFN-y in vitro than cells from infected mice not treated with poly I:C. These data show that NK cells play an important role for the control of parasite multiplication in the early, non-specific phase of the infection.

24th Meeting of the Society of Immunology . 183 Fraunhofer-Institut, Dept. of Immunobiology, Hannover, Germany

L.32 Evidence for development of distinct T helper cell responses in murine Leishmania donovaniinfection

J. LEHMANN, s. HOCKERTZ, and M.-L. LOHMANN-MATTHES In Leishmania major infection it has been shown, that a THI response leads to healing or resistance, whereas a T H2 response causes progressive disease. In visceral Leishmaniasis, induced by Leishmania donovani the situation has not yet been clearly analyzed. To investigate whether a T HI or a T H2 mediated immune response predominates, splenic CD4+ cells derived from healer strain C3H/HeJ and non-healer strain BALB/c were tested for their ability to secrete IFN-y and other cytokines. It was found, that C3H/HeJ spleen cells produced significantly higher titers of IFN-y after stimulation with Concanavalin A at all time points tested compared with BALB/c. Furthermore, as a correlate for IL4 production serum IgE levels were measured over the initial phase of infection up to day 120 p.i. While in BALB/c mice IgE were detected at days 60 and 120 p.i., in C3H/HeJ no IgE were found at any investigated time point. These data imply, that also in Leishmania donovani infection distinct TH cell responses can be developed. C3H/HeJ mice seem to establish a THI mediated immune response which results in a very low parasite burden in spleen and liver. In contrast, BALB/c develop and T H2 response which leads to fatal disease with high parasite numbers in spleen and liver.

Fraunhofer Institute for Toxicology, Dept. of Immunology, Hannover, Germany

L.33 The effect of liposomal antimony therapy on the secretory capacity of macrophages and T-lymphocytes from L. donovaniinfected B10D2/N mice H. LUBBING, P. KLEMPEL, M.-L. LOHMANN-MATTHES, and S. HOCKERTZ Leishmania donovani infected BIOD2/n mice were treated with Pentostam® encapsulated in multilamellar vesicles composed of phosphatidylcholine. This treatment led to a complete parasite reduction in spleen and liver, the most invaded organs in visceral leishmaniasis. The aim of the study was to determine changes in lymphokine production of splenic macrophages and T-lymphocytes from healthy donors, infected untreated and infected treated animals. During the infection IL-l production of splenic macrophages showed a rapid decrease and was unchanged after liposomal Sb treatment. IL-IO, secreted from macrophages, increased to a 2-fold higher amount in cause of a L. donovani infection. After inoculation of liposomal antimony IL-I0 production declined to the value of healthy mice. IL-2, produced by T -helperl-cells, showed a rapid decrease caused by L. donovani infection. Administration of the liposomal drug led to an IL-2 production which was similar to uninfected animals. In conclusion the impairment of the physiological balance of cytokine production from macrophages and T -lymphocytes during visceral leishmaniasis was restored by liposomal antimony therapy.

184 . 24th Meeting of the Society of Immunology Neurologische Klinik der Universitat, Gottingen, Germany

L.34 Alkaline phosphatase-binding antibodies in severe bacterial infections M. MADER and W. BEUCHE Antibodies highly specific to alkaline phosphatase (AP) were found in sera of patients with acute and chronic bacterial infections. They were neither observed in other diseases nor in healthy subjects. These antibodies occurred in about 60 % of the patients studied (n = 284) which suffered from severe bacterial infections. The presence of AP-binding antibodies was independent of the infected organ or the bacterial species causing infection. Using immunoaffinity chromatography and Western blotting or enzymelinked immunosorbent assay (ELISA) the AP-binding antibody was identified as immunoglobulin class G (IgG). Because elution of this IgG from AP coupled to Sepharose was achieved but with high concentrations (3M) of magnesium chloride a high affinity antigen-antibody interaction was suggested. Since the AP-binding IgG shows a monoclonal to oligoclonal pattern upon isoelectric focusing a regulatory function in Bcell proliferation and differentiation, where AP is expressed on the plasma membrane, is assumed rather than an autoimmune process.

Institute of Immunology, Philipps University, Marburg, Germany

L.35 Limited effect of influenza A virus on human C04+ lymphocytes K. MAYER, A. BENDER, D. GEMSA, and M. NAIN The specific cellular immune response against influenza A virus infection is carried out by cytotoxic CD8+ T lymphocytes whose generation depends on help delivered by CD4+ cells. To study a possible direct effect of influenza virus A/PR8 on T helper cells, we negatively selected CD4+ cells by MACS from the mononuclear cell fraction of healthy blood donors. Following exposure of CD4+ lymphocytes to A/PR8, an adsorption of virus to the cell membrane was demonstrated by immunofluorescence. Upon further incubation with and without PWM stimulation, neither viral replication nor de novo viral protein synthesis was observed. However, mitogen-stimulated CD4+ lymphocytes responded to A/PR8 exposure by releasing enhanced amounts of IL-2, TNF, and IFN-y but failed to produce IL-4. These data support the notion that CD4+ lymphocytes per se are non-permissive to influenza A virus replication. However, CD4+ cells apparently react to virus by a selective release of those cytokines which could aid in generating an influenza A virus-specific cytotoxic T cell response.

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Depts. of !Clin. Immunology, 21ntensive Care Unit and 3Nephrology of the Medical Faculty (Charite), Humboldt University, Berlin, Germany

L.36 Functional characterization of an inhibitory factor in the plasma of septic patients with fatal prognosis - new aspects for therapeutic intervention A. MEINECKE!, K. KLUG 2 , H. ZUCKERMANN 2, P. REINKE 3, W. D. DOCKE!, R. VON BAEHR!, and H.-D. YOLK! Septic disease can lead to immunodepression, in the course of which reduced monocyte activation (HLA-DR < 30 %, impaired cytokine production and antigen presentation) is, in particular, closely associated with fatal outcome. Because of this observation, we designed in vitro experiments to reconstitute monocytic activation using IFN-gamma or GM-CSF. As a result, a factor present in plasma of patients with fatal prognosis, which inhibits monocytic activity was characterized functionally. This factor inhibits HLADR-expression as well as the cytokine production of monocytes from patients or healthy controls. Plasmapheresis but not ultrafiltration or hemodialysis reduced the in vivo inhibitory activity, which resulted in a reconstitution of monocytic function. The survival rate was increased from 15 % to 48 % (n=36).

Institut fur Klinische Mikrobiologie der Universitat Erlangen-Nurnberg, Erlangen, Germany

L.37 Experimental cutaneous leishmaniasis: infected lymph node dendritic cells in immune hosts stimulate a T-cell immune response H. MOLL, N. DONHAUSER, and M. ROLLlNGHOff Upon infection with Leishmania major, a cause of human cutaneous leishmaniasis, mice of resistant strains are able to control the disease with lesions resolving spontaneously. A long-lasting cell-mediated immunity protects the host from subsequent infections. We have recently documented that epidermal Langerhans cells can ingest L. major and have the unique ability to transport viable parasites from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Thus, we analyzed whether the well-documented phenomenon of parasite persistence in immune hosts is associated with dendritic cells (DC). Immunohistological studies showed that in the lymph nodes of mice that have recovered from infection with L. major, both macrophages and DC harbor parasites. However, only lymph node DC were able to induce a vigorous T-cell immune response to L. major in the absence of exogenous antigen. In vivo tracking experiments suggested that the infected DC are derived from Langerhans cells that have emigrated from the skin. The data indicate that L. major-infected lymph node DC in immune animals represent long-term host cells that may be required for maintenance of a memory population of protective T cells.

186 . 24th Meeting of the Society of Immunology lInstitute of Immunology and Transfusion Medicine, University of Lubeck, Medical School, Lubeck; 2German Cancer Research Center, Heidelberg, Germany

L.3S V~-specific stimulation of T-cells by the Mycoplasma arthritidis-derived superantigen (MAS) is dependent on the MHC class-II haplotype L. RINK!, W. NICKLAS 2, L. ALVAREZ-OSSORIO!, M. KOESTER!, and H. KIRCHNER! Mycoplasma arthritidis (M.a.) is a pathogen for mice and rats which induces an acute arthritis in these species. The pathogenicity of M.a. in human rheumatoid arthritis is still a matter of discussion. In cultures of M.a., a superantigen, MAS, is produced. Superantigens are known to stimulate T cells V~-specifically by crosslin king the MHC II with the V~-chain of the TCR. MAS is also specific for I-Ea in the MHC II of mice. In contrast to other investigators, we used flow cytometry to determine amplification as well as depletion of V~-subfamilies of the T cell receptor. In our experiments, we observed a V~­ specific T cell stimulation which was strain dependent. In spleen cell cultures of C3HI He] mice (H-2k) there were a depletion of V~ 2 and 10 T cells and an amplification of Vr) 3, 6, 9, 12, and 14 T cells. In contrast, we observed an amplification of V~ 5, 9, 13, and 17 T cells in cultures of RIllS mice (H-2r) spleen cells. Other T cells of both strains were not altered significantly, although both strains were MAS-responsive (I-Ea+). In man, we found a complex situation with respect to the MAS response. In healthy individuals V~­ specific amplification or depletion of the same V~-type were observed for V~ 8, 13, and 19. However, in the percentage of V~ 2, 3, and 17 T cells there were no changes detectable in all individuals tested. In conclusion, the V~-specific amplification is dependent on the alleles of the MHC II haplotype as shown by two H-2 different, I-Ea+ mouse strains. In man there seems to be a stimulation if the HLA-DR is able to present MAS and a depletion if the HLA-DR is defective for presentation. Therefore, HLA-DR phenotypes divide human individuals into MAS-responders and non-responders, whereas all humans seem to be responsive for staphylococcal superantigens.

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

L.39 Oral immunization with bacteriallysates increases immune response in the respiratory tract C. RUEDL, G. WICK, and H. WOLF We investigated the immunomodulating and protective effect of a bacterial lysate (LUIVAC@), consisting of seven common respiratory pathogens, on various mucosal tissues, with particular relevance to the respiratory tract, where bacterial infections frequently begin. Because of the importance to develop new immunization strategies against respiratory infections, we examined whether this bacterial lysate is able to improve the local immune response in the respiratory tract following initial oral immunization at a remote site, i.e. the intestinal tract. Analysis of lymphocyte kinetics demonstrated an enhanced localization of intestinal donor cells (lamina propria -LPL and Peyer's patch lymphocytes -PPL) in the respiratory tract of syngeneic recipient mice

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which had received these cells intravenously (RUEDL et a!., Int. Immunol. 5, 29-36, 1993). In addition, the rate of immunoglobulin synthesis (IgG, IgM, and IgA) in the gut (LP, PP's), lungs, and spleen of mice orally immunized with the immunomodulator, measured by time resolved fluorometry, revealed and increased total IgA (150%) and bacterialspecific IgA (180 %) in the lungs of the animals. In contrast, the IgG and IgM secretion rate did not show remarkable differences between the two groups investigated. We conclude that priming of the GALT with bacterial immunomodulators results in an enhanced cellular and humoral (IgA) response in the respiratory tract.

Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University and IBiometec GmbH, Greifswald, Germany

L.40 LPS activation of C014-negative human monocytes is mediated by soluble C014 T. SCHILLING, C. KRUGER, F. STELTER,]. SCHLETTER, XIAOLONGFAN, S. WITTI, U. GRUNWALD, H. DIETZ, and C. SCHUTT Membrane bound CD14 (mCD14) antigen, which is usually expressed by more than 90 % of populations of normal monocytes and macrophages, has been reported to be a receptor for complexes of lipopolysaccharide (LPS) and LPS-binding protein (LBP), and has an important function in the pathophysiology of endotoxic shock. Soluble CD14 (sCDI4) has recently been shown to be required for activation of mCD14-negative endothelial and epithelial cells by LPS. We found that even in monocytes lacking mCD14 from a patient suffering from paroxysmal nocturnal hemoglobinuria (PNH) anti-CD14 antibodies (e.g. My4, biG14) inhibit the serum dependent LPS-induced activation of these cells. To confirm a role of sCD14 in the activation of mCD14-negative monocytes, experiments were carried out in which serum is replaced by sCD14 isolated from serum, recombinant human sCD14 (rsCD14) and purified LBP to study the endotoxin-inducible oxidative burst response by these cells. We observed that sCD14 binds to LPS alone as well as enhanced after addition of LBP, and that PNH monocytes can be activated by LPS in the presence of rsCD14, but not of LBP alone. In contrast to these effects, normal mCD14-positive monocytes respond to LPS in the presence of purified LBP. These studies suggest that LPS/LBP-complexes transfer LPS to rsCD14 and the formed LPSI rsCD14-complex then bind to a unknown cellular molecule. This work was supported by grants of the Fond der Chemischen Industrie to C.S. and C.K.

188 . 24th Meeting of the Society of Immunology Institut fur Mikrobiologie der Universitat Ulm, Ulm; Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie der Universitat Hamburg, Hamburg, Germany

L.41 Immunization of mice with the N-terminal (1-272) fragment of simian virus 40 large T-antigen (without adjuvants) specifically primes cytotoxic T lymphocytes R. SCHIRMBECK,]. ZERRAHN, A. KUHROBER, W. DEPPERT, and]. REIMANN Immunization of C57BLl6 (B6) mice (H-2 b) with the <
Dept. of Medical Immunology, Medical School (Charite), Humboldt University, Berlin, Germany

L.42 Binding of Neisseria meningitic/is by the natural human monocionallgM antibody CB03 - hints for first line defense by polyreactive antibodies G. SCHOENHERR, D. ROGGENBUCK, U. MARX, and T. PORSTMANN Natural polyreactive antibodies are suggested to play an important role as first line defense against bacteria and viruses. This could be especially of importance in early childhood when the specific immune system has not been well developed. Neisseria meningitidis (N.m.) is one of the most important pathogenic bacteria in younger children causing severe diseases such as meningitis and W aterhouse-FriedrichsenSyndrome. We wanted to examine whether natural poly reactive antibodies can bind to meningococcal membranes and contribute to protection against meningococcal infection. To test this we purified the DB03 natural polyreactive monoclonal human IgMantibody as described previously (ROGGENBUCK et aI., JIM, submitted). Binding of Neisseria meningitidis were demonstrated by indirect immunofluorescence using the complete CB03 antibody and FITC labelled anti human [!-specific antibody. Specificity of this reaction was demonstrated with control antibodies and with purified Fab from CB03 in competition experiments. The great majority of N.m. showed bright

24th Meeting of the Society of Immunology . 189 fluorescence. The CB03 antibody reacts in Western blot with bacterial lysate forming a precipitation line at molecular mass of less than 10 KD which corresponds to meningococcal lipooligosaccharide (LOS). Reactivity was specified with meningococcal LOS (types 1, 2, 4, 7) purified from strains 126E, 61 mise, M136, and 6155. The biological effect of this binding will be reported by demonstration of results from in vitro assays.

Med. Mikrobiologie und Immunologie, AG Infektabwehr, Ruhr-Universitat Bochum, Bochum, Germany; 1Institut Pasteur, Paris; 2Laboratoire de Bacterio!" Universite L. Pasteur, Strasbourg, France

L.43 Protein tyrosine kinases are involved in cellular protection against different bacterial toxins (alveolysin, leukocidin) in human leukocytes A. SCHREINER, M. KOLLER,]. E. ALOUF!, G. PREV6sT2, Y. PIEMONT2, and W. KONIG Cytolytic toxins (leukocidin, alveolysin) bind and integrate into the cellular membranes of human leukocytes. Sublytic concentrations of these toxins lead to membrane activation which involves signal transduction processes. High toxin doses lead to cellular damage. We analyzed toxin-induced cellular function at sub lytic as well as lytic toxin concentrations by monitoring membrane integrity (LDH-test, trypan blue exclusiontest), synthesis of different cytokines (IL-8, IL-6, TNF-a), release of lipid mediators [Ieukotriene (LT) B4J, and the fragmentation of genomic DNA. The toxin-induced effects (lytic concentration) include a decrease in IL-8, IL-6, and TNF-a release in Iymphocytes/monocytes/basophils (LMBs) and polymorphonuclear granulocytes (PMNs). Under these conditions a release of LTB4 was observed. Additionally, a marked DNA-degradation occurred. These toxin-related effects were reduced by preincubation with protein tyrosine kinase (PTK)-inhibitors (Lavendustin A, Tyrphostin). Similar cellular protection was obtained by priming with GM-CSF. Lavendustin A inhibits the GM-CSF-mediated enhancement of fMLP-induced IL-8 production and LTB4 generation. Thus cellular protection induced by PTK-blockers may involve a different mechanism than cellular protection induced by growth factors. These data provide evidence that the biological activity of distinct cytolytic toxins is mediated by the activation of PTKs.

Dept. of Medical Immunology, Medical School (Charite), Humboldt University of Berlin, Berlin, Germany

L.44 Strategies for the generation and characterization of human monoclonal antibodies to common determinants of gramnegative bacteria M. SEIFERT, R. RUBIKAITE, H. BOHME, and R. VON BAEHR Life-threatening infections in hospitalized patients are mainly caused by different gramnegative pathogens. To generate human monoclonal antibodies (mAbs) to lipopolysaccharides (LPS) of gram-negative bacteria we immortalized B-Iymphocytes from different

190 . 24th Meeting of the Society of Immunology sources by PEG-fusion or Epstein-Barr-virus(EBV)-transformation: 1. peripheral blood of patients with gram-negative sepsis 2. human spleen, and 3. peripheral blood of patients with chronic lymphocytic leukemia (CLL). EBV -transformation of septic B-cells and normal spleen cells resulted in a high percentage of lymphoblastoid cell lines (LCL) secreting anti-LPS-reactive human mAbs. LCL with an interesting reaction pattern in ELISA screening systems were fused with heteromyeloma cell lines (CB-F7, K6H6-B5) to stabilize and enhance antibody production. For ELISA screening we used Lipid A (Salmonella minnesota R595) and different LPS preparations of rough (E. coli J5, Salmonella minnesota R5 and R595, Salmonella thyphimurium: Ra, Rc, Re-type) and smooth (E. coli 0111 :B4, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria strains. Positive-selected lines had to be screened in the following sequence of test systems: 1. Western blotting with LPS 2. TNF-release test with human monocytes and 3. behavior in LAL-test. All antibodies we describe in our study are of IgM (kappa or lambda)-isotype and the majority reacts with the very conserved core/lipidA-region of the LPS-molecule. Although the therapeutical potential of a single anti-core/lipidA mab is discussed controversially, cocktails with a set of those mAbs may have protective capacities.

Dept. of Medical Immunology, Medical Academy Magdeburg, Magdeburg, Germany

L.45 Generation of superoxide and hydroxyl radicals by stimulated neutrophils studied by electron spin resonance (ESR) H. STRUY, D. SCHMIDT, R. GARTNER, and]. MORENZ Neutrophils undergo a respiratory burst in response to stimulants. The increase in oxygen consumption was found to be associated with production of oxygen-centered radicals. Electron spin resonance spectrometry and spin trapping techniques are being applied to investigate the production of free radicals. Using the spin trap 5,5-1 dimethyl1-pyrroline-N-oxide (DMPO) responses of neutrophils to opsonized zymosan were investigated. DMPO reacts with superoxide to form 5,5-dimethyl-2-hydroperoxy-1pyrroline-N-oxide (DMPO-OOH) and with OH to form 5,5-dimethyl-2-hydroxy-1pyrroline-N-oxide (DMPO-OH). Neutrophils showed increases concentrations of (DMPO-OH) only within the first ten minutes after stimulation. The concentration of DMPO-OOH decreases within the first five minutes and thereafter increases with a maximum at about 30 minutes. No spin trap ad ducts were seen with unstimulated neutrophils. In patients with pancreatitis superoxide production is markedly increased. Yet a parallel increase of hydroxyl radical adducts could hardly be detected. Reasons for the lack of the hydroxyl adduct increase at the peak of superoxide production and in patients with pancreatitis are discussed.

24th Meeting of the Society of Immunology . 191 Depts. of IClin. Immunology and 2Intensive Care Unit of Medical School (Charite), Humboldt University, Berlin, Germany

L.46 Shift from inflammatory to anti-inflammatory monocytic cytokine production is critical for outcome in septic disease U. SYRBEl, C. LIEBENTHALl, w. D. DocKEl, H. ZUCKERMANN2 , and H.-D. VOLK I Inflammatory cytokines like TNF-alpha are thought to have deleterious effects in septic disease. However, in our study prognostic ally negative severe immunodepression in the late phase of sepsis was found to be associated with diminished production of inflammatory cytokines. Monocytic cytokine production in response to LPS in whole-blood and PBMNCcultures was repeatedly tested throughout the course of septic disease. Concomitantly with a in vivo-decrease of monocytic HLA DR-antigen expression as a sign of immunodepression, we generally found decreased endotoxin-induced production of proinflammatory monocytic cytokines such as IL-l~, IL-6, and TNF-a (ELISA) and increased production of anti-inflammatory factors such as IL-IRA and soluble TNFreceptor in vitro. Persistent immunodepression was associated with a poor clinical prognOSIS.

Dept. of Immunology, University of Ulm, Ulm; IInstitute for Biology II and Microbiology, Albert-Ludwigs-University, Freiburg, Germany

L.47 MHC class I antigen presentation of and cytoplasmic invasion by Listeria monocytogenes is independent from listeriolysin secretion G. SZALAY,]. HESS,]. GOLECKIl, and S. H. E. KAUFMANN Virulence of the intracellular bacterium Listeria monocytogenes (L.m.) depends on a cytolysin, the listeriolysin (Hly). Recent studies provided evidence that Hly facilitates recognition of listerial antigens, in association with MHC class I molecules, by CDS T lymphocytes. Here we show, that the Hly deficient strains, (i) SLCCS3, where a deletion lies in the regulatory gene prfA, (ii) M3, where the Tn 916 inserts in the promotor region of the hly gene and (iii) M20, where the Tn 916 insert in the hly structural gene, are avirulent for mice and unable to replicate inside of bone marrow derived macrophages (BMM). However, BMM infected with M3, M20 of SLCCS3 were as efficiently lysed as BMM infected with Hly expressing wild type strain EGD by MHC class I dependent CDS CTL. Moreover, M3 and SLCCS3 could be localized in the cytoplasm of infected BMM at a time point, where infected BMM were used for cytotoxicity assays. Because we used a rather long infection time, we tried to exclude potential reversion into a Hly + phenotype, by analysing infected BMM with PCR for possible hly mRNA. mRNA was detectable in BMM infected with EGD, SLCCS3, a shortened hly mRNA in the case of M20, but mRN A is totally absent in M3 infected BMM. On the one hand, these findings confirm the central role of Hly in virulence and intracellular replication. On the other hand, they dispute the notion that only Hly is exclusively required for (i) presentation of listerial antigens in context with MHC class I molecules and (ii) invasion into the cytoplasm. Hence virulence of L.m. can be dissociated from MHC class I presentation to CDS T cells.

192 . 24th Meeting of the Society of Immunology Dept. of Immunology, University of Ulm, Ulm, Germany; 'National Institutes of Health, Bethesda, USA

L.48 Listeriosis in T-cell receptor delta-chain deficient mice. Immunohistological analysis S. THOMA-USZYNSKI,j. ARNOLDI, P. MOMBAERTS', S. TONEGAWA" and S. H. E. KAUFMANN In normal mice, sublethal i.v. infection with the facultative intracellular pathogen Listeria monocytogenes causes a characteristic cell-mediated immune response. Granulomatous lesions arise in infected livers and spleens, and mice acquire protective immunity within less than one week. Listeriosis in mice deficient for the Ii-chain of the T-cell receptor and consequently lacking functional y/li T -cells was comparable to controls as assessed by CFU analysis in spleens. Also vaccination induced protection appeared equally strong as in controls. In contrast, prominent tissue reactions developed in Ii-TCR deficient mice, as compared to normal littermates which were composed of mainly granulocytes and macrophages. Initially single, partially necrotic, hepatic lesions which almost exclusively consisted of MAC-1 + cells developed. In the course of infection, lesions became abscesslike, and CD4+ and CDS+ T-cells appeared in the periphery. Complete reconstitution of liver parenchyma was not achived. These results indicate exacerbated histopathology in Ii-TCR deficient mice suffering from listeriosis, suggesting a regulating effect of y/li Tcells on cell-mediatd immunopathological tissue reactions.

'Institute of Immunology and Transfusion, University of Lubeck, Medical School, Lubeck, Germany; 20rganon Teknika, AB Boxtel, Netherlands

L.49 Failure to detect Epstein-Barr virus (EBV) nuclear antigen (EBNA) 1 and EBNA 2 in Blymphocytes of EBV-ONA positive, latently infected individuals H.j. WAGNER', M. HORNEF!,j. MlDDELDORp2, and H. KIRCHNER! It has been assumed that all EBV -infected cells are EBNA positive. For our studies, we modified an anti-complement indirect immunofluorescence (ACIF) test for detecting EBNA in cytocentrifuge preparations of cells to a more reliable and sensitive one by using monoclonal antibodies and alkaline phosphatase anti-alkaline phospatase (APAAP) technique. We found EBNA 1 and EBNA 2 positive cells in B-lymphocyte preparations of 6 patients undergoing active EBV -induced infectious mononucleosis in a range of 2 to 0.09 'Yo. However, taking these slides as positive controls, we could not find any EBNA 1 or EBNA 2 positive cells in 2 x 106 B-lymphocytes of each latently EBV-infected individual, although samples of another 106 B-cells of the same probes were EBV-DNA positive in a polymerase chain reaction (PCR) assay developed in our laboratory and previously reported. Our data indicate that there are EBV -positive cells in the peripheral blood of latently EBV-infected individuals being EBNA 1 and 2 negative, at least on an immunocytochemically detectable level. The expression of Epstein Barr nuclear antigens in latency may be regulated differently as compared to active infection.

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Max-Planck-Institut fur Immunobiologie, Freiburg, Germany

L.5D Altered C04+ T cell responses to Plasmoclium chabaucli in B-cell-deficient mice T. VONDER WElD and]. LANGHORNE Mice depleted of B cells from birth by treatment with anti-It antibodies can control but not clear an infection with the malaria parasite Plasmodium chabaudi chabaudi (AS). Splenic CD4+ T cells from these mice were unable to mount a significant Th2 response to the parasite in vitro as shown by much lower precursor frequencies of T helper cells for antibody production and of IL-4-producing cells compared with the response of controltreated mice. CD4+ T cells of the anti-It-treated mice which respond to antigens of P. chabaudi chabaudi maintained a Th1 phenotype throughout primary infection, in contrast to control mice in which a sequential appearance of Th1 and Th2 responses was observed. These data show that Th1 responses in anti-It-treated mice are sufficient to control parasitemia but not to eliminate an infection. The data further suggest that depletion of B cells by treatment with anti-It antibodies reduces the generation of the Th2 subset during a primary response to P. chabaudi chabaudi.

Dept. of Immunology, V niversity of Vim, Vim, Germany

L.51 Epitope analysis of an in vivo generated murine cytolytic COS T cell clone specific for mycobacterial hsp60 and recognizing self epitopes on IFN-y stressed macrophages V. ZOGEL, B. SCHOEL, and S. H. E. KAUFMANN Crossreactive CDS T cells with specificity for a mycobacterial heat shock protein (hsp60) can lyse stressed autologous target cells. These crossreactive T cells are activated in vivo by immunization of mice with mycobacterial hsp60 in ISCOM. Cloned CDS T cells not only lyse macrophages labelled with a tryptic digest of hsp60 (trp hsp60) but also macrophages stressed with IFN-y in the absence of foreign peptides. The trp hsp60 was separated by HPLC and fractions were tested with the hsp60 specific T cell clone. A 23mer peptide was identified as the responsible epitope. This peptide contains a 9-mer strong H-2D b-binding motif. Interestingly this H-2D b-motif failed to stimulate the T cell clone but competed for H2-Db with the synthetic 23-mer peptide. Treatment of the peptide-transporter mutant lymphoma celline RMA-S with either trp hsp60, 23-mer peptide or H-2D b-motif induced a strong H-2Db expression. These findings suggest H2D h-restricted recognition of the epitope. The 23-mer peptide is presented to the T cell clone independent from ~2 micro globulin (~2m). Macrophages from ~2m-deletion mutant mice pulsed with the 23-mer peptide or stressed with IFN-y were lysed by the hsp60 specific T cell clone under serum-free conditions.