workshop 8: immunology of infectius disease

9
Workshop 8 Immunology of Infectious Disease Hcinrich-Pette-InsritUl fUr Experimentelle Virologic und Immunologie a n def Un ivers itat Hamburg. FRG 1. Effect of adoptive immunization on elimination of LCM virus from the spleen of acutely infected mice URSULA ASSMANN, R. SCHWACHENWA l.D, and F. LEHMANN-G RUBE In general, for t he con trol of virus infections in higher animals two modes arc discussed. (1 ) Dir ec t of cytotoxic T lymphocytes (eTL), which are assumed to lyse infected celis, thereby bl ock ing further virus replication and exposing intracellularly matured infectious pa rti cles to antibodi es, macrophagcs and the like. (2) T l y mp hocyte-mediated activa tion of ce ll s of the mononuclear phagocyte system (MPS). which acq uire in creased potencial to in gest and diges t vi rus and to release manokin cs that i nterfe re with virus re pli cation; according ro th is latter view, elements of the MPS, es pec iall y monocytes, are the real effector cells. We have tested thes e propositions by study in g the effect of adoptive immunization on lymphocyti c choriome ni ngit is (LCM) virus in the spleens ofacutely infected mice. From the numbers of T lym phocytes in oculated, t he pro portions settJi ng in the s p lee n, and the proportions lysing virus· infect ed target cells in limiting di lution experiments, it can be calcu lated that as few as 200 LCM virus-specific CTL dimini sh virus in the recipients' spleens if meas ured 40 hours after transfe r. With higher cell numbers marked reduction can be de tect ed as ea rl y as 6 hours later. B o th th e lo w number of cell s an d th e short inl erval need ed to curtai l virus repli ca tion make direct effects of CTL an u n likely mec ha ni sm fo r eli mination. O n the o th er hand, macro phages arc no t activated in a dopti vely imm unized in fected mice, although they arc during the ti me a prima ry infection i s te rminated without the help of exogeno usly s u pp li ed immu ne cells. Furthermore, irradiation of rec ipients prior to cell transfer does nOl impair their ab ility to get ri d ofthe agen t. These find ings arc difficult to reconcile with a mechanism based on activation of cdls of the MP S. At present our findings are bcst cx plained by assuming [h at the spleen of the mouse is frce d of LCM virus by the action of lymp ho ki nes whic h are secreted by activa ted T lymphocytes an d in terlcre with replication of the virus. Iins ti tutc of Medical Biology, Univers ity of Perugia, Italy, 2 Max -Pla nck -lnslitut f ur lmmun- biologic, fre ib urg, FRG, J Fraunbofer Inst itut fUr Toxikologie, Abtcilung Imm unb iologie, H annover, fR G 2. Natural cytotoxicity against candida albicans yeast particles. Cells of mouse and human macrophage lineage as effector cells M. B ACCARINI\ D. KRUMW IEH 2 , and M .- L. LOHMANN-MAITHES} Cell s derived from mouse liquid bone marrow cultures displayed natu ral cytotoxic ac tiv it y against the yeast fo rm of candida albicans. The effector cells were functionally characterized as

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Page 1: Workshop 8: Immunology of Infectius Disease

Workshop 8 Immunology of Infectious Disease

Hcinrich-Pette-InsritUl fUr Experimentelle Virologic und Immunologie a n def Universitat Hamburg. FRG

1. Effect of adoptive immunization on elimination of LCM virus from the spleen of acutely infected mice

URSULA ASSMANN, R. SCHWACHENWA l.D, and F. LEHMANN-G RUBE

In general, for the con trol of virus infections in higher animals two modes arc discussed. (1 ) Direct effect.~ of cytotoxic T lymphocytes (eTL), which are assumed to lyse infected celis, thereby blocking further virus replication and exposing intracellularly matured infectious particles to antibodi es, macrophagcs and the like. (2) T l ymphocyte-mediated activation of cells of the mononuclear phagocyte system (MPS). which acq uire increased potencial to ingest and digest virus and to release manokincs that interfere w ith virus replication; according ro th is latter view, elements of the MPS, espec ially monocytes, are the real effector cells. We have tested these propositions by studying the effect of adoptive immunization on lymphocytic choriomeningitis (LCM) virus in the spleens of acutely infected mice. From the numbers of T lym phocytes inoculated, the proportions settJi ng in the s pleen, and the proportions lysing virus· infected target cells in limiting dilution experiments, it can be calculated that as few as 200 LCM virus-specific CTL diminish virus in the recipients' spleens if measured 40 hours after transfer. With higher cell numbers marked reduction can be detected as early as 6 hours later. B oth the low number of cells and the short inlerval needed to curtai l virus repli cation make direct effects of CTL an u nlikely mechani sm fo r elimination. O n the other hand, macrophages arc not activated in a doptively immunized infected mice, although they arc during the time a primary infection i s terminated without the help of exogenously s upp li ed immune cells. Furthermore, irradiation of recipients prior to cell transfer does nOl impair their ability to get rid of the agen t. These findings arc difficult to reconcile with a mechanism based on activation of cdls of the MPS. At present our findings are bcst cxplained by assuming [hat the spleen of the mouse is frced of LCM virus by the action of lymphoki nes which are secreted by activated T lymphocytes and interlcre with replication of the virus.

I institutc of Medical Biology, University of Perugia, Italy, 2 Max-Planck-lnslitu t f ur lmmun­biologic, freiburg, FRG, J Fraunbofer Inst itut fUr Toxikologie, Abtcilung Immunbiologie, Hannover, fRG

2. Natural cytotoxicity against candida albicans yeast particles. Cells of mouse and human macrophage lineage as effector cells

M. B ACCARINI\ D. KRUMW IEH2, and M.-L. LOHMANN-MAITHES}

Cells derived from mouse liquid bone marrow cultures displayed natu ral cytotoxic activity against the yeast fo rm of candida albicans. The effector cells were functionally characterized as

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68 XVI. Meeting of the Society of Immunology

nonadherent, nonphagocytic cells, which lack the surface antigen Asialo-GMI. This cell population was further separated by Percoll gradient centrifugation into a band containing macrophage precursors and another one containing young granulocytes and granulocyte precursors. The macrophage precursors, purified up to 95 'Yo, were analysed for candidacidal activity at a single cell level. They proved to actively bind and kill the target without being damaged during the interaction. In a long-term colony forming unit inhibition assay they proved to be even more active than granulocytes which are considered to be the most active candidacidal effectors.

Human macrophages were obtained by cultivating peripheral blood monocytes in Teflon bags. Cells were harvested at different stages of maturation and tested for their candidacidal activity in the colony forming unit inhibition assay. Freshly isolated young monocytes displayed good levels of activity. While the early stages of the culture showed a very low killing efficiency a strong candidacidal response was observed with the later stages of the culture containing monocyte-derived mature macrophages. The kinetics of activity and the possibility of modulating it by lymphokine was investigated.

Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat, Bochum, FRG

3. Monoclonal rat antibodies against antigens of the parasitic nematode Nippostrongylus brasiliensis and dinitrophenol

A. BOHN and W. K ()NIG

Monoclonal rat antibodies against surface antigens of Nippostrongylus brasiliensis (N.b .) and Dinitrophenyl-(DNP)-ovalbumin were generated by fusion of spleen cells from Lou/MI Wol rats which have been immunized with DNP-ovalbumin and infested with 3000 third stage larvae of N.b. , with the IR 983F rat myeloma cell line. 33 antibodies reacted with the worm surfaces and 25 antibodies also bound to larvae as was revealed by enzyme linked immunosor­bent assays (ELISA). 13 antibodies were specific for DNP. The antibodies were further characterized with respect to their binding specificity with worm and larvae homogenates and secretions: three antibodies reacted with worm homogenates and one antibody worm secre­tions. furthermore the binding capacity of the monoclonal antibodies with acetylcholine esterase from electric eel (one specific antibody) and from bovine erythrocytes (eight specific antibodies) was investigated by specific ELISAs. The isotypes and subclasses of the mono­clonal antibodies were determined by ELISAs with protein A and concanavalin A and by immunodiffusion against monospecific antisera. The epitope specificity of the antibodies was tested b y the inhibition of binding to worm surfaces in the presenc of different carbohydrates and pbosphorylcholine. Our results demonstrate L all larval antigens which are recognized by our antibodies are also present on adult worms, 2. most of the antibodies specifically react with worm and larvae surfaces, 3. the binding of some antibodies to worm surfaces is inhibited by phosphorylcholine, 4. all antibodies which react with DNP are crossreactive with worm and larvae surfaces.

rorschungsinstitut Borstel, 2061 Borstel, FRG

4. Further immunochemical characterization of a common lipopolysaccharide specificity

H. BRADE, 1. BRADE, and E. TH. RIETSCIlEL

Previously we have described a new type of common antigen that is present in all lipopolysaccharides (LPS) of gram-negative bacteria eXCept in those of the Re-type. Prelimi-

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nary immul10chemical investigations indicated that the antigenic determinant requires the presence of 2-kero-3-deoxyoctonic acid (KDO) and at least one adjacent neutral sugar. However, two observations were made wh ich seemed to be in contradiction to our hypothcsis, i) the fact that LPS of Vibrio cholerae (which has been reponed to lack KDO) is active as an inhibitor in a serological test system specific for the new type of antigen and ii) the absence of reacti vity in a LPS from Acincwbaacr calcoacclicus which fulfiUs the afore-mentioned compositional requi rements for expressing the specificity (KDO and rhamnose were found). Our pn::st:n t results show that both observations are not contrad ictory. First, it was shown despite earlier reports that the LPS of V. cholerae con tains a KDO derivative which was identified as KDO-5-phosphate by combined gas-liquid chromatography/mass spectrometry, and, second, methylation anal ysis of the core ol igosaccharide of tbe i\cillecobacrer LPS indicattd that KDO in this c ase is substituted in pos ition 7. Since in all enterobacterial LPS studied the co re oligosaccharide is substituring the KDO residue in position 5 it is assumed that the antigen requires for its expression an appropriate s\lbstitut'ion of KDO. Since 4-0-substi tuted KDO (as present in Re-type LPS) does also not ex press thi s antigtn, it is supposed that S-O-substituted KDO is ntcessary for this type of antigen.

This work was supported by a grant of the Deutsche Forschungsgemeinschaft to H . B. (B r. 73 1/3).

Forschungsinstitu t Borstd, 206 1 Borstd, FRG, and National Pub lic Health Inst,itute, IIel­sinki , Finland

5. Antigenic properties of chlamydiallipopolysaccharidc (LPS)

L. nRADE, M. NURMfNEN, P. H. MAKELA, E. TH. RI ETSCHEL, and H . BH.ADE

Chlamydia a rc bacteria which arc obl igatory intracellular paras ites being of considerable relevance in human and veterinarian medicine. Tht:y cause a number of - predominantly chronic - infections, e.g. pneumonia, trachoma, and non-specific infections of the genito­urinary trac!. little is known on the biochemical properties of virulence faclOrs and surface antigens in Chlamydiae. Recently it has been shown that the ch lamydial group antigen cross reacts with tbe Re-type LPS of Acinecobacrer calco3cericus and Sa.lmonella minnesO!a (RS9S). These studies were carried out by immuno-blotting technique using PAGE-separated LPS and antisera against Chlamydia, Acinetobacter and Salmonella LPS.

Here we prtsent data that show that the chlamydial antigen i s a typical LPS ; I. chemical analysis rtvealtd the presence of characteristic LPS constituents, i.t. glucosamine, 3-hyd roxy fatty acid, and 2-keto-3-deoxyocronic acid and 2. by serological investigations demonstrating the presence of Re- type LPS specific ity and of lipid A sptcificity, both cross reacting with the corresponding antige ns of enterobacterial LPS.

The results indicate that chlamydial cells product 3. typical LPS as it is known from gram­negati vt bacteria, and that thi.~ LPS is of the Re-type. This is further supported by the fac t that the chlamyd ial LPS docs not ex press tht common LPS sptcific ity which is present in all LPS except in those of the Rc- type.

This work was supported by a grant of the Deutsche Forschungsgemeinschaft to H. B. (Br. 73 1/31.

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70 XVI. Meeting of the Society of Immunology

I Fraunhofer- Institut fu r Toxikologie und Aerosolforschung, Abteilung Immunbiologie, Han­nover, FRG, 2 Max-Planck-Institut fur Immunbiologie, Freiburg, FRG

6. The murine Kupffer cell as cytotoxic effector cell in intra- and extracellular killing systems

T. H. DECKER!, A. KIDERLEN1, and M.-L. L OHMANN-MA'ITHI-'..sl

Murine Kupffer cells were isolated from the liver by perfusion with pronase and purified by differential sedimentation over Metrizamid followed by plastic adherence. The resultant population consisted of more than 9 0 % phagocytosing cells, bearing macrophage specific antigens as defined by anti macrophage monoclonal antibodies. These Kupffer cells were h ighly susceptible for in virro activation. Kupffer cells activated either with Iymphokines containing native Interferon y or with r-Interferon y (kindly supplied by Boehringer, Wien) developed strong cytotoxiciry against extracellular tumor targets and intracellular Leishmania parasites. When cultured in the presence of L 929 derived C';F Kupffer cells showed a strong prolifera­tive capacity. They can be harvested in large numbers from such proliferating c ulrures and show similar functional and antigenic properties as the freshly isolated liver Kupffer cell. These data strongly suggest an active contribution of the resident Kupffer cell to resistance against tumor and infectious disease.

Max-Planck-Institut fur lmmunbiologie, Freiburg, foRG

7. Protective effects of an antiserum with c1onotypic activity

S. H. E. KAUFMANN and r. MttLLER

Mice (C57BlJ6) were immunized subcutaneously (s.c.) with live organisms of Listeria monocytogenes strain EGO and lymph node cells collected after seven days. Lymph node cells were cultured in the presence of heat-killed Listeriae (HKL) and syngeneic accessory cells and long-term cultured T cells cloned at limiting dilution conditions. Cloned T cells could confer protection when given locally together with live L. monocytogenes organisms. Cloned T cells were fused to the AKR thymoma line BW 5147 and hybridomas selected in HAl' medium. Cloned T cell hybridomas were stimulated by accessory cells plus HKL or by Concanavalin A (Con A) and supernatantS tested for interleukin 2 (CL2)-activity on an IL2-dependend cytotOxic T cell line. Hybridoma cells were stimulated by HKL of strain EGD but not by HKL of strain ATCC 19114 and required accessory cells of H-21_Ab haplotype. (C57B1! 6 x DBA/2)Fl mice were immunized with irradiated hybridoma cells at weekly intervals and sera from individual mice collected after the fihh to ninth immunization. An antiserum which blocked antigen- but not Can A-induced IL2-production of the homologous T cell hybridoma but not of three other L. monocytogenes-specific hybridomas was identified. Mice were immunized with this antiserum using the following regimes: (a) intravenous injection, (b) S.c.

injection, (c) s.c. injection following treatment with cyclophosphamide, and (d) s.c. injection in Freund's complete adjuvant (foCA). After five to eight days mice received a challenge infection with viab le L. monocytagenes and protection in spleens was detennined after another two days. Mice which had been immunized with antiserum in FCA were significantly protected against listeriosis suggesting that c1onotypic antibodies directed against protective T cells can induce acquired resistance.

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I Max-Planck-lnstitut fur Immunbio!ogie, Freiburg, und 2 Fraunhofer-Institut fur Toxikologie und Aerosolforschung, Abteilung Immunbiologie, 3000 Hannover, PRG

8. Protection against intracellular pathogens exerted by recombinant immune interferon (r-IFN-y) - in vivo and in vitro activation of macrophages by r-IFN-y is a radioresistant function

A. F. KIDERLEN\ J. MAUEL1, s. H . E. KAUFMANNl

, and M.-L. LOHMANN-MATfHES2

In an in virro infection model with Leishman ia enrietti, tropica and donovan i, we demon­strate that periwneal and bone-marrow derived macrophage.~ arc equally susceptible to the activation with r- IFN -y. Also when applied in vivo, r-IFN-y is very effi cient in activating macrnphages to intracellular cytowxicity. Furthermore r-IPN-y can protect mice in vivo against the intracellular pathogen L. monocytogenes in a local as well as in a systemic infection model. The susceptibility of macrophages to activation with r-IFN-y is not impai red by in vivo sublethal irradiation with 500 r or by ill vitro irradiation with 2000 r. Peritoneal macrophages of miee irradialed 48 h previously are as effectively activated by r-IFN -y applied in vivo as macrophages from contro l mice. These data demonstrate a great potential of r-IFN -y for immunotherapy.

Arbeitsbereich Mikrobiologie und Immunologic der Universitat, 7400 Tiibingen, FRG

9. Immunomodulation by synthetic analogues of bacterial lipoprotein and by fragments of bacterial protein I

ANGELIKA LEX, H.-M. VORDERMEIER, R. SPRENGER, BARBARA SUIIR, and W. G. BESSl.ER.

Lipoprotein and Protei.n I from the outer membrane of Escherichia coli are potent mitogens and poly clonal activators of immunoglobulin synthesis for murine B-cells and lymphocytes of other species . The minimal molecular structure of these membrane proteins required for leukocyte activation was prepared either by chemical synthesis or by CnBr-c1eavage of the molecules (1, 2, and K. H. WmsMOLLER, C. l UNG, W. BESSLER: in preparation). Several segments of the N-terminal part of lipoprotein were synth esized and exhibited biological activity comparable to the native molecule: S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N -pal­mitoyl-(R)-cyteinyl-serine, -sery l-serine, -serlyl-seryl-asparagin, and -seryl-seryl-asparaginyl­alanine all constitute potent mitogens, as monitored by the incorporation of -lH-thymidine into DNA, and ntrned out to be potent polyclonal activators, as shown by hemolytic plaque assays and by measuring Ig-secretion jn vitro. The synthetic lipoprotein analoga were shown to markedly enhance, in submitogenic doses, the antigenic response against underivati:.:.ed and highly trinitrophenylated sheep red blood cells (SRBC, TNP-SRBC) JJJ vitro. TIle potent adiuvant activity of the synthetic mitogens could be demonstrated for primary as well as for secondary immune responses. Pragments of protein 1 also exhibited mitogenic and adiuvant properties, which were comparable to ones induced by the lipoprotein segments. Our results show, that defined molecular regions of two bacterial surface components are responsible for their marked mitogenic, polyclonally activating and adiuvant properties. These potent bacte­rial compounds may have a significant influence on the host parasite relationship before and during the establishment of the specific humoral or cellular jmmune responses .

1. K. H. WIESMOLLER, W. G. B~SSLER, G. lUNG; Hoppe-Seyler's Z. Physiol. Chern. 364: 593 (1983).

2. R. B. JOHNSON, S. KOHL, K. WIESMUU.F.R, G. JUNG, W. G. BESSLER: Immunobiol. 165: 27 (1983).

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72 . XVI. Meeting of the Society of Immunology

H einrich-Pette-Institut f. Experimentelle V irologie u. Imm unologie a. d. Universitat Ham­burg, FRG

10. Atopic production of antibodies against lymphocytic choriomeningitis (LCM) virus in LCM virus carrier mice

D. MOSKOPHIDIS,]. LOHLER, and F. LEHMANN-GRUBE

After connatal or neonatal infection of a mouse the LCM virus persists lifelong in high concentrations in essemially all organs. Inability to eliminate the virus is assumed to res ult from LCM virus-specific immunologic tolerance of the T cell compartment; antibodies against the virus are often produced and are held responsible for a wasting disea.~e in aging carrier mice. They are thought to form pathogenic immune complexes with virus which are deposited in tissues with high fi ltration rates such as kidney glomerula, plexus choroidei, and walls of blood vessels. The propensity to form LCM-viral antibodies and to develop immune complex disease (ICD) varies among mouse strains, and while for instance persistently infected NMRI mice are severly affected, in others such as C3H/HeJ and CBAI] as well as grey house mice neither alHibod ies nor pathologic alterations can be detected. By usc of a solid-phase immunoenzymatic technique devcloped by SEDGWICK and HOLT (1) for the enumeration of single rat cells producing antibodies against ovalbumin and adapted by us to mouse cells and LCM virus IgM and IgG antibody-producing cells (APC) were detected ill spleens of carrier mice strain NMRI but not C3H/He], CBAlI, and Mus musculus. Thus, a correlation exists between APC in the spleens, antibodies in the circulalion, and lCD. In carrier mice undergoing l eD many tissues contain focal accumulalions of plasma cells which may be so extens ive as to resemble plasma cell granuloma. This finding has induced us to search for APC in parenchymatous organs. Leukocytes were isolated by trypsin ization fo llowed by Ficoll­Isopaque separation. In kidneys, livers, and brains TgG and TgM APC were read ily detected, even in individuals of strains free of ICD and previously thought not to produce LCM-viral antibod ies, with highest numbers in the brains. To our knowledge, this is the first example of localization of cells producing antibodies against the virus during a persistent virus infection in organs not belonging to the immune system.

1. SEDGWICK, ]. D., and P. G. HOLT. 1983. A solid-phase immunoenzymacic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Meth. 57: 30 1.

GBF-Gesellscbaft fur Biotechnologische Forschung mbH , 3300 Braunschweig, PRG

11. The effects of pyocyanine, a pigment produced by Pseudomonas aeruginosa, on in vitro interleukin-2 (IL-2) production and differentiation of cytolytic T cells

P. F. MtrHLRADT, H . TSAI, and D. A. MaNNER

/\·eudomonas aeruginosa is a common cause of opportunistic infections in immunosuppres­sed patients. It has long been known that the bacteria produce a bluish pigment, pyocyanine. This pigmen t was recently reported to inhibit lymphocyte proliferation (R. U. SORENSEN et aL 1983. Infect. and Imm unity 41 :321). We investigated the effects of pyocyanine on prolifera­tion, lL-2 production, and development of cytolytic T cells in mitogen-stimulated murine­splenocyte cultures. At doses which inhibit proliferation, an enhanced IL-2 production was measured. Lack of lL-2 can therefore not be the cause of the inhi bi tory effects of pyocyanine

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on proliferation. The formation of cytolytic effector 'J' cells was also totally inhibited by pyocyanine, although inhibition of proliferation by mitomycin did not have this effect. It is discussed that the presence of pyocyanine at the focus of an infection with Pseudomonas aerllginosa may cause a partial local immuno.suppression which favors the further spreading of the infection.

Institut fiir Medizinische Mikrobiologie, Freie Universitat, Berlin, West Germany

12. Essential role of Ly 2+ H-2 K restricted T cells for granuloma formation in infection with facultative int racellular bacteria

H . NAHER, U. SPERLING, and H. HAHN

Cell ular, genetic and antigenic requirements for granuloma formation, the essential feature of immunity to facultative intracellular bacteria were investigated. When 5 x 106 Listeria monocytogenes specific peritoneal exudate T lymphocytes enriched cells (PETLEs) were tran.sferred together with 5 X 104 bacteria into various congeneic and mutant recipient mice, adoptive transfer of granuloma formation was associated with a high degree of protection. Markedly less protection was observed under conditions of H-2 1-A homology between donor and recipient mice. Treatment of specific PETLEs with anti-iy 1 antiserum plus C or anti-Ly 2 anti.serum plu.s C abolished the capacity of these cells to transfer granuloma formation. Recombination of the Ly I and Ly 2 depleted populations did not reconstitute the effect. Thus a Ly 1+2+ T cell is critically involved in adoptive granuloma formation. When labeled anti­Ly 1 of anti-i y 2 antiserum were used to stain T cells within granulomas equal numbers of cells were of the Ly 1 or Ly 2 phenotype, early in formation of granulomas. At a later stage a preponderance of Ly 1+ over Ly 2 ' T cells within granu lomas was observed indicating recruitment of Ly 2 · T cells into the granulomatous lesions. Expression of granuloma formation in adoptively immunized mice depended on the injection of living bacteria and heat­killed L. monocytogenes, even if administered in a 2000 fold higher amount did not suffice. It is concluded that twO T cell populations are involved in protection against L. monocytogenes . Protection based on granuloma formation depends on H -2 K restricted Ly 2' (Ly 1+2+) T cells eresumably requiring living antigen for activation and H-2 I-A restricted protection is due to macrophage activation hy T helper cells. Within granulomas both T cell populations arc present.

Institute of Medical Microbiology, freie Universitat, Berl in, and I Immunology Research Unit, Klinikum Steglitz, freie Universitat, Berlin, West Germany

13. Characterization of listeria monocytogenes specific rat T c ell clones in vitro and study of dynamics of IL-2 receptor expression

R. M. SrOLPMANN, H. NAHER, H. OSAWA1, H. HAHN, and T. DIAMANTSTEIN1

Listeria monocytogenes specific rat T cell dones were established and phenotypically characteriz.ed u.sing the monoclonal antibodies \Yl3125 and MRC Ox 8. Functional differences between the T cell clones existed with respect to MIF-, MAF- or I L~2-production. further-

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more, the dynamics of 11.-2 receptor expressing of two T helper cell clones were studied. It is generally thought that interaction with the antigen receptor induces IL-2 receptor expression and interaction of IL-2 with the 11.-2 recepto r results in cell proliferation. T helper cell clones require the presence of IL-2 and antigen for continuous growth. Antigen induced IL-2 receptor expression is a transient event and the presence of antigen is required for maintenance of IL-2 receptor expression (1 ). Expression of lL-2 receptors at various days after antigenic stimulation was determined by an indirect radioactive antibody binding assay using the monoclonal anti-rat lL-2 receptor antibody ART1S (2). In parallel, proliferative response of T celis was measured in the lymphocyte proliferation assay. Our resultS demonstrate that the decrease of IL-2 receptor expression coincided with the decrease of the pro life rative response. The influence of antigenic restimulation on proliferarion and expression of the 11-2 receptor was also studied . The data show that the antigen induced expression of the 1L-2 receptor on T cells is a transient event and has its maximum at d 2 or 3, reaching background levels on d 14. Antigenic restimulation of 1 4-d old T cells causes an increase of IL-2 receptor expression the prerequisite for 11.-2 dependent continuous growth of T cells.

1. DIAMA?-.TTSTEIN, T., and H. OSAWA. 1984. Molecular Immuno!. in press. 2. OSAWA, H., and T. DrAMANfSTEIN. 1983. J. Immuno!. 30: 51.

Institut fur Chirurgische Porschung der Universitiit Munchen und Max-v. -Pettenkofer­Institut der Universitiit Munchen, FRG

14. Discrimination of pathogenic and environmental strains of Legionella pneumophila by means of monoclonal antibodies

B. U. V. SPECHT, W. EHRn, and W. BRENDEl.

Since 1977 a new family of gram-negative bacteria called LegionelJae is known to cause severe pneumonia in man. from the 11 known species of LegioneHae, the species L. pneumophila with 8 characterized different serogroups is clinically most imponant, because the majority of infections is caused by serogruup une, 4 monoclonal antibodies against this serogroup were prepared and tested against 30 environmental isolates and 9 clinic isolates of L. pneumophila serogroup 1.

Methods: Balblc mice were immunized 3 times i.v. with heat inactivated L. pneumophila serogroup one. Antibody titers were measured by ELISA with a peroxydase conjugated antimouse IgG (NEN). immune spleen cells were fused with NSI myeloma cells (obtained by Dr. KOH1.ER), grown in culture and doned by limiting dilutation as described by KOHLER and MTI.~iTETN. Immunofluorescence was measured on slides of either heat or fonnaldehyd inactivated L.

Results: AI1 4 monoclonal antibodies showed binding to heat or fo rmaldehyde inactivated L. pneumopbila serogroup one. Only 2 reacted with the 9 isolal'es obtained from patients. One antibody (133/AS) showed binding to the isolates Kassell and 3; there was no binding to 7 different isolates form Munich, lledin, Augsburg, Zurich and Stockholm, belonging all to serogro up 1 of L. pneumophila. Three of the monoclonal antibodies (81, 132/A4 and 133/A5) showed no reactivity with 30 different environmental strains. One (G I) showed binding to 5 out of the 30 isolates. The results show that the monoclonal antibodies produced can clearly discriminate between pathogenic and environmental isolates of l. pneumophila serogroup 1. The molecular basis for this discrimination is currently under investigation.

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I Dept. of Neurology, University Clinic of Gottingen, 2 Dept. of Internal Medicine, Div. of H ematology/Oncology, University Clinic of Gottingen, Robert-Koch-Str. 40, 3400 Goningen, FRG

15. Phenotypic evaluation of peripheral blood lymphocytes in acute viral and bacterial infections of the central nervous system

Ttl. WEBER', P . SClTUFF-WERNER2, H . WISMANN', and G. OHMS2

Peripheral blood lymphocytes were investigated in five cases of encephalitis and in five cases of meningitis. Ten cases of polyneuropathy and 14 healthy volunteers served as controls. All cases were analyzed within five days of admission by flow cytometry with a FACS™ analyzer. Encephalitis c ases showed a significant reduction in pan-T -cells (Leu 1 + ILeu 4 + IT 11-1 ) (41.8 % ± 27.38 %; 681 ± 618 cells per Ill) and in thehelper/ inducer subset (Leu 3a + ILeu 3a+b + /

T4A+) (20.86% ± 15.11 %; 327 ± 306 cells per Ill). Neither in cases of meningitis nor in poly­neuropathies significant changes were seen as compared to the control group. Alterations in the Leu 2a+/T8, Leu r and the l.eu 12+/B 1 subsets were insignificant in all three groups as compared to the control group. From these findings we conclude that significant alterations are associated with viral but not with bacterial in fections of the central nervous system nor with presumed auroimmune disorders of the peripheral nervous system. Peripheral blood lymphocyte subset analysis with monoclonal anti bodies seems to give a d iagnostic clue with regard to the viral or bacterial nature of an infection of the central nervous system. The use of double fluorescence may help to identify antigens of lymphotropjc viruses on peripheral blood lymphocytes, thereby allowing rapid viral diagnosis.

Supported by BMrT-project no. 0384100.