Workshop L B Cell Activation

Workshop L B Cell Activation

Institut für Genetik der Universität zu Köln, Weyertal121, 5000 Köln 41, FRG

L.t Properties of hapten binding of anti-NP antibodies investigated by site-specific mutagenesis D.

J. ALLEN and K. RAJEWSKY

The primary response anti-NP (4-hydroxy-3-nitrophenylacetyl) monoclonal antibody BI-8 expresses the germline V186.2 Vh gene in combination with the DFL16.1 D element. In the secondary response to the hapten, extensive somatic mutation throughout the Vh gene accompanies an increase in hapten binding affinity (CUMANO and RAJEWSKY. 1986. EMBO J. 5: 2459-2468). We have investigated the structural basis of both hapten binding specificity and affinity by performing site-specific mutagenesis on antibody BI-8. We have shown that a thyrosine residue at position 99 (CDR3) of the antibody is essential for hapten binding, which reflects its invariance in all anti-NP antibodies so far sequenced. We have also shown that although secondary response antibodies are highly mutated, a sonsistently-observed mutation in CDRI (tryptophan to leucine at position 33) is sufficient to increase the affinity for hapten to a level typical of a secondary response antibody.

Abt. Immunologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61, FRG

L.2 Human NK clones upregulate antibody production

J.

BECKER, TH. WERFEL, C. SCHREIBER, and R. E. SCHMIDT

There is increasing evidence for the immunoregulatory role of natural killer (NK) cells. Several studies with NK enriched preparations from peripheral blood suggest a role of NK cells in B cell differentiation. Enhancement and suppression have been described. Using human NK clones, both NKH1+, T11+, T3- OT BI8, CNK6, NKC4) and NKH1+, Tl1+, TH OT9, JT10) cells, we studied the regulatory effects in a Staphylococcus aureus Cowan strain l/rIL-2 activated B cell system. After coculture of highly purified B cells with NK clones, IgG and IgM were determined in an ELISA. Both IgG and IgM production were significantly increased in the presence of NK clones. Enhancement was found to be dependent on the

364 . XIXth Meeting of the Society of Immunology number of effector ceils added. Kinetic studies revealed optimal antibody production, when NK clones were added to day 2 preactivated B ceils. For studying the mechanisms involved in B cell differentiation by NK ceils, we performed cocultures in the presence of monoclonal antibodies blocking ceil-ceil adhesion (anti LFA-l, CD lla) or specific target recognition (anti TNKtar). Since such antibodies inhibited B ceil differentiation by NK ceils, ceil-ceil interaction via specific ceil surface structures appears to be necessary. Whether soluble factors are released during this interaction and involved in mediation of B ceil differentiation by these effector ceils is addressed in further studies. Supported by DFG Schm 596/2-1.

Institut für Virologie und Immunbiologie, Versbacher Str. 7, D-8700 Würz burg, FRG

1.3 Regulation of Ig transcription in variously stimulated B cells I. BERBERICH, E. WECKER, and A. SCHIMPL Resting B ceils stimulated with LPS proliferate and eventuaily differentiate to Ig secreting ceils. This differentiation process is characterized in respect to IgM-specific RNA as foilows: 1. shift !!m to !!" 2. large increase in the transcription and the amount of !!- and k-chain specific RNAs, 3. induction of the J-chain gene. Antireceptor antibodies (F(ab')2) suppress the differentiation step in LPS stimulated ceils without affecting the proliferative response. Molecular analysis showed that Ig gene transcription per se is hardly affected, but 2 days post stimulation there is a selective loss of!!s mRNA. At later times both mature!! mRNA levels (!!s and !!m) as weil as k mRNA are greatly reduced though the transcription rate only goes down by a factor of 2 to 3 (CHEN-BETIECKEN et a1.1985. PNAS 82: 7384). Since crosslinking of the IgM-receptor leads to hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) we looked for the influence of ionomycin (1) and phorbol 11,12-dibutyrate (P) (both analogues of the hydrolysis products of PIP 2) added simultaneously with LPS to B ceils at the ceilular and molecular level. DNA synthesis, Ig secretion and !!, k- and J-chain specific RNA were analyzed at day 3. We show that I, P and both IP together again hardly affect DNA synthesis, but individuaily and, particulady, in combination drasticaily reduce Ig secretion in LPS activated ceils. The block of differentiation exerted by 1 and/or P in LPS stimulated B ceil cultures detectable at the ceilular level is already established at the RNA level. Whereas in respect to J- and k-chain RNA transcriptional as weil as post-transcriptional mechanisms might be responsible and necessary for changes in the specific RNA contents, in the case of the !!-chain a posttranscriptional mechanism alone is sufficient to lead to the specific loss of mRNA of the !!s-form.

Institut für Genetik, Weyertal121, 5000 Köln 41, FRG

1.4 Methylation pattern of IgG loci: supermethylation of IgG constant region genes in B lymphocytes prior to activation C. BURGER and A. RADBRUCH The molecular basis of differential activation of immunoglobulin genes during B cell ontogeny is still poody understood. In most systems accessibility of a gene correlates with an

XIXth Meeting of the Society of Immunology . 365 active chromatin structure as defined by demethylation and DNase I sensitivity. This model is strongly supported by the present analysis of the methylation pattern of the Cf.!, cyl, and cy3 constant region genes du ring B cell differentiation. We compared the Mspl/Hpall restriction enzyme patterns of splenic B cells before and after activation with LPS in the presence and the absence of interleukin 4 (IL4). In splenic B cells the Cf.llocus is hypomethylated as compared to liver cells. In addition, the gene of the secreted form of the [.I chain becomes demethylated at distinct sites after LPS activation. The cy genes are supermethylated in normal cells as compared to nonlymphoid cells and thereby probably protected from unspecific activation. After stimulation of splenie B cells to Ig dass switching certain cy genes become demethylated again. The pattern of demethylation is dependent of the type of stimulation. The importance of methylation for dass switching is emphasized by the finding that 5' Azacytidine, a methylase inhibitor, can overcome the suppressive effect of interleukin 4 on the switch to cy3.

Institut für Immunbiologie der Universität, D-7800 Freiburg, and Institut für Organische Chemie der Universität, D-7400 Tübingen, FRG

1.5 Immunoglobulin subclass-specific responses to synthetic B lymphocyte-activating lipopeptides covalently linked to antigens T. BÖLTZ, K. SCHAUERTE, P. KOCH, W. G. BESSLER

J.

METZGER, K.-H. WIESMÜLLER, G. JUNG, and

Synthetic analogues of the N-terminal part of lipoprotein from the outer membrane of

Escherichia coli constitute potent polydonal B lymphocyte activators. Using tripalmitoyl-S-

glyceryl-cysteinyl-serine (Pam3Cys-Ser) and related compounds as carriers for antigens, the lipopeptide conjugates induced specific immunoglobulin production in vivo without the addition of any further adjuvant. In contrast to the conjugates, neither the lipopeptides, the low-molecular-weight antigens alone, nor mixtures of these compounds elicited a significant specific response. By means of an isotype-specific enzyme-linked immunoassay, we determined the magnitude of antibody production and isotype pattern induced by stimulation with the lipopeptide analogues alone, and the antigen-specific responses following immunization with the lipopeptide-antigen conjugates in euthymie as weil as in athymic BALB/c mice. Primary and secondary responses to digoxin (Boehringer, Mannheim, F.R.G.), and to a synthetic peptide coded for by the HIV -envelope gene, were compared to evaluate the influence of the chemical structure of the antigen on the immunogenic properties of the lipopeptide and possible immune mechanisms involved in these responses. Supported by BMFT grant PBE/03 8768 FKZ: 11-002-86.

Institute for Immunology, Univ. of Heidelberg, and Institute for Med. Microbiology, Univ. of Mainz, FRG

1.6 Stringent requirement for the complement component C3 du ring the induction of the humoral immune response S. KLEINDIENST, E. BÖTTGER, D. BITTER-SUERMANN, R. SCHÄFER, and R. BURGER

The role of C3 fragments in the immune response was previously analyzed in vitra and also in vivo by decomplementation. Some of the results obtained were controversial. We described

366 . XIXth Meeting of the Society of Immunology recently an inherited C3 deficiency (C3D) in inbred strain 2 guinea pigs. The animals provide a valuable model for studying the role of C3 in immune response in a direct fashion without artificial pretreatment. The C3D animals showed a markedly impaired humoral immune response against a limited dose of the T cell dependent antigen bacteriophage
Institute for Hygiene, Innsbruck, Austria

1.7 Stimulation of the C3d/EBV-receptor, CR2, results in the enhanced growth of a Burkitt-Iymphoma cellline G. PERNEGGER, T. F. SCHULZ, B. L. MYONES, A. EIGENTLER, A. PETZER, and M. P. DIERICH

The receptor for the C3d-fragment of the third complement component and the EpsteinBarr virus, CR2, has been shown to be involved in the control of B cell proliferation (1). In the case of murine B cells grown und er serum-free conditions, polymerized C3d acts like a macrophage-derived growth factor allowing the B cell to go through the S-phase. We wanted to establish whether malignant human B-Iymphoblastoid cells growing permanently in serum containing media without any further stimuli are still responsive to control mechanisms operating at the level of CR2, i.e. the transition between G 1 and S-phase. We established a cell culture system which allowed us to grow the human Burkitt-Iymphoma cellline RA}I und er serum-free conditions. Under these conditions RA}I cells grow to a maximal concentration of 7 x lOS/rn!. In the presence of four different HPLC-purified monoeIonal antibodies to CR2 growth of RA}I cells in this culture system was enhanced by a factor of 2-3. Incorporation of 3H-thymidine also increased by the same factor at an antibody concentration of 0.1 [!g/m!. This growth enhancing effect was obtained both with soluble antibodies and antibodies coated onto the culture plate or sepharose beads to obtain cross-linking of CR2 on the RA}I cell surface. Similar effects were obtained with purified human C3d. These effects could not be observed if even trace amounts of FCS were present in the culture medium. These results could suggest that the restriction point in the B cell cyeIe which is controlled by CR2 is still operative in malignant B-Iymphoblastoid cells with the capacity to grow permanently in cell culture. 1. MELCHERS, F., et a!. 1985. Nature 317: 264. Supported by FWF P-6054.

XIXth Meeting of the Society of Immunology . 367 1 Institut für Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstr. 10, D-6100 Darmstadt, and 2 Institut für Immunologie, Johannes-Gutenberg-Universität, Obere Zahlbacherstr. 67, D-6500 Mainz, FRG

L.S Participation of T cells in the enhancement of IgG subclass responses to lipopolysaccharide (LPS) produced by complexing LPS with a major outer membrane pro tein from Proteus mirabilis S. SCHELL!, C. PETERS2, F. ZIMMERMANN2, E. RÜDE 2, and K. NIXDORFF 1

We have previously shown that a major outer membrane protein (39 KD protein) from

Proteus mirabiJis, complexed with lipopolysaccharide (LPS) before administration to mice, greatly enhanced the induction of LPS-specific IgG antibody-producing cells when compared with responses to LPS alone. Our studies further revealed that the numbers and subclasses of IgG-producing cells induced were determined by the primary stimulus (induction of LPSspecific IgG memory cells). Terminal differentiation of these memory cells into antibodyproducing cells was achieved by a secondary stimulus. In the present report, the involvement of T cells in the enhancement of IgG responses to LPS by complexing LPS with the 39 KD protein was investigated. Comparison of the responses in normal and in nu/nu mice showed that enhancement of LPS-specific IgG responses did not occur in nu/nu mice, indicating aT cell dependence. Treatment of spleen cells with antibodies to Thy 1 plus complement to eliminate T cells before secondary stimulation in vitra also greatly reduced the IgG responses. These results suggest that, although LPS-specific IgG memory cells are induced by the primary stimulus, T cells are still necessary for the terminal differentiation of these IgG memory cells into antibody-producing cells by the secondary stimulus in our system.

Universität Konstanz, Fakultät für Biologie, Abt. Immunologie, Konstanz, FRG

L.9 Monoclonal anti a(1-3)dextran antibodies from dextran-defective XID mice H.-C. SELINKA and R. BÖSING-SCHNEIDER CBA/N mice and their male hybrid progeny (CBA/N X BALB/c) bear an X-linked B cell defect (XID) leading to unresponsiveness against type 2 antigens. After repeated immunizations with a(1-3)dextran (dex), male F1 mice fai! to produce specific antibodies, whereas female littermates express idiotype-positive anti-dex antibodies. In addition, by immunization with anti-idiotypic antibodies, a dex-specific response could be induced only in females. However, male (CBA/N X BALB/c)F1 mice carry the genetic potential to produce such antibodies. After several booster injections spleen cells of serum antibody negative male F1 mice were fused with SP-2 myeloma cells. Supernatants were screened for binding to a(1-3)dextran by hemagglutination and Elisa techniques. Eight out of 99 antibody secreting hybridomas were specific for the a(1-3)glucosidic linkage of dextran and recloned by limiting dilution. All eight isolated hybrids were found to be of IgM isotype, bearing the A light chain. Experiments are und er way to determine the expression of idiotopes on these hybrid oma proteins in order to gain information about idiotype regulation of anti-dex antibodies. This work was supported by Deutsche Forschungsgemeinschaft, SFB 156.

368 . XIXth Meeting of the Society of Immunology Institute for Immunology and Genetics, German Cancer Research Center, INF 280, 6900 Heidelberg, FRG

1.10 Monoclonal antibodies against cell surface antigens on a malignant B cell-line associated with cytokine induced immunoglobulin secretion B. TRAUTH, R. HEDERER, H. KRAFFT-CZEPA, A. PETERS, K.M. DEBATIN, and P. H. KRAMMER

J.

FISCHER, H. LAHM, W. FALK,

Interleukin-4 (IL-4, B-cell-stimulating-factor-l, BSF-l) is a T-cell product initially described as a cofactor required for proliferation of resting mouse B-cells stimulated with antiIgM antibodies. IL-4 also induces Ia antigen expression on resting B-cells and prornotes the secretion of IgG 1 and IgE by lipopolysaccharide-stimulated B-cells. IL-4 also has T -cellgrowth-factor (TCGF) and mast-cell-growth-factor (MCGF) activities. Human IL-4 also shows pleiotropic activities, induding B-cell-growth-factor (BCGF) and TCGF activities, induction of FCE-receptor II and dass II MHC antigen expression. We report he re that human IL-4 also shows B-cell-differentiation-factor (BCDF) activity on two human lymphoblastoid cell-lines SKW6.4 (sIgM, x) and CESS (sIgG, A) inducing IgM and IgG secretion, respectively. At optimal concentrations IL-4 induces a twofold stimulation of Ig secretion of both cell-lines. In addition B-cell-stimulating-factor-2 (BSF-2, Interferon-ß2) causes a fourfold stimulation of IgM secretion of SKW6.4 cells and a threefold stimulation of IgG secretion of CESS. Monodonal antibodies were raised against the cytokine reactive cell-line SKW6.4. Four hybridomas were selected that inhibited cytokine induced IgM secretion of SKW6.4. These monodonal antibodies may recognize cell surface epitopes on SKW6.4 expressed on receptors for B-cell-tropic cytokines or on antigens functionally associated with such receptors.

Institut für Immunbiologie der Universität, Stefan-Meier-Str. 8, D-7800 Freiburg, FRG

1.11 Early signals during lymphocyte activation triggered by mitogenic lipopeptide or lipopolysaccharide (LPS) L. WAGNER-Roos, B. KLEINE, S. HAUSCHILDT, and W. G. BESSLER Hydrolysis of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP3) and diacylglycerol (DAG) has been observed during activation of many different cell types, and IP3 and DAG are widely accepted as second messengers. Stimulation of B cells by Igcrosslinking ligands also results in IP3 and DAG formation. However, during polydonal mitogenic activation of B cells by lipoprotein (LP) and LPS, increased phosphatidylinositide (PI) metabolism could not be found, suggesting different signalling pathways leading to the same biological responses, e.g. proliferation, enhanced la-expression. In consequence, IP3induced mobilization of calcium from intracellular stores could not be observed. Incubation of small resting B cells with a synthetic analog (TPP) of LP resulted in a dose-dependent influx of extracellular Ca into the stimulated cells whereas LPS failed to augment Ca influx in this system. Furthermore, we have studied mitogen induced phosphorylation of membrane proteins. 50 far we have identified an acidic 60 kDa protein phosphorylated only in LP5 activated cells. Attempts to reveal membrane kinases as mitogen receptor structures are in progress. Supported by the Deutsche Forschungsgemeinschaft (Be489/4-1 and 4-2).

XIXth Meeting of the Society of Immunology . 369 Basel Institute for Immunology, CH-4005 Basel, Switzerland, and ". Institute of Immunology and Rheumatology, University of Oslo, 0172 Oslo, Norway

1.12 B lymphomas process and present their endogenous idiotype to MHC-restricted T cells S. WEISS, and B. BOGEN'"

B cells are able to process and present exogeneously added antigen to major histocompatibility complex (MHC) restricted T cells. We addressed the question whether B cells also spontaneously process and present self molecules like endogenous immunoglobulin (Ig) in the context of their class II molecules. To that end, we have transfected the gene for the 1.2 light chain of MOPC 315 into B-Iymphoma cell lines. The A2 J15 chain differs from germline encoded 1.2 chains by several amino acids (AA) including AA position 94-96, due to somatic point mutations. Recently established 1-Ed restricted T cell clones recognize a processed idiotypic determinant of the 1.2 315 chain involving AA 94-96 (1). We could show that the transfectants, which express the A2 J15 light chain on the cell surface constitutively present this endogenous idiotypic determinant to the 1-Ed restricted T cell clones. Presently we are testing whether this idiotypic determinant is also presented when the 1.2 315 chain is not expressed on the cell membrane but in different cellular compartments. 1. BOGEN, B., R. SNODGRASS, J. P. BRIAND, and K. HANNESTAD. 1986. Eur. 1379-1384.

J. Immunol.

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Institut für Genetik, Universität zu Köln, Weyertal121, 5000 Köln 41, FRG

1.13 Deletions within the intron 5' of Cf! and their significance for class-switch recombination E. WINTER, A. RADBRUCH, and U. KRAWINKEL

B Iymphocytes of the mouse rearrange their heavy chain genes upon stimulation with lipopolysaccharide (LPS). We have analyzed the extent of heavy chain gene re arrangement in hybridomas derived from LPS-blasts and have found both inter-intro nie S-S-recombination leading to immunoglobulin (Ig) class-switch and deletions within the intron between the Ig enhancer and the CIl gene (WINTER et al. 1987. EMBO J. 6, in press). The intra-intronic deletions were observed 1) independently of Ig class-switch in some IgM-positive cells, 2) in addition to class-switch recombination on almost all allelically excluded IgH loci which retained the CIl gene. In order to determine the precise location of the deletions we have examined a panel of hybrid omas derived from LPS-blasts by Southern-blot analysis. Our results allow to define the extension of functionally essential parts of the SIl region.