Workshop 3 Regulation of the Immune Response II

Institute for Genetics, University of Cologne, WcycrtaJ 121,5000 Koln, FRG

1. Mechanisms which create antibody diversity

TH. BLANKENSTElN, G. ZOEBELEIN, and U. KRAWINKEL

OUT group works on the question of how diversity in antibody V-region genes is generated in the germ-line and somatically. Germ-line V wregion genes weTe isolated from the genome of hybridoma Bt-S.V1. The evolution of gene VA lU 04 tentatively can be fe-constructed: Duplication of the BALB/C derived germ-line gene VMPCIl-11J.4 generated a precursor of gene VAR104 which recombined with gene VARIOC, This recombination appears to have involved heteroduplex formation benveen the recombining genes followed by mismatch repair. Repeti­tive elements andior DNA-sequences being able to convert from B into Z-conformation upstream of the genes VAR100 and VMPCl l_100i may have promoted the recombination,

Hygiene-Institut, Westfalische Wilhelms-Universitat, 4400 Munster, PRG

2. Cellular interactions between T cell clones, antigen presenting cells and T helper cells

R. DI PAULI and T. BROCKNER

We have isolated several T cell clones, specific for minor histocompatibility antigens (HA) and restricted to the K end of the H-l. These clones, although sensitive to IL2, depend for continuous growth on the presence of irradiated spleen cells, bearing the specific minor H A and the H-l to which the clones are restricted. The stimulator spleen cells arc radioresistant but have to be metabolically active to induce proliferation by the clones. We have separated the stimulating spleen cells into plastic adherent, Thy-l - cells and into NW nonadherent T cells. None of the two populations, taken individually, is capable to induce the T cell clones to

proliferate, however when mixed they regain their original stimulatory capacity. Further characterization of the cdl types involved showed that, one is a splenic adherent, Thy-1 - and I-A + cell (SAC). the other a NW nonadhering, Thy-t t and Lyt-l tr cell (Th) . To study the level of interaction between the T cell clones and the stimulating cells, we combined SAC and Th of different minor HA. but identical B-2 to restore the stimulatory capacity. Only combinations where both of the cells, SAC and Th, carried the original stimulating minor H A, recognized by the dones, were capable to induce a proliferation in the dones. These results suggest that certain T cells, not only interact with accessory cells presenting the antigen but also. through specific recognition, with T helper cells in the stimulator cell population.

Workshop 3: Regulation of the Immune Response II 23

Max~Planck-Institut fur Immunbiologie, f<reihurg, FRG

3. Analysis of the human in vitro immune response to carbohydrate of A streptococci. Failure of anti-A-CHO producing B cells to switch to IgG in polyclonally or antigen activated cultures

F. EMMRlCH and K. EICHMANN

To study immunoregulation in a human in vitro model system streptococcal A carbohydrate (A-CHO), mainly directed to the terminal sugar hapten N~acetyl-D-glucosamine (GlcNAc): (1) A-CHO is a structurally well defined antigen of (2) physiological importance_ Compared to protein antigens (3) only limitt!d information is available on the response to carbohydratt! antigens in vitro, but (4) the response is T cell dependent, (5) perhaps as limited as the B cell response regarding the number of specific clones involved. (6) In addition, remarkable amounts of anti-A-CHO specific antibodies are found in nearly all human sera reaching in some cases nearly 10 % of the total immunoglobulin. Accordingly, large numbers of presen­sitized T as well as B cells should be expected among peripheral blood lymphocytes. This was confirmed by frequt!ncy analyses of polyclonally activated human B cells producing total IgM versus specific JgM (anti-A-CHO): Among lymphocytes of a single healthy donor up to one out of six activated B cells produced IgM anti-A-CHO antibodies. Anti-A-CHO antibodies arc mainly of the IgM isotype whereas only minimum amounts of IgG anti-A-CHO are produced by polyc1onal as well as by antigen-specific activation which is in contrast to the anti-A-CHO antibodies produced in vivo and, on the other hand, also in contrast to in vitro produced antibodies of other specificities. Therefore, anti-A-CHO antibodies seem to be produced by a B-cell subpopulation which is functionally distinct from B cells of other specificities. The considerable amounts of IgM anti-A-CHO found in serum and in activated macrocultures arc paralleled by equivalent B cell frequencies for IgM anti-A-CHO in PBL suggesting that the B cell precursors are equally distributed in peripheral blood compared to

lymphoid tissue and, in addition, that the amount of Ig produced per B cell equals the production rate for B cells of other spt!cificicies. Purified T4 cells providt!d a bystander help to the IgM response which was not influenced by the addition of T8 cells. Distinct in vitro differentiation or switch requirements may be postulated for IgG anti-A-CHO producing B cells in contrast to the bulk of IgG producing B cdls with other specificities. This functional criterium might be helpful in defining human B cell subpopulations.

Institut fur Immunologic der Universitat Main;t, 6500 Main;t, FRG *Institut fur Virusforschung, Deutsches Krebsforschungszentrum, 6900 Heidelberg, FRG

4. In vitro activation by y-IFN triggers long-term cultured bone marrow-derived macrophages to antigen presenting function equivalent to that of in vivo activated peritoneal macrophages

H.-G. FISClIER, A. B. RESKE-KUNZ, E. SPAETH, H. KIRCHNER\ and E. RODE

Bone marrow-derived macrophages (BMM4I), grown in liquid culture with L-cell CSF as differentiation and growth faclar, were previously reported to have the capacity for antigen presentation to primed lymph node T cells (1). Upon long-term culture such BMM¢llose their presentation function as shown by a reduction of their ability to induce an antigen-dependent proliferation of primed lympb node T cells or of the cloned T cell line ST2/K.9 (2). However, the accessory cell function of these agt!d BMM¢l could be completely restored by a shan-term

24 . XVI. Meeting of the Society of Immunology

pulse-treatment with lymphokines (LK). Several supernatants obtai.ned from ConA-stimu­lated rat spleen cell s and from T cell lines stimulated by antigen or mitogen activated aged BMMw for antigen presentation, whereas control supernatants from unstimulated T cells were ineffective. All LK preparations active in our system proved to cont.lin y-IFN as determined in a virus p laq ue reduction assay. Their effect could be substituted by a low dose (S-10 units/ml) of gemechnologically produced murine y-IfN. In direct comparison in vitro LK-triggered BMM~ presented antigen as efficiently as peritoneal exsudate macrophages which had been activated in vivo by ConA. Therefore, these findings obtained with long-term cultured cells can be expected to reflect a physiological mechanism for the ampl ification of the inunune response.

1. Rr.sKE-KuNz, A. n., E. SPAETH, K. REsKE, M.-L. LOHMANN-MAlTHES, and E. RODE. 1981. induction of anamnestic T cell proliferation by antigen-pulsed, bone marrow derived macrophages. Eur. J. Immuno!. 11: 745.

2. FISCHER, H.-G., A. B. R ESKE-KUNZ, E. SPAETH, H. KIRCHN'.R, and E. R ODE. 1984. Characterization of lymphokine-mediated activation of macrophages fo r antigen presenta­tion: srudies with long-tenn cultured bone marrow-derived macrophagcs and cloned T cells. lmmunobiology, in press.

Institute for Genetics, University of Cologne, Weyertal 121 ,5000 Koln 41, FRG

5. Deletion of the JgH enhancer does not reduce immunoglobulin heavy chain production of a mouse hybridoma cell line

S. KLEIN, F. SABLlTZKY, and A. RADBRUCH

Recently DNA sequences have been identified tbat ensure tissue-specific transcription of immunoglobulin genes. They are called enhancers or activators. We have isolated a variant hybridoma cell that has deleted the Ig heavy chain gene enhancer in vitro upon elass switching from TgM to IgD and has no other IgH enhancer, because the active IgH locus is the only one, the cell contains. We have quantitated the amount of Ig produced by these cells and compared it to the parental cell line and to other hybridoma cell lines expressing IgM or IgD. In the absence of any 19 heavy chain gene enhancer 19 heavy chain production is not reduced. Therefore, one explanation is that the IgH enhancer is probably needed fo r activation of IgH transcription rather than for maintenance or regulation of high rate transcription. Another possibility is the creation of an enhance r dement de novo upon recombination.

Max Planck Society, C li nical Research Unit for Multiple Sclerosis, POB 6120, 8700 WUrLburg, FRG

6, Production of antibodies against soluble antigen after intravenous injection of antigen-specific T cells

W. E. 1'. KLrNKERT

In vitro p roduction of antibodies can be achieved by immunization of soluble antigen emulsified in complete Freund's ad juvant. Here, r report that a rat T -helper cell line specific for ovalbumin is sufficient for the production of antibodies if injected intravenously without

Workshop 3: Regulation of the Immune Response II . 25

soluble antigen. Serum levels of antibodies can be detected as early as day 4 if the injected cells were restimulated in vitro with the relevant antigen or Concanavalin A 3 days before injection. Antibodies against ovalbumin were not detected when irradiated cells were in jected indicating an active participation of the T A cel1line. In addition, the serum derived from animals injected with restimulated T -cells which were both irradiated or non-irradiated enhanced the prolifer­ation of the T-cell line in vitro if restimulated in presence of antigen. Th is effect was not specific for the T-cell line wh ich was injected. Thus, evidence has accumulated mat injected antigen-specific T-cell lines induce antibodies against the original amigen and that the sera augment the antigen-response of long term T-cell lines. Therefore, injection of antigen-specific T-cells appears to influence the regulation of both T and B cells through idiotypic network.

I I. Medizinische Klinik und Poliklinik, Joh. Gutenberg Universitiit Mainz, PRG 1 Department of Medicine, Harvard Medical School, Boston, USA

7. A functional role for the Tll sheep erythrocyte receptor of human T lymphocytes

S. c. MEUERI, s. P. SCHLOSSMAN2, and E. 1. REINHERZ2

A SO KD (kilo-dalton) glycoprotein appears at the earliest level of human T lineage ontogeny and demonstrates a remarkable structural conservation on T cells of phylogenetically distinct primates. This structu re, termed Tt I, was recently described to represem the receptor for sheep erythrocytes (S RBC). To characterize the physiologic function of the TIl surface molecule a series o f monoclonal antibodies was produced which detected three distinct epitopes of this SO KD Structu re: Tl110 the SRBC binding site expressed on all T lymphocytes and thymocytes ; T l h with an identical distribution as 1111 - however unrelated to SRBC­binding; and TIl l, a neoepitope expressed only upon '1' cell activation. Importantly, simul­taneous triggering of TI1 2 and Til) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen Or antigen presenting cells. This antigen independent mode of T cell activation is clearly distinct from that involving the T3-Ti antigen receptor complex and, therefore, represents a previously uniden­tified alternative pathway of T cell activation. Given the early expression of TIl in T cell ontogeny - prior to the T3· Ti antigen receptor complex - the former may be involved in clonal expansion and/or differentiation of immature T -lymphocytes.

Inst. f. Med. Mikrobiologie und Immunologic, Ruhr-Universitiit Bachurn, PRG

8. IgE-synthesis in vitro: suppression by mouse serum factor(s) and the interaction with IgE

PH. P FEIFFER, I. RAUSCHEN, A. BOliN, and W. KONIG

Analyzing the regulation of the 19E antibody synthesis we previously described that the addition of normal mouse serum suppressed the IgE production of lymphoid mouse cells in vitro. After fractionation of the serum by ammoniumsu1fate-precipitation it is demonstrated that only the serum fractions obtained at 20-50 % sah saturation inhibited the IgE antibody synthesis by 63 %. In contrast, the 20 % amOloniumsulfate-precipitate and the fractions

26 . XVI. Meeting of the Society of Immunology

obtained with 70-80 % salt saturation could not suppress IgE synthesis in vitro. Ultrafiltration (Amicon XM50) and gelfiltration (Fractogel TSK/HW55) of the 50 o/v-precipitate demon­strated the dependence on serumfactor(s) with molecular weight of more than 50,000 Dalton. Immunoabsorption of the suppressive fraction with aIgE-Sepharose diminished the inhibitory effects significantly. In contrast, IgE-synthesis of cultured mouse cells was not suppressed by addition of monoclonal, DNP-specific IgE alone or with the 20 %-precipitate, which has been enriched with IgE. Thus, the suppression is dependent on factors present in the 50 %­precipitate of normal mouse serum. Studying the role of factor interaction, we demonstrate that a cellfree culture supernatant (> 50,000 Dalton) of mouse lymphocytes, which had been incubated with 20 Ilg 19E for 24 hr5, suppressed the in vitro TgE-synthesis. In contrast, low molecular weight supernatants « 50,000 Dalton) or the supernatants which were prepared by incubation of mouse cells without IgE failed to inhibit in vitro IgE-synthesis. The binding of l2'J -Iabelied anti-mouse 19E to ox erythrocytes, which wt!re coated with IgE, was inhibited by the 50 %-serumfraction as well as the high molecular weight supernatants but not by the low molecular weight supernatant~ « 50,000 Dalton). The Jatter, however, as well as the serum fraction and tht! high molt!cular weight supern~tant (> 50,000 Dalton) reduced the rosette formation of mouse lymphocytcs with ox crythrocytes, coated with IgE. Our results indicate, that 1) high molecular factor(s) which are present in defined serumfractions suppress the IgE-synthesis in vitro and 2) IgE antibodies may trigger the suppression, while IgE­binding factors might regulate IgE antibody production at the cellular level as well.

Department of Dem1atology I., University of Vienna, Vienna, Austria

9. The role of Langerhans cells in the induction of cytotoxic T cell activity against keratinocyte-bound surface antigens

G. STEINER, K. WOLFF, E. TSCIlACHLER, and G. STINGL

The capacity of epidermal cells (EC) to activate proliferative and cytotoxic T cell responses is dependent upon the presence of la-bearing Langerhans cells (LC). In all assay systems tested so far, LC display antigen presentation function, i.e. they express surface-bound foreign or altered-self structures and thereby stimulate T cdl activation. In contrast, numerous attempts to demonstrate an accessory cell function of EC have so far yielded negative results. This means that EC containing no foreign antigens could nOt restore H-2 restricted cytotoxic T cell reactions in an la+-adherent cell-depleted culture system.

Reasoning that the accessory cell function of EC might be dept!ndent on a close physical contact between Le, stimulator cells and responder T cells, we used - instead of conventional flat-bottom cultures - culture conditions which allow a better interaction between the cell types involved. Under these modified, but not under conventional culture conditions, EC proved to be potent accessory cells in the induction of alloreactive and hapten-specific cytotoxic T cell responses. Whereas C3H/He keratinocytes did not induce alloreactive cn activity in purified Balb/c T cells, the addition of Balblc EC restored this functional capacity. Similarly, the failure of TNP-modified C3H/He keratinocytes to evoke TNP-specific H-2 restricted CTL in purified C3H/He T cells was restored by the addition of unmodified C3HI He Le-containing EC. Removal of LC by relevant anti-Ia antibodies plus complement abolished tlle accessory cell capacity of EC.

These data demonstrate that LC-containing EC are capable of inducing CTL activity against KC-bound antigens and, thus, bear important implications with regard to the role of LC in the induction of specific effector mechanisms against allo-, neo- and differentiation antigens on tbeir epidermal symbiollts.

Workshop 3: Regulation of the Immune Response II 27

Inst. of General and Experimental Pathology, Univ. of Vienna, Austria

10. Macrophages as suppressor cells in mitogen induced spleen cell proliferation

E. TRAVNICZEK, G. BOI:l"l-N ITULESCU, Ch. HOLZINGER, and O. FORSTER

The role of macrophages in lectin-induced lymphocyte proliferation is still controversial. There are data from rat, mouse, guinea pig and man, which show that macrophages have accessory functions. On the other hand there is some evidence for suppressive influences of alveolar macrophages from rat, rabbit, dog and cattle. Most likely different cell subpopulations are responsible for these fum:t ions, and they may have distinct markers. We isolated rat macrophages by pulmonary lava~e and separated them into plastic-adherent and -nonadherem cells. Spleen cells were isolated by density gradient centrifugation and depleted of adherent cell s by passage over a Sephadex G- IO column. Addition of 1 to 10% adherent alveolar macrophages to total spleen cells decreased the PHA and ConA iuduced proliferat ion in a dose dependent manner. These cells were esterase positive, phagocytic, had Fc IgG receptor and «ganglioside-receptor» (GR). Addition of 1 % nonadherent alveolar macrophages to Sephadex G-l0 depleted spleen cel ls increased the stimulation, on the other hand 5 % and to % were again suppressive. These cells were heterogeneous in size and morphology and a lower number of them expressed the markers of the adherent cell population. Further separation of cells according to expression of Ia antigens and GR showed that these twO markers were mutually exclusive. Ia + /GR - -cells were found to suppOrt the mitogen induced stimulation of lympho­cytes whereas Ia - /GR + -cells were strongly suppressive. The GR, a structure which recognizes sialic acid containing carbohydrates, may be a useful marker to define the phenotype of suppressor macrophages.

Supported by Austrian Science Research Fund Project 5056.

Forschungsinstitut Borstel, 2061 Borstel, .FRG

11. Human T-Iymphocytes can be stimulated by PHA in absence of HLA-DR + accessory cells when cultured in presence of Leu-7 positive cells

A. J. ULMER, w. SCHOLZ, and H.-D. F LAD

Recently we have reported that DNA-synthesis of highly purified (hp) 'f-cells from human peripheral blood can be stimulated by PHA in an ultra micro culture (1 !Alar 2 Ill) in the total absence of ANAE positive mOllocytes (1). Further experiments along this line gave the following results: 1. A rabbit anti-serum against human leukocytic pyrogen (also containing anti IL-l activity)

reduced but did not abrogate tlle stimulation of hp T -cells by PHA. 2. Depletion of either HLA-DR+ cells (classical accessory cells) or of Leu-7+ cells (a

population of medium to large lymphocytes possessing NK cell activity) from hp T-cells by cell sorting also reduced but did not abrogate the stimulation of the hp T -cells by PHA.

3. However, in absence of HLA-DR+ and of Leu-7+ cells the hp T-cells totally failed to respond to PHA.

4. This abrogation of the response to PHA was not found when hp T-cells were depIcted of HLA-DR I and Leu-l1a+ cells (a population of Fe receptor bearing lymphocytes with NK cell activity).

28 XVI. Meeting of the Society of lmmunology

From these results we conclude that human T-Iymphocytes can be stimulated by PHA in the total absence of HLA-DR+ cells on condition that Leu-7+ cells arc present. It has to be discussed wether L eu-7+ (but not Leu-11a l

) cells are able to provide an alternative activation pathway during mitogenic stimulation which is at least partially independent of the classical pathway initiated by fl -l providing HLA-DR+ accessory cells.

(Supported by Deutsche Forschungsgemcinschaft. FL 10414-1.)

1. A. J. U LMER, W. SCIIOLZ, M. ERNST, and H.-D. FLAD. Europ. J. Cell 8;01. 1983. Suppl. 2: 42.

I lost. for Brain Research, Austrian Academy of Science.~, and zInst. of General and Experi­mental Pathology. Univ. of Vienna, Austria

12. Possible participation of gang~oside-like sugar-structures in the macrophage-lymphocyte interaction

M. WALGRAMl, G. BOLTz - NITULf.'iCU2, and O. P6 RSTER2

Interaction between macrophages (Mw) and unprimed lymphocy tes ( Ly) by cluster forma­tion is known for some time as a transitory event which may be important for the induction of an immune response. The nature of membrane st ructures which mediate this weak binding is unknown. 1berefure we initiated studi es upon the interaction between adherent alveolar (AMt'Jl) or peritoneal macrophages (PMtp) and Ly of sp leen. thym us or lymph nodes from syngeneic Lewis rats. Macrophagcs were allowed to adhere to glass (2 hrs. }70) and after removing nonadherent cells Ly-suspensions (4 X 106 cells/ml) were added and incubation continued for various time periods. Maximal cluster formation was found at 1 hr incubation. The average of cluster-forming Mil> with sp lt:cn-Ly was 30 %, with thymic-Ly 15 % and with lymph node-Ly 10 %. A moderate increase of cluster-formation was seen with proteose­peptone eli cited cells, an intense increase with cells harvested 4 days after Corynebacterium parvum stimulation. A dose dependent reduction of cluster formation was noted after preincubation of Ly with trypsin or neuraminidase. Preincubation of M<l) with various concentrations of gangliosides (GMh GM2, Gt-n. GDla, G01 b, GfIb). sialyUactose and N -acetyl­galactosamine also inhibited cluster formation in a dose dependent fashion, but never completely. Lactose up to 10-2M and mannan up to 20 mg/ml were ineffective. Our data point to a possible importance of the previously described ... ganglioside receptOh of rat McJ.> in M¢l­Ly interaction.

This study was supported by Austrian Science Research Fund Project 5056 and Hochschul­jubilaumsstiftung der Stadt \Vien.

Institute for Genetics, University of Cologne. Cologne, FRG

13. Analysis of immunoglobulin gene rearrangement in mouse B­lymphocytes stimulated with LPS

E. WfNfER, A. RADBRUCH, F. SABLITZKY, and U. KRAWINKEL

As recently reported deletion of C!! genes on one or both chromosomes occurs in mouse B­lymphocytes upon stimulation with LPS (Radbruch and Sablitzky, EMBO J., 2, 1929-1935

Workshop 3: Regulation of the Immune Response II . 29

[1983 J) . In order to further investigate these events at the molecular level, we prepared genomic libraries from DNA of LPS blasts expressing surface IgM or IgG, respectively. Seven clones have been obtained, each one comprising a constant region gene together with a rearranged JH gene. It is possible to decide whether the cloned genes come from the active or the inactive chromosome. Switch recombination appears to have taken place within S regions in clones containing the Cy3 gene. Restriction maps and DNA sequences of the regions of interest are presented.

Max-Planck-Institut fur Immunbiologie, Freiburg, FRG

14. Isotype-specific suppression of phosphocholine specific antibody formation in the immune defective CBA/N mouse by heterologous antiidiotypic antisera

B. ZARNACK, H. MOSSMANN, K. BARTSCH, and D. K. HAM.MER

CBAIN mice produce IgE and IgG antibodies after low doses of p-diazophenylphos­phocholine-conjugated keyhole limped hemocyanin (PC-KLH) but their response is virtually devoid of IgM and IgA antibodies. In order to study, if the IgE and IgG responses are regulated independently, heterologous anti-idiotypic antisera against PC-specific BALBlc myeloma proteins T 15 and M 167 were passively administered to CBA/N mice in the course of an anti-PC response. A single injection of IgG anri-TIS, given one day prior or 7 days after the secondary challenge with PC-KLH, resulted in a long-lasting suppression of the formation of IgE bm 110t IgG antibodies. The administration of IgG anti-M 167, however, was without any effect on the formation of PC-specific antibodies of both isotypes. This indicates that anti­PC IgE but not IgG antibodies consist mainly of the T t 5 idiotype or of cross-reacting idiotypes. Thus, the CBA/N mouse offers an excellent model to demonstrate the independent regulation of the IgE and IgG response.