Institute of Immunology, University, 6900 Heidelberg, Germany

Workshop N T-Cells I

N.1 Identification and characterization of a new monoclonal antibody (23012) against the human y/b T-cell receptor

THOMAS ACKERMANN, BRIGITTE HECKL-OSTREICHER, and DIETER KABELITZ

In an attempt to generate monoclonal antibodies (mAb) against the human y/6 T-cell receptor (TCR), mice were immunized with peripheral blood lymphocytes (PBL) from a patient with chronic T-cell leukemia of y/6-TcR phenotype. PBL from patient BU stained with mAb A13 and 6TCSl but failed to react with mAb BB3,7 A5 or TiyA, and thus expressed V6l but neither V62 nor Vy9. One of the mAb obtained from the fusion reacted with PBL of the patient as well as with certain cloned y/6+ cells but not with peripheral blood a/B+ T -cells. Preliminary evaluation of this mAb (termed 23D12) revealed the following characteristics: (1) In addition to its reactivity with the immunizing leukemic y/6+ T-cell population, mAb 23D12 recognizes some but not other V6l+ (i.e. 6TCS1+ or A13+) y/6+ clones; (2) in rare instances, mAb 23D12 also reacts with V61-negative y/6+ clones; (3) among normal peripheral blood T­cells 23D12+ cells are < 1 %; (4) 23D12+ T-cells are readily detectable among CD4-Cds­postnatal human thymocytes; in these instances, 23D12+ cells are confined to both V6l + andV6l- subsets of y/6+ thymocytes. (5) mAb 23D12 activates 23D12+ y/6+ clones when offered in a cross-linked manner; activation results in cytokine production (IL-2, TNF-a, IFN-y), autocrine proliferation, and cytolytic effector activity against FcR-positive target cells; (6) mAb 23D12 precipitates the TCR molecule from the surface of 23D12+ y/6+ clones. Experiments in progress are aimed at the molecular characterization of the specificity of mAb 23D12.

Dept. Internal Medicine, Div. Experimental Immunology, Med. Academy, 3090 Magdeburg, and 1 Dept. Immunology and Cell Biology, Forschungsinstitut, 2061 Borstel, Germany

N.2 Functional significance of dipeptidyl peptidase IV (OP IV, CD26) and aminopeptidase N (AP N, COB) in peripheral blood mononuclear cells - influence of peptidase inhibitors on IL-6 production

U. BANK, D. REINHOLD, F. BUHLING, D. KUNZ, E. SCHON, H.-D. FLADI, and S. ANSORGE

The dipeptidyl peptidase IV (DP IV) and aminopeptidase N (AP N), ectopeptidases of the plasma membrane of lymphocytes, have been found to be involved in regulation of lympho-

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cyte activation and proliferation in vitro. To study the influence of DP IV inhibitors on the IL-6 production of peripheral blood mononuclear cells (PBMC) in comparison to DNA synthesis we used PWM induction as an alternative approach to PHA/TP A stimulation. We could show, that the DP IV inhibitor Lys-Z-nitro-thiazolidide and also amastatin and bestatin - inhibitors of the AP N - decreased the IL-6 production of PWM stimulated PBMC in a dependent manner. These data collelated with the decreased ability of PWM stimulated PBMC to synthesize DNA (measured by eH]thymidine incorporation) in the presence of these inhibitors and of monoclonal and polyclonal anti-DP IV and anti-AP N antibodies. Using flow cytometric methods we could detect, that these inhibitors arrested the cells in the GO/G1

phase of the cell cycle.

Max-Planck-Society, Clinical Research Units for Rheumatology/Immunology, Institue for Clinical Immunology, University of Erlangen-Niirnberg, 8520 Erlangen, Germany

N.3 CD3 and CD4-mediated signalling and proliferation in resting and activated human T cells

B. M. BROKER, A. Yu. TSYGANKOV, A. H. GUSE, U. MEINKE, E. ROTH, and F. EMMRICH

We wanted to investigate the response of preactivated human T cells to stimuli given via TCR/CD3 and CD4. Six CD4+ T cell clones and resting T cells (RT) were stimulated by soluble anti-CD3 and/or anti-CD4. In some cases the mAbs were crosslinked on the cell surface using F (ab')2 fragments of goat anti-mouse Ig in solution. Proteintyrosine phosphory­lation (Tyr-P) and Ca2+i were determined and compared with the proliferative response.

The general level of Tyr-P correlated with increases of Ca2+i but not with DNA synthesis. However, the phosphorylation of two substrates (100 KDa and 36 KDa) paralleled the increases of DNA synthesis in clones. In both RT and T cell clones Tyr-P and Ca2+i increases were strikingly similar, whereas the proliferative responses differed. In RT anti-CD3 cross­linking abolished [1] and cocrosslinking with CD4 enhanced the anti-CD3 induced prolifera­tion. This was not observed in clones even in a quiescent state. We conclude that preactivated T cells and RT receive similar <early' signals via TCR/CD3 and CD4 but that they interpret them in a different way.

1. TSYGANKOV, BROKER, EMMRICH: Eur. J. Immunol., submitted.

1 II. Dept. of Internal Medicine and 2 Institute of Med. Microbiology and Hygiene, Technical University, 8000 Munich 80, 3 Clin. Research Group for Rheumatology, University Hospital, 7800 Freiburg, Germany

NA yo T<2:R+ Human intestinal intraepitheliallymphocytes exhibit restricted V-gene segment usage and marked oligoclonality

KAI DEUSCH!, GERD PLUSCHKE3, MEINHARD CLASSEN!, ULRICH KRAWINKEL3, HERMANN WAGNER2

, and KLAUS PFEFFER2

Intraepithelial T lymphocytes (lEI.) have been suggested to playa crucial role in the immune surveillance of surface epithelia. We have previously shown that a major fraction of human

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colonic intraepithelial lymphocytes (c-lEL) expresses a yO T cell receptor with preferential usage of the VOl gene segment. Moreover, yO T cells have been implicated in anti-bacterial immune responses. Since c-IEL are constitutively confronted with a large variety of microbial antigens, we were intrigued to characterize the yO T cell receptor repertoire of c-lEL at the molecular level. To this end, we sequenced PCR amplified VOl-Co cDNA derived from c-IEL of three individuals. Surprisingly in donor 1, 18 out of 19 VI'Jl-CO transcripts were identical and in donor 2, a limited set of canonicalO-chain sequences were observed, whereas in donor 3, the VI'Jl-Co transcripts were diverse. PCR analysis of the y chain V gene segment usage revealed a predominance of the Vy Subgwupl transcripts in donors 2 and 3. In contrast, in c-IEL of donor 1 the four known Vy subgroups were more evenly expressed. Sequence analysis of PCR products obtained with a VYsubgwupI specific primer revealed in donor 2 canonical Vy sequences whereas in donor 1 most transcripts were out of frame indicating pairing of the dominating chains with other Vy gene products. Thus our results provide evidence for a clonal dominance of yO T cell receptors expressed on c-IEL and suggest that certain c-IEL may be clonally expanded or selectively retained during intraepithelial immune responses. Finally, our data indicate that human intestinal IEL may have a restricted TCR repertoire. If the IEL expressing particular (oligo-) clonal TCR chains are involved in responses to invading pathogens, detailed analysis of these cells will be extremely advantageous in understanding immune surveillance at physiologic body surfaces.

Max-Planck-Institut fur Immunbiologie, 7800 Freiburg, Germany

N.S Genomic organization of the murine T cell-specific serine proteinase MTSP-l

K. EBNET, J. CHLUBA-DE TAPIA, and M. M. SIMON

Cytolytic T lymphocytes (CTL) elicit their effector functions by the release of cytoplasmic granules. In addition to perforin these granules contain 7 serine proteases, called MTSP-1/ Granzyme A, Granzyme B, C, D, E, F, and G. From these enzymes only MTSP-1 and Granzyme B show strong amidolytic activity. Although many experiments suggest the involvement of MTSP-1 and Granzyme B in different functions of the T cell - migration, cytolysis of target cells, regulation of B cells - final proof for their participation in any of these functions is still lacking. Therefore, we are planning to inactivate the MTSP-1 gene in embryonic stem (ES) cells. As a prerequisite for this technique we had to determine the genomic oroganization of the MTSP-1 gene. The screening of a genomic library resulted in one clone containing the whole coding region and 6 kb of the 5' regulatory region. The clone has been mapped with respect to several restriction enzyme sites. Using cDNA-derived oligonu­cleotides we have amplified the introns by PCR and have cloned the resulting PCR-products. Subsequent double-strand sequencing revealed the exact location of the exon/intron-bound­aries. The genomic organization of MTSP-1 is very similar to that of the two related serine proteases Granzyme Band C. Weare now constructing a targeting vector containing part of the MTSP-1 gene, which has been interrupted by the neomycin-resistance gene. Transfection of ES cells and injection of the ES cells into blastocysts may result in MTSP-1-deficient, mice which would allow to analyse the biological function(s) of MTSP-l.

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1 Institute of General and Experimental Pathology, Medical School, University, 6020 Innsbruck, Austria, 2 Dept. of Med. Microbiology, University, Turku, Finland

N.6 The numbers of CD4/CD8 chicken lymphocytes are regulated by MHCgenes

KAREL HALA 1, GUNTHER BOCK 1, OLLI V AINI02, and ROSWITHA SGONC i

CD4 and CDS antigens, expressed on peripheral T cells, define functional subsets of mature T cells. To investigate the percentage of these cells in chickens, we have used the CB and CC congenic lines (differing only in the major histocompatibility complex-MHC) and have studied peripheral blood lymphocytes (PBL), spleen cells and thymocytes by flow cytometer analysis. Murine monoclonal antibodies to CD4 and CDS antigens as a first antibody and rabbit anti-mouse antibodies labelled with fluorescein as a second antibody were used in indirect immunofluorescence tests. In previous experiments we have found that significant differences between these two chicken lines with respect to the percentage of CD4+ and CDS+ PBL are under the control of the MHC (HALA et al. 1991). In the present experiments, we analyzed lymphoid cells from spleen and thymus. In spleen cells we observed the same relationship between CD4 and CDS as in PBL. CB chickens have a higher number of CD4+ splenocytes than CC chickens. In the case of CDS+ splenocytes the percentage was not significantly different. We did not find any influence of regression or progression of Rous sarcoma virus induced tumors on the percentage of peripheral T cells and on the IL-2 production in vitro.

K. HALA, O. VAINIO, J. PLACHY, and G. BOCK: Animal Genetics 1991, in press.

This work was supported by the Jubilaumsfonds of the Austrian National Bank (Grant No. 3615).

Abt. Angewandte Immunologie, Deutsches Krebsforschungszentrum, 6900 Heidelberg, Ger­many

N.7 Signals in human T cell activation: Evidence for a common intracellular pathway induced through CD2, CD4, and CD8 but not TcRlCD3

S. HENNING, A. BADER, B. SCHRAVEN, S. MEUER, and Y. SAMSTAG

Comparing total protein phosphorylation we have recently identified a novel 19 kDa cytosolic phosphoprotein (pp19) that is dephosphorylated following CD2 but not T cell receptor (TcR)/CD3 stimulation of peripheral human T lymphocytes (SAMSTAG et aI., J. Immunol. 147, in press). Dephosphorylation occurs on serine residues and is clearly independ­ent of expression of a TcR. The extent of pp19 dephosphorylation is dose dependent and correlates with functional responses as DNA synthesis and lymphokine production. To investigate whether this early intracellular event is exclusively linked to the alternative pathway of T cell activation via CD2 we have analyzed protein phosphorylation after triggering of other membrane molecules which are known to exert a positive synergistic signal to T cell activation through the TcR/CD3 complex, namely CD4 and CDS. In marked contrast to TcR triggering alone approximation of CD4 and/or CDS to the TcR/CD3 complex by monoclonal antibodies (mAb) leads to pp19 dephosphorylation indicating that at least some intracellular signalling events are shared between CD4, CDS, and CD2 but not the TcR. Further comparison of

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protein phosphorylation events revealed a second intracellular process, namely serine phos­phorylation of a 67 kDa cytosolic protein (pp67), which is also observed following stimulation with both soluble anti-CD2 mAb or crosslinked anti-CD3/anti CD4/CD8 mAb but not anti­CD3 mAb alone. The existence of a functionl relationship between CD2, CD4, and CD8 was further supported by the finding that triggering through these surface molecules leads to induction of T cell responsiveness to IL-l and IL-6. None of these reactions could be initiated through TcR/CD3 alone. Finally, following chemical crosslinking of sterically associated surface molecules with DSP, both CD4 and CD2 molecules are detectable in immunoprecipi­tates obtained with Cd45 mAb. These data indicate the existence of common intracellular signalling pathways for CD2, CD4, and CD8 which are not linked to the TcR/CD3 complex.

Institute of Immunology, University, 6900 Heidelberg, Germany

N.S In vitro induction of programmed cell death (apoptosis) in short­term T -cell lines and T-cell clones

OTTMARJANSSEN, SEBASTIAN WESSELBORG, and DIETER KABELITZ

We have recently reported that T-cell receptor (TCR) signaling induces death by apoptosis in human yb+ T cell clones OANSSEN et aI., J. Immunol. 146,35, 1991). Here we have extended this investigation to a~+ (and additional yb+) T-cell clones as well as to short-term polyclonal T-cell lines. So far, 25 T-cell clones expressing the a~ [12] or yb [13] TCR were tested. 18 out of these IL-2-dependent clones underwent apoptosis upon restimulation. In these instances, 50 % to 90 % of the clonal population died (as evidenced by propidium iodide staining) when incubated with anti-TCR/CD3 mAb, ionomycin, or PHA in the presence of rIL-2. Current experiments address the issue as to why some (7 out of 25) T-cell clones are seemingly resistant to induction of apoptosis. In addition, we show that apoptosis can also be induced in polyclonal short-term T-cell lines. Time course studies revealed that the susceptibility to apoptosis increased with time after culture initiation. Thus PHA did not affect the viability of freshly isolated mononuclear cells but induced death by apoptosis in up to 90 % of cells expanded (after initial PH A-stimulation) for 19 days in the presence of rIL-2. In these experiments anti-TCR/CD3 mAb were less efficient than PHA or ionomycin in triggering cell death. Taken together, our data indicate that the susceptibility of a given T-cell to undergo receptor-induced suicide depends on at least three parameters: i) the state of activation (cell cycle?, age?); ii) the quality of the receptor signal, and iii) costimulatory signals provided by cytokines.

Institute for Immunology and Genetics, German Cancer Research Center, 6900 Heidelberg, Germany

N.9 Reduction of the binding avidity is still sufficient for negative selection

M. KNOBLOCH, G. SCHON RICH, J. SCHENKEL, G. J. HAMMERLlNG, and B. ARNOLD

Besides the antigen-specific contact between the T cell receptor (TCR) and antigenic peptides presented by major histocompatibility antigens (MHC) the additional interaction

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between the CD8 molecule and the a3-domain of MHC class I antigens is also necessary for transduction of an activation signal. In previous studies we have established that the mouse CD8 molecule cannot interact with the a3-domain of human HLA-A2. To test the contribu­tion of CD8 to negative selection in the thymus we generated two types of transgenic mice. One set of mice expresses an H-2Kb antigen, inwhich the a3-domain is exchanged by human HLA-A2 sequences (Kb A2). The second set of mice expresses an H-2Kb-specific T cell receptor. Using double-transgenic mice (TCR x Kb A2) we demonstrate here that despite the reduction of the avidity due to the interruption of the CD8-a3-interaction, we still obtain negative selection of TCR expressing thymocytes. These results show that a lower binding avidity is sufficient for tolerance induction in the thymus in contrast to T cell activation in the periphery.

Max-Planck-Society Research Unit for Immunology/Rheumatology, Institute for Clinical Immunology, University of Erlangen-Niirnberg, 8520 Erlangen, Germany, 1 Squibb Institute for Medical Research, Princeton, NJ 08543-4000, USA

N.lO Identification and initial characterization of a novel steroid receptor involved in early T-cell activation

R. A. KROCZEK, R. BRAVO!, F. EMMRICH, and H. W. MAGES

We have constructed a cDNA library from human peripheral blood T-cells activated by PMA and ionophore. By differential screening a collection of novel T-cell activation genes was established. Sequence analysis of clone 19913 revealed structural characteristics common to the steroid receptor family: a DNA-binding domain with zinc-fingers and a ligand binding domain. Several 19913 subregions were found to be closely homologous to known steroid receptors, however, the overall structure indicates that 19913 belongs to a new family of steroid receptors. The 19913 receptor is encoded by a mRNA of 4.2 kb and is expressed in T­cells following stimulation by ionophore or PMA in the presence of cycloheximide. Using the synergistic combination of PMA + ionophore 19913 is expressed in T-cells within 30 min, maximal expression is at 1 h, but mRNA is still detectable at 24 h. Expression of 19913 in T­cells is partially inhibited by Cyclosporin A. Our data thus indicate that the 19913 receptor belongs to the group of ligand-activated transcription factors involved in early gene regulation of activated T -cells. Our present efforts are aimed at defining the expression characteristics of 19913 in T -cells stimulated with physiological stimuli and at analyzing the expression of this receptor in various tissue compartments.

Behringwerke AG, Research Laboratories, 3550 Marburg/Lahn, Germany

N.ll Regulation of humoral responsiveness by anti-TCR antibodies

R. KURRLE, W. SEYFERT-BRANDT, H. U. SCHORLEMMER, and F. R. SEILER

Monoclonal antibodies (MAb) directed to constant domains of the human or murine a/B-T cell receptor (TCR) have turned out to be highly effective in regulating immuneresponsiveness in patients as well as in various experimental in vitro and in vivo model systems. Nevertheless,

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the mode of action of anti-TCR MAb is not yet completely understood. Previously, we could demonstrate, that at least in experimental transplantation and autoimmune models elimination of T cells by anti-TCR MAb is not a crucial pre-requisite for induction of immunosuppres­sion. We now analyzed the in vivo effects of the anti-TCR MAb H57-597 (generated by R. KUBO; Denver) on humoral responsiveness in mice. As antigen either SRBC or tetanus toxoid (IT) was used. Anti-SRBC antibody titers were determined by hemagglutination tests and by cytofluorometric assays, whereas anti-IT antibodies were measured in an ELISA-system. The results indicate that two-fold application of H57-597 (1 mg/kg) during the induction phase of an immunereaction (e.g. dO + d2) is sufficient to suppress completely the anti-TT IgG response; even booster injections 5-6 weeks later do not result in a significant anti-IT IgG response. In contrast, anti-TCR treatment suppress the anti-IT IgM response only weakly or even results in stimulation. Surprisingly, immunization of mice with SRBC resulted in completely suppressed hemagglutination titers and in a moderate suppression of the IgM and IgG response. However, the degree of suppression was variable for the different IgG subclasses and dependent on the mouse strain used. Treatment of MRLl1 mice with H57-597 resulted in suppression of anti-dsDNA autoantibodies (IgG) and rheumatoid factor (IgM). Experiments are in progress to analyze whether these results can be explained by selective activation of T cell subsets as measured by cytokine release (Il-2, IL-3, IL-4, GM-CSF, TNF, y-IFN) and thymidine-incorporation.

Forschungsinstitut Borstel, 2061 Borstel, Germany

N.l2 Anti-CD26 monoclonal antibodies block T cell proliferation in the late G1 phase of cell cycle

T. MATTERN, H.-D. FLAD, and A. J. ULMER

Within the last years it became evident that the CD26 antigen dipeptidyl peptidase IV (DPPIV), an activation marker of T cells, might be involved in the process of T cell activation. We have, therefore, explored the effects of the anti-CD26 monoclonal antibody (moAb) TIl 19-4-7 on the proliferation and expression of activation markers (CD25, CD71 and HLA-DR) of human mononuclear cells after mitogenic stimulation with PHA.

We can show that the CD26 moAb inhibited the proliferation of T cells after mitogenic stimulation in a dose-dependent manner. By addition of purified DPPIV to this system the inhibitory effect of the antibody could be abrogated. In our hands, DPPIV or anti-CD26 moAb alone were not mitogenic for the cells. We can further show that the expression of the T cell activation markers CD25, CD71 or HLA-DR was not affected by anti-CD26 moAb. From these observations we conclude that the addition of anti-CD26 moAb resulted in an arrest of the cells in the cell cycle somewhere between the late G1 phase and the S phase. Propidium iodide staining of the cells corroberated this hypothesis. The G1 phase of mitogen­stimulated T lymphocytes was found to be prolonged in cultures with anti-CD26 moAb. Whether these effects of anti-CD26 moAb are due to an inhibition of the enzymatic activity of DPPIV or whether the antibody/antigen binding sets inhibitory signals, is still under investiga­tion.

This work was supported by BMFT, grant no. III-006-89.

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I. Med. Klinik, Universitat Mainz, Germany

N.B The T-cell receptor-CD3 complex on human intestinal T -lymphocytes is uncoupled from phospholipase C activation

U. PIRZER, I. KAISER, and K.-H. MEYER ZUM BOSCHENFELDE

Triggering of the TCR-CD3 complex as well as the CD2 glycoprotein of human T­lymphocytes has been shown to induce inositol phospholipid breakdown via activation of an inositol specific phospholipase C. PLC-dependent hydrolysis of phosphatidylinositol-4,5-biphosphate generates two intracellular messengers, IP3, which regulates intracellular calcium levels, and diacylglycerol (DAG), the natural activator of protein kinase C. In human lamina propria T-lymphocytes an impaired proliferative response upon activation through the TCR­CD3 complex has been reported, whereas CD2-dependent stimulation seems to be functional. Since the initiation of particular activation-dependent cellular responses has been associated with the generation of biochemical second messengers, we investigated those in parallel both in lamina propria- and peripheral blood-derived T-lymphocytes after selective stimulation through individual pathways. Data will be presented which indicate that TCR-CD3 mediated activation does not induce a detectable rise in both IP3 and intracellular calcium in LPL as opposed to PBL. In contrast, triggering of the CD2 molecule initiates a substantial rise in IP3

and (Ca)i in both LPL and PBL. Since the generation of inositol phosphates represents a measure of PLC activation, a potential lack of inositol-specific phospholipase C can so far be excluded. However, the apparent lack of TCR-CD3 coupling to PLC activation is further demonstrated by an impaired production of DAG in human lamina propria T cells. Since distinct effector functions like lymphokine production do occur upon TCR-CD3 induced activation, our results raise questions about the putative causal relationship of early biochemi­cal changes and late biological responses in human intestinal T-lymphocytes.

Dept. of Pathology, Harvard Medical School and Dana-Farber Cancer Institute, Boston, MA 02115, USA

N.14 Characterization of a glycoprotein concerned with signal transduction through the T cell receptor (TCR) CD3 complex

HANS REISER, BIRGIT MASCHEK, and JESUS GONzALEZ CABRERO

We have derived a hamster monoclonal antibody (mAb) which affects murine T cell function when added in vitro to cell cultures. This mAb is directed against a lymphocyte cell surface molecule which we provisionally termed sgp-60. We have characterized sgp-60 biochemically as a single-chain glycoprotein with an apparent molecular weight of approxi­mately 60 kd that is anchored to the cell surface via a glycosylphosphatidylinositol anchor. Higher-order crosslinking of sgp-60, with the anti-sgp-60 mAb and a second-step antibody reagent, results in the activation of resting T cells in the presence of a second signal. Most interestingly, monovalent or bivalent engagement of sgp-60 results in profound and direct inhibition of antigen-presenting cell-independent, anti-CD3 + PMA- or Con A + PMA-dri­ven, T cell activation. These findings suggest that sgp-60 may be involved in signal transduc­tion through the TCR/CD3 complex. Further experiments show that the anti-sgp-60 mAb affects T cell activation early, by inhibiting the generation of second messengers. Our results therefore indicate an important physiologic role for sgp-60 in T lymphocytes.

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Dept. Internal Medicine, Div. Experimental Immunology, Medical School, 3090 Magdeburg, Germany

N.15 Role of dipeptidyl peptidase LV (DP IV) in T cell activation: Inhibitors of DP IV modulate expression of activation molecules and T cell proliferation induced by different stimuli

EKKEHARD SCHON and SIEGFRIED ANSORGE

Dipeptidyl peptidase IV (DP IV; EC 3.4.15.5.), recently clustered as CD26 membrane antigen of human leukocytes, has been shown to be involved in activation and proliferation of T cells by using inhibitors of DP IV [1] or monoclonal antibodies against CD26 [2]. Two approaches are used in further investigating the role this ectopeptidase plays in the immune system. Firstly, purified T cells were activated by different stimuli in presence and absence of DP IV inhibitor. Immobilized anti-CD3, IL-2 and different pairs of other stimuli like phorbol ester, calcium ionophore and lectins, were used to induce T cell activation. As a result, different pathways of T cell activation yielding in DNA synthesis are shown to be sensitive to DP IV inhibitors. Secondly, the expression of surface proteins known to be involved in signal transduction in T cells like CD2, CD4, CD7, CD25 and others was studied and found to be modulated in presence of DP IV inhibitors. Thus the conclusion is drawn that this membrane peptidase is linked to signaling pathways involving the expression of activation molecules in the plasma membrane of human T cells.

1. Schon, E., et al.: Eur. J. Immunol. 17, 1821 (1987). 2. Dang, W., et al.: J. Immunol. 145,3963 (1990).

Angewandte Immunologie, Deutsches Krebsforschungszentrum, 6900 Heidelberg, Germany

N.16 A functional complex is formed in human T lymphocytes between the CD45 protein tyrosine phosphatase, the P56lck protein tyrosine kinase and PP32, a possible common substrate

B. SCHRAVEN, H. KIRCHGESSNER, B. GABER, Y. SAMSTAG, and S. C. MEUER

Monoclonal antibodies directed at the extracellular domains of CD45, the major protein tyrosine phosphatase expressed in human T lymphocytes, were found to coprecipitate a 32 kDa phosphoprotein (pp32) from 32p labelled resting human T lymphocytes lysed in the nondisrupting detergent Digitonin. The same protein became phosphorylated in vitro on tyrosine residues if the CD45 immunoprecipitates were subjected to in vitro protein kinase assays. This suggested that a protein tyrosine kinase had been coprecipitated by CD45 mAb. The protein tyrosine kinase was identified as p56lck by means of reprecipitation experiments and by Western blotting of whole CD45 immunoprecipitates employing p56kk antiserum. Together with the finding that in vitro kinase assays of p56lck immunoprecipitates also resulted in phosphorylation of pp32 these data indicated that a functional complex is formed between CD45, p56lck, and pp32. This assumption was further provided by the observation that in vitro phosphorylated pp32 became completely dephosphorylated by purified CD45 (suggesting that it could serve as a substrate for the CD45 protein tyrosine phosphatase in vivo) and by the

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finding that in vitro phosphorylation of pp32 by p56kk was dependent on expression of the CD45 molecules. Detailed biochemical analysis of pp32 demonstrated that its electrophoretic mobility became rapidly (within minutes) altered following human T cell activation. However, the molecular modifications of pp32 markedly differed in T lymphocytes stimulated by either CD2 mAb, CD3 mAb, or PMA. Taken together the present data strongly suggest that the CD4S/p56kk/pp32 complex serves as an important part of the signalling machinery in human T lymphocytes.

Institute for Immunology and Genetics, German Cancer Research Center, 6900 Heidelberg, Germany

N.l7 Positive and negative selection of the T cell repertoire by the human DR3 molecule in transgenic mice

G. STRAUSS, D. VIGNALI, G. SCH<')NRICH, and G. J. HAMMERLING

In human lymphocyte antigen (HLA) DR3 transgenic mice on the B10.Q background only DRa~ molecules are expressed but not mixed mouse/human heterodimers because B10.Q is deficient for Ea and E~ and because I-Aq chains do not pair with DR chains. In these B10.Q.DR mice positive selection of v~6 and deletion of v~l1 thymocytes was found to occur as efficiently as in I-E positive mice. From additional studies we have evidence that the interaction of the mouse CD4 with the human DR molecule is very inefficient. Thus, these data suggest that a low avidity interaction between mouse T cell receptor and DR, which in vitro is not sufficient for activation, is still sufficient for positive and negative selection in the thymus. These data also have implications for the establishment of disease models in HLA transgenic mice.

Institut fur Immunologie, Universitat, 6500 Mainz, Germany, 1 Virginia Commonwealth University, Richmond, VA, USA

N.l8 The transferrin receptor targets ovalbumin into the antigen processing compartment of an Ia+ T cell clone

U. TSCHOETSCHEL, S. FROSCH, K. L. McCoy!, and A. B. RESKE-KuNZ

Helper T cells are induced to proliferate and to secrete lymphokines upon recognition of processed antigen presented by antigen presenting cells (APC). APC internalize the antigen and degrade it to peptides that are associated with MHC class II molecules and transported to the cell surface. The murine T cell clone BK-BI-2.6.04.1.15 (BII04.1.15) synthesizes and expresses Ia molecules constitutively. These cells are able to present various protein antigens to antigen-specific CD4+ T cells. However, a higher concentration of antigen is needed by BII 04.1.15 cells in comparison with spleen cells or with the more homogeneous population of bone marrow-derived macrophages (BMM<I» to induce a similar T cell response. To test whether the reduced APC potential of BII04.1.1S cells was due to less efficient uptake of antigen by pinocytosis and targeting into the processing compartment, we used ovalbumin (OVA) coupled to human ferric transferrin to achieve transferrin receptor-mediated uptake of

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OVA. The conjugate was about SO-240 times more efficient than native OVA in inducing proliferation of an OVA-specific T cell clone. The presence of ferric transferrin in the cultures competitively inhibited the enhancement of the T cell response. BMM<I>, used as a control, processed and presented the conjugate with similar efficiency as native OVA. These data suggest that the reduced APC potential of BII04.1.15 T clone cells is due to inefficient uptake of OVA by pinocytosis and delivery into the processing compartment.

Institute of Med. Microbiology and Hygiene, Technical University, SOOO Munich SO, Ger­many

N.19 Cotriggering via CDS co receptors rescues CDS T lymphocytes from a/~ T cell receptor induced programmed cell death (apoptosis)

ANJA WELWERT-KoSSIK, HERMANN WAGNER, and KLAUS HEEG

Opposed to immature thymocytes, triggering of mature peripheral T cells via a/~ T cell receptors (TCR) is considered to result in T cell activation rather than inactivation or even clonal deletion. Crosslinking of coreceptors (CDS, CD4) in addition is thought to further strengthen the activating signal, thus supplementing «weak» signals perceived via tha a/~ TCR not capable of inducing activation. To analyze the coreceptor requirement, we have triggered resting muring CDS T lymphocytes with defined quantities of immobilized anti-a/~ TCR monoclonal antibodies (mAb). Anti-a/~ mAb induced proliferation of CDS T cells over a wide dose response curve (10.000 - 20 ng/ml), yet failed to activate T cells at lower concentrations (10 - 1 ng/ml). Cotriggering with immobilized anti-CDS mAb restored the capability of low-dose anti-a/~ triggered CDS T cells to proliferate. Since triggering with anti­CDS mAb alone had no effect on T cells, we concluded that low-dose engagement of a/~ TCR provides a signal to the T cell, which by its own is not sufficient to induce proliferation. In defining the quality of this signal, we found that low-dose anti-a/~ mAb triggered T cells to programmed cell death (apoptosis) accompanied with specific DNA-fragmentation. Cell death occurred within 24 h and could neither be influenced by activators (PMA) nor by inactivators of the PKC (H7). However, cotriggering with immobilized anti-CDS mAb completely inhibited apoptosis. Hence, «weak» a/~ TCR mediated signals induce programmed cell death rather than activation in peripheral CDS+ T cells. Furthermore, costimulator signals such as CDS triggering rescue T cells from programmed death and permit thus T cell activation.

Institut fur Immunologie, Universitat, 6500 Mainz, Germany

N.lO Generation of RT1.B associated conformation isomers in a cell-free system

U. WENDLING, J. SYHA-jEDELHAUSER, and K. RESKE

Structurally interrelated subsets of RTl.B specific class II molecules of the LEW rat were dcefined on the basis of biosynthetic studies and use of the none ross reactive mab OX3 and OX6. Two serologically distinct a,~ heterodimers - OX3- and OX6-reactive respectively -

290 . XXIInd Meeting of the Society of Immunology

were found to be expressed at the cell surface of constitutively I-A + cells. The two molecular forms were suggested to represent conformation isomers derived from an OX6-reactive biosynthetic intermediate containing terminally glycosylated a-, ~- and t-chains. A novel experimental approach has been used to study the assembly of class II constituents in the presence and absence of the invariant y-chain by a coupled in vitro transcription-translation system (BUJARD et aI., Meth. Enzymol. 155; 416, 1987). Full length cDNA clones of RT1.Bl a­and ~-chains including the rat invariant y-chain recently described by our laboratory (Nucl. Acids Res. 18,4598,1989; Biochim. Biophys. Acta, in press, 1991; Nucl. Acids Res. 16, 11822, 1988) were placed in transcription vectors and the constructs were used to produce large amounts of clone-specific mRNA. The transcripts possessing a capped 5'terminus were employed in a eukaryotic cell-free translation system to generate class II specific a-, ~-, and y­chains. Because under conditions where no y-chain mRNA was added to the system, OX3-and OX6-reactive a, ~ heterodimers were detectable it appears that the invariant chain is not a prerequisite for the occurrence of RT1.B specific conformation isomers.

Supported by SFB 31, C2.