Workshop D T Lymphocytes, Microenvironment and Autoimmunity

Workshop 0 T Lymphocytes, Microenvironment and Autoimmunity

II. Medizinische Klinik, Technische Universitat, Ismaninger StraGe 22, D-SOOO Miinchen SO, FRG

D.l The CD45 molecule differentially regulates the activation of human intestinal lamina propria lymphocytes induced by CD3 crosslinking K. DEUSCH, F. LULING, K. REICH, and M. CLASSEN

The CD45 molecule is a transmembrane protein expressed on virtually all hematopoetic cells and has been shown to exert significant tyrosine-phosphatase activity. Several isoforms of this molecule are differentially expressed in T lymphocytes and this behavior appears to correlate with the functional activity of these cells. In this study, we were interested as to whether antibodies directed to CD45, CD45RO or CD45RA can influence the activation of human colonic lamina propria T lymphocytes (LPL) upon crosslinking to the CD3/TCR-complex employing suboptimal doses of anti-CD3 antibodies coated onto plastic dishes. We found that anti-CD45RO antibody (UCHL-l) most potently enhanced the proliferative response of LPL, stimulation index (5.1.)=65. Surprisingly, anti-CD45 antibody that recognizes all forms of CD45 augmented LPL-proliferation to a much lesser extent (5.1. = IS). Anti-CD45RA antibodies had only little effect (5.1. = 3). However, this may be due to the low frequency of these cells within the LPL-population in the lamina propria « 15 %). Immunohistochemical and FACS analysis revealed that almost 90 % of LPL cells expressed both CD45 and CD45RO. Therefore, we conclude that our data support the notion that the CD45RO molecule plays a distinct functional role during the activation processes incited by the perturbation of the T-cell antigen receptor complex expressed on human lamina propria T-lymphocytes. Supported by Deutsche Forschungsgemeinschaft (De-3S4/3-1)

III. Medizinische Klinik und 2Institut fUr Mikrobiologie und Hygiene, Technische Universitat, Ismaninger StraGe 22, D-SOOO Miinchen SO, FRG

D.l A distinct set of human gamma/delta (TCRl+) cells homes to the intestinal epithelium K. DEUSCH\ K. REICHl, F. LULING \ K. PFEFFER2 , and M. CLASSEN I

We have found that TCRl+ T lymphocytes preferentially home to the human intestinal epithelium. The majority of these cells were shown to express CDS molecules on their surface.

152 . XXIst Meeting of the Society of Immunology In this study, we demonstrate that, in normal human colonic epithelium, TCR1+ intraepitheliallymphocytes (IEL) represent a distinct subset of T -cells using a restricted set of variable region genes to synthesize their y/b-T-cell receptor (TCR1). Employing monoclonal antibodies directed to the surface products of defined variable regions and subsequent FACS analysis, we will show that TCR1+-IEL are markedly restricted to the use of the V-delta-l region (64 %) assembling their TCR-complex. In marked contrast, peripheral blood-TCRI + cells obtained from the same individuals preferentially expressed the product of the V-gamma9/V -delta-2 (79 %) region on their surface. The frequency of TCRI + cells in the lamina propria (LPL) using V-delta-lor V-gamma-9 antibodies were 42% and 49%, respectively, which indicates that these cells represent a "transitional" population. The products of V-delta-l and V-gamma-9 represent polypeptides that have been described to not associate to form a functional TCRI-complex and therefore appear to delineate distinct sets of TCRI + cells. Taken together, our findings clearly demonstrate that TCR1+-IEL not only differ from TCRI +-PBL with regard to CD8 expression but also, and perhaps more importantly, by virtue of distinct usage of TCR-variable regions. In conclusion, these data suggest that a unique subset of TCRI + cells exhibiting a restriced receptor repertoire home to the colonic intestinal epithelium. Alternatively, V-delta-l cells could have been locally expanded due to the presence of unique antigens on the luminal surface of the intestine that are normally excluded from the peripheral blood. Supported by Deutsche Forschungsgemeinschaft (De-384/3-1)

Institute for Immunology, University of Munich, GoethestraBe 31,8000 Miinchen 2, FRG

D.3 T cells express the B isoform of FCERII after stimulation with allergen or PHA N. ENDRES, ]. C. PRINZ, and E. P. RIEBER For the low affinity IgE receptor CD23 two isoforms have been described that differ in the six N-terminal cytoplasmic amino acids. Fc€RIIa is constitutively expressed on B cells, while the expression of Fc€RIIb can be selectively induced by IL-4 in B cells and monocytes (1). CD23 is also found on few T cells after PHA stimulation, whereas a major portion of T blasts express CD23 when PBL from allergic individuals are activated by the specific allergen (2). In order to characterize the CD23 isoforms expressed in T cells, PBL from normal and allergic individuals were stimulated, the T cells were purified at the time of maximal CD23 expression, RNA was isolated, transcribed into cDNA and analysed by PCR with CD23a- or b-specific primers. Only Fc€IIb could be detected in purified T cells activated either by PHA or in T cells from allergic donors stimulated with the specific allergen. No CD23 message was identified by PCR in ConA- or tetanus toxoid-induced T blasts. Fcdla was never found in T cells. IL-4 alone was not capable to induce CD23 on T cells, but was found to enhance the expression of Fc€RIIb after stimulation with PHA or allergen. 1. YOKOTA, A., H. KIKUTANI, T. TANAKA, R. SATO, E. L. BARSUMIAN, M. SUEMURA, T. KISHIMOTO. 1988. Two species of human Fc€ receptor II (FcdIlCD23). Cell, 55: 611. 2. PRINZ, J. c., N. ENDRES, G. RANK, J. RING, E. P. RIEBER. 1987. Expression of Fc€ receptors on activated human T lymphocytes. Eur. J. Immunol. 17: 757.

XXIst Meeting of the Society of Immunology· 153 IAbt. fur Immunologie, 1. Medizinische Klinik, Universitatskrankenhaus, 2000 Hamburg, FRG, and 2Department of Pathology, Stanford University, U.S.A.

D.4 Role of adhesion molecules in lymphocyte migration A. HAMANN!, CORNELIA BERLIN!, B. HOLZMANN2, ANDRIJKA KASHAN!, DOROTHEE JABLONSKI-WESTRICH!, and H.-G. THIELE!

Selective immigration of lymphocytes into different organs seems to be regulated by different adhesion molecules. Inhibitory effects of monoclonal antibodies on in vitro binding of lymphocytes to high endothelial venules (HEV) in the frozen section assay have led to the postulation of at least two types of "homing receptors": a peripheral node-specific receptor, represented in the mouse by gp90 MEL- 14 , and a Peyer's patch (PP)-specific, for which the integrin LPAM 112 has been proposed to be a candidate. Additional molecules like LFA-l are supposed to playa more general accessory role in lymphocyte-endothelium interactions. Our data show some organ-specific effects in migration for this molecule also. In vivo homing studies indicate that the functional role of the "homing receptors" is still far from being clear. MEL-14 antibodies in vivo also inhibit the immigration of lymphocytes into PP, albeit only partially. It is still unclear, whether gp90 MEL- 14 may also recognize PP-HEV in vivo, or whether this finding points to additional functions of gp90 MEL- 14 in the regulation of general adhesion events. Antibodies against LPAM 112 inhibit in vitro binding to PP-HEV, but in first experiments, no significant inhibition was seen for homing into PP in vivo. Further complications arise in the interpretation of the migration patterns of activated lymphocytes. Their behavior cannot sufficiently be explained by the function (or lack of function) of one of these molecules. Binding studies using transformed mouse endothelial cell lines point to the relevance of further, so far unidentified interaction molecules. Data from this experimental model confirm findings in the human system, showing an up regulation of binding mechanisms on endothelium by inflammatory mediators like TNF.

Institute for Clinical Chemistry and Pathobiochemistry, RWTH Aachen, Pauwelsstra6e, D-5100 Aachen, FRG

D.S Analysis of activated T-cell populations in synovial fluid of patients with rheumatoid arthritis H.-D. HAUBECK Several lines of evidence suggest the involvement of specific autoreactive T lymphocytes in the pathogenesis of rheumatoid arthritis (RA). In the synovium of patients with rheumatoid arthritis dense infiltrates of mononuclear cells (lymphocytes, monocytes) can be found. A major subpopulation of these infiltrating cells are CD4+ T-helper (Th) cells which can be shown to express activation antigens (Ag). The concept that autoreactive Th cells are involved in the induction and perpetuation of the inflammatory process in RA is further supported by the marked' HLA disease association. By using a panel of monoclonal antibodies (mAb's) we have analyzed the expression of activation Ag's on T lymphocytes of synovial fluid from patients with RA. For a detailed analysis mAb's for differentiation Ag's, activation Ag's, Ag's of the different T cell receptors (TCR alpha, beta and TCR gamma, delta) and of memory T-cells were used. Usually 80-90 %

154 . XXIst Meeting of the Society of Immunology of the lymphocyte population were of the T cell phenotype. About 95 % of these T cells used the classical T cell receptor (TCR alpha, beta) whereas only 1-5 % used the alternate receptor (TCR gamma, delta). The percentage of CD4+ Th cells ranged from 20--60 %. The expression of activation antigens varied from 0-60 % depending on mAb's for the different activation Ag's. About 80-90 % of the lymphocytes expressed antigens of memory cells (CD 29, CD 45RO). Activated CD4+ Th cells will now be sorted and used in antigen-dependent T-cell proliferation assays for isolation and characterization of antigens which are responsible for the induction and maintenance of the chronic autoimmune process in RA.

Division of Clinical Immunobiology, Department of Internal Medicine and Institute of Blood Transfusion, University of Innsbruck, Innsbruck, Institute for Blood Group Serology, Vienna, Austria, and Department of Internal Medicine III, Ludwig-Maximilians-University, Munich, FRG

D.6 Cytotoxic lymphocyte precursor (CTL-p) frequencies in allogeneic bone marrow recipients E. IRSCHICK, F. HLADIK, D. NIEDERWIESER, D. SCHONITZER, A. HAJEK, E. HOLLER, H. ]. KOLB, AND CH. HUBER

CTL-p frequencies were determined by limiting dilution analysis in twelve recipients of allogeneic HLA sibling bone marrow grafts. Eleven patients received HLA identical grafts, one had one HLA-DR mismatch. Tests were set up in graft-versus-host (GvH) direction. Recipient-pre-transplantation (Tx) specific and thirdparty reactive CTL-p were assessed before and one month after grafting or at the time of GvH-disease (GvH-D). Additionally, split-well analyses were performed and tested against recipient-pre-Tx and donor targets. Very low recipient-pre-Tx specific frequencies were detected in two out of five patients without GvH-D. Six patients developed GvH-D but only one patient with a severe GvH-D showed a high recipient-pre-Tx specific CTL-p frequency at the time of disease. Third-party specific CTL-p frequencies were detectable in all patients and were significantly reduced in eleven of twelve cases after transplantation. Donor specific CTL-p were not detected at any tested time point in split-well analysis.

Max-Planck-Society, Clinical Research Unit of Rheumatology/Immunology; Department of Nuclear Medicine; University of Erlangen-Niirnberg, 8520 Erlangen, FRG

D.7 T-cell migration in rat adjuvant arthritis R. KINNE, H. REINECKE, S. BLOCH, U. VORDERWULBECKE, W. BECKER, and F. EMMRICH

The relevance of T cells for the induction and perpetuation of rat adjuvant arthritis (AA) has been demonstrated by a) adoptive transfer of AA by T cells and b) successful treatment of AA with anti-T-cell monoclonal antibodies. However, little is known about the kinetics of T-cell migration into arthritic joint tissue in AA.

XXIst Meeting of the Society of Immunology . 155 Therefore, radioactively (111Indium) labeled lymph node T-cells from normal donor rats were injected i.v. into normal and arthritic rats at day 16 after induction of AA. In vivo distribution of the radiolabeled cells in anesthetized recipients was registered using a gammacamera. In addition, the radioactivity contained in various organs was measured in a gammacounter at different time points (between 15 min and 26 h) after injection. At 4.5 h after injection, a significantly (p < 0.05) increased accumulation of donor T cells (expressed as specific activity/total body counts) was observed in foot and ankle joints of arthritis recipients in comparison to normals. The difference between arthritic and normal joints was less pronounced in the fore paws (0.05 < P < 0.1). In contrast, spleen, liver, lung, and blood of arthritic recipients did not show significantly different values from normals at 4.5 h after injection. An estimated 33 % of the injected T cells accumulated in the total bone marrow of both AA and normal rats until 4.5 h after injection. These results show that joints of arthritic rats favor the entry of T cells, possibly mediated by a higher permeability of the endothelium for T cells. The changes in the joints of arthritic recipients are not paralleled by corresponding changes in blood and central organs. High numbers of T cells migrating into the bone marrow might interact locally with hemopoiesis, thus supporting a systemic component of AA.

Berufsgenossenschaftliches Institut fiir Arbeitsmedizin (BGFA), 4360 Bochum, FRG

D.8 Stimulation of human T lymphocytes by the insect allergen Chi t I V. LIEBERS, G. MAZUR, X. BAUR, and S. MODROW

Using the lymphocyte transformation test we measured the proliferation of human peripheral blood lymphocytes (PBL) after incubation with chironomid hemoglobins (Chi t I), single Chi t I components, fragments and synthetic peptides. Chi t I component III, which has been structurally analyzed, shows between positions 117-136 amino acid sequences which, according to the predictions of Rothbard and also Berzofsky, represent T cell-epitopes. We synthesized this peptide and got significant PBL-proliferation in six out of seven patients. We also cleaved this peptide at position 121 and tested the two peptides obtained. They both still show reactivity. At the present time we are studying the relevance of HLA-DR, -DQ, and -DP molecules by using antibodies against non-polymorphic and polymorphic HLA-regions. Preliminary results indicate an involvement of the HLA-DR- and DP-regions.

I. Medizinische Klinik, Joh.-Gutenberg-Universitat, 6500 Mainz, FRG, and Dept. of Cell Biology, Weizmann Institute of Science, Rehovot, Israel

D.9 Reduction of the mixed lymphocyte reaction by T-cell vaccination A. W. LOHSE, E. MOR, T. RESHEF, K. H. MEYER ZUM BOSCHENFELDE, and I. R. COHEN

T-cell vaccination has been applied successfully in preventing and treating experimental autoimmune disease. DTH reactions can also be reduced by T-cell vaccination. The aim of the present study was the ellucidation of the effect of T-cell vaccination on alloantigen responses

156 . XXIst Meeting of the Society of Immunology to learn about its possible applications to transplantation immunology. MHC-reactive lymphocytes were induced in Lewis rats and in BALB/c mice by priming with irradiated splenocytes from Brown-Norway (BN) rats or C57BLl6 mice, respectively. Primed lymph node cells were activated in vitro by ConA stimulation, crosslinked with 0.3 % glutardialdehyde and injected into naive Lewis rats or BALB/c mice. Vaccination was repeated rwice in weekly intervals. The mixed lymphocyte reaction (MLR) was then tested against the priming MHC-bearing cells and an unrelated MHC type (Wistar-Furth or C3H). The MLR could be markedly reduced (between 50 % and 80 %) by T-cell vaccination. The MLR against unrelated MHC-antigens was similarly reduced, presumably due to crossreactive epitopes. The presence of cross reactive epitopes was underlined by an increase of the MLR against unrelated MHCbearing cells (e.g. C3H and C57BLl6) after priming with cells of only one MHC type (e.g. C57BLl6). Vaccination with naive syngeneic activated T cells led to an increase in the MLR, showing that the suppression of the MLR by T cell vaccination was not due to an antiergotypic effect. The presence of suppression could be demonstrated by mixing cells from vaccinated animals with naive cells. T cell vaccination can thus suppress alloantigen responses and may therefore be useful in preventing and treating transplant rejection.

III. Medizinische Klinik, Technische Universitiit, D-8000 Miinchen 80, FRG, and 2Dana Farber Cancer Institute, Boston, U.S.A.

D.IO Crosslinking of IF7, a novel lymphocyte surface molecule, with the CD3 complex strongly enhances CD3-induced stimulation of human intestinal T lymphocytes F. LOLINGl, K. REICHl, C. MORIMOT0 2 , M. CLASSEN!, and K. DEUSCH 1

Anti-1F7 antibody recognizes a novel cell surface molecule involved in helper function of CD4+ T lymphocytes. The most prominent structure defined by this antibody is a 110 kD glycoprotein that appears to define CD4+ cells that can respond to soluble recall antigens such as tetanus toxoid. The latter subset of CD4+ cells is also defined by anti-CDw29 and antiCD45RO antibodies, however, the latter rwo antibodies recognize distinct surface structures also termed accessory molecules. A number of accessory molecules (CD2, CD4, CD28 etc.) expressed on the surface of human T lymphocytes have been shown to upregulate the proliferative response of peripheral blood T lymphocytes when crosslinked to CD3/TCRcomplex. In this study, we isolated lamina propria T lymphocytes (LPL) from normal intestines and examined their proliferative response following crosslinking of their CD3/TCRcomplex with the 1F7 target structure. We observed that anti-1F7 most potently upregulates CD3-induced proliferation of LPL (60X) even in the absence of autologous monocytes. The magnitude of this enhancement was comparable to that induced by CD3:CD2-crosslinking (55X) yet much greater than that observed, in decreasing order, for CD3:C45 (lOx) or CD3:CD4 (5X) crosslinking. FACS analysis and immunohistochemistry revealed that the majority (> 85 %) of human LPL express 1F7, CDw29, and CD45RO and therefore support the notion that these cells are memory T cells. Taken together, our findings clearly demonstrate that the structures recognized by the monoclonal antibody anti-1F7 are functional molecules that can exert most potent costimulatory activity during the activation process of monocyte depleted human lamina propria derived intestinal T lymphocytes. Supported by Deutsche Forschungsgemeinschaft (De-384/3-1)

XXIst Meeting of the Society of Immunology· 157 Institute for Immunology and Genetics, German Cancer Research Center, D-6900 Heidelberg, FRG

D.II T cell repertoire selection by targeting T cell receptors (TCRs) to cortical epithelial cells in situ K.-P. MOLLER and B. A. KYEWSKI

The intrathymic development of the T cell repertoire is shaped by two selection processes: positive selection of those thymocytes whose T cell receptor( s) [TCRs] recognize intrathymic self-MHC and negative selection of those thymocytes whose TCRs recognize intrathymic complexes of self-antigens and self-MHC. Both events are known to be mediated by thymocyte-accessory cell interactions, to operate at the same T cell differentiation stage and to involve the same or similar receptor/ligand complexes, yet their outcome is diametrically opposite: selective survival or deletion of the respective thymocytes. Several explanations have been offered to account for this conundrum; a) thymocytes pass through distinct stages at which they are exclusively susceptible to either positive or negative selection, b) low affinity interactions lead to positive and high affinity interactions to negative selection, c) different microenvironments, e.g. epithelial cells versus bone marrow-derived antigen presenting cells provide distinct signals possibly via cell type-specific cytokines. In order to more precisely define microenvironment(s) and molecular requirements for T cell selection we targeted TCRs of the V~8 family to non-MHC surface molecules of thymic cortical epithelial cells in vivo via bispecific hybrid antibodies. This targeting results in specific overselection of the particular T cell subset whereas a mixture of each of the parental antibodies results in specific suppression. These data suggest that crosslinking of TCRs to cortical epithelial cells is sufficient for positive selection. In addition, we will report on the effect on T cell repertoire selection of targeting VB6 TCRs, CD4, and CD8 receptors to thymic cortical epithelial cells in situ.

Institute for General and Experimental Pathology, Medical School, University of Innsbruck, and Immunoendocrinology Unit of the Austrian Academy of Sciences, A-6020 Innsbruck, Austria

D.12 Autoreactivity of intra-thymic nurse cell lymphocytes JOSEF PENNINGER and GEORG WICK Thymic nurse cells (TNC) are epithelial cells containing intact T cells engulfed within membrane lined vacuoles in their cytoplasm. Since thymic epithelial cells are strongly MHC class I and class II antigen positive, these lymphoid-epithelial complexes are good candidates where the process of self-recognition and/or clonal deactivation of T cells may take place. Assessing micromanipulated, single chicken TNC in the chorionallantoic membrane (CAM)assay, we previously showed at a clonal level that intra-TNC-lymphocytes (TNC-L) react against foreign MHC molecules with high efficiency and induce a specific graft-versus-host reaction (GvHR). Now we demonstrate that TNC-L also possess a strong GvH-reactivity in syngeneic combinations. The efficiency to induce a GvHR was significantly higher for TNC-L (1137) as compared to auto reactive thymocytes (11380000) or peripheral blood lymphocytes (1/150000) of the same donor. In serial transfer experiments onto appropriate syngeneic, MHC-congeneic or allogeneic secondary embryonic hosts we also demonstrate that TNC-L

158 . XXIst Meeting of the Society of Immunology react specifically against self MHC molecules. The implication of our data for positive and negative selection will be discussed. The work was supported by the Austrian Research Council (project S7391).

Klinische Forschergruppe fiir Rheumatologie, Mooswaldallee 1-9, 7800 Freiburg, FRG

D.13 T cell antigen receptor repertoire of synovial fluid T cells in rheumatoid arthritis GERD PLUSCHKE, HElKE TAUBE, GESA RICKEN, and ULRICH KRAWINKEL

Rheumatoid arthritis (RA) is a disease of unknown etiology, which manifests as chronic inflammation of the lining of multiple joints and leads to the destruction of cartilage and bone. Widespread infiltration of the synovium with activated T lymphocytes, and linkage of RA to specific class II major histocompatibility determinants, which control antigen presentation to CD4+ T cells suggest a potential pathogenic role for T lymphocytes. It has been postulated, that proliferation of autoreactive T cells in the affected joints might lead to the selection of a limited number of clones expressing a restricted T cell antigen receptor (TCR) repertoire. Due to the uncertainty regarding the nature of the target antigens several attempts have been made to demonstrate dominant rearrangements of TCR genes in synovial infiltrate T cells. For most of these studies synovial T cells expanded in vitro prior to analysis of TCR rearrangements have been used. Outgrowth of distinct cell subpopulations may explain why conflicting results were obtained in these studies. In an attempt to define the degree of clonal diversity in synovial infiltrate T cells from patients with RA we have therefore extracted RNA from synovial fluid T cells without prior culturing. RNA was transcribed into cDNA and TCR specific cDNA was amplified employing conventional or anchored polymerase chain reaction (PCR). The diversity of TCR structures was analyzed by hybridisation analysis and sequencing of cloned PCR products. Some evidence for a non-random usage of TCR gene segments was obtained. We also found V and J sequences that had not been detected previously in cDNA libraries derived from thymocytes or peripheral blood T cells.

lClinic for Dermatology and 2Inst. for Immunology, University of Munich, 8000 Munich, FRG

D.14 In vitro IL-4-induced IgE secretion requires activated FcERlI CD23+ T lymphocytes

J. C. PRINZ!, N. ENDRES2, and E. P. RIEBER2 Recently we have demonstrated that Fc,R2!CD23 expression is a selective property of allergen-specific T lymphocytes (1). We now intended to analyze the role of FcE R2!CD23+ T cells in IgE secretion. T cells were prepared from peripheral blood mononuclear cells (PBMC) by rosette formation with AET -treated sheep red blood cells (SRBC) followed by density

XXIst Meeting of the Society of Immunology· 159 gradient centrifugation and NH 4 CI-mediated lysis of SRBC. These T cells were used either untreated, or treated with mitomycin C to block proliferation, or after having been incubated with the Fc fR2/CD23-specific mAb M-L25. For IgE secretion, 2x 105 /well non-T-cells together with 5x 104 T cells treated as described were cultured in 96 well microtiter plates either alone or in the presence of rabbit F(ab')z anti-human IgM antibodies (RaHuIgM) and/or IL-4. After 10 days, supernatants were harvested and analyzed for IgE content using an IgEspecific ELISA developed in our laboratory. High IgE levels were detected in samples of non-T cells cultured with untreated T cells and RaHuIgM and IL-4. If, however, prior to addition the T cells had been treated with either or both mitomycin C and M-L25, IL-4 and RaHuIgM were insufficient to induce IgE-synthesis by non-T cells. No IgE was produced by either cell population alone. Moderate IgE-leveis were detected in the supernatants of unseparated PBMC cultured with IL-4 and RaHuIgM. Collectively, our data demonstrate that, in addition to IL-4, initiation of in vitro IgE secretion essentially requires the presence of activated Fc fR2/CD23+ T lymphocytes. They furthermore stress that allergen-induced Fc fR2/CD23 expression on T cells (1) is a pivotal event in the generation of the allergic IgE response. 1.

J.

C. PRINZ, X. BAUR, G. MAZUR, E. P. RIEBER. 1990. Allergen-directed expression of FcfRII (CD23) on human T lymphocytes is modulated by IL-4 and IFN-y. Eur. J. Immunol. 20: 1259.

II. Medizinische Klinik, Technische Universitat, Ismaninger StraGe 22, SOOO Miinchen SO, FRG

D.tS Human gamma/delta-T cells (TCRt+) selectively home to the intestinal epithelium K. REICH, F. LULING, M. CLASSEN, and K. DEUSCH

Recently, it has been reported that TCR1+ T lymphocytes represent a population of cells that follow a unique pathway of differentiation, exhibit an intriguing specificity for heat shock proteins and localize preferentially in specialized tissue sites. Murine TCR1+ cells were shown to preferentially localize in the gastro-intestinal epithelium (IEL) and the skin. In this study, we demonstrate that CDS+TCR1 + T lymphocytes represent a major cell type within the human intestinal IEL-population. Specifically, employing monoclonal antibodies directed towards the constant region of the TCR-delta (TCR-d-1) and TCR-alpha/beta chains (BMA031, b-F1), respectively, we found by FACS- and immunocytochemical analysis that a large fraction of highly purified (> S5 %) human IEL, isolated from normal colonic mucosa, express TCR1 (42 ± 9 %) on their surface. Of these the majority are CDS+ (66 ± 6 %) and less than 1 % are CD4+. Similarly, the majority of TCR2+ IEL were CDS+ (SO %). In marked contrast, the frequency of TCR1 + T cells within the intestinal lamina propria T cell population (LPL) ofthe same individuals were 5.S ± 3.3 % (TCR2+ =94.1 ± 3.3 %) akin to their reported frequency in the peripheral blood T cell population. In addition, with regard to CD4 and CDS expression TCRl+-LPL resembled TCR1+-PBL-T in that they were mostly double negative. In summary, our data show that TCR1+CDS+ T lymphocytes selectively home to the gastrointestinal epithelium. These findings suggest a specific functional role of TCR1 + T lymphocytes during the mounting of the local immune response towards exogenous antigens. Furthermore, TCR1 + human IEL may function, as has been suggested by others, to eradicate phenotypically altered epithelial cells. Supported by DFG (De 3S4/3-1)

160 . XXIst Meeting of the Society of Immunology Institute for Immunology, Goethestra6e 31, 8000 Munchen 2, FRG

D.l6 Differential manipulation ofT cell functions by a panel of monoclonal CD4 antibodies C. REITER, U. PIRRON, and E. P. RIEBER

The CD4 molecule plays a crucial role in the antigen-dependent T cell activation. Therefore, monoclonal CD4 antibodies (CD4 mAb) may represent suitable tools to modulate T cell functions in vitro and in vivo. In order to compare the biological effectiveness of mAb recognizing different epitopes on the human CD4 molecule, a panel of 104 different CD4 mAb was generated and characterized with regard to binding specificity. An initial crude epitope mapping was performed by competitive binding assays using 5 reference CD4 mAb which were typed on CD4 mutants with single amino acid exchanges and clustered according the CD4 domain recognized (1). Extended cross competition experiments revealed an individual binding specificity for each of the 104 CD4 mAb. The mAb were then tested for their ability to induce modulation of the CD4 molecule from blood T cells and T cell lines and to block antigen induced Ca2+ -mobilization in tetanus toxoid specific T cell clones. Results indicated a great variation in the functional capacities of different CD4 mAb. However, modulatory capacity did not coincide with the ability to inhibit T cell activation and no clearcut correlation was found between the biological effects of a CD4 mAb and its isotype, binding affinity or the CD4 domain recognized. 1. A. PETERSON, B. SEED. 1988. Genetic analysis of monoclonal antibody and HIV binding sites on the human lymphocyte antigen CD4. Cell 54: 65-72.

lInstitute of Immunology and Genetics, German Cancer Research Center, 6900 Heidelberg, FRG, 2INSERM, Marseille-Luminy, France

D.l7 Tissue specific expression of MHC class I antigens in transgenic mice and its implication for tolerance induction GUNTHER SCHONRICH 1 , FRANK MOMBURG 1, BERNARD MALISSEN2, ANNE-MARIE SCHMITTVERHULsr, GUNTER]' HAMMERLINGl, and BERND ARNOLD 1

Tolerance to selfantigens expressed in the thymus is due to intrathymic deletion of specific T cells. Whether the same holds true for antigens found only on lymphocytes is unknown. Moreover, it is an open question how the immune system becomes tolerant to non-thymic molecules. Therefore, several types of transgenic mice expressing an MHC class I antigen (Kb) in a tissue specific fashion on an allogenic background were created. To get expression in defined tissues the following promoters have been used: The human CD2 promoter (lymphocytes), the glial acidic protein promoter (glial cells), the albumin promoter (liver) and the keratinIV promoter (epithelial cells). In addition, we introduced the T cell receptor (TCR) genes from an Kb specific alloreactive T cell clone (KB5-C20) into fertilized eggs. The resulting TCR transgenic mice were crossed to the mice described above. Presently we are analyzing the fate of the Kb specific (autoreactive) T cells in mice expressing simultaneously Kb under the control of the various promoters. Our data indicate that tolerance to antigens expressed on lymphocytes and non-thymic tissue is not due to intrathymic deletion.

XXIst Meeting of the Society of Immunology· 161 Abt. Klinische Immunologie und Transfusionsmedizin, Medizinische Hochschule Hannover, 3000 Hannover, FRG

D.tS Clonal analysis of the phosphatidyl-inositolglycan-(PIG)anchoring defect in PNH lymphocytes JORG SCHUBERT, PETER UCIECHOWSKI, WALDEMAR KOLANUS, PATRICIA DELANY, and REINHOLD E. SCHMIDT

Paroxysmal nocturnal hemoglobinuria (PNH) has recently been demonstrated to be a clonal disorder of bone marrow derived cells deficient in surface expression of proteins which are attached to the cell membrane via a phosphatidyl-inositolglycan-(PIG)-anchor. Using monoclonal antibodies against newly defined PIG-linked surface structures such as CD48, CD55, and CD59 we demonstrated in previous studies for the first time the involvement of lymphocytes in the disease. In these studies we present T and NK cell clones from one PNH patient which exhibit a differential expression of PIG-linked surface molecules. Phenotypic analysis revealed 7 PIG-molecule deficient, in contrast to 40 PIG-molecule positive T cell clones and 7 deficient, in contrast to 3 positive NK cell clones. The clonal analysis revealed that surface expression of PIG-molecules was always in parallel, i.e., the clones either coexpressed CD48, CD55, and CD59 or they were completely negative for all of them. In functional tests PIG-molecule negative clones showed an increased susceptibility to lysis by homologous complement when compared to positive clones. In contrast, we could not detect any difference in the sensitivity to cell-mediated cytotoxicity. Furthermore, both types of clones had similar levels of mRNA transcription for the PIG-linked molecules CD55 and CD59. Although PIGnegative cells lack the molecules at the cell surface the protein appears to be produced since the CD59 antigen could be detected in both types of clones. When analyzing CD59 protein bands by Western blotting we could detect an additional band of 28 kD, i.e., 8 kD longer than the usual 18-20 kD bands. This band is hardly detectable in the positive clones but is abundant in the deficient clones and might correspond to an aberrant CD59 precursor protein. In summary, we present ex vivo mutant cell lines from the T cell and NK cell fraction which express the defect leading to PIG-molecule deficiency in PNH. These cell lines exhibit an increased sensitivity to autologous complement and appear to synthesize aberrant PIGmolecule precursor proteins.

Division of Immunology, Medical Institute of Environmental Hygiene at the Heinrich Heine University Dusseldorf, 4000 Dusseldorf, FRG

D.t9 Mercuric chloride induces mouse T cells to produce interleukin-4 in vitro E. VAN VLIET, D. THlSSFN, and F.. GLEICHMANN

Mercuric chloride is a potent inducer of autoimmunity and immunoglobulin E production in vivo. The present study analyses the in vitro effects of mercuric chloride on mouse lymphocytes. Mercuric chloride induces enhancement of expression of major histocompatibility complex (MHC) class II antigens on mouse splenic lymphocytes in vitro, as demonstrated with flowcytometry. Comparison of the effects of mercuric chloride on unseparated and T

162 . XXlst Meeting of the Society of Immunology cell-depleted spleen cells demonstrates that this induction is T cell-dependent. Blocking studies with anti-interleukin-4 (IL-4) antibody llBll confirm this finding and show that the T cells mediate this effect through HgCb-induced IL-4 production.

Klinik fur Abdominal- und Transplantationschirurgie, Zentrum Chirurgie, Medizinische Hochschule Hannover, 3000 Hannover, FRG

D.20 Expression of CD45RA and CD45RO on T cells of liver grafted patients with acute rejection M. WINKLER, R. SCHWINZER, and K. WONIGEIT

Based on the expression of different isoforms of the CD45 molecule, human PBL can be separated in "naive" T cells expressing the CD45RA isoform and in antigen-primed "memory" T cells expressing the CD45RO isoform. In order to study the effect of in vivo activation on CD45RA and CD45RO expression, CD4+ and CD8+ T cells of patients in the early course after liver transplantation were investigated using two color flow cytometry and appropriate monoclonal antibodies. Results: In 20 patients with acute graft rejection the percentage of CD4+ T cells coexpressing CD45RA declined from 57 ± 20 % (mean ± S.D.) in clinical quiescense to 32 ± 17 % before anti-rejection treatment. The decrease in the expression of CD45RA was more pronounced in the CD4 than in the CD8 subset. These results prompted a subsequent study in which the expression of CD45R isoforms on CD4+ T cells was regularly monitored. 38 liver recipients were studied. In 15 of these patients 18 episodes of a relative and absolute increase in the frequency of CD45RO+ CD4+ T cells were detected. 10 of these episodes were associated with acute graft rejection. In 6 of the remaining 8 episodes a transient increase in liver enzyme levels was observed, indicating mild rejection which responded to increased basic immunosuppression. Conclusion: These data demonstrate that an increase in the frequency of CD45RA(CD45RO+) CD4+ "memory" T cells is detectable during acute rejection and during episodes of subclinical immune activation. The question whether the observed increase of CD45RA- T cells in fact represents the rise of a "memory" subset of T cells specifically sensitized by graft alloantigens remains to be determined.