486 Intervention in autoimmunity 25 June 1997- Poster presentations

such as iodide-induced thyroiditis. When a high iodide dose is administered to goitrous NOD mice the iodide-induced thyroid cell necrosis is followed by diffuse infiltration by macrophagas and CD4 and CD8 T cells, leading to follicular destruction similar to the Hashimoto's thyroiditis in the human.

In this autoimmune thyroiditis model, injection of IL-9 completely abrogated T lymphocyte and macrophage infiltration. In addition, IL-9 induced an increase in germinal center formation in draining lymph nodes, indicating that the inhibition of the cell-mediated response is accompanied by B cell activation. However, no significant change in TH 1 or TH2 cytokine expression was detected in the thyroid or lymph nodes of IL-9-treated NOD mice. A similar inhibition of cellular infiltrate was observed in the pancreatic islets of NOD mice, where a 4-6 day treatment with IL-9 significantly suppressed the insulitis. By contrast, IL-4 is inactive in the thyroiditis model and needs more prolonged administration to inhibit insulitis, suggesting the involvement of distinct mechanisms in the activity of these TH2 cytokines.

Taken together, these data indicate that, in NOD mice, IL-9 favors humored auto-immunity but inhibits cellular mediated auto-immune processes.

P.5 .22.20 1 Compar i son of co l l agenase and I lberase in is let i

t ransp lan ta t ion : Role of endo tox ln and CD14 In the fa i lure of Is let graf ts

F. Vargas, J.F. Llamazares I J.F. Julian 1, F. Garcia 1 p. Armengol, N. Somoza, A. Sanmadf 2, R. PujoI-Borrall, M. Vivas-Pi. Immunology Unit, Hospital Germans Trias i Pujol. Universitat Autdnoma de Barcelona, Badalona, Spain, 1 Surgery Dept., Hospital Germans Tries i Pujol. Universitat Autdnoma de Barcelona, Badalona, Spain, 2 Endocrinology Dept., Hospital Germans Trias i Pujol. Universitat ALrYmoma de Barcelona, Badalona, Spain

Islet transplantation is a method to restore insulin secretion in IDDM patients but unfodunately the rate of graft failure is until now unacceptably high. We have recently reported the presence of high endotoxin activity in collagenase P, the enzyme commonly used to isolate islets for transplantation; this, together with the finding of CD14 (andotoxin/LPS receptor) on the islet cell surface led us to propose that endotoxin in collagenase could play an important role in islet graft failure.

We have tested this hypothesis in alloganaic islet transplantation in rats by using collagenasa preparations with different levels of endotoxin activity and monitoring rejection by immunohistochemistry. 800 islets from Wistar rats iso- lated by enzymatic dispersion of the gland and discontinuous density gradient were transplanted under the left kidney capsule of Lewis rats. After one week, kidneys were removed and consecutive tissue sections from the tissue contain- ing the graft ware stained using monoclonal antibodies to immunological and cell type markers. Islets from groups of 15 animals were prepared with either ccllaganase P (360 EU/ml), Liberase TM (a new preparation made of a mixture of enzymes with very low endotoxin activity, 2.8 EU/ml) or Liberase TM supple- mented with exogenous LPS (5,000 EU/ml). Survival of the islet cells, intensity of MHC and ICAM-1 expression, area and type of infiltration and the presence of lymphocyte activation markers were assessed using a standardisod scoring system (0 to 4+).

Results show that the intensity of the rejection was parallel to that of sndo- toxin activity of the digestion solution and followed the hierarchy: exogenous LPS plus Liberase TM > Collagenase P > Liberasa TM.

In conclusion, the results suggest that the low sndotoxin content in the enzyme Liberasa TM may be advantageous for islet transplantation. Work is in progress to elucidate also whether the cytokine pattern is also different.

I P.5.22.21 I Decrease in CD4 + CD45RC+ T cel ls af ter an I

ef fect ive t rea tment of ad juvan t ar thr i t is wi th ant l -CD4 monoc lona l a n t i b o d y

C. Palegrf, M. Rodrfguez-Palmero, M. Serra, C. Castallote, M. Castell, A. Franch. Unit of Physiology and Physiopathology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Introduction: Thl and Th2 pattems of cytokines can play an important role in the autoimmune disease processes, such as rheumatoid arthritis. Recently, it has been shown that there is a dominant Thl response in collagen-induced adhdtis developed in mice. On the other hand, we have demonstrated both preventive and curative affects of anti-CD4 monoclonal antibody in rat adjuvant adhdtis (AA). Thus, the aim of the present study was to analyse the CD45RC + and CD45RC- CD4 + T cells, which are thought to be representative for the Thl and Th2 subsets, after a preventive anti-CD4 treatment of AA.

Materiels and Methods: In order to induce AA, Wistar rats were intreder- mally injected in the base of the tall with a suspension of heat-inactivated Mycobacterium butyricum. Animals received 7 doses of W3/25 monoclonal an- tibody dudng the latency padod of AA (10-14 days). PBL were obtained by retroorbital puncture and erythrocyta lysis, Phanotype of lymphocytas was es- tablished by means of cytometry analysis using anti-CD45RC MoAb (OX22) combined with anti-CD4, CD8, CD5 and Ig MoAb.

Results: On days 14 and 21 of AA, rats treated with anti-CD4 did not have inflammation and showed a decrease in CD45RC+CD4 + T cells whereas an increase in CD45RC-CD4 ÷ T cells was observed. On day 28, percentages of these T cell populations showed a tendency to reach those levels of healthy and adhdtic rats, although the antlinflammatory effect of the antJ-CD4 MoAb remained.

Conclusion: The effective treatment with anti-CD4 monoclonal antibodies in adjuvant adhdtis could be related to a switch from Thl to a Th2 response.

P.5.22.22 1 Man ipu la t ing T-cell cy tok lne pro f i les wi th a l tered i

pep t lde I igands: Effects on pr la tane Induced arthr i t is

J.N. Francis, A. Lamont ~, T. Johnson 1, S.J, Thompson. Immunology Unit, Dept. Pathology & Microbiology, Medical School, University of Bristol, UK, 1Pepb'de Therapeutics Ltd. Cambridge Science Park, UK

Introduction: This work aims to establish the optimal route of administration of immunodominant bacterial heat shock protein peptides and altered paptide ligands with differential ability to stimulate Thl or Th2 responses in vitro on the disease course of the CD4+ Thl mediated medal pdstane induced arthritis.

Materials and Methods: CBA (H-2k) mice immunisad ip with hsp65 are protected from the development of pdstane induced arthdtis (PIA) and such protection is associated with a Th2 response. An immunodominant epitope 261-271 has been identified which also protects mice from PIA. Using a panel of related and ovedapping peptides we have identified T-cell epitopes and altered peptide ligands capable of inducing diverse in vitro T-call proliferation and cytokine profiles. These peptides have been administered ip, sc, im and intranasally and their affect on immune responses and on the modulation of PIA monitered.

Results: Peptides that elicit good Th2 responses on immunisation can pro- tect from PIA whereas intranasal administration of peptidas generally decrease both T cell proliferative responses and cytokine production for up to 40 days post inhalation.

Conclusion: These results show the potential for peptida therapy in the treatment of autoimmune disease.

P.5.22.23 [ proliferative responses to HSP65 or to PBMC HSP65-derived peptides correlate with remission or l ow g rade of RA

L.A. De Netto I V. Grover ~, A.G. Lemont 2, T. Johnson 2, S.J. Thompson ~. 1 Dept. Pathology, University of Bristol, UK, 2 Peptide Therapeutics Ltd, Cambridge, UK

Introduction: Mycobasterial HSP65 or the HSP65-dedved peptide 261-271 induce Th2 cytokine responses upon intraparitoneal injection into mice which protects against onset of arthdtis after subsequent administration of pdstane i.p. Preliminary assessments of the ability of 261-271 to beth bind HLA-DR and to elicit T-cell proliferation of normal donor peripheral blood T-cells reveal that this peptide may be efficacious as an immunotherapeutic for RA. This study aims to assess the ability of 261-271 to elicit PBMC proliferation in normal donors or RA patients of varying clinical grade. The effects of steroid treatment upon the proliferative response of PBMCs was assessed as it may be necessary to take patients off these general immunosuppressants prior to immunotherapy.

Materials and Methods: PBMCs were isolated by fractionation on ficoll and were utilised in daily in vitro proliferation assays over an 11 day pedod to assess immunogenicity of 261-271 or HSP65.

Results: The PBMCs of 5/9 normal donors proliferated in response to 261-271. The PBMCs of 2/9 normal donors did not respond to any pep- tide utilised. Peptide negative responders had low proliferative responses to HSP65, whereas peptida respondars had large proliferative responses to HSP65. PBMCs derived from two normal donors unresponsive to 261-271, did however proliferate in response to related peptidas from this region. RA patients appeared to be segregated into two clear groups, namely peptide responders (8/13) and peptida non-responders (5/13). The peptida responders also had re- activity to HSP65, whilst the non-responders lacked HSP65 reactivity. 6/13 gave proliferative responses to 261-271, and a further 2/13 lacked 261-271 reactivity but gave responses to p259-270 and p263-274. Clinical grade was assessed by early moming stiffness, degree of synovitis, blood analysis, and clinicians opinion at the time of blood sampling on a scale of 0-3, (0 = remission and 3 = severe). Strikingly, those patients responding to 261-271 or related peptidas were either in remission or had grades of 1. Those patients not responding to peptides or to hap65 had grades of 2-3. There is a statistically significant difference between these two groups of patients with respect to grade of RA and with respect to the magnitude of hsp65 responsiveness. Treatment with the steroid prednisolone abolished peptide responses and lowered mitogan, KLH, PPD and hsp65 responses in the RA group.

Conclusion: Reactivity to 261-271, or to related peptides appears to cor- relate with both reactivity to hsp65 and low grade RA or remission. The results

25 June 1997- Poster presentations Novel immunosuppressives and xenotransplantation 487

also demonstrate that this is not due to general hyporesponsivaness in the high grade RA subset.

09:00-18:30112:00-14:00 Forum lounges

P.5.23 Novel immunosuppressives and xenotransplantation

P.5.23.01 I M l togen lc i t y o f ant i -TCR/CD3 m A b s a t t r ibu tab le to I

inef fect ive c ross- l ink ing of low-af f in i ty Fc~ recep to rs

J. Pischei 1, U. Zengerie 1, T. Takai 2, R. Mailhammer 3 S. Thierfelder 1, U. Kummer 1.1GSF-Institut fOr Immunologie, Marchioninistr. 25, 81377 MOnchen, FRG, 2 Department of Biotechnology, Faculty of Engineering, Okayama Universi~ Tsushima-Naka 3-1-1, Okayama 700, Japan, 3 GSF-Institut for Experimentelle Hdmatologie, Marchioninistr. 25, 81377 MOnchen, FRG

Introduction: OKT3, a mAb reacting with the CD3 complex, has proved ef- feotive for immunosuppression in clinical practice for about 10 years. However, parallel with this beneficial effect, toxic effects which can result in significant morbidity have been reported upon initial treatment of the patients. In mice, anti-CD3 therapy has been mainly based on the use of the hamster anti-CD3 mAb 145-2Cll which shares many properties with OKT3. In this context lit- tle attention has been given to the therapeutic and/or toxic profile of anti-CD3 isotypas different from 145-2Cll.

Materials end Methods: In this study we have compared the effects of in vivo administration of 145-2Cll and 17A2 (a rat IgG2b anti-mouse CD3 mAb) on several parameters, including TCR/CD3 modulation, T cell depletion, T cell activation and activation-induced apoptosis. We have extended these in vivo expedmants using mice genetically deficient in FcyRII. The strength of antl-CD3 mAb binding to low-affinity FcyR has been quantitated in an anti-CD3-induced T cell proliferation assay using the Fc~,RII/lll-blocking mAb 2.4G2.

Results: We have found that both anti-CD3 mAbs have similar kinetics of TCR/CD3 down-regulation, albeit 145-2Cll was significantly more efficient in this respect. In addition, 17A2 exhibits depleting properties, whereas 145-2Cll tdggers a complete signal of cellular activation which subsequently leads to a massive expansion of the T cell pool followed by a marked T cell depletion due to apoptosis. The depleting and mitoganic properties of 17A2 and 145-2Cll, respectively, are both dependent upon interaction with low-affinity FcyR and could be totally abrogated by administration of mAb 2.4G2. However, FcyRII/lll blockade by 2.4G2 induced only a partial inhibition of 17A2 induced IL-2 pro- duction of T cells in vitro. In contrast, in an analogous in vitro expedment 2.4G2 completely neutralized the ability of 145-2Cll to tdgger IL-2 secretion. Further- more, we have also observed that 17A2 and 145-2Cll retain their mechanisms of action in FcyRII KO-mico.

Conclusion: Taken together, these results suggest that the number of pro- ductive contacts between antibody-coated target cells and FcyR-bearing ef- fector cells significantly impacts on the nature of the immunosuppression of anti-CD3 mAbs. In effect, nonmitogenic anti-CD3 mAbs with a high affinity for low-affinity FcyR may be effectively trapped on effector cells and therefore directly removed from the circulation. In contrast, the mitoganic properties of anti-CD3 mAbs may be a result of their low FcyR binding affinities. The results and conclusions may open major therapeutic perspectives for the human situa- tion. First, the immunosuppressive effects of nonmitoganic anti-CD3 mAbs may be as potent as those of mitogenic anti-CD3 mAbs though without the risk of severe side effects. Second, mitoganic anti-CD3 mAbs might be exploited clini- cally to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myeiodysplastic disorders.

P.5 .23.02 J Inh ib i t ion of DPP IV/CD26 enzymat i c ac t iv i ty 1

ab roga tes acce le ra ted re ject ion of rat card iac a l logra f ta

I. De Meester 1 , S. Korom 2, T.H.W. Stadlbauer 2, F. Goossans 1 , A. Haemers 1, J.W. Kupiec-Weglinski 2, S. Scharp61.1 Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium, 2 Surgical Research Lab. Harvard Med. School, Dept of Surge~ Brigham and Women's Hospital, Boston, MA, USA

Introduction: Dipeptidyl peptidase IV (EC 3.4.14.5) is an ecto-enzyme that occurs as an integral membrane protein on the cell surface. It cleaves off

dipeptides from the aminoterminus of pepfides with a proline, hydroxyproline or alanine at the penultimate position. DPP IV is identified as the activation antigen CD26, one of the major costimulatory molecules inveived in T cell activation. Although in vitro inhibitor-studies provide substantial evidence for a role of the catalytic activity in costimulation, this issue remains a matter of debate. Recently, we showed that in vivo inhibition of DPP IV abrogates acute cardiac allogreft rejection at day 7, and prolongs cardiac transplant (l"x) survival to 14.0 +/ - 0.9 days (p < 0.0001). In this study we investigated the effect of DPP IV inhibition on the accelerated rejection in sensitized allograft recipients.

Matedata and Methods: Following accelerated rejection model was used: LEW rats sensitized with BN skin Tx (day -7) receive LBNF1 cardiac Tx (trans- planted at day 0). In untreated animals rejection occurs within 36 hr. Prodipine (Pro-Pro-diphenylphosphonate) was given sc 30 mg/ret at day -7, followed by 20 mg/rat at days -4, -1 and +4. AIIo-antibody responses were measured in the serum by flow cytometry.

Results: In vivo targetting DPP IV activity with the low molecular weight specific inhibitor Prodipine reduced plasma DPP IV activities to <15% of pre- treatment values. Treatment abrogated accelerated rejection. Cardiac Tx sur- vival was prolonged in 7 out of 13 recipients (mean survival time +/-SD = 5.1 +/- 2.1 days, p < 0.01). Prodipine prevented systemic IgM allo-Ab responses and the effect was so endudng that the usual second IgM peak by day 21 post "Ix was not observed, despite the recovery of DPP IV (>70% of pretreatmant value) at that time. The effects of Prodipine treatment on serum cytokine levels are currently under investigation.

Conclusion: Our findings on prolonged graft acceptance in sensitized trans- plant recipients (accelerated cardiac rejection model in the rat) upon DPP IV inhibition demonstrate the involvement of CD26/DPP IV in the in vivo immune response upon solid organ transplantation.

1:).5.23.03 1 Dualistic capacity of the novel immunosuppresslve drug mycopheno la te mofet l l (CellCept ®)

R.A. Blaheta 1, K. Leckel 1, B. Wittig 2, M. Scholz 1, D. Zenker 1, S. Weber 1, A. Encke 1 , B.H. Markus 1.1j. W. Goethe-Universi~ Dept. Gen. Surge~ Frankfurt am Main, Germany, 1DKFZ, Heidelberg, Germany

Introduction: Cellular rejection is charactedzed by 1) proliferation and differen- tiation of the recipient lymphocytes and 2) infiltration of the lymphocytas via the endothelium into the donor organ. Immunosuppressive drugs as Cyclospodne A or tacrolimus (FK 506) block cellular activation but have no effect on the infiltration process. The novel drug mycophenotate mofetil (MMF) effectively suppresses cell mitosis by blocking inosine-monophosphate-dehydrogenase (IMD), the key enzyme for the pudne biosynthesis. Because IMD additionally regulates the glycoprotein synthesis, some of which are adhesion molecules, an influence of MMF on the cellular infiltration process might also be possible. We therefore investigated in vitro, whether and in what way MMF interferes into the lymphocytic infiltration cascade.

Materials and Methods: CD4 and CD8 T-lyrnphocytes were added to en- dothelial cells (EC), derived from human umbilical veins, together with MMF (0-100 /zM). Lymphocyte binding was analyzed by the monolayer invasion assay, expression of endothelial adhesion receptors by FACS measurement. Activity of the lymphocytic counter receptors in presence of MMF was evaluated by the adhesion spot assay, using isolated chimeric proteins containing the extrecallular domain of E-seleotin, P-selectin, ICAM-1 or VCAM-1. Horizontal locomotion of CD4 and CD8 T-ceils was investigated by videomicroscopy.

Results: MMF dose-dependently suppressed loose (moderately) and firm (strongly) attachment of CD4 and CD8 T-lymphocytes to allogeneic EC. The effect was more pronounced when EC were pretreated with ~,-IFN, IL-1 or IL-I+TNF(~. Moreover, MMF-incubation of both lymphocytes and EC lead to a stronger reduction of cell binding than MMF-incubafion of either lymphocytes or EC. Fluorometdc analysis of the endothelial adhesion receptors ICAM-I, VCAM-I, E-selectin and P-salectin showed that MMF moderately influenced ICAM-I (optimal dosage: 1-10 /~M), VCAM-I (optimal dosage: 0.1-I /~M) and P-saleotin (optimal dosage: 0.1 /~M) expression but strongly reduced de novo synthesis of E-seleotin (0.001-10 /~M, p < 0.05). Invastigafion of the lymphocytic counter receptors by the adhesion spot assay demonstrated that MMF did not downregulate binding of CD4 or CD8 T-lymphocytes to E-salectin, but differentially suppressed the cellular interaction via ICAM-1, VCAM-1 or P-selectin proteins. Videomicroscopic analysis of the locomotory activity of CD4 and CD8 T-cells revealed an additional mode of action of MMF on the intmcellular cytoskeleton (optimal dosage: 1/~M).

Concluelon: The results presented clearly demonstrate that MMF -in strong contrast to CsA or tacrolimus- blocks organ rejection in a dualistic way. Beside its antiproliterative effect MMF also possesses distinct suppressive properties with regard to the cellular infiltration process. The unique charaotedstice of MMF might allow new immunosuppresslve strategies. Fur~er experiments should evaluate the therapeutic value of MMF on other infiltration-dopendant processes such as tumor metastasis.