An alternative conditioning regimen for induction of specific allograft tolerance

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An alternative conditioning regimen for induction of specific allograft tolerance
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  152 Tolerance induction 23 June 1997 Poster presentations LP.5.01.17 Hematopoietic and immune effects of blood and plasma transfusion I. Gtzelak, M. Zaleska, W.L. Olszewski. Surg Ras Tmnspf Da@, Med Ras Ctr, Pof Acad Sci, Warsaw, Poland Blood transfusions (BT) should be viewed as the transplantation of blood cellular and soluble elements. They evoke various alterations in the immune responsive- ness of blood recipients, contributing to increased risk of infection and cancer occurrence. The exact mechanism by which BT induce a state of the reduced immune responsiveness remains unclear. Blood cells as well as humoral plasma components may play a role in this phenomenon. Although relatively much is known about post-transfusion changes in various populations of recipient circu- lating blood cells, few data are availabte on the immune changes in the organs these cells otiginate from. In this study the effect of transfusion of whole blood (BT) and plasma (PT) was examined in a rat model. The effects of syngenaic (SBT) and allogenaic (ABT) blood and plasma (SPT and APT, respectively) were compared. Normov- olemic WAG rats were transfused i.v. with either syngeneic or allogeneic (AUG) heparinized venous blood or plasma (3 x 2 ml, every other day). Three, 7 and 14 days after the last transfusion blood (B), spleen (SPL), mesenteric lymph nodes (MLN) and bone marrow (BM) cells were examined. Re8u)ts: of this study, on blood and plasma transfusion, point to the de- velopment of specific post-transfusion changes in B. SPL. MLN and BM of the recipients. The most characterfstic nclude, in case of whole BT, he changes in the hemopoietic system, reflected by a significant decrease in BM etythroid and increase in myeloid and lymphoid lineage cell populations, and with a rise in the percentages of BM 0X7+ stem cells and their subsequent release to the blood circulation. Furthermore, a significant drop in BM 0X6+ class II+ cell population in both SBT and ABT rats and in Band MLN of ABT rats was seen. Besides, the responsiveness of B, SPL, MLN and BM lymphocytes was significantly reduced. Transfusions of syngeneic plasma, almost doubling the circulating plasma vol- ume, caused a decrease in the number of 0X7+ stem cells and 0X6+ cells in the BM of SPT rats. No changes in 0X7+ cells while a significant drop in 0X6+ cells in BM of SPT rats and BM, SPL, MLN and B of APT rats was noted. Furthermore, a significant decrease in the responsiveness of 8, SPL and BM lymphocytes was seen following both SPT and APT. Surprisingly, the responsiveness of MLN cells was increased in both rat groups. Summary: The data conclusively demonstrate that plasma factors partici- pate in evoking the changes in immune responsiveness of blood recipients. The effect of whole blood transfusion is a total of blood cells and plasma. Subtract- ing the changes developing after PT from those after whole BT. we can infer that suppression of B, SPL and BM leucocyte responsiveness and decrease in BM 0X7+ stem and BM, B and MLN 0X6+ cell frequency may be caused by an excess of transfused plasma soluble factors (cytokines released from platelets and leukocytes, shed receptors for intetteukins, inhibitors of cytokines and blockers of their receptors, soluble histocompatibility antigens and various proteins with blocking properties) in the recipient circulation. P 5.01 18 Oral tolerance in mice: induction with multiple, small doses of antigen T.M.R. Jergensen, H. Frehr. Department of Biochemistry nd Nutrition, Build. 224, Technical UnivatMy of Denmark, 2600 Lyngby, Denmark Introduotlon: Specific and systemic immunological hyporesponsiveness after oral administration has been demonstrated with vatfous dietary proteins and is termed oral tolerance (OT). Usually OT is achieved by feeding one (or a few) large dose(s) of protein, whereas ng-fig doses are believed to be beneath a threshold where OT can not be induced. To mimic a naturally occurrtng situation we studied the effect of a dally intake of very low doses of antigen on OT induction. Materials nd Methods: roups of mica ((BALB/c x CFl)Fl) were fed pure ovomucoid (OM) or OM in the context of egg white extract in varying amounts (1 @day-IO &day) for 30,50,75, and 100 days. For evaluation of the immune response mice were immunized intraperttonally with 10 pg of OM on day 7 and 14 after the last feeding day (day 0). The humoral immune response was measured in blood samples on day 14 by ELISA, and the cellular immune response was evaluated on day 21 by proliferation of s&en cells cultured with OM. OM positive B-cells in Peyers Patches (PP) and spleens were measured using a FACStar plus (Beckton Dickinson). Results: It was shown that it is possible to induce OT with relatively small doses (ng-&day) of OM when administrated for a prolonged period of time. Induction of OT was only achieved for some amounts of OM. As an example, feeding for 100 days with 1 pg OM/day gave a significantly reduced humoral and cellular immune response compared to the control, whereas feeding IO Fg OM/day for 100 days did not induce OT. Feeding for 30 days gave a significantly reduced humoral immune resoonse when usino IO no/dav. but neither the humoral nor the cellular immune response was reduced when using 0.1 or I &lay. Moreover, the induction of OT is accomplished at different doses of OM when using pure OM compared to OM in egg white extract. The group of mice that induced OT. when fed for 30 days, had an enhanced level of OM positive B-cells in both PP and spleen compared to the control; while groups that induced OT after feeding for 50 and 75 days had a lowered level of OM positive B-cells in PP and an unchanged level in the spleen. This indicates that the mechanisms of OT induction are different depending on the period of feeding. Conclusion: In an attempt to mimic a natural situation it has been demon- strated that it is possible to induce oral tolerance with relatively small dcees of OM when administrated for a prolonged period of time. I .5.01 19 Histological changes in the thymus after intrathymic injection of alloantigen A.S. Wubbena, F.A. Klatter, H.-P. Rat& J. Razing, P. Nieuwenhuis. Dept. of Histology & Call &dog) University of Groningen, Oos~atsingal69-1, 9713 EZ Gronin&, The Neih&lan& Inboduotlon: lntrathymic injection of donor type splenocytes in combination with a treatment with antilymphocyte setum (ALS) (1 ml i.p., day 0) followed by short course of cyclospotin A (CsA) (15 mg/Kg i.m., days 1.2 & 3) induces long-ten acceptance of cardiac allografts set on day 0. To urther elucidate the mechanism responsible for the Induction and maintenance of intrathymically induced tolerance we have followed the fate of allogeneic cells after injection in the host thymus using immuno-histochemical methods. Mamrlals & Methods: A0 (RTlu Rl7a) animals were divided in three groups. On day 0 all animals received an injection of 2.5e7 PVG (RTlc RT7b) splenocytes into the left lobe of the thymus. The various groups were then treated as follows: group 1, no further treatment; group 2, 1 ml ALS i.p. on day 0; group 3, 1 ml ALS i.p., CsA on days 1, 2 and 3. Two animals from each group were sacrificed on days 4, 7 and 15 respectively. Additionally, animals in groups 2 and 3 were killed 21 and 26 days after inbathymic injection. At autopsy the thymus was removed and frozen. For analysis 7 pm cryostat sections were sequentially stained with the monoclonal antibodies MRC-OX27 (RTlc), HIS41 (RT7b), ED1 (macrophages), HIS56 (B-cells). Reautts: In untreated animals injected cells can be detected at 4 and 7 days but not at 15 days after the inoculation. In the ALS treated animals the allogenic cells are no longer present at 21 days while in the ALS & CsA treated animals hey disappear between 21 and 26 days after inoculation. This suggests a protonged state of intrathymic tolerance in these animals. The allogeneic cells are located in a very restricted area of the thymus initially in both the cortex and medulla but are later found almost exclusively in the medulla. Also a marked increase in the number of thymic B-cells was observed in the ALS and ALS & CsA treated animals afterthe inoculation. These B cells are organized in follicular structures comprised of both donor and recipient type cells. Furthermore there is an influx of macrophages, which localize adjacent to the allogeneic cells and remain present some time after these cells are gone. Conclusions: ntrathymic injection of allogeneic cells induces major histo- logical changes in the thymus and ALS as well as a combination of ALS and CsA treatment results in the prolongation of these changes. The exact signifi- cance of these changes in the induction of tolerance is uncertain at this time. However, although we can not exclude the possibility that intrathymic deletion or inactivation of alloreactive clones plays a role during the induction of tolerance it is unlikely that this plays a major role in the maintenance of intrathymlcally induced tolerance as the allogeneic material is restricted to a small area of the thymus and disappears within a four week period. 1 P.5.01.20 1 An alternative conditlonlng reglmen for induction of specific allograft tolerance A. de Vries-van der Zwan, M.A. van der Pol, L.P. da Waal, C.J.P. Boog. Dept. of Tmnsplantation Immunolcg~ Central Lab. of fha Nathadands Red Cross Blood Tmnsfusion Service, Amsterdam, The Narherlands Introduction: he induction of donor-specific transplantation tolerance is a ma- jor goal in organ transplantation, in order to eliminate he requirement for lifelong immunosuppressive herapy. Recently, we have developed a murine transplan- tation model in which recipient mice were treated with antKD3, anti-CD4. low dose total body irradiation (TBI; %6 GY) and allogeneic donor bone marrow cells. This well-tolerated protocol results in the development of stable mixed chimerism and pement transplantation tolerance across MHC barriers, with- out any clinical evidence of GvHD. Thusfar, the clinical application, however, of our and other experimental models has been hampered by the toxicity of conventional conditioning, which is currently used for bone marrow transplanta- tion, and the relatively long interval between tolerance induction and the actual allotransplantatfon. Materlate and Method% In the present study, we have investigated if low concentrations of dimethyl myleran (DMM) in combination with low dose cy- clophophamide (Cv) can be used as an alternative for TBI in our tolerance inducing protocol. Furthermore, we have shortened the whole procedure and performed skin transplantation within one hour following conditioning.  23 June 1997 Poster presentations Effector phase of he allergic response 153 Reeuh Our data cleady show that conditioning with low dose TBI can be substiid by a combined single low dose DMM/CY therapy, resulting in mixed chime&m and specific tolerance as well. Finally, we show that skin transplantation cfm be successfully performed directly following conditioning. Conclusion: Since skin is more immunogenic than most other graft types, we suggest that our protocol can be used for donor-specific tolerance induction in other organ transplantations. P.5.01.21 lntrathymlc injection of staphylococcal enterotoxin B Induces T cell depletion and unresponsiveness In adult mice Reeuh No side-effects have been observed dudng the treatment of CsA and mAb 87-24. Until day 7 after transplantation (TX), high levels of mAb 87-24 could be detected in the blood. From day 7, all animals developed a RAMA-response. lmmunohistology of biopsy material demonstrated that at day 5 after TX the mAb 87-24 was detectable on the dendritic-like cells and macmphages in the lymph node and grafted skin. The skin graft sulyival time in Rhesus monkeys treated with CsA and mAb B7-24 (14 days) was significantly increased compared to Rhesus monkeys treated with CsA alone or control (IO days both). Eventually, skin grafts in all Rhesus monkeys were rejected which coincided with an increase in helper T-cell and cytotoxic T-cell frequency and the induction of an antibody response directed against donor antigens. Conclusion: Short term immunosuooression. resulting in prolonged skin graft sutival could be obtained using ‘a’ suboptimal doses of CsA and mAb B7-24 as a prophylactic treatment in a Rhesus monkey skin transplant model. . Goettelfinger, R. Roussin, F. Lecerf, S. BenihAknin, M. Fattal-Geman. Labomtoire d’lmmunobgie CNRS ERS 588, Hopi&d Marie Lannelongue, Le Plessis-Robinson, France Introduction: Induction of specific and long-lasting immunologic tolerance to donor antioens bv intrathvmic (Lt.1 mute is thouoht to be the best strateav for allograft a&eptance due*to mbdiiications of T “cell reactivity, even in adults. Using superantigens in a mouse model to explore the possible mechanisms of tolerance induced by Lt. injection, we have investigated the modifications of the V@+ T cell subsets and the lymphocyte mactivlty in the thymus and in the spleen following SEB i.t. injection. OkOO-18:30/12:00-14:00 Forum lounges MaterlaIr and Methods: C57Bl16 mice were injected with SEB on day 0. P.5.02 Effector Phase of the allergic Thymocyte and splenocyte subsets expressing the Vfi8 antigen were i&es- tigated by cytofluorimetric analysis using triple-stained immunofluorescence method. The in vitro reactivity of thymocytes and splenocytes to SEB was studied by the proliferative response to SEB in the presence or absence of responsi - exogenous 11-2. ReeulB: An early but transient increase in the percentage of CDB+VBB+ thymocytes day 2) was observed followed by a significant decrease in the CD8+VB8+ thymocytes from day 7 to day 13. By contrast, the percentage of CW+VB& splenocytes was significantly decreased from day 7 to day 28. Thymocyte proliferative response was significantly decreased from day 2 to day 13. By contrast, an enhanced proliferative response of the splenocytes brecedd a dramatic unresponsiveness from day 7 to day 28. This unresponsiveness was SE&specific. It could be reversed by exogenous IL-2 and partially by anti-CD28 monoclonal antibody. P.5.02.01 Expression of FcERI by dendritic cells from normals and asthmatlcs J.A. Hartley, S.T. Holgate, A.E. Semper. University edicine, General Hospital, Southampton, UK Introduction: The high affinity receptor for IgE, F-RI, was first shown to be present on mast calls and basoohils. In atooic diseases, FcERI surface expression has been shown on Langehans cells in’the skin, on blood monocytes from both normals and atopics, and on dendtitic cells (DC) in the blood and lungs of normals and asthmatics. Furthermore. numbers of FcsRla+ DCs are greater onclusion: I.t. injection of SEB induced a clonal deletion of the V@+ T cells both in the central and peripheral compartments with involves the mature CD8+ thymocytes and CD4+ splenocytes. Concomitantly, a specific IL-2-dependent unresponsive state was observed. This model of T cell clonal activation using SEB inoculation should prove useful for determining the optimal condiiions to induce tolerance to alloantigens by the i.t. route. 1 P.5.01.22 1 Use of anti-B7.1 monoclonal antibody in combination with cyclorporlne A to prevent skin transplant rejection in a rhesus monkey model in the lungs of atopic asthmatics than in control subjects. The purpose of this study was to investigate whether levels of mceotor exoression in the pelipheral bloob of normals and asthmatics were similar increased. MaterlaIr and Methods: Peripheral blood mononuclear cells (PBMC) were isolated from blood by centtifugation over Lymphopmp. Cells were simultane- ously labelled with (i) a cocktail of phycoerythrin (PE) conjugated antibodies against T cells (CD3), monocytes (CDl4), B cells (CD20) and NK cells (CD58); (ii) an FITC conjugated anti-MHC class II DR antibody; (iii) various biotinylated neutralising antibodies directed against the FeRI-cr subunit (15.1 was a kind oift from Dr Kinet). These were visualised with streotavidin quantum red. Calls were also labelI& with appropriate fluorochrome’wnjugated isotype control antibodies. DCs were identified by flow cytometry as cells showing no labelling for CD3, CD14, CD20 and CD58, but strong labelling for HIA-DR. Using three colour flow cytometric analysis, the number of Fc&Rla+ cells within this popula- tion were determined. PBMCs were also treated with 0.01 M buffered lactic acid for 1, 2, 5 or IO minutes prior to staining to ship IgE bound to FaRI, allowing analysis of the level of receptor occupancy. MA. Cssevoort I, L. Boon 2, K. Lo& 3, M. de Boer 2, M. Jonker I. ’ Dept. Immunotherap) Biomedical Primate Research Cenwe, R@wijk The Netherlands, 2 PanGenetics BV; Amsterdam, The Netherlands, 3 nnogenetics NV; Gent, Belgium Introduction: The optimal therapy to prevent graft rejection should be based on the induction of specific non-responsiveness to the donor tissue. Current con- cepts on induction of immunological tolerance hold that non-responsiveness is the result of intracellular signalling after T cell receptor/Major Hi&compatibility Complex (MHC)-peptkte interaction (signal 1) in the absence of a costimulatory signal (signal 2). One of the co-stimulatory signals is provided by the interaction of 87 wstimulatory molecules on the antigen presenting cell and CD28 on the T lymphocytes. It has been demonstrated that the alloantiaen-specific proliferative and cytotoxic response of rhesus monkey pedpheral L&d ‘mononwlear cells (PBMC) stimulated by Rhesus monkey B cell lines or Rhesus monkey PBMC can completely be abrogated by adding CsA in combination with anti Cl%0 mAb (87-24 (87.1) mAb, lnnogenetics NV, Gent). In this study the efficacy of the prophylactic treatment of mAb 87-24 and CsA was tested in a Rhesus mon- key skin transplant model. Graft survival times were taken as indicator for the immunosuppr&sive potency of the therapy combining mAb 87-24 with CsA. Materlats and Methods: The rhesus monkeys received an allogeneic MHC mismatched skin graft on day 0. The mAb 87-24 was given daily for IO days starting at day -1 (serum rough level = f20 &ml). The suboptimal doses of CsA wasgiven intramuscular starting at day-2 until rejection has been observed (CsA blood trough level f 100 @ml). At several times points during the experiment, blood samples were taken to determine the blood level of CsA, serum levels of mAb 87-24, the Rhesus monkey anti-mouse antibody (RAMA-) response and anti-donor antigen antibody response. For the measurement of the cellular response hepatinbed Mood samples were taken prior to the transplantation and at several time points after rejection to isolate PBMC and perform the combined helper T-cell and cytotoxic T-cell frequency (HTLp/CTLp) assay. Rew4ts: Fc&RI s expressed on the surface of peripheral blood DCs from both normal and asthmatic subjects, although normals have lower levels of un- occupied receptor. Treatment of cells with lactic acid prior to staining, increases the level of unoccupied receptor detected. Conclusion: We have previously shown that numbers of Fc&Rla+ DCs are greater in the lunos of atooic asthmatics than in control subjects. This study ~ __ shows that the leiels of unoccupied receptor were higher in atopic asthmatic subjects. despite the higher levels of IaE in the serum. and so there were fewer uno&upied r&tots. ?his couM be-accounted for ‘by an increase in levels of the receptor on circulating DCs in asthmatics compared to normal subjects. Further studies on Fc&RI+ DCs are in progress. P.5.02.02 Reduced thymulin (ZnFTS) activlty In allergy: Role of IgG immunoglobulln E. Mocchegiani, L. Santarelli. /mmuno/oy Ctr., Res. Dept., institute National Research Centers on Aging (I. N. R.C.A.), Ancona, Italy Introduction: Reduced thymulin (ZnFTS) activity has been suggested to be a concause of the impaired T-suppressor function in allergy. Contradictory data exist in thymulin activity in allergy. A marginal zinc deficiency has been reported in atopy. Patient We have tested thymulin (which is a strictly zinc-dependent thymic hormone) and zinc in 80 asthmatic and 25 atopic children.
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