Donor-specific hyporeactivity in lung transplant recipients

Donor-specific hyporeactivity in lung transplant recipients

150 23 June 1997 - Poster presentations Tolerance induction ~98% pure by FACS. Bone marrow macrophages were isolated from BALB/c mice and cultured ...

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150

23 June 1997 - Poster presentations

Tolerance induction

~98% pure by FACS. Bone marrow macrophages were isolated from BALB/c mice and cultured for 18 days in GM-CSF before used in experiments. SEC and CD4+ cells were coincubated with 10 &ml specific peptide for five days. Primed T cells were restimulated for 48 hours with anti-CD3 antibody crosslinked by anti-rat-lg. Cytokine procuction (IFN-g, 11-4, IL-lo) was determined by specific ELISA as measure of T cell activation. Resu& SEC could induce a weak Thl-like antigen-specific activation of naive CD4+ T cells (no peptide 12.5 U/ml f 1.4 U/ml IFN-g, BSA 8.5 U/ml f 2.2 U/ml and specific peptide 20.1 U/ml f 3.4 U/ml). Addition of exogenous IL-12 (50 pa/ml) significantly increased T cell activation (128 U/ml f 4.8 U/ml), whereas other cytokines such as IL-l, IL-8 and TNFa did not show any effect. Bone marrow macrophages (BM-Me) as fully competent antigen presenting cells were used as controls (no peptide 13.1 U/ml, specific peptide 82 f 2.4 U/ml IFN-g). Upon restimulation with anti-CD3 T cells prfmed by SEC produced IL-4 (43 @ml f 2.1) which could not be overcome by addition of IL-12 during priming. BM-Me primed T cells produced IFN-g upon restimulation (388 U/ml f 13.2). Conclualon: We have recently shown that SEC can act as antigen presenting cells and activate T cell clones. Now we demonstrate that SEC could prime naive T cells and that a Th2 shit was Induced in the primed T cells. Professional APC’s, e.g. BM-Me, induced a Thl type immune response in naive T cells. Our results suggest that the liver microenvironment modulates the immune response which may lead to the induction of toterance. In chronic liver inflammation (e.g. viral hepatitis) our findings could explain the presence of antibodies to the inflammatory agent and the absence of effective cell mediated immune response leading to chronicity of infection.

01 10 1The interactlon between CWOL and CD40 as a r.P 5 .-. target for lmmunosuppresslon in vitro and in vivo Anne George l, Ahmad Kasran l, Joost van den Oord 2, Mark de Boer 3, Jan L. Ceuppens I. l Laboratory of Experimental Immunology, Catholic University of Leuven, Belgium, 2Laboratory of HistoQtochemistr)5 Catholic University of Leoven, Belgium, 3PanGenetics NY Amsterdam, The Netherlands Introduction: CD40 is a 45-50 kDa glycoprotein, a member of the TNF superfamily, and expressed on B cells, monocytes, dendrftic cells and thymic epithelial ceils. The ligand for CD40 is CD4OL, which is rapidly induced on T cells after TCR triggering. CD40CD4OL interaction is required for activation of B cells and for induction of APC functions. We have studied the effect of blocking CD4OCD4OL interaction on T cell activation in vitro and in vivo. Materlals and Methods: Purified human T cells are stimulated with atlogeneic monocytes in the presence or absence of anti-CD40 mAb, and then restimulated with the allogeneic monocytes to study the induction of energy or hyporesponsiveness. For in vivo effect, the EAE model was used with SJUJ female mice, sensitised with PLPrsg+r and then treated with a gelonine-coupled antibody to CD40L. Brain sections obtained during the acute phase of EAE or after recovery are stained for 87-l and 87-2. Similarly, splenocytes isolated from these mice are tested for their responsiveness upon in vitro restimulation with PLPI~~,~, . Results: (1) In cultures of T cells with aflogeneic monocytes. the addition of a blocking anti-CD40 mAb partly inhibits allogeneic T cell activation, and moreover leads to hyporesponsiveness as evidenced upon restimulation of these primed T cells. (2) The in vivo studies are going on, and results on clinical evolution, brain inflammation, and residual sensifsation will be shown. Conclusion: Blocking of the CD4OCD40L interaction has strong immunosuppressive properties. The effect of antiCD40L mAb in vivo in an animal model of autoimmunity will be shown.

P-5.01 .ll

Induction of immune tolerance by oral adminlstration of drug delivery system “antlgen plus immunosuppressant”

S.V. Panko, A. Lyzikov’, S.S. Panko ‘, A. Karpitzkyl, M. Sachek, V. Anytchkin ‘, S. Zhavoronok, A. Sanin *, L. Tikhonova’ Research Institute for Ridiation Medicine of Vitebsk Belarus, Russia, ’ Medicalinstitute of Vitebsk Belarus, Russia, 2Gamaley Research Institute of Epidemiology and MiMobrblogy;Moscow, Russia Introduction: Oral administration of antigens in different experimental models may induce the production of secretory antibodies andlor systemic unresponsiveness with suppression of delayed-type hypersensitivity or specific IgG and IgE antibody production or both. Matwlals and Methods: We studied the immunogenicity of thyroglobulin and tolerogenicity of liposome-entrapped soluble thyroglobulin with Cyclosporyne A (DDS-Tg + CsA) for suppression of rat experimental autoimmune thyroiditis (EAT). EAT induced by the immunization of responders in the foot pad with a mixture of rat thyroglobulin (RTg) emulsified in complete Freund’s adjuvant (control group).

Reauftm Oral administration of DDS-Tg + CsA each other day, after immunization of responders, resulted in reduced EAT severity and markedly anti-RTg antibody decrease in those animals compared with rats received a similar dose of the liposome-entrapped RTg without CsA. Table 1. Antibodies titre to thyroglobulin in different methods of induction of Immune Toferante (*P = 0.0431 to 2.5 “b”) Group charactersitic

Liposoma-entrappedTg (1 mg) with CSA (1 mg) per 05 Liposome-entrappedTg (1 mg) per OS Liposome-entrappedCSA (1 mg) per 08 Commercial CSA (1 mg) per 0s Control group (untreated)

Group Ab-Tg titre “a” No (15 days,) (7 doses)

Ab-Tg titre “b” Ab-Tg title “c” (30 days,) (45 day%) (30 doses) (15 doses)

1 2

l/l22 11185

I/ 55; l/l84

I/ 36 II 39

3

11137

l/136

II 55

4 5

11156 11115

11123 l/263

II 53 1I204

The morphology data obtained durfng two months showed that the more severe granulomatous form of EAT was also suppressed by feeding DDS-Tg + CsA but not by feeding liposome-entrapped RTg without CsA to responders rats. Conclusion: We conclude, that the results suggest that this method of Induction of Immune Tolerance by oral administration of Drug Delivery System- “immunosuppressant plus antigen” with subtherapeutic doses CsA or FK-508 may be used, in prospect, for treatment autoimmune disorders such as Hashimoto’s thyroiditis, multiple sclerosis and others, various allergy and prevention of Graftversus-host reaction and allograft rejection, if a major histocompatfbility complex allopeptides used as antigen.

P.5.01.12 n

Donor-specific hyporeactlvity in lung transplant recipients

A. de Haan’, B.F.T.van der Gun*, B.G. Hepkema3, W. van der Bij’, L.F.M.H de Leij 2, J. Prop ‘. ’ Cardiopulmonary Surgery Research Division, %epamnent of Clinical lmmunolq~ 3Labomtory of Trensp/antImmunology, 4Department of Pulmonary Diseases, Gnmingen Lung Tiansptantation Group, University Hospital Groningen, Hanzeplein 1.9700 R& Groiingen, The Netfredands Introduction: Chronic graft dysfunction (bronchiolitis obliterans: BO) remains the major complicatkm in long term survivors after lung transplantation. In some studies, the risk for chronic graft dysfunction was reduced in those patients that developed donor-specific hyporeactivity. This hyporeactfvity was determined in vitro using mixed lymphocyte cultures as a qualitative assay. In order to study the development of donor-specific hyporeactivity in a more quantitative fashion, we specifically determined precursor frequencies of donor-specific helper T lymphocytes (pHTL) and cytotoxlc T lymphocytes (pCTL) in peripheral blood lymphocytes of lung transplant recipients. Mate&Is and Method% pHTL and pCTL precursor frequencies were assessed before and one yearafter transplantation by limiting dilution assay using donor spleen cells as stimulators cells. As a control, pHTL and pCTL were determined against unrelated third party spleen cells. The studied group consisted of 13 patients: 10 patients were BO-free at time of study and during follow-up (14-25 months post-b& 3 patients were diagnosed with BO before or at one year post-transplantation. Results: In BO-free patients, pCTLfrequencies were significantly decreased at one year post-transplantation while third party-specific pCTL frequencies remained constant, indicating the development of donor-specific hyporeactivity. A similar decrease in donor-specific pHTL frequencies, however, was not noticed: both donor-spectfic and third party specific pHTL frequencies were lower at one year post-tx. In BO patients, donor-specific pHTL and pCTL frequencies as well as third party specific pHTL and pCTL frequencies were lower at one year post-transplantation, probably reflecting increased immunosuppression. Conclusion: We conclude that donor-specific hyporeactfvky is prominently reflected in decreased pCTL frequencies. It remains to be established whether decreased donor-specific pCTL frequencies reduces the risk for development of chronic graft dysfunction.

P.5.01 .13

The effect of a panel of cytokines on activation, tolerance induction and maintenance of human Th2 and ThO clones

A. Verhoef, JR. Lamb. Department of Biology; imperial Cottege of Science, Technology and Medicine, Prince Consort Road, London, UK Introduction: Populations of specific T cells may be rendered non-responsive (anergic or tolerant), charactertsed by their inability to respond to a subsequent antigenic challenge. In vitro, this may be achieved by exposing T cells to a supraoptimal dose of their cognate antigen in the absence of antigen presenting