64 Abstracts

VI-85 Donor Specific Cytotoxic T Lymphocytes

In Allografted Human Heart

H.Z. Hu, N. de Jong, M. Robertus, S. Plomp, F. C. van Reijsen, M.G.J. Tilanus, F. Gmelig- Meyling, H-J. Schuurman, & R.A. de Weger

Department of Pathology, Internal Medicine, Molecular Genetics, Clinical Immunology & Dermatology, University Hospital, Utrecht, The Netherlands

Rejection of transplanted allografts results from T- cell recognltion of histocompatibity antigens expressed by the cells of donor graft. The rejection responses are initiated by activation of lymphokine secreting T helper (Th) cells and are mediated by the cellular destruction inflicted by lymphokine dependent cytolytic T (Tc) cells. Previous studies of T cell repertoire in the human grafts of end stage rejection demonstrated heterogeneous population, and this was interpreted as nonspecific recruitment. In this study, we analyzed the early enterlng T ly~phocytes in biopsies from 4 allo- grafted human hearts by the specifity, function and phenotypes. Biopsles were taken once a week in the first month and once in two weeks in the second and third month, and T cells were propagated in culture medium containing interleukin-2 and interleukin-4. In 3 cases, the donor specific cytotoxicity of the T cells was nicely correlated with rejection reactions. T cells from the fourth case were cytotoxic, but no clinical rejection occured. T cells from one of the bulks were cloned. Among the 96 clones grown, 66 clones were cytotoxic to the donor ly~phoblastoid cells; 22 out of the 66 clones were specific To, as they were phenotypi- cally CD8 positive, and did not show any cytotoxicity to a third-party spleen cells. Furthermore, bulk T cells and T cell clones were analyzed by T cell recep- tor V6 genes at mRNA level, and limited TCR VB gone usage was demonstrated. In conclusion, infitrating T cells in allografted human heart at the early stage contain a high density of cytotoxic T cells. These T cells are heterogenous and responsible for graft rejec- tion.

VI-87 Dave Roelen, Simone van Bree, Urs Schanz. EIs van Bcelen. Jon van Rood, Frans Claas Dept. of lmmunohaematology and Blood Bank University Hospital Leiden Rijnsburgerweg 10. 2333AA Leiden

QUANTITATIVE AND QUALITATIVE DIFFERENCES BETWEEN CTLP AGAINST HLA-A ANTIGENS AND CTLP AGAINST HLA-B ANTIGENS In healthy individuals, like in highly sensitized patients, cytotoxic T cell precursors (CTLp) frequencies against HLA-A antigens (mean 67 CTLp/10E6 PBL) were found to be significantly lower than CTLp frequencies against HL.A-B antigens (mean 126 CTLp/,0E6 PBL)(p=0.0033). Limiting dilution assays performed in the absence or presence of antibodies against CD8 showed that CTLp directed against HLA-B were significantly less inhibited by anti-CD8 (mean inhibition 61%) compared to CTLp against HLA-A (84 % mean inhibition)(p=0.0011). The low anti-CD8 inhibition of the CTLp directed against HLA- B antigens, which is very hcterogcnous, may be explsined by a higher affinity of the CTLp for HLA-B antigens on the target cells. If this high-affinity is due to previous priming, then differences between CTLp directed against HLA-A and HLA-B should also be retie*ted in inhibition-studies with Cyclosporine A (CsA). Preliminary studies seem to confirm this. A clinical relcvance of these findings is suggested by the observation that matching for HLA-B antigens is of greater importance for kidney graft survival than matching for HLA-A.

VI-86 Gert Datema', Len M.B. Vaessen', C. Rene Daane', Carla C. Bean',

Willem Weimar', Frans H.J. Class" and Peter J. van den Elsen"

"Department of Immunohaematology and Bloodbank, University

Hospital Leiden, Building i, E3-Q, P.O. Box 9600, 300 RC, Leiden,

The Netherlands

'Department of Internal Medicine, University Hospital Dykzigt-

Rotterdam, Building 299, P.O. Box 1738, 3000 DR, Rotterdam,

The Netherlands

Functional and Molecular Characterization of Graft-lnfiltrating

T-LyJphocytes Following Cardiac Transplantation

Alloresctive T-lymphocytes play an important role in graft

rejection. In the present study we have analyzed the T-cell

receptor (TCR) V~- and V~-gene se91nent usage of endomyocard

tissue infiltrating T-lymphocytes during a rejection crisis and

at the time before and after the episode. In parallel, we have

analyzed the cytolytic capacity of these graft infiltrating T-

lymphocyte cell lines that were propagated from the various

endomyocard biopsies against donor cells. Furthermore, T-cell

clones were generated from the rejection biopsy and analyzed for

their cytotolytic Capacity and nucleotide composition of the TCR

e and ~ chain. The results of these studies revealed a restricted

usage of both Vo- and Va-gene se~ents and a shsred umage of

defined TCR V-genes by these biopsy derived T-cells taken at

different time points following transplantation. A strong

cytolytic activity against donor cells by T-cells derived from

the rejection biopsy was observed. T-cells that were responsible

for this activity, as detected by T-cell cloning and TCR-gene

analysis could not been identified in earlier biopsies indicating

that these cytolytic cells were recently recruted towards the

endomyocard.

VI-88 TCR a/[~ VARIABILITY IN PERYPHERAL BLOOD LYMPHOCYTES AND INTESTINAL MUCOSA OF COELIAC AND NON-COELIAC PATIENTS

~ , Nicola Migone, Gennaro De Libere*, Giuseppina Oderda#, Nicoletta Ansaldi# and Ginlia Casorati Dipartimento di Genetica, Biologia e Chimica Medica, Universit~ di Torino e Centro CNR Immunegenetica e Istocompatibilit~, Torino; *Dept. of Research, Kantonsepital, Basel; #Gastroenterologia Pediatrica, Universit~ di Torino

Coeliac disease is a disorder characterized by malabsorption, abnormal small-bowel epithelial structure, and intolerance to gluten. Jejunal biopsy specimens from patients show characteristic lesion with blunting and flattening of the mucesal surface and infiltration of inflammatory cells in the epithelium layer.

In our study we analyzed the V~ repertoire of the T lymphocytes obtained from jejunal biopsy and perypheral blood of coellac and non-coeliac patients. Since only few human V-specific antibody are available, the study of the cd~ T lymphocytes repertoire has been so far carried out using the Polymerase Chain Reaction (PCR) with V specific oligonucleotides. This approach, however, raises some problems, since the precise quantification of the PCR products is difficult to assess due to the variable amplification efficiency of each oligonucleotide and the entire V~ repertoire is not known.

In order to bypass these problems we used the anchor-PCR (APCR) to amplify all the expressed V~ transcripts of the lymphocytes present in the perypheral blood and jejunal biopsy. The APCR products are cloned in Bluescripts plasmids and introduced into bacteria cell. The identification of the expressed V region is performed through colony ibridization on replica filters, using V specific oligonucleotides as probes.

The identification of the expansion of particular V~ region in pathologic tissues, might reflect a clonal selection mediated by an antigen involved in the etiology of the disease. Our preliminary data obtained from intestinal and blood lymphocytes cultured in vitro show differencies in the V~ repertoire; however this phenomenon does not seem to be related to the disease status. Further studies of the predominant V~ populations using the heteroduplex analysis of the PCR products obtained using V~ specific oligonucleotides, showed an oligoclonai expanmon in cultured samples compared to the freshly isolated intestinal and perypheral lymphocytes, which show a policlonal pattern. We are currently studying the APCR products of TCR VB from fresh jejunal biopsy specimens.