The objectives were:
1) To find tests in vitro to predict the risk of graft rejection after transplantation organ or of graft versus host disease (GvHD) after Bone Marrow Tranplantation (BMT).
2) To study the mechanisms of action of new immunosuppressive compounds (monoclonal antibodies, and peptides) or of immunological manoevers (such as pegraft blood transfusions) aimed at decreasing allogeneic immune response. The primary approach was to use graft recipient blood cells or cells extracted directly from the graft (rejection/tolerance) or skin (GvHD) to study their characteristics and functions in vitro.
Most of the aims have been fulfiled and often extended. Productive collaborations have been set up and will continue.
1) Before grafting: new in vitro tests for typing major or minor alloantigens have been defined and their results correlated the outcom of to organ and Bone Marrow (BM) grafts. In vitro (HTLp, CTLp) and in vivo (graft outcome) effects of recipient conditioning with blood have been studied in various protocols.
2) After grafting: we have developed new semi-automatic technologies to measure cytokine transcriptions (RT-PCR) and TCR repertoire (CDR3 size in Vbeta families). These techniques and the measurement of informative soluble factors (sCD30, cytokines) have been applied to blood and graft infiltrating (or skin GvH lesion) cell samples and the results correlated to graft outcome. In vitro correlates of the effect of immunossupressive agents (anti-LFA1, anti CD2, anti CD4 and HLA derived peptides) have been described.
MAJOR SCIENTIFIC BREAKTHROUGHS:
1) TCR repertoire analysis: Vbeta assignement and RT-PCR with the 3.0 version of the Immunoscope sofware allowing an automatic analysis of CDR3 sizes within a Vbeta family have been optimized and applied to the study of the repertoire of skin infiltrating cells and peripheral cells in long lasting BM grafts. Major TCR biases have been indentified after BM and correlated to clinical outcome.
2) Study of the polymorphims of HLA molecules has been refined by various methods, including allele specific PCR amplification (268 class II alleles, 64 class I alleles, LPM, DMA alleles) and correlated to graft outcome. The role of minor and HLA-DP incompatibilities has been assessed in BM graft outcome and correlation between DMA alleles and kidney rejection (DMA-0102) or glomerulopathy (DMA-103) identified. In vivo effects of potentially immunossuppressive HLA-derived peptide have been described. We have also described the functional alterations resulting from blood transfusion matched to kidney recipient or donor HLA when administrated alone or with an anti CD4.
3) New methods of identifying functional T cell population from the cytokines production pattern of and soluble factors have been described, optimised and correlated to graft outcome: LDA of HTLp-ILbeta producers according to HLA-DRbeta1, amino acid mismatches, THI/TH2 profile (quantitative and qualitative correlates) of clones (n=702) from graft infiltrates have been described in grade I, II or bordeline rejection episodes. Increased soluble CD30 have been correlated to c GvH.
4) In vitro correlates of in vivo activity of various reagents, tested for the first time in organ recipients (anti-CD2, anti-LFA1 and HLA-derived peptide), have been described. They have provided guidelines for pharmacokinetic, optimal doses, and efficacy monitoring.
Funding SchemeCSC - Cost-sharing contracts
141 86 Huddinge
2300 RC Leiden