Mohamed R. Daha

T-Cell Receptor V-Gene Usage in T-Cell Lines Propogated from Graft-Infiltrating T Lymphocytes in Needle Biopsies of Rejecting Renal Allografts

Needle biopsies taken from renal allografts of 42 renal transplant patients were tested for in vitro propagation of graft-infiltrating T-lymphocytes (GITL). From 30 out of 42 needle biopsies T-lymphocyte cell lines could be established. There was a significant correlation between in vitro outgrowth of T cells and histological signs of graft rejection. The majority of GITL cell lines displayed cytotoxicity both against donor proximal tubular epithelial cells (PTEC) and PHA stimulated donor splenocytes in an MHC class I restricted fashion. However, six cell lines were only cytotoxic against donor PTEC which is suggestive of recognition of tissue-specific antigens by these GITL derived T-cell lines. Analysis to the level of diversity of the T-cell receptor repertoire by PCR with TCRBV-family specific-oligonucleotides of a selection of these GITL derived cell lines revealed that the majority of the T-cell lines tested were polyclonal in nature on the basis of TCRBV gene family usage. A clear dominance of TCRBV genes was observed in only three GITL derived T-cell lines. There was no apparent correlation with the diversity of the TCRBV repertoire of GITL derived T-cell lines and the number of HLA-mismatches between the recipient and donor derived graft. Furthermore, the time interval between the transplantation and biopsy sampling did not contribute to the level of diversity of the TCRBV repertoire nor the tissue-specificity of these GITL derived T-cell lines.

Involvement of Minor Histocompatability Antigens in the Rejection of an HLA Identical Renal Transplant from a Living Related Donor

Graft-infiltrating T-lymphocytes (GITL) were isolated from two successive biopsies of a patient who had received an HLA-identical kidney from his brother. Both T-cell lines were highly cytotoxic against cultured proximal tubular epithelial cells (PTEC) and PHA stimulated peripheral blood lymphocytes (PBL), both of donor origin. In addition paired samples of PBL isolated at the time of the second rejection and cultured in a similar fashion as GITL displayed cytotoxicity against PTEC, although to a lesser extent. Cytotoxicity was not due to LAK activity since HLA typed target cells were specifically lysed. By using PBL from several members of the patient’s family as target it was demonstrated that cytotoxicity was related to the haplotype HLAA25, B18, CW7. Susceptibility to lysis was inherited independently from the HLA haplotypes as would be expected of minor histocompatibility antigens (mH). Using a panel of HLA typed PBL not related to the patient, a single HLA specificity could not be demonstrated. Cytotoxicity was T cell mediated and MHC class I restricted as could be shown by inhibition experiments. Adhesion between GITL and PTEC was almost completely dependent on the LFA-1/ICAM-1 adhesion pathway. The diversity of the T-cell receptor repertoire in both T-cell lines was reduced on the basis of usage of certain TCRBV gene families in comparison to paired control PBL. However, the T-lymphocyte repertoire of GITL propagated from the second biopsy was more extensive when compared to the repertoire of GITL propagated from the first biopsy. These results demonstrate that mH antigens could play a critical role in renal allograft rejection in HLA-identical siblings and that they are expressed on PTEC. Furthermore the T-cell response against the renal allograft seems to be mediated initially by T cells with a restricted TCRBV gene family usage. As the rejection process progresses, the diversity of the T lymphocytes which accumulate into the renal allograft increases.