Endothelial cells as targets of allograft rejection.

Endothelial cells harbor many antigenic determinants that may be targets for antibodies and, as such, induce acute or chronic antibody-mediated rejections. In certain cases of organ transplantation, antibodies reacting with endothelial cells, but not with ABO or human leukocyte antigens, can be observed and these are probably responsible for the rejection of the graft. The antigenic targets, however, are still poorly defined and the mechanisms of action of such antibodies would appear to be diverse, leading to the lack of a relevant in vitro assay for the detection of those antibodies. Increasing data suggest that, apart from direct alloimmune responses, autoimmune mechanisms might be triggered by alloreactivity and also play a significant role in the pathogenesis of the vascular lesions, the so-called chronic rejection, observed in organ allografts.

Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes.

Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.

Intravenous immunoglobulins and transplantation for patients with anti-HLA antibodies.

Transplantation for patients possessing allo-antibodies against HLA antigens can be delayed for years, and, once the graft has been transplanted, its survival is significantly reduced. We and others have shown that administration of intravenous immunoglobulins (IVIgs) can induce a profound and sustained decrease in the titres of the anti-HLA antibodies, thus greatly enhancing the chances of those patients to obtain a transplant. In a number of cases, pre-treatment sera contained anti-donor antibodies that disappeared after IVIg administration. A similar approach, combining plasmapheresis and low-dose IVIgs, has shown similar results and has been successfully applied to ABO-incompatible transplantations. Patient and graft survival are excellent, despite a rather high rate of rejections, most notably humoral ones. These protocols thus demonstrate that the presence of anti-donor antibody, once an absolute contra-indication to transplantation, can, nowadays, be considered as an immunological hurdle that can be managed through appropriate immunological manipulation.