Identification of Non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.

The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.

Lymphocyte activation induces cell surface expression of an immunogenic vimentin isoform.

High titers of anti-vimentin antibodies after transplantation are known to be associated with poor long-term graft survival. Vimentin is an intracellular protein which is present in different isoforms in the cell. In a previous study with sera from hemodialysis patients on the kidney transplantation waiting list we could show that only a 49 kDa and a 60 kDa isoform are recognized by patients' anti-vimentin antibodies while the other isoforms remain undetected. However, it is still unclear whether antibodies against this intracellular protein can bind to intact cells. Here we show that vimentin can be present on the cell surface under certain conditions. Lymphocytes from healthy volunteers were used as a model for allogeneic cells. We could show by immunofluorescence microscopy, flow cytometry and Western blot experiments that concanavalin A (Con A) activated lymphocytes express a 49 kDa vimentin isoform on their cell surface while the 60 kDa isoform remains inaccessible from the outside. This expression is associated with an increased binding of sera from hemodialysis patients which were positive for anti-vimentin antibodies. These results suggest that cell activation enhances binding of anti-vimentin antibodies to intact cells which might contribute to chronic allograft nephropathy.