Donor-derived leukocyte antigen class I proteins in the serum of heart transplant recipients
ABSTRACT Human leukocyte antigen class I proteins are expressed on most cell types in all organ allografts but are constitutively secreted only by certain organs, for example, the liver. We hypothesized that detectable levels of donor-derived human leukocyte antigen proteins would be released from transplanted cardiac allografts only when the allograft was immunologically stimulated, that is, during rejection and perhaps during viral infection. If so, then the release of donor human leukocyte antigen might be a noninvasive monitor of these events.
We used an enzyme-linked immunosorbent assay to detect donor-derived human leukocyte antigen-A2 in the serum of 21 human leukocyte antigen-A2 negative recipients of human leukocyte antigen-A2-positive heart transplants. The level of donor human leukocyte antigen-A2 during the first 100 days after transplantation was correlated with the clinical status of the patient.
We found little or no donor human leukocyte antigen in the serum of heart transplant recipients whose postoperative clinical course was unremarkable for infection or rejection. We did find donor-derived human leukocyte antigen in the serum of heart transplant recipients transiently in the week immediately after transplantation, continuously from patients in whom chronic rejection was developing, during cytomegalovirus infection, and during some, but not all, acute rejection episodes as determined by endomyocardial biopsy.
These findings are consistent with the hypothesis that the donor human leukocyte antigen serum level reflects vascular diseases, rather than myocardial disease in the transplanted heart. Therefore, the serum level of donor human leukocyte antigen cannot be used as a monitor of cellular infiltration and myocyte damage as currently assessed by endomyocardial biopsy but may be an early indicator of the development of vascular disease such as chronic rejection.
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ABSTRACT: The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in beta 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of beta 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 177 micrograms/liter of f beta 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human beta 2m produced in fungi (f beta 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified f beta 2m from culture supernatants appeared biochemically similar to beta 2m obtained from human urine (u beta 2m) as seen in immunoblot analysis. Functionally, f beta 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELISA for detecting properly folded HLA class I heavy chain and in assays showing cell-surface beta 2m exchange into the mouse class I MHC H-2Kd. In these experiments the biological activity of f beta 2m was indistinguishable from u beta 2m. The successful expression of biologically active beta 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.Human Immunology 01/1997; 51(2):63-72. DOI:10.1016/S0198-8859(96)00224-8 · 2.28 Impact Factor
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ABSTRACT: Increased levels of both donor and recipient derived HLA molecules can be found in serum and plasma of transplanted patients during rejection. Recent data suggest that levels of donor specific soluble HLA Class I (sHLA-1) correlate better with graft rejection than total sHLA Class I [1, 2]. Therefore, quantification of donor specific soluble counterparts of HLA Class I in the serum of the recipient may be a new way for non-invasive monitoring of rejection after organ transplantation. Up to now, only a limited number of mouse monoclonal antibodies (alpha HLA-A2, and alpha HLA-B7) has been used in enzyme linked immunosorbent assays (ELISAs) to detect donor specific HLA molecules in the plasma of transplant recipients. To monitor other donor-recipient combinations, we tested some of our HLA Class I specific human monoclonal antibodies, routinely used in complement dependent cytotoxicity, for their suitability in ELISA based assays. In the present model system, we used alpha HLA-A9 (BvK5C4) or alpha HLA-A3 (OK2F3) hybridoma-supernatant to set up a sHLA-A9 and sHLA-A3 specific ELISA. In a pilot study we show that these assays were sensitive enough to detect an increase of donor specific sHLA-I during rejection in the plasma of two heart transplant recipients. Use of a large set of human hybridoma's will enable monitoring most recipient/donor combinations in the near future.Human Immunology 02/1998; 59(2):106-14. DOI:10.1016/S0198-8859(97)00253-X · 2.28 Impact Factor
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ABSTRACT: We and others have found donor-derived soluble beta2m-associated HLA class I proteins (sHLA/beta2m) in the serum of allograft recipients with acute and chronic rejection. Whether appearance of sHLA/beta2m and upregulated expression of donor cell-bound HLA/beta2m during allograft rejection are related events is unknown. Activation-induced upregulation of in vitro HLA/beta2m expression correlates with the surface expression of another form of HLA class I, namely beta2m-free HLA heavy chains (beta2m-free HC). We have shown that beta2m-free HC, but not beta2m-associated HC, are then cleaved by a specific membrane-bound metalloproteinase and released into supernatants as soluble 36 kDa proteins. We show now that activated peripheral blood lymphocytes produce predominantly the 36 kDa form of sHLA proteins which is present in supernatants as both beta2m-free HC and sHLA/beta2m. Importantly, the metalloprotease inhibitor BB-94 blocked not only the release of soluble beta2m-free HC, but also the appearance of sHLA/beta2m in cell supernatants. Low levels of 36 kDa beta2m-free HC were also present in human plasma of healthy donors. These data suggest an important role for the HLA class I-specific metalloproteinase in vivo in healthy individuals and during allograft rejection in the generation of soluble beta2m-free and beta2m-associated HLA proteins.Human Immunology 08/1998; 59(7):426-34. DOI:10.1016/S0198-8859(98)00032-9 · 2.28 Impact Factor