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My research interests cover three areas: 1) the response of CD8 T lymphocytes to donor HLA antigens after transplantation: regulatory cells or killer cells; and in collaboration with Paul Warner, 2) identification of the elements generated from clinical laboratory assays that make up the profile of a recipient of a successful solid-organ transplant and, 3) immunomodulation following transfusion.
T lymphocytes of the lineage identified by the CD8 marker have receptors for class I MHC plus peptide. Their role in response to transplanted organs has historically been thought to be limited to mediators of acute rejection via direct cytotoxicity or interactions between receptors and proteins of the TNF family. However, the larger group of CD8 T cells includes cells with the capacity to down regulate the activity of other T cells either via direct contact, via secreted cytokines or via action on antigen presenting cells. These CD8 cells with regulatory capacity can be differentiated by the expression of the cytokine IL-10 as opposed to the expression of granzymes or other cytotoxic mediators. We have been analyzing peripheral blood samples taken prior to and following transplantation for CD8 cells that proliferate in response to re-exposure to donor HLA antigens ex vivo. The cytoplasm of proliferating cells has been stained to identify expression of IL-10, IFN-gamma or granzymes to identify cells with regulatory or cytotoxic phenotypes. Our hypothesis is that increases in cells with regulatory capacity after re-stimulation with donor tissue is a marker for a reduced risk of rejection.
The rapid translation of research techniques into clinically-applicable assays and methods is enabling us to individualize the management of patients after transplantation. The clinical immunogenetics/HLA laboratory at the Puget Sound Blood Center is a well-respected and recognized leader in this field. The assays we employ allow snapshots of the immune response to HLA and tissue–specific antigens of the transplant donor. Data from these snapshots are then assembled to make up a profile that can be correlated with real-time data on the status of the transplanted organ. Elements associated with stable function or pending rejection will in the future be used to guide selection of immunosuppressive strategies or monitor tolerance-induction protocols.
A. Identification of antibodies to HLA in solid organ transplantation.
Recent advances in microparticle-based flow cytometry assays now allow for the detection and identification of antibodies against single HLA molecules.
a. We are participating in research to identify the clinical correlates of different titers of antibodies detected by these assays: immune memory or active response. Data from these assays may form the basis for allocation of organs from deceased donors. We are partnering with other HLA laboratories in piloting this change. These new tools are not completely understood – there is ongoing research to identify artifacts and holes created by the nature of the recombinant molecules.
b. Data from these assays are also among the elements of the profile of an organ recipient. It was previously assumed that any donor-specific antibody generated after transplant was a predictor of rejection. Another focus of our studies is the hypothesis that antibodies with low titer or directed against antigens with a lower copy number may give survival signals and not mediate rejection.
B. Identification of cytokine profiles of lymphocytes responding to donor tissue.
Lymphocytes responding to transplanted organs include the CD8 T lymphocytes described above as well as CD4 T lymphocytes and NKT cells. Changes in these populations after transplant are elements of the organ recipient profile. NKT studies are performed in Dr. Warner’s laboratory.
C. Identification of peptides and tissue-specific antigens that are targets of rejection.
This is a new area of investigation that Paul Warner and I are pursuing in collaboration with Brad Stone and Chris Kuhr.
A. Impact of transfusion component processing.
Filtration of transfusion components has been advanced as a solution to post-transfusion immunomodulation. However, most of the studies on allosensitization have used immunosuppressed subjects and studies on immunosuppression have used clinical outcomes as the endpoints. Our studies are targeting cardiac surgery patients (non-immunosuppressed) in collaboration with Dr. Gabriel Aldea at the University of Washington. We are measuring antibodies to HLA with the microparticle assays described above. These assays have the advantage of identifying changes in antibodies in patients previously allosensitized. To study immunosuppression, Paul Warner is analyzing the effects of transfusion on NKT cells and Yvette Latchman is studying CD4 T regulatory cells.
B. Transfusion-Associated Acute Lung Injury (TRALI).
In collaboration with Dr. Theresa Nestor and Dr. Terry Gernsheimer, we are measuring antibodies to HLA antigens in transfusion components as will as in recipients with clinical symptoms meeting the criteria for TRALI. |
