Mixed Lymphocyte Reaction in a Standardized Flow Cytometry Panel

Abstract

Introduction: Donor-specific antibody (DSA) and antibody (Ab)-mediated rejection are a challenge for long-term outcomes accelerating graft vasculopathy and limiting options for re-transplantation. Sensitizing events such as homograft use in surgical procedures, assist device implantation, and transfusions may lead to sensitization and development of HLA-Ab. Thymectomy is routinely performed during pediatric heart surgery; our overarching goal is to study the impact of thymectomy and its role in de novo DSA. In order to study donor-recipient allorecognition, we evaluated the mixed lymphocyte reaction (MLR) for alloreactive T cell proliferation combined with a standardized flow cytometry lymphocyte phenotyping panel (Duraclone IM, Beckman Coulter) widely used in the Canadian National Transplant Research Program and the ONE Study, providing standardized data across sites. Methods: Pre- and post-transplant de novo DSA data from our clinical laboratory were analysed (n = 117; data not shown). MLR and flow phenotyping were performed using adult and pediatric control peripheral blood mononuclear cells and irradiated pooled HLA-mismatched third-party splenocytes. Cell proliferation was visualized using CellTrace Violet dye combined with the flow panel (Duraclone Immunophenotyping panel) or BrdU incorporation ELISA (n = 12). Results: The BrdU assay demonstrated proliferation in response to non-self HLA but lacked the ability to identify which cell populations proliferated. Proliferation dye was readily detected within the standardized panel. Unstimulated cells did not proliferate whereas mitogen stimulated cells showed strong proliferation. Phenotypic changes comparing pre- to post-MLR analysis included decreased %T cells (although %CD4 and %CD8 remained consistent), increased %B cells, increased %NK cells, decreased % NKT cells and monocyte disappearance. Small sample size (0.2 × 106 cells) was sufficient for phenotyping thus enabling use in a pediatric population. Conclusion: Our preliminary results show this flow panel, in combination with CellTrace proliferation dye, is a novel way to update detection of cell proliferation in an MLR assay; the standardized assay can allow detection of alloimmune responses in individual patients in a reproducible manner from patient to patient and centre to centre, and provide an opportunity to standardize clinical investigation of immune responses.