Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells.


Tanimine N(1), Burrell BE(2), Deng K(1), Rickert C(1), Lee KM(1), Feeney N(1), Pardo J(2), LeGuern C(1), Markmann JF(3).
Author information:
(1)Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
(2)Immune Tolerance Network, Bethesda, MD, USA.
(3)Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA. Electronic address: [Email]


Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.