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

J Immunol Methods. 2021 Apr;491:112987. doi: 10.1016/j.jim.2021.112987. Epub 2021 Feb 6.


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.

Keywords: Alloreactive T cells; CD137; CD154; Peripheral blood mononuclear cell; Transplantation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD / analysis
  • Antigens, Differentiation, T-Lymphocyte / analysis
  • B-Lymphocytes / immunology
  • CD40 Ligand / analysis
  • Cryopreservation
  • Humans
  • Lectins, C-Type / analysis
  • Leukocytes, Mononuclear / immunology*
  • Lymphocyte Activation
  • Lymphocyte Culture Test, Mixed
  • T-Lymphocytes / immunology*
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / analysis


  • Antigens, CD
  • Antigens, Differentiation, T-Lymphocyte
  • CD69 antigen
  • Lectins, C-Type
  • TNFRSF9 protein, human
  • Tumor Necrosis Factor Receptor Superfamily, Member 9
  • CD40 Ligand