Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs

Jiro Miyamae, Hayato Yagi, Keita Sato, Masaharu Okano, Kohei Nishiya, Fumihiko Katakura, Manabu Sakai, Tomohiro Nakayama, Tadaaki Moritomo, Takashi Shiina

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1–10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms.

Original languageEnglish
Pages (from-to)635-645
Number of pages11
Issue number10
Publication statusPublished - 1 Nov 2019


  • Dog
  • Dog leukocyte antigen
  • Major histocompatibility complex
  • Mixed lymphocyte reaction with flow cytometer
  • Quantification of alloreactive T cells in dogs


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