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

Abstract

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.

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Acknowledgments

We would like to thank Dr. Kiyoshi Sakata, Dr. Ichiro Hirahara, and Dr. Hideki Satou, Terumo Corporation R&D Center (Japan), for supporting funding and sampling for this work.

Funding

This work was supported by the Sasakawa Scientific Research Grant from The Japan Science Society (2018-4052) and a grant of International joint research and training of young researchers for zoonosis control in the globalized world (S1491007) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Japan.

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Correspondence to Jiro Miyamae.

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Supplementary Fig. 1

Each primer location for the determination of (A) DLA-DRB1, (B) -DQA1, and (C) -DQB1 cDNA allele sequences. Each primer location is highlighted with an underline and the primer name is described below the line. Each coding sequence is highlighted with a green background. The information on each DLA class II sequence shown in the figure is as follows: DLA-DRB1 (Accession No. M29611), DLA-DQA1 (Accession No. NM_001011726), and DLA-DQB1 (Accession No. AH006318). SP: Signal peptide, TM: Transmembrane region, CY: Cytoplasmic region (DOCX 147 kb)

Supplementary Fig. 2

Inheritance of DLA haplotypes based on cDNA analysis in four beagle families. A total of 38 beagles from four families were used for obtaining haplotype information including five DLA loci. Colored bars show the DLA haplotypes that are indicated in the table below the four pedigree charts (PPTX 108 kb)

Supplementary Fig. 3

Method for quantifying alloreactive T cell proliferation in CytoTell-based MLR assay. After finishing MLR culture, a CytoTell fluorescent histogram of CD3+, CD3+CD4+, or CD3+CD8+ T cell populations was created to represent the history of T cell proliferation upon reacting to allo-antigens. Each gate on the CytoTell fluorescent histogram was constructed in accordance with the CytoTell fluorescent intensity decreasing by half from the highest intensity peak. Each cell division round was labeled based on each gate from 0 to n. The number of daughter T cells (Nc) was obtained from the flow cytometer. The number of precursor cells (Npc) was calculated by dividing the number of daughter cells in each gate by 2n. Total number of proliferation events was obtained by subtracting the total of precursor T cells from the total of daughter T cells. The proliferation indices (PI) were calculated by dividing the total number of precursors by proliferation events. Precursor frequency was calculated by dividing the total of precursor T cells that had divided once or more (sum of Npc1 to Npc8) by the total of all precursors including non-dividing ones (sum of Npc0 to Npc8) (PPTX 79 kb)

Supplementary Fig. 4

Comparisons of estimated amino acid sequences and protein structure between DLA-DRB1*001:01 and DLA-DRB1*001:02. (A) Alignment of estimated amino acid sequences in exon 2. Dots indicate matched sites with the HLA-DRB1 sequence, and a red colored background indicates the different residue between two DLA-DRB1 alleles at position 47. (B) Estimated protein structures of DLA-DRB1*001:01, DLA-DRB1*001:02, and HLA-DRB1*01:01:01. Green and purple colors indicate β-sheet and α-helix structures, respectively. Carbon skeletons at position 47 are shown in each structure. Red circle in the HLA-DRB1*01:01:01 structure represents an approximate location of peptide-binding site pocket 7 (Stern et al. 1994) (PPTX 1460 kb)

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Miyamae, J., Yagi, H., Sato, K. et al. Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs. Immunogenetics 71, 635–645 (2019). https://doi.org/10.1007/s00251-019-01147-4

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Keywords

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