흉선 T 세포 발달과 증식에서 CD36의 역할 연구

Abstract

Background: CD36, also known as fatty acid translocase and scavenger receptor, is a transmembrane molecule which functions as signaling molecule and lipid transporter. CD36 binds oxidized low-density lipoprotein (ox-LDL), long chain fatty acid, damage-associated molecular pattern (DAMP), and pathogen-associated molecular pattern (PAMP). CD36 is expressed not only in fat cells, muscle cells, and endothelial cells, but also in immune cells such as platelets, red blood cells, macrophages, dendritic cells, and T cells. CD36 expressed in immune cells is known to play a role in immune metabolism, but its function in T cell development has been less elucidated.

Methods: CD36-floxed mice and Lck-cre mice were crossed to generate T-cell-specific CD36 knock-out (KO) mice, which were named CD36 conditional KO (cKO) mice. The thymus, spleen, and bone marrow cells of 3-4 week-old CD36 cKO mice and thymocytes of 5-8 week- old CD36 cKO mice were analyzed by flow cytometry. T cell proliferation was assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution and cell counting kit 8 (CCK8) assay in the presence of IL-2, anti-CD3, and anti-CD28 monoclonal antibodies. To investigate the mechanism underlying increased T cell proliferation, the activation of AKT, mTOR, and ERK was observed by flow cytometry. RNA was extracted from thymus and bone marrow cells and mRNA sequencing was performed.

Results: Three- to four-week-old CD36 cKO mice displayed increases in CD3+, double negative (DN), DN3, CD3+ double positive (DP) and CD8 single positive (SP) cells and reduction in DN4, total DP, CD3- DP and CD4 SP cells in the thymus. CD36 cKO splenocytes had lower frequency of CD4 SP cells, compared with WT. The frequency and mean fluorescence index (MFI) of CD3+ bone marrow cells from CD36 cKO mice were lower than those of WT. Thymic T cells from CD36 cKO mice were more proliferative than those of WT upon CD3 and CD28 stimulation. mRNA-sequencing data showed that the expressions of Ccl17 and Ccr7 were upregulated, and those of Ccl22 and Ccr9 were downregulated in CD36 cKO thymus. It was confirmed by quantitative real-time PCR (qRT-PCR) that the expressions of Ccl22 and Ccr9 were significantly decreased in CD36 cKO thymocytes, compared with those of WT. Ccl17 and Ccr7 expressions were higher and Ccl22 and Ccr9 expressions were lower in the CD36 cKO bone marrow than in WT, although the differences were not statistically significant.

Conclusion: CD36 cKO 3-4 week-old mice had defects in DN3 to DN4 transition. CD36 cKO thymic T cells were more proliferative than WT. The developmental changes of thymic T cells might result from the changes of the expression of chemokines and chemokine receptors. This study suggests that CD36 is a potential contributor to thymic T cell development from DN stage and has a regulatory function in proliferation.