Mitochondria are mainly involved in ATP production to meet the energy demands of cells. Researchers are increasingly recognizing the important role of mitochondria in the differentiation and activation of hematopoietic cells, but research on how mitochondrial metabolism influence different subsets of lymphocyte at different stages of differentiation and activation are yet to be carried out. In this work, the mitochondrial functions of lymphocytes were compared at different differentiation and activation stages and included CD8+ T lymphocytes, CD4+ T lymphocytes, B lymphocytes, NK cells as well as their subsets. For this purpose, a complete set of methods was used to comprehensively analyze mitophagy levels, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the mitochondrial mass (MM) of subsets of lymphocytes. It is expected that this will provide a complete set of standards, and drawing the mitochondrial metabolic map of lymphocyte subsets at different stages of differentiation and activation. Of all lymphocytes, B cells had a relatively high mitochondrial metabolic activity which was evident from the higher levels of mitophagy, ROS, MMP and MM, and this reflected the highly heterogeneous nature of the mitochondrial metabolism in lymphocytes. Among the B cell subsets, pro-B cells had relatively higher levels of MM and MMP, while the mitochondrial metabolism level of mature B cells was relatively low. Similarly, among the subsets of CD4+ T cell, a relatively higher level of mitochondrial metabolism was noted for naive CD4+ T cells. Finally, from the CD8+ T cell subsets, CD8+ Tcm had relatively high levels of MM and MMP but relatively low ones for mitophagy, with effector T cells displaying the opposite characteristics. Meanwhile, the autophagy-related genes of lymphoid hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells and lymphocyte subsets were analyzed, which preliminarily showed that these cells were heterogeneous in the selection of mitophagy related Pink1/Park2, BNIP3/NIX and FUNDC1 pathways. The results showed that compared with CD4+ T, CD8+ T and NK cells, B cells were more similar to long-term hematopoietic stem cell (LT-HSC) and short-term hematopoietic stem cell (ST-HSC) in terms of their participation in the Pink1/Park2 pathway, as well as the degree to which the characteristics of autophagy pathway were inherited from HSC. Compared with CLP and B cells, HSC are less involved in BNIP3/NIX pathway. Among the B cell subsets, pro-B cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with pre-B, immature B and immature B cells. Among CD4+ T cell subsets, nTreg cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with naive CD4+ T and memory CD4+ T cells. Among the CD8+ T cell subsets, compared with CLP and effector CD8+ T cells, CD8+ Tcm inherit the least characteristics of HSC in participating in Pink1/Park2 pathway. Meanwhile, CLP, naive CD4+ T and effector CD8+ T were more involved in BNIP3/NIX pathway than other lymphoid hematopoietic cells. This study is expected to provide a complete set of methods and basic reference values for future studies on the mitochondrial functions of lymphocyte subsets at different stages of differentiation and activation in physiological state, and also provides a standard and reference for the study of infection and immunity based on mitochondrial metabolism.
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