The Development And Differentiation Of Spleen CD49b~-NK Cells

NK cells are a group of innate immune cells with cytotoxic ability and play an important role in anti-infective and anti-tumor immunity.For a long period of time,CD49b-NK1.1+NKp46+cells were regarded as immature NK(iNK)cells,while CD49b+NK1.1+NKp46+cells were considered as mature NK cells.However,with the deepening of research,the concept of NK cell heterogeneity is widely accepted.It has been found that,in addition to the conventional NK(cNK)cells recirculating in the peripheral organs,tissue-specific NK cells exist in various tissues,and these NK cells usually have the tissue-resident property and often exhibit a CD49b-phenotype.Liver CD49b-NK cells,once considered as iNK cells,have been found to be a CD49a+CD49b" phenotypically stable cell population with liver-resident character.Bone marrow CD49b-NK cells were also found to develop primarily into liver-resident NK cells but not cNK cells.There has been increased emphasis on the differences between cNK cells and tissue-resident NK cells.Whether the development stage of iNK cells exist,and how to phenotypically define iNK cells,have become a confusing question.To address this question,we conducted the following research.1.Spleen,but not bone marrow,CD49b-NK cells have diverse developmental potentials in peripheral tissuesWe first sorted bone marrow CD49b-NK cells and adoptive transferred them into sublethally-irradiated WT mice.We found that these cells were difficult to survive in WT recipient mice.Next,we sorted the spleen CD49b-NK cells for similar experiments and found that these cells could generate different NK cell subsets in various tissues,including the cNK and liver-resident NK cells.2.Spleen CD49b-NK cells are divided into two subsets:CD49a+and CD49a-cellsThrough phenotypic analysis,we found that spleen CD49b-NK cells could be divided into two subsets:CD49a+and CD49a-cells.We named CD49a+CD49b-NK cells as 49A NK cells and CD49a-CD49b-NK cells as DN NK cells.We found that spleen 49A NK cells had a unique phenotype and transcription factor expression:CD127+CD27+CXCR3+T-bet+Eomes-.The spleen DN NK cells showed an immature phenotype.They expressed T-bet but partially expressed Eomes.In addition,the DN NK cells did not express KLRG1,a marker of NK cell maturation.These cells were mainly at the immature stage:CD11b-CD27+stage.By comparing various tissues,we found that only the spleen had a large number of DN NK cells.3.Spleen 49A NK cells tend to develop into liver-resident NK cellsThe spleen 49A NK cells migrated mainly to the liver after adoptive transfer,giving rise to CD49a+CD49b-liver-resident NK cells.They down-regulated the expression of CD127 and CD27,but kept CXCR3 and T-bet positive and Eomes negative,consistent with liver-resident NK cells.In addition,a small amount of spleen 49A NK cells returned to the spleen after transfer,maintaining their original phenotype,but it was difficult to detect the donor cells in bone marrow or peripheral blood.Additionally,although spleen 49A NK cells were CXCR3 positive,CXCR3 deficiency did not affect their migration to the liver and generation of liver-resident NK cells.4.Spleen DN NK cells have diverse developmental potentialsAfter adoptive transferred into recepient mice,the spleen DN NK cells could generate different NK cell subsets in each tissue.They could migrate to the liver,up-regulating the expression of Trail,CD200R and CXCR6 and developing into CD49a+CD49b-liver resident NK cells.CXCR3 deficiency had no impact on this process.They could also return to the spleen and up-regulate the expression of CD49b,CD62L,CD11b,KLRG1 and Eomes,developing into mature cNK cells.In addition,some cells that returned to the spleen maintained the phenotype of DN NK cells.5.The spleen has no critical effect on the maintenance of liver-resident NK cellsAfter surgical removal of the mouse spleen,there was no change in the proportion,number and phenotype of NK cells in the liver.By performing parabiosis experiments between the T-bet-deficient mice and WT mice,we found that the T-bet-deficient mice could obtain peripheral-derived liver-resident NK cells in this way.However,splenectomy of WT mice in the parabiosis experiment still did not affect the peripheral source of liver-resident NK cells of T-bet deficient mice.6.Spleen DN NK and 49A NK cells are spleen-resident.Through parabiosis experiment,we found that spleen DN NK and 49A NK cells had few peripheral sources and were spleen-resident.7.T-bet intrinsically affects the development of DN NK cells into mature cNK cellsWe found that the number and proportion of cNK cells in the spleen of T-bet-deficient mice significantly reduced,and the proportion of terminal maturation also reduced.In contrast,the number and proportion of DN NK cells in the spleen significantly increased.By co-transferring the T-bet deficient and WT spleen DN NK cells at 1:1 ratio,we found that T-bet deficiency impeded the development of DN NK cells into mature cNK cells.Conclusion:We discovered two new spleen NK cell subsets:49A NK(CD49a+CD49b-)and DN NK(CD49a CD49b-)cells.The spleen 49A NK cells tended to develop into liver-resident NK cells after adoptive transfer.The transferred DN NK cells had diverse developmental potentials.They could migrate to the liver,developing into liver-resident NK cells,and return to the spleen,developping into mature cNK cells or maintaining DN NK cell phenotype.However,through parabioisis experiments,we found that these two NK cell subsets were spleen-resident.This indicates that,under normal physiological conditions,spleen DN NK cells can only reside in the spleen and develop into mature cNK cells.Therefore,spleen DN NK cells can be considered as iNK cells in peripheral tissues.In addition,CXCR3 deficiency did not affect the the development of the two subsets into liver-redident NK cells after adoptive transfer.On the other hand,T-bet deficiency intrinsically impeded the development of DN NK cells into mature cNK cells.