| 研究概要 |
It is generally believed that natural killer T (NKT) cells expressing an invariant Vα14Jα18 receptor are generated by CD1d-selection at the CD4+CD8+ double-positive (DP) thymic stage, called "mainstream theory". Here we identified the NKT precursor in the CD4-CD8- double-negative DN fraction with the CD44+/CD24lo/CD127+ surface phenotype in the adult and fetal thymus of CD1d-/- and wild-type mice. These DN precursors reside in so called "DN1e" thymic fraction and have unique gene expression profiles that differ significantly from mature thymic NKT cells verified at the single-cell level. The major finding is that these DN precursor cells already express key Th1-like signature genes including transcription factors such as Runx3, Eomes, Tbet even before the CD1d selection event in the CD1d-deficient mouse, and preferentially develop into Th1-type NKT cells in the presence of CD1d. We found that these DN NKT precursors are derived from the DN4 stage thymocytes but not from DP thymocytes in vivo and in vitro. Furthermore, the development of these DN precursors is dependent on IL-7R, PLZF and SAP, while that of DP precursors is not. Thus, our findings reveal a novel NKT cell developmental pathway that is different from the DP pathway and is of significant importance in the field of NKT cell development in the thymus.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
Currently held view of the NKT cell developmental pathway holds that NKT precursors share common intermediates with conventional αβT cells and diverge at the thymic DP stage. This model also postulates “linear differentiation” of NKT cells, where the CD1d-selected cells mature through sequential stages, CD44lo/NK1.1- (st1) NKT cells with Th2-character to CD44hi/NK1.1- (st2) NKT cells with Th2 and Th17-biased features, with final differentiation into terminally matured CD44hi/NK1.1+ (st3) NKT cells with Th1-biased signature. However, generation of these NKT subsets cannot be adequately explained by the linear differentiation model. Here, we identify the novel type of NKT precursor cell in the thymic DN fraction (DNP) before CD1d selection that gives rise to mature NKT cells in the presence of CD1d. Strikingly, the DNP cells in the CD1d-/- mouse already have a Th1-biased gene expression profile, which results in preferential development of Th1-biased NKT cells. Also, the development of DNP cells is abrogated, while that of DP precursors is intact in IL-7R-/-, SAP-/- and PLZF-/- mice. These data strongly suggest the presence of an alternative developmental pathway, which is distinct from the DP pathway, for the Th1-type NKT cells originating from DNP cells. In summary, our data demonstrates the presence of multiple NKT cell developmental pathways with specialized precursor cells underlies the development of different functional NKT cell subsets.
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| 今後の研究の推進方策 |
The following experiments would be aimed to further substantiate our finding on the presence of DN pathway of NKT cell development:
1) Fate mapping experiment: ROSA26-YFP reporter mouse (loxP-flanked “STOP” sequence in the YFP-fluorescent reporter construct) will be used together with the DP-specific CD8III-Cre mouse in order to investigate the origin of DNP cells. If the DNP cells would have the YFP-negative phenotype, it would directly provide the genetic evidence of DNP pathway is distinct from the DP pathway. Currently, these mice are under breeding.
2) Generation of conditional Rag2 knockout mouse at the DP stage: floxed Rag2 mouse (Rag2f/f) will be used together with the DP-specific CD8III-Cre mouse. Because it is generally thought that NKT cells are the progenies of DP thymocytes, the deletion of Rag2 specifically in DP thymocytes will provide direct evidence for the presence of the independent pathway for the generation of NKT cells by the DN pathway. If the NKT cells would be present in these DP-specific Rag2-deleted mice, the further functional analyses of DN-pathway originated NKT cells would be pursued using next generation sequencing and cytokine production assay.
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