2014 Fiscal Year Research-status Report
胸腺ダブルネガティブ前駆細胞からのＮＫＴ細胞新規分化経路の解明
| Project/Area Number |
24790490
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| Research Institution | The Institute of Physical and Chemical Research |
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Principal Investigator |
D NYAMBAYAR 独立行政法人理化学研究所, 統合生命医科学研究センター, 研究員 (50443057)
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| Project Period (FY) |
2012-04-01 – 2016-03-31
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| Keywords | NKT / CD1d / thymus / development / precursor |
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| Outline of Annual Research Achievements |
The current view of the Vα14 invariant natural killer T (NKT) cell development holds that NKT precursors share common intermediates with conventional αβT cells and diverge at the thymic CD4+CD8+ double-positive (DP) stage, where the polymorphic major histocompatibility complex (MHC) molecule selects conventional T cells, while monomorphic CD1d selects NKT cells. This model also postulates “linear differentiation” of NKT cells, where the initial CD1d-selected cells mature through sequential stages, CD44lo NK1.1- stage-1 (st1) NKT cells with T helper (Th)2-biased features to CD44hi NK1.1- stage-2 (st2) NKT cells with Th2 and Th17-biased features, with the final differentiation into the terminally matured CD44hi NK1.1+ stage-3 (st3) NKT cells with a Th1-biased signature.
Here we found an alternative developmental pathway of NKT cells from thymic CD4-CD8- double-negative (DN) stage that is before transition to the DP stage. This DN pathway preferentially generates Th1-type NKT cells, raising possibility that the NKT cell developmental stages are likely to represent distinct lineages and not simply maturation stages. Furthermore, our findings suggest a possible role of the developmental pathway in determining specific differentiation programs underlying the development of diverse functional NKT cell subsets.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
It is still not fully understood whether NKT cells develop exclusively by DP pathway in a manner closely resembling that of conventional T cells, or some alternatives to this pathway exist. It is widely believed that invariant NKT cells are generated from CD4+CD8+ double-positive (DP) thymocytes by selection on CD1d, a process termed the "mainstream" or DP pathway, where it is widely accepted that the RAG-1 and RAG-2 mediated Vα14Jα18 rearrangements are initiated exclusively on DP thymocyte precursors.
Here, we provide genetic evidence of the presence of an alternative NKT cell developmental pathway from DN thymocytes using several approaches. The fate mapping experiment with ROSA26-YFP reporter mouse crossed with the DP stage-specific Cre transgenic mouse revealed the presence of NKT cells that were not developed through the DP pathway. To further substantiate our findings, we have generated another mouse model, where the expression of RAG was restricted to the DN stage by conditional deletion of Rag2 at the DP stage. Again, our data revealed the presence of Vα14Jα18 rearrangements on DN4 stage cells but not on DP cells, indicating that the residual NKT cells developed in this mouse were generated from the DN pathway. These DN pathway derived NKT cells show predominantly Th1-type responses that are different from those generated by the DP pathway.
Our present findings provide new insights in understanding the developmental pathway of Th1-type NKT cells, which might be of use for future studies aimed to manipulate NKT cells for human benefits.
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| Strategy for Future Research Activity |
The following experiments would be aimed to further substantiate our findings and characterize NKT cells derived by the DN pathway:
1) Functional analyses of DN-pathway originated NKT cells both in vivo and in vitro.
2) Total transcriptome analyses of DN-pathway derived NKT cells using next generation sequencing technology.
3) Analysis of CD1d1, CD1d2-double-deficient mice for the analysis of DN precursor cells and their characteristics at the single-cell level using in-house developed multiplex single-cell RT-qPCR method.
4) Analysis of pTα-deficient mice aimed to test the requirement for a beta-selection process on the development of DN precursors of NKT cells.
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