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The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor.

Yu X, Wang Y, Deng M, Li Y, Ruhn KA, Zhang CC, Hooper LV - Elife (2014)

Bottom Line: Clonal differentiation studies revealed that CXCR6(+) cells within the αLP population differentiate into all ILC lineages but not T- and B-cells.We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX.These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Innate lymphoid cells (ILCs) are recently identified lymphocytes that limit infection and promote tissue repair at mucosal surfaces. However, the pathways underlying ILC development remain unclear. Here we show that the transcription factor NFIL3 directs the development of a committed bone marrow precursor that differentiates into all known ILC lineages. NFIL3 was required in the common lymphoid progenitor (CLP), and was essential for the differentiation of αLP, a bone marrow cell population that gives rise to all known ILC lineages. Clonal differentiation studies revealed that CXCR6(+) cells within the αLP population differentiate into all ILC lineages but not T- and B-cells. We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX. These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.

No MeSH data available.


Related in: MedlinePlus

Experimental design and gating strategy for the Tox rescue experiment.(A) Schematic illustrating the experimental design. Nfil3−/− LSK (CD45.2+) cells were retrovirally transduced with empty (MSCV-IRES-hCD2), TOX-encoding (MSCV-Tox-IRES-hCD2) or NFIL3-encoding (MSCV-Nfil3-IRES-hCD2) vectors. Cells were then transplanted into lethally irradiated wild-type mice (CD45.1+) and ILCs were examined 4–6 weeks later. (B) Gating strategy for examining ILCs in the recipient mice. Live cells were first electronically gated as ZombieGreen-negative and cells transduced by retrovirus were identified as CD45.2+ hCD2+. ILC2, ILC3, cNK and non-NK ILC1 were gated as Lineage (CD3, CD19, CD5, TCRβ, TCRγδ)− CD127+ GATA3+, Lin− CD127+ RORγt+, Lin− NK1.1+ T-BET+ EOMES+ and Lin− NK1.1+ T-BET+ EOMES−, respectively. The results of the experiment are summarized in Figure 5F.DOI:http://dx.doi.org/10.7554/eLife.04406.014
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fig5s2: Experimental design and gating strategy for the Tox rescue experiment.(A) Schematic illustrating the experimental design. Nfil3−/− LSK (CD45.2+) cells were retrovirally transduced with empty (MSCV-IRES-hCD2), TOX-encoding (MSCV-Tox-IRES-hCD2) or NFIL3-encoding (MSCV-Nfil3-IRES-hCD2) vectors. Cells were then transplanted into lethally irradiated wild-type mice (CD45.1+) and ILCs were examined 4–6 weeks later. (B) Gating strategy for examining ILCs in the recipient mice. Live cells were first electronically gated as ZombieGreen-negative and cells transduced by retrovirus were identified as CD45.2+ hCD2+. ILC2, ILC3, cNK and non-NK ILC1 were gated as Lineage (CD3, CD19, CD5, TCRβ, TCRγδ)− CD127+ GATA3+, Lin− CD127+ RORγt+, Lin− NK1.1+ T-BET+ EOMES+ and Lin− NK1.1+ T-BET+ EOMES−, respectively. The results of the experiment are summarized in Figure 5F.DOI:http://dx.doi.org/10.7554/eLife.04406.014

Mentions: Because Tox is known to be essential for cNK and ILC3 development (Aliahmad et al., 2010), we postulated that lowered Tox expression leads to the broad ILC deficiency in Nfil3−/− mice and that restoring Tox expression would rescue ILC development. To test this idea, we cloned Tox coding sequences into a bicistronic vector (MSCV-IRES-hCD2), which allowed expression of the native form of TOX and also marked cells with the cell surface marker hCD2. We then delivered the TOX-encoding plasmid or the empty vector into purified Nfil3−/− LSK cells (CD45.2+) by retroviral transduction (Zheng et al., 2012; Spencer et al., 2014), followed by transfer of these cells into lethally irradiated wild-type mice (CD45.1+) (Figure 5—figure supplement 2). Compared to the empty vector control, transduction of the TOX-encoding plasmid led to increased numbers of cNK cells in spleen, non-NK ILC1 cells in liver, and ILC2 and ILC3 in the small intestines of recipient mice (Figure 5F). We observed that rescue of ILC development from Nfil3−/− LSK cells by Tox was largely comparable to rescue by Nfil3 in the same setting, supporting the idea that Tox acts downstream of Nfil3 in ILC development. Though ILC2 cells developing from Tox-rescued LSK cells were generally fewer than those from Nfil3-rescued LSK cells, the difference between the two groups was not statistically significant. Thus, ILC development is rescued by restoring Tox expression in Nfil3−/− progenitors, indicating that NFIL3 drives ILC development in part by regulating Tox expression.


The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor.

Yu X, Wang Y, Deng M, Li Y, Ruhn KA, Zhang CC, Hooper LV - Elife (2014)

Experimental design and gating strategy for the Tox rescue experiment.(A) Schematic illustrating the experimental design. Nfil3−/− LSK (CD45.2+) cells were retrovirally transduced with empty (MSCV-IRES-hCD2), TOX-encoding (MSCV-Tox-IRES-hCD2) or NFIL3-encoding (MSCV-Nfil3-IRES-hCD2) vectors. Cells were then transplanted into lethally irradiated wild-type mice (CD45.1+) and ILCs were examined 4–6 weeks later. (B) Gating strategy for examining ILCs in the recipient mice. Live cells were first electronically gated as ZombieGreen-negative and cells transduced by retrovirus were identified as CD45.2+ hCD2+. ILC2, ILC3, cNK and non-NK ILC1 were gated as Lineage (CD3, CD19, CD5, TCRβ, TCRγδ)− CD127+ GATA3+, Lin− CD127+ RORγt+, Lin− NK1.1+ T-BET+ EOMES+ and Lin− NK1.1+ T-BET+ EOMES−, respectively. The results of the experiment are summarized in Figure 5F.DOI:http://dx.doi.org/10.7554/eLife.04406.014
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356142&req=5

fig5s2: Experimental design and gating strategy for the Tox rescue experiment.(A) Schematic illustrating the experimental design. Nfil3−/− LSK (CD45.2+) cells were retrovirally transduced with empty (MSCV-IRES-hCD2), TOX-encoding (MSCV-Tox-IRES-hCD2) or NFIL3-encoding (MSCV-Nfil3-IRES-hCD2) vectors. Cells were then transplanted into lethally irradiated wild-type mice (CD45.1+) and ILCs were examined 4–6 weeks later. (B) Gating strategy for examining ILCs in the recipient mice. Live cells were first electronically gated as ZombieGreen-negative and cells transduced by retrovirus were identified as CD45.2+ hCD2+. ILC2, ILC3, cNK and non-NK ILC1 were gated as Lineage (CD3, CD19, CD5, TCRβ, TCRγδ)− CD127+ GATA3+, Lin− CD127+ RORγt+, Lin− NK1.1+ T-BET+ EOMES+ and Lin− NK1.1+ T-BET+ EOMES−, respectively. The results of the experiment are summarized in Figure 5F.DOI:http://dx.doi.org/10.7554/eLife.04406.014
Mentions: Because Tox is known to be essential for cNK and ILC3 development (Aliahmad et al., 2010), we postulated that lowered Tox expression leads to the broad ILC deficiency in Nfil3−/− mice and that restoring Tox expression would rescue ILC development. To test this idea, we cloned Tox coding sequences into a bicistronic vector (MSCV-IRES-hCD2), which allowed expression of the native form of TOX and also marked cells with the cell surface marker hCD2. We then delivered the TOX-encoding plasmid or the empty vector into purified Nfil3−/− LSK cells (CD45.2+) by retroviral transduction (Zheng et al., 2012; Spencer et al., 2014), followed by transfer of these cells into lethally irradiated wild-type mice (CD45.1+) (Figure 5—figure supplement 2). Compared to the empty vector control, transduction of the TOX-encoding plasmid led to increased numbers of cNK cells in spleen, non-NK ILC1 cells in liver, and ILC2 and ILC3 in the small intestines of recipient mice (Figure 5F). We observed that rescue of ILC development from Nfil3−/− LSK cells by Tox was largely comparable to rescue by Nfil3 in the same setting, supporting the idea that Tox acts downstream of Nfil3 in ILC development. Though ILC2 cells developing from Tox-rescued LSK cells were generally fewer than those from Nfil3-rescued LSK cells, the difference between the two groups was not statistically significant. Thus, ILC development is rescued by restoring Tox expression in Nfil3−/− progenitors, indicating that NFIL3 drives ILC development in part by regulating Tox expression.

Bottom Line: Clonal differentiation studies revealed that CXCR6(+) cells within the αLP population differentiate into all ILC lineages but not T- and B-cells.We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX.These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Innate lymphoid cells (ILCs) are recently identified lymphocytes that limit infection and promote tissue repair at mucosal surfaces. However, the pathways underlying ILC development remain unclear. Here we show that the transcription factor NFIL3 directs the development of a committed bone marrow precursor that differentiates into all known ILC lineages. NFIL3 was required in the common lymphoid progenitor (CLP), and was essential for the differentiation of αLP, a bone marrow cell population that gives rise to all known ILC lineages. Clonal differentiation studies revealed that CXCR6(+) cells within the αLP population differentiate into all ILC lineages but not T- and B-cells. We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX. These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.

No MeSH data available.


Related in: MedlinePlus