Kruppel-like factor 4 (KLF4) promotes the survival of natural killer cells and maintains the number of conventional dendritic cells in the spleen - PubMed (original) (raw)

Kruppel-like factor 4 (KLF4) promotes the survival of natural killer cells and maintains the number of conventional dendritic cells in the spleen

Chun Shik Park et al. J Leukoc Biol. 2012 May.

Abstract

The development and survival of NK cells rely on a complex, spatiotemporal gene expression pattern regulated by specific transcription factors in NK cells and tissue-specific microenvironments supported by hematopoietic cells. Here, we show that somatic deletion of the KLF4 gene, using inducible and lineage-specific cre-transgenic mice, leads to a significant reduction of NK cells (NK1.1(+) TCR-β(-)) in the blood and spleen but not in the BM, liver, or LNs. Functional and immunophenotypic analyses revealed increased apoptosis of CD27(+/-) CD11b(+) NK cells in the spleen of KLF4-deficient mice, although remaining NK cells were able to lyse tumor target cells and produce IFN-γ. A normal recovery of adoptively transferred KLF4-deficient NK cells in WT hosts suggested that the survival defect was not intrinsic of NK cells. However, BM chimeras using KLF4-deficient mice as donors indicated that reduced survival of NK cells depended on BM-derived hematopoietic cells in the spleen. The number of CD11c(hi) DCs, which are known to support NK cell survival, was reduced significantly in the spleen of KLF4-deficient mice, likely a result of a lower number of precDC progenitor cells in this tissue. Taken together, our data suggest that the pluripotency-associated gene KLF4 is required for the maintenance of DCs in the spleen and consequently, survival of differentiated NK cells in this tissue.

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Figures

Figure 1.

Figure 1.. Loss of KLF4 function leads to a reduction of NK cells in peripheral blood.

(A) KLF4 transcript levels were measured by real-time quantitative PCR in granulocytes (CD11b+ Gr1+), Mo (CD11b+ Gr1−), CD8+ T cells, CD4+ T cells, B cells (B220+), NK cells (NK1.1+ TCR-β−), and DCs (CD11chi; _n_=3). (B) Deletion of the Klf4 gene was confirmed by PCR using genomic DNA from BM cells isolated from _Klf4_fl/fl _Mx1_-Cre− (fl/fl) and _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) mice, 3 months after poly (I:C) administration, and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) and _Klf4_fl/fl _Vav_-iCre− mice (fl/fl). (C) KLF4 expression was measured by real-time quantitative PCR in purified NK cells from _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) and _Klf4_fl/fl _Mx1_-Cre− (fl/fl) mice, 3 months after gene deletion. (D) Percentages of NK cells, Mo, B cells, and T cells were measured in blood 5 months after poly (I:C) administration in _Klf4_fl/fl _Mx1_-Cre− (fl/fl) and _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) mice (_n_=12). (E) Determination of NK cells, Mo, B cells, and T cells in the blood of 3-month-old _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) and _Klf4_fl/fl _Vav_-iCre− mice (fl/fl). Each symbol represents an individual mouse (_n_=5). *P < 0.05; **P < 0.005; ***P < 0.0005 (unpaired Student's t test).

Figure 2.

Figure 2.. KLF4 regulates homeostatic maintenance of NK cells in the spleen.

(A) Flow cytometric analysis of NK1.1+ TCR-β− NK cells in the BM, iLNs, spleen, and liver of _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) and _Klf4_fl/fl _Mx1_-Cre− (fl/fl) mice. Numbers represent the percentage of cells in an outlined gate of representative dot plots. (B) Total numbers (BM, iLN, spleen) and percentages (liver) of NK cells were determined in _Klf4_fl/fl carrying the _Mx1_-Cre (Mx1) or _Vav_-iCre (Vav) transgenes (fl/fl, closed bars; Δ/Δ, open bars; _n_=8). n.d., Not determined. (C) Total numbers of T cells (TCR-β+; left panel) and B cells (B220+; right panel) in BM, iLN, and the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed bars) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open bars) mice (_n_=6). **P < 0.005 (unpaired Student's t test).

Figure 3.

Figure 3.. mNK cell subsets are reduced in blood and the spleen of KLF4-deficient mice.

(A) Flow cytometric analysis of NK cell subsets based on the expression of CD27 and CD11b in NK1.1+ TCR-β− cells: CD27+ CD11b− (Subset I), CD27+ CD11b+ (Subset II), and CD27− CD11b+ (Subset III). A representative dot plot and distribution are shown for BM, liver, blood, and the spleen of _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) and _Klf4_fl/fl _Mx1_-Cre− (fl/fl) mice (_n_=6). (B) Flow cytometric analysis of NKP (CD11b− CD122+ TCR-β− NK1.1−), iNK (CD11b− CD122+ TCR-β− NK1.1+), and mNK (CD122+ NK1.1+ TCR-β− CD11b+) cells. Total numbers of NKP, iNK, and mNK cells are shown in the BM of _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed bars) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open bars) mice (_n_=8). *P < 0.05; **P < 0.005; ***P < 0.0005 (unpaired Student's t test).

Figure 4.

Figure 4.. Increased apoptosis in KLF4-deficient mNK cells.

(A) Apoptosis of NK Subsets I–III in the spleen was determined by Annexin V staining in _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open bars) and _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed bars) mice. Representative data are shown on the left and statistical analysis on the right (_n_=6). FSC, Forward-scatter. (B) Intracellular detection of proapoptotic proteins Bim and Noxa in CD11b− and CD11b+ NK cells isolated from the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl, dotted lines) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, solid lines) mice. (C) Statistical analysis of Bim and Noxa in CD11b− and CD11b+ NK cells from the _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed circles) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open circles) mice (_n_=8). MFI, Mean fluorescence intensity. *P < 0.05; **P < 0.005; ***P < 0.0005 (unpaired Student's t test).

Figure 5.

Figure 5.. BM-derived hematopoietic cells support survival of NK cells in the spleen of KLF4-deficient mice.

(A) NK cells were purified from the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) mice (CD45.2+) and adoptively transferred into nonirradiated WT mice (B6.SJL, CD45.1+) to study NK cell dependency on impaired survival. Numbers of transferred NK cells recovered in the spleen and BM after 10 days are shown on the right. (B) To study the role of KLF4 on radio-resistant stromal cells, BM cells from WT mice (B6.SJL, CD45.1+) were transplanted into lethally irradiated _Klf4_fl/fl _Mx1_-Cre− (fl/fl) or _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) mice (both CD45.2+). The numbers of donor-derived NK cells (CD45.1+ NK1.1+ TCR-β−) were determined in blood, BM, and the spleen, 2 months after transplant (_n_=4–6). (C) BM cells from _Klf4_fl/fl _Vav_-iCre− (fl/fl) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) mice (CD45.2+) were transplanted into lethally irradiated WT mice (B6.SJL, CD45.1+) to examine the role of hematopoietic cells in NK cell survival (_n_=4–5). Three months after transplantation, the contribution of donor-derived NK cells (CD45.2+ NK1.1+ TCR-β−) was monitored in the spleen, blood, and BM by flow cytometry. *P <0.05 (unpaired Student's t test).

Figure 6.

Figure 6.. KLF4 regulates the pool of cDCs in the spleen.

(A) Representative flow cytometric determination of cDC (CD11chi CD11b+), Mo (CD11clo CD11bint), and Mφ (CD11clo CD11bhi; left panel). Total numbers of cDCs, Mo, and Mφ in the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl) and _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) mice are shown on the right (_n_=8–9). (B) Annexin V staining of cDCs, Mo, and Mφ isolated from the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed circles) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open circles) mice (_n_=8–9). (C) Numbers of precDCs (CD11cint CD45RAlo CD43int SIRPαint CD11b− CD4− CD8− cells) were determined in the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl, closed circles) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ, open circles) mice by flow cytometry (_n_=8–9). (D) Percentage of donor-derived cDCs in the spleen of WT recipient mice (B6.SJL), transplanted with _Klf4_fl/fl _Vav_-iCre− (fl/fl) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) BM cells (_n_=5). (E) KLF4 expression in cDCs isolated from the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) mice (_n_=3). (F) Transcript levels of IL-15 in cDCs isolated from the spleen of _Klf4_fl/fl _Vav_-iCre− (fl/fl) or _Klf4_fl/fl _Vav_-iCre+ (Δ/Δ) mice (_n_=3). **P < 0.005; ***P < 0.0005 (unpaired Student's t test).

Figure 7.

Figure 7.. KLF4 is dispensable for the expression of effector molecules and cytotoxicity in NK cells.

(A) Intracellular levels of IFN-γ, granzyme B, and perforin and cell-surface expression of CD107a were analyzed in _Klf4_fl/fl _Mx1_-Cre− (fl/fl, dotted lines) and _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ, solid lines) NK cells by flow cytometry. The gray, filled histograms correspond to an unstained control sample. (B) Representative flow cytometric analysis of the in vitro NK cell cytolytic assay using a mixture of CFSE-labeled RMA-S cells (targets) and EL4 cells (control), incubated NK1.1+ cells purified from the spleen of _Klf4_fl/fl _Mx1_-Cre+ (Δ/Δ) and _Klf4_fl/fl _Mx1_-Cre− (fl/fl) mice. (C) Specific lysis calculated based on the percentage of CFSElow (RMA-S) and CFSEhigh (EL4) cells at different E:T ratios. Data are representative of two independent experiments (two mice/genotype).

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