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. 2015 Dec 1;195(11):5272-84.
doi: 10.4049/jimmunol.1501367. Epub 2015 Oct 30.

V体育官网入口 - Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

Kyle L O'Hagan  1 , Jie Zhao  1 , Olga Pryshchep  1 , Chyung-Ru Wang  1 , Hyewon Phee  2
Affiliations

"VSports在线直播" Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

Kyle L O'Hagan et al. J Immunol. .

Abstract

NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery VSports手机版. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression. .

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Figures

FIGURE 1.
FIGURE 1.
Loss of Pak2 in T cells results in a block in the development of NKT cells. (A) Flow cytometric analysis of TCRβ+Tet+ NKT cells from the thymus, liver, spleen, and lymph nodes from Pak2F/F (WT) and Pak2F/F;Cd4-Cre (KO) mice. (B) Total percentage (upper panel) and cell numbers (lower panel) of NKT cells (TCRβ+Tet+) from the thymus, liver, and spleen of WT and KO mice. (C) Flow cytometric analysis of CD24, CD44, and NK1.1 expression within total NKT cells (TCRβ+Tet+) from the thymus of WT and KO mice. To distinguish NKT cell developmental stages, we separated TCRβ+Tet+ gated cells by CD44 and NK1.1 (upper panel). CD44−NK1.1− NKT cells were further separated by side scatter (SSC) and CD24 (lower panel). Gating strategies for stages 0, I, II, and III are indicated on the right of each panel. (D) Total percentage (upper panel) and cell numbers (lower panel) of TCRβ+Tet+ NKT cells at stages 0–III from WT and KO mice (upper panel). (E) Flow cytometric analysis of NK1.1 and CD44, CD122 and CD127 expression within TCRβ+Tet+ gated NKT cells from the thymus (upper panel) and liver (lower panel) from WT and KO mice. (F) Flow cytometric analysis of CD122 (upper panel) and CD127 (lower panel) at stages 0–III. Graphs in this figure show mean ± SD (n = 3). Results are representative of at least three independent experiments. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001 (unpaired two-tailed Student t test).
FIGURE 2.
FIGURE 2.
The developmental defect in NKT cell development is T cell intrinsic. (A) Flow cytometric analysis of 1:1 mixed BM chimeras generated by transferring WT (Pak2+/+; CD45.1+CD45.2+) and KO (Pak2F/F;Cd4-Cre; CD45.2+) donor BM cells containing hematopoietic stem cells (HSCs) into sublethally irradiated C57BL/6 WT hosts that expressed CD45.1. Cells from WT (upper panel) and KO (lower panel) donor compartments were separated by TCRβ and CD1d/αGC tetramer to gate on NKT cells from the thymus, liver, spleen, and lymph nodes. (B) Ratio of the percentage of TCRβ+Tet+ NKT cells generated from KO BM cells relative to cells generated from WT BM cells in the thymus, liver, spleen (SPL), and lymph nodes (LN). (C) Flow cytometric analysis of TCRβ+Tet+ NKT cells from WT and KO donor compartments separated by CD44 and NK1.1 expression from the thymus of chimeric mice (left panel). The right panel indicates the ratio of the percentage of TCRβ+Tet+ NKT cells generated from KO BM cells relative to cells generated from WT BM cells at different developmental stages [double negative (DN), DP, stages I–III] within the thymus of chimeric mice. (D) Flow cytometric analysis of CD44 and NK1.1 expression within TCRβ+Tet+ NKT cells from WT and KO donor compartments from the thymus. (E) Flow cytometric analysis of CD122 (upper panel) and CD127 (lower panel) expression within TCRβ+Tet+ NKT cells from WT and KO donor compartments from the thymus of chimeric mice. (F) Flow cytometric analysis of BrdU incorporation within total TCRβ+Tet+ NKT cells from the thymus, liver, spleen, and lymph nodes of chimeric mice. Mice were administered a single i.p. injection of BrdU followed by 3 d BrdU-pulsed water. Graphs in this figure show mean ± SD (n = 6). Results are representative of at least six independent chimeras. ****0.00001 < p < 0.0001 (unpaired two-tailed Student t test).
FIGURE 3.
FIGURE 3.
Increased NKT cell apoptosis in the absence of Pak2 is independent of Bcl2- or Bcl-xL–dependent survival signals. (A) Flow cytometric analysis of TCRβ+Tet+ gated NKT cells from the thymus of Pak2F/F (WT) and Pak2F/F;Cd4-Cre (KO) mice separated by 7-AAD and Annexin V. Total NKT cells were also separated by developmental stage based on CD44 and NK1.1 expression (as described previously). (B) Flow cytometric analysis of Bcl-2 and Bcl-xL expression within total thymic NKT cells and NKT cells at stages I–III from WT and Pak2 KO mice. (C) Flow cytometric analysis of TCRβ+Tet+ gated NKT cells generated within the thymus of Het (Pak2F/+;Cd4-Cre;Bcl2-Tg) and KO (Pak2F/F;Cd4-Cre;Bcl2-Tg) mice overexpressing Bcl2. Heterozygous Pak2 mice that express Bcl2 transgene (Pak2F/+;Cd4-Cre;Bcl2-Tg) were used as positive controls. TCRβ+Tet+ NKT cells were separated into developmental stages I–III by CD44 and NK1.1 expression. (D) Flow cytometric analysis of NK1.1, CD44, CD122, and CD127 expression within TCRβ+Tet+ NKT cells from the thymus of Het and KO mice overexpressing Bcl2. (E) Absolute cell number of stages I–III thymic NKT cells generated in Het and KO mice overexpressing Bcl2. (F) Flow cytometric analysis of TCRβ+Tet+ NKT cells generated within the liver (upper panels), spleen (middle panels), and lymph nodes (lower panels) of Het and KO mice overexpressing Bcl2. Results are representative of at least three independent experiments. ***0.0001 < p < 0.001 (unpaired two-tailed Student t test).
FIGURE 4.
FIGURE 4.
Pak2-deficient mice possess fewer NKT1 and NKT2 cells and exhibit reduced IFN-γ– and IL-4–producing capacity. (A, upper panels) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and RORγt expression within TCRβ+Tet+ gated NKT cells from the thymus of WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice. (A, lower panels) Flow cytometric analysis of NKT2 cells based on PLZF and GATA3 expression within TCRβ+Tet+ gated NKT cells from the thymus of WT and Pak2 KO mice. (B) Total percentage (upper panels) and absolute cell numbers (lower panels) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF and RORγt from (A). (C, upper panels) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and T-bet expression within TCRβ+Tet+ gated NKT cells from the thymus of WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice. (C, lower panels) PLZFhiT-betlow NKT cells were separated by PLZF and RORγt, to distinguish NKT2 (RORγt−) and NKT17 (RORγt+) cells. (D) Total percentage (upper panels) and absolute cell numbers (lower panels) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF, T-bet, and RORγt from (C). (E) Flow cytometric analysis of IFN-γ expression within TCRβ+Tet+ gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. (F) Flow cytometric analysis of IL-4 expression within TCRβ+Tet+ gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. (G) Flow cytometric analysis of IL-17A expression within TCRβ+Tet+ gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. Graphs in this figure show mean ± SD (n = 3). Results are representative of at least three independent experiments for transcription factor (A–D) staining and four independent experiments for cytokine staining (E–G). *0.01 < p < 0.05 (unpaired two-tailed Student t test).
FIGURE 5.
FIGURE 5.
TCR signaling is normal in Pak2-deficient NKT cells. (A) Flow cytometric analysis of TCRβ+Tet+ NKT cells from the thymus (upper panel) of WT (Pak2F/F;Nur77-GFP) and KO (Pak2F/F;Cd4-Cre;Nur77-GFP) mice divided into their developmental stages by CD44 and NK1.1 expression. (B) Flow cytometric analysis of TCR signaling intensity by monitoring GFP signal expressed under the promoter of Nur77. GFP fluorescence was determined in total TCRβ+Tet+ NKT cells and NKT cells between developmental stages 0–III of WT and KO mice. Results are representative of at least two independent experiments for stage 0 NKT cells and five independent experiments for NKT cells at stages I–III.
FIGURE 6.
FIGURE 6.
Loss of Pak2 impairs the expression of PLZF and Egr2 in developing NKT cells. (A) Flow cytometric analysis of PLZF expression within TCRβ+Tet+ NKT cells from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice. Left panel shows PLZF expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the PLZF expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. (B) Flow cytometric analysis of Egr2 expression within TCRβ+Tet+ NKT cells from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice. Left panel shows Egr2 expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the Egr2 expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. (C) MFI of PLZF from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. (D) MFI of Egr2 from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE (n = 3). *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001.
FIGURE 7.
FIGURE 7.
Absence of Pak2 impairs the expression of SLAM6 on developing NKT cells. (A) Flow cytometric analysis of side scatter (SSC) and SLAM6 within total NKT cells (TCRβ+Tet+) from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. Cells were gated as SLAM6− and SLAM6+. (B) MFI of SLAM6 within total NKT cells (TCRβ+Tet+) from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. (C) Flow cytometric analysis of SLAM6 expression within SLAM6+ NKT cells [gated as in (A)] from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. (D) MFI of SLAM6 within SLAM6+ gated NKT cells (TCRβ+Tet+) from WT (Pak2F/F) and KO (Pak2F/F;Cd4-Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE (n = 3). *0.01 < p < 0.05, **0.001 < p < 0.01.
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