1　Materials and methods

1.1　Mice

All animal procedures were approved by the University of Science and Technology of China (USTC) Institutional Animal Care and Use Committee. All experiments were performed in accordance with the approved guidelines. To generate LCK ^cre Ei24 ^fl/fl mice, mice harboring LoxP sites flanking exon 3 of the Ei24 gene were crossed with mice expressing Cre under the control of the LCK (lymphocyte protein tyrosine kinase) promoter (LCK ^cre). The LCK-Cre-Flox strategy was used to delete the Ei24 gene specifically in T cells.

1.2　Preparation of immune cells

LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice were sacrificed, thymus and spleen were minced and passed through 100 μm stainless steel meshes. Livers were minced and passed through 100 μm stainless steel meshes and centrifuged to remove the supernatant. The cell pellets were resuspended in 40% percoll and centrifuged. Red blood cells were lysed by erythrocyte lysis buffer (Solarbio, Beijing, China).

1.3　Cell enrichment

LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice were sacrificed and spleens were minced and passed through 100 μm stainless steel meshes. Cells were blocked with anti-mouse CD16/32 for 15 min, then stained with anti-CD4-PE for 30 min on ice. CD4⁺ T cells were enriched with anti-PE microbeads (Miltenyi Biotec, Germany).

1.4　Western blot

CD4⁺ T cells were harvested and lysed with sample buffer and boiled for 10 min. Proteins were separated by electrophoresis and detected by Western blot. The antibodies used were as follows: rabbit anti-Ei24 (Sigma, HPA047165), mouse anti-β-actin (Transgen, HC201), HRP-conjugated anti-rabbit immunoglobulin G (Jackson IR, 111-035-144) and HRP-conjugated anti-rabbit immunoglobulin G (Jackson IR, 111-035-146).

1.5　Activation of iNKT cells in vivo

LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice were injected intraperitoneally with α-GC (2 μg/mouse, Avanti Polar Lipids, Alabama), 4 h before tissue collection. Immune cells were harvested for flow cytometric analysis.

1.6　Flow cytometry

Cells were blocked with purified anti-mouse CD16/32 for 15 min on ice, then were stained with fluorochrome-conjugated monoclonal antibodies against CD44(IM7), NK1.1(PK136), CD24(M1/69), TCR-β(H57-597), CD1d-PBS57 tetramer. To detect intracellular transcription factor PLZF, T-bet, RORγt, and cytokine IL-4, IFN-γ, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) and permeabilized with PBS buffer containing 0.1% saponin (Sigma-Aldrich, Munich, Germany) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich, Munich, Germany). Then, cells were stained with antibodies against PLZF (9E12), T-bet (4B10), RORγt (AFKJS9), IFN-γ (XMG1.2), IL-4 (11B11) for 1 h on ice. All antibodies were purchased from BioLegend (USA). CD1d-PBS57 tetramer is provided by National Institute of Health (NIH) Tetramer Core Facility. Cells were acquired on a FACSVerse flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (TreeStar).

1.7　Statistical analyses

Error bars represent SEM. Statistical analyses were performed using student’s t-test (GraphPad Software). *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

2　Results

2.1　Ei24 is crucial for iNKT cell development in thymus

Ei24 has been shown to negatively regulate cell growth^[26]. To investigate whether Ei24 plays a similar role in iNKT cell development, we used Cre/loxP system to generate mice with T cells-specific knockout of Ei24. Homozygous loss of Ei24 is embryonically lethal^[27]. In LCK ^cre Ei24 ^fl/fl mice, Ei24 protein in CD4⁺ T cells was almost fully deleted (Figure 1a). We analyzed iNKT cells from thymus of LCK ^cre Ei24 ^fl/fl mice and Ei24^{f l/fl} mice, and found a significantly decrease in frequency and absolute cell number in the absence of Ei24 (Figure 1b). Next, we investigated which stage was affected by the deficiency of Ei24 during the development of iNKT cells. LCK ^cre Ei24 ^fl/fl mice had a lower frequency and absolute number of stage 3 iNKT cells (CD24^- NK1.1^hi CD44^hi) than littermate controls, whereas there was no difference in stage 1 iNKT cells (CD24^- NK1.1^lo CD44^lo) and stage 2 iNKT cells (CD24^- NK1.1^lo CD44^hi) (Figure 1c, 1d). Taken together, these data suggest that Ei24 regulates maturation of iNKT cells in thymus.

Fig. 1 Development of iNKT cells is dependent on Ei24 expression

NOTE: (a) Western blot analysis of Ei24 protein from enriched CD4⁺ T cells in spleen from LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice. β-Actin served as the control. (b) Frequencies of stage 1—3 iNKT cells in the thymus from LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice. In the FACS plots, numbers adjacent to outlines indicate the percentages of cells in the gate. The frequency (c) and absolute number (d) of iNKT cells at stage 1—3 among CD24^- CD1d-tetramer⁺ iNKT cells in indicated mice. Data are mean ± SEM of nineteen mice per group. Statistical analysis was performed using a student’s t-test (GraphPad Software). **P < 0.01; ***P < 0.001.

2.2　Ei24 is required for the development of iNKT1 and iNKT17 cells

iNKT cells can be divided into three different subsets: iNKT1, iNKT2 and iNKT17^[28]. Next, we investigated if Ei24 controlled the development of iNKT cell subsets. We found the absolute number of iNKT1 and iNKT17 cells were lower in LCK ^cre Ei24 ^fl/fl mice, and no difference in the absolute number of iNKT2 cells was observed (Figure 2a, 2b). These data demonstrate that Ei24 is important for iNKT1 and iNKT17 cells development.

Fig. 2 Ei24 is required for the development of iNKT1 and iNKT17 cells

NOTE: (a)Representative FACS plots of iNKT1,iNKT2 and iNKT17 cells in the thymus,compared between LCK ^cre Ei24 ^fl/fl mice and Ei24^fl/fl mice. In the FACS plots,numbers adjacent to outlines indicate the percentages of cells in the gate. (b)The frequency (left) and absolute number (right) of iNKT subsets among CD24^- CD1d-tetramer⁺ iNKT cells in indicated mice. Data are mean ± SEM of nine mice per group. Statistical analysis was performed using a student’s t-test (GraphPad Software). *P < 0.05; **P < 0.01; ***P < 0.001.

2.3　Ei24 deficiency results in decreased splenic and hepatic iNKT cells

During the development in thymus, immature stage 2 iNKT cells can leave the thymus and colonize in peripheral organs. A final maturation step that occurs either in the thymus or in the peripheral organs is accompanied by the expression of NK1.1^[13,29]. In agreement with the data in the thymus, iNKT cell frequencies and numbers were also decreased in the liver and spleen of LCK ^cre Ei24 ^fl/fl mice. However, there was no difference of iNKT cells in the lymph node (Figure 3). These data demonstrate impaired maturation of splenic and hepatic iNKT cells in LCK ^cre Ei24 ^fl/fl mice.

Fig. 3 Deletion of Ei24 results in diminished splenic and hepatic iNKT cell numbers

NOTE: (a)Representative FACS plots of iNKT cells in the spleen,liver and lymph node,compared between LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice. In the FACS plots,numbers adjacent to outlines indicate the percentages of cells in the gate. The frequency (b)and absolute number (c)of iNKT cells in the spleen,liver and lymph node of indicated mice. Data are mean ± SEM of eight to nine mice per group. Statistical analysis was performed using a student’s t-test (GraphPad Software). ***P < 0.001.

2.4　Ei24 deficiency results in impaired IFN-γ production in splenic and hepatic iNKT cells

Next, we studied whether deletion of Ei24 affected peripheral iNKT cell functions. Remarkably, the majority of the splenic and hepatic iNKT cells are iNKT1 cells (Figure 4a). Upon activation by α-GC, hepatic iNKT cells from LCK ^cre Ei24 ^fl/fl mice showed significantly impaired IFN-γ production compared to Ei24 ^fl/fl mice. However, Ei24 deficiency showed no influence on IL-4 production in hepatic iNKT cells (Figure 4b, 4d). Consistently, we found similar reduction of IFN-γ in splenic iNKT cells in the absence of Ei24 (Figure 4c, 4e). These results indicate that Ei24 promotes IFN-γ production in iNKT cells.

Fig. 4 Ei24 deficiency results in impaired IFN-γ production in iNKT cells

(a—c)Representative FACS plots of iNKT1 cells among hepatic and splenic iNKT cells (a),intracellular staining of IFN-γ and IL-4 in hepatic (b)and splenic(c)iNKT cells 4 h after i.p. administration of α-GC,from LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice as indicated. (d,e)Frequencies of IFN-γ⁺ iNKT cells and IL-4⁺ cells iNKT cells from the liver (d) and spleen(e)of LCK ^cre Ei24 ^fl/fl mice and Ei24 ^fl/fl mice with or without α-GC injection. Data are mean ± SEM of six mice per group. Statistical analysis was performed using a student’s t-test (GraphPad Software). *P < 0.05.

3　Discussion

Our data demonstrated a critical role of Ei24 in regulating maturation and function of iNKT cells. In the absence of Ei24, thymic, splenic, and hepatic iNKT cell numbers were significantly reduced (Figure 1 and 3). In thymus, only stage 3 but not stage 1 or stage 2 iNKT cells were influenced by deletion of Ei24 (Figure 1d). In spleen and liver, most of the iNKT cells are at stage 3. Therefore, Ei24 controls the terminal maturation of iNKT cells. Ei24 has been reported to mediates autophagy signaling pathways^[30]. Autophagy regulates cell differentiation, proliferation, and survival in multiple immune cells, including iNKT cells. It has been reported that, in the absence of autophagy, the number of iNKT cells is reduced in the thymus. Autophagy defect results in the accumulation of mitochondrial superoxide species, leading to apoptotic cell death. Moreover, autophagy is especially required for the Th1 response of iNKT cells, the production of IFN-γ in iNKT cells is reduced in the absence of autophagy^[24,25]. Here, deficiency of Ei24 inhibited development and function of iNKT cells in a similar manner as deficiency of autophagy. It is possible that Ei24 may regulate iNKT cell development through modulating autophagy, detailed mechanism study is required. Moreover, we identified Ei24 as a key player in controlling differentiation of iNKT1 and iNKT17 rather than iNKT2 cell lineages in the thymus (Figure 2). However, we did not observe impaired expression of T-bet and RORγt in these two iNKT subsets when Ei24 was deleted. The mechanisms how Ei24 regulate differentiation of iNKT1 and iNKT17 remain unclear.

iNKT cells play important roles in regulating immune responses. Majority iNKT cells in mice are iNKT1 cells. Upon activation by lipid antigens, iNKT1 cells can produce large amounts of Th1 and Th2 cytokines, such as IFN-γ and IL-4. IFN-γ is a key player in body defense against tumor cells and bacteria, whereas IL-4 helps to suppress inflammation and maintain homeostasis. Th1 or Th2 biased cytokine production of iNKT cells has been related to progression of many diseases^{[10,31,32,33]}. In our studies, deletion of Ei24 inhibited IFN-γ production in iNKT cells but did not influenced IL-4 production (Figure 4). These results suggested a functional polarization of iNKT cells toward Th2 response in the absence of Ei24. Taken together, our findings suggest that Ei24 is required for the development and Th1 function of iNKT cells.

白丽. Tel:18656156426，E-mail: baili@ustc.edu.cn

References