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. 2019 Feb;49(2):255-265.
doi: 10.1002/eji.201847755. Epub 2018 Dec 3.

JNK2 modulates the CD1d-dependent and -independent activation of iNKT cells

Jianyun Liu  1 , Richard M Gallo  1 , Masood A Khan  1   2 , Abhirami K Iyer  1 , Ian M Kratzke  1 , Randy R Brutkiewicz  1
Affiliations

JNK2 modulates the CD1d-dependent and -independent activation of iNKT cells

Jianyun Liu et al. Eur J Immunol. 2019 Feb.

Abstract

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti-tumor, and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKT cell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild-type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKT cell activation and loss during viral infections.

Keywords: Antigen processing and presentation; CD1d; NKT cells; Signal transduction; Viral infection.

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Conflict of interest statement

Conflict of Interest Disclosure

The authors declare no commercial or financial conflict of interest.

Figures

Figure 1.
Figure 1.
JNK negatively regulates CD1d-mediated Ag presentation. (A) LMTK-CD1d1 cells were infected with UV-inactivated VV or live VV for 4 h. The cells were lysed and the lysates were analyzed by Western blot using Abs specific for either phospho-JNK1/2 or total JNK1/2. The relative level of phospho-JNK to total JNK in each treatment is shown in the graph below the blot. (B) Murine LMTK-CD1d1 cells were transfected with plasmids containing a JNK1/2-targeting shRNA or a scrambled sequence for the negative control (NC). Stable transfectants were co-cultured with the mouse type II NKT cell hybridoma, N37–1A12, for 24 h. Culture supernatants were harvested and IL-2 production was measured by ELISA. Human HEK293-CD1d cells were transfected with plasmids containing shRNA specific for JNK1/2 (C), MKK4 (D) or MKK7 (E). Stable transfectants were co-cultured with human iNKT cells for 48 h. Culture supernatants were harvested and GM-CSF production was measured by ELISA. The data shown are representative of at least three independent experiments. **, p<0.01; ***, p<0.001.
Figure 2.
Figure 2.
JNK2 (but not JNK1) negatively regulates Ag presentation by CD1d. (A) HEK293-CD1d cells were transfected with plasmids containing JNK1 (left) or JNK2 (right) shRNA. Stable transfectants were lysed and analyzed by Western blot using a JNK1- or JNK2-specific mAb. HEK293-CD1d cells expressing scrambled shRNA (NC) were included as a control. The relative expression of JNK1 or JNK2 is shown in the graphs below the blots. (B) HEK293-CD1d cells expressing JNK1, JNK2 or NC shRNA were co-cultured with human iNKT cells for 48 h. Culture supernatants were harvested for measuring GM-CSF production by ELISA. BMDCs from JNK1 KO (C) or JNK2 KO (D) mice were co-cultured with the mouse type I NKT hybridoma N38–2C12 for 24 h. IL-2 production in the supernatants was measured by ELISA. (A-C) The data presented are representative of at least three independent experiments. The data shown in (D) are pooled from three independent experiments. Each dot represents an individual mouse, and plotted as mean ± SEM. **, p<0.01.
Figure 3.
Figure 3.
Reduced iNKT cell numbers in thymi and spleens of JNK2 KO mice. (A) Thymocytes, splenocytes and liver mononuclear cells from WT and JNK2 KO mice were stained with α-GalCer-loaded CD1d tetramers and a TCR-β-specific mAb for the identification of iNKT cells by flow cytometry. (B) The percentages (upper graphs) and total numbers (lower graph) of iNKT cells are shown for thymus, spleen and liver. Pooled data from two independent experiments are shown. Each dot represents an individual mouse. The data are plotted as the mean ± SEM. (C) Thymocytes from WT and JNK2 KO mice were stained with α-GalCer-loaded CD1d tetramers. The cells that were detected with α-GalCer-loaded CD1d tetramers were gated and the forward scatter (FSC) is shown in the histogram. (D) The region 1 (R1) and R2 gating strategy of the thymocytes for (E) is shown. (E) Thymocytes from WT and JNK2 KO mice were stained with α-GalCer-loaded CD1d tetramers and a TCRβ-specific mAb for the identification of iNKT cells. The ratio of iNKT cells localized in the R2 gate relative to those in R1 was calculated and normalized (WT=1). Pooled data from three independent experiments are shown; each dot represents an individual mouse. The data are plotted as the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 4.
Figure 4.
JNK2 (but not JNK1) contributes to the VV-induced loss of iNKT cells. (A) JNK2 KO mice and their WT littermates were infected i.p. with VV. On day 4 p.i., LMNC were harvested and the cells were stained with α-GalCer-loaded CD1d tetramers and a TCRβ-specific mAb. (B) On day 4 p.i., the percentage of liver iNKT cells in VV-infected mice was compared to mock-infected mice. The change in the percentage of liver iNKT cells was normalized (mock=1) and plotted. Pooled data from four independent experiments are shown, with each dot representing an individual mouse. The data are plotted as the mean ± SEM (n=9–12); *, p<0.05. (C) WT and JNK1 KO mice were infected i.p. with VV. On day 4 p.i., liver MNC were harvested and the cells were stained with α-GalCer-loaded CD1d tetramers and a TCRβ-specific mAb. The percentage of liver iNKT cells is shown, consisting of pooled data from two independent experiments, with each dot representing an individual mouse. The data are plotted as the mean ± SEM (n=6); *, p<0.05.
Figure 5.
Figure 5.
Increased DC and macrophage populations in JNK2 KO mice post-VV infection. JNK2 KO mice and their WT littermates were infected with 1×106 pfu VV, i.p. On day 4 p.i., spleens were harvested and the splenocytes were stained with an MHC II- and a CD11c-specific mAb (A; DCs), an F4/80-specific mAb (B; macrophages) or a B220-specific mAb (C; B cells). The percentage of each cell type in VV-infected mice was compared to mock-infected and the change was normalized (mock=1) and plotted. Pooled data from four independent experiments are shown, with each dot representing an individual mouse. The data are plotted as the mean ± SEM (n= 9–12); **, p<0.01.
Figure 6.
Figure 6.
IL-12-mediated iNKT cell loss is JNK2-dependent. (A) JNK2 KO mice and their WT littermates were injected i.v. with 0.5 μg/mouse of recombinant murine IL-12. LMNCs were harvested on day 2, and the cells were stained with α-GalCer-loaded CD1d tetramers, and a TCRβ-specific mAb. The change in liver iNKT cells was normalized (vehicle=1) and plotted. Pooled data from two independent experiments are shown, with each dot representing an individual mouse. The data are plotted as the mean ± SEM (n= 4). (B, C and D) JNK2 KO mice and their WT littermates were injected i.v. with recombinant murine IL-12. LMNCs and splenocytes were harvested on day 1. The cells were stained with α-GalCer-loaded CD1d tetramers and mAbs against TCRβ and IL-12Rβ1. Splenic iNKT cells (tetramer+/TCR-β+) were gated and the levels of IL-12Rβ1 are shown in (B). The percentage of IL-12R+ splenic or liver iNKT cells is plotted in (C) and (D). (E) LMNCs were harvested from JNK2 KO mice and their WT littermates. These cells were co-cultured with α-GalCer-pulsed WT BMDCs for two days for the analysis of iNKT cell function. The production of GM-CSF into the supernatants was measured by ELISA and normalized (WT=1). Pooled data from two independent experiments are shown, with each dot representing an individual mouse. The data are plotted as the mean ± SEM (n= 6); **, p<0.01; ***, p<0.001.
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