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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: J Hepatol. 2017 Jul 14;67(4):791–800. doi: 10.1016/j.jhep.2017.05.024

Crosstalk between type II NKT cells and T cells leads to spontaneous chronic inflammatory liver disease

Xiufang Weng 1,2, Ying He 1, Lavanya Visvabharathy 1, Chia-Min Liao 1,3, Xiaosheng Tan 2, Arjun Balakumar 1, Chyung-Ru Wang 1
PMCID: PMC5605413  NIHMSID: NIHMS882253  PMID: 28596110

Abstract

Background & AIM

NKT cells are CD1d-restricted innate-like T cells that modulate innate and adaptive immune responses. Unlike the well-characterized invariant/type I NKT cells, type II NKT cells with a diverse TCR repertoire are poorly understood. This study defines the pathogenic role of type II NKT cells in the etiology of chronic liver inflammation.

Methods

Transgenic mice with the Lck promoter directing CD1d overexpression on T cells in Jα18 wild-type (Lck-CD1dTgJα18+; type I NKT cell sufficient) and Jα18-deficient (Lck-CD1dTgJα18o, type I NKT cell deficient) mice were analyzed for liver pathology and crosstalk between type II NKT cells and conventional T cells. CD1d expression on T cells in peripheral blood samples and liver sections from AIH patients and healthy individuals were also examined.

Results

Lck-CD1dTgJα18o and Lck-CD1dTgJα18+ mice developed similar degrees of liver pathology resembling chronic autoimmune hepatitis in humans. Increased CD1d expression on T cells promoted the activation of type II NKT cells and other T cells. This resulted in Th1-skewing and impaired Th2 cytokine production in type II NKT cells. Dysfunction of type II NKT cells was accompanied by conventional T cell activation and pro-inflammatory cytokine production, leading to a hepatic T/B lymphocyte infiltration, elevated autoantibodies and hepatic injury in Lck-CD1dTg mice. A similar mechanism could be extended to humans as CD1d expression is upregulated on activated human T cells and increased presence of CD1d-expressing T cells was observed in AIH patients.

Conclusions

Our data reveals enhanced crosstalk between type II NKT cells and conventional T cells leads to a Th1-skewed inflammatory milieu, leading to the development of chronic autoimmune liver disease.

Keywords: CD1d, NKT cells, Autoimmune hepatitis, Liver inflammation, Transgenic/knockout mice

Graphical abstract

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Introduction

The liver is constantly exposed to large varieties of antigens derived from the gastrointestinal tract as well as from systemic circulation, a possible explanation for why liver tissue is subject to a greater degree of immune tolerance than some other organs [1]. Despite this, disruption of tolerance leads to inflammatory liver injuries during chronic diseases such as persistent viral infection [2], drug toxicity [3] and autoimmune hepatitis (AIH) [4]. As one of the predominant lymphocyte populations in the liver, CD1d-restricted NKT cells contribute to the maintenance of immune tolerance but also direct adverse immune responses in the liver [5, 6]. NKT cells are divided into two subsets based on TCR usage: type I NKT cells expressing a semi-invariant TCR (Vα14Jα18 in mice and Vα24Jα18 in human), and type II NKT cells having a diverse TCR repertoire. While type I NKT cells are the dominant NKT cell subset in mice, type II NKT cells appear to be more prevalent in humans [7]. Unlike type I NKT cells which can be unambiguously detected by α-galactosylceramide-loaded CD1d tetramer, type II NKT cells are more difficult to study because of the lack of specific markers that can uniquely define them. Although NK1.1 and CD161 are expressed in most NKT cells in mice and humans, NK1.1/CD161 positive T cells also comprise several other innate-like T cell subsets including the newly discovered mucosal associated invariant T (MAIT) cells [8, 9]. Therefore, we have utilized a variety of markers such as NK1.1+ CD8− and functional phenotypes to define type II NKT cells more accurately in order to determine the contributions of type II NKT cells to inflammatory liver disease.

CD1d is a non-polymorphic MHC class I-like molecule which presents self- and foreign lipid antigens to NKT cells. CD1d is expressed on a variety of hematopoietic and non-hematopoietic cells and its altered expression levels are correlated with several disease conditions. While certain viral infections such as human immunodeficiency virus and herpes simplex virus type-1 infection down-regulate CD1d expression as a viral immune evasion strategy [10, 11], CD1d expression is enhanced during chronic inflammatory and autoimmune diseases. In experimental autoimmune encephalomyelitis, CD1d is upregulated on macrophages and T cells invading inflamed CNS tissue [12]. Furthermore, CD1d is strongly upregulated in the liver during hepatitis C infection, which might contribute to liver injury during chronic infection [7, 13]. This raises the possibility that functional differences in NKT cells may be partially mediated by differential interactions between NKT cells and different types of antigen presenting cells (APC) expressing elevated levels of CD1d.

Several transgenic mouse models have been used to examine the effect of altered CD1d expression patterns on NKT cell development and function. By manipulating CD1d expression under the control of a MHC I, MHC II or proximal Lck promoter in the CD1d-deficient background [14–17], we and others have shown that only mice with transgenic CD1d driven by Lck promoter (Lck-CD1dTg mice) are sufficient to support NKT cell development [15, 16]. Interestingly, the Lck-CD1dTg mouse model we generated in which both thymocytes and peripheral T cells express high levels of CD1d develops liver pathology in the absence of any exogenous manipulation [16]. Although peripheral T cells express low levels of CD1d in both humans and mice, CD1d could be upregulated on T cells by activation in vivo or in vitro [18]. Apart from the critical role of thymocytes in NKT cell selection, it is not yet clear whether T cells can function as CD1d-restricted APCs.

Our previous study showed that type I NKT cells in Lck-CD1dTg mice are hypo-responsive to α-GalCer stimulation [16]. However, it is unclear whether altered CD1d expression also affects the function of type II NKT cells and by extension whether type II NKT cells contribute to the development of liver pathology. We utilized Lck-CD1d transgenic mice to determine whether enhanced crosstalk between type II NKT cells and conventional T cells in the liver affects the development of chronic hepatic inflammation.

Materials and Methods

Mice

Jα18o, Kb-CD1dTg, Lck-CD1dTg, IL-4 GFP enhanced transcript (4get), and 24αβTg mice have been described elsewhere [16, 19–23]. Lck-CD1dTg mice were crossed with Jα18o and 24αβ Tg mice to obtain Lck-CD1dTgJα18o and Lck-CD1dTg24αβ Tg, respectively and then further crossed with 4get mice to track IL-4-expressing cells. Both male and female mice were used in this study. All animal studies and procedures were approved by the Northwestern University Institutional Animal Care and Use Committee.

Human samples

Blood samples from healthy donors and AIH patients were collected from Tongji Hospital, Wuhan, China. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Paraffin-embedded human normal and AIH liver sections were obtained from tissue bank of Tongji Hospital. Individuals with viral hepatitis, HIV infection, or alcoholic liver disease were excluded. The characteristics of AIH patients included in this study were detailed in Supplementary Table 2. The project and protocols involving human samples were approved by the ethics committee of Tongji Medical College. Informed consent was obtained from all subjects.

Cell preparations and analysis

Mouse thymocytes, splenocytes, and hepatic leukocytes were prepared by mechanical dispersion as described previously [24]. Type II NKT, conventional T, 24αβT and B cell populations were purified by FACS or magnetic-activated cell sorting (MACS) using specific antibodies. Bone marrow-derived DCs (BMDCs) were prepared as previously described [19]. Cell surface staining, T cell activation and intracellular cytokine staining were perform as described [25]. Flow cytometry, proliferation assay, cytokine release detection, and RT-qPCR were performed to determine the phenotype and function of various cell types.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Data were analyzed using unpaired Student’s t test for 2 group comparisons or one way ANOVA for more than two group comparisons, followed by Bonferroni post-hoc test. Values are mean + SEM. Statistically significance is indicated by the following annotation: *P < 0.05; **P < 0.01; ***P < 0.001.

Additional descriptions of methodology and reagents are provided in the Supplementary Materials and Methods section.

Results

Lck-CD1dTgJα 18o mice develop chronic inflammatory liver disease spontaneously

We have previously shown that Lck-CD1dTg mice develop liver hypertrophy spontaneously [16], regardless of whether the endogenous CD1d is present or not. The liver hypertrophy and the elevated liver-to-body weight ratio found in Lck-CD1dTgJα18+ mice were also observed in Lck-CD1dTgJα18o mice (Figure 1A, B). Additionally, the Lck-CD1dTgJα18o mice exhibited splenomegaly, which is often caused by shunting of blood from the liver to the spleen as a result of portal hypertension during chronic liver disease [26]. In contrast, Lck-CD1dTgJα18o kidneys were normal in size (Figure 1A). Both Lck-CD1dTgJα18+ and Lck-CD1dTgJα18o mice had elevated hepatic leukocyte numbers and ALT levels when compared to wild-type (WT) and Jα18o mice (Figure 1B). Furthermore, H&E stained liver sections showed that hepatocytes from Lck-CD1dTgJα18o mice exhibited cytomegaly similar to Lck-CD1dTg mice (Figure 1C). In fact, we observed no significant difference in liver-to-body weight ratio, the degree of leukocyte infiltration and changes in liver histology (Figure 1C) between Lck-CD1dTgJα18+ and Lck-CD1dTgJα18o mice. These data suggest that type I NKT cells are dispensable for the development of liver pathology in this model.

Figure 1. Lck-CD1dTgJα 18o mice develop chronic inflammatory liver disease spontaneously.

Figure 1

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(A) Gross morphology of spleens, livers and kidneys from 6-mo-old Jα18o and Lck-CD1dTgJα18o (Tg+ Jα18o) mice; scale bar, 1cm. (B) Liver-to-body weight ratios, hepatic leukocyte numbers and ALT levels among B6 (WT, n=23), Lck-CD1dTgJα18+ (Tg+ Jα18+, n=19), Jα18o (n=27) and Lck-CD1dTgJα18o (Tg+ Jα18o, n=24) mice. (C) Representative H&E-stained liver sections from 5-mo-old mice. Scale bars, 40 μm. (D–F) Oval cell-specific staining (D), TUNEL staining (E) and anti-CD45 staining (F) of liver sections. Scale bars, 40 μm. (G) Percentage and number of different leukocyte subsets in Jα18o and Tg+Jα18o mice (n=5). (H) Gross morphology of the spleen, liver and kidney, 11-mo-old Lck-CD1dTgJα18o mice. White arrows: liver nodules. (I) Representative H&E-stained liver sections from 11-mo-old Lck-CD1dTgJα18o mice. Left image: portal inflammation (arrow), ballooned hepatocytes (asterisk), and steatosis of hepatocytes (arrowhead). Right image: regenerative nodules surrounded by fibrous connective tissue (arrow). Scale bars, 100 μm. ***P < 0.001; **P < 0.01; *P< 0.05; n.s. = not significant. Statistical analysis was performed using One-Way ANOVA followed by Bonferroni post-hoc test for 4 group comparisons and Student’s t-test for 2 group comparisons.

To gain a deeper insight into the liver pathology in Lck-CD1dTgJα18o mice, immunohistochemical analysis of liver sections was performed. Staining with oval cell-specific antibody and TUNEL staining unveiled significant oval cell hyperplasia and cell death in Lck-CD1dTgJα18o livers (Figure 1D, E). Anti-CD45 staining demonstrated leukocyte infiltration and formation of inflammatory foci in livers (Figure 1F). Flow cytometry revealed T and B cells to be two major infiltrating leukocyte populations in Lck-CD1dTgJα18o livers, though myeloid cells were also slightly increased in number (Figure 1G). Additionally, the severity of liver disease in Lck-CD1dTgJα18o mice progressed with age and was more profound in female mice. At 11 months of age, most of the female Lck-CD1dTgJα18o mice had macroscopically visible liver nodules (Figure 1H) while this was largely absent in male mice (data not shown). H&E staining of liver sections from these mice revealed portal inflammation, hepatocyte injury, hepatocyte steatosis (Figure 1I, left panel), and the presence of regenerative nodules surrounded by fibrous connective tissue (Figure 1I, right panel), which bear resemblance to AIH in humans (Supplementary Figure 1).

CD1d-restricted type II NKT cells are reduced in number and dysfunctional in Lck-CD1dTgJα 18o mice

Type II NKT cells are the major CD1d-restricted T cell population in Lck-CD1dTgJα18o mice. Because NK1.1, CD122 and IL-4 transcript are expressed in most type II NKT cells in WT mice [23], we sought to evaluate type II NKT cells using NK1.1, CD122 and IL-4-eGFP (4get-GFP) as surrogate markers. Because most CD1d-restricted NKT cells in mice exhibit a CD4+ or CD4−CD8− (DN) phenotype, we focused our analysis on CD8 negative (CD8−) T cell population. We observed a significant decrease in the percentage and number of TCRβ+NK1.1+CD8−, TCRβ+CD122+CD8− and TCRβ+4get-GFP+CD8− cells in hepatic lymphocytes from Lck-CD1dTgJα18o mice compared to those from Jα18o mice (Figure 2A–C). The percentage and number of type II NKT cells in the thymus of Lck-CD1dTgJα18o and Jα18o mice were comparable (Figure 2D–F), indicating the reduction of type II NKT cells in Lck-CD1dTgJα18o mice was mainly a peripheral event.

Figure 2. The frequency and number of type II NKT cells are reduced in the liver of Lck-CD1dTgJα 18o mice.

Figure 2

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Hepatic lymphocytes and thymocytes were isolated from Jα18o (n=23), Lck-CD1dTgJα18o (n=17), Jα18o4get (n=7), and Tg+ Jα18o4get (n=5) mice. CD8− cells were gated and analyzed for TCRβ, NK1.1, CD122, and 4get-GFP expression. (A and D) Dot plots: percentages of TCRβ+NK1.1+, TCRβ+CD122+ or TCRβ+4get+ in CD8− hepatic lymphocytes (A) or CD8−thymocytes (D). Quantification of TCRβ+NK1.1+CD8−, TCRβ+CD122+CD8−, or TCRβ+4get+CD8− T cells in the liver (B and C) and thymus (E and F). Values are mean + SEM. Statistical analysis was performed using Student’s t-test. ***P < 0.001; **P < 0.01; *P< 0.05.

FACS sorted type II NKT cells (defined as TCRβ+NK1.1+CD8−) from Jα18o and Lck-CD1dTgJα18o livers exhibited CD1d-dependent IFN-γ production upon stimulation with CD1d-overexpressing BMDCs which express CD1d under the control of the MHC-Ia promoter [19] (Figure 3A). However, these type II NKT cells produced lower levels of IFN-γ than those from Jα18o mice. Further, type II NKT cells from Lck-CD1dTgJα18o mice were completely defective in IL-4 and IL-13 production upon stimulation with Kb-CD1dTg BMDCs (Figure 3B). Analysis of TCR Vα and Vβ usage by type II NKT cells showed only subtle differences between Jα18o and Lck-CD1dTgJα18o mice (Supplementary Figure 2), indicating that the functional impairments observed were unlikely to be attributed to changes in the TCR repertoire of type II NKT cells.

Figure 3. Type II NKT cells in Lck-CD1dTgJα 18o mice have increased cell death and impaired IL-4 producing capacity.

Figure 3

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Hepatic TCRβ+NK1.1+CD8− T cells were FACS sorted and stimulated with Kb-CD1Tg BMDCs. (A) Levels of IFN-γ production ± anti-CD1d mAb (n=6). (B) Levels of IL-4, IL-13, TNF-α, IL-6, and IL-17 production (n=4). (C) Activation marker expression on TCRβ+NK1.1+CD8− T cells from indicated mice. (D) Percentages of dead (7AAD+) cells in type II NKT and conventional T cells (n=5). Data shown are from 3 independent experiments. Statistical analysis was performed using Student’s t-test. ***P < 0.001; **P < 0.01.

We have previously shown that type II NKT cells have an activated phenotype as CD69hiCD44hiCD62LloCD122+ [23]. Although most type II NKT cells in Lck-CD1dTgJα18o mice expressed similar levels of CD122 and CD62L as those from Jα18o mice, they expressed higher levels of CD69 and CD44, suggesting their hyper-activated status in vivo (Figure 3C). Hepatic type II NKT cells in Lck-CD1dTgJα18o mice also had a higher percentage of cell death compared with Jα18o mice (Figure 3D). This suggested that activation induced cell death may account for the decreased number of type II NKT cells in Lck-CD1dTgJα18o mice. In contrast, conventional T cells in Lck-CD1dTgJα18o mice did not show increased cell death when compared to that of Jα18o mice. Collectively, these data suggested the type II NKT cells in Lck-CD1dTgJα18o mice are chronically activated and thus hyporesponsive to antigen stimulation, resulting in impaired cytokine responses.

CD1d overexpression on conventional T cells is sufficient to drive type II NKT cell activation and proliferation

To further determine the effects of enhanced T cell-specific CD1d expression on the functionality of type II NKT cells, we crossed monoclonal type II NKT cell TCR transgenic mice (24αβTg) [22] into the Lck-CD1dTg background (Lck-CD1dTg/24αβTg). Although total liver leukocytes increased, the percentage and number of Vα3.2+Vβ9+ 24αβ T cells significantly decreased in the liver of Lck-CD1dTg/24αβ Tg mice compared to 24αβ Tg mice (Figure 4A, B). This is consistent with the decrease in polyclonal type II NKT cells observed in the Lck-CD1dTgJα18o liver (Figure 2A–C). Moreover, 24αβ T cells similarly lost the capacity to produce IL-4, as shown by the decreased 4get-GFP expression (Figure 4C). The elevated CD69 and CD44 expression in 24αβ T cells in Lck-CD1dTg/24αβ Tg mice suggested that dysfunctional monoclonal type II NKT cells were also hyperactivated in vivo (Figure 4C).

Figure 4. Enhanced interaction between type II NKT cells and Lck-CD1dTg+ T cells leads to bidirectional activation of type II NKT and conventional T cells.

Figure 4

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(A–C) Numbers in representative dot plots show percentage of 24αβ (Vα3.2+Vβ9+) T cells (A). Percentage and absolute number of 24αβ T cells in indicated mice (n=6) (B). 4get-GFP, CD69, and CD44 expression on 24αβ T cells (n=4) (C). (D) CD69 upregulation and proliferation of 24αβ T cells were determined in 24α β T cells stimulated with the indicated APCs (n=4). (E and F) CFSE-labeled 24αβ T cells were adoptively transferred to Jα18o or Lck-CD1dTgJα18o (Tg+Jα18o) mice. 7 days post-transfer, proliferation of donor 24αβ T cells was determined (E). One month post-transfer, T cells in Lck-CD1dTgJα18o mice that received 24αβ T cells (AT) or PBS (No AT) were analyzed for Teff (CD44hiCD62Llo) cells (F) (n=4). Statistical analysis was performed using Student’s t-test. ***P < 0.001; *P< 0.05.

To determine which APC is primarily responsible for activating type II NKT cells in Lck-CD1dTg mice, we compared the stimulatory capacity of T cells, B cells, and BMDCs from Lck-CD1dTg mice on 24αβ T cells. 24αβ T cells upregulated CD69 expression and underwent extensive cell proliferation following stimulation with Lck-CD1dTg-expressing T (Tg+ T) cells, but not WT T (Tg− T) cells (Figure 4D). This effect was abolished in the presence of anti-CD1d mAb, confirming these responses to be CD1d-dependent. In contrast, neither BMDCs nor B cells from Lck-CD1dTg mice, which expressed WT levels of CD1d (Supplementary Figure 3), had comparable stimulatory capacities (Figure 4D). This suggests that increased CD1d expression on T cells alone is sufficient to induce type II NKT cell activation.

Enhanced interaction between type II NKT cells and conventional T cells leads to conventional T cell activation in Lck-CD1dTgJα 18o mice

NKT cells are known to modulate T cell responses [23, 27]. In order to study the reciprocal effects between type II NKT cells and Lck-CD1dTg-expressing T cells in vivo, CFSE-labeled 24αβ T cells were adoptively transferred into 1-month-old Jα18o and Lck-CD1dTgJα18o mice. The proliferative capacity of 24αβ T cells and the percentage of CD44highCD62Llow effector T (Teff) cells in recipient mice were compared 7 days and 1-month post-transfer. We chose one-month-old mice as recipients because liver pathology in Lck-CD1dTgJα18o mice becomes apparent at 2 months of age. We consistently found that naïve 24αβ T cells exhibited a higher degree of proliferation when adoptively transferred to Lck-CD1dTgJα18o mice, compared to that of Jα18o mice (Figure 4E). Moreover, there was a significant increase in Teff cells in the liver of Lck-CD1dTgJα18o mice that received 24αβ T cells as compared to the liver of Lck-CD1dTgJα18o mice that had not received 24αβ T cells (Figure 4F).

To address whether polyclonal type II NKT cells had similar effects on conventional T cells, we evaluated CD4+ and CD8+ T cells in Lck-CD1dTgJα18o mice. CD4+ and CD8+ T cell numbers increased in the liver of Lck-CD1dTgJα18o mice compared to Jα18o controls (Figure 5A). Foxp3+ regulatory CD4+ T cells (Treg) also increased in Lck-CD1dTgJα18o mice compared to controls (Figure 5B), suggesting that inflammation in Lck-CD1dTgJα18o livers was not due to impaired Treg cell differentiation. However, Lck-CD1dTgJα18o mice had a higher frequency of Teff cells in both the CD4+ and CD8+ T cell compartments compared to age and sex-matched controls (Figure 5C, D).

Figure 5. Conventional T cells are more activated and produce more pro-inflammatory cytokines in Lck-CD1dTgJα 18o mice.

Figure 5

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(A) The total number of CD4+ and CD8+ conventional T cells in the liver (n=8). (B) The percentage of CD4+FoxP3+ Treg cells in indicated mice (n=4). (C and D) Representative dot plots (C) and bar graphs (D) depict the percentage of Teff (CD44hiCD62Llo) cells in CD4+ and CD8+ T cells (n=6). (E) The percentage of IFN-γ producing (left panel) and IL-4 producing (right panel) T cells. (F–G) FACS sorted CD8+ (F) and CD4+ (G) T cells were stimulated with anti-CD3/CD28 for 48h. Cytokine levels in supernatants were determined by CBA (n=5). Statistical analysis was performed using Student’s t-test. ***P < 0.001; **P < 0.01; *P< 0.05.

Functional analysis of conventional T cells in Lck-CD1dTgJα18o mice showed that IFN-γ producing cells, but not IL-4 producing cells, increased significantly in Lck-CD1dTgJα18o mice compared with Jα18o controls (Figure 5E). Multiplex cytokine assays showed that CD8+ T cells from Lck-CD1dTgJα18o livers produced more Th1-like cytokines upon TCR stimulation (Figure 5F), and conventional CD4+ (NK1.1−) T cells produced more IL-17 and less IL-4, when compared with their counterparts from Jα18o livers (Figure 5G). Imbalanced Th1/Th2 responses and/or increases in Th17 proportions in the liver have long been thought to be associated with persistent inflammation and chronic liver pathology [28]. Thus, increased pro-inflammatory cytokine production by conventional T cells could partially account for T-cell mediated liver injury in Lck-CD1dTgJα18o mice.

B-cell activation and autoantibodies are increased in Lck-CD1dTgJα18o mice

It has been suggested that hyperactive T cells promote autoreactive B cell responses through increasing costimulatory molecule signaling, cytokines, and/or other stimulatory factors [29]. We found CD4+ T cells from Lck-CD1dTgJα18o liver expressed higher mRNA levels of the B-cell activating factors BAFF and IFN-α (Figure 6A). IFN-α upregulates BAFF, and BAFF has been shown to promote autoreactive B cell activation [30]. Indeed, B cells were greatly increased in Lck-CD1dTgJα18o livers (Figure 1G) and were mainly mature B-2 cells (Supplementary Figure 4). They upregulated CD69 to a higher degree and produced more pro-inflammatory IL-6 upon LPS stimulation as compared to B cells from Jα18o livers (Figure 6B–C). Serum IgM levels (but not IgG) were also increased in Lck-CD1dTgJα18o mice that were older than 4 months (Figure 6D and data not shown). Additionally, elevated anti-dsDNA antibodies were detected in Lck-CD1dTgJα18o mice, with IgM and IgG2a/c being notably elevated (Figure 6E). Collectively, our data suggests that the increased inflammatory T cell response in Lck-CD1dTgJα18o mice promotes B cell activation and autoantibody production.

Figure 6. Increased B-cell activation and autoantibody production in Lck-CD1dTgJα 18o mice.

Figure 6

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(A) Expression of various B-cell activation factors in sorted hepatic CD4+ T cell. All mRNA levels relative to mRNA expression in CD4+ T cells from Jα18o mice (n=7). (B) CD69 expression on LPS-activated B cells (n=6). (C) Cytokine production in supernatants from FACS sorted hepatic B cells stimulated with LPS for 48h, determined by CBA (n=5). (D) Levels of serum IgM in indicated mice at different ages (n=7–16 per group). (E) Levels of dsDNA-specific IgG1, IgG2a/c, IgG2b, IgG3 and IgM (n=8). Statistical analysis was performed using Student’s t-test. ***P < 0.001; **P < 0.01; *P< 0.05.

Dysregulation of type II NKT and T cells is also observed in Lck-CD1dTgJα18+ mice

A recent study described a perturbed TCR-α repertoire with lower TCR diversity in Jα18o mice [31]. To assess whether the dysfunction of type II NKT, T and B cells was influenced by changes in TCR repertoire in Lck-CD1dTgJα18o mice, we evaluated the functional status of these cell subsets in Lck-CD1dTgJα18+ mice. Type II NKT cells, defined as CD1d-αGalCer tetramer−CD8−TCRβ+NK1.1+, were significantly reduced in Lck-CD1dTgJα18+ mice compared to Jα18+ WT mice (Figure 7A). Moreover, impaired IL-4 production and over-activation of type II NKT cells in Lck-CD1dTgJα18+ mice were also observed (Figure 7B). These results suggested similar dysregulation of type II NKT cells in Lck-CD1dTgJα18+ mice as seen in Lck-CD1dTgJα18o mice.

Figure 7. Lck-CD1dTgJα 18+ mice exhibited similar dysregulation of type II NKT, T and B cells as Lck-CD1dTgJα 18o mice.

Figure 7

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Hepatic leukocytes and serum were prepared from Jα18+ (n=13), Lck-CD1dTgJα18+ (Tg+Jα18+, n=12), Jα18+4get (n=4), and Tg+ Jα18+4get (n=4) mice. (A) Representative dot plots show percentages of TCRβ+NK1.1+ type II NKT cells in CD1d Tet−CD8− hepatic lymphocytes. (B) 4get-GFP (left panel) and CD69 expression (right panel) on type II NKT cells. (C) Absolute number of various leukocyte subsets (n=4). (D) Levels of dsDNA-specific IgM (n=8–10/group). (E) Percentage of Teff (CD44hiCD62Llo) cells in conventional CD4+ and CD8+ T cells (n=6). (F) The percentage of IFN-γ producing (left panel) and IL-4 producing (right panel) T cells (n=6). Statistical analysis was performed using Student’s t-test. ***P < 0.001; *P< 0.05.

In further support of the congruence of these two models, conventional T and B cells remained the two major populations of infiltrating leukocytes in Lck-CD1dTgJα18+ mice (Figure 7C). Enhanced activation and Th1-biased cytokine production in conventional T cells were also observed in Lck-CD1dTgJα18+ mice, along with elevated levels of circulating autoantibodies (Figure 7D–F). Collectively, these results showed that the dysfunctional phenotypes of type II NKT, conventional T and B cells observed in Lck-CD1dTgJα18o mice were also found in Lck-CD1dTgJα18+ mice.

Overexpression of CD1d on T cells are found in AIH patients

Since the human liver contains a high frequency of type II NKT cells, it will be of great interest to investigate whether elevated CD1d expression can be detected in AIH patients. Consistent with a previous report that activated T cells upregulate CD1d expression [18], we found in vitro activation of human T cells with anti-CD3 or IFN-γ treatment resulted in CD1d upregulation (Figure 8A). In addition, ex vivo analysis of CD1d expression on peripheral blood T cells revealed higher CD1d expression on pre-activated CD69+ T cells than that on CD69− T cells (Figure 8B). Interestingly, PBMC from human AIH patients have increased frequency of activated CD69+ T cells, suggesting more circulating CD1d-expressing T cells in AIH patients (Figure 8C). Furthermore, in situ immunofluorescence co-staining of CD3 and CD1d unveiled a proportion of infiltrated T cells expressed high level of CD1d in AIH liver (Figure 8D). These data suggest that upregulation of CD1d on T cells and possibly aberrant activation of NKT cells may contribute to the development of clinical AIH.

Figure 8. Increased CD1d expression on activated T cells that were more prevalent in AIH patients.

Figure 8

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(A) CD1d expression on unstimulated, anti-CD3 stimulated, and IFN-γ-treated T cells from human PBMCs (n=6/group). (B) CD1d expression on CD69+ and CD69− human T cells. (C) Percentage of CD69+ T cells from healthy donors (HD, n=8) and AIH patients (AIH, n=8). (D) Representative immunofluorescent staining of normal liver sections from hepatic hemangioma patients (n=2) and inflammatory liver sections from AIH patients (n=3) with anti-CD3 (green) and anti-CD1d (red). Statistical analysis was performed using Student’s t-test. **P < 0.01.

Discussion

NKT cells have been shown to play either a pathogenic or protective role in inflammatory liver disease [32, 33]. However, most of these studies used a model of ConA-induced hepatitis which causes acute liver inflammation but does not reflect chronic disease seen in human AIH. Here, we showed that aberrant CD1d expression on T cells resulted in hyperactivation of type II NKT cells and conventional T cells, leading to the development of chronic inflammatory liver disease in the absence of any exogenous antigenic or pharmacological stimuli. Liver pathology observed in Lck-CD1dTgJα18o mice showed some features reminiscent of human AIH (e.g. portal inflammation, hepatocyte injury, hepatic steatosis and fibrosis), while no signs of pathology were observed in the kidney, stomach, small intestine and colon of these mice. In addition, the disease severity is more profound in female Lck-CD1dTgJα18o mice as female Lck-CD1dTgJα18o mice tended to have higher liver to body weight ratios and more severe histopathology than male Lck-CD1dTgJα18o mice. This is consistent with the finding that AIH is more common in female patients. Further, hypergammaglobulinemia and autoantibody production commonly associated with AIH were also found in Lck-CD1dTgJα18o mice. Our study thus suggests a potential role for type II NKT cells in the pathogenesis of AIH.

Our finding that Lck-CD1dTgJα18+ and Lck-CD1dTgJα18o mice had similar degrees of liver pathology suggests either that type I and type II NKT cells play a redundant role or that type II NKT cells play a dominant role in liver pathogenesis in this mouse model. The Jα18o mice used in this study have been shown to have lower TCR diversity due to suppressed transcription of TRAJ gene segments upstream of Traj18 (Jα18) [31]. However, the influence of this change on liver inflammation in Lck-CD1dTgJα18o mice is limited because similar liver pathology, immune cell infiltration/activation, and B cell autoantibody production were found in Lck-CD1dTgJα18+ and Lck-CD1dTgJα18o mice. Additionally, we have shown that type II NKT cells in Jα18o mice closely resemble those from wild-type mice in terms of their surface phenotype and cytokine-producing capacity [23], despite having lower TCR diversity.

Previous studies have shown the importance of conventional T cell activation in the etiology and pathogenesis of AIH [34, 35]. Several studies have suggested that impaired Treg function is responsible for over-activation of CD4+ and CD8+ T cells, which can lead to a breakdown in tolerance to liver antigens and a progression towards liver disease [36, 37]. Here, we showed that type II NKT cells may also play a role in the establishment of AIH. NKT cells from Lck-CD1dTgJα18o mice were able to produce IFN-γ but not IL-4/IL-13 in a CD1d-dependent manner (Figure 3A, B). Though total IFN-γ production by type II NKT cells in Lck-CD1TgJα18o mice was lower than that in Jα18o mice, the complete lack of IL-4 and IL-13 production suggests that Th2-mediated inhibition of the Th1 response in these mice is impaired. This could lead to chronic, low-level production of IFN-γ which can, over time, activate conventional T cells to produce pro-inflammatory cytokines leading to liver pathology. It is unknown why type II NKT cells were unable to produce Th2 cytokines in Lck-CD1dTgJα18o mice, but we speculate that overexpression of CD1d on T cells induces a chronic, sub-optimal stimulation of type II NKT cells, leading to their aberrant activation and preferential Th1 polarization.

Finally, increased CD1d expression has been reported in several disease settings and in response to cytokine treatments [7, 12, 38, 39]. Here, we provide evidence that elevated CD1d expression on T cells promotes enhanced interaction between type II NKT and T cells, which leads to Th1-biased inflammatory liver disease in Lck-CD1dTgJα18o mice. We also present data showing that increased CD1d expression on T cells can be seen in AIH patients. The findings in this study are the first to show the effect of altered CD1d expression on the function of type II NKT cells at the polyclonal level and demonstrate their role in the pathogenesis of spontaneous inflammatory liver disease. Taken together, these results suggest that type II NKT cells, which are enriched in the human liver, could be an important clinical target in the treatment of AIH.

Supplementary Material

supplement

Highlights.

  1. CD1d overexpression alters type II NKT cell activation/polarization polyclonally.

  2. Dysfunctional type II NKT cells trigger an inflammatory loop with T and B cells.

  3. Enhanced cross-talk among type II NKT/T/B cells leads to autoantibody production.

  4. A pathogenic role of type II NKT cells in autoimmune liver diseases is proposed.

Acknowledgments

Financial support

This work was supported by the National Institutes of Health [NIH grant R01AI43407 to C.-R.W.] and National Nature Science Foundation of China [NSFC grant 31570913 to X. W.]

We thank the Northwestern University flow cytometry core facility for cell sorting; mouse histology & phenotyping laboratory for histology services; the NIH tetramer facility for CD1d tetramer; Sreya Bagchi for critical reading of the manuscript, Dr. Guang-Yu Yang for advice on histological assessment of liver pathology and Dr. Susanna Cardell for providing 24αβ Tg mice.

Footnotes

Conflict of interest

The authors have declared that no competing interests exist with respect to this manuscript

Authors contributions

X.W and C.-R. W. conceived and designed the experiments. X.W., Y.H., L.V., C.L., X.T. and A.B. performed the experiments. X.W., Y.H., L.V., C.L., X. T. and C.-R.W. analyzed the data. X.W., L.V. and C.-R.W. wrote the manuscript.

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Supplementary Materials

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