DX5+NKT cells display phenotypical and functional differences between spleen and liver as well as NK1.1-Balb/c and NK1.1+ C57Bl/6 mice
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Natural killer T cells represent a linkage between innate and adaptive immunity. They are a heterogeneous population of specialized T lymphocytes composed of different subsets. DX5+NKT cells are characterized by expression of the NK cell marker DX5 in the context of CD3. However, little is known about the phenotype and functional capacity of this unique cell population. Therefore, we investigated the expression of several T cell and NK cell markers, as well as functional parameters in spleen and liver subsets of DX5+NKT cells in NK1.1- Balb/c mice and compared our findings to NK1.1+ C57Bl/6 mice.
In the spleen 34% of DX5+NKT cells expressed CD62L and they up-regulated the functional receptors CD154 as well as CD178 upon activation. In contrast, only a few liver DX5+NKT cells expressed CD62L, and they did not up-regulate CD154 upon activation. A further difference between spleen and liver subsets was observed in cytokine production. Spleen DX5+NKT cells produced more Th1 cytokines including IL-2, IFN-γ and TNF-α, while liver DX5+NKT cells secreted more Th2 cytokines (e.g. IL-4) and even the Th17 cytokine, IL-17a. Furthermore, we found inter-strain differences. In NK1.1+ C57Bl/6 mice DX5+NKT cells represented a distinct T cell population expressing less CD4 and more CD8. Accordingly, these cells showed a CD178 and Th2-type functional capacity upon activation.
These results show that DX5+NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity.
KeywordsMouse Strain iNKT Cell Intracellular Cytokine Staining Interstrain Difference Maturation Marker CD103
Natural killer T (NKT) cells represent a small but important subset of T lymphocytes with characteristics of both T and NK cells. They have potent immunoregulatory function that reportedly can promote cell-mediated immunity to tumors and infectious organisms and, paradoxically, suppress cell-mediated immunity associated with autoimmune disease and allograft rejection . In mice, these cells express NK cell markers such as NK1.1 and CD94, as well as T-cell receptors (TCR) α/β with a restricted repertoire [2, 3]. The invariant T cell receptor α chain Vα14-Jα18 with a conserved CDR3 region is associated with Vβ8.2, Vβ7 or Vβ2 gene segments [3, 4].
In contrast to conventional T-lymphocytes, the TCR of NKT cells does not interact with antigens presented by classical major histocompatibility complex (MHC)-encoded class I or II molecules. Instead, their TCR recognizes glycolipids presented by CD1d, which is a MHC class-I-like glycoprotein that belongs to a group of CD1 molecules associated with β2-microglobulin [5, 6, 7]. CD1d is known to present lipids including glycosylceramides and glycosylphosphatidylinositol [8, 9]. Activation via CD1d initiates the production of both Th1 (IFN) and Th2 cytokines (IL-4, IL-5, IL-13) , and increases the cytolytic activity of NKT cells .
NKT cells do not represent a homogeneous population. So far, three types of NKT cells have been described. First, there is an invariant Vα14-NKT cell (iVα14-NKT) also called a type I NKT cell or iNKT cell. This group can be further differentiated into CD4+ single positive and CD4-CD8- double-negative variants. Second, a population of CD1d-reactive NKT cells expressing diverse non-Vα14TCRs, referred to as type II NKT cells, has also been characterized. A third category has been termed NKT-like cells, which are CD1d-independent and express diverse TCRs .
Despite many years of NKT cell research, controversy remains about defining these cells. The expression of several surrogate markers, such as NK1.1 in C57Bl/6 mice and CD161 in humans, co-expressed with the TCRα/β, have been frequently used for NKT cell identification . There are also NK1.1+ T cells which do not express the semiinvariant Vα14-Jα18 T cell receptor and are not CD1d-dependent, excluding their consideration as NKT cells.
A common marker for NKT cells in NK1.1- mice strains is the antibody DX5, which recognizes the α2-integrin CD49b . DX5 was initially characterized as a marker for NK cells  and more recently DX5 co-expressing CD3+ lymphocytes have been described . Several studies, including some from our group, revealed evidence for immunoregulatory properties of these cells [15, 16, 17, 18]. DX5+NKT cells produce Th1 and Th2 cytokines after stimulation like other NKT cells and seem to play a central role in anti-tumor immunity [13, 19]. Recently published studies suggest an immune modulatory function of DX5+NKT cells after bone marrow and solid organ transplantation [20, 21]. Although all these studies describe typical characteristics of NKT cells, Pellecci et al. showed that DX5+ T cell numbers were normal in CD1d-/- and even in TCR Jα18-/- mice . Therefore, it is more likely that DX5+NKT cells belong to the third group of NKT-like cells [22, 23].
In the present study we have further characterized DX5+CD3+ T cells referred to as DX5+NKT cells in NK1.1- (Balb/c) and NK1.1+ (C57Bl/6) mice. For this purpose DX5+NKT cells were isolated from spleen and liver and several T and NK cell surface markers as well as maturation and activation markers were studied by flow cytometry. Distinct differences could be found between both subsets and between mouse strains.
T cell marker expression is different between spleen and liver DX5+NKT cells
Thus far, surface receptor expression was only examined in freshly isolated cells. Since DX5+NKT cells display their immunoregulating function especially upon activation , expression patterns after stimulation were analyzed. For this purpose, splenic and hepatic mononuclear cells were stimulated for 4 hours with anti-CD3 and anti-CD28 antibodies. Activation of spleen DX5+NKT cells from Balb/c mice (Figure 1C) resulted in a down-regulation of TCRα/β (90 ± 2% vs. 51 ± 2%, P = 0.004) and an up-regulation of CD25 (14 ± 2.6% vs. 27 ± 3%, P = 0.0411). However, stimulation had little effect on liver derived cells of Balb/c mice. DX5+NKT cells revealed an expression pattern similar to that of freshly isolated cells: there was no down-regulation of TCRα/β in liver derived DX5+NKT cells upon stimulation.
NK cell and activatory markers are expressed differently in spleen and liver DX5+NKT cells
We also analyzed if the distinct phenotype of spleen and liver DX5+NKT cells was based on differences in the maturation status. Therefore, freshly isolated cells were stained with anti-CD103 and anti-CD62L antibodies (Figure 2B). Spleen DX5+NKT cells were positive for CD103 in 15 ± 2.4% of cells. In contrast, freshly isolated liver DX5+NKT cells expressed this maturation marker less frequently (4.5 ± 1.7%, P = 0.014). Freshly isolated spleen DX5+NKT cells were even more frequently positive for the maturation marker CD62L (34 ± 5.5%) compared to liver DX5+NKT cells (3 ± 2.2%, P = 0.0079). Upon 4 h stimulation, liver DX5+NKT cells displayed an increase in CD62L expression (14 ± 1.7%, P = 0.0317), but still significantly less than the spleen subset (36 ± 4%, P = 0.0028).
Next, differences in activation markers were evaluated. In freshly isolated DX5+NKT cells the activation marker CD38 was expressed on approximately 80% of splenic and hepatic cells and displayed no significant change upon stimulation (data not shown). In contrast, upon 4 h stimulation, there was a strong up-regulation of the activating marker CD154 (3 ± 2.2% vs. 39 ± 5.8%, P = 0.0238) in spleen DX5+NKT cells (Figure 2C). However, liver DX5+NKT cells did not display a similar up-regulation of CD154 (4 ± 1%, P = 0.0498). Another activating marker, CD178, was also not significantly expressed on freshly isolated DX5+NKT cells, but upon 4 h stimulation, there was an up-regulation of CD178 in the spleen subset (2 ± 1.3% vs. 19 ± 1.4%, P = 0.0087).
Spleen and liver DX5+NKT cells display a distinct cytokine secretion pattern in Balb/c mice
Results thus far suggest distinct phenotypic differences between spleen and liver DX5+NKT cells. Next, the production of Th1 and Th2 cytokines were compared to further assess functional differences between these subsets. Lymphocytes were isolated from spleen and liver of Balb/c mice and cultured for 4, 24 or 48 h in the presence of anti-CD3 and anti-CD28 antibodies. The cells were additionally incubated with PMA, ionomycin and GolgiPlug for intracellular cytokine staining.
Next, Th2 cytokines such as IL-4, IL-6, IL-10 and IL-13 were analyzed. Only very few spleen DX5+NKT cells produced IL-4 after 4, 24 and 48 h. However, the number of IL-4 positive liver DX5+NKT cells increased after 48 h (11 ± 1.2%, P = 0.0286). Neither spleen nor liver DX5+NKT cells were significantly positive for IL-6 at any time point (data not shown). Spleen DX5+NKT cells showed a significant increase in the frequency of IL-10-producing cells after 24 h (13 ± 2.1%) compared to 4 h (0.4 ± 0.2%, P = 0.0187), and compared to liver DX5+NKT cells after 24 h stimulation (1 ± 0.6%, P = 0.0357). In the spleen the number of IL-13-producing DX5+NKT cells significantly increased after 48 h compared to 4 h (20 ± 3% vs. 10 ± 1.2%, P = 0.0061), whereas in liver the number of IL-13 positive DX5+NKT cells peaked after 4 h compared to 24 h (16 ± 2.1% vs. 3 ± 1.2%, P = 0.0286). Furthermore, we tested for IL-17a as a Th17-type cytokine. Liver DX5+NKT cells after 4 h more frequently produced IL-17a (7 ± 1%) compared to the splenic-derived cells (1 ± 0.7%, P = 0.0061); after 24 h, the frequency of liver-derived IL-17a-producing DX5+NKT cells decreased (0.3 ± 0.3%, P = 0.286).
NK1.1+C57Bl/6 mice reveal distinct surface marker expression on DX5+NKT cells
Cytokine production of spleen DX5+NKT cells is different in NK1.1-Balb/c and NK1.1+ C57Bl/6 mice
All freshly isolated DX5+NKT cells from NK1.1 deficient Balb/c mice were positive for TCRα/β, as expected for type III NKT cells [2, 3, 24]. Most of them additionally expressed CD4, and a few expressed CD8a and CD25. As mentioned in other reports for spleen and thymus , a difference between spleen and liver subsets was observed. Liver DX5+NKT cells expressed less CD4 and CD62L. Since expression of CD62L is associated with naivety or homing to peripheral lymph nodes in iNKT-cells , and CD4 becomes down-regulated after repeated activation , this suggests a higher maturational status of liver DX5+NKT cells in Balb/c mice. This is supported by the finding that Vβ8.2-containing Vα 14i TCRs in NKT cells have a higher antigen affinity than those containing Vβ7 or Vβ2 [28, 29]. Consistent with the idea of a negative selection, liver DX5+NKT cells displayed a higher expression of Vβ8.1/8.2.
Upon activation, a down-regulation of TCRα/β was observed in spleen DX5+NKT cells in Balb/c mice. A decrease of TCRα/β-expression was also reported for NK1.1+NKT cells . Due to a lack of detection, a rapid apoptotic death of this cell was initially claimed upon stimulation . More recently TCRα/β-down-regulation in spleen and liver NK1.1+NKT cells of C57Bl/6 mice was discovered to be the reason for this assumed cell disappearance [31, 32]. However, our study does not confirm this finding for liver DX5+NKT cells in Balb/c mice, supporting evidence that there are distinct subsets of DX5+NKT cells in the spleen and liver. CD154 expression was also increased upon stimulation. As the ligand of CD40, CD154 represents a co-stimulatory molecule and is expressed by activated and regulatory T cells [33, 34]. Furthermore, CD178, the ligand of Fas responsible for the extrinsic induction of apoptosis (e.g. expressed on CD8+ cytotoxic T cells) was up-regulated . This up-regulation was more evident in spleen than in liver DX5+NKT cells and is also reported for NK1.1+NKT cells . Taken together, liver DX5+NKT cells are more mature and display a different activating phenotype upon stimulation compared to the spleen subset.
Since NKT cells are supposed to link innate and adaptive immunity by rapid secretion of cytokines, including IFNγ, TNFα, IL-4, IL-10, IL-13, GM-CSF and IL-2 [24, 37, 38], we further assessed differences between spleen and liver DX5+NKT cells on a functional basis. Fewer liver DX5+NKT cells in Balb/c mice produced Th1 cytokines such as IL-2, IFN-γ, and TNF-α. In terms of Th2 cytokines, more liver DX5+NTK cells produced IL-4, but fewer produced IL-10 compared to the spleen subset. Charbonnier et al. described an induction of IL-10 production in liver CD49b+CD4+ T cells by immature dendritic cell vaccination . However, we did not confirm this finding for liver DX5+NKT cells in Balb/c mice after anti-CD3/anti-CD28 stimulation. Collectively, the limited Th1 cytokine secretion upon stimulation confirmed the observation that a different activating phenotype of DX5+NKT cells exists in the liver of Balb/c mice.
Recently, type I NKT cells have been shown to be capable of producing IL-17, potentially implicating this cell type in inflammatory conditions [39, 40]. We showed that a subset of DX5+NKT cells in the liver of Balb/c mice has to be taken into account as an IL-17 producing cell. These results are consistent with our result regarding a more mature phenotype of liver-derived DX5+NKT cells, with a different phenotypical profile upon stimulation compared to the spleen subset.
In NK1.1+ C57Bl/6 mice fewer spleen DX5+NKT cells revealed expression of CD4, but more of CD8a. These findings are consistent with data from Pellici et al., who observed similar differences in C57Bl/6 mice . In accordance with Conzalez et al., 40-50% of DX5+NKT cells express NK1.1 . Taking these results together with the higher CD4 expression in NK1.1-deficient Balb/c mice, we suggest that these mice compensate for a reduced number of NK.1.1+NKT cells with a higher prevalence of CD4+DX5+NKT cells. In contrast to results from Gonzalez et al. , about one-third of these cells in the spleen of C57Bl/6 mice, and 11% in Balb/c mice, were CD8+.
Upon activation a down-regulation of TCRα/β was observed in spleen DX5+NKT cells in Balb/c and in C57Bl/6 mice, as it has been reported for NK1.1+NKT cells . Furthermore, upon a 4 h stimulation there was a notable up-regulation of CD25 in Balb/c versus C57Bl/6 mice. CD25, the IL-2 receptor α-chain, is proposed to be a phenotypical marker of regulatory T cells [41, 42, 43]. The up-regulation we observed especially for spleen DX5+NKT cells in Balb/c mice could be either due to activation and increased receptor expression based on the regulatory background of DX5+NKT cells or due to a natural selection because of a higher apoptotic resistance in CD4+CD25+ T cells . Another marker for activation on regulatory T cells is CD154 . CD154 expression was increased upon stimulation in spleen DX5+NKT cells in Balb/c mice, but was significantly less prominent in C57Bl/6 mice. Upon 4 h stimulation, CD178 (ligand of Fas) was similarly up-regulated  on splenic DX5+NKT cells in both mice strains. However, after 24 h stimulation CD178 expression was decreased in Balb/c versus C57Bl/6 mice, and CD154 was further up-regulated in Balb/c, but down-regulated in C57Bl/6 mice (data not shown). These changes suggest a more regulatory CD4+CD25+ phenotype in NK1.1- Balb/c and a more CD8+ and CD178+ driven cytotoxic component in the NK1.1+ C57Bl/6 mouse strain .
High IL-2 production by DX5+NKT cells in the early phase of activation was observed only in Balb/c mice, further supporting our findings of interstrain differences. Moreover, splenic DX5+NKT cells in C57Bl/6 mice displayed a more one-sided Th1 cytokine profile, with mostly IFNγ and TNFα producing cells, which is also described for CD8+ T cells . DX5+NKT cells in Balb/c mice displayed a more polyfunctional cytokine profile. In the late phase of stimulation, IFNγ and TNFα production decreased and IL-10 and IL-13 secretion increased. In contrast to liver CD49b+CD4+ cells , and supporting our data regarding splenic DX5+NKT cells, Kassiotis et al. revealed no IL-10 production for splenic CD49b+CD4+ T cells in C57Bl/6 mice . Furthermore, Pellicci et al. could not detect any significant IL-10 production in DX5+TCRα/β+CD1d-Tet+ cells in the same mouse strain . Confirming our finding and giving again evidence for a difference between both mouse strains, we showed a significant increase in IL-10 production after 24h stimulation for splenic DX5+NKT cells in Balb/c mice. Taken together, these results suggesting a more one-sided Th1 cytokine production from DX5+NKT cells in C57Bl/6 mice confirm their more CD8 and CD178 expressing phenotype.
In conclusion, our data show that distinct subsets of DX5+NKT cells exist within NK1.1- Balb/c mice. In the spleen these cells appear more naive with a higher CD62L expression and an up-regulation of CD154 and CD178 upon activation. In contrast, in the liver DX5+NKT cells are more mature and display a different regulatory potency upon stimulation. Furthermore, there are remarkable inter-strain differences. In NK1.1+ C57Bl/6 mice, fewer DX5+NKT cells are CD4+ and more are CD8+, confirming their CD178 and Th1-sided functional behavior upon activation.
Cell harvesting and isolation
All experiments were approved by the institutional animal care committee at the University Hospital Regensburg. Different lymphocytes subsets were purified from splenic or hepatic mononuclear cells isolated from Balb/c mice or C57Bl/6NCrl mice (Charles River Laboratories, Wilmington, MA, USA). If necessary, further isolation was performed by magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) or by FACS (FACSAria I, BD Bioscience, San Jose, CA, USA). Briefly, cell suspension of the spleen was prepared by cutting the tissue into small pieces and gently pressing through a 100 μm wire mesh. For preparation of the liver the portal vein was flushed with 10 ml sodium chloride until the organ became pale. Then the liver was cut into small parts and passed through a 100 μm wire mesh. Hepatic lymphocytes were further isolated by density gradient centrifugation using 80% and 40% Percoll (Biochrom, Berlin, Germany). For further isolation, DX5+ cells were purified using anti-mouse-DX5+ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS-column (type LS) attached to a Midi-MACS-magnet (Miltenyi Biotec). DX5+ cells were labeled with FITC-conjugated anti-mouse CD3 (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further CD3+DX5+ enrichment by FACS, and are referred to as DX5+NKT cells throughout the study.
Antibodies and flow cytometry
The following reagents were used for cell surface labeling in multiparameter flow cytometric analysis (FACS Calibur, BD Bioscience): FITC or Alexa Fluoar 647-conjugated anti-mouse CD3 (clone: 17A2, rat IgG2b), APC-conjugated anti-mouse-CD4 (clone: GK1.5, rat IgG2b), APC-conjugated anti-mouse-CD8a (clone: 53-6.7, rat IgG2a), PE-conjugated anti-mouse-CD38 (clone: 90, rat IgG2a), PE-conjugated anti-mouse-CD49b (clone: DX5, rat IgM), APC-conjugated anti-mouse-CD62L (clone: MEL-14, rat IgG2a), PE-conjugated anti-mouse-CD103 (clone: M290, rat IgG2a), PE-conjugated anti-mouse-CD178 (Fas-Ligand) (clone: MFL3, hamster IgG1), PE-conjugated anti-mouse-Vβ 8.1, 8.2 TCR (clone: MR5-2, rat IgG2a) all BD Biosciences. APC-conjugated anti-mouse-CD49b (clone: DX5, rat IgM), FITC-conjugated anti-mouse-CD49b (clone: DX5, rat IgM) all obtained from Miltenyi Biotec. FITC-conjugated anti-mouse-CD94 (clone: 18d3, rat IgG2a), APC-conjugated anti-mouse-CD154 (CD40L) (clone: MR1, hamster IgG), APC-conjugated anti-mouse-NK1.1 (clone: PK136, mouse IgG2a) all obtained from eBioscience (San Diego, CA, USA). APC-conjugated anti-mouse-CD25 (clone: PC61 5.3, rat IgG1), FITC-conjugated anti-mouse-TCRα/β (clone: HM 3601, hamster IgG) from Caltag (Towcester, UK).
Activation of DX5+NKT cells
For lymphocyte stimulation, 96 well cell culture plates (Corning costar, Sigma Aldrich) were coated with anti-mouse-CD3e (Clone: 145-2C11, BD Biosciences) at 10 μg/ml and stored overnight at 4°C. Wells were washed twice with PBS. And up to 1 × 106 isolated lymphocytes from spleen and liver were plated in 200 μl RPMI culture medium (Biochrom). For activation, 5 μg/ml of anti-mouse-CD28 (clone: 37.51, BD Biosciences) and 2000 IU/ml IL-2 (Peprotech, Rocky Hill, NJ, USA) were added. Plates were incubated for the indicated time at 37°C and 5% CO2
Intracellular cytokine staining
4 × 105 DX5+-NKT cells were incubated in 200 μl culture medium in a 96 well plate as mentioned above. Additionally, 50 ng/ml PMA (InvivoGen, San Diego, CA, USA) was added at the beginning, with 750 ng/ml ionomycin (Sigma-Aldrich, St. Louis, USA) being added for the last 4h; 1 μg/ml GolgiPlug (BD Bioscience) was added 2 h before cell harvesting. Culture supernatants were harvested and stored at -20°C for IFNγ ELISA. Cells were fixed in 1ml Fix/Perm (eBioscience) for 60 min at 4°C. After incubation with permeabilization buffer (eBioscience) cells were stained with PE-conjugated anti-mouse-cytokine Abs (IL-2, clone: JES6-5H4/IL-4, clone: BVD4-1D11/IL-6, clone: MP5-20F3/IL-10, clone: JES5-16E3/IFNγ, clone: XMG1.2/TNFα, clone: MP6-XT22) from BD Bioscience and with PE-conjugated anti-mouse-Abs (IL-13, clone: eBio13A/IL-17a, clone: eBio17B7) and FITC-conjugated anti-mouse-IFNγ (clone: XMG1.2) (all eBioscience).
IFNγ analysis by Enzyme-Linked Immunosorbent Assay
Stimulation of DX5+NKT cells was confirmed by IFNγ ELISA. IFNγ concentration from harvested supernatants was analyzed using a commercially available sandwich ELISA kit (BD Bioscience). Tetramethylbenzidine dihydrochloride was used for detection. ELISA readings were determined by OD scanning at 450 nm using an Emax precision microplate reader from Molecular Devices (Downingtown, PA, USA).
All in vitro experiments were repeated at least 3 times and data are presented as the mean value ± SEM. Statistical analyses were performed using either a student's t-test or the Mann-Whitney-U-test. Differences were considered significant at P < 0.05.
We wish to thank Joachim Schweimer for his excellent technical assistance. This work was supported by funds from the "Regensburger Forschungsförderung Medizin (ReForM)".
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