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Infection and Immunity, October 2006, p. 5903-5913, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00311-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid Development of a Gamma Interferon-Secreting Glycolipid/CD1d-Specific V14⁺ NK1.1^– T-Cell Subset after Bacterial Infection

Masashi Emoto,^1,2* Izumi Yoshizawa,¹ Yoshiko Emoto,^1,2 Mamiko Miamoto,¹ Robert Hurwitz,³ and Stefan H. E. Kaufmann¹

Department of Immunology, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany,¹ Laboratory of Immunology, Department of Laboratory Sciences, Gunma University School of Health Sciences, Maebashi, Gunma 371-8511, Japan,² Central Support Unit Biochemistry, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany³

Received 24 February 2006/ Returned for modification 1 May 2006/ Accepted 16 July 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The phenotypic and functional changes of glycolipid presented by CD1d(glycolipid/CD1d) specific V14⁺ T cells in the liver of mice at early stages of bacterial infection were investigated. After Listeria monocytogenes infection or interleukin-12 (IL-12) treatment, -galactosylceramide/CD1d tetramer-reactive (-GalCer/CD1d⁺) T cells coexpressing natural killer (NK) 1.1 marker became undetectable and, concomitantly, cells lacking NK1.1 emerged in both euthymic and thymectomized animals. Depletion of the NK1.1⁺ subpopulation prevented the emergence of -GalCer/CD1d⁺ NK1.1^– T cells. Before infection, NK1.1⁺, rather than NK1.1^–, -GalCer/CD1d⁺ T cells coexpressing CD4 were responsible for IL-4 production, whereas gamma interferon (IFN-) was produced by cells regardless of NK1.1 or CD4 expression. After infection, IL-4-secreting cells became undetectable among -GalCer/CD1d⁺ T cells, but considerable numbers of IFN--secreting cells were found among NK1.1^–, but not NK1.1⁺, cells lacking CD4. Thus, NK1.1 surface expression and functional activities of V14⁺ T cells underwent dramatic changes at early stages of listeriosis, and these alterations progressed in a thymus-independent manner. In mutant mice lacking all -GalCer/CD1d⁺ T cells listeriosis was ameliorated, suggesting that the subtle contribution of the NK1.1^– T-cell subset to antibacterial protection is covered by more profound detrimental effects of the NK1.1⁺ T-cell subset.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Natural killer (NK) T cells represent a unique T-cell population, which shares characteristic features with NK cells. Notably, both cell types surface express type II C-type lectin, NKR-P1B, and C (NK1.1) (3). In the mouse, the majority of NKT cells express an invariant T-cell receptor (TCR) chain encoded by V14 gene segments paired with J18 and a highly biased TCRVß toward Vß8.2, Vß7, and Vß2 (3). The development of V14⁺ NKT cells depends on CD1d, which is surface expressed together with ß₂-microglobulin (ß2m) (3, 7, 39, 48). The -galactosylceramide (-GalCer), which is derived from a marine sponge, is recognized by all V14⁺ NKT cells in the context of CD1d (29), and microbial ligands have recently been identified (19, 31, 38). The V14⁺ NKT cells are abundant in the liver, where the majority of cells express CD4 and few cells lack both CD4 and CD8 (12). Liver V14⁺ NKT cells rapidly secrete high concentrations of both gamma interferon (IFN-) and interleukin-4 (IL-4) upon TCR ligation (12, 15, 16, 17).

Listeria monocytogenes is a gram-positive facultative intracellular bacterium that preferentially replicates in macrophages and liver parenchymal cells (28). Type I cytokines, notably IL-12 and IFN-, play a pivotal role in protection against experimental listeriosis of mice (1, 2, 26, 27, 41, 45, 54, 55, 57), whereas type II cytokines such as IL-4 exacerbate disease (22, 50, 53, 56). After systemic infection, the vast majority of L. monocytogenes organisms are rapidly trapped in the liver (34). Hence, immunocompetent cells, which reside in the liver, are critical for the control of infection (20, 28). Although sterile eradication of this pathogen is ultimately achieved by conventional T cells (28), IFN--secreting NK1.1⁺ cells seem to participate in protection against L. monocytogenes infection (1, 2, 26, 28, 41, 45, 47, 55).

We have previously shown that cells stained with monoclonal antibodies (MAbs) to CD4 and NK1.1 (CD4⁺ NKT cells) become undetectable in the liver of mice after L. monocytogenes infection (17, 18), which could be due to downmodulation of the NK1.1 marker upon activation (6, 44). In the present study, we assessed NK1.1 expression on V14⁺ NKT cells in the liver of mice at early stages of listeriosis by using -GalCer-loaded CD1d (-GalCer/CD1d) tetramers. We found that during listeriosis, an -GalCer/CD1d tetramer-reactive (-GalCer/CD1d⁺) NK1.1^– T-cell population developed from an NK1.1⁺ subpopulation in a thymus-independent manner. These cells secreted IFN- but not IL-4. We assume that this -GalCer/CD1d⁺ NK1.1^– subset contributes to early antilisterial resistance, thus bridging the gap between early resistance mediated by professional phagocytes and subsequent acquired immunity mediated by conventional T cells. However, listeriosis was ameliorated in mice lacking -GalCer/CD1d⁺ T cells. It is possible that the NK1.1⁺ subset, which produces IL-4 in addition to IFN-, is a detriment to the infected host and covers protective effects of the NK1.1^– subset, which exclusively produces IFN-.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. Female adult thymectomized (ATX, 8 weeks after birth) C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Breeding pairs of J18^–/– (29) and C57BL/6 Vß^a (Vß^a) mice were kindly provided by M. Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) and A. M. Livingstone (Imperial College of Science, Technology, and Medicine, London, Great Britain), respectively. These mutants backcrossed onto C57BL/6 mice (J18^–/– and J18^+/–, 8th generation; Vß^a, 18th generation), and C57BL/6 mice were maintained under specific-pathogen-free conditions, and weight-matched female mice were used at 8 to 12 weeks of age.

Antibodies. MAbs to TCR/ß (H57-597), TCR/ (GL3), CD3 (145-2C11), NK1.1 (PK136), CD4 (YTS.191.1), CD8 (YTS169.4), Fc receptor (FcR) (2.4G2), IL-12 (p40/p70) (C17.8), IL-4 (11B11, BVD6-24G2), and IFN- (R4-6A2, XMG1.2) were purified from hybridoma culture supernatants. MAbs to IFN- (XMG1.2) and IL-4 (BVD6-24G2) were biotinylated, and MAbs to TCR/ß and CD3 were conjugated with fluorescein isothiocyanate (FITC) by conventional methods. FITC-conjugated MAbs to CD11a (M17/4), CD54 (H9.2B8), CD25 (7D4), CD122 (TM-ß1), CD49d (R1-2), CD69 (H1.2F3), CD4 (H129.19), NK1.1 (PK136), and mouse immunoglobulin G2a (IgG2a; R19-15); biotinylated MAbs to NK1.1 (PK136), rat IgG2b (G15-337), and mouse IgG2a (R19-15); and biotinylated mouse IgG2a (G155-178) were purchased from BD PharMingen (Hamburg, Germany). Rabbit anti-asialo GM1 (ASGM1) antibody and rabbit IgG were obtained from Wako Chemicals (Neuss, Germany) and Sigma-Aldrich (Schnelldorf, Germany), respectively.

Bacteria and infection. L. monocytogenes (strain EGD) recovered from infected liver were grown in tryptic soy broth (Difco Laboratories, Detroit, MI) at 37°C for 18 h, and aliquots were frozen at –80°C until used. The final concentration of viable bacteria was enumerated by plate counts on tryptic soy agar (Difco). Mice were infected intravenously with 2 x 10³ L. monocytogenes.

In vivo treatment. Mice were treated intraperitoneally (i.p.) with 1 µg of recombinant IL-12 (rIL-12; Genzyme, Alzenau, Germany). To deplete NK1.1⁺ cells, mice were treated i.p. with anti-NK1.1 MAb (300 µg) 4 and 2 days before infection. For depletion of NK cells, mice were treated i.p. with anti-ASGM1 antibody (5 mg) 3 days before infection. To deplete CD4⁺ cells, mice were treated i.p. with anti-CD4 MAb (300 µg) 4 and 2 days before infection. Depletion of NK1.1⁺ cells (>95%), NK cells (>95%), and CD4⁺ cells (>98%) was verified by immunohistochemistry and/or flow cytometry. For endogenous IL-12 neutralization, mice were treated i.p. with anti-IL-12 MAb (500 µg) 2 h before infection. Isotype-matched MAbs purified by the same procedure as for specific MAbs or phosphate-buffered saline (PBS) used for MAb purification were used as controls. Rabbit IgG served as a control for anti-ASGM1 antibody.

Mouse CD1d/ß2m tetramer. Mouse CD1d/ß2m tetramers were prepared by using the baculovirus expression system using a CD1d construct with a BirA biotinylation site, followed by a His₆ tag as described previously (37). In brief, Sf9 insect cell line (BD Biosciences, Heidelberg, Germany) was infected with mouse CD1d/ß2m-expressing virus stock kindly provided by M. Kronenberg (37) at a multiplicity of infection of 0.1 for expanding the viral stock. Culture supernatants were harvested on day 4 postinfection (p.i.) and used at high multiplicity of infection of 1 to 5. These large-scale cultures were performed in Sf-900 II serum-free medium (Gibco/Invitrogen Corp., Karlsruhe, Germany) and harvested on day 3 or 4 by centrifugation. Supernatants were concentrated by passing through a hollow-fiber tangential flow module (MiniKros 1,100 cm²; Spectrum; MembraPure, Boddenheim, Germany). The CD1d molecules were purified by metal-chelating chromatography (Chelating Sepharose Fast Flow; Amersham Pharmacia Biotech, Uppsla, Sweden) on an NTA-Sepharose column (Amersham Pharmacia Biotech) charged with cobalt chloride (Roth, Karsruhe, Germany). Protein was eluted with 200 mM imidazole (Merck, Darmstadt, Germany), and pooled fractions were concentrated to 0.5 ml by ultrafiltration (Ultrafree units; Millipore, Bedford, MA). Purity and protein amount were assessed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Bradford reagent (Bio-Rad, Munich, Germany), respectively. Purified CD1d protein was subsequently biotinylated with BirA enzyme (Avidity, Denver, CO) according to the manufacturer's instructions. Biotinylated CD1d proteins were purified by gel filtration (Superdex 200 HR10/30; Amersham Pharmacia Biotech). Soluble biotinylated CD1d/ß2m protein was loaded with or without -GalCer (kindly provided by Kirin Brewery Co., Ltd., Tokyo, Japan), which was dissolved in PBS containing 0.5% Tween 20 at 220 µg/ml at a molar ratio of 1:3 (protein-lipid) overnight at room temperature. For tetramerization, streptavidin (SA)-conjugated phycoerythrin (PE) (MobiTec, Göttingen, Germany) was added to -GalCer/CD1d/ß2m monomers at a 1:4 (monomer-SA-conjugated PE) molar ratio. The purification of PE-labeled CD1d/ß2m tetramers loaded with or without -GalCer was performed by gel filtration (Superdex 200 HR10/30; Amersham Pharmacia Biotek).

Cell preparation and flow cytometry. Mice were killed by cervical dislocation and organs were collected. Hepatic leukocytes (HLs) were prepared as described previously (15). Bone marrow (BM) plugs in the femurs were eluted by flushing with RPMI 1640 containing 10% fetal calf serum (FCS). T cells were enriched by passage through a nylon wool column. Splenocytes were prepared by conventional methods. For staining with MAbs, cells were incubated with anti-FcR MAb and then stained with conjugated MAbs at 4°C for 15 min. Biotinylated MAbs were visualized by using SA-conjugated CyChrome (BD PharMingen). Stained cells were washed with PBS containing 0.1% bovine serum albumin (Serva, Heidelberg, Germany) and 0.1% sodium azide (Merck, Darmstadt, Germany), fixed with 1% paraformaldehyde (Merck), and acquired by FACScan or FACSCalibur (BD Biosciences, Mountain View, CA), and lymphoid cells were analyzed with CellQuest software. For staining with -GalCer/CD1d tetramer, cells were stained with PE-labeled -GalCer/CD1d tetramers for 15 min at room temperature after blocking.

Determination of CFU. Mice were killed by cervical dislocation on day 4 p.i. The liver was perfused with 10 ml of sterile PBS to wash out bacteria in the blood vessels, and CFU counts in the liver were determined by plating serial dilutions of liver homogenates on tryptic soy agar plates.

Cell sorting. -GalCer/CD1d⁺ cells were positively sorted by magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. In brief, HLs were stained with PE-labeled -GalCer/CD1d tetramers for 15 min at room temperature after blocking and subsequently incubated with anti-PE microbeads (Miltenyi Biotec) at 6°C for 15 min. Cell suspensions were applied to an MS+/RS+ column (Miltenyi Biotec), and then -GalCer/CD1d⁺ cells were enriched. The purity of -GalCer/CD1d⁺ cells was consistently >95%.

ELISPOT assay. IFN- and IL-4 production was measured by the enzyme-linked immunospot (ELISPOT) method as described previously (15, 17) with a slight modification. Briefly, cells were cultured for 18 h in the presence or absence of phorbol myristate acetate (PMA; 10 ng/ml; Sigma-Aldrich) and ionomycin (1 µg/ml) in ELISPOT plates (Millipore, Eschborn, Germany) precoated with anti-IFN- MAb (R4-6A2) or anti-IL-4 MAb (11B11). After being washed, the plates were incubated with biotinylated anti-IFN- MAb (XMG1.2) or biotinylated anti-IL-4 MAb (BVD6-24G2), respectively. For developing spots, SA-conjugated alkaline phosphatase (Dianova, Hamburg, Germany) and BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium tablets (Sigma-Aldrich) were used. The numbers of cytokine-secreting cells were estimated by counting spots using a dissecting microscope.

Statistical analysis. Statistical significance was determined by using a Student t test, and P values of <0.05 were regarded as significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential appearance of NK1.1⁺ and NK1.1^– subsets of -GalCer/CD1d⁺ T cells in the liver after L. monocytogenes infection. C57BL/6 mice were infected with L. monocytogenes, and the kinetics of V14⁺ T cells in the liver were monitored using -GalCer/CD1d tetramers. High proportions of -GalCer/CD1d⁺ T cells, as well as NK1.1⁺ TCR/ß cells, were detected in the liver before infection (Fig. 1A). Consistent with recent findings (4), the majority of -GalCer/CD1d⁺ T cells coexpressed NK1.1, and a small population lacked this marker. The -GalCer/CD1d⁺ T cells were undetectable in J18^–/– mice, verifying that V14⁺ T cells are specifically detected by -GalCer/CD1d tetramers (data not shown). The proportions of -GalCer/CD1d⁺ T cells, as well as NK1.1⁺ TCR/ß cells, were markedly reduced by day 2 p.i. and were still low on day 4 p.i. (Fig. 1A). At these time points, only a few -GalCer/CD1d⁺ T cells coexpressed the NK1.1 marker.

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FIG. 1. Appearance of -GalCer/CD1d+ T cells in the liver of C57BL/6 mice in response to L. monocytogenes infection or rIL-12 treatment. Mice were infected with L. monocytogenes (A, B, and C) or treated with rIL-12 (D) on day 0, and HLs were prepared on the days indicated in the figure. Cells were stained with FITC-conjugated anti-TCR/ß MAb, biotinylated anti-NK1.1 MAb, and PE-labeled -GalCer/CD1d tetramers, followed by SA-conjugated CyChrome. (A) Data are expressed as dot plots after gating on lymphoid cells. The numbers in the dot plots represent the percentages of each cell population within the square. Representative staining patterns from three to six mice at each time point are shown. (B) Data represent recovery numbers of HLs and are expressed as means of three to six mice at each time point. Vertical lines represent error bars. (C and D) Data represent absolute numbers of -GalCer/CD1d+ NK1.1+ T cells (), -GalCer/CD1d+ NK1.1– T cells (), and total -GalCer/CD1d+ T cells () and are expressed as means of three to six mice at each time point. *, P < 0.05; **, P < 0.01 (before infection versus after infection).

The numbers of HLs gradually increased after infection (Fig. 1B). The absolute numbers of -GalCer/CD1d⁺ NK1.1⁺ T cells were markedly diminished by day 2 p.i., whereas the numbers of -GalCer/CD1d⁺ NK1.1^– T cells increased subsequently, and the latter were dominant among the -GalCer/CD1d⁺ T-cell population on days 2 and 4 p.i. (Fig. 1C). No inverse relationship was found in the appearance of NK1.1⁺ and NK1.1^– -GalCer/CD1d⁺ T cells after L. monocytogenes infection. Similar results were obtained in Vß^a mice (data not shown). Thus, the NK1.1⁺ and NK1.1^– subsets of -GalCer/CD1d⁺ T cells showed differential kinetics during listeriosis.

Selective expansion of the -GalCer/CD1d⁺ NK1.1^– T-cell subset after L. monocytogenes infection involves IL-12. IL-12 has been shown to contribute to the compression of the NKT cell population coexpressing CD4 in the livers of L. monocytogenes-infected mice (18). Here we determined whether IL-12 is involved in the expansion of the -GalCer/CD1d⁺ NK1.1^– T-cell subset. Consistent with previous findings (44), the absolute numbers of the -GalCer/CD1d⁺ NK1.1⁺ T-cell subset were markedly diminished by day 2 after rIL-12 treatment, whereas the NK1.1^– T-cell subset was virtually unaffected at this time point and expanded thereafter (Fig. 1D). Consistent with this, anti-IL-12 MAb treatment reversed the reduction of the NK1.1⁺ T-cell subset and the subsequent increase of the NK1.1^– T-cell subset of the -GalCer/CD1d⁺ population during listeriosis (Fig. 2A). Similar results were obtained in Vß^a mice (data not shown). Consistent with previous findings (54), the CFU counts in the liver were significantly, although slightly, reduced by anti-IL-12 MAb treatment (Fig. 2B). Hence, IL-12 plays a critical role both in the reduction of the NK1.1⁺ subset and in the subsequent increase of the NK1.1^– subset of the -GalCer/CD1d⁺ T-cell population.

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FIG. 2. Influence of endogenous IL-12 neutralization on emergence of -GalCer/CD1d+ NK1.1– T cells after L. monocytogenes infection and on bacterial growth in the liver. Mice were treated with anti-IL-12 MAb or PBS 2 h before L. monocytogenes infection. (A) HLs were prepared on day 4 p.i., and cells were stained with PE-labeled -GalCer/CD1d tetramers and biotinylated anti-NK1.1 MAb, followed by SA-conjugated CyChrome. The data are expressed as dot plots after gating on lymphoid cells. The numbers in the dot plots represent percentages of each cell population within the square. Representative staining patterns from three mice in each group are shown. (B) CFU in the liver were determined on day 4 p.i. The data are from five mice, and each symbol represents CFU in an individual animal. Horizontal bars represent mean CFU. Because no significant difference was found between rat IgG2a (isotype-matched MAb for anti-IL-12 MAb)-treated and PBS-treated group in another experiment, PBS was used as a control. , P < 0.05 (PBS-treated group versus the anti-IL-12 MAb-treated group).

The NK1.1^– T-cell subset is derived from the NK1.1⁺ subset of -GalCer/CD1d⁺ T-cell population. To determine whether the appearance of the NK1.1^– T-cell subset during listeriosis was due to downmodulation of the NK1.1 marker, the consequences of NK1.1⁺ cell depletion on the emergence of -GalCer/CD1d⁺ NK1.1^– T cells were assessed. In addition to NK (CD3^–NK1.1⁺) cells, total NKT (CD3⁺NK1.1⁺) cells, as well as -GalCer/CD1d⁺ NK1.1⁺ T cells, became virtually undetectable in the liver after anti-NK1.1 MAb treatment, whereas the proportion of -GalCer/CD1d⁺ NK1.1^– T cells increased (Fig. 3A). Labeling with FITC-conjugated anti-mouse IgG2a MAb (secondary MAb for detection of anti-NK1.1 MAb) did not stain cells from MAb-treated mice, excluding the residual coating of the cells with MAb (data not shown). It is likely that some cells, which were slightly stained with anti-NK1.1 MAb, still remained in the liver even after anti-NK1.1 MAb treatment. This was probably caused by a biotin-mediated nonspecific reaction, because (i) similar staining patterns were obtained when cells were stained with biotinylated mouse IgG2a (isotype-matched MAb for anti-NK1.1 MAb; Fig. 3B) and (ii) NK1.1 staining was completely negative when FITC-conjugated MAb to NK1.1 was used (Fig. 3C). The increase in both the proportion and absolute numbers of -GalCer/CD1d⁺ NK1.1^– T cells after L. monocytogenes infection was prevented by NK1.1⁺ cell depletion (Fig. 3A and D), suggesting that NK1.1⁺ cells are critical for the development of -GalCer/CD1d⁺ NK1.1^– T cells in the livers of L. monocytogenes-infected mice.

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FIG. 3. Influence of anti-NK1.1 MAb or anti-ASGM1 antibody treatment on -GalCer/CD1d+ T cells in the L. monocytogenes-infected liver of C57BL/6 mice. Mice were treated with anti-NK1.1 MAb or PBS on days –4 and –2 (A, B, C, and D), or with anti-ASGM1 antibody or rabbit IgG on day –3 (E and F), infected with L. monocytogenes on day 0, and the HLs were prepared on day 0 (A, B, C, and E) and/or day 4 (A, C, D, E, and F). Cells were stained with FITC-conjugated anti-CD3 MAb, biotinylated anti-NK1.1 MAb and PE-labeled -GalCer/CD1d tetramers, followed by SA-conjugated CyChrome (A, D, E, and F), biotinylated mouse IgG2a, and PE-labeled -GalCer/CD1d tetramers, followed by SA-conjugated CyChrome (B), or FITC-conjugated anti-NK1.1 MAb and PE-labeled -GalCer/CD1d tetramers (C). (A) Data are expressed as dot plots after gating on lymphoid cells. The numbers in the dot plots represent percentages of each cell population within the square. Representative staining patterns from eight mice in each group are shown. Because no significant difference was found between the anti-human transferrin receptor MAb (isotype-matched MAb for anti-NK1.1 MAb)-treated and PBS-treated groups in another experiment, PBS was used as a control. (B) The data are expressed as dot plots after gating on lymphoid cells. The numbers in the dot plots represent percentages of the cell population within the square. Representative staining pattern from two mice is shown. (C) The data are expressed as dot plots after gating on lymphoid cells. The numbers in dot plots represent the percentages of cell populations within the square. Representative staining patterns from two mice in each group are shown. (D) The data represent absolute numbers of NK1.1+ and NK1.1– -GalCer/CD1d+ T cells. Each symbol represents an individual animal, and horizontal bars represent the means of eight mice. *, P < 0.01 (anti-NK1.1 MAb-treated group versus the PBS-treated group). (E) The data are expressed as dot plots after gating on lymphoid cells. The numbers in the dot plots represent percentages of each cell population within the square. Representative staining patterns from five mice per group are shown. As a control for polyclonal anti-ASGM1 antibody (rabbit IgG), rabbit IgG was used. (F) The data represent the absolute numbers of NK1.1+ and NK1.1– -GalCer/CD1d+ T cells. Each symbol represents an individual animal, and horizontal bars represent the means of five mice. *, P < 0.01 (anti-ASGM1 antibody-treated group versus the rabbit IgG-treated group).

Because some T cells bearing CD8 and/or TCR/ also coexpress NK1.1 (11, 13), we determined the involvement of these cells in the expansion of the -GalCer/CD1d⁺ NK1.1^– T-cell subset. No measurable alterations were found in the numbers of -GalCer/CD1d⁺ NK1.1^– T cells in the liver of L. monocytogenes-infected mice after CD8⁺ or TCR/⁺ cell depletion (data not shown). These results exclude the involvement of NK1.1⁺ cells expressing CD8 and/or TCR/ in the expansion of the -GalCer/CD1d⁺ NK1.1^– T-cell population.

Because both NKT cells and NK cells express NK1.1, we examined whether NK cells participate in the increase of the -GalCer/CD1d⁺ NK1.1^– T-cell population. C57BL/6 mice were treated with anti-ASGM1 antibody and infected with L. monocytogenes, and the appearance of -GalCer/CD1d⁺ NK1.1^– T cells was assessed. Consistent with previous findings (14, 40), NK (CD3^– NK1.1⁺) cells became undetectable in the liver after anti-ASGM1 antibody treatment, whereas -GalCer/CD1d⁺ T cells were virtually unaffected (Fig. 3E). In contrast to anti-NK1.1 MAb treatment, both the proportion and the absolute number of -GalCer/CD1d⁺ NK1.1^– T cells during listeriosis were increased by anti-ASGM1 antibody treatment (Fig. 3E and F). These results indicate that NK cells prevent the development of -GalCer/CD1d⁺ NK1.1^– T cells in the livers of L. monocytogenes-infected mice. We conclude that the NK1.1^– T-cell subset, which appears in the L. monocytogenes-infected liver, is primarily derived from the NK1.1⁺ subpopulation of -GalCer/CD1d⁺ T cells.

-GalCer/CD1d⁺ NK1.1⁺ T cells in the thymus are not affected by L. monocytogenes infection. Mice were treated with anti-NK1.1 MAb, and the numbers of -GalCer/CD1d⁺ NK1.1⁺ T cells in the thymus and BM were determined. A small but distinct population of -GalCer/CD1d⁺ T cells was detected in the BM, and the majority of these coexpressed NK1.1 (Fig. 4A). In contrast, virtually all -GalCer/CD1d⁺ T cells in the thymus coexpressed NK1.1 on their cell surface. Similar to the liver, -GalCer/CD1d⁺ NK1.1⁺ T cells in BM and thymus became virtually undetectable after anti-NK1.1 MAb treatment (Fig. 4A). This was also true in other organs, including the spleen (data not shown). In parallel to the compression of the NK1.1⁺ subset, the proportion of the NK1.1^– subset of -GalCer/CD1d⁺ T cells increased in both organs after anti-NK1.1 MAb treatment (Fig. 4A), although the absolute numbers remained virtually unchanged (data not shown). Slight staining with anti-NK1.1 MAb was due to a biotin-mediated nonspecific reaction (data not shown). Thus, anti-NK1.1 MAb treatment efficiently depleted -GalCer/CD1d⁺ NK1.1⁺ T cells from the lymphoid organs.

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FIG. 4. Influence of NK1.1+ cell depletion or L. monocytogenes infection on -GalCer/CD1d+ T cells in the BM and thymus of C57BL/6 mice. (A) Mice were treated with anti-NK1.1 MAb or PBS on days 0 and 2, and BM cells and thymocytes were prepared on day 4. Cells were stained and analyzed as in Fig. 3. Representative staining patterns from eight mice in each group are shown. Because no significant difference was found between anti-human transferrin receptor MAb (isotype-matched MAb for anti-NK1.1 MAb)-treated and PBS-treated groups in another experiment, PBS was used as a control. (B) Mice were infected with L. monocytogenes, and BM cells and thymocytes were prepared on days 0 and 2 p.i. Cells were stained and analyzed as in Fig. 1. Representative staining patterns from three to six mice in each group are shown.

Similar to the liver, -GalCer/CD1d⁺ NK1.1⁺ T cells became virtually undetectable in the BM of L. monocytogenes-infected mice (Fig. 4B). In contrast, in the thymus of L. monocytogenes-infected mice considerable numbers of the NK1.1⁺ subset persisted, and the subset of -GalCer/CD1d⁺ T cells did not enlarge. This discrepancy is probably due to differential IL-12 levels in different organs: high frequencies of IL-12-producing cells were detected in BM and liver during listeriosis, whereas IL-12 producers were virtually absent from the thymus (data not shown). Thus, thymic -GalCer/CD1d⁺ NK1.1⁺ T cells were apparently unaffected by L. monocytogenes infection.

Thymus-independent emergence of -GalCer/CD1d⁺ NK1.1^– T cells. ATX mice were infected with L. monocytogenes, and the numbers of liver V14⁺ T cells were determined by using -GalCer/CD1d tetramers. Similar to euthymic mice, high numbers of -GalCer/CD1d⁺ T cells were detected in the liver before infection, and the majority of these cells coexpressed NK1.1 (Fig. 5). Absolute numbers of the NK1.1⁺ subset were markedly diminished by day 4 p.i., whereas the NK1.1^– subset of -GalCer/CD1d⁺ T cells was numerically increased (Fig. 5). Thus, numerical alterations in the NK1.1⁺ and NK1.1^– subsets of the -GalCer/CD1d⁺ T-cell populations, in response to L. monocytogenes infection, were thymus independent.

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FIG. 5. Appearance of -GalCer/CD1d+ T cells in the L. monocytogenes-infected liver of ATX mice. Mice were infected with L. monocytogenes on day 0, and HLs were prepared on days 0 and 4 p.i. Cells were stained as described in Fig. 1. The data represent absolute numbers of -GalCer/CD1d+ NK1.1+ T cells () and -GalCer/CD1d+ NK1.1– T cells () and are expressed as the means of three mice at each time point. *, P < 0.01 (day 0 versus day 4).

Phenotypic changes of -GalCer/CD1d⁺ T cells during listeriosis. We compared the surface expression of various markers on -GalCer/CD1d⁺ T cells before and after L. monocytogenes infection. In uninfected mice, the vast majority of -GalCer/CD1d⁺ T cells expressed CD122 and high levels of CD11a, and this expression pattern remained unchanged during infection (data not shown). In contrast, CD69, CD54, CD25, and CD49d were upregulated after infection (Fig. 6). These results suggest that -GalCer/CD1d⁺ NK1.1^– T cells are highly activated.

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FIG. 6. Cell surface phenotype of -GalCer/CD1d+ T cells in the liver of C57BL/6 mice before and after L. monocytogenes infection. Mice were infected with L. monocytogenes, and HLs were prepared on days 0 and 4 p.i. Cells were stained with PE-labeled -GalCer/CD1d tetramers and the FITC-conjugated or biotinylated MAbs indicated in the figure, followed by SA-conjugated CyChrome. The data are expressed as histograms after gating on -GalCer/CD1d+ T cells. Representative staining patterns from three to six mice are shown.

Similarly, we compared CD4 expression on -GalCer/CD1d⁺ T cells in the liver during listeriosis. Before infection, the majority (80%) of -GalCer/CD1d⁺ T cells coexpressed CD4 and a minority (20%) lacked this marker (Fig. 7A). In contrast, the percentages of CD4⁺ and CD4^– cells among -GalCer/CD1d⁺ T cells shifted to 65 and 35%, respectively, on day 4 p.i. Hence, L. monocytogenes infection primarily compressed the CD4⁺ rather than CD4^– CD8^– (CD4^–8^–) -GalCer/CD1d⁺ T-cell population.

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FIG. 7. CD4 surface expression on -GalCer/CD1d+ T cells in the liver of C57BL/6 mice before and after L. monocytogenes infection and influence of CD4+ or NK1.1+ cell depletion on IFN- and IL-4 production by -GalCer/CD1d+ T cells. (A) Mice were infected with L. monocytogenes, and HLs were prepared on days 0 and 4 p.i. Cells were stained with FITC-conjugated anti-CD4 MAb, biotinylated anti-NK1.1 MAb, and PE-labeled -GalCer/CD1d tetramers, followed by SA-conjugated CyChrome. The data of NK1.1 versus -GalCer/CD1d tetramers are expressed as dot plots after gating on lymphoid cells. Profiles of CD4 are expressed as histograms after gating on -GalCer/CD1d+ T cells. Representative staining patterns from five mice per group are shown. (B) Mice were treated with anti-CD4 MAb, anti-NK1.1 MAb, or PBS on days –4 and –2 and infected with L. monocytogenes on day 0, and HLs were prepared on day 0 and/or day 4. Cells were stained with PE-labeled -GalCer/CD1d tetramers, followed by anti-PE microbeads. -GalCer/CD1d+ cells were positively sorted by magnetic cell sorter and cultured in the presence or absence of PMA and ionomycin on ELISPOT plates coated with either anti-IFN- MAb or anti-IL-4 MAb. The data are expressed as means of duplicate cultures (standard deviation of <10%). Experiments were performed three times with comparable results. +, In the presence of PMA and ionomycin; –, in the absence of PMA and ionomycin. Because virtually all -GalCer/CD1d+ T cells in the liver on day 4 p.i. lacked NK1.1, anti-NK1.1 MAb treatment was not performed in infected mice. Because no significant difference was found in the anti-human transferrin receptor MAb (isotype-matched MAb for anti-NK1.1 MAb)-treated, rat IgG2b MAb (isotype-matched MAb for anti-CD4 MAb)-treated, and PBS-treated groups in another experiment, PBS was used as a control.

Preferential IFN- production by CD4^–8^– -GalCer/CD1d⁺ T cells lacking CD4, CD8, and NK1.1 during listeriosis. We previously reported that IL-4-secreting cells in the liver become virtually undetectable in parallel with the disappearance of CD4⁺ NK1.1⁺ T cells after L. monocytogenes infection (17, 18). Because -GalCer/CD1d⁺ NK1.1^– T cells, which developed in the liver after L. monocytogenes infection, are phenotypically different from the NK1.1⁺ subpopulation that resides in naive mice, we wondered whether the NK1.1^– and NK1.1⁺ subpopulations differ functionally. To address this issue, the -GalCer/CD1d⁺ T cells were purified from the liver before and after infection, and IFN- and IL-4 secretion were determined by ELISPOT assay. Substantial numbers of IFN- and IL-4 producers were detected among -GalCer/CD1d⁺ T cells from uninfected animals after in vitro stimulation with PMA and ionomycin (Fig. 7B). Considerable numbers of IFN- producers were found among -GalCer/CD1d⁺ T cells from L. monocytogenes-infected mice, whereas IL-4-producing cells became virtually undetectable. These results suggest that -GalCer/CD1d⁺ NK1.1^– T cells, which emerged in the L. monocytogenes-infected liver, fail to produce IL-4 and hence express a Th1-like phenotype.

The -GalCer/CD1d⁺ T cells segregate into several subsets on the basis of CD4/NK1.1 surface expression. Moreover, the NK1.1⁺ and NK1.1^– subsets are differentially influenced by L. monocytogenes infection. Finally, the CD4⁺ and CD4^–8^– -GalCer/CD1d⁺ T-cell populations are differentially influenced by L. monocytogenes infection. Hence, we wondered whether the functional activities of V14⁺ T cells coexpressing CD4 and/or NK1.1 differ from those lacking CD4 and/or NK1.1. To address these issues, mice were treated with anti-CD4 or anti-NK1.1 MAb and infected with L. monocytogenes, and the frequencies of IL-4 and IFN- producers among -GalCer/CD1d⁺ T cells were determined by ELISPOT assay. NK1.1⁺ cells and CD4⁺ cells became virtually undetectable in the liver after anti-NK1.1 Mab and anti-CD4 MAb treatment, respectively (data not shown). Labeling with FITC-conjugated anti-mouse IgG2a MAb (secondary MAb for detection of anti-NK1.1 MAb) or FITC-conjugated anti-rat IgG2b MAb (secondary MAb for detection of anti-CD4 MAb) did not stain cells from MAb-treated mice, excluding the residual coating of the cells with MAbs (data not shown). In uninfected mice, CD4⁺ or NK1.1⁺ cell depletion markedly reduced the numbers of IL-4-producing -GalCer/CD1d⁺ T cells, whereas the numbers of IFN- producers were comparable in both groups (Fig. 7B). In contrast, after infection, the frequencies of IFN--producing -GalCer/CD1d⁺ T cells were 3.5-fold higher in CD4⁺ cell-depleted mice compared to nondepleted mice. These results suggest that -GalCer/CD1d⁺ NK1.1^– T cells in the L. monocytogenes-infected liver, which produce IFN-, are preferentially CD4^–8^–.

J18^–/– mice are more resistant than control animals to L. monocytogenes infection. IFN- is essential for protection against L. monocytogenes infection (1, 2, 26, 27, 41, 45, 54, 55, 57), whereas IL-4 exacerbates the disease (22, 50, 53, 56). Because high numbers of IFN--secreting cells were detected in the livers of L. monocytogenes-infected mice, we determined whether V14⁺ T cells participate in resistance against L. monocytogenes infection. To this end, J18^–/– and J18^+/– mice were infected with L. monocytogenes, and bacterial burdens were determined on day 4 p.i. Listerial CFU in both livers and spleens were significantly lower in J18^–/– mice than in J18^+/– mice (Fig. 8). Thus, J18^–/– mice were more resistant to L. monocytogenes infection than heterozygous littermates. These findings suggest that -GalCer/CD1d⁺ T cells comprise a heterogeneous population, which in its entirety does not contribute to antilisterial protection and may even exacerbate disease. We consider it likely that the IFN--producing NK1.1^– subset of -GalCer/CD1d⁺ T cells ameliorates, while the IL-4-producing NK1.1⁺ subset exacerbates, disease.

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FIG. 8. Bacterial growth in livers of J18–/– and J18+/– mice after L. monocytogenes infection. Mice were infected with L. monocytogenes, and CFU counts in livers and spleens were determined on day 4 p.i. The data are from five mice, and each symbol represents the CFU in an individual animal. Horizontal bars represent mean CFU. *, P < 0.02 (J18–/– versus J18+/–). Experiments were performed twice with comparable results.

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Our findings reveal the disappearance of the IL-4-producing NK1.1⁺ subset of -GalCer/CD1d⁺ T cells and the appearance of IFN--producing NK1.1^– subset in the livers of L. monocytogenes-infected mice. Thus, NK1.1 surface expression and functional activities of glycolipid presented by CD1d-specific V14⁺ T cells are markedly and rapidly influenced by listerial infection.

Similar kinetics of -GalCer/CD1d⁺ T cells were seen in Vß^a mice, in which V14⁺ T cells express TCRVß7 or TCRVß2, but not TCRVß8 (42). Moreover, most -GalCer/CD1d⁺ NK1.1^– T cells in the L. monocytogenes-infected liver expressed TCRVß8, and only few expressed TCRVß7 and TCRVß2 (I. Yoshizawa and M. Emoto, unpublished results). We conclude that the compression of the -GalCer/CD1d⁺ NK1.1⁺ T-cell population and the subsequent expansion of NK1.1^– subpopulations occur independently of TCRVß usage.

IL-15 plays a central role in the proliferation of both NK cells and NKT cells (30, 33, 43). Recently, it was shown that the depletion of NK cells by anti-ASGM1 antibody treatment promotes the expansion of thymus-derived V14⁺ T cells in the liver due to increased local concentrations of IL-15 (35). Because NK cells are numerically increased in the liver of mice during L. monocytogenes infection (53), we consider it likely that similar mechanisms underlie the numerical increase of -GalCer/CD1d⁺ NK1.1^– T cells after NK cell depletion.

The NK1.1⁺ subset in the periphery was recently found to be derived from a thymic NK1.1^– subpopulation of -GalCer/CD1d⁺ T cells (5). In that study, NK1.1 surface expression on V14⁺ T cells was acquired in the periphery after the cells had left the thymus during ontogeny. In our study, however, the NK1.1^– subset that emerged in the L. monocytogenes-infected liver developed from the NK1.1⁺ subpopulation of -GalCer/CD1d⁺ T cells. Hence, the accumulation of V14⁺ T cells in the liver is probably differentially regulated under physiological and under inflammatory conditions (10).

Expansion of V14⁺ T cells in response to -GalCer in vivo has been reported (8, 25, 58). Because the numbers of NK cells were increased after L. monocytogenes infection (53) and the expansion of V14⁺ T cells was severely impaired in the presence of NK cells (35, 46), it is unlikely that the -GalCer/CD1d⁺ NK1.1^– T-cell population was expanded in situ. An increase of the -GalCer/CD1d⁺ NK1.1^– T-cell population during listeriosis depended on IL-12. Hence, it is likely that stimulation by a specific antigen (-GalCer) and by the cytokine IL-12 have differential outcomes. Although we cannot formally exclude in situ expansion of V14⁺ T cells, we consider the following scenario most likely. After having left the BM, -GalCer/CD1d⁺ NK1.1⁺ T cells encounter endogenous IL-12 at sites of inflammation, resulting in loss of the NK1.1 surface marker.

CD4⁺ NKT cells differ from CD4^–8^– NKT cells in their cytokine production profile (9, 21, 23, 24, 32, 52). Generally, CD4⁺ rather than CD4^–8^– NKT cells are responsible for IL-4 production, albeit at various levels in different organs. In the liver, CD4⁺ cells dominate over CD4^–8^– cells among V14⁺ T cells (12, 15). Moreover, IL-4-producing cells among HLs are markedly reduced by NK1.1⁺ cell depletion (15). Finally, large numbers of IL-4 producers are detected among purified liver CD4⁺ NK1.1⁺ cells after TCR ligation (17). In our experiments, the numbers of IL-4-producing -GalCer/CD1d⁺ T cells from CD4⁺ or NK1.1⁺ cell-depleted mice were minute. Thus, it appears that under physiological conditions the CD4⁺NK1.1⁺ subset is mainly responsible for IL-4 production in the liver, although -GalCer/CD1d⁺ T cells express both IL-4 and IFN- mRNA upon stimulation (36, 49). Differential stimulation, i.e., specific antigen versus IL-12, is probably responsible for distinct cytokine production with inflammation driving IFN- production by V14⁺ T cells (17, 18; the present study).

At first sight, the finding that listeriosis in V14⁺ T-cell-deficient mice was ameliorated could be taken as an argument against a protective role of the V14⁺ T cells in listeriosis. However, as shown here, two subsets of V14⁺ T cells exist. (i) The first is the CD4⁺NK1.1⁺ subset, which produces IL-4 and hence should be of no benefit or even be a detriment in listeriosis. At early stages of infection, exacerbation by this T-cell population seems to dominate because depletion of the total V14⁺ T-cell population ameliorated listeriosis. This notion is consistent with previous findings showing that anti-CD1 MAb treatment improves listeriosis (51). (ii) The CD4^– NK1.1^– subset produces IFN-, suggesting its beneficial role in listeriosis. The contribution of the NK1.1^– subset to resistance, however, occurs later and seems supportive but not essential, because conventional T cells can assume the burden of protection. These more subtle effects of V14⁺ NK1.1^– T cells could not be visualized in mouse mutants devoid of the entire V14⁺ T-cell population because gene knockout mutants only allow identification of essential phenotypic features of gene deletion. However, recent studies revealed a contribution of V14⁺ T cells in protection against enteric listeriosis, suggesting a prevailing role of protective V14⁺ T cells in mucosal defense (47).

In conclusion, we show here the influences of bacterial infection on NK1.1 surface expression by V14⁺ T cells. Although V14⁺ T cells produce both IFN- and IL-4 in naive mice, the majority of this cell population produces IFN-, but not IL-4, during listeriosis due to abundant IL-12 production. Therefore, it is tempting to assume that distinct V14⁺ T-cell populations play unique roles in infection. Of these, the NK1.1⁺ subset seems ineffectual or even harmful, whereas the NK1.1^– subset appears to contribute to antilisterial protection by means of IFN-, thus probably bridging the gap between early resistance and subsequent acquired immunity.

.

 
This study was supported by grants from the German Science Foundation (SFB 421); a Grant-in-Aid for Scientific Research (grant 17590383) from the Japan Society for the Promotion of Science; The Waksman Foundation of Japan, Inc.; and the Japan Research Foundation for Clinical Pharmacology.

We are grateful to M. Taniguchi, A. M. Livingstone, M. Kronenberg, and the Kirin Brewery Co., Ltd., for providing J18^–/– mice, Vß^a mice, mouse CD1d/ß2m-expressing baculovirus, and -GalCer, respectively. We thank M. Stäber for MAb purification and Daniela Groine-Triebkorn for the screening of mice.

 
* Corresponding author. Mailing address: Laboratory of Immunology, Department of Laboratory Sciences, Gunma University School of Health Sciences, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Phone: 81-27-220-8935. Fax: 81-27-220-8935. E-mail: memoto{at}health.gunma-u.ac.jp.

Editor: J. L. Flynn

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Infection and Immunity, October 2006, p. 5903-5913, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00311-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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