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Originally published online as doi:10.1189/jlb.0108038 on September 4, 2008

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(Journal of Leukocyte Biology. 2008;84:1462-1471.)
© 2008 by Society for Leukocyte Biology

Bacterial infection alters the kinetics and function of iNKT cell responses

Hak-Jong Choi*,{dagger},1, Honglin Xu*,1, Yanbiao Geng*,1, Angela Colmone*, Hoonsik Cho*,{dagger} and Chyung-Ru Wang*,{dagger},2

* Department of Pathology, University of Chicago, Chicago, Illinois, USA; and
{dagger} Department of Microbiology and Immunology, Northwestern University, Chicago, Illinois, USA

2 Correspondence: Department of Microbiology and Immunology, Northwestern University, 320 E. Superior Street, Searle 3-401, Chicago, IL 60611, USA. E-mail: chyung-ru-wang{at}northwestern.edu


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ABSTRACT
 
CD1d-restricted V{alpha}14 invariant NKT cells (iNKT) are innate-like, immunoregulatory lymphocytes that play critical roles in autoimmunity, tumor surveillance, and infectious disease. Although iNKT cells are activated during microbial infection, the impacts of infection on the function of iNKT cells have not been fully characterized. Using a Listeria monocytogenes (LM) infection model, we found that iNKT cells failed to expand after infection, resulting in prolonged loss in the spleen, in contrast to the typical expansion and contraction of conventional T cells. iNKT cells from LM-infected mice responded more rapidly to secondary LM infection; however, they became functionally hyporesponsive to antigenic challenge for at least 1 month. This infection-induced hyporesponsiveness was also induced by Mycobacteria infection and was more profound in LM-infected, thymectomized mice, suggesting that infection-primed iNKT cells might have altered functionality. Interestingly, activation with {alpha}-galactosylceramide-loaded dendritic cells was able to overcome infection-induced hyporesponsiveness of iNKT cells, suggesting a role for extrinsic factors in this functional deficit. Taken together, these findings suggest that infection affects iNKT cell responses quantitatively and qualitatively. As humans are under constant microbial insult, predictions of iNKT cell function based on naïve animal models may not accurately reflect iNKT cell behavior in a clinical setting.

Key Words: CD1 • immunomodulation • immunotherapy


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INTRODUCTION
 
NKT cells are a unique subset of T lymphocytes that were defined initially as coexpressing TCR and NK cell receptors, such as CD161 in humans and NK1.1 in mice. A significant population of NKT cells is restricted to the nonclassical MHC molecule CD1d. These cells are functionally distinct from conventional CD4+ and CD8+ T cells, responding rapidly to lipid antigens rather than peptide antigens and secreting large amounts of Th1 and Th2 cytokines [1 , 2 ].

CD1-restricted NKT cells comprise at least two subsets. The majority of NKT cells, invariant NKT (iNKT) cells, expresses a restricted TCR repertoire (V{alpha}14J{alpha}18/Vβ8.2, Vβ7, or Vβ2 in mice; V{alpha}24J{alpha}18/Vβ11 in humans) and can be activated by the marine sponge-derived glycolipid {alpha}-galactosylceramide ({alpha}-GalCer) [1 , 3 ]. Another subset of the NKT cell, variant NKT (vNKT) cells, expresses diverse TCR {alpha} and β chains [4 ]. iNKT cells have been implicated in the control of various types of immune responses, including activation of responses against pathogens and tumors, as well as repression of responses that lead to autoimmune diseases [1 , 2 ]. vNKT cells, by contrast, have been shown to have a suppressive role in the regulation of tumor immunosurveillance [5 ].

{alpha}-GalCer has been used to specifically track and target iNKT cells in mice and humans [6 ]. In several mouse models, {alpha}-GalCer or its analogs have been shown to prevent tumor metastases, reduce autoimmunity, and enhance responses to infections [3 ]. In addition, {alpha}-GalCer has been tested recently in clinical trials with cancer patients [7 8 9 10 ]. As a result of its clinical potential, the kinetics and dynamics of iNKT cell responses to {alpha}-GalCer have been well characterized [11 12 13 ]. {alpha}-GalCer-stimulated iNKT cells respond much more rapidly than conventional T cells and down-modulate TCR and NK1.1 expression within 3–12 h of stimulation, thereby rendering these cells undetectable by flow cytometry. By 24 h after stimulation, iNKT cells can again be detected in peripheral organs, expanding at least ten- to 15-fold by Day 3. These cell numbers then contract to prestimulation levels by Days 7–10. Interestingly, a single administration of {alpha}-GalCer induces long-term hyporesponsiveness in iNKT cells (at least 1 month) in terms of their capacity to proliferate, produce cytokines, and transactivate other cell types [12 ].

{alpha}-GalCer and its structural analogs have not been found endogenously in mice or most infectious agents, however, making it possible that the kinetics and function of iNKT cells in physiological conditions may differ. Although the roles of iNKT cells in host defense against several microbial pathogens are well established [14 ], the effects of infection on the function of iNKT cells have not been fully understood. Two nonexclusive mechanisms have been shown to elicit iNKT cell activation during microbial infection. iNKT cells may directly recognize bacterial lipid antigens, as is the case against the LPS-negative bacterium Sphingomonas and the spirochete Borrelia burgdorferi. Alternately, bacterial infection may signal through TLRs, as is the case with the LPS-positive bacterium, Salmonella [15 16 17 ]. This signaling may result in altered CD1d expression, differential processing of endogenous antigens, or changes in accessory molecules and inflammatory cytokine production, therefore affecting iNKT cell responses [18 ].

Infection may play a key role in regulating iNKT cell number and function. Indeed, the differences in frequency and function of human and murine iNKT cells may in part be a result of previous exposure to microbial infection in humans [19 ]. Consistent with this notion, the numbers of iNKT cells are decreased significantly in tuberculosis patients and HIV-1+ individuals [20 , 21 ]. To study the effect of infection on the functionality of iNKT cells and their subsequent responses to antigenic challenge, we chose to use the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model organism. The kinetics of MHC class Ia- and class Ib-restricted T cell responses is well characterized in the LM system, allowing a direct comparison with iNKT cell responses [22 ]. In addition, our use of the CD1d/{alpha}-GalCer tetramers (CD1d-tet) in this system to unequivocally identify iNKT cells, in contrast to NK1.1+ TCRβ+, which also stains activated, conventional T cells in infected mice, will allow us to expand the current knowledge of the kinetics and function of iNKT cells in response to infection [23 , 24 ]. A recent study has shown that LM infection altered the phenotype and polarized the function of iNKT cells at the early stage of infection [25 ]. However, the long-term effects of LM infection on the dynamics, functions, and therapeutic activities of iNKT cells have not been explored thoroughly.

Here, we show that LM infection leads to a prolonged loss of iNKT cells in the spleen. Compared with naïve iNKT cells, remaining infection-primed iNKT cells are hyporesponsive to in vivo {alpha}-GalCer challenge in terms of cytokine production and expansion. This infection-induced hyporesponsiveness is not observed when iNKT cells are stimulated with {alpha}-GalCer-loaded dendritic cells (DCs), suggesting that extrinsic factors such as increasing antigen presentation/costimulation can overcome this functional deficit. As human iNKT cells, unlike naïve, murine iNKT cells, are exposed repetitively to infection, these observations may relate directly to human iNKT cell responses.


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MATERIALS AND METHODS
 
Mice
Eight- to 12-week-old C57BL/6 (B6) and thymectomized B6 (Tx) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Anita Chong (The University of Chicago, Chicago, IL, USA) kindly provided IFN-{alpha} knockout (KO) mice in the B6 background. All experiments involving animals were performed in a specific pathogen-free facility at the University of Chicago in compliance with institutional guidelines and have been approved by the Institutional Animal Care and Use Committee.

Reagents
{alpha}-GalCer (KRN7000) was obtained from Kirin Brewery Co. Ltd. (Gunma, Japan) and was reconstituted in PBS containing 0.5% polysorbate-20 (Sigma-Aldrich, St. Louis, MO, USA). Fluorescently labeled tetrameric CD1d molecules loaded with {alpha}-GalCer were prepared as described previously [6 ]. Anti-TCRβ-FITC, anti-CD69-PE, anti-NK1.1-PE, anti-IFN-{gamma}-FITC, anti-IFN-{gamma}-allophycocyanin, and anti-IL-4-PE were obtained from BD PharMingen (San Diego, CA, USA).

Bacteria, infection, and {alpha}-GalCer challenge procedure
The recombinant (r)LM strain, rLM-OVA, which secretes OVA and contains a erythromycin marker, was provided by Leo Lefrançois (University of Connecticut Health Center, Farmington, CT, USA) and grown in brain-heart infusion (BHI) broth (BD Difco) containing 5 µg/ml erythromycin (Sigma-Aldrich). For primary LM infections, mice were i.v.-infected with 5 x 104 CFU (0.1 LD50) rLM-OVA. Secondary LM infection with 1 x 106 CFU was performed 1 month after primary infection. The dose of rLM-OVA was verified by plating on BHI agar supplemented with 5 µg/ml erythromycin. For Mycobacterium bovis bacillus Calmette Guerin (BCG) infection, mice were i.v.-infected with 1 x 106 BCG (strain Pasteur, kindly provided by Chris Dascher, Mount Sinai School of Medicine, New York, NY, USA). The concentration of viable bacteria was enumerated by plate counts of CFU with Middlebrook 7H11 agar (BD BBL) supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase enrichment (BD BBL). For {alpha}-GalCer challenge, mice were i.p.-injected with 5 µg {alpha}-GalCer in 100 µl PBS containing 0.025% polysorbate-20 (vehicle).

Flow cytometry analysis
Single-cell suspensions of the spleen and liver were prepared as described previously [26 ]. Cells were preincubated with anti-Fc{gamma}RII/III (2.4G2, hybridoma supernatant) to avoid unspecific binding of antibodies and stained with various fluorescence-conjugated antibodies on ice for 30 min in HBSS containing 2% FBS and 0.1% sodium azide. Flow cytometric analysis was performed with a FACSCanto (BD Biosciences, San Jose, CA, USA), and the acquired data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Intracellular cytokine staining
Splenocytes (2x106) were treated with {alpha}-GalCer (100 ng/ml) for 5 h in U-bottom 96-well plates. Monensin (10 µM) was added 2 h before harvest. Cells were stained with anti-TCRβ-PerCP and allophycocyanin-conjugated CD1d-tet. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.15% saponin and then stained with anti-IFN-{gamma}-FITC and anti-IL-4-PE antibodies.

Cytokine capture assay
Naïve or LM-infected mice were i.v.-injected with 2.5 µg {alpha}-GalCer in 100 µl vehicle. Forty-five minutes after injection, IFN-{gamma} and IL-4 production by iNKT cells was measured using mouse IFN-{gamma} or IL-4 secretion assay detection kits according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA).

RNA/DNA extraction and quantitative real-time PCR
Total RNA and genomic DNA isolation was performed using the RNeasy mini kit and DNeasy blood and tissue kit, respectively (Qiagen, Valencia, CA, USA). Single-stranded cDNA was generated with Superscript II RT (Invitrogen Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using the ABI Prism 7700 instrument (Applied Biosystems, Foster City, CA, USA). Each PCR was run in duplicate, and the level of V{alpha}14J{alpha}18 expression was normalized to GAPDH using Sequence Detector software (Applied Biosystems). PCR of cDNA and genomic DNA specimens was conducted in a total volume of 50 µl using SYBR Green PCR Master Mix (Qiagen) with the following primers: V{alpha}14J{alpha}18 forward, 5'-TCTAGAATTCTAAGCACAGCACGCTG-3'; V{alpha}14J{alpha}18 reverse, 5'-CAATCAGCTGAGTCCCAGCT-3'.

DC culture and isolation
Single-cell suspension isolated from bone marrow (BM) was cultured for 7 days in the presence of GM-CSF (10 ng/ml) and IL-4 (2 ng/ml). BM-derived DCs (BMDCs) were purified using CD11c microbeads (Miltenyi Biotec). The purified BMDCs (>95% purity) were pulsed overnight with 100 ng/ml {alpha}-GalCer and then used for adoptive transfer (6x105 cells/mouse). To isolate splenic DCs, the spleen was digested with 0.2 mg/ml collagenase D (Roche Applied Science, Indianapolis, IN, USA) in HBSS, and DCs were then purified with CD11c microbeads (>85% purity).

iNKT cell purification, stimulation, and adoptive transfer experiments
Splenocytes and hepatic leukocytes were stained with anti-TCRβ-FITC and CD1d-tet-allophycocyanin. iNKT cells were then sorted with FACSAria (BD Biosciences). Sorted iNKT cells (5x104) were cultured with splenic DCs at a 1:1 ratio in the absence or presence of {alpha}-GalCer (100 ng/ml) for 48 h in a U-bottom 96-well plate. The levels of IFN-{gamma} and IL-4 in the culture supernatant were quantitated by sandwich ELISA (BD PharMingen).

Statistical analysis
All mice were examined individually in each experiment, and data were analyzed using Student’s t-test. All statistical analyses were performed with the Prism program (GraphPad Software, Inc., San Diego, CA, USA). A P value of <0.05 was considered statistically significant.


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RESULTS
 
iNKT cell kinetics during primary and secondary LM infection
Although the kinetics of iNKT cell responses to {alpha}-GalCer has been well characterized, the effects of primary and recall infection on the dynamics and function of iNKT cell responses have yet to be explored comprehensively. To address these issues, B6 mice were i.v.-infected with LM, and the cell number and surface phenotype of iNKT cells were examined by flow cytometry at different time-points following infection (1–30 days). As indicated by NK1.1 down-regulation and CD69 up-regulation, a significant proportion of iNKT cells was activated as early as Day 1 postinfection (Fig. 1 A and B, left panel ), concurrent with the innate immune response. Early activation of iNKT cells accompanied a substantial decrease in the total number of iNKT cells (Fig. 1 A and C) . Indeed, during the first 3 days of LM infection, iNKT cells showed a similar decrease in cell number as other T cells in the spleen (Fig. 1C) [27 , 28 ]. Notably, iNKT cells did not expand during primary LM infection in the liver or spleen (Fig. 1C) . This lack of expansion contrasted with conventional T cells, which showed a typical expansion (peaking at Day 8) and contraction during primary LM infection (Fig. 1C) . A similar lack of iNKT cell expansion has been reported in Salmonella infection, possibly as a result of the inability of iNKT cells to compete with the proliferation of antigen-specific, conventional T cells in these models [13 , 29 ]. In fact, the iNKT cell number in the spleen did not recover to preinfection levels, even at Day 30 postinfection (Fig. 1C) . This prolonged loss of iNKT cells was not simply a result of down-regulation of the TCR, as V{alpha}14J{alpha}18 RNA levels were reduced in LM-infected mice (Fig. 1D) . Moreover, the genomic levels of the V{alpha}14J{alpha}18-rearranged gene showed a similar reduction as those of RNA (Fig. 1E) . iNKT cell numbers in the liver recovered more rapidly (Fig. 1C) , likely as a result of organ-specific cytokines/chemokines or the greater number of residual iNKT cells in the liver. Indeed, organ-specific differences in immune response to LM infection are well documented [30 ]. Nevertheless, the prolonged loss of iNKT cells in both organs of LM-infected mice may have direct downstream effects on subsequent iNKT cell responses.


Figure 1
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  		Figure 1. Kinetics of iNKT cell response during primary and secondary LM infection. (A) Dynamics of the iNKT cells during primary or secondary infection. Numbers represent the percent of iNKT cells and NK1.1-expressing iNKT cells at indicated time-points. (B) The surface CD69 expression level on iNKT cells was measured by flow cytometry at indicated time-points. Data are presented as mean fluorescence intensity (MFI) after subtraction of isotype control. (C) Kinetics of the total number of iNKT cells versus conventional T cells during primary LM infection. (D and E) Decreased levels of V{alpha}14J{alpha}18 RNA and a rearranged gene at early stages of LM infection. Total RNA and genomic DNA were prepared from LM-infected splenocytes at indicated time-points. The V{alpha}14J{alpha}18 mRNA levels (D) and V{alpha}14J{alpha}18-rearranged gene levels (E) were measured by real-time PCR and normalized to GAPDH. Un, uninfected. (F) Kinetics of the total number of iNKT cells after secondary LM infection. Data are presented as the mean ± SD of two to four mice in each group.

One potential effect of infection-induced changes to iNKT cell numbers would be differences in the quality or quantity of secondary iNKT cell responses. To examine potential disparities in the primary and secondary iNKT cell response, LM-infected mice were rechallenged 1 month later with a higher dose of LM. Spleen cells and hepatic leukocytes were isolated from mice infected for various times and were analyzed by flow cytometry. As shown in Figure 1A , iNKT cells from spleen and liver had an increased rate of NK1.1 down-regulation during secondary LM infection. In contrast to primary infection, no appreciable decrease in the frequency and number of splenic iNKT cells was observed during secondary infection (Fig. 1 A and F) . In addition, the CD69 expression level on splenic iNKT cells was not changed by secondary infection (Fig. 1B , right panel). However, the changes of liver iNKT cell number resembled those following a primary infection, further highlighting the differences between these organs (Fig. 1 A and F) .

By comparison, conventional T cells showed a typical memory response with a three- or sixfold expansion on Day 5 postinfection in the spleen and liver, respectively (data not shown). Given the rapidity and strength of the conventional T cell memory response, a highly expansive and rapid memory iNKT cell response is likely redundant. Nevertheless, our data suggest that although these cells do not display a conventional memory response, they also do not behave like naïve iNKT cells.

Function of iNKT cells during primary and secondary bacterial infection
Cytokine secretion, especially IL-4 and IFN-{gamma} secretion, is a hallmark of iNKT cell activation [1 ]. A recent study has described the functional polarization of iNKT cells to a Th1-like phenotype at the early stage of primary LM infection [25 ]. However, it is unknown whether bacteria infection may have long-term, functional consequences and influence the response of iNKT cells to subsequent infection. We therefore compared the intracellular IL-4 and IFN-{gamma} profile of iNKT cells from naïve and LM-infected mice during primary and secondary infection. As expected, naïve iNKT cells in the presence of vehicle alone expressed neither cytokine, although {alpha}-GalCer stimulation resulted in the rapid production of IL-4 and low levels of IFN-{gamma} (Fig. 2 ). However, at Day 1 after primary LM infection, a significantly higher proportion of vehicle-treated and {alpha}-GalCer-stimulated iNKT cells produced IFN-{gamma}, and fewer produced IL-4, suggesting that infection may alter the functionality of iNKT cells (Fig. 2) . At Day 2 after infection, the cytokine response of residual iNKT cells was abolished almost completely, concurrent with the drastic reduction in cell number (data not shown).


Figure 2
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  		Figure 2. Altered iNKT cell function during secondary LM infection. Splenocytes from naïve and LM-infected mice were prepared at the indicated time-points after infection. iNKT cell function was examined by intracellular cytokine staining for IL-4 or IFN-{gamma} 5 h after in vitro restimulation with {alpha}-GalCer or vehicle. Values represent the percentage of total iNKT cells. A representative of four separate experiments is shown.

In secondary LM infection, few vehicle-treated iNKT cells (<1%) secreted IFN-{gamma} on Day 1 postinfection, highlighting the disparity between primary and secondary iNKT cell responses (Fig. 2) . In addition, a greater proportion of {alpha}-GalCer-stimulated iNKT cells from mice with secondary LM infection secreted IL-4 rather than IFN-{gamma}, in contrast to iNKT cells derived from primary, LM-infected mice that predominately produced IFN-{gamma} after {alpha}-GalCer stimulation (Fig. 2) . Therefore, bacterial infection results not only in a different iNKT cell function than {alpha}-GalCer challenge but also in functional differences between primary and secondary iNKT cell responses.

Bacterial infection results in prolonged functional alteration of iNKT in vivo
The decrease in IFN-{gamma} production by iNKT cells after secondary LM infection could be a result of differences in the inflammatory milieu during primary and secondary LM infection or reflect altered iNKT cell function as a result of prior exposure to LM. We therefore used {alpha}-GalCer as a surrogate, secondary antigen to directly evaluate the effects of infection on the function of iNKT cells. We challenged LM-infected mice with {alpha}-GalCer or vehicle on Days 3 and 7 and 1 month after LM infection. As expected, {alpha}-GalCer administration of naïve mice induced an increase in proportion as well as expansion of the total number of iNKT cells in spleen and liver on Day 3 after challenge (Fig. 3 A and B ). At Days 3 and 7 after LM infection, however, {alpha}-GalCer stimulation led to little increase in the proportion of splenic iNKT cells and a five- to tenfold decrease in the proportion of hepatic iNKT cells. At 1 month postinfection, iNKT cells are able to expand in response to {alpha}-GalCer; however, the expansion was still significantly lower than in naïve animals (Fig. 3B) . These data suggest that bacterial infection results in long-term hyporesponsiveness of iNKT cells to subsequent antigen stimulation.


Figure 3
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  		Figure 3. Bacterial infection results in long-term iNKT cell hyporesponsiveness to {alpha}-GalCer challenge. (A) Naïve or LM-infected mice were challenged at the indicated time-points postinfection with {alpha}-GalCer or vehicle. After 3 days, the iNKT cell number was determined. Results are representative of three mice at each time-point. (B) Naïve mice or mice infected with LM or BCG 1 month previously were challenged with {alpha}-GalCer or vehicle. The total number of iNKT cells was determined on Day 3 after injection. Data shown are mean ± SEM of six to nine mice for naïve and LM-infected groups and three mice for the BCG-infected group. Numbers indicate fold increase in total number of iNKT cells in response to {alpha}-GalCer stimulation. *, Statistically significant differences (P<0.05). (C–E) Naïve or LM-infected mice were challenged with {alpha}-GalCer. iNKT cell responses were determined by various assays. (C) After 45 min, a cytokine capture assay was performed. Histograms depict cytokine production from iNKT cells. (D) Serum IL-4 and IFN-{gamma} levels from indicated mice were quantitated by ELISA at 2 h and 12 h postchallenge, respectively. *, Statistically significant differences (P<0.05). Serum cytokine levels from unstimulated mice were comparable with background. (E) Transactivation of other T cells (TCRβ+NK1.1–) and B cells in naïve (black lines) and LM-infected mice (gray lines) was examined 24 h after {alpha}-GalCer injection by FACS staining for CD69 up-regulation. Data shown are representative of four mice per group from two independent experiments.

The altered iNKT cell response in infection-primed mice was substantiated further by functional analysis. Splenic and liver iNKT cells from naïve mice and mice infected with LM 1 month earlier were assayed directly by an ex vivo cytokine capture assay for IL-4 and IFN-{gamma} production. Only 20–30% of splenic iNKT cells from LM-infected mice were able to produce both cytokines after in vivo stimulation with {alpha}-GalCer compared with 50–70% in naïve mice (Fig. 3C) . Similarly, the percentage of IL-4- and IFN-{gamma}-producing iNKT cells in the liver of LM-infected mice was consistently lower than that of naïve mice. In addition, serum levels of IL-4 and IFN-{gamma} were lower in LM-infected mice than naïve mice after {alpha}-GalCer injection (Fig. 3D) , suggesting that the decreased number of cytokine-secreting iNKT cells in the spleen and the liver described above was not a result of migration of activated iNKT cells to other sites in LM-infected mice. Indeed, the observed differences in iNKT function had downstream consequences. Transactivation of other lymphocytes, as determined by early CD69 expression, was lower upon {alpha}-GalCer activation of LM-primed iNKT cells than unprimed iNKT cells (Fig. 3E) .

This infection-altered response of iNKT cells to {alpha}-GalCer is not limited to LM infection. Mice infected with BCG 1 month earlier also had a less-robust expansion of iNKT cells in spleen and no expansion of iNKT cells in liver in response to {alpha}-GalCer administration (Fig. 3B) . These results suggest that alterations in iNKT cell kinetics and function could be a general phenomenon after bacterial infection.

iNKT cell hyporesponsiveness induced by LM infection is more profound in Tx mice
The iNKT cell compartment in LM-infected mice consists of two populations: the cells that survive from infection and new thymic emigrants. To determine the contribution of new thymic emigrants to the residual, functional responses that are seen in mice at 1 month post-LM infection, we examined iNKT cell responses to {alpha}-GalCer challenge in Tx, LM-infected mice that lack newly developed iNKT cells. B6 mice were thymectomized at 6 weeks of age when the peripheral iNKT cell pool is fully established. Three weeks after thymectomy, mice were infected with LM and then challenged with {alpha}-GalCer or vehicle at 1 month after infection. iNKT cells from naïve, Tx mice responded similarly to control B6 mice upon {alpha}-GalCer challenge (Fig. 4A ). However, in Tx, LM-infected mice, the {alpha}-GalCer-induced proliferation and IFN-{gamma} production were abrograted completely (Fig. 4B) . In addition, the serum IL-4 level was reduced significantly in Tx, LM-infected mice, as compared with naïve, Tx mice (Fig. 4B) . Lack of cytokine production was not just a result of lack of iNKT cells in Tx, LM-infected mice, as a significant number of iNKT cells (~5%) were present in the liver of Tx, LM-infected mice prior to {alpha}-GalCer challenge (Fig. 4A) . These data suggest that the infection-primed iNKT cells have an impaired response to subsequent antigenic challenge. Furthermore, the long-term nature of hyporesponsiveness to {alpha}-GalCer in LM-infected mice may be a result of the relatively long interval for new thymic emigrants to repopulate the periphery. Indeed, at 10 weeks after LM infection, splenic and hepatic iNKT cell numbers returned to preinfection levels (Fig. 4C) . In addition, the extent of {alpha}-GalCer-induced expansion from LM-infected mice at this time was comparable with naïve mice (Fig. 4C) , consistent with the notion that newly emigrated, thymic iNKT cells may be responsible for this recovery.


Figure 4
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  		Figure 4. New thymic emigrants are essential for restoring iNKT cell responsiveness in LM-infected mice. (A and B) Tx mice and B6 mice were left untreated or infected with LM. After 1 month, mice were challenged with {alpha}-GalCer or vehicle. (A) After 3 days, splenocytes and hepatic lymphocytes were stained with anti-TCRβ antibody and CD1d-tet to identify iNKT cells. (B) Serum IL-4 and IFN-{gamma} levels were measured by ELISA at 3 and 24 h postimmunization, respectively. Data shown are mean ± SEM of three mice per group. *, P < 0.05, compared with naïve mice. (C) Naïve mice and mice infected with LM 10 weeks earlier were challenged with vehicle or {alpha}-GalCer. Bar graphs depict mean ± SEM for the total number of iNKT cells for naive and LM-infected mice (n=5) on Day 3 after injection.

Infection-induced iNKT cell hyporesponsiveness can be rescued by enhanced antigen presentation
{alpha}-GalCer-induced hyporesponsiveness is iNKT cell autonomous, as iNKT cells remain hyporesponsive when stimulated in vitro with {alpha}-GalCer-pulsed DCs derived from naïve or {alpha}-GalCer-injected mice [12 ]. To further examine the mechanisms leading to iNKT cell hyporesponsiveness after LM infection, we isolated splenic DCs and iNKT cells from naïve and LM-infected mice and performed coculture experiments in the presence of {alpha}-GalCer. Naïve and infected DCs induced a similar level of iNKT cell activation, as indicated by IFN-{gamma} secretion after {alpha}-GalCer stimulation (Fig. 5A ). These data suggest that infection-primed DCs sufficiently stimulate iNKT cells and that the hyporesponsiveness of iNKT cells in LM-infected mice is not likely a result of the induction of tolerogenic DC. Notably, in this in vitro system, iNKT cells isolated from LM-infected mice secreted more IFN-{gamma} (three- to fourfold) than naïve iNKT cells in response to {alpha}-GalCer presented by naïve or LM-primed DCs (Fig. 5A) . Therefore, LM-primed iNKT cells are not only capable of responding to {alpha}-GalCer, but consistent with the more rapid down-regulation of NK1.1, they may also have an enhanced effector function. It is noteworthy that LM-primed iNKT cells, but not naïve iNKT cells, secrete low but detectable amounts of IFN-{gamma} when stimulated with {alpha}-GalCer in the absence of DCs. These data suggest that LM-primed iNKT cells might be able to respond to CD1d expression on nonprofessional APC types, resulting in suboptimal activation and the apparent hyporesponsiveness to {alpha}-GalCer challenge in vivo.


Figure 5
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  		Figure 5. Infection-induced iNKT cell hyporesponsiveness can be overcome by stimulation with {alpha}-GalCer-pulsed DC. (A) Splenic DCs and iNKT cells were isolated from naïve and LM-infected mice for coculture experiments in the presence or absence of {alpha}-GalCer. After 2 days, the amount of IFN-{gamma} present in the culture supernatant was quantitated by ELISA. Data shown are representative of two independent experiments. IFN-{gamma} levels from cultures in the absence of {alpha}-GalCer were comparable with background. (B) Naïve and LM-infected mice were challenged with 6 x 105 {alpha}-GalCer-pulsed BMDCs. The serum IFN-{gamma} level was quantitated by ELISA at 24 h postchallenge (n=7). (C) Bar graphs depict mean ± SEM for the total number of iNKT cells for naive and LM-infected mice 72 h after stimulation with {alpha}-GalCer-pulsed DC (n=4 per group). Data shown are representative of two independent experiments.

To determine if infection-induced hyporesponsiveness of iNKT cells could be overcome in vivo, we challenged naïve mice and mice infected with LM 1 month earlier with {alpha}-GalCer-pulsed BMDCs. One day after immunization, serum IFN-{gamma} levels were comparable between naïve and LM-infected mice groups (Fig. 5B) . In addition, on Day 3, these two groups had comparable iNKT cell expansion in spleen and liver (Fig. 5C) . This functional compensation by {alpha}-GalCer-loaded DC is not observed with {alpha}-GalCer-induced hyporesponsiveness [12 ]. These results, therefore, support the suggestion that extrinsic factors, such as enhanced antigen presentation, can overcome iNKT cell hyporesponsiveness caused by LM infection.

Taken together, these data indicate that bacterial infection can alter the kinetics and function of the iNKT cell response and that this effect is not solely by iNKT cell-intrinsic. Differences in antigen presentation, competition with other cell types, changes in cell-surface accessory molecule levels, and cytokine microenvironment in vivo during infection could all play a role in inducing/maintaining iNKT cell hyporesponsiveness.


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DISCUSSION
 
iNKT cells play an important role in regulating innate and adaptive immune responses. Although much work has been done in manipulating iNKT cell responses through the use of {alpha}-GalCer, the actual physiological responses of iNKT cells to infection are less well characterized. Here, we report that unlike conventional T cells, iNKT cells do not undergo typical expansion and contraction in response to LM infection. In fact, LM infection results in long-term loss of iNKT cells, especially in the spleen. The infection-primed iNKT cells are hyporesponsive to subsequent antigenic challenge, as reflected by their decreased capacity to produce cytokines and proliferate compared with naïve iNKT cells. Although infection-induced iNKT cell hyporesponsiveness resembles that reported for {alpha}-GalCer, there are some key differences that may be critical to consider when manipulating iNKT cell responses. First, {alpha}-GalCer stimulation leads to robust iNKT cell expansion followed by contraction [12 , 13 ], whereas iNKT cells are activated in the absence of expansion during LM infection. Indeed, the expression of early activation markers with subsequent loss of iNKT cell numbers mimics bystander activation rather than antigen-specific CD8+ T cell activation in LM infection [27 ]. Second, {alpha}-GalCer-induced, anergic iNKT cells are not able to down-regulate TCR when rechallenged with {alpha}-GalCer [12 ], unlike infection-induced hyporesponsive iNKT cells, which show significant TCR down-regulation following in vivo injection of {alpha}-GalCer (data not shown). Furthermore, {alpha}-GalCer-induced hyporesponsiveness is iNKT cell-intrinsic, and our finding that {alpha}-GalCer-loaded DCs can rescue the hyporesponsiveness of iNKT cells induced by LM infection suggests that extrinsic factors may be involved in regulating the function of iNKT cells after infections. Thus, the mechanisms underlying the hyporesponsiveness in these two systems may be somewhat different.

A recent report by Kim et al. [31 ] has shown that iNKT cells activated by multiple microorganisms and bacterial products become unresponsive to subsequent activation with {alpha}-GalCer. Consistent with this observation, we and others [32 ] found that iNKT cells from BCG-infected mice also have altered responses to {alpha}-GalCer restimulation (Fig. 3) . These data suggest that infection-induced iNKT cell hyporesponsiveness may be a common consequence of bacterial infection. Recent studies have shown that different subsets of CD1d-restricted NKT cells have opposing effects in parasitic diseases and tumor immunity [33 34 35 ]. In addition, iNKT cells and vNKT cells have been demonstrated to counter-regulate each other in tumor-challenged mice [36 ]. However, it remains to be seen if bacteria infection affects the function of vNKT cells and alters the balance between iNKT cells and vNKT cells.

Some bacteria, such as Sphingomonas and Borrelia, activate iNKT cells through direct recognition of pathogen-specific glycolipid antigens [15 16 17 ]. Bacteria that lack cognate iNKT cell antigens induce DCs to produce IL-12 in a TLR-dependent manner, which alters the phenotype and polarizes the function of iNKT cells at the early stage of infection [14 ]. Although we cannot eliminate the possibility that some LM-derived glycolipid antigens may contribute to iNKT cell activation, we found that IL-12 plays an important role in LM-induced iNKT cell activation, as anti-IL-12 inhibited IFN-{gamma} production by iNKT cells stimulated with heat-killed, LM-pulsed BMDCs (data not shown). In addition, IL-12 has been shown to be required for iNKT hyporesponsiveness induced by heat-killed Escherichia coli, LPS, and flagellin [31 ]. These findings highlight the critical role of IL-12 in regulating iNKT cell activity during bacteria infection.

Prior studies have shown that LM infection induces type I IFN production, which sensitizes lymphocytes to undergo apoptosis during LM infection [27 , 37 38 39 ]. A similar role of type I IFN on iNKT cells has been implicated in lymphocytic choriomeningitis virus infection, which results in long-term loss of iNKT cells [40 ]. To assess the effect of type I IFN on the initial loss of iNKT cells during LM infection, we compared the number of iNKT cells in wild-type and IFN-{alpha}βR KO mice on Day 3 post-LM infection. We found that the decrease of iNKT cells in LM-infected IFN-{alpha}βR KO mice was comparable with wild-type mice, suggesting that type I IFN might not be involved in the loss of iNKT cells at an early stage of LM infection (Supplemental Fig. 1 ). Nevertheless, type I IFN signaling has been shown to down-regulate the expression of IL-12 [41 ], which consequently may control the extent of iNKT cell activation and responsiveness in LM-infected mice.

It is noteworthy that the iNKT response in the liver does not directly mimic the response in the spleen. Although lack of iNKT cell expansion is observed in the liver, the percentage of iNKT cells returns to levels observed in naïve mice by Day 30. These differences could potentially be a result of differences in cell surface molecules or chemokine and cytokine expression patterns induced by infection in the splenic and liver microenvironments. Alternatively, the higher number of residual iNKT cells in the liver could result in faster repopulation. Indeed, splenic iNKT cell numbers return to preinfection levels at 10 weeks postinfection, supporting this hypothesis (data not shown). In addition, although the frequency and number of liver iNKT cells after secondary infection revealed a similar reduction to that seen after primary infection, no appreciable change in the number of splenic iNKT cells or CD69 expression was observed after secondary infection. The distinct kinetics observed in splenic iNKT cells during primary and secondary LM infection suggests that they may contribute differently to antilisterial immunity during primary and secondary infection.

Although the mechanism of LM infection-induced iNKT hyporesponsiveness has yet to be fully delineated, our findings have several important implications. {alpha}-GalCer has been used to activate iNKT cells with the intention of ameliorating infections or tumor growth. However, most of these studies use pretreatment or treatment shortly after infection or the onset of disease [42 , 43 ]. Our data clearly revealed that from Day 2 to Day 30 postinfection, iNKT cells actually become nonresponsive or hyporesponsive to in vivo {alpha}-GalCer challenge. Therefore, the timing and efficacy of this treatment may need to be reconsidered.

In addition, humans are infected more frequently by various microbes than mice housed in specific pathogen-free facilities. Therefore, manipulating iNKT cell function by {alpha}-GalCer stimulation could be less efficacious in humans. This view is supported by results from a recent clinical trial in which injection of {alpha}-GalCer alone to patients results in only transient, immunological activation in a subset of individuals examined [8 ]. Our data would suggest that the use of {alpha}-GalCer-pulsed DCs might rescue the hyporesponsiveness of iNKT cells in individuals in which the iNKT cell function may be compromised as a result of infection. Consistent with this notion, another clinical trial has shown that injection of {alpha}-GalCer-pulsed, mature DCs can lead to sustained expansion of glycolipid-reactive iNKT cells in vivo in cancer patients [7 ].

Polarization of the cytokine secretion profile of iNKT cells has been found in various disease systems, including allergy and autoimmunity. In addition, iNKT cell skewing is thought to play a role in tumor immunosurveillance, where IFN-{gamma} secretion is deemed beneficial and IL-13 detrimental [44 , 45 ]. Alteration of iNKT cell functions through infection may result in dysregulation of these processes. This information should be taken into consideration for etiological analyses of human diseases. Furthermore, in some tumor-bearing [46 ] or asthmatic mice and patients [47 , 48 ], iNKT cells are only able to secret Th2 cytokines but not IFN-{gamma} after restimulation in vitro with {alpha}-GalCer. These responses could represent differential reactivity of infection-primed or antigen-experienced iNKT cells, as indicated by our study. Thus, it would be of interest to determine whether {alpha}-GalCer-pulsed DC could alter iNKT cell function in these conditions. Understanding the effects of bacterial infection on iNKT cell function would therefore be important for manipulating iNKT cells in the clinical setting.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institute of Health grants AI40310, AI43407, and AI57460 (to C-R. W.). We thank Kirin Brewery Co. Ltd. for providing {alpha}-GalCer; Dr. Leo Lefrançois for providing rLM-OVA; Dr. Anita Chong for providing IFN-{alpha}βR KO mice; Dr. Chris Dascher for providing BCG; Kyrie Felio for assistance in BCG infection; and Dr. Michael Zimmer and Stephen Wood for critical review of the manuscript.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received January 15, 2008; revised July 31, 2008; accepted August 14, 2008.


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