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The Journal of Immunology, 2004, 173: 4976-4984.
Copyright © 2004 by The American Association of Immunologists

Essential Role of LFA-1 in Activating Th2-Like Responses by {alpha}-Galactosylceramide-Activated NKT Cells1

Goichi Matsumoto2,*, Eiro Kubota*, Yasushi Omi*, Ushaku Lee* and Josef M. Penninger{dagger}

* Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, Kanagawa, Japan; and {dagger} Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-{gamma}) and Th2 (IL-4) responses following stimulation of their invariant V{alpha}14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the V{alpha}14 ligand {alpha}-galactosylceramide ({alpha}-GalCer) remains unclear. To address this issue, LFA-1–/– (CD11a–/–) mice were used to investigate IL-4 and IFN-{gamma} production by NKT cells following {alpha}-GalCer stimulation. Intriguingly, LFA-1–/– mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to {alpha}-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in {alpha}-GalCer-activated NKT cells from LFA-1–/– mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer T cells belong to an unusual lymphoid lineage distinct from conventional T, B, or NK cells (1). These cells are characterized by the expression of a single invariant Ag receptor encoded by V{alpha}14-J{alpha}281 segments paired preferentially with V{beta}8.2, V{beta}2, or V{beta}7, and recognize glycolipid Ags in the context of the MHC class Ib molecule CD1d (1, 2, 3). Moreover, NKT cells are stimulated by the glycolipid {alpha}-galactosylceramide ({alpha}-GalCer),3 a potent inducer of antitumor activity in mice (4). When stimulated through their TCR by anti-CD3 or {alpha}-GalCer, NKT cells can produce large amounts of cytokines, including IL-4 and IFN-{gamma} (4, 5, 6, 7). Therefore, it has been postulated that NKT cells influence the differentiation of naive CD4+ T cells into functional Th cell subsets (8). However, loss of CD1d in mice had no effect on the differentiation of naive CD4+ T cells into functional Th cells (9, 10), implying that NKT cells are not absolutely required for polarized Th responses. Because NKT cells can produce both IL-4 as well as IFN-{gamma}, the molecular mechanisms by which NKT cells can regulate the development of Th1 vs Th2 cells have not yet been clarified. Recognition of {alpha}-GalCer by NKT cells depends on the interaction of the V{alpha}14 TCR on NKT cells with {alpha}-GalCer presented by CD1d on dendritic cells (DCs) (2). DCs express both adhesion and costimulatory molecules, which play crucial roles in determining the responses of conventional T cells to Ag. Several studies have shown that ICAM-1/LFA-1, B7/CD28, and/or CD40/CD154 interactions are important for driving Th1 or Th2 cell development from naive T cells (11, 12, 13, 14). Major efforts have been made to elucidate the molecular basis of Th1 vs Th2 cell subset differentiation and to identify transcription factors controlling Th1 and Th2 cytokine expression. The T-box transcription factor T-bet is involved in the commitment of Th1 cells by inducing IFN-{gamma} synthesis and repressing IL-4 and IL-5 production, even in fully polarized Th2 cells (15). In contrast, the transcription factor GATA-3 is necessary and sufficient for expression of the Th2 cytokines IL-4, IL-5, and IL-13 and the commitment of Th2 cells (16, 17).

It has been reported that LFA-1 ligation on activated human naive Th cells leads to a shift toward Th1 cell differentiation, accompanied by a rapid increase in the T-bet:GATA-3 mRNA expression ratio (12). Similarly, during Ag presentation, the B7/CD28 and CD154/CD40 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of {alpha}-GalCer-activated NKT cells (18, 19). How adhesion receptors contribute to the regulation of Th1 and Th2 functions of {alpha}-GalCer-activated NKT cells is not clear.

In this study, we investigated the role of adhesion receptor LFA-1 in {alpha}-GalCer-activated NKT cells using LFA-1–/– mice (20). We report that LFA-1 is critical for {alpha}-GalCer-activated NKT cells to produce IL-4 but not IFN-{gamma}. The LFA-1-mediated interaction between DCs and NKT cells promotes negative regulation of IL-4 production by NKT cells. These results provide the first genetic evidence that adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice deficient in LFA-1 (LFA-1–/–) have been previously described (20). Mutant mice were backcrossed to a C57BL/6 (H-2b/b) background for at least six generations. As wild-type (LFA-1+/+) controls, we used age-matched C57BL/6 mice (purchased from Japan SLC, Shizuoka, Japan). All mice used were 6–12 wk old. All mice were maintained at Kanagawa Dental College under specific pathogen-free conditions in accordance with the animal study guidelines of the college.

In vivo NKT cell activation with {alpha}-GalCer and anti-CD3

To activate NKT cells with {alpha}-GalCer, {alpha}-GalCer (2 µg) was i.p. administered to LFA-1+/+ and LFA-1–/– mice. {alpha}-GalCer was purchased from Kirin Brewery (Gunma, Japan) (21) and dissolved in 0.9% NaCl solution containing 0.5% polysorbate 20 (Nikko Chemical, Tokyo, Japan). Vehicle solution was used in all experiments as a control. Following in vivo administration of {alpha}-GalCer, serum IL-4 and IFN-{gamma} cytokine levels were determined at different time points using specific ELISA kits (BioSource International, Camarillo, CA) according to the manufacturer’s instructions. The amount of serum IgE was determined by an IgE-specific ELISA (BD Pharmingen, San Diego, CA). For IgG1- and IgG2-specific ELISAs, microtiter plates were coated with PBS containing 4 µg/ml anti-IgG1 and anti-IgG2 (clones A85-3 and R11-89, respectively) and kept overnight at 4°C. Unspecific binding to plates was blocked with PBS containing 10% FCS for 1 h followed by a wash with 0.05% Tween 20 in PBS. Serum samples were then incubated for 2 h at 37°C, washed with 0.05% Tween 20 in PBS, and for 1 h overlaid with biotin-conjugated isotype-specific anti-mouse IgG1 (clone A85-1) and anti-mouse IgG2 (clone R19-15) mAbs followed by avidin-HRP (mAbs were from BD Pharmingen). After the color reaction was terminated with 2 N H2SO4, Ab titers were determined at OD450 nm on a microplate reader (Bio-Rad, Hercules, CA) using Ig isotype control standards (BD Pharmingen). To directly activate the TCR of NKT cells (5), we i.v. injected a single dose of 1.5 µg of anti-CD3 mAb (clone 145-2C11; BD Pharmingen). After 90 min, spleens were removed, and single cell suspensions were incubated at appropriate cell densities in 24-well tissue-culture plates (Sumitomo Bakelite, Osaka, Japan) at 37°C for 2 h without additional stimulus. Supernatants were then collected and analyzed for IFN-{gamma} and IL-4 production by ELISA.

In vitro stimulation with {alpha}-GalCer

Single cell suspensions were prepared from spleens and livers of LFA-1+/+ and LFA-1–/– mice. Mononuclear cells (MNCs) from spleen (4 x 106) or liver (2 x 106) were cultured in 96-well U-bottom plates (Sumitomo Bakelite) with 100 ng/ml {alpha}-GalCer or vehicle in complete medium (CM; RPMI 1640 medium supplemented with 10% heat-inactivated FCS) in a humidified atmosphere containing 5% CO2 at 37°C. After culture for 72 h, supernatants were harvested to measure cytokine levels. Proliferation was measured after a 12 h pulse of [3H]thymidine (1 µCi/50 µl) using an Aloka LSC-5100 liquid scintillation counter (Tokyo, Japan).

Isolation of NKT cells and DCs

Spleen cells were incubated on nylon-wool columns for 45 min and nonadherent cells were stained with anti-NK1.1 (PE conjugated, clone NKR-PC1) and anti-TCR {alpha}{beta} (FITC conjugated, clone H57-597) (BD Pharmingen). NK1.1+TCR {alpha}{beta}+ NKT cells were further purified by cell sorting on a FACSCalibur (BD Biosciences, San Diego, CA). The purity of the sorted cells was >90%. DCs were prepared from spleens as described (22). Briefly, collagen-digested spleen cells were suspended in a dense BSA solution in PBS (p = 1.080; Sigma-Aldrich, St. Louis, MO), overlaid with FCS-free RPMI 1640 medium, and centrifuged at 9500 x g for 15 min at 4°C. Cells in the low density fraction at the interface were collected, washed, and allowed to adhere to plastic dishes for 2 h at 37°C. Cells were incubated for an additional 20 h to allow DCs to detach from the plastic dishes. DCs and other nonadhering cells were then positively selected with HL-3 microbeads (anti-CD11c) using an Auto MACS (Miltenyi Biotec, Sunnyvale, CA). More than 90% of purified cells were CD11c+ and I-A+ DCs. For NKT cell activation, DCs (5 x 104) were cocultured with NKT cells (5 x 104) in the presence of 50 ng/ml {alpha}-GalCer in 96-well U-bottom plates (23). Cells were incubated for 48 h and then supernatants harvested to measure cytokine levels. To measure the proliferation of {alpha}-GalCer-specific NKT cells, cells were pulsed with 1 µCi of [3H]thymidine for 12 h. In some experiments, anti-LFA-1 (clone M17-4; BD Pharmingen) mAbs were added to the cell cultures for DC-NKT cell adhesion blockade.

Flow cytometry

Surface phenotypes of cells were determined using two- or three-color immunofluorescence. MNCs of spleen and liver were resuspended in an immunofluorescence-staining buffer (PBS, 4% FCS, 0.1% NaN3) and incubated with the appropriate mAbs for 30 min. To block unspecific binding via Fc{gamma}Rs, all samples were preincubated with a nonconjugated CD16/CD32 mAb for 15 min. We used the following anti-mouse mAbs: PE-anti-NK1.1, CyChrome-anti-TCR{alpha}{beta}, FITC-anti-CD69, FITC-anti-ICAM-1, FITC-anti-ICAM-2, PE-anti-B220, PE-anti-I-Ab, FITC-anti-CD23, biotin-anti-CD11c, biotin-anti-Ly49A, biotin-anti-Ly49C/I, FITC-anti-Ly49G2, FITC-anti-CD40, FITC-anti-CD1d (obtained from BD Pharmingen), and PE-{alpha}-GalCer-loaded CD1d/tetramers (a gift from Dr. M. Taniguchi, Chiba University, Chiba, Japan) (24). Detection of apoptotic cells was performed using Annexin V (FITC conjugated) after staining with PE-{alpha}-GalCer-loaded CD1d/tetramers and CyChrome-anti-TCR {alpha}{beta} mAb. Flow cytometric analysis of intracellular IL-4 and IFN-{gamma} was performed with Cytofix/Cytoperm reagents and Golgi-stop (BD Pharmingen), according to the manufacturer’s specifications. Cells were stained with PE-{alpha}-GalCer-loaded mCD1d/tetramers, fixed, permeabilized, and then incubated with biotin-anti-IL-4 (BD Pharmingen) or biotin-anti-IFN-{gamma} Abs (BD Pharmingen). Biotin-conjugated mAbs were visualized with streptavidin CyChrome (BD Pharmingen). Cells were analyzed using a FACSCalibur and CellQuest software (BD Biosciences).

DC and NKT cell adhesion assays

Purified DCs (5 x 104) were labeled with 50 µg/ml hydroethidine (HE; Polysciences, Warrington, PA) and NKT cells (1 x 105) labeled with 2 x 10–6 mol/ml PKH2 (Zynaxis Cell Science, Malvern, PA) for 5 min at room temperature. The cells were then washed six times using CM to remove unbound fluorochromes. Two-hundred-fifty microliters of labeled DCs were combined with 250 µl of labeled NKT cells in polystyrene tubes, and then centrifuged at 1,500 rpm for 2 min. Pellets were incubated for different time points at 37°C to allow for conjugate formation between cells. Resuspended cells were then fixed by the addition of 500 µl of ice-cold PBS/4% paraformaldehyde to stop conjugate formation and to avoid bleaching of the fluorescence dyes. Binding between DC target and NKT cells was analyzed using a FACSCalibur. DC and NKT cells were gated in the forward scatter (FSC)/side scatter (SSC) profiles and analyzed for the presence of HE and PKH2 double-positive cells. At least 15,000 gated events were collected. Percentage of adhesion was calculated as the number of double-positive cells divided by the sum of the double-positive and the single-positive cells. In addition, fluorochrome-labeled conjugates between DCs and NKT cells were directly visualized using fluorescence microscopy.

Quantitative RT-PCR

Total RNA was extracted using Isogen (Nippon Gene, Toyama, Japan). Single-stranded cDNAs were synthesized from total RNA by reverse transcriptase using the Super Script II (Invitrogen Life Technologies, Carlsbad, CA). Quantitative RT-PCR was conducted with specific primers for IL-4, IFN-{gamma}, IL-5, IL-13, GATA-3, and T-bet. Primer sequences were as follows: IL-4 (401 bp), 5' primer-atgggtctcaacccccagctggt and 3' primer-gctctttaggctttccaggaagtc; IFN-{gamma} (231 bp), 5' primer-gctctgagacaatgaacgct and 3' primer-aaagagataatctggctctgc; IL-5 (331 bp), 5' primer-ttgacacagctgtccgctca and 3' primer-tgtcaccatggagcagctca; IL-13 (327 bp), 5' primer-ctttgctgccttggtggtctcgc and 3' primer-gcagttttgttataaagtgggct; GATA-3 (246 bp), 5' primer-gaaggcatccagacccgaaac and 3' primer-acccatggcggtgaccatga; T-bet (348 bp), 5' primer-cgccaggaagtttcatttgg and 3' primer-gttgacagttgggtccaggt. To detect the amounts of the GAPDH mRNA, RT-PCR was conducted with FAM-labeled primers for GAPDH (Invitrogen Life Technologies). The expression of the housekeeping gene GAPDH was used to normalize for input cDNA. Real-time monitoring of PCR products was done with fluorescence of SYBR green I (Takara Bio, Shiga, Japan) during every PCR cycle at the extension step. RNA from Th1- or Th2-conditioned CD4+ T cells (25) was used as positive control of IFN-{gamma} and T-bet, or IL-4, IL-5, IL-13, and GATA-3, respectively. Each mRNA expression induced by {alpha}-GalCer is indicated in the figure as induction index, calculated as follows: induction index = mRNA expression of {alpha}-GalCer-stimulated sample/mRNA expression of unstimulated sample (vehicle).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{alpha}-GalCer-activated LFA-1–/– NKT cells produce increased amounts of IL-4 in vivo

To analyze the contribution of the LFA-1-mediated adhesion pathway to IL-4 and IFN-{gamma} production in vivo, we administered {alpha}-GalCer to control LFA-1+/+ and mutant LFA-1–/– mice. In pilot experiments in LFA-1+/+ mice, the serum IL-4 concentration rapidly peaked at 3 h and serum IFN-{gamma} concentrations peaked at 9 h after i.p. {alpha}-GalCer injecting (not shown). In LFA-1–/– mice, the serum IFN-{gamma} concentration was comparable to that of LFA-1+/+ mice, but serum IL-4 concentrations were markedly increased following an in vivo challenge with {alpha}-GalCer (Fig. 1A). Despite increased IL-4 levels, there was no apparent shift in the time courses of IL-4 or IFN-{gamma} induction in the LFA-1–/– mice (not shown). We next examined {alpha}-GalCer-induced IL-4 and IFN-{gamma} production of splenic (Fig. 1B) and hepatic (not shown) MNCs isolated from LFA-1+/+ or LFA-1–/– mice in vitro. Similar to the in vivo challenge, in vitro {alpha}-GalCer activation resulted in enhanced IL-4 production of splenic and hepatic MNCs from LFA-1–/– mice. Both splenic and hepatic MNCs from LFA-1–/– mice produced amounts of IFN-{gamma} that were comparable to LFA-1+/+ mice. In addition to increased IL-4 production, loss of LFA-1 resulted in enhanced proliferation of {alpha}-GalCer-activated NKT cells (Fig. 1B and not shown).



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  		FIGURE 1. Increased IL-4 production in {alpha}-GalCer-activated LFA-1–/– NKT cells. A, Serum IFN-{gamma} and IL-4 levels in LFA-1+/+ and LFA-1–/– mice. Sera were obtained at 3 and 9 h after i.p. administration of {alpha}-GalCer (2 µg/100 µl) or vehicle. IL-4 and IFN-{gamma} levels were determined by ELISA; n = 5 per group. B, {alpha}-GalCer-induced proliferation, IFN-{gamma}, and IL-4 production by splenic MNCs from LFA-1+/+ and LFA-1–/– mice. Freshly isolated splenic MNCs were stimulated by {alpha}-GalCer (100 ng/ml) in 96-well U-bottom plates for 72 h. Culture supernatants were harvested to measure IFN-{gamma} and IL-4 by ELISA. Proliferation of responder cells was measured by [3H]thymidine incorporation. Data are represented as the mean ± SD of triplicate cultures. Similar results were obtained in three independent experiments and in liver-derived NKT cells. C, Intracellular cytokine production by CD1d/{alpha}-GalCer tetramer-positive NKT cells. Splenic MNCs from LFA-1+/+ and LFA-1–/– mice were stimulated with {alpha}-GalCer (100 ng/ml) or cultured in a vehicle control. After 48 h, cells were stained for surface Ag receptor expression (TCR{alpha}{beta} and CD1d/{alpha}-GalCer tetramer) and intracellular cytokine contents. Numbers are the percentages of IL-4- or IFN-{gamma}-positive cells within gated TCR{alpha}{beta}+CD1d/{alpha}-GalCer tetramer+ cell populations. Experiments were repeated three times with similar results.

 
We next investigated intracellular IL-4 and IFN-{gamma} levels in {alpha}-GalCer-stimulated NKT cells using flow cytometry. When NKT cells were analyzed by gating on CD1d/{alpha}-GalCer tetramer-positive cells, we reproducibly detected a 2-fold increase in the percentage of IL-4-producing LFA-1–/– splenic NKT cells following in vitro {alpha}-GalCer restimulation. The percentage of IFN-{gamma}-producing splenic NKT cells from LFA-1–/– mice was comparable to LFA-1+/+ mice (Fig. 1C). These data show that LFA-1 plays an important negative regulatory role for NKT cells to produce Th2 cytokines.

Activation of NKT cells in LFA-1–/– mice

It has been reported that NKT cells are rapidly activated and depleted due to apoptosis in response to {alpha}-GalCer (26). However, recent evidence suggests that TCR down-regulation rather than apoptosis is the primary cause of NKT cell disappearance after {alpha}-GalCer treatment (27, 28). Therefore, we examined whether splenic and hepatic NKT cell disappearance involves apoptosis after {alpha}-GalCer administration by FACS analysis. The proportion of naive NKT cells in spleen (but not liver) of LFA-1–/– mice was comparable to LFA-1+/+ mice (29, 30). By 24 h after {alpha}-GalCer administration, the numbers of CD1d/{alpha}-GalCer tetramer-positive NKT cells were markedly reduced from spleens and liver of both LFA-1+/+ and LFA-1–/– mice (Fig. 2A and not shown). However, splenic and hepatic NKT cells from {alpha}-GalCer-injected mice did not exhibit significantly increased annexin V staining compared with those from vehicle-injected mice; moreover, annexin V staining on NKT cells was also comparable among LFA-1+/+ and LFA-1–/– mice (Fig. 2B and not shown). The proportion of activated splenic and hepatic NKT cells as detected by induction of the CD69 activation marker significantly increased 24 h after {alpha}-GalCer administration in both LFA-1+/+ and LFA-1–/– mice (Fig. 2C). Surface CD69 expression levels and kinetics of induction on NKT cells were comparable among LFA-1+/+ and LFA-1–/– mice at all time points analyzed (not shown). Thus, loss of LFA-1 expression has no apparent effect on {alpha}-GalCer-induced activation and cell death of {alpha}-GalCer-specific NKT cells.



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  		FIGURE 2. Phenotypes of {alpha}-GalCer-activated splenic NKT cells. A, TCR{alpha}{beta} and CD1d/{alpha}-GalCer tetramer-staining profiles of splenic MNCs in LFA-1+/+ and LFA-1–/– mice. Splenic MNCs were isolated 24 h after {alpha}-GalCer or vehicle injection (i.p.). Numbers are the percentages of TCR{alpha}{beta}+CD1d/{alpha}-GalCer tetramer+ NKT cells. B, Splenic MNCs were isolated 24 h after {alpha}-GalCer or vehicle i.p., stained with mAbs, and analyzed by FACS. Gated TCR{alpha}{beta}+CD1d/{alpha}-GalCer tetramer+ NKT cells were analyzed for binding to annexin V. NKT cells from {alpha}-GalCer-injected mice are indicated by the solid lines, and those of vehicle-injected mice by dotted lines. Experiments were repeated twice with similar results. C, Induction of CD69 expression on gated splenic TCR{alpha}{beta}+CD1d/{alpha}-GalCer tetramer+ NKT cells isolated at 12 h after {alpha}-GalCer or vehicle injection. Solid lines indicate MNCs from {alpha}-GalCer-injected mice and dotted lines MNCs from vehicle-injected mice. D, Expression of Ly49A on splenic NK1.1+TCR{alpha}{beta}+ cells. Splenic MNCs were stained for TCR{alpha}{beta}, NK1.1, and Ly49A, and gated NK1.1+TCR{alpha}{beta}+ cells were analyzed for Ly49A expression. Bold lines indicate staining with anti-Ly49, and thin lines staining with isotype-matched control IgG. One result representative of three independent experiments is shown.

 
The Ly49 receptor family is comprised of 10 members of homodimeric C-type lectin-like receptors that interact with specific alleles of MHC class I molecules (31). The majority of Ly49 receptors has an ITIM motif in their cytoplasmic domains and can function as inhibitory receptors. Although all Ly49 receptors were cloned from NK cells, expression of Ly49 is not restricted to NK cells but it is also detected in CD8+ T (32) and NKT cells (33). It has been reported that Ly49-negative NKT cells can be stimulated with {alpha}-GalCer-pulsed DCs, whereas Ly49-positive NKT cells do not respond (34). Thus, we examined the expression of Ly49A, Ly49C/I, and Ly49G2 on NKT cells from LFA-1+/+ and LFA-1–/– mice. The frequency of splenic NKT cells that express these Ly49 receptors was again comparable among LFA-1+/+ and LFA-1–/– mice (Fig. 2D and not shown). These results indicate that increased proliferation and production of large amounts of IL-4 by {alpha}-GalCer-activated NKT cells in LFA-1–/– mice are not due to differences in Ly49-positive NKT cells.

LFA-1 expression on NKT cells controls IL-4 production in response to the cognate {alpha}-GalCer Ag presented on DCs

Using conventional T cells, several studies have revealed that LFA-1/ICAM-1 interaction can have important roles for driving Th1 cell polarization, as blockage of this interaction leads to increased Th2 cell development from naive Th cells (11, 35). Therefore, we hypothesized that during {alpha}-GalCer presentation by DCs, inhibition of LFA-1/ICAM-1 interactions enhances IL-4 production by NKT cells. We first examined the expression of the LFA-1 ligands ICAM-1 and ICAM-2 on DCs. ICAM-1 but not ICAM-2 was expressed on DCs from both LFA-1+/+ and LFA-1–/– mice. Expression levels of ICAM-1 were comparable among LFA-1+/+ and LFA-1–/– DCs. CD1d and CD40 expression on DCs from LFA-1+/+ and LFA-1–/– mice were also comparable (Fig. 3A). NKT cells from LFA-1+/+ mice expressed high levels of LFA-1 on their cell surface (not shown),



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  		FIGURE 3. LFA-1 expression on NKT cells controls proliferation and IL-4 production in response to {alpha}-GalCer presented on DCs. A, Phenotypic analysis of splenic DCs. Purified DCs were stained with anti-I-Ab (PE), anti-CD11c (biotinylated), and FITC-labeled mAbs reactive against ICAM-1, ICAM-2, CD1d, and CD40. I-Ab+CD11c+ cells were gated and analyzed for the expression of ICAM-1, ICAM-2, CD1d, and CD40. Thin lines indicate the staining with isotype-matched control IgG. One result representative of three independent experiments is shown. B and C, Loss of LFA-1 expression on NKT cells results in increased proliferation (B) and IL-4 production (C) in response to {alpha}-GalCer-loaded DCs. LFA-1+/+ or LFA-1–/– NKT cells and DCs were cocultured in various combinations with {alpha}-GalCer. B, Proliferation of LFA-1+/+ or LFA-1–/– NKT cell responder cells was measured by [3H]thymidine incorporation at 48 h. C, IFN-{gamma} and IL-4 levels in culture supernatants from cells activated as in B were measured by ELISA. D, Inhibition of LFA-1/ICAM interaction using an anti-LFA-1 mAb (clone M17-4) enhanced IL-4, but not IFN-{gamma}, production by wild-type NKT cells. Anti-LFA-1 mAb was added at 10 µg/ml into the culture. After 48 h, culture supernatants were harvested and the levels of IFN-{gamma} and IL-4 were determined by ELISA. Data are represented as mean values ± SD of triplicate wells. Similar results were obtained in three independent experiments.

 
Next we examined whether NKT cells and/or DCs are affected by the loss of LFA-1 NKT cells and whether DCs were purified from LFA-1+/+ and LFA-1–/– mice and cultured in various combinations in the presence of {alpha}-GalCer. The proliferation of LFA-1–/– NKT cells was ~2-fold higher than that of NKT cells from LFA-1+/+ mice when the cells were cocultured with LFA-1+/+ DCs (Fig. 3B). Moreover, LFA-1–/– NKT cells produced increased IL-4 when cocultured with LFA-1+/+ or LFA-1–/– DCs in the presence of {alpha}-GalCer (Fig. 3C). By contrast, LFA-1–/– DCs did not induce enhanced IL-4 production in wild-type NKT cells (Fig. 3C). In addition, we cultured DCs and NKT cells separately in a Transwell culture system with {alpha}-GalCer to determine whether soluble factors play a role in NKT cell triggering. In these Transwell cultures, NKT cells failed to produce detectable levels of IL-4 and IFN-{gamma} (not shown) suggesting that DC-mediated NKT cell activation requires intimate cell-to-cell contact. Experiments using specific mAb to block LFA-1/ICAM interaction in vitro have revealed that signaling through LFA-1 plays a major role in the regulation of IL-4 production by CD4+ T cells (11, 12). Therefore, we next examined whether anti-LFA-1 mAb treatment can augment the in vitro production of IL-4 by {alpha}-GalCer-activated NKT cells from LFA-1+/+ mice. Blocking the LFA-1/ICAM interaction between LFA-1+/+ NKT cells and DCs indeed resulted in increased IL-4 production (Fig. 3D). Thus, blocking LFA-1/ICAM interactions between NKT cells and DCs contributes to enhanced IL-4 production by NKT cells in response to {alpha}-GalCer. These results indicate that LFA-1 expression on NKT cells is a critical component that determines IL-4 production in response to the cognate {alpha}-GalCer Ag presented on DCs.

LFA-1–/– mice normally produce IL-4 and IFN-{gamma} in response to in vivo anti-CD3 challenge

NKT cells whose TCR are directly activated by cross-linking with anti-CD3 mAb produce large amounts of IL-4 and IFN-{gamma} within 90 min after i.v. Ab injection (5). Therefore, we analyzed whether bypassing the Ag-specific V{alpha}14 TCR on NKT via anti-CD3 mAb stimulation would also induce increased IL-4 by LFA-1–/– NKT cells. However, following anti-CD3 stimulation, the levels of IL-4 and IFN-{gamma} production by splenic MNCs from LFA-1–/– mice were comparable to those of LFA-1+/+ mice (Fig. 4), indicating that NKT cells from LFA-1–/– mice retain a normal capacity to produce IL-4 and that the effects of LFA-1 are specific for TCRV{alpha}14 stimulation.



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  		FIGURE 4. Normal in vivo activation of LFA-1–/– NKT cells with anti-CD3 mAb. LFA-1+/+ and LFA-1–/– mice were injected with PBS or 1.5 µg of anti-CD3 mAb. Splenic MNCs were harvested 90 min later and plated in 96-well plates at the indicated cell numbers at 37°C for 2 h without additional stimulus. The amounts of IL-4 and IFN-{gamma} released in the culture supernatants were measured by ELISA. Three mice were used in each group.

 
LFA-1 regulates in vivo Th2-like functions of {alpha}-GalCer-activated NKT cells

Because {alpha}-GalCer-activated NKT cells produced markedly increased levels of IL-4 in vitro and in vivo, we analyzed whether deregulation of this pathway affects Th2 cell functions in LFA-1–/– mice. For instance, it has been reported that IL-4 produced by {alpha}-GalCer-activated NKT cells can activate B cell (36). Therefore, we investigated whether B cells were activated in LFA-1–/– mice following an in vivo challenge with {alpha}-GalCer. The expression of the early activation marker CD69 on B cells increased by 24 h after {alpha}-GalCer was administered to LFA-1+/+ and LFA-1–/– mice (Fig. 5A). However, the expression level of CD69 on B cells in LFA-1–/– mice was increased compared with that of B cells in LFA-1+/+ mice. Because IL-4 increases expression of I-Ab and CD23 on B cells (37), we also investigated whether expression of I-Ab and CD23 on B cells were up-regulated in LFA-1–/– mice. After in vivo administration of {alpha}-GalCer, the expression of I-Ab and CD23 was markedly increased in B cells from LFA-1–/– mice (Fig. 5A).



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  		FIGURE 5. Involvement of LFA-1 in {alpha}-GalCer-induced B cell activation and serum Ig responses. A, Expression of CD69, I-Ab, and CD23 on B220+TCR{alpha}{beta}– B cells. Mice were i.p. administered with {alpha}-GalCer (2 µg/100 µl) or vehicle. Twenty-four hours after treatment, gated splenic B220+TCR{alpha}{beta}– cells were electronically gated and analyzed for the expression of CD69, I-Ab, and CD23. Solid lines indicate cells following {alpha}-GalCer stimulation, and dotted line indicate cells from vehicle administered mice. One result representative of three independent experiments is shown. B, Serum Ig isotype levels in {alpha}-GalCer-treated mice. LFA-1+/+ or LFA-1–/– mice were administered with {alpha}-GalCer (2 µg/100 µl; i.p.) or vehicle. Total IgE, IgG1, and IgG2 serum levels were measured by ELISA at 7 days after the administration of {alpha}-GalCer or vehicle. Data are represented as the mean ± SD of three mice in each group. Similar results were obtained in two independent experiments.

 
IL-4 promotes IgE and IgG1 responses and IFN-{gamma} favors B cell switching to IgG2a (38). Because loss of LFA-1 on NKT cells results in increased IL-4 production, we investigated the contribution of LFA-1 to {alpha}-GalCer-induced Ig class switching. In LFA-1–/– mice, 7 days after a single dose of {alpha}-GalCer, serum IgE levels were ~2-fold higher whereas IgG1and IgG2 levels were comparable to those of LFA-1+/+ mice (Fig. 5B). These results show that LFA-1 acts as a negative regulator of NKT cell-regulated Th2-like immunity in response to the cognate Ag {alpha}-GalCer.

LFA-1 mediates the adhesion of NKT cell with {alpha}-GalCer-pulsed DCs

To address whether LFA-1 has a role in {alpha}-GalCer-specific cell adhesion between DCs and NKT cells, we examined whether LFA-1–/– NKT cells are able to form conjugation with {alpha}-GalCer-pulsed DCs. {alpha}-GalCer-pulsed DCs were mixed with naive NKT cells and {alpha}-GalCer-pulsed DCs/NKT cell conjugate formation was analyzed by flow cytometry. As described above in Fig. 3A, DCs express a high level of ICAM-1 but not ICAM-2 on the cell surface. We observed cell conjugation between DCs and NKT cells isolated from LFA-1+/+ and LFA-1–/– mice (Fig. 6). The percentage of NKT cells in conjugates increased with a similar kinetic in both LFA-1+/+ and LFA-1–/– mice. LFA-1–/– NKT cells formed less conjugates between DCs and NKT cells, although the observed reduction was not statistically significant at any of the time points analyzed. Moreover, baseline adhesion between LFA-1–/– NKT cells and DCs was slightly reduced (Fig. 6). These data suggest that the {alpha}-GalCer-dependent DC/NKT cell conjugation can occur in the absence of LFA-1; however, LFA-1 appears to contribute to optimal cell adhesion.



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  		FIGURE 6. Conjugate formation between NKT cells and {alpha}-GalCer-pulsed DCs. A, NKT cells from LFA-1+/+ or LFA-1–/– mice were labeled with PKH2 fluorochrome and incubated with HE-labeled {alpha}-GalCer pulsed wild-type DCs to form conjugates. Conjugate formation was determined by FACS at the indicated time points. DCs and NKT cells are distinguishable by their size differences detectable in the FSC/SSC profiles. Large cells (DCs) and small cells (NKT cells) were gated and analyzed for the presence of dual PKH2+HE+-positive cells (upper right quadrant). Numbers are the percentages of unconjugated DCs (upper left quadrant), unconjugated NKT cells (lower right quadrant), and conjugated DCs and NKT cells (upper right). B, Kinetics of conjugate formation. At different time points, conjugates were determined by flow cytometry by measuring the percentage of NKT cells that adhered to {alpha}-GalCer-pulsed DCs. Data from triplicates ± SD are shown.

 
Expression of Th1/Th2-specific transcription factors and cytokines in LFA-1–/– NKT cells

It has been reported that NKT cells but not conventional T cells spontaneously express IFN-{gamma} and IL-4 mRNA (39). We first examined the levels of IFN-{gamma} and IL-4 in naive NKT cells to that of purified naive CD4+ T cells from LFA-1+/+ and LFA-1–/– mice using quantitative RT-PCR. Naive NKT cells but not naive CD4+ T cells from LFA-1+/+ and LFA-1–/– mice contained constitutive IFN-{gamma} and IL-4 mRNA without cognate stimuli. The expression levels of IFN-{gamma} and IL-4 were comparable between LFA-1+/+ and LFA-1–/– NKT cells (Fig. 7A). These results demonstrate that naive NKT cells constitutively express IFN-{gamma} and IL-4, but loss of LFA-1 expression has no apparent effect on constitutive expression of IFN-{gamma} and IL-4 in naive NKT cells.



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  		FIGURE 7. {alpha}-GalCer-induced expression of Th1 or Th2 transcription factors cytokines in NKT cells. A, Naive NKT and CD4+ T cells were purified from spleens of LFA-1+/+ or LFA-1–/– mice. Quantitative RT-PCR for IL-4 and IFN-{gamma} was performed on total RNA. Expression levels of cytokine mRNA are represented as ratios to that of control GAPDH. Data represent one of three comparable experiments. B, NKT cells from LFA-1+/+ or LFA-1–/– mice and {alpha}-GalCer-pulsed DCs from LFA-1+/+ mice were cocultured for 48 h. Quantitative RT-PCR for T-bet, IFN-{gamma}, GATA-3, IL-4, IL-5, and IL-13 was performed on isolated total RNA and the abundance of each mRNA normalized to GAPDH. Data of mRNA levels are presented as induction index described in Materials and Methods. One result representative of three independent experiments is shown.

 
The T-box transcription factor T-bet is involved in the commitment of Th1 cells by inducing IFN-{gamma} synthesis and repressing IL-4 and IL-5 production, even in fully polarized Th2 cells (15). In contrast, GATA-3 is essential for Th2 lineage commitment, as it plays a critical role for IL-4, IL-5, and IL-13 expression (16, 40). To elucidate the consequences of LFA-1 deficiency in {alpha}-GalCer-activated NKT cells for the expression of these transcription factors, T-bet and GATA-3 mRNA levels were determined by quantitative RT-PCR analysis. We purified splenic NKT cells from LFA-1+/+ or LFA-1–/– mice and cocultured them in the presence or absence of {alpha}-GalCer-loaded DCs for 48 h. The up-regulations of T-bet and IFN-{gamma} were comparable between LFA-1+/+ and LFA-1–/– NKT cells. By contrast, GATA-3 and IL-4 mRNA were highly induced in LFA-1–/– NKT cells (Fig. 7B). The genes for IL-4, IL-5, and IL-13 are closely linked on murine chromosome 11 (41), which has led to considerable speculation that these genes are coordinately regulated. We indeed observed increased IL-5 and IL-13 mRNA expression in {alpha}-GalCer-activated NKT from LFA-1–/– mice. Thus, loss of LFA-1 affects induction and expression of the Th2 lineage transcription factor GATA-3 and the Th2 cytokines IL-4, IL-5, and IL-13 in {alpha}-GalCer-activated NKT cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that loss of LFA-1 in NKT cells leads to a shift toward Th2-like differentiation, accompanied by increased B cell activation and enhanced GATA-3 and Th2 cytokine mRNA expression. LFA-1 seems to play an important role in the initiation of immune responses by establishing contact between APCs and T cells, as well as in effector functions such as CTL/target interactions, T cell proliferation, and migration of leukocytes through endothelial cell layers into inflamed tissues (42, 43). The role of LFA-1/ICAM-1 interactions in the induction of Th responses by DCs has been studied using blocking mAbs to ICAM-1 or ICAM-1–/– APCs and these experiments indicated abrogation of Th2 responses as a consequence of LFA-1/ICAM-1 interactions (11, 35). It has been also reported that LFA-1 ligation on activated human naive Th cells leads to a shift toward Th1 cell differentiation (12). Therefore, LFA-1 seems to be one of the key molecules in Th2 development. However, it has been also reported that LFA-1–/– mice developed high Th1 immune responses caused by accumulation of IL-12 produced by granulocytes into sites of inflammation after Listeria monocytogenes infection (44). Thus, whether LFA-1 is indeed involved in Th1 or Th2 polarization in T cells remains elusive. Our genetic data for the first time show that that LFA-1 negatively regulates Th2 cytokine production by NKT cells in response to their cognate {alpha}-GalCer Ag presented by DCs in vitro and in vivo.

Recognition of Ag presented by DCs to conventional T cells requires the formation of a specialized junction between these cells; the first step requires low-avidity interactions of adhesion molecules such as LFA-1 and CD2 on T cells to assure positional stability, thus facilitating TCR engagement (45). TCR triggering subsequently initiates strong adhesive LFA-1-mediated interactions through avidity alterations (46, 47). LFA-1 binding to ICAM-1 helps to stabilize the physical interaction between DCs and Th cells, thereby enhancing or prolonging TCR-dependent signals (48, 49). We have previously reported that IL-12-activated NKT cells showed impaired conjugate formation with target tumor cells (50). In contrast, we observed that loss of LFA-1 does not significantly affect the conjugate formation between {alpha}-GalCer-loaded DCs and naive NKT cells. It could be speculated that LFA-1 on IL-12-activated NKT cells is in a high avidity state that drives stable conjugate formation even in the absence of TCR ligation. Whereas the mechanism of the regulation of LFA-1 avidity is unclear, a change in the conformation of ICAM binding site or redistribution in the membrane seems most likely to regulate LFA-1-dependent adhesion (51). Although the mechanism by which the loss of LFA-1 augments the IL-4 production of {alpha}-GalCer-activated NKT cells remains to be investigated, we hypothesize that change of specialized junction(s) between DCs and NKT cells in the absence of LFA-1 may lead to qualitatively different interactions between DCs and NKT cells, resulting in a different TCRV{alpha}14 signal and altered IL-4 production by NKT cells.

Our genetic results show that LFA-1 negatively regulates gene expression of GATA3 and Th2 cytokines in NKT cells. The transcription factors found to regulate IL-4 expression and the commitment of Th2 cells include GATA-3 (16), NFAT (52), c-Maf (53), NF-{kappa}B (54), or JunB (55). We examined the expression of GATA-3 in {alpha}-GalCer-activated NKT cells. Our data show that the loss of LFA-1 results in the up-regulation of GATA-3 mRNA expression and increased mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13 in {alpha}-GalCer-activated NKT cells. c-Maf has been shown to be critical for IL-4 gene expression and directly activates the IL-4 promoter (53). However, we did not observe any induction or changes in c-Maf mRNA expression in our experimental system (not shown). IL-4 and IL-13 are of critical importance to trigger B cell Ig class switching to IgE (56, 57). Indeed, we observed that serum IgE was markedly elevated after administration of {alpha}-GalCer in LFA-1–/– mice. The signaling intermediates that couple LFA-1 to the GATA-3 pathway in NKT cells remain to be identified.

Recent studies have suggested that NKT cells can regulate autoimmunity (58, 59, 60) and the induction of the cardiac allograft tolerance (61). It has also been reported that repeated administration of {alpha}-GalCer to nonobese diabetic mice can prevent type 1 diabetes due to Th2-mediated adaptive immune responses (62, 63). Furthermore, analogues of {alpha}-GalCer can induce predominant production of IL-4 by NKT cells, leading to suppression of experimental autoimmune encephalomyelitis (64). It has been also reported that statins, commonly used for treatment of hypercholesterolemia, can block LFA-1-mediated costimulation of lymphocytes (65). Our current data may point to new approach for prevention of onset and recurrence of Th1-associated diseases such as rheumatoid arthritis, Crohn’s disease, and psoriasis or the induction of transplantation tolerance. However, further studies are needed to unravel how LFA-1 participates in the interactions between DCs and NKT cells during infection and autoimmunity in vivo and how LFA-1 negatively controls Th2-cytokine production by NKT lymphocytes.


   Acknowledgments
 
We thank Kirin Brewery and Dr. Masaru Taniguchi for providing {alpha}-GalCer and PE-{alpha}-GalCer-loaded mCD1d/tetramers.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the IMBA, the Jubilaeumsfonds of the Austrian National Bank, the Austrian Ministry of Education and Science, and EuroThymaide (Sixth European Union framework). Back

2 Address correspondence and reprint requests to Dr. Goichi Matsumoto, Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, 82 Inaoka, Yokosuka, Kanagawa 238-8580, Japan. E-mail address: gmatsu{at}kdcnet.ac.jp Back

3 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galactosylceramide; DC, dendritic cell; MNC, mononuclear cell; CM, complete medium; HE, hydroethidine; FSC, forward scatter; SSC, side scatter. Back

Received for publication November 24, 2003. Accepted for publication August 5, 2004.


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