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Originally published online as doi:10.1189/jlb.0307163 on August 28, 2007

Published online before print August 28, 2007
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(Journal of Leukocyte Biology. 2007;82:1455-1465.)
© 2007 by Society for Leukocyte Biology

Modulation of CD1d-restricted NKT cell responses by CD4

Xiuxu Chen*, Xiaohua Wang*, Gurdyal S. Besra{dagger} and Jenny E. Gumperz*,1

* Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA; and
{dagger} School of Biosciences, University of Birmingham, Birmingham, United Kingdom

1Correspondence: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., Madison, WI 53705, USA. E-mail jegumperz{at}wisc.edu


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ABSTRACT
 
CD4+ and CD4– NKT cell populations have been shown to be functionally distinct, but the role of CD4 molecules in NKT cell activation is not clear. Here, we have used human CD1d-restricted NKT cell clones to investigate the contribution of CD4 to NKT cell functional responses. Coligation of CD4 with the TCR/CD3 complex resulted in enhanced cytokine secretion and increased calcium flux by CD4+ NKT cell clones, indicating that CD4 is functionally active in these cells. CD4 blockade specifically inhibited cytokine secretion and proliferation of CD4+ NKT cell clones in response to CD1d+ APCs but did not affect cytotoxicity, suggesting that CD4 preferentially modulates some NKT cell functional responses and not others. Anti-CD4 mAb treatment inhibited NKT cell responses to both MHC class II+ and MHC class II– APCs, indicating that its effect was not due to blocking CD4 binding to MHC class II molecules on APCs. The inhibitory effect of the anti-CD4 mAb also did not require recognition of CD1d by the NKT cell, since calcium flux was reduced in response to anti-CD3 mAb stimulation. Western blot analysis revealed that anti-CD4 treatment resulted in increased phosphorylation of an inhibitory site of p56lck (tyrosine 505). Thus, CD4 blockade interferes with the course of CD3-mediated signaling events in NKT cells. These results indicate that CD4 can contribute to NKT cell activation independently of the presence of a CD4-ligand on APCs and suggest that it preferentially modulates cytokine and proliferative responses.

Key Words: costimulation • cytotoxicity • TCR • CD3 • p56lck • MHC class II


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INTRODUCTION
 
NKT cells are a subpopulation of T lymphocytes that recognize lipids and glycolipids presented by CD1d molecules [1 ]. Remarkably, in addition to recognizing specific microbial lipids, NKT cells can recognize self lipids as antigens [2 3 4 ]. NKT cells are also distinctive in that they can secrete both Th1- and Th2-type cytokines rapidly upon stimulation and appear to have regulatory effects on downstream immune responses [5 6 7 8 ]. Paradoxically, NKT cells have been shown to have contrasting immunological effects in several disease models [7 , 9 ]. It remains unclear how the diverse effects of NKT cells in different systems are determined.

Because NKT cells can respond to self antigens, and therefore their activation is not solely regulated by the presence or absence of foreign antigenic challenge, one possibility is that their functional responses are determined by the levels of costimulating factors in the local environment. Indeed, previous studies have shown that IL-12 production by dendritic cells (DCs) that have been exposed to microbial products enhances NKT cell IFN-{gamma} secretion but does not affect IL-4 [10 , 11 ]. A substantial fraction of NKT cells express CD4, and this subset has been shown to be functionally distinct from those that are CD4-negative [12 , 13 ]. CD4+ NKT cells were found to produce both Th1 and Th2 cytokines, whereas CD4– NKT cells were found mainly to produce Th1 cytokines [12 , 13 ]. Additionally, CD4+ and CD4– NKT cells showed different patterns of perforin expression, and CD4– NKT cells expressed the costimulatory NK-receptor NKG2D, whereas expression of FasL appeared limited to CD4+ NKT cells [12 ]. Thus, expression of CD4 distinguishes two phenotypically and functionally distinct subsets of NKT cells. However, it is not clear whether CD4 is simply a lineage marker for NKT cells or whether CD4 directly contributes to the functional properties of NKT cells.

The CD4 molecule plays multiple roles in the activation of MHC class II-restricted T cells, including signaling functions and providing an adhesive effect [14 , 15 ]. CD4 binds a conserved epitope formed from the {alpha}2 and β2 domains of MHC class II molecules via its N-terminal D1 domain, and this binding stabilizes TCR/MHC class II interactions [16 , 17 ]. Current models suggest that binding of CD4 to the same MHC class II molecule that is bound by the TCR may be important for signaling, but it is also clear that CD4 molecules bind MHC class II molecules that are not bound by TCRs, since the TCR and CD4 molecular populations can be seen to segregate during T cell activation [18 ]. Thus, CD4 molecules on NKT cells could bind to MHC class II molecules on antigen-presenting cells (APCs), even though the TCR binds to CD1d molecules. Additionally, a recent analysis indicates that CD4 may be able to bind directly to CD1d molecules [19 ]. The affinity of CD4 for CD1d, as compared with MHC class II is not known. Moreover, since physiological levels of CD1d expression are much lower than those of MHC class II [20 ], the relative importance of CD1d and MHC class II molecules as ligands for CD4 during NKT cell activation remains unclear.

CD4 contributes to T cell intracellular signaling through recruitment of the p56lck tyrosine kinase, which binds directly to a cysteine-containing motif in the cytoplasmic tail of CD4 molecules [21 , 22 ]. p56lck is a member of the Src family kinases and phosphorylates the CD3 {zeta}-chain, an event that is critical for propagation of the TCR signaling cascade [23 24 25 ]. Like other Src family kinases, the activity of p56lck is both positively and negatively regulated by phosphorylation at different sites [26 ]. In particular, phosphorylation of tyrosine 394 (Y-394) is required for the activating effects of p56lck, while phosphorylation of tyrosine 505 (Y-505) is inhibitory [27 ]. During T cell activation Y-394 becomes autophosphorylated, and there is evidence that the degree to which CD4 molecules are dimerized is critical for this step [28 ]. Phosphorylation of Y-394 permits binding of the C-terminal Src kinase Csk, which preferentially phosphorylates the inhibitory Y-505 site [29 , 30 ]. Phosphorylation of this site induces a conformational change in the p56lck molecule that renders it inactive [27 , 31 ]. Further regulation of p56lck occurs through the activity of multiple phosphatases, including CD45, PEP, SAP-1, and SHP-1, which can dephosphorylate the Y-505 and Y-394 sites, resulting in either restored kinase function or deactivation, respectively [32 33 34 35 ]. Thus, the activity of p56lck is sensitively regulated by transient phosphorylation of the Y-394 and Y-505 sites, resulting in either positive or negative modulation of T cell activation.

Given the complexity of p56lck activation, the functional impact of CD4 expression on NKT cells is not clear. Moreover, the requirements for APC expression of MHC class II, as compared with CD1d, as potential ligands for CD4 are not known. Here, we have investigated the role of CD4 in the functional responses of human NKT cell clones and its effects on TCR-mediated activation.


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MATERIALS AND METHODS
 
Antibodies
The anti-human CD3{epsilon} antibody (clone SPVT3b, murine IgG2a) was purified from hybridoma supernatant by protein A affinity chromatography. The anti-human CD4 antibody (clone RPA-T4, murine IgG1) was purchased from Biolegend (San Diego, CA, USA). Negative control antibodies (clone MOPC-21, murine IgG1, and clone UPC10, murine IgG2a) were purchased from Sigma (St. Louis, MO, USA). Mouse anti-human total p56lck antibody (clone 28) and mouse anti-human phosphotyrosine 505 specific p56lck antibody (clone 4) were obtained from BD Biosciences (San Jose, CA, USA). Secondary cross-linking antibody (goat anti-mouse IgG) was purchased from Invitrogen (Carlsbad, CA, USA).

NKT cell clones
Human NKT cell clones were established as described previously [10 , 36 ] and maintained in T cell medium [RPMI 1640 medium; 2 mM L-glutamine; 100 µg/ml each of penicillin and streptomycin; 10% FBS; 5% bovine calf serum; 5% human AB serum from Gemini, West Sacramento, CA, USA; and 400 U/ml recombinant human IL-2 from Chiron (Emeryville, CA, USA)], with periodic restimulation by irradiated allogeneic feeder PBMCs and PHA-p as described [10 , 36 ].

Antigen-presenting cells
Human monocyte-derived DCs were generated as follows: fresh monocytes were purified from peripheral blood mononuclear cells (PBMCs) of healthy donors by magnetic sorting using CD14 microbeads (Miltenyi Biotec, Auburn, CA, USA). The resulting monocytes were differentiated for 3 days in medium (RPMI 1640, FBS, Fe2+ supplemented calf bovine serum, 2 mM L-glutamine, 100 µg/ml penicillin, and streptomycin each, pyruvate, HEPES), containing 300 U/ml recombinant human GM-CSF and 200 U/ml recombinant human IL-4 (Berlex Labs, Richmond, CA, USA) and then treated with 250 ng/ml Salmonella typhimurium LPS (Sigma-Aldrich) for an additional 2 days. The resulting cells were confirmed to have a mature DC phenotype by flow cytometric analysis (CD14low, CD1a+, CD1b+, CD86+, CD83+, DC-SIGN+). The lymphoblastoid cell line 721.221 and the myelomonocytic cell line K562 were transfected with human CD1d cDNA, drug selected, and sorted by flow cytometry to obtain homogeneously CD1d+ lines, as described previously [37 ]. The resulting transfectants ("721.221-d" and "K562-d") were maintained in culture medium [RPMI 1640 medium; 2 mM L-glutamine; 100 µg/ml each of penicillin and streptomycin; 1 mg/ml G418 sulfate from Mediatech (Herndon, VA, USA); 10% Fe2+ supplemented/defined bovine calf serum from Hyclone, Logan, UT, USA). The 3023 and 2001 cell lines were a kind gift from Dr. Robert DeMars and are derived from the MHC class II deletion mutant lymphoblastoid cell line 721.174 [38 ], by transfection with HLA-DR{alpha} (3023), and HLADR{alpha} and DR7β (2001). These cell lines were stably transfected with cDNA encoding human CD1d, drug selected, and sorted as described above. The resulting CD1d+ cell lines ("3023-d" and "2001-d") were maintained in culture medium (RPMI 1640 medium; 2 mM L-glutamine; 100 µg/ml each of penicillin and streptomycin from Mediatech; 5% FBS; and 10% bovine calf serum from Hyclone). For the 3023-d line, the culture medium was supplemented with 0.5 mg/ml G418 sulfate (Mediatech) and 0.5 µg/ml puromycin (Sigma), and for the 2001-d cells, 10 µg/ml mycophenolic acid and 7.6 µg/ml xanthine (both from Sigma) were also included.

Preparation of lipid antigen {alpha}-GalCer
The glycolipid antigen {alpha}-galactosylceramide ({alpha}-GalCer) was prepared from D-lyxose as described [39 ] and dissolved in DMSO at a concentration of 100 µg/ml, and stored frozen in glass vials at –20°C. Prior to use, the lipid was sonicated in a heated water bath for 15 min at 37°C.

Stimulation of NKT cell cytokine secretion
High protein binding flat-bottomed 96-well plates were coated at 4°C overnight with 5 ng/well anti-CD3 antibody (SPVT-3b) in the presence of a titrated concentration of anti-CD4 mAb (RPA-T4) or an isotype-matched negative control mAb (MOPC21). Alternatively, 0.5 µg human CD1d-Fc fusion protein was coated per well in the presence of a titrated concentration of anti-CD4 or isotype control mAb at 4°C overnight. The human CD1d fusion protein was loaded with 50 ng/well {alpha}-GalCer or an equivalent volume of vehicle (DMSO) in 10 mg/ml PBS/BSA buffer at 37°C overnight, as described previously [36 ]. The plates were washed, and NKT cell clones (5x104/well) were added in a final volume of 200 µl/well in T cell culture medium lacking IL-2. For stimulation of NKT cell cytokine secretion by DCs, the APCs were pulsed for 4 h with the indicated concentrations of {alpha}-GalCer or DMSO as a vehicle control. NKT cell clones (5x104/well) were coincubated for 16 h with the DCs (5x104/well), in a final volume of 200 µl/well in T cell culture medium lacking IL-2. Where indicated 10 µg/ml anti-CD4 antibody (RPA-T4) or mouse IgG1 (MOPC-21) were added to the NKT cell and APC cocultures. All assays were performed in triplicate. Supernatants were tested for the indicated cytokines by commercially available ELISAs [GM-CSF, IL-2, IL-4, and IL-13 capture and detection antibodies were from Biolegend (San Diego, CA, USA); IFN-{gamma} capture and detection antibodies were from Endogen (Rockford, IL, USA), and quantified by comparison to the relevant recombinant human cytokine standards (all from Peprotech, Rockyhill, NJ, USA).

Calcium flux assays
Calcium flux was determined by assessing the change in fluorescence of two different calcium-sensitive dyes: Fluo-4 and Fura Red. Because calcium binding results in increased Fluo-4 fluorescence signal and decreased intensity of Fura Red, the use of these two dyes together has been shown to provide equivalent calcium flux sensitivity to single ratiometric dyes such as Indo 1 [40 ]. NKT cells (4x105/well) were labeled with 1 µM Fluo-4 and 2 µM Fura Red in PBS for 30 min at 37°C, then washed with medium lacking IL-2. The NKT cells were incubated with 0.5–4 µg/ml anti-CD3 mAb (clone SPVT3b, IgG2a), and 1 µg/ml anti-CD4 (clone RPA-T4, IgG1) or IgG1 negative control mAb (clone MOPC21) for 10 min at room temperature; then 20 µg/ml cross-linking antibody was added, and the cells were immediately analyzed by flow cytometry. For cocross-linking of CD3 with CD4, a goat anti-mouse pan-IgG polyclonal antibody was used (GAM). For cross-linking CD3, but not CD4, a rat anti-mouse IgG2a specific antibody was used (RAM). For analysis of calcium flux without CD3 cross-linking, 1 µg/ml anti-CD4 antibody (RPA-T4) or mouse IgG1 isotype control (MOPC-21) were added to the NKT cells and preincubated for 10 min at room temperature; then 0.5–4 µg/ml anti-CD3 antibody was added, and the cells were immediately analyzed by flow cytometry. The data were analyzed using FlowJo analysis software, and the percentage of cells in the flux gate was determined for 10-s intervals at the times shown.

NKT cell proliferation
NKT cells were labeled with 2 µM CFSE in RPMI 1640 for 10 min at 37°C, then washed twice with T cell medium lacking IL-2. The NKT cells (5x104 per well) were coincubated at a 1:1 ratio with DCs pulsed either with the indicated concentrations of {alpha}-GalCer or with vehicle (DMSO) alone, in the presence of 10 µg/ml anti-CD4 antibody (RPA-T4) or mouse IgG1 isotype control (P3). Proliferation was measured after 5 days by flow cytometric analysis of CFSE dilution. The percent of the starting NKT cells that divided was determined using the proliferation analysis platform of the Flowjo software program (Treestar, Inc., Ashland, OR, USA).

NKT cell cytotoxicity
Target cells were pulsed with 5 ng/ml or the indicated concentrations of {alpha}-GalCer or vehicle (DMSO) for 16 h, then washed, and labeled with 100 µCi/ml chromium-51 (51Cr) for 4 h, then washed 3 times in cold RPMI medium. Target cells (2x103/well) were incubated with 20:1 or the indicated E:T ratios of NKT cells for 4 h, and 51Cr released into the supernatant was measured using a PerkinElmer Microbeta Trilux plate reader. Spontaneous release was determined by analysis of 51Cr labeled target cells in the absence of effector cells, and total release was determined by analysis of 51Cr labeled target cells treated with 0.1% Triton-X 100 buffer. The specific killing was calculated as follows:

Formula
To evaluate the effect of CD4 blockade on cytotoxicity, 10 µg/ml anti-CD4 antibody (RPA-T4) or mouse IgG1 isotype control (MOPC-21) was added during the NKT cell and target cell coincubation. All assays were performed in triplicate.

Analysis of p56lck phosphorylation
NKT cells were stimulated with the indicated antibodies (anti-CD3 or anti-CD4) with or without cross-linking, for the indicated times (2, 5, or 10 min). To stop the reaction, 1 ml cold PBS/BSA was added to the NKT cells at the specified time point, and the cells were immediately transferred onto ice, spun down at 4°C, and lysed by addition of a nondenaturing detergent buffer (Cell LyticTM M Cell Lysis Reagent; Sigma). Insoluble material was removed by centrifugation, and clarified lysates from 5x105 NKT cells were loaded per lane and resolved by 10% SDS-PAGE. Proteins were transferred from PVDF membranes by semi-dry electrophoresis at 20 V for 45 min, and the membranes were incubated with a phosphotyrosine-505 p56lck specific antibody (clone 4) (BD Biosciences, San Jose, CA, USA) in blocking buffer, followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Biolegend Inc.). Bands were quantified by Image J software (National Institutes of Health, Bethesda, MD, USA) after densitometric scanning. Total p56lck was subsequently detected from the same blots by stripping the membranes for 30 min at 50°C in stripping buffer (2% SDS, 0.7% β-mercaptoethanol in Tris/HCl buffer, pH 6.7), then incubating the membranes with mouse anti-human p56lck (clone 28) (BD Biosciences, San Jose, CA, USA), and quantifying as described above.


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RESULTS
 
CD4 can costimulate NKT cell function
Human CD1d-restricted NKT cell clones that were CD4+ or CD4– were derived using {alpha}-GalCer loaded CD1d tetramers, as described previously [10 , 36 ]. To investigate whether CD4 was functionally active on these cells, CD4+ and CD4– clones were tested for their responses to a suboptimal concentration of plate-bound anti-CD3 mAb in the presence of increasing concentrations of plate-bound anti-CD4 mAb or a nonspecific isotype matched negative control mAb. The anti-CD4 mAb specifically enhanced cytokine secretion by CD4+ NKT cell clones in a concentration-dependent manner but had no effect on cytokine secretion by CD4– clones (Fig. 1A ). Similarly, when plate-bound human CD1d-Fc fusion protein (hCD1d-Fc) loaded with a suboptimal concentration of {alpha}-GalCer was used as the primary stimulus, plate-bound anti-CD4 mAb specifically enhanced the responses of CD4+ NKT cell clones in a concentration-dependent manner but did not affect the responses of CD4– NKT cell clones (Fig. 1B) . Similar results were also obtained using antibody-coated microbeads instead of tissue culture plates (data not shown). Thus, ligation of CD4 along with either CD3 or the TCR on a rigid substrate resulted in increased cytokine secretion by CD4+ NKT cell clones.


Figure 1
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  		Figure 1. Plate-bound anti-CD4 antibody costimulates NKT cell cytokine secretion in response to a suboptimal TCR stimulus. (A) A CD4+ (left) and a CD4– (right) NKT cell clone were incubated in microtiter plate wells that were coated with 100 ng/ml anti-CD3 antibody and the indicated concentrations of anti-CD4 antibody (triangles) or isotype-matched negative control antibody (squares). Solid circles indicated NKT cells that were incubated in wells without anti-CD3 antibody. (B) Microtiter plate wells were coated with human CD1d-Fc fusion protein and the indicated concentrations of anti-CD4 antibody (triangles) or negative control antibody (squares), and then pulsed with 50 ng/well {alpha}-GalCer. Solid circles show results from wells that were not pulsed with {alpha}-GalCer. The plots show mean GM-CSF secretion in the culture supernatants, as quantified by ELISA of three replicate wells, and error bars show the standard deviations of the means (not visible for some data points on the scales shown). Results are representative of 6 independent experiments.

To investigate whether the enhanced responses of the CD4+ NKT cell clones were due to a signaling effect mediated by CD4 or to increased adhesion of the clones to the substrate presenting the primary stimulus, we tested the effect of CD4 coligation with CD3 on NKT cell calcium flux. CD4+ or CD4– NKT cells were labeled with the intracellular calcium indicator dyes Fluo-4 and Fura Red, and the number of cells that fluxed calcium after anti-CD3 stimulation was assessed by flow cytometric analysis (Fig. 2A ). The NKT cells were stimulated with anti-CD3 mAb in combination with anti-CD4 or an isotype-matched negative control mAb, in the presence of polyclonal goat-anti-mouse IgG (GAM) antiserum to provide cross-linking, and calcium flux was measured over 4 or 5 min following the addition of the stimulus. In the presence of the anti-CD4 mAb, the calcium flux appeared accelerated and amplified compared with cells that were treated with anti-CD3, and the negative control mAb (Fig. 2B , left). NKT cells that were treated with anti-CD4 mAb and GAM alone (without anti-CD3) showed little or no calcium flux (Fig. 2B) . Addition of the anti-CD4 antibody did not enhance the calcium flux of CD4– NKT cells, demonstrating that its effect on CD4+ clones was specific (Fig. 2B , right). These results show that ligation of CD4 with CD3 can costimulate intracellular signaling of NKT cell clones, indicating that CD4 is functionally active on these cells.


Figure 2
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  		Figure 2. NKT cell calcium flux in response to cross-linked CD3 and CD4. (A) Flow cytometric contour plots of Fluo-4 and Fura Red-labeled NKT cells that were unstimulated (left) or treated with anti-CD3 antibody cross-linked with a polyclonal goat-anti-mouse Ig anti-serum (GAM) to induce calcium flux (right). The polygon shows the gate used to quantify cells that have shifted in fluorescence intensity as a result of intracellular calcium flux. (B) A CD4+ (left) and a CD4– (right) NKT cell clone were treated with anti-CD3 and anti-CD4 antibodies (squares), or anti-CD3 and an isotype-matched negative control antibody (triangles), or anti-CD4 antibody alone (circles), followed by cross-linking with GAM. The plots show the percentage of cells that had moved into the flux gate during 10-s intervals at the indicated times after stimulation. Results are representative of more than 6 independent experiments.

Effect of CD4 blockade on NKT cell-functional responses
Monocyte-derived DCs have previously been shown to efficiently activate NKT cell-functional responses, including cytokine secretion and proliferation [20 ]. We tested the responses of CD4+ and CD4– NKT cell clones to monocyte-derived DCs that were pulsed with varying concentrations of {alpha}-GalCer or vehicle alone, in the presence of anti-CD4 mAb or an isotype-matched negative control mAb. Cytokine secretion by the CD4+ clones was markedly diminished by the addition of anti-CD4 mAb compared with the negative control mAb (Fig. 3A ). Addition of anti-CD4 had no effect on cytokine secretion by CD4– NKT cell clones (data not shown). The inhibitory effect of CD4 blockade appeared somewhat greater in the absence of {alpha}-GalCer (i.e., auto-antigenic stimulation) and at low {alpha}-GalCer concentrations but was also observed at higher {alpha}-GalCer concentrations, suggesting the contribution of CD4 to NKT cell activation was not completely overcome by strong TCR signaling (Fig. 3A) . These results suggest that CD4 can contribute to the activation of cytokine secretion by NKT cell clones, and its impact may be greatest on NKT cell autoreactive responses or in the presence of low antigen doses.


Figure 3
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  		Figure 3. CD4 blockade inhibits NKT cell cytokine production and proliferation. (A) A CD4+ NKT cell clone was coincubated with fresh monocyte-derived dendritic cells that were pulsed with the indicated concentrations of {alpha}-GalCer (open symbols) or vehicle alone (solid symbols), in the presence of an anti-CD4 antibody (triangles) or an isotype-matched negative control antibody (squares). The plots show the mean concentrations of IL-13, IL-4, IFN-{gamma}, and IL-2 in the culture supernatants, as measured by ELISA of three replicate wells, and error bars show the standard deviations of the means (not visible for some data points on the scales shown). Similar results were obtained in 6 independent experiments. Addition of the CD4 antibody did not affect the cytokine secretion responses of CD4– NKT cell clones (not shown). (B) Compiled results from the analysis of the effect of anti-CD4 blockade on cytokine secretion by 5 CD4+ and 2 CD4– NKT cell clones. The clones were incubated with CD1d-transfected APCs in the presence of a CD4 blocking antibody or an isotype-matched negative control antibody, and secretion of the indicated cytokines was assessed by ELISA. Each point represents an independent analysis, and the y-axis shows the percent inhibition observed in the presence of the anti-CD4 mAb compared with the isotype control. (C) The effect of CD4 blockade on NKT cell proliferation. CD4+ and CD4– NKT cell clones were labeled with CFSE and coincubated with monocyte-derived dendritic cells that had been pulsed with the indicated concentrations of {alpha}-GalCer, in the presence of anti-CD4 antibody or an isotype-matched negative control antibody for 5 days. The histograms show the CFSE-staining results for CD3+DAPI– cells, and numbers above the gate markers indicate the percentage of cells with reduced CFSE intensity as a result of proliferation. The plots show the percentage of the starting NKT cells that underwent cell division, as calculated using the proliferation platform of the Flowjo data analysis software. Similar results were obtained in more than 6 independent experiments.

We have previously observed that NKT cell IFN-{gamma} production can be selectively enhanced by IL-12 secreted by DCs [10 ]. Additionally, we have previously observed that all of our NKT cell clones efficiently secrete GM-CSF, but this cytokine can also be produced by monocyte-derived DCs, and hence it is difficult to accurately quantitate GM-CSF secretion by NKT cells in response to these APCs. Therefore, to further investigate the effect of CD4 blockade on NKT cell cytokine secretion, we tested the responses of CD4+ or CD4– NKT cell clones to CD1d transfected APCs, in the presence of the anti-CD4 mAb or a negative control mAb. Addition of anti-CD4 blocking mAb resulted in a significant reduction in secretion of GM-CSF, IL-4, and IFN-{gamma} by CD4+ NKT cells but did not inhibit cytokine secretion by CD4– clones (Fig. 3B) . The mean inhibition of IFN-{gamma} secretion was 44% (±19.07 SD), whereas the mean for GM-CSF was 24% (±21.98 SD) and that for IL-4 was 27% (±28.71 SD). On the basis of these results, CD4 blockade resulted in a significantly greater inhibition of IFN-{gamma} than of GM-CSF (P=0.042), but although the mean inhibition of IL-4 secretion was close to that of GM-CSF, the difference between IL-4 and IFN-{gamma} was not significant.

We next investigated the effect of CD4 on NKT cell proliferative responses. The CD4+ and CD4– NKT cell clones were labeled with CFSE, and coincubated with {alpha}-GalCer-pulsed or vehicle-treated DCs in the presence of an anti-CD4 blocking mAb or an isotype-matched negative control mAb. After 5 days, the cells were stained with an anti-CD3 mAb and with DAPI to allow gating on the live T cells, and NKT cell proliferation was assessed by flow cytometric analysis of the dilution of the CFSE fluorescence as a result of cell division. Little proliferation was observed in response to vehicle-treated DCs or to those that were pulsed with less than 25 ng/ml {alpha}-GalCer (Fig. 3C) , suggesting that a relatively strong TCR stimulus was required to activate significant proliferative responses by the NKT cell clones. DCs that were pulsed with high concentrations of {alpha}-GalCer stimulated marked proliferation, and anti-CD4 mAb addition reproducibly diminished proliferation by CD4+ clones but had no effect on CD4– clones (Fig. 3C) . Analysis of the CFSE dilution profiles suggested that CD4 blockade reduced the fraction of NKT cells within the starting population that divided (Fig. 3C , far right column) but had less effect on the number of rounds of division by those cells that did divide (data not shown). Hence, CD4 may contribute to the activation threshold required for proliferation by CD4+ NKT cell clones.

To investigate the cytotoxic responses of our NKT cell clones, we tested their ability to lyse two HLA class I-deficient target cell lines, 721.221 and K562, that are known to be efficiently killed by natural killer (NK) cells [41 ]. We first determined whether lysis by the NKT cell clones was dependent on stimulation by CD1d, or could occur independently of TCR-mediated signaling, as is the case for NK cells. The NKT cell clones were able to kill CD1d-transfected 721.221 and K562 cells but showed little or no specific lysis of the untransfected parent cells, suggesting that activation of their cytolytic responses required TCR-mediated signaling and was not simply dependent on activation through NK receptors (Fig. 4A ). There was little specific lysis of CD1d transfected target cells in the absence of {alpha}-GalCer, but even relatively low levels of {alpha}-GalCer (5 ng/ml) were sufficient to induce maximal killing responses (Fig. 4B) . Cytotoxic responses varied substantially among the clones. The clones that demonstrated the highest cytotoxicity were the CD4– ones, whereas the CD4+ clones ranged from very low to relatively high cytolysis of MHC class II+ 721.221 CD1d transfectants (Fig. 4B and 4C) . In contrast to its effect on cytokine secretion and proliferation, the addition of the anti-CD4 mAb did not significantly alter the cytotoxic responses of CD4+ NKT cell clones to the 721.221 and K562 target cells (Fig. 4D) .


Figure 4
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  		Figure 4. Cytotoxic responses of CD4+ and CD4– NKT cell clones. (A) Cytolysis is CD1d dependent. CD1d transfected (CD1d+) and untransfected (UT) target cell lines (721.221 lymphoblastoid and K562 myelomonocytic cells) were pulsed with 5 ng/ml {alpha}-GalCer and tested for killing by a CD4+ and a CD4– NKT cell clone (20:1 E:T ratio) by a standard 4-h chromium release assay. (B) Cytolysis is {alpha}-GalCer dependent. CD1d-transfected 721.221 cells were pulsed with the indicated concentrations of {alpha}-GalCer, or treated with vehicle alone, and tested for cytotoxicity by a series of CD4+ NKT cell clones (open symbols) compared with a CD4– clone (solid symbols) at a 20:1 E:T ratio. (C) Effector to target titration. CD1d-transfected 721.221 cells were pulsed with 50 ng/ml {alpha}-GalCer and tested for specific killing by CD4+ NKT cell clones (open symbols) and a CD4– NKT clone (solid symbols) at the indicated effector to target cell ratios. (D and E) CD4 blockade does not affect cytotoxicity. CD1d-transfected 721.221 (MHC II+) and K562 (MHC II–) cells (D), or CD1d-transfected 2001 (MHC II+) and 3023 (MHC II–) cells (E), were pulsed with 5 ng/ml {alpha}-GalCer and were tested for specific killing by the indicated NKT clones, in the presence of anti-CD4 antibody (solid bars) or an isotype-matched negative control antibody (gray bars). The plots show the mean specific lysis of at least 3 replicate samples, and error bars represent the standard deviations of the means (in some cases not visible on the scales shown).

To further investigate the role of CD4 in NKT cell cytotoxicity, we tested the ability of CD4+ NKT cell clones to lyse a matched pair of CD1d-transfected cell lines that differ only in MHC class II expression. The MHC class II {alpha}β-deficient human lymphoblastoid cell line 721.174 was transfected with DNA-encoding HLA-DR{alpha} to generate the 3023 subline (surface MHC class II negative), and 3023 was further transfected with HLA-DRβ7 to generate the 2001 subline, which is surface MHC class II positive [38 ]. We stably transfected these "sister" cell lines with human CD1d to generate 3023-d and 2001-d [42 ]. Two CD4+ NKT cell clones that showed relatively strong cytotoxic responses (J24L.17 and J3N.4) were tested for killing of 3023-d and 2001-d in the presence of anti-CD4 or negative control mAbs. There was no significant reduction of cytotoxicity in the presence of the anti-CD4 mAb, and there was also no evidence of increased lysis of the MHC class II+ transfectant (Fig. 4E) . Thus, CD4 blockade did not impede the cytotoxic response, and the presence of a CD4 ligand did not enhance it. Together, these results show that anti-CD4 mAb treatment affects cytokine secretion and proliferation but not cytotoxicity, suggesting that CD4 contributes to the activation of some functional responses but not others.

MHC class II expression by APCs is not required
We hypothesized that the inhibitory effect of anti-CD4 mAb treatment on NKT cell cytokine secretion and proliferation might be due to blockade of CD4 binding to MHC class II molecules on APCs. To investigate this possibility, we tested whether anti-CD4 mAb treatment selectively inhibited NKT cell responses to MHC class II+ APCs. NKT cell cytokine secretion was tested in response to CD1d-transfected 721.221 cells (MHC class II+) and K562 cells (MHC class II–), or the 2001-d (MHC class II+) and 3023-d (MHC class II–) cell lines, in the presence of the anti-CD4 mAb or an istotype-matched negative control mAb. The addition of the anti-CD4 mAb specifically inhibited cytokine secretion by CD4+ NKT cells in response to all of the APCs, regardless of MHC class II expression, but did not affect the responses of a CD4– NKT cell clone (Fig. 5A and 5B ). Thus, MHC class II expression by the APC did not seem to be required for the inhibitory effect of anti-CD4 treatment, and therefore, the effect of the anti-CD4 mAb is unlikely to be due to blockade of an interaction between CD4 and MHC class II molecules on the APCs.


Figure 5
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  		Figure 5. CD4 blockade inhibits NKT cell cytokine production in response to both MHC class II+ and class II– antigen presenting cells. (A) The indicated CD4+ or CD4– NKT cell clones were coincubated with CD1d-transfected 721.221 (MHC class II+) or K562 (MHC class II–) cells (each pulsed with 5 ng/ml {alpha}-GalCer), in the presence of anti-CD4 antibody (solid bars) or isotype-matched negative control antibody (gray bars). (B) NKT cell responses to a matched pair of APCs: the parental cell line 3023-d (MHC class II deficient) and the daughter line 2001-d (HLA-DR7 transfected). Results are representative of 6 independent experiments.

CD4 blockade directly affects CD3-mediated signaling events
To further investigate the mechanism of the inhibition, we tested the effect of soluble anti-CD4 mAb on NKT cell calcium flux. In this system, CD3 molecules on the NKT cell are ligated by an anti-CD3 mAb to generate a calcium flux, in the presence or absence of non-cross-linked anti-CD4 mAb. Thus, because the primary signal is mediated by the anti-CD3 mAb and there are no APCs, any effect of the anti-CD4 mAb is independent of a ligand on the APC. We first tested the effect of the anti-CD4 mAb on non-cross-linked CD3, since we have observed that rested NKT cell clones can flux calcium in response to the weak stimulus provided by dimeric anti-CD3 alone. In this system, the CD3-activated calcium flux of CD4+ NKT cell clones was almost completely abrogated by the addition of the anti-CD4 mAb (Fig. 6A ). To further assess the effect of the anti-CD4 mAb on CD3-mediated calcium flux, we used an isotype-specific second step antibody to cross-link the anti-CD3 mAb, while the anti-CD4 mAb remained non-cross-linked. In this case, in the presence of the anti-CD4 mAb the calcium flux was reduced, but not abrogated (Fig. 6B) . Thus, the presence of non-cross-linked anti-CD4 mAb had an inhibitory effect, even when a strong primary stimulus was used. These results show that CD4 blockade can interfere directly with the primary signaling response of CD4+ NKT cells and that a ligand on the APC is not necessary for this effect.


Figure 6
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  		Figure 6. The effect of noncross-linked anti-CD4 antibody on NKT cell calcium flux. (A) Calcium flux by a CD4+ NKT cell clone that was treated with noncross-linked anti-CD3 and anti-CD4 antibodies (squares), or anti-CD3 and IgG1 negative control antibody (triangles), or anti-CD4 antibody alone (circles). (B) Calcium flux when the CD3 was cross-linked, but the CD4 was not. The anti-CD3 antibody was specifically cross-linked using IgG2a-specific rat anti-mouse (RAM) antibody, in the presence (squares) or absence (triangles) of noncross-linked CD4 mAb (IgG1 isotype). The plots show the percentage of cells that moved into the flux gate during 10-s intervals at the indicated times after stimulation. Results are representative of 2–4 independent experiments.

To investigate the mechanism of inhibition further, we analyzed the effect of the anti-CD4 mAb on p56lck phosphorylation. It is known that p56lck is phosphorylated at multiple sites following TCR stimulation of MHC class II-restricted T cells, including sites that promote its kinase activity (e.g., Y-394), and sites such as Y-505 that have an inhibitory effect [27 ]. Under strongly stimulating conditions, phosphorylation of the Y-505 inhibitory site of p56lck is rapidly removed by cellular phosphatases, which facilitates continued TCR signaling [26 ]. To investigate this process for NKT cells, CD4+ clones were stimulated by anti-CD3 mAb in the presence or absence of anti-CD4, or a secondary antibody (GAM) to provide cross-linking, then lysed and analyzed by Western blot analysis with an antibody that was specific for Y-505 phosphorylated p56lck, or an antibody against total p56lck. Unstimulated NKT cells showed no detectable phosphorylation of Y-505, whereas Y-505 phosphorylation was clearly detected from lysates of NKT cells that were treated with anti-CD3 without cross-linking (Fig. 7A ). Treatment with anti-CD3 in the presence of the GAM cross-linking antibody resulted in a transient increase in Y-505 phosphorylation, consistent with the occurrence of a dephosphorylation event under strongly activating conditions, as has been observed previously [32 , 33 , 35 , 43 ]. In contrast, in the presence of the anti-CD4 mAb, the Y-505 phosphorylation signal appeared to increase over time (Fig. 7A) , suggesting that CD4 blockade either leads to increased phosphorylation of the inhibitory Y-505 site or reduces the dephosphorylation of this site. Although the absolute signal for phosphorylated Y-505 varied from experiment to experiment, the ratio of the signal for the 5-min time point divided by that of the 2-min time-point was consistently increased in the presence of the anti-CD4 mAb, compared with the anti-CD3 mAb alone (Fig. 7B) . Hence, these results demonstrate that in addition to reducing the magnitude of the CD3-mediated calcium flux, CD4 blockade has an inhibitory effect on p56lck, a critical early component of the TCR-signaling cascade.


Figure 7
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  		Figure 7. Western blot analysis of NKT cell p56lck phosphorylation of tyrosine-505. (A) A CD4+ NKT cell clone was treated with the indicated antibodies in the presence or absence of a cross-linking antibody (GAM) for 2, 5, or 10 min. Clarified lysates were analyzed by Western blot analysis with an antibody specific for tyrosine-505 phosphorylated p56lck, as shown in the upper blot panels. The lower blot panels show the same membranes after they were stripped and reprobed for total p56lck. The intensity of the bands was quantified by Image J software after densitometric scanning. The plot below shows the tyrosine-505 phosphorylated p56lck signal normalized by the total p56lck signal for the indicated stimulation conditions. (B) Anti-CD4 antibody treatment results in increased duration of p56lck tyrosine-505 phosphorylation. The normalized p56lck-tyrosine 505 signals from NKT cells stimulated with anti-CD3 antibody alone (triangles) or anti-CD3 and anti-CD4 antibodies (squares) were plotted as ratio of the signal at 5 min divided by the signal at 2 min. The plot shows the results from four independent experiments, with each treatment pair represented as two linked points. Statistical analysis by two-sided paired Student’s t test demonstrated with a significance of P = 0.01 that the 5 min/2 min ratio was higher for NKT cells that were exposed to anti-CD3 in the presence of anti-CD4 than for those that were treated with anti-CD3 alone, indicating that Y-505 was phosphorylated for a longer time in the presence of the anti-CD4 mAb.


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DISCUSSION
 
We show here that CD4 is functionally active in CD1d-restricted NKT cells. CD4 engagement affected NKT cell calcium flux responses and altered the phosphorylation status of p56lck, indicating that CD4 can contribute to NKT cell signaling pathways similarly to its costimulation of signaling in MHC class II-restricted T cells. However, whereas CD4 functions as a coreceptor on MHC class II-restricted T cells, it is not clear that this is the case for NKT cells. The effect of CD4 in our system did not depend on MHC class II expression by the APC and also did not require TCR ligation by CD1d. Thus, our results show that CD4 can affect NKT cell activation independently of a CD4-ligand on the APC.

CD4 blockade inhibited NKT cell signaling responses that were induced by anti-CD3 mAb treatment, and the effect was mediated in part through p56lck, suggesting that CD4 plays a role in recruiting p56lck to sites of molecular signal transduction. Our results show that upon NKT cell activation through the CD3 complex, p56lck molecules are transiently phosphorylated and that CD4 blockade may prevent efficient dephosphorylation of the inhibitory Y-505 site. This could occur through the anti-CD4 antibody blocking an association between the extracellular domains of CD4 and cellular phosphatases, such as CD45, that mediate Y-505 dephosphorylation, which might prevent the intracellular access of the phosphatase to p56lck. Alternatively, anti-CD4 antibody treatment might block dimerization of CD4 molecules. CD4 molecules that have been mutated to prevent dimerization show increased p56lck Y-505 phosphorylation levels after T cell activation [28 ], suggesting that the ability to form dimers is important for the removal of Y-505 phosphorylation. Finally, it is also possible that the effect of the anti-CD4 antibody is mediated through blocking the binding of CD4 to another ligand on the NKT cell surface, such as MHC class II, as it is known that activated human T cells express MHC class II, and we have found that this is present on our NKT cell clones (data not shown).

Consistent with the results we have presented here, it has been previously observed that cocross-linking of CD4 molecules with the CD3 complex enhances the response of classical CD4+ T cells, whereas treatment with noncross-linked anti-CD4 antibody is inhibitory [44 45 46 ]. However, the physiological relevance of a ligand-independent effect of CD4 on MHC class II-restricted T cells has remained unclear, since such T cells require MHC class II molecules for TCR recognition, and therefore, a strong CD4 ligand will always be present on physiological APCs. In contrast, as the affinity of CD4 for CD1d remains unclear and physiological expression levels of CD1d are very low, it is not certain that all APCs for NKT cells will provide efficient ligation of CD4. Thus, the ability of CD4 to affect CD3-mediated signaling responses in the absence of a strong ligand on the APC may be particularly relevant for NKT cells.

However, it is important to note that our results do not rule out the possibility that engagement by MHC class II or CD1d molecules on APCs could further enhance the effects of CD4 on NKT cells. Antibody-mediated coligation of CD4 with the TCR/CD3 complex clearly increased NKT cell cytokine secretion and led to accelerated and amplified calcium flux responses. Cocross-linking of CD3 and CD4 also appeared to result in reduced phosphorylation of the inhibitory Y-505 site of p56lck (data not shown). Thus, NKT cell activation is augmented when CD4 molecules are brought into close proximity with the CD3 signaling complex. This could be due to CD4 binding to CD1d [19 ] or could occur as a result of CD4 binding to MHC class II, as a fraction of CD1d molecules have been found to be associated with MHC class II molecules at the cell surface [47 ].

The role of MHC class II as a potential ligand for CD4 is of particular interest, because some CD1d+ APCs are normally class II negative (e.g., keratinocytes), and others show markedly varying levels of MHC class II expression (e.g., monocytes, immature myeloid DCs, and mature myeloid DCs). Thus, MHC class II expression levels could differentially modulate NKT cell responses, depending on the APC type. Our analysis of CD1d transfected cell lines that were either positive or negative for MHC class II did not consistently reveal greater responses by CD4+ NKT cell clones to MHC class II+ APCs compared with MHC class II– APCs, and therefore, our experiments do not directly support a role for MHC class II as an NKT cell CD4 ligand. However, CD1d is greatly overexpressed in the transfected cell lines compared with the levels found on physiological APCs such as myeloid dendritic cells, and therefore, the relative contributions of CD1d and MHC class II molecules might be substantially altered in the transfectants compared with physiological APCs.

To further investigate the role of MHC class II in CD4-mediated costimulation of NKT cells, we tested the effect of MHC class II blocking antibodies (mAb clones IVA12 and L243) on the activation of CD4+ NKT cells in response to myeloid DCs and CD1d transfected lymphoblastoid cell lines. We did observe an inhibitory effect in some cases, particularly at low {alpha}-GalCer doses, but the results were not highly consistent among different CD4+ NKT cell clones (data not shown). The results of this analysis are also difficult to interpret because it has been observed that cross-linking of MHC class II molecules leads to rapid apoptosis of lymphoblastoid cell lines, and can also affect monocyte-derived DCs [48 49 50 51 ]. We have observed a similar anti-MHC class II antibody-mediated killing of our APCs (data not shown), and therefore, it is difficult to determine whether inhibitory effects of anti-MHC class II treatment on NKT cell activation are due to blocking an interaction with CD4 or to a separate effect on the APC. Thus, our data do not conclusively rule out a role for MHC class II, and it remains possible that class II expression by APCs could engage CD4 molecules on NKT cells and thereby provide an enhanced costimulatory signal.

We found that CD4 blockade did not inhibit cytotoxicity by CD4+ NKT cell clones. Previous studies have established that human NKT cells can kill certain target cells, particularly leukemic cells of monocytic origin, but the stimulation requirements for the induction of cytotoxicity are not well understood [52 53 54 55 ]. Consistent with most previous observations, we found that both CD1d expression and presentation of {alpha}-GalCer were required for significant cytotoxic responses by our NKT cell clones. However, whereas a recent analysis presented data suggesting that CD4 blockade can inhibit cytotoxic responses by human iNKT cells [19 ], we saw no effect of anti-CD4 antibody treatment on cytotoxicity by any of our NKT cell clones. The reason for the discrepancy between our results and those of Thedrez et al. is not clear, but it may relate to the use of different target cells in the two systems. Notably, we observed that anti-CD4 treatment reproducibly did diminish cytokine secretion by our NKT cells in response to the same APCs that were used as target cells for the cytotoxicity assays, and therefore our results suggest that CD4 blockade can differentially impact NKT cell functional responses.

It has been previously reported that CD8+ cytotoxic T cells required fewer cognate peptide-MHC complexes to activate killing than cytokine secretion, suggesting that cytolysis requires a lower activation threshold than cytokine secretion [56 ]. Hence, CD4 blockade might fail to inhibit cytotoxicity because the activation threshold for this function is lower than those required for cytokine secretion and proliferation. However, we observe that NKT cell cytokine secretion can be quite efficiently stimulated in the absence of exogenous antigen (i.e., auto-antigenic stimulation) and is further enhanced by the addition of even very low levels of the strong agonist {alpha}-GalCer, whereas cytotoxic responses appear to require addition of at least moderate levels of {alpha}-GalCer, and proliferation requires exposure to high levels of {alpha}-GalCer. Hence, the antigen dose required for activation of NKT cell cytotoxicity seems to fall between those of cytokine secretion and proliferation. Thus, the differential effect of CD4 blockade on cytotoxic responses compared with cytokine secretion and proliferation does not seem to correlate directly with a lower activation threshold. Thus, our results suggest that the signaling pathways involved in the activation of NKT cell functional responses may differ and that CD4 preferentially modulates cytokine secretion and proliferation.

We previously observed that activated CD4+ CD1d-restricted NKT cells in human peripheral blood generally demonstrated brighter intracellular cytokine staining than the CD4– subset but showed less perforin expression, suggesting that CD4+ NKT cells are more oriented toward cytokine secretion than perforin-mediated cytotoxicity in vivo [12 ]. Our current analysis was performed using cultured human NKT cell clones, which may have important differences in activation requirements compared with primary NKT cells, and therefore, the importance of CD4 for the functions of CD1d-restricted NKT cells in vivo remains to be determined. However, the results presented here are consistent with the distinctions that we have previously observed between the CD4+ and CD4– NKT cell subsets in peripheral blood, in that CD4 does not appear to costimulate the cytotoxic function of NKT cell clones, but it does seem to play a role in their cytokine secretion and proliferative responses. Hence, taken together, these results support the possibility that CD4 expression may contribute directly to functional differences between CD4+ and CD4– NKT cells, by playing a signaling role during NKT cell activation that selectively enhances particular functional responses.


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ACKNOWLEDGEMENTS
 
This study was supported by National Institutes of Health grant R01-AI060777 to J.E.G., and a DOD hypothesis development award (grant CM030014) to J.G. and X.C. J.E.G. is a Pew Scholar in the Biomedical Sciences. G.S.B. acknowledges support from the Medical Research Council, the Wellcome Trust, and Mr. James Bardrick in the form of a personal research chair.

Received March 15, 2007; revised August 3, 2007; accepted August 4, 2007.


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