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CD4^–CD8 Subset of CD1d-Restricted NKT Cells Controls T Cell Expansion¹

Ling-Pei Ho^2,*,, Britta C. Urban^*, Louise Jones^*, Graham S. Ogg^* and Andrew J. McMichael^*

^* Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, and Oxford Center for Respiratory Medicine, Churchill Hospital, Oxford, United Kingdom

	Abstract

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V24 invariant (V24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4^–CD8⁺ subset of V24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4^– and CD4⁺ V24i NKT clones and demonstrate that a CD4^–CD8⁺ subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.

	Introduction

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CD1-restricted cells respond to glycolipid Ags presented on CD1a, b, c, or d molecules on APCs. CD1a-c molecules present foreign glycolipid like that from Mycobacterium tuberculosis (1), and therefore, are important in expanding the repertoire of the adaptive host defense system. In contrast, CD1d-restricted NKT cells (also known as V24 invariant (V24i)³ NKT cells due to its invariant TCR repertoire) are perceived to be self-reactive, possibly recognizing a self lipid (2, 3). The actual ligand for CD1d molecules is not known, but the effect of recognition of the CD1d-glycolipid complex is considerable. Upon activation by a mimic (the marine sponge lipid, -galactosyl ceramide (GC)), V24i NKT cells produce large amounts of both Th1 and Th2 cytokines and are known to influence or provide help for NK cells, macrophages, B lymphocytes, and dendritic cells (DCs) (4, 5, 6). Transfer and activation of these cells (V14 in mice) to disease prone animal models (e.g., nonobese diabetic and experimental allergic encephalitis mice) can prevent development of disease (7, 8, 9); they have anti-cancer properties (10), and more recently were shown to be required for appropriate protection against infectious diseases like Pseudomonas aeruginosa infection of the lungs, Lyme disease, malaria, and several protozoan infections (11). In humans, CD1d-restricted NKT cell deficiency correlates with autoimmune diseases like type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, and systemic sclerosis (12, 13, 14). Due to these data, and the possession of an invariant TCR repertoire and an effector phenotype when activated, it is widely regarded that CD1d-restricted NKT cells orchestrate the other arms of the immune system rather than directly contribute to host defense. There is evidence that in adaptive immunity, protective function afforded by these cells results from their immediate cytokine production and their ability to contribute to activation of other cells in the immune system. Brigl et al. (15) recently demonstrated that the converse is also true—IL-12 secreted by DCs after exposure to microbial products amplified the response of CD1d-restricted NKT cells to self Ags yielding greater effector function. In tumor immunity, CD1d-restricted NKT cells are thought to potentiate the effect of anti-tumor CTL and NK cells by releasing IL-4 and IFN- and interacting with DCs inducing the production of IL-12 (16). Less is known about the mechanism by which CD1d-restricted NKT cells prevent autoimmune disease; indeed, it is not well understood how this regulatory effect is mediated and there is little evidence that these cells actually curb or control T cell activities. Therefore, we were keen to explore this area further.

In the last year, it has become apparent that CD1d-restricted NKT cells in humans can be divided into functionally distinct groups—CD4^– subsets which produce mainly Th1 cytokines and possess cytotoxic activity; and CD4⁺ subsets that secrete both Th1 and Th2 cytokines in large quantities (17, 18, 19). We hypothesize that these subsets are likely to perform different functions and that at least one of them may have a suppressive or regulatory effect on T cell proliferation or cytokine production. We first examined how activation of naturally occurring human CD1d-restricted NKT cells might influence the activation and expansion of Ag-specific T cells in PBMCs. For this, we tracked the behavior of EBV peptide-specific T cells (with EBV/MHC I tetrameric complexes) when they were pulsed with an immuno-dominant EBV peptide in vitro in the presence of activated CD1d-restricted NKT cells. We found that when PBMCs were pulsed with both GC and EBV peptide, the expansion of EBV-specific T cells were significantly decreased compared with when no GC was added. To delineate which subset may be responsible and how these cells curbed the expansion of T cells, we derived a panel of CD1d-restricted NKT clones with the aid of GC-loaded CD1d tetramer and used these clones to examine their individual effects on proliferation and cytokine production of T cells responding to a recall Ag, and on Ag-specific memory T cell expansion. We found that a highly efficient cytolytic V24i NKT clone with a CD4^–CD8⁺ phenotype was able to uniquely inhibit the proliferation of activated T cells. We observed that CD4⁺ and CD4^–CD8⁺ V24i NKT cell clones followed different proliferative kinetics after stimulation, with the latter expanding at a slower rate. This reflected the rate of expansion in the subsets within naturally occurring V24i NKT cell population during a recall response. We propose that the CD4^–CD8⁺ subset of CD1d-restricted NKT cells may have a specific immunoregulatory function and exert this effect by suppressing proliferation of activated T cells at a later time point during a T cell response.

	Materials and Methods

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Generation of CD1d and MHC class I tetramers

CD1d-GC tetramers were generated by in vitro oxidative refolding chromatography, as described (20). Briefly, synthesized CD1d H chain and ₂-microglobulin were refolded with oxidative refolding chromatography in the presence of GC, and concentrated soluble proteins were biotinylated directly.

MHC I tetramerically complexed to EBV peptide was made as previously described (21).

Abs for flow cytometry

Fluorochrome conjugates of mAbs against the following were obtained as detailed: CD11a, TGF-, V24, and V11 (Serotec, Oxford, U.K.); CD1d, CD8, CD3, HLA-DR, CXCR3, CD62l, CD83, CD86, CD161, IL-4, IFN-, IL-2, IL-10, TNF-, IL-13, perforin, granzyme A (all from BD Biosciences, San Jose, CA); CD8, and CD56-PC5 (Immunotech, Marseille, France); CXCR5 (R&D Systems, Abingdon, U.K.) and CD4 (DAKO, Ely, U.K.).

Flow cytometric studies

For characterization of the NKT cell clones, FACS staining was performed at the plateau phase of growth; all stainings were performed on fresh cells. For perforin and granzyme A staining, cells were fixed, then permeabilized with 0.1% saponin/0.1% BSA (Sigma-Aldrich, Dorset, U.K.) for 15 min, before washing and staining for 15 min at room temperature. For studies involving intracellular staining, cells were incubated with 10 µg/ml brefeldin A (Sigma-Aldrich) in the last 4–6 h of incubation, washed, and then fixed with 2% paraformaldehyde/1% BSA, before permeabilization with saponin (as above) and staining with appropriate Abs. Stimulation was performed separately, with 10 ng/ml PMA and 1 µg/ml ionomycin (Sigma-Aldrich); and with the CD1d-specific Ag, GC (a gift from Kirin Brewery Company, Gunma, Japan). For GC stimulation in NKT clone functional studies, autologous immature monocyte-derived DCs were used as CD1d-bearing APCs at a 1:20 DC:NKT clone ratio.

For GC-CD1d and EBV/MHC I tetramer stainings, samples were incubated with optimally titrated amount of tetramer at 37°C for 15 min, followed by the addition of a panel of titrated Abs at 4°C.

Generation of CD1d-restricted and control T cell clones

Peripheral blood lymphocytes were derived by Ficoll-Hypaque density centrifugation. V24⁺ T cells were positively selected from freshly isolated PBMCs from two normal donors, by labeling with V24 mAbs and then with the appropriate anti-IgG1 MACS beads, according to manufacturer’s protocol (Miltenyi Biotec, Bisley, U.K.). These cells were seeded onto 96-well plates at a density of 0.1 and 0.5 cells per well together with 10,000-irradiated allogeneic feeder cells from three donors, and suspended in 200 µl per well of RPMI cell culture medium, PHA (1/200 dilution; Abbottmurex, Dartford, U.K.) and 10% human serum (First Link, Brierley Hill, U.K.). On day 3, 200 U/ml IL-2 was added and cells were screened on day 15 and day 20 for V24⁺V11⁺ CD1d tetramer⁺ cells. Cells positive for all three markers were then expanded with gamma-irradiated allogeneic feeders and PHA mixture as above. They are referred to from here forward as V24i NKT clones.

A CD8⁺ cytotoxic T cell line specific for an influenza viral peptide (ASCMGLIY) and a non-CD1d-restricted CD4 T cell clone bearing V11-TCR, were cloned in the same manner as the NKT clones to provide control T cell clones. Both were determined to be GC/CD1d tetramer^– on staining.

CTL (killing) assay

DCs or .221 B cell line (MHC class I negative) stably expressing CD1d was used as target cells for 51 chromium (⁵¹Cr) release assay. CD1d-transfected .221 B cell lines were made by transfecting .221 cell line with a full-length cDNA in the pcDNA3.1 vector (Invitrogen, Paisley, U.K.) by electroporation and culturing in the presence of the selection antibiotic, hygromycin (Calbiochem, Nottingham, U.K.). The cells were then cloned manually by limiting dilution in culture medium containing 300 µg/ml hygromycin. The positive clone was then FACS sorted to obtain cells of high CD1d expression.

Standard ⁵¹Cr release assays (22) were performed and the assays counted on a flatbed scintillation counter (Wallac, Gaithersburg, MD). Background chromium release was always <20%. Percentage of lysis was calculated from the formula 100 x (E – M/T – M), where E is the experimental release, M is the release in the presence of medium, and T is release in the presence of 5% Triton X-100 detergent. Target cells were used at an optimized concentration of 10/1 E:T ratio. ⁵¹Cr labeling was performed for 45 min, following which, cells were incubated for 4 h with various NKT clones. Controls included target cells incubated with medium or 5% Triton X-100 only; and target cells that did not bear CD1d molecules (untransfected .221 cells).

Generation of monocyte-derived DCs

This was performed as previously described (23). Briefly, CD14⁺ cells were derived with MACS beads and cultured in medium supplemented with 1% pooled human serum (First Link), 100 ng/ml IL-4 (PeproTech, Rocky Hill, NJ), and 100 ng/ml granulocyte macrophage-stimulating factor (Leucomax; Schering-Plough, Hertfordshire, U.K.) for 5–6 days. All DCs were checked for maturation markers (CD83, CD86, and HLA-DR) and determined to be CD1b and CD1d⁺ and CD14^– at time of use.

Ag-specific T cell expansion

For the first experiment, expansion of EBV-specific T cells in 1.5 x 10⁶ PBMCs (with or without GC) was determined on days 1, 3, 5, 7, 10, and 15 following stimulation with 100 µM of an EBV-specific peptide BMLF-1 (Genosys, Haverhill, U.K.), for an hour, in 24-well plates. Cells were cultured in RPMI 1641 supplemented with penicillin, streptomycin, and 10% FCS. On the third day of culture, 200 U/ml IL-2 (Proleukin; Chiron, Warwickshire, U.K.) was added. Expansion was determined by staining with EBV(BMLF-1)/MHC I tetramers and analyzed with flow cytometry. The phenotype and expansion of V24i NKT cells, and CD1d expression on CD69⁺ and EBV tetramer-positive T cells in the wells were observed simultaneously at the described time points using multicolor flow cytometry analysis.

In the second experiment, gamma-irradiated V24i NKT clones (CD4⁺ and CD4^– phenotype) were added at the beginning of the culture at 1:10 ratio (NKT clones:PBMC). The cultures were stimulated and observed as described above. This provided a greater than normal effect of either the CD4^– or CD4⁺ NKT cell subsets on T cell responding to EBV peptide stimulation.

Frequencies of EBV tetramer⁺CD8⁺ cells were expressed as a percentage of CD8 population and also as absolute numbers.

T cell proliferation assays

Standard tritiated thymidine ([³H]) incorporation assay was used. All assays were performed in 96-well plates with 10 x 10⁵ freshly derived human PBMC. 0.5 µCi of tritiated thymidine (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) was added for the last 16 h on the designated days. Incorporated [³H]thymidine was measured with the TopCount software on a microplate beta counter (TopCount-NXT; Packard Bioscience, Boston, MA).

To determine the effect of NKT clones on proliferation kinetics ofAg-responding T cells, PBMC and gamma-irradiated V24i NKT or autologous control clones (at 10:1 ratio) were stimulated with either medium alone, 0.1 µg/ml GC, 100 U/ml streptokinase/streptodornase (Wyeth Pharmaceuticals, Taplow, U.K.), or GC and streptokinase/streptodornase together at described doses. T cell proliferation was determined on the 3rd, 5th, and 7th day after stimulation. In a separate study, the dose of T cell clones added to PBMCs was titrated, and thymidine incorporation measured on the 5th day.

	Results

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Stimulated CD1d-restricted NKT cells inhibit the expansion of Ag-activated T cells

To examine the effect of stimulating CD1d-restricted NKT cells on the expansion of activated T cells, we pulsed PBMCs from a donor, known to have had glandular fever, with an EBV peptide and cultured the cells with or without an optimized dose of GC.

Expansion of 1) CD8⁺ EBV-specific T cells, and 2) V24i CD1d-restricted NKT cells and their phenotype were tracked on days 1, 3, 5, 7, 10, and 15 with EBV/MHC I tetrameric complex, and V24 mAb and GC-CD1d tetrameric complexes, respectively. As controls, PBMCs were cultured with medium alone or with GC alone without pulsing with EBV peptide. This assay allowed us to visualize the expansion of an Ag responsive population and the concomitant effect of activation of NKT cells on this proliferation. We found that addition of GC resulted in a decreased expansion of EBV-specific T cells compared with cultures where no GC was added. In addition, there was a more rapid loss of these Ag-specific T cells (Fig. 1A). CD1d-restricted NKT cells were seen to expand with GC stimulation, with or without stimulation with EBV peptide (Fig. 1B). CD4^–CD8⁺ cells comprised between 5 and 10% of V24i NKT cells at the start (before stimulation) but this subset significantly increased by the 5th day in those wells where GC was added. There was an even greater expansion in wells that were also pulsed with peptide (Fig. 1C), suggesting a contribution from Ag-activated T cells or pathogenic stimuli to the acquisition of CD8⁺ subset of V24i NKT cells. This observation supports a recent study showing the additive effect of microbial influence on the expansion of V24i NKT cells (15).

View larger version (50K): [in this window] [in a new window]   FIGURE 1. A, Activation of CD1d-restricted NKT cells suppresses expansion of Ag-specific CD8+ T cells. Shown are FACS plots for the starting population of CD1d-restricted NKT cells (as a percentage of lymphocytes) and EBV/MHC class I tetramer (expressed as a percentage of CD8 population and absolute numbers) in the PBMC sample. After stimulation with EBV peptide for 7 days, the EBV tetramer population increased to 7.6% (very little expansion before the 7th day for either GC-stimulated or unstimulated cultures). This persisted for the next 8 days (upper panel; plots show CD8+EBV/MHC I tetramer+ population on 7th, 10th, and 15th day after stimulation). A parallel set of culture plates (lower panel) showed inhibition of this expansion together with a quicker resolution of the Ag-specific cells when the naturally occurring CD1d-restricted NKT population in the PBMC was stimulated with addition of GC. Plots are representative of four experiments. B, Activation of PBMCs with GC is accompanied by expansion of V24I NKT cells. While PBMCs were cocultured with different combinations of culture medium alone, GC, and EBV peptide, the proportions of CD4– and CD4+ subsets in the V24i NKT population changed, favoring the increase of CD4–CD8+ subsets in those wells with GC stimulation (B); with or without peptide. V24i NKT cells refer to V24+ CD1d tetramer+ cells. This population of cells disappeared in the first few days but recovered in frequency by day 5. There was no statistical difference in V24i NKT cell numbers between the wells with GC alone and those with GC and EBV peptide, but both wells contained significantly higher numbers of V24i NKT cells compared with those with no GC (p = 0.03, Student’s t test; n = 4 experiments). C, Expansion of V24i NKT cells during EBV peptide stimulation favors the acquisition of CD4–CD8+ subset. Graph shows the serial changes in CD4–CD8+ subset within the V24i NKT cell population. GC addition alone resulted in a near 3-fold increase in CD8+CD4– V24i NKT cells. There was an augmented increase in this subset with the addition of EBV peptide to GC, which was not seen with EBV-peptide stimulation alone. PBMC + 0 = PBMC alone with culture medium, PBMC + GC = PBMC with 0.1 mcg/ml GC added to its culture on day 1, PBMC + peptide = PBMC pulsed with 100 µM EBV peptide on day 1; PBMC + peptide + GC = PBMC pulsed with 100 µM EBV peptide and with 0.1 µg/ml GC added to its culture on day 1. Experiments shown are representative of four consecutive experiments. Error bars = SEM.

Generation and characterization of CD4^– and CD4⁺ V24i NKT clones

To substantiate the above findings, and to determine whether a particular subset was responsible for the inhibitory effect, we generated CD1d-restricted NKT clones from a human donor. This was done by deriving V24⁺ cells from PBMCs using magnetic bead selection and then manually cloning with limiting dilution. Cells were then screened for V24 and V11 staining by flow cytometry and finally, V24⁺V11⁺CD1d tetramer⁺ cells were expanded with PHA and irradiated feeder cells. From a starting PBMC of 40 x 10⁶, we obtained 12 CD1d-restricted T cell clones, here forward called V24i NKT clones.

The phenotypic spectrum of our NKT cell clones reflected that seen in the natural population of V24i NKT cells in human PBMCs as observed by Lee and Gumperz (18, 19). Thus, our clones can be grouped into: 1) CD4⁺/predominantly CD8^–, 2) CD4^–/predominantly CD8⁺, and 3) CD4^–CD8^– ("DN NKT" clone) clones (Fig. 2A). All clones were V24⁺ and V11⁺; CD45RO⁺ and CD25^–. We chose two representative clones from the CD4^–CD8⁺ and CD4⁺CD8^– subsets (called here LH18 and LH22) for additional experiments. Further phenotypic characteristics of these clones are described in Table I. Absence or presence of CD4 expression definitively defined the CD4⁺CD8^– and CD4^–CD8⁺ NKT clones, but we noted that CD8 expression varied with the time of growth, blast populations, and degree of activation of both T cell clones. Due to this spectrum of CD8 expression, we were keen to ensure these were T cell clones rather than lines. Therefore, we subcloned the CD4⁺ NKT and CD8⁺ NKT clones twice. This resulted in the same phenotypic profile as described. In addition, CD8^high and CD8^– populations were FACS sorted from the NKT clones and cultured separately. Kinetic experiments over 15 days showed that these cells subsequently adopted the overall spread of CD8 expression after 10 days in culture. However, at any one time, the CD4^–CD8⁺ NKT clone was predominantly CD8⁺ (at least 70%) and CD4⁺ NKT clone was predominantly CD8^–. The CD4^–CD8⁺ clone was determined to be CD8 (Fig. 2B).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Characterization of the NKT clones. The salient phenotypic and functional characteristics of the CD4–CD8+ NKT clone (A, top panel) and CD4+ NKT clone (bottom panel) are shown. Presence or absence of CD4 expression definitively differentiated the clones; but CD8 positivity was seen as a spectrum which varied with time of growth and activation (although with predominantly CD8+ staining in "CD4–CD8+ NKT clone" and CD8– in "CD4+ NKT clone"). B, CD8 staining of the CD8+ NKT clone compared with a positive control (PBMC). C, Differences in cytolytic ability between CD4+ and CD4–CD8+ clone. Result shown is with DCs as target cells. D, Graph for [3H]thymidine incorporation for CD4– and CD4+ NKT clones over a 7-day period. CD4+ NKT clones showed a very high rate of proliferation within the first few days of culture followed by a quick decline while CD4–CD8+ NKT clones peaked on the 4th to 5th day. All error bars are SEM of three consecutive experiments. CD4 NKT clone = CD4+ NKT clone; CD8 NKT clone = CD4–CD8+ NKT clone.

Activated NKT clones curb Ag-induced expansion of T cells in vitro

To determine how different subsets of CD1d-restricted NKT cells affected T cell responses, we examined the effect of adding different subsets of CD1d-restricted NKT clones to PBMCs responding to streptokinase or to EBV peptide (BMLF-1). For streptokinase recall response, we examined the kinetics of tritiated thymidine incorporation after stimulation. Gamma-irradiated NKT or autologous control clones were added to PBMC at 1:10 ratio (clone: PBMC), and the coculture stimulated with either medium alone, GC, 100 U/ml streptokinase, or GC and streptokinase. T cell proliferation was measured on days 3, 5, and 7. For EBV peptide response, we tracked the expansion of EBV/MHC I tetramer⁺ cells on days 1, 3, 6, 10, and 15 with addition of either of the two contrasting gamma-irradiated V24i NKT clones after the PBMCs were pulsed with 100 µM EBV peptide. The cocultures were stimulated with either medium alone or GC in addition to the EBV peptide. As controls, PBMCs with either of the V24i NKT clones were cocultured with GC or EBV peptide alone.

We found that when activated, CD4^–CD8⁺ NKT clones suppressed the magnitude of the streptokinase-induced proliferative response (Fig. 3A); up to 70% reduction on day 5 of proliferation. This inhibition was not seen with a control non-CD1d-restricted CD4 T cell clone or CD8 CTL line (Fig. 3A), suggesting that this function is unique to CD4^–CD8 CD1d-restricted NKT cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. CD8CD4– NKT clone suppressed the Ag-induced proliferation of other T cells in vitro. A, Streptokinase-induced T cell proliferation (measured by [3H]thymidine incorporation) on the 5th day of coculture of PBMCs and irradiated, autologous CD4+, CD4–CD8+ NKT clones, or control autologous T cell clones (CD4 non-CD1d-restricted T cell clone and CD8 CTL line specific for an influenza peptide) with or without specific NKT (or control) clone activation by GC (or influenza peptide). Coculture with CD4–CD8+ NKT clone resulted in a statistically significant reduction in Ag-induced T cell proliferation (p = 0.01; Student’s t test). B, Dose-dependent suppressive effect with increasing amounts of NKT clones in the coculture. Results shown are for 5th day of proliferation. All error bars are SEM of three consecutive experiments. "CD4 NKT clone" = CD4+ NKT clone; "CD8 NKT clone" = CD4–CD8+ NKT clone.

Expansion of EBV/MHC I tetramer⁺ cells was observed to be inhibited to a greater degree with addition of activated CD4^–CD8⁺ V24i NKT clones compared with when no clones (data not shown) or when V24i CD4⁺ NKT clones were added (Fig. 4). It was interesting to note that when absolute cell numbers were calculated, the CD4⁺CD8^– NKT clones were observed to enhance proliferation of EBV/MHC I tetramer⁺ cells (results for absolute cell count shown in Fig. 4 is the average of 3 experiments). This is in keeping with published results showing amplification of T cell responses by NKT cells. No difference in Ag-specific T cell expansion was noted when GC was not added to the cocultures (and therefore, the NKT clones or cells were not activated). This indicates that activated V24i NKT clones, rather than the mere presence of an extra population of cells induced the suppressive effect on T cell activation. As expected, the irradiated V24i NKT cells did not expand during the coculture period (Table II).

View larger version (68K): [in this window] [in a new window]   FIGURE 4. Addition of CD8+CD4–, but not CD4+ V24i NKT clones to PBMC stimulated with EBV peptide, inhibits the expansion of Ag-specific T cells. Representative FACS plots are shown for serial changes in CD8+ EBV/MHC I tetramer+ cells (from a starting population of 1%; all values in FACS plot expressed as a percentage of CD8 cells) when PBMCs were stimulated with EBV peptide in the presence of CD4–CD8+ (LH 18) or CD4+CD8– (LH 22) V24i NKT clones, with or without stimulation with GC. There was a loss of tetramer-positive cells in the early days after EBV peptide pulsing. This is likely to be due to down-regulation of the TCR upon activation; n = 3 experiments were conducted. Absolute cell counts are also shown and values are mean of three experiments.

View this table: [in this window] [in a new window]   Table II. Changes in frequency of gamma-irradiated V24i NKT clones during coculture experiment with PBMCs and EBV peptide ± GCa

View larger version (22K): [in this window] [in a new window]   FIGURE 5. CD1d expression on activated T cells. Figure shows a representative FACS histogram plot of CD1d expression on activated T cells during activation of PBMCs with EBV peptide. Plots demonstrate cells from CD69+CD3+ gate at different time points throughout the coculture. Peptide refers to BMLF-1 EBV peptide, top panel shows samples from a well with no GC added, bottom panel where 0.1 µg/ml GC was added. Values refer to mean fluorescence intensity.

NKT clones did not secrete IL-10 or TGF-, and did not affect IFN- or IL-2 production in proliferating Ag-stimulated T cells

Since there is a possibility that these cells may behave like regulatory T cells and conventional T regulatory cells are reported to secrete IL-10 and/or TGF- (24), we examined the ability of the NKT clones themselves to produce these upon activation by GC. We found no IL-10 or TGF- production at 6th, 12th, or 18th hour of stimulation either by the clones on their own (with DC as APCs) (Table I) or by naturally occurring CD1d-restricted NKT cells (on 3rd, 5th, 7th, and 10th day) in PBMCs stimulated with streptokinase or EBV peptide (data not shown).

Although it is known that CD1d-restricted NKT cells affected the production of cytokines from NK cells and DCs (16), it is unknown if Ag-specific T cells were subjected to the same influence. We examined the production of cytokines that might directly affect the proliferation of T cells (IFN- and IL-2) from Ag-specific T cells during a recall immune response while CD1d-restricted NKT cells were concomitantly stimulated by GC. PBMCs were cocultured with EBV peptide BMLF-1 compared with appropriate controls and cytokine production by EBV/MHC Itetramer-positive cells and other activated T cells was determined on days 1, 3, 5, 7, and 10 after activation (by intracellular staining).Four-color FACS staining, using a combination of EBV/MHC I tetramer, CD4, CD8, and CD69 mAbs with multiple selection of mAbs to intracellular cytokines was used. We found no difference in production of IFN- or IL-2 by Ag-specific or -activated CD4 or CD8 T cells at these time points compared with PBMCs stimulated with EBV but not cocultured with GC (data not shown).

These findings show that changes effected by CD4^–CD8 NKT clones in the proliferation of activated T cells during a recall response is probably not mediated by variation in IFN- or IL-2 production by the activated T cells after the initial program of T cell activation has been triggered.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper, we demonstrate a potential mechanism by which NKT cells could regulate T cell activity. A specific subset of CD1d-restricted NKT cells, bearing a CD4^–CD8 phenotype, was found to inhibit the proliferation of Ag-activated T cells. This effect was seen with two different antigenic responses (EBV peptide and streptokinase). The involvement of this particular subset was strengthened by the observation of a concomitant expansion in this CD4^–CD8⁺ V24i NKT cells during this process, a dose-dependent increase in inhibition of streptokinase-induced T cell proliferation with the addition of the CD4^–CD8⁺, but not CD4⁺ V24i NKT clone, and a suppression of EBV peptide-activated T cells with the addition of stimulated CD4^–CD8⁺, but not CD4⁺ V24i NKT clone to PBMCs. Activated CD1d-restricted NKT clones did not affect cytokine production from activated T cells, but the CD4^–CD8 V24i NKT clone was able to kill CD1d-bearing cells including DCs, raising the possibility that the suppression of the expanding Ag-specific T cells was mediated via lysis of APCs or CD1d-expressing T cells. CD1d expression was not increased on activated T cells or EBV/MHC I tetramer⁺ cells, supporting the possibility that DCs, rather than the activated T cells, form the targets of these cytolytic NKT cells. We propose that CD4^–CD8⁺ subset of V24i NKT cells could be the suppressive arm of CD1d-restricted NKT cells and be involved in modulating a T cell response. Its slower proliferative capacity (Fig. 2D) compared with the CD4⁺ V24i NKT clone suggest that it probably acts at a later phase of the immune response, possibly in preventing an overwhelming response and ensuring contraction of the immune response. This is likely to be important in preventing nonspecific bystander activations of autoreactive T cells. It is conceivable that the containment of T cell activation could also prevent the "over-spill" of an immune response into destructive inflammatory process during host defense against pathogens. Thus, it is envisaged that the CD4⁺ V24i subset would augment the initiation of immune response (as has been shown by many studies, and in Fig. 4; Refs. 16 and 25) while the CD4^–CD8⁺ subset kills DCs or other APCs that have completed their tasks during the later phase of a primary or secondary T cell response. This dual function attributed to different subsets of V24i NKT cells that act at distinct stages of an immune response could reconcile the apparent opposing observations that these cells potentiate immune responses and also suppress autoimmune diseases. In this respect, CD4^–CD8⁺ V24i NKT cells could be another arm of an intricate regulatory system which is necessary to ensure a clean and effective immune response to an exogenous Ag.

Several studies have already implicated the interaction between DCs and V24i NKT cells and subsequent shaping of the immune response as a mechanism for preventing autoimmune disease (26, 27). It is thought that under normal circumstances, V24i NKT cells maintain a population of "tolerogenic" DC which promotes the anergisation of autoreactive T cells (26). In the absence of V24i NKT cells (as in some autoimmune diseases), this control is lost and activated DCs engage autoreactive T cells. Our study suggests that V24i NKT may also be able to control the frequency of activated DCs by directly lysing these cells via the employment of this specific cytolytic subset of the V24i NKT cells in a timely fashion.

The inhibitory effect of the CD8 NKT subset on T cell proliferation raises the possibility that these cells may behave like the T regulatory cells, as described by Powrie and colleagues (24). However, there are few similarities beyond this ability to inhibit T cell proliferation. In contrast to these established T regulatory cells, these inhibitory CD8⁺ V24i NKT cells are CD4^– and can proliferate upon stimulation by a specific Ag. In addition, our NKT clones do not secrete TGF-, IL-10, or express CD25. Nevertheless, it is possible that CD8⁺CD4^– V24i NKT cells form one component of an immune regulatory network that influence the fate of T cells after Ag activation. Thus, it could belong to a category that comprises T regulatory type 1, T cells, and Th3 cells (24, 28). This group of cells are thought to provide an extrinsic mode of regulation against activation of self-reactive T cells; and therefore, prevent autoimmune diseases. Indeed, both the "conventional" T regulatory cells and CD1d-restricted NKT cells have been shown to prevent the development of autoimmune diabetes mellitus and experimental allergic encephalitis in animal models (7, 8, 29, 30). Beaudoin et al. (31) provided a potential mechanism for this regulatory concept by demonstrating that NKT cells prevented the differentiation of naive disease-causing T cells into Th1 effectors cells. NKT cells were also found to induce a state of anergy in these anti- islet T cells thereby abrogating development of type I diabetes in recipient mice.

It is noteworthy that this subset of inhibitory cells expresses the atypical CD8 homodimeric rather than the more common CD8 isoform. CD8 is also found on intraepithelial lymphocytes in the gut, NK cells, and T cells. Like the CD8 heterodimer, it can bind to peptide-MHC class I complexes but there is evidence that it CD8 may also exert a lymphocyte regulating role upon interaction with a nonclassical MHC I molecule. Leishman et al. (32) showed that in mice, CD8 can regulate the behavior of T cells through interaction with thymus leukemic Ag, a MHC class I-like molecule. CD8 interaction with MHC class I on its own is likely to be too weak and brief to effect a signaling cascade (33); but CD8 engagement with thymus leukemic Ag was shown to be of significantly stronger affinity, and may trigger TCR clustering, resulting in either enhancement or suppression of intraepithelial lymphocyte responses depending on varying degree of cytokine signals in the vicinity (32). If a nonclassical MHC I-like molecule is involved in the interaction with CD8⁺ subset of V24i NKT cells, then cytokine variation could potentially be one of the means of determining the point of engagement of this subset of V24i NKT cells and when a suppressive action is appropriate during an immune response. Currently, the ligand for CD8 in humans is unknown.

In summary, our findings provide a mechanism by which a subset of V24i NKT cells could regulate activated T cells. It defines the presence of a subset of CD4^– V24i NKT cells which express CD8 and which exert a suppressive effect on proliferation of activated T cells, in contrast to other subsets that augmented T cell response. The findings suggest that multiple subsets within the V24i NKT cell population may exist in humans to serve different functions at different stages of the immune response.

	Acknowledgments

 
We thank Prof. V. Cerundolo for providing the CD1d tetramer, critical reading of the manuscript and discussions, Dr. Gavin Screaton for discussions, Dr. Tao Dong for initial help with T cell cloning, and Nan Chen for .221-transfected cell line.

	Footnotes

 
¹ The work was funded by the Medical Research Council (U.K.).

² Address correspondence and reprint requests to Dr. Ling-Pei Ho, Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, OX3 7DS, U.K. E-mail address: Ling-pei.ho{at}imm.ox.ac.uk

³ Abbreviations used in this paper: V24i, V24 invariant; DC, dendritic cell; GC, -galactosyl ceramide.

Received for publication January 9, 2004. Accepted for publication April 12, 2004.

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