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The Journal of Immunology, 2005, 175: 5087-5094.
Copyright © 2005 by The American Association of Immunologists

Human Invariant NKT Cells Are Required for Effective In Vitro Alloresponses1

Scott Patterson*, Ioannis Kotsianidis*, Antonio Almeida*, Marianna Politou*, Amin Rahemtulla*, Bini Matthew{dagger}, Richard R. Schmidt{dagger}, Vincenzo Cerundolo{ddagger}, Irene A. G. Roberts* and Anastasios Karadimitris2,*

* Department of Haematology, Imperial College London, Hammersmith Hospital, London, United Kingdom; {dagger} Department of Chemistry, University of Konstanz, Konstanz, Germany; and {ddagger} Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells are a small subset of regulatory T cells conserved in humans and mice. In humans they express the V{alpha}24J{alpha}18 invariant chain (hence invariant NKT (iNKT) cells) and are restricted by the glycolipid-presenting molecule CD1d. In mice, iNKT cells may enhance or inhibit anti-infectious and antitumor T cell responses but suppress autoimmune and alloreactive responses. We postulated that iNKT cells might also modulate human alloreactive responses. Using MLR assays we demonstrate that in the presence of the CD1d-presented glycolipid {alpha}-galactosylceramide ({alpha}GC) alloreactivity is enhanced (37 ± 12%; p < 0.001) in an iNKT cell-dependent manner. iNKT cells are activated early during the course of the MLR, presumably by natural ligands. In MLR performed without exogenous ligands, depletion of iNKT cells significantly diminished the alloresponse in terms of proliferation (58.8 ± 24%; p < 0.001) and IFN-{gamma} secretion (43.2 ± 15.2%; p < 0.001). Importantly, adding back fresh iNKT cells restored the reactivity of iNKT cell-depleted MLR to near baseline levels. CD1d-blocking mAbs equally reduced the reactivity of the iNKT cell-replete and -depleted MLR compared with IgG control, indicating that the effect of iNKT cells in the in vitro alloresponse is CD1d-dependent. These findings suggest that human iNKT cells, although not essential for its development, can enhance the alloreactive response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer T cells are a small but potent subset of regulatory T cells highly conserved in humans and mice (1). Human NKT cells comprise <0.1% of blood T cells and are characterized by a unique TCR made up of an invariant V{alpha}24J{alpha}18 (V{alpha}14J{alpha}18 in mice) and a diverse V{beta}11 chain (2), hence the term invariant NKT (iNKT)3 cells. iNKT cells also express the NK cell marker CD161 (NK1.1 in mice); however, its expression varies with the activation status of the cell (1, 3). In humans only a small proportion of CD161+ T cells are iNKT cells, whereas in mice ~50% of NK1.1+ T cells are iNKT cells as identified by CD1d/{alpha}-galactosylceramide ({alpha}GC) tetramer staining (1, 4). Upon engagement of their TCR by the glycolipid-presenting MHC class I-like molecule CD1d, iNKT cells are rapidly activated and secrete large amounts of Th1 and Th2 cytokines (1). {alpha}GC, a marine sponge glycolipid presented by CD1d, potently activates iNKT cells in vitro as well as in vivo (1, 5). Activated iNKT cells play a pivotal role in modulating all aspects of the innate and adaptive immune responses mainly through interactions with APCs (1). For example, in response to {alpha}GC, iNKT cells regulate Ag-specific in vivo Th1 cell responses by increasing dendritic cell (DC) expression of surface molecules important in Ag presentation such as HLA, CD80, CD83, and CD86 (6, 7). NKT cell activation often leads to enhanced responses against pathogens (e.g., viruses) and tumors and suppression of autoimmunity (reviewed in Refs.1 , 8 , and 9). However, depending on the experimental context, NKT cell activation may also be associated with increased susceptibility to viral disease and loss of tumor immunosurveillance (reviewed in Refs.1 , 8 , and 9).

Alloreactivity, one of the most powerful immune responses, is a Th1 cell- and cytokine-mediated response directed against disparate major or minor histocompatibility Ags. As in other types of immune responses, it is likely that the magnitude and the quality of the alloreactive response is modulated by networks of regulatory T cells that may enhance or dampen the effects of cytokine and cell alloeffectors. Understanding these networks may offer a basis for development of rational and novel therapeutic approaches for the control of the alloresponse in the clinical arena.

The role of iNKT cells in the modulation of alloreactivity has been studied in murine models of acute graft-vs-host disease (GVHD). Host iNKT cells appear to protect from lethal acute GVHD in systems involving myeloablative (10, 11, 12) as well as nonmyeloablative host preparation protocols (13, 14, 15). In addition, bone marrow-derived donor NKT cells as identified by expression of NK1.1 (or CD161 in humans) suppress the alloresponse (16, 17).

The modulatory effect of human NKT cells, as they are defined by the expression of the invariant V{alpha}24J{alpha}18 chain, in the alloreactive response has not been studied. In this study, using MLR assays, we dissect the role of human iNKT cells, {alpha}GC, and CD1d in the modulation of the in vitro alloresponse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
CD3+ T cell selection

Buffy coats from normal blood donors were supplied by the North London Blood Transfusion Service under Local Research Ethics Committee approval. PBMC were obtained after layering over Ficoll. For selection of CD3+ cells, the EasySep negative selection kit was used as per manufacturer’s instructions (StemCell Technologies). Purity of CD3+ cells was always >90% (data not shown).

Flow cytometry and flow sorting

Cells were stained with mAbs using standard protocols (18). For the identification and flow sorting of iNKT cells, selected CD3+ cells were stained with mAb against TCR V{alpha}24 and V{beta}11. With this approach the vast majority but not all bona fide iNKT cells as defined by CD1d/{alpha}GC tetramer staining are identified (18, 19). Therefore, depletion of iNKT cells using TCR V{alpha}24 and V{beta}11 mAbs will remove most CD1d/{alpha}GC tetramer-positive iNKT cells except a very small population of CD1d/{alpha}GC tetramer-positive TCR V{alpha}24-negative iNKT cells (19). The following mAbs were used: mouse anti-human TCR V{alpha}24 and TCR V{beta}11 either FITC- or RPE-labeled, or biotin-labeled (Serotec); mouse anti-human biotin (Beckman Coulter); CD69-FITC, CD3-allophycocyanin, IgG1-FITC, IgG1-RPE, and IgG1-biotin (Caltag Laboratories); and CD4-PerCP, HLA-DR-allophycocyanin, and steptavidin-allophycocyanin (BD Biosciences). Multicolor flow cytometry was performed using a FACSCalibur, whereas flow sorting was performed using a FACSDiva (BD Biosciences). Data analysis was performed with the CellPro or FlowJo software.

MLR and proliferation assays

For all cultures, T cell medium consisting of RPMI 1640, 5% heat-inactivated human serum supplemented with 1% of L-glutamine and penicillin/streptomycin was used. For MLR, generally 30–50 x 103 CD3+ cells were placed in triplicates against autologous or allogeneic irradiated (3000 rad) PBMC in 96-well plates at responder to stimulator (R:S) ratios as indicated. For long-term MLR, 30–50 x 104 CD3+ cells were plated in 48-well plates. [3H]Thymidine was added at 1 µCu/well for the last 16 h of the MLR. Proliferation was measured using a liquid scintillation counter after harvesting with a cell harvester. Parallel cultures were set for cytokine release assays. In indicated experiments, {alpha}GC (100 ng/ml) or its diluent vehicle (NaCl 150 mM/Tween 20 0.05%) were added to the MLR. The following mAbs were used for blocking MLR: anti-CD1d (clone 42.1; BD Pharmingen); anti-HLA class II (clone TU39; BD Pharmingen); and IgG1 isotype (BD Pharmingen) at indicated concentrations.

CFSE (Molecular Probes) staining of CD3+ cells was performed in PBS for 8 min at room temperature using CFSE at 5 µM with occasional mixing. After washing with an equal volume of FCS, cells were extensively washed with complete T cell media.

ELISA

Quantitation of IFN-{gamma} and IL-4 in the supernatants of the MLR was performed with the Quantikine kit (R&D Systems).

Statistical analysis

The Wilcoxon signed ranks test was used to compare differences in proliferation between iNKT cell-replete and cell-depleted MLR and between {alpha}GC or vehicle-treated MLR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Invariant NKT cell-dependent enhancement of MLR by {alpha}GC

In in vivo murine models, treatment of irradiated recipient mice with {alpha}GC induces host-derived, iNKT cell-dependent IL-4 secretion and protection from acute GVHD (11, 12). We tested the effect of {alpha}GC in human in vitro alloresponses. For this purpose we performed 4-day MLR, using as responders purified negatively selected T cells and as stimulators irradiated PBMC pulsed with {alpha}GC or vehicle. As shown in Fig. 1a, top, addition of {alpha}GC to the MLR of responder 86 against a panel of five stimulators caused a 25–73% increase in proliferation. To rule out nonspecific T cell activation that may be induced by {alpha}GC (20), autologous MLR was performed in parallel with the allogeneic MLR. In the presence of either {alpha}GC or vehicle, minimal proliferation was observed in autologous as compared with allogeneic MLR (Fig. 1a, top), suggesting thus that the enhancing effect of {alpha}GC in the allogeneic MLR is alloreactive T cell-specific. In addition to responder 86, we tested another three responders against a panel of five stimulators and found that treatment of the MLR with {alpha}GC resulted in a 37 ± 12% (p < 0.001) increase in proliferation as compared with MLR treated with vehicle only (Fig. 1a, bottom). Consistent with this, IFN-{gamma} production was also comparably increased in the MLR performed in the presence of {alpha}GC; no IL-4 was detected at 96 h (Fig. 1b) or at 4 and 24 h of the MLR (data not shown). Furthermore, the enhancing effect of {alpha}GC was iNKT cell-dependent, as upon iNKT cell depletion (see below) and in the presence of {alpha}GC, proliferation was equally affected as in iNKT cell-depleted MLR treated with vehicle (Fig. 1c).



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  		FIGURE 1. {alpha}GC enhances alloreactivity in an iNKT cell-dependent manner. a, MLR was performed using T cells from responder 86 against a panel of five stimulators in the presence of vehicle or {alpha}GC (top). In the presence of {alpha}GC, proliferation increased by 48% (range from 25 to 73%). Minimal reactivity was observed in autologous MLR. In total, MLR of four responders against five stimulators (each repeated twice) were tested and in all cases addition of {alpha}GC (bottom) had an enhancing effect on the alloreactive response (37 ± 12%; p < 0.001). b, IFN-{gamma} production was increased in {alpha}GC-treated allogenic compared with vehicle-treated MLR but not in autologous MLR. No IL-4 production was detected under these conditions. Data are representative of four independent MLR. c, iNKT cell-replete or cell-depleted MLR were performed in the presence of vehicle or {alpha}GC. In iNKT cell-depleted allo-MLR, proliferation was equally reduced under both conditions, indicating that the enhancing effect of {alpha}GC on the alloresponse is iNKT cell-dependent (CD3+ indicates iNKT cell-replete MLR, and CD3+NKT– indicates iNKT cell-depleted MLR). d, Proliferation as determined by CFSE staining (upper) and absolute numbers (middle) of iNKT cells (as determined by flow cytometry and total cell count) in a 96-h MLR in the presence of {alpha}GC or vehicle. In parallel, proliferation was measured by 3H incorporation (lower). Blots are gated on iNKT cells identified by anti-TCR V{alpha}24 and V{beta}11 staining. iNKT cells proliferate more in the presence of {alpha}GC; however, their increase in absolute numbers does not account for the 50% increase in proliferation observed in the presence of {alpha}GC. Data are representative of two independent experiments and are presented as mean ± SEM of triplicate assays.

 
To further elucidate the relative role of iNKT cells in the enhancement of MLR by {alpha}GC, we correlated proliferation of iNKT cells (as assessed by CFSE labeling) and absolute counts (as determined by flow cytometry and total cell count) with the proliferation of all T cells (as assessed by 3H incorporation). CFSE staining showed that iNKT cells proliferated more in the presence of {alpha}GC compared with vehicle (Fig. 1d, upper). This proliferation corresponded to an absolute increase from a baseline of 300 iNKT cells on day 0 (corresponding to 2 x 106 plated total T cells) to 450 and 690 cells 4 days later in the presence of vehicle and {alpha}GC, respectively (Fig. 1d, middle). At the same time, the MLR reactivity of total T cells (5 x 104 responder T cells corresponding to ~10–15 iNKT cells) increased by almost 50% (corresponding to ~10,000 cpm) in the presence of {alpha}GC (Fig. 1d, lower). It is very unlikely that this difference is the result of proliferation of the few NKT cells activated on day 4 by {alpha}GC; instead, it reflects the proliferation of the alloreactive T cells.

Dynamics of iNKT cell activation in MLR

{alpha}GC is a pharmacological agent and its effect on iNKT cell expansion and activation may not reflect the mechanisms of iNKT cell activation by natural CD1d ligands. The dynamics of iNKT cell expansion and activation (as determined by the surface activation marker CD69) in response to natural ligands were monitored during the course of long-term MLR (Fig. 2). In the presence of {alpha}GC the relative frequency of iNKT cells did not increase significantly on day 4, but it did increase by at least 10-fold by day 8 (Fig. 2a, top). As well as proliferation, {alpha}GC also induced increasing activation of iNKT cells over time (Fig. 2a, bottom). The lack of relative expansion of {alpha}GC-treated iNKT cells on day 4 might be more apparent rather than real because upon {alpha}GC exposure, the TCR of iNKT cells is known to be initially down-regulated but re-expressed later (21, 22). In the presence of vehicle, the relative frequency of iNKT cells during the 8-day MLR did not change significantly from baseline (Fig. 2a, top). Despite the lack of significant proliferation, vehicle-treated iNKT cells were progressively activated between days 0, 4, and 8 of the MLR (Fig. 2a, bottom). iNKT cells therefore are activated during the course of the MLR in the absence of {alpha}GC, presumably by natural ligands presented to them by CD1d-expressing APC, although they do not proliferate significantly. On day 8 of the same series of MLR, we studied IFN-{gamma} production by iNKT cells as well as total T cells. IFN-{gamma} production was higher in iNKT cells treated with {alpha}GC rather than vehicle and, importantly, IFN-{gamma} production by total T cells was considerably higher in the presence of {alpha}GC (Fig. 2b). In addition, in the presence of {alpha}GC there was increased activation of non-iNKT cells as determined by forward scatter characteristics (Fig. 2b) and CD69 expression (data not shown). These findings provide further evidence that increased reactivity of MLR in the presence of {alpha}GC does not merely reflect activation of iNKT cells but is primarily due to activation of alloreactive T cells.



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  		FIGURE 2. Expansion and activation of iNKT cells in long-term MLR. a, Three independent MLR were performed in the presence of {alpha}GC or vehicle without exogenous IL-2 supplementation. On indicated time points the frequency as well as the activation of iNKT cells was determined by flow cytometry after staining with anti-TCR V{alpha}24, V{beta}11, and CD69. At least 5 x 104 events were collected for analysis. The frequency of iNKT cells in the MLR treated with vehicle (top, solid line) remained similar to baseline throughout the course of the MLR. In the presence of {alpha}GC (dashed line), the frequency of iNKT cells on day 4 was similar to baseline; however, it increased by at least 10-fold by day 8. The frequency of naturally activated iNKT cells progressively increases during the course of the MLR (bottom). Greater activation of iNKT cells is seen when cells are pharmacologically activated by exogenous {alpha}GC. b, IFN-{gamma} production by iNKT cells and non-iNKT T cells in MLR treated with {alpha}GC or vehicle on day 8. {alpha}GC induces more IFN-{gamma} production by total T cells as well as iNKT cells. In addition, activation of non-iNKT cells as assessed by forward scatter characteristics is also higher in the presence of {alpha}GC. Gates for iNKT cells blots have been set on TCR V{alpha}24+V{beta}11+ events. Gates for total CD3 cells for both blots and histograms exclude iNKT cells.

 
Role of iNKT cells in the absence of exogenous ligand

The findings discussed above suggest that human iNKT cells are an integral part of the in vitro alloresponse and are required for its efficient development. This predicts that depletion of iNKT cells would attenuate the alloreactivity. To test this, we performed 4-day MLR using either iNKT cell-replete or cell-depleted responders. Rigorous iNKT cell depletion of negatively selected T cells was performed by staining with anti-TCR V{alpha}24 and V{beta}11 mAbs followed by flow sorting (Fig. 3a). iNKT cell-replete T cells were also flow sorted on the basis of their physical characteristics. To rule out preactivation of iNKT cells triggered by staining with the anti-TCR V{alpha}24 and V{beta}11 mAbs we established that engagement of the invariant TCR by the anti-TCR mAbs during the sorting procedure did not have a mitogenic effect on iNKT cells and did not alter their Th1/Th2 cytokine secretion balance (data not shown). When iNKT cells were depleted, the MLR reactivity of responder 49 against a panel of stimulators decreased by ~65% (Fig. 3b, left). In an extended panel in which we tested another three responders against three to five stimulators, the proliferation rate in iNKT cell-depleted compared with iNKT cell-replete MLR was reduced (by 58.8 ± 24%; p < 0.001) in every responder to stimulator pair (Fig. 3b, right). This suppressive effect was mirrored by a comparable reduction of IFN-{gamma} secretion in the supernatants of the iNKT cell-depleted MLR compared with baseline (by 43.2 ± 15.2%; n = 7; p < 0.001) (Fig. 3c). No IL-4 was detected. The effect of iNKT cell depletion was observed in MLR performed with varying R:S ratios (Fig. 4a) and also in longitudinal MLR (Fig. 4b). Finally, add-back of highly purified fresh iNKT cells to iNKT cell-depleted MLR restored proliferation close to the levels of the baseline iNKT cell-replete MLR (Fig. 4c). These findings suggest that although iNKT cells are not essential, they do contribute to the development of full MLR reactivity.



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  		FIGURE 3. iNKT cell depletion attenuates in vitro alloresponse. a, iNKT cell depletion of CD3+ T cells by flow sorting. Negatively selected CD3+ cells with a purity of >90% (data not shown) were stained with anti-TCR V{alpha}24 and V{beta}11 mAbs. Using a FACSDiva flow sorter, TCR V{alpha}24+V{beta}11+ NKT cells were depleted from CD3+ cells. Pre- and postsorting analysis of at least 5 x 104 events revealed that the procedure consistently generated highly iNKT cell-depleted CD3+ cells and highly purified iNKT cells. In addition, CD3+ iNKT cell-replete cells were sorted on the basis of their physical characteristics (data not shown). b, iNKT cell-replete MLR (indicated as CD3+) using T cells from responder 49 against a panel of five stimulators was compared with iNKT cell-depleted MLR (indicated as CD3+NKT–) (left). In total, MLR of four responders were tested against three to five stimulators (each panel repeated twice with similar results), and in every responder to stimulator pair, iNKT cell depletion significantly diminished the alloreactive response ranging from 36 to 85% (p < 0.001; right). Data are presented as mean ± SEM of triplicate assays. c, In accordance with the proliferation data, IFN-{gamma} secretion was comparably reduced in iNKT cell-depleted MLR compared with baseline. No IL-4 was detected (representative of seven independent MLR).

 


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  		FIGURE 4. Dynamics of the alloresponse inhibition by iNKT cell depletion. a, iNKT cell depletion reduces MLR reactivity at varying R:S ratios and at different time points and a fixed R:S ratio of 1:2 (b). c, iNKT cells were ex vivo purified by flow sorting as shown in Fig. 3a and were used in an add-back MLR assay. Addition of 50 responder iNKT cells (corresponding roughly to the number of responder iNKT cells in the iNKT cell-replete MLR) were added to iNKT cell-depleted MLR. MLR reactivity increased by >50% and even further when 5- and 10-fold more iNKT cells were added back. Each result is representative of three independent MLR.

 
Role of CD1d in MLR

CD1d is the restricting element of iNKT cells and is expressed on monocytes, B cells, and APC. We therefore tested the effect of anti-CD1d mAb on the MLR. Anti-CD1d caused a dose-dependent inhibition of MLR as compared with Ig isotypic control (Fig. 5a) and anti-CD1d used at a fixed dose inhibited MLR at different R:S ratios (Fig. 5b). In longitudinal MLR, T cell proliferation and activation as determined by CFSE and HLA-DR staining, respectively, were significantly and dynamically decreased in the presence of anti-CD1d compared with Ig isotypic control (Fig. 5c). Further, T cells stimulated in primary MLR in the presence of anti-CD1d were less responsive in secondary MLR as compared with T cells derived from primary MLR treated with isotypic Ig control (Fig. 5d), suggesting that CD1d blocking may have rendered responder T cells anergic.



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  		FIGURE 5. Role of CD1d in the MLR. a, MLR were set up at a R:S ratio of 1:2 in the presence of varying concentrations of anti-CD1d or IgG. At an anti-CD1d concentration of as low as 5 µg/ml, MLR reactivity was significantly reduced. b, MLR performed in the presence of anti-CD1d 40 µg/ml was significantly inhibited at varying R:S ratios as compared with MLR treated with medium only. c, MLR were performed in the presence of anti-CD1d or IgG (40 µg/ml each). Proliferation and activation as assessed by CFSE and HLA-DR staining, respectively, were monitored at different time points by flow cytometry. Significant reductions in both proliferation and activation of T cells were observed over time in the presence of anti-CD1d as compared with anti-IgG. d, Proliferation was measured in primary 4-day MLR set up at a R:S ratio of 1:2 and treated with either anti-CD1d or IgG isotype (20 µg/ml each). After 2 days of rest, equal numbers of T cells were placed in a secondary MLR against the same stimulators at a R:S ratio of 1:2 but without mAb. Comparison of the primary with the secondary MLR revealed that CD1d treatment reduces T cell proliferation in the secondary as well as the primary MLR. Each result is representative of three independent MLR.

 
CD1d is required for the iNKT cell-mediated effect on MLR

In principle, the effect of anti-CD1d mAb on MLR might reflect either inhibition of an enhancing effect of NKT cells on the MLR or enhancement of an inhibitory effect. To discriminate between these two effects, MLR were performed using iNKT cell-replete or cell-depleted responders in the presence of anti-CD1d, anti-HLA class II, or Ig isotype (Fig. 6). As expected, the iNKT cell-replete MLR was inhibited by anti-CD1d and by anti-HLA class II mAbs either alone or in combination. iNKT cell depletion resulted in reduction of proliferation but the presence of anti-CD1d in the iNKT cell-depleted MLR did not result in any further significant reduction of proliferation. These results indicate that CD1d is required for the iNKT cell-mediated effect on MLR.



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  		FIGURE 6. CD1d is required for the iNKT cell-mediated effect on MLR. iNKT cell-replete and cell-depleted MLR were set up in the presence of IgG, anti-CD1d, anti-HLA class II (at 20 µg/ml each), or a combination of anti-CD1d and anti-HLA. Reactivity, as expected, was reduced in the absence of iNKT cells. Anti-CD1d and anti-HLA class II treatment of iNKT cell-replete MLR inhibited reactivity by 48 and 57%, respectively. Treatment of the iNKT cell-depleted MLR with anti-CD1d did not have any significant effect compared with IgG treatment (12% inhibition), whereas anti-HLA class II blocking, as in the iNKT cell-replete MLR, resulted in a significant (60%) reduction of proliferation. Data are representative of three independent MLR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Invariant NKT cells are emerging as an important subset of immunoregulatory cells. Unlike the exclusively suppressive immunomodulation mediated by the CD4+CD25+ subset of T cells (23), they either enhance or inhibit an Ag-specific immune response through direct interaction with APC (1). This is the first report dissecting the role of human iNKT cells (as defined by the coexpression of the TCR V{alpha}24 and V{beta}11 chains), CD1d (the restricting element of iNKT cells), and {alpha}GC (the iNKT cell ligand) in the alloresponse. Our results suggest that the iNKT cell/CD1d axis positively regulates the alloresponse in humans. This idea is consistent with an earlier report documenting the ability of human iNKT cell clones to drive maturation of DC that can subsequently support efficient alloreactive responses (24). It is also consistent with studies showing that like the alloresponse, other Th1 responses against a variety of pathogens (for example, CTL responses against viruses) can diminish in the absence of iNKT cells or CD1d (25, 26). Conversely, in the presence of {alpha}GC, Th1 innate and adaptive immune responses are considerably enhanced in an iNKT cell- and CD1d-dependent manner (6, 7).

Our data contrast with a number of in vivo studies in which iNKT cells appear to inhibit rather than enhance the alloresponse. In murine models of acute GVHD involving myeloablative irradiation of the host, animals that receive {alpha}GC injections at the time of irradiation are protected from acute GVHD (10, 11, 12). This protective effect of {alpha}GC requires the presence of host iNKT cells and CD1d and is mediated by IL-4 secreted by host cells (10, 11). In our studies, in an attempt to mimic this in vivo set up, the stimulators (PBMC) of the MLR were irradiated and pulsed with {alpha}GC. However, we consistently observed increased reactivity of the MLR both in terms of proliferation and IFN-{gamma} secretion, an effect that was iNKT cell-dependent. Crucially, no IL-4 was detected at any time point tested during a 4-day MLR. This is in line with the observation that administration of {alpha}GC-loaded DC to normal human subjects is associated with increased IFN-{gamma} and IL-12 but decreased IL-4 levels in the serum (27).

A second group of studies have reported that protection from lethal acute GVHD in the context of a nonmyeloablative, tolerance-inducing regimen is associated with persistence of CD1d/{alpha}GC tetramer-positive host iNKT cells (13, 14). Similarly, donor-derived NKT cells (identified as NK1.1+ T cells in C57BL/6 and DX5+ cells in BALB/c mice) protect from lethal acute GVHD after their adoptive transfer to a lethally irradiated host (16, 28). IL-4 and IL-10 seem to be the cytokine mediators of these effects (16, 28). However, NK1.1+ cells were not implicated as being the regulatory T cells that protect from acute GVHD following donor lymphocyte infusion (29, 30).

By performing MLR in the presence and absence of human peripheral blood iNKT cells, we established the importance of responder iNKT cells in enhancing the in vitro alloresponse in steady state and without exogenous pharmacological activation. This effect was documented by the specific decrease in proliferation and IFN-{gamma} production in iNKT cell-depleted MLR and by restoration of these parameters upon adding back highly purified fresh iNKT cells. Furthermore, the effect of iNKT cells on the alloresponse was clearly CD1d-dependent. Interestingly, the degree of inhibition of the MLR upon iNKT cell depletion was remarkably uniform for a given responder tested against a panel of stimulators but it varied between responders (Fig. 3a, right). This could reflect the variable numbers of TCR V{alpha}24 iNKT cells that were not removed by our iNKT cell depletion strategy (see Materials and Methods).

It is not clear why iNKT cells have an enhancing effect in the human in vitro alloresponse and the opposite effect in the murine in vivo alloresponse. It is possible that because only 50–70% of murine bone marrow and spleen NK1.1+ T cells are iNKT cells, as defined by CD1d/{alpha}GC tetramer staining (4), NK1.1 marks two groups of CD1d-restricted subsets of regulatory T cells. A subset of cells that may or may not express NK1.1 but are stained with the CD1d/{alpha}GC tetramer (iNKT cells) and a subset of cells that always express NK1.1 but are not stained with the CD1d/{alpha}GC tetramer (non-iNKT cells). In support of this, Exley et al. (17) have reported that human CD161+ T cells, like their murine NK1.1+ counterparts, are enriched in bone marrow, secrete IL-4, and suppress the MLR (17). However, these CD161+ T cell lines were devoid of iNKT cells. Indeed, only 2 of 12 lines contained any measurable numbers of TCR V{alpha}24+V{beta}11+ NKT cells (17).

The cell and cytokine networks responsible for the enhancing effect of iNKT cells in the course of an Ag-specific immune response have been studied in in vivo models of microbial infections (31, 32, 33). Initial activation of iNKT cells requires a CD1d-dependent cell contact with the APC (1). This leads to increased IFN-{gamma} production by iNKT cells, which in turn enhances the Ag-presenting capability of APC. This positive feedback loop of iNKT cell and DC activation is dependent on DC- and pathogen-derived ligands structurally resembling {alpha}GC (31, 32, 33). Our data support the existence of a similar network steering the enhancing effect of iNKT cells in the context of the human alloresponse in which iNKT cells can be activated by both exogenous and natural ligands. The structure of the natural ligands driving activation of iNKT cells in the context of the alloresponse remains to be determined.

Because of their flexible immunomodulatory role, iNKT cells hold promise for manipulation and therapeutic use in clinic including modulation of acute GVHD. It is not clear whether addition or depletion or activation or inactivation of iNKT cells would be beneficial: in vivo murine studies would favor adoptive transfer of iNKT cells (13, 14) or treatment of the host with {alpha}GC (11), whereas human in vitro studies (24 and this work) would favor iNKT cell depletion of donor T cells. Studies of the dynamics of iNKT cell activation in the setting of human acute GVHD will be beneficial in understanding more on the role of iNKT cells in vivo and how to exploit them for therapy.

In conclusion, we have shown that iNKT cells are naturally activated during the course of in vitro human alloreactivity in a CD1d-dependent manner and although not essential they contribute to the mounting of an efficient alloresponse.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 is supported by Cancer Research U.K. Grant C399-A2291 and the Cancer Research Institute (to V.C.). A.K. is a Leukaemia Research Fund Bennett Senior Fellow. Back

2 Address correspondence and reprint requests to Dr. Anastasios Karadimitris, Department of Haematology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K. E-mail address: a.karadimitris{at}imperial.ac.uk Back

3 Abbreviations used in this paper: iNKT, invariant NKT; {alpha}GC, {alpha}-galactosylceramide; DC, dendritic cell; GVHD, graft-vs-host disease. Back

Received for publication April 28, 2005. Accepted for publication August 4, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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