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Granulocyte Colony-Stimulating Factor Modulates -Galactosylceramide-Responsive Human V24⁺V11⁺ NKT Cells¹

Tania Crough^*, Mie Nieda and Andrew J. Nicol^2,*,

^* Department of Medicine, University of Queensland, and Queensland Institute of Medical Research, Brisbane, Australia; and Yokohama City University School of Medicine, Yokohama, Japan

	Abstract

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Despite more than a 10-fold increase in T cell numbers in G-CSF-mobilized peripheral blood stem cell (PBSC) grafts, incidence and severity of acute graft-vs-host disease (GVHD) are comparable to bone marrow transplantation. As CD1d-restricted, V24⁺V11⁺ NKT cells have pivotal immune regulatory functions and may influence GVHD, we aimed to determine whether G-CSF has any effects on human NKT cells. In this study, we examined the frequency and absolute numbers of peripheral blood NKT cells in healthy stem cell donors (n = 8) before and following G-CSF (filgrastim) treatment. Effects of in vivo and in vitro G-CSF on NKT cell cytokine expression profiles and on responsiveness of NKT cell subpopulations to specific stimulation by -galactosylceramide (-GalCer) were assessed. Contrary to the effects on conventional T cells, the absolute number of peripheral blood NKT cells was unaffected by G-CSF administration. Furthermore, responsiveness of NKT cells to -GalCer stimulation was significantly decreased (p < 0.05) following exposure to G-CSF in vivo. This hyporesponsiveness was predominantly due to a direct effect on NKT cells, with a lesser contribution from G-CSF-mediated changes in APC. G-CSF administration resulted in polarization of NKT cells toward a Th2, IL-4-secreting phenotype following -GalCer stimulation and preferential expansion of the CD4⁺ NKT cell subset. We conclude that G-CSF has previously unrecognized differential effects in vivo on NKT cells and conventional MHC-restricted T cells, and effects on NKT cells may contribute to the lower than expected incidence of GVHD following allogeneic peripheral blood stem cell transplantation.

	Introduction

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Allogeneic peripheral blood stem cell (PBSC)³ transplantation offers the only curative treatment for many hematological malignancies (1, 2), but it is frequently complicated by graft-vs-host disease (GVHD). Donor-derived T lymphocytes are major mediators of GVHD (3), and while depletion of T cells from the graft can reduce the incidence and severity of GVHD, this increases the risk of engraftment failure and tumor relapse, due to the loss of beneficial graft-vs-leukemia (GVL) effects (4, 5). Therefore, maximizing beneficial GVL activity while preventing or reducing GVHD following allogeneic PBSC transplantation remains a major therapeutic goal.

G-CSF-primed peripheral blood (PB) is widely used as an alternative to bone marrow as a source of hemopoietic stem cells(HSC) (6, 7). G-CSF administration results in significant increases in most cell types, including T cells in addition to mobilization of CD34⁺ HSCs (8, 9). Despite the fact that at least 10-fold more allogeneic T cells are administered with PB HSCs than with bone marrow, the incidence and severity of acute GVHD are comparable (8, 10, 11). The reasons for this remain unclear. Possibilities include immunomodulatory effects of G-CSF on donor T cells or quantitative and qualitative differences of other cell types such as monocytes (Mo) and dendritic cells (DC) in the G-CSF-mobilized allografts (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). One potentially important unexplored possibility is an effect on V24⁺V11⁺ NKT cells.

Human NKT cells are a subset of T lymphocytes defined by the presence of the invariant V24 V11 TCR, and are distinct from CD56⁺ T cells also frequently labeled as NKT cells (26, 27). V24⁺ NKT cells and their murine counterpart, V14⁺ NKT cells, are activated and induced to proliferate in response to specific stimulation by -galactosylceramide (-GalCer), an effect enhanced by the addition of IL-2 and dependent on CD1d-expressing cells such as Mo and DC (28, 29, 30, 31). Activated NKT cells rapidly secrete large amounts of IL-4 and IFN- (29, 32), and can exert potent direct and indirect antitumor activity against tumor cells in murine models (33, 34), a range of tumor cell lines (35, 36), and human tumor cells (37, 38, 39). They also have pivotal immune regulatory functions and are reported to have both immune stimulatory (40, 41, 42, 43, 44, 45, 46) and immune suppressive (47, 48, 49, 50) activity depending on the circumstance or model system evaluated. There is a significant body of evidence indicating that NKT cells are protective against autoimmunity (51, 52) and GVHD (53, 54); however, interpretation of much of this data is difficult because NKT cell subpopulations other than the CD1d-restricted, -GalCer-responsive population have been included in the majority of analyses, and most studies have focused on nonactivated NKT cells rather than those activated through their TCR.

Of particular interest is the increasing awareness of the immune adjuvant properties of NKT cells. NKT cells have been shown to increase NK cell activation and NK-mediated cytotoxicity (40, 42, 44, 55, 56, 57), as well as having secondary effects on conventional Ag-specific T cells (32, 43, 46, 56, 57), B cells (45, 57), and DC (41, 46, 49, 58). A corollary of this is that reduced numbers or reduced responsiveness of NKT cells would be predicted to have an immune suppressive effect.

Only a single study has evaluated the effects of the administration of G-CSF to normal PBSC transplant donors on PB V24⁺ NKT cells (9). This study demonstrated that CD3⁺ V24⁺CD161⁺ cells were not preferentially mobilized in G-CSF-primed PB. However, an effect of G-CSF on CD1d-restricted NKT cells, if any, has not been conclusively determined, as the cell population evaluated included conventional T cells (expressing the V24 TCR, but expressing V chains other than V11 and hence non-CD1d reactive), did not include V24⁺V11⁺ NKT cells without high levels of CD161, and no functional effects were assessed.

We aimed to examine the effects of G-CSF on the number and proliferative potential of human V24⁺V11⁺ NKT cells in healthy donors, the goal of which was to determine whether modulation of NKT cells may have a role in the lower than expected incidence of acute GVHD following administration of G-CSF-primed PBSC transplantation.

	Materials and Methods

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Patient samples

All samples were collected from healthy allogeneic PBSC donors after informed consent. The study was approved by the Royal Brisbane Hospital and Health Services Districts Human Research Ethics Committee (Brisbane, Australia), Queensland Institute of Medical Research Human Ethics Committee (Brisbane, Australia), and University of Queensland Medical Research Ethics Committee (Brisbane, Australia). Blood samples were collected before (pre-G-CSF) and following (post-G-CSF) 4 days of in vivo G-CSF (neupogen, filgrastim; Amgen, Thousand Oaks, CA) administration at a dose of 10 µg/kg to nine subjects. One subject recruited was removed from analysis as an outlier; thus, all subsequent data reported are based on n = 8 subjects.

Assessment of NKT cell percentages by flow cytometry

PBMC collected by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density centrifugation were washed with 0.9% sodium chloride (Baxter Healthcare, Sydney, Australia) and resuspended in PBS supplemented with 5% normal human AB serum (a gift from Australian Red Cross Blood Transfusion Service, Brisbane, Australia). The PBMC were stained with FITC-conjugated anti-V24 (IgG1: C15), PE-conjugated anti-V11 (IgG2a: C21), and PE-cyanin 5.1 (PC5)-conjugated anti-CD3 (IgG1, UCHT1), fixed with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), and analyzed within 24 h by three-color flow cytometry using a FACSCalibur (BD Biosciences, San Diego, CA) or FC500 (Beckman Coulter, Miami, FL). NKT cells were defined as V24⁺V11⁺CD3⁺ cells located within the lymphocyte region as determined by forward scatter and side scatter, and, to ensure accurate quantitation of this small population of cells, a minimum of 120 triple-positive events was collected. An isotype control sample with CD3 PC5, IgG1 FITC, and IgG2a PE was used to ensure optimal control of the infrequent V24⁺ and V11⁺ positive events.

Selected samples were also analyzed with a CD1d dimer (CD1d:Ig DimerX; BD Pharmingen, San Diego, CA) to assess the reproducibility of NKT cell quantitation by anti-V24/anti-V11/anti-CD3 staining. Briefly, the CD1d:Ig DimerX was incubated overnight with -GalCer (KRN7000; a gift from Pharmaceutical Division, Kirin Brewery, Gunma, Japan) in excess. PBMC were incubated with human Ig to block any nonspecific reactions before incubation with 2 µg of unloaded or -GalCer-loaded CD1d dimer. The cells were then stained with secondary biotin (Beckman Coulter) and streptavidin PC5 (Beckman Coulter) mAbs and anti-V24 FITC-, anti-V11 PE-, and PE-cyanin 7-conjugated anti-CD3 (IgG1; UCHT1) cell surface markers.

Calculation of NKT cell numbers

NKT cell numbers were expressed as a percentage of the CD3⁺ lymphocyte population, as determined by flow cytometry. To determine the number of NKT cells per liter of blood, the fraction of lymphocytes that were NKT cells according to flow cytometry was multiplied by the number of lymphocytes per liter, as determined by an automated full blood count on the same sample.

Proliferative capacity of V24⁺V11⁺ NKT cells

To assess the effect of G-CSF administration on the proliferative capacity of NKT cells, the fold expansion of NKT cells in pre-G-CSF and post-G-CSF PBMC in the presence of -GalCer was determined. Pre-G-CSF and post-G-CSF PBMC, containing Mo and B cells as CD1d-expressing APC, were assessed for NKT cell numbers and cultured in AIM-V medium with 10% AB serum (Invitrogen Life Technologies, Melbourne, Australia), 10 U/ml IL-2 (Chiron, Melbourne, Australia), and 100 ng/ml -GalCer for 7 days, after which the cells were harvested, counted, and analyzed by flow cytometry, as described above. The results are expressed in terms of fold expansion, as determined from the respective NKT cell numbers at days 0 and 7.

Effect of in vitro added G-CSF on NKT cell expansion

Pre-G-CSF NKT cells in pre-G-CSF PBMC were activated and expanded with -GalCer, as described above, in the presence of in vitro added G-CSF at 0, 10, and 100 ng/ml in five donors, and the respective fold expansion was calculated.

Evaluation of possible effects of G-CSF on APC stimulation of NKT cells

CD14⁺ Mo were positively isolated and CD3⁺ T cells negatively isolated from pre-G-CSF and post-G-CSF PBMC by MiniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) immunomagnetic separation, according to the manufacturer’s instructions. Pre-G-CSF and post-G-CSF Mo were each combined with pre-G-CSF and post-G-CSF CD3⁺ cells (giving four permutations) at a responder:stimulator ratio of 10:1. The NKT cells were expanded for each of the four combinations by -GalCer stimulation, as already described, and the fold expansion was calculated.

Flow cytometric analysis of NKT cell subsets

NKT cell subpopulations were assessed by five-color flow cytometry with a FC500 flow cytometer (Beckman Coulter) following staining with anti-V24 FITC, anti-V11 PE, PE-Texas Red-X-conjugated anti-CD8 (IgG1, SFCI21Thy-2D3G8), anti-CD4 PC5 (IgG1, 13B8.2), and anti-CD3 PE-cyanin 7. Results are expressed as CD4⁺ NKT cells (comprising CD4⁺CD8^– and CD4⁺CD8⁺ NKT cells) and CD4^– NKT cells (comprising CD4^–CD8^– and CD4^–CD8⁺ NKT cells). NKT cell subsets were analyzed on baseline (day 0) pre-G-CSF and post-G-CSF NKT cells. Pre-G-CSF and post-G-CSF V24⁺ NKT cells, purified by positive (V24) immunomagnetic separation (miniMACS; Miltenyi Biotec), were also cultured with autologous pre-G-CSF Mo (not exposed to G-CSF), 100 ng/ml -GalCer, and IL-2 for 7 days to expand NKT cells, after which NKT subset analysis was again conducted (day 7). These pre-G-CSF and post-G-CSF NKT cell lines were used for subsequent cytokine secretion assays, as described below.

Analysis of NKT cell cytokine production

Purified pre-G-CSF (not exposed to G-CSF) and post-G-CSF (exposed to in vivo G-CSF) NKT cell lines (1 x 10⁵) were cultured with 1 x 10⁴ autologous irradiated -GalCer-pulsed Mo-derived DC, generated by the culture of adherent Mo (not exposed to G-CSF) with 500 U/ml IL-4 (R&D Systems, Sydney, Australia) and 800 U/ml GM-CSF (Schering-Plough, Sydney, Australia) for 5 days, in 96-well plates. After 72 h, the culture supernatant was collected from each well, and the concentrations of IFN- and IL-4 in the supernatants were measured by ELISA, according to the manufacturer’s protocol (OPTEIA anti-human ELISA kits; BD Pharmingen, San Diego, CA).

Statistical analysis

Data represent mean ± SEM. The statistical significance was analyzed by Wilcoxin’s test. Differences with a p value <0.05 were considered significant.

	Results

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G-CSF administration increases T cells, but not V24⁺V11⁺ NKT cells

In each donor following G-CSF treatment, V24⁺V11⁺CD3⁺ NKT cells as a percentage of CD3⁺ cells were substantially reduced with mean NKT cell percentages significantly decreasing from 0.24 ± 0.09% to 0.09 ± 0.03% (p < 0.05), as shown in Fig. 1. Although we used anti-V24/anti-V11/anti-CD3 staining to evaluate CD1d-restricted NKT cells in this study, the reliability of this assay for detection of CD1d-restricted cells before and after periods in culture was validated using a -GalCer-loaded CD1d dimer (-GalCer dimer). As shown in Fig. 2, the frequency of NKT cells determined by -GalCer dimer staining correlated with the proportion of V24⁺V11⁺CD3⁺ NKT cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Proportion of V24+V11+ T cells as a percentage of CD3+ lymphocytes (mean ± SEM; n = 8) in the PB. The percentage of NKT cells is significantly decreased following in vivo treatment with G-CSF (*, p < 0.05).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Representative plot demonstrating identification of NKT cells by CD1d dimer staining as compared with V24+V11+CD3+ staining. PBMC samples were stained with A, unloaded CD1d dimer and anti-V11 mAb; B, -GalCer-loaded dimer and anti-V11 mAb; or C, anti-V24 and anti-V11 mAb. The frequency of NKT cells determined by -GalCer dimer equated to that by V24+V11+CD3+ staining.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Mean (± SEM) of absolute numbers of V24+V11+ NKT cells and CD3+ T cells in the PB of healthy donors before (pre-G-SCF) and following G-CSF treatment (post-G-CSF) (n = 8). CD3+ T cells/L (*, p < 0.05), but not NKT cells/L PB, are modulated by G-CSF treatment.

Effect of G-CSF on -GalCer-induced NKT proliferation

NKT cell-induced proliferation was measured as fold expansion, as determined by the proportions of NKT cells that expanded from mononuclear cell cultures following presentation of -GalCer by CD1d-expressing APC (Mo and B cells in the respective PBMC). Assessment of NKT cell proliferative capacity G-CSF administration to normal donors resulted in a significant reduction in the in vitro proliferative capacity of NKT cells in response to -GalCer stimulation. This was observed in eight of eight donors, as displayed in Fig. 4A, with G-CSF administration resulting in an average reduction in fold expansion of 65.01 ± 4.09% (p < 0.05). Similar to the in vivo G-CSF-induced effect, the addition of G-CSF to the in vitro proliferation assays resulted in a significant reduction in expansion capacity of NKT cells in a dose-dependent manner, with 10 ng/ml G-CSF leading to an average 48.36% reduction (p < 0.05) and 100 ng/ml G-CSF an average 76.39% reduction (p < 0.05) in fold expansion when compared with cultures not containing in vitro added G-CSF (Fig. 4B).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. A, Effect of in vivo administration of G-CSF on the proliferative response (fold expansion) of NKT cells to specific stimulation by -GalCer. Reduced responsiveness to -GalCer stimulation was observed in all donors (n = 8) following G-CSF administration (post-G-CSF). B, Reduction in fold expansion of NKT cells in response to -GalCer stimulation. G-CSF added to in vitro cultures reduced the ability of V24+ NKT cells to expand following -GalCer stimulation in a dose-dependent manner (*, p < 0.05; n = 5).

To determine whether the apparent inhibitory effect of G-CSF on NKT cell proliferation was via direct effects on NKT cells or resulted from G-CSF effects on APC such as Mo, we evaluated the effect of G-CSF on the capacity of Mo to stimulate NKT cell proliferation. The expansion of NKT cells exposed in vivo (post-G-CSF) or not exposed (pre-G-CSF) to G-CSF following in vitro stimulation by Mo exposed (post-G-CSF) or not exposed to G-CSF (pre-G-CSF) is shown in Fig. 5. Greatest expansion was observed when pre-G-CSF Mo were combined with pre-G-CSF CD3⁺ cells, with a fold expansion of 375.6. Substitution of pre-G-CSF Mo with post-G-CSF Mo resulted in a considerable reduction in the fold expansion (235.05), and this was further diminished in the combination of pre-G-CSF Mo and post-G-CSF CD3⁺ cells (40.10). The least expansion was observed in cultures with post-G-CSF Mo and post-G-CSF T cells (24.37). This trend was also observed in cultures using a responder:stimulator ratio of 5:1 (data not shown). These results indicate that in addition to G-CSF indirectly impairing NKT cell proliferation through effects on APC, G-CSF also has direct effects on NKT cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Crossover experiment evaluating the effects of in vivo treatment of G-CSF on the expansion of NKT cells and on the stimulatory ability of Mo. Pre-G-CSF, cells collected before G-CSF administration to normal donors. Post-G-CSF, cells collected from normal donors following 4 days of in vivo G-CSF treatment.

G-CSF alters NKT cell subpopulations following -GalCer stimulation

As NKT cells are a heterogeneous population and G-CSF can clearly impact directly on NKT cells, we evaluated whether the various NKT cell subpopulations were modulated by G-CSF administration. Subpopulation analysis of baseline pre-G-CSF (day 0, before administration of G-CSF to the donor) NKT cells compared with post-G-CSF (after 4 days of G-CSF administration) NKT cells revealed significant differences in responsiveness to stimulation with -GalCer. The results of analysis of subpopulations of pre-G-CSF and post-G-CSF NKT cells before and after 7 days in vitro stimulation with -GalCer are shown in Table I. Administration of G-CSF resulted in significant preferential in vitro expansion of the CD4⁺ subset of post-G-CSF NKT cells following -GalCer stimulation, with the median proportion increasing from 54.12 to 79.24% (p < 0.05).

View this table: [in this window] [in a new window]   Table I. Proportions of NKT cell subpopulations after G-CSF administration and -GalCer stimulationa

Th2 polarization of NKT cells by G-CSF

The effect of G-CSF administration on IFN- and IL-4 production by purified NKT cells was examined, with the results shown in Fig. 6, respectively. NKT cells not exposed to in vivo G-CSF secreted high levels of IFN- and low levels of IL-4, whereas NKT cells following G-CSF exposure secreted significantly lower levels of IFN- and increased levels of IL-4. These results indicate polarization of post-G-CSF NKT cells toward a Th2 cytokine phenotype.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Comparison of IFN- (A) and IL-4 production (B) by purified pre-G-CSF and post-G-CSF NKT cell lines, as measured by ELISA (n = 8). Post-G-CSF NKT cells are biased toward a Th2 phenotype, secreting large amounts of IL-4. Conversely, pre-G-CSF NKT cells have a predominant Th1 profile, secreting large amounts of IFN- and very little IL-4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies have focused on the possible effects on donor T cells and APC, particularly Mo as potential mechanisms for the less than expected acute GVHD following allogeneic PBSC transplants. G-CSF has been shown to inhibit the secretion of Th1 cytokines by T cells, thus polarizing them toward a Th2 cytokine (IL-4 and IL-10) phenotype (9, 12, 13, 15, 59). We (our unpublished results) and others have also demonstrated that G-CSF suppresses responsiveness of conventional T cells, resulting in impairments in the ability of T cells to proliferate, and to differentiate in response to IL-2, mitogens (PHA and Con A), and allo-Ags (14, 16, 18, 20, 59, 60, 61, 62). These effects on T cell function are thought to result via direct actions of G-CSF (12, 13, 15) or through indirect mechanisms involving cell populations capable of suppressing T cell function such as Mo (9, 16, 20). Mo-mediated suppression of T cell activity has been attributed to IL-10 production by the large numbers of Mo present in G-CSF-mobilized PBSC (14, 60, 62), and also decreased expression of the costimulatory molecule CD86 on the Mo (14), as well as impaired induction of the CD28-responsive complex on CD4⁺ T cells in G-CSF-primed PBSC (18). However, other phenotypic and functional differences in G-CSF-primed Mo suggest that G-CSF also indirectly modulates Mo function (20, 24). In addition to the effects on Mo, G-CSF has been shown to lead to the preferential mobilization of lymphoid (DC2) DC (23, 25), with a predominant Th2 cytokine response, and also modulate NK cell number and function (9, 17, 63, 64).

We have demonstrated in this study that in contrast to conventional T cells, absolute PB NKT cell levels are unaffected by G-CSF administration, resulting in altered ratios between these two cell types. Immunological events are substantially controlled by relative numbers of immune subsets, at least partly due to the differences in the cytokine milieu that results. Similar to the G-CSF effects on T cells was suppression in NKT cell responsiveness exemplified by the reduction in proliferative potential to specific stimulation by -GalCer. This may further increase the balance of immune effectors and regulators in favor of conventional T cells. Furthermore, as NKT cells activated by -GalCer have been shown to enhance NK activation and NK cytotoxicity (40, 42, 44, 55, 56, 57), the decreased responsiveness of G-CSF-primed NKT cells is also a plausible mechanism for the decreased NK cell activity observed following G-CSF administration (9, 64). These events may in turn have important influences on GVHD manifestation and GVL activity.

Clearly, the immunological effects of G-CSF are complex and multifactorial, and modulation of GVHD can potentially involve effects on APC, T cells, and NK cells. Our studies indicate that NKT cells also need to be considered, particularly in view of the increasing evidence of their role in coordinating and controlling both T and NK cells immune responses (32, 40, 42, 43, 44, 45, 46, 55, 56, 57) and their effects on DC function (41, 46, 49, 58). We have clearly demonstrated that Mo of G-CSF-treated subjects have a reduced ability to stimulate NKT cell expansion, and that NKT cells from G-CSF-treated donors have a reduced capacity to expand in vitro (Fig. 5). It is therefore evident that G-CSF suppression of NKT cell responsiveness to -GalCer stimulation appears to involve both a direct action on NKT cells and indirect effects through G-CSF-mediated effects on APC, such as Mo. The precise mechanism by which G-CSF alters the expansion potential of NKT cells remains to be determined, but may involve effects on the G-CSFR on NKT cells or other downstream receptors and pathways, considering G-CSFR expression has recently been detected on conventional T cells (65) and at the mRNA level in V24⁺ NKT cells (66). Although there is currently no information available as to the constitutive cell surface expression or expression of G-CSFR on NKT cells following activation with -GalCer or in vivo treatment with G-CSF, the observation that NKT cell proliferation is directly inhibited by G-CSF implies that NKT cells do have functional G-CSFR. It appears, however, that receptor engagement does not lead to recruitment into PB as occurs with myeloid cells.

Various subpopulations of NKT cells have now been identified that are functionally distinct in terms of their cytokine secretion profile. It is now well established that the CD4⁺ NKT cell subset, while producing some IFN-, are the exclusive producers of the Th2 cytokine IL-4 (67, 68). CD4^– NKT cells, in contrast, have a strict Th1 (IFN-) profile (67, 68). The secretion of Th1 and Th2 cytokines by NKT cells is thought to be a mechanism of their immune regulatory properties, particularly in view of their ability to regulate some Th1-mediated autoimmune disease by a selective induction of IL-4 secretion (69, 70). CD4⁺ NK1.1 murine NKT cells suppress GVHD in an IL-4-dependent fashion (53). It is therefore of particular interest that we have demonstrated not only that administration of G-CSF results in preferential expansion of CD4⁺ NKT cells to subsequent specific stimulation (by -GalCer), but also that NKT cell lines generated following G-CSF treatment are Th2 biased with a greater capacity to produce IL-4 and reduced IFN- production than NKT cell lines generated without prior exposure to G-CSF. Our results indicate that despite an unchanged number of NKT cells in donors following G-CSF administration, NKT cells retain the potential to modulate immune responses (through alterations in cytokine production) and have the potential to suppress GVHD through the production of IL-4. It is plausible that these G-CSF-mediated alterations in NKT cells contribute to the lower than expected acute GVHD following allogeneic G-CSF-mobilized PBSC transplants.

In conclusion, our studies demonstrate that G-CSF has different effects on conventional peptide Ag-specific T cells and immunoregulatory V24⁺V11⁺ NKT cells, resulting in altered ratios between T and NKT cells, suppression of NKT cell expansion in response to specific stimulation, modulation of NKT cell subsets, and Th2 polarization of NKT cells. Additional studies are required to further characterize the mechanisms behind these differences and to determine whether alterations in human NKT cells can influence the frequency or severity of GVHD. Our results add weight to the possibility that therapeutic manipulation of NKT cells may allow reduction in GVHD in the allogeneic stem cell transplant setting.

	Acknowledgments

 
We thank Judy Cummings, bone marrow transplant coordinator at the Royal Brisbane and Women’s District Hospital, for her assistance in the collection of the samples for this study.

	Footnotes

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

¹ This work was funded by the Leukemia Foundation of Queensland, Royal Brisbane Hospital Research Foundation, and University of Queensland.

² Address correspondence and reprint requests to Dr. Andrew J. Nicol, H Floor, Clive Berghofer Cancer Research Centre, Queensland Institute of Medical Research, Herston, Queensland, Australia, 4029. E-mail address: anic9909{at}bigpond.net.au

³ Abbreviations used in this paper: PBSC, peripheral blood stem cell; -GalCer, -galactosylceramide; DC, dendritic cell; GVHD, graft-vs-host disease; GVL, graft-vs-leukemia; HSC, hemopoietic stem cell; Mo, monocyte; PB, peripheral blood; PC5, PE-cyanin 5.1.

Received for publication November 20, 2003. Accepted for publication August 5, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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