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* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia;
School of Biosciences, University of Birmingham, Edgbaston, United Kingdom; and
Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
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Abstract |
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-chain cytokine, IL-21, is produced by CD4+ T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag 
-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with -galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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14-J18 chain coupled with V
8.2, V7, or V2, whereas human NKT cells express the homologous TCR genes, V24-J18 in conjunction with V11 (2). This biased TCR usage appears to confer on NKT cells the ability to recognize glycolipids presented in the context of the MHC class I-like molecule, CD1d (3).
The most commonly used glycolipid for the study of NKT cell activation is -galactosylceramide (-GalCer)3 (4). -GalCer is presented by CD1d-expressing APCs and potently activates NKT cells to rapidly produce both Th1 and Th2 cytokines such as IFN- and IL-4 (1). Importantly, activation of NKT cells with -GalCer leads to potent downstream activation of CD8 T cells (5, 6), NK cells (7, 8), and APC such as dendritic cells (DC) (9, 10) and B cells (11). Bystander activation of these cells is crucial to the protective antitumor and microbial immunity mediated by -GalCer (12, 13, 14). The potent immunoregulatory activity of NKT cells in mice and humans has led to -GalCer being used in clinical trials as an antitumor agent (15, 16, 17, 18).
Recently, we demonstrated that the stimulatory effects of - GalCer could be dramatically enhanced when used in combination with the common -chain (c) receptor cytokine IL-21, resulting in enhanced protection in a number of different tumor models (19). IL-21 enhanced the antitumor effects of -GalCer by optimizing NK cell cytotoxicity through increased IFN- and perforin production, although the direct impact of IL-21 on NKT cells was not investigated (19). IL-21 shares sequence homology with IL-2 and IL-15, and acts through a unique IL-21R, coupled to the c receptor (20, 21). IL-21R is expressed by multiple lymphoid cell types, including NKT cells (22). Since its discovery, the influence of IL-21 on a number of cell types has been investigated, including B, T, NK cells, and DC, but not NKT cells. The findings indicate that IL-21 may play a very significant role in immunity by triggering T and B cell proliferation (22, 23, 24), B cell Ig class switching to IgG production (25), dampening DC function (26, 27), and enhancing the differentiation of effector and central memory T cells (28, 29). IL-21 also exhibits potent antitumor function by virtue of enhancing NK and CD8 T cell cytotoxicity and IFN- production (30, 31, 32, 33), and this has led to its use in the first phase of clinical trials in advanced cancer patients (34). Increased IL-21 responsiveness has also been associated with impaired T cell homeostasis and type 1 diabetes in NOD mice (35), and an absence of IL-21 may contribute to the B cell phenotype observed in X-linked SCID (25). Although -GalCer is the most common reagent used for NKT cell stimulation, NKT cells are also susceptible to activation and regulation by cytokines, including c receptor cytokines such as IL-2, IL-7, and IL-15 (36, 37, 38, 39, 40), as well as DC-derived cytokines such as IL-12 and IL-18 (41, 42, 43, 44, 45). These cytokines have been found to enhance NKT cell cytokine production and some are also known to stimulate NKT cell cytotoxicity against tumors in vitro and in vivo (43, 46).
Given that NKT cells constitutively express the IL-21R, we hypothesized that IL-21 would influence NKT cell function. Furthermore, although the main source of IL-21 is considered to be activated conventional CD4+ T cells (24), it is known that NKT cells are one of the most potent subsets of CD4+ T cells, responsible for production of many different cytokines. The contribution of NKT cells to CD4+ T cell-derived IL-21 production has not been tested. In this study, we examine the influence of IL-21 on NKT cell function, and also test NKT cells for their ability to produce this cytokine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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C57BL/6 wild-type mice were bred in-house at the Department of Microbiology and Immunology Animal House, Melbourne University or the Peter MacCallum Cancer Centre Animal Facility (Melbourne, Victoria, Australia). IL-12-deficient mice were bred at the Peter MacCallum Cancer Centre Animal Facility. Mice age 7–14 wk were used in all experiments. All experiments were conducted with the approval of appropriate institutional animal ethics committees.
Lymphocyte isolation
Lymphocytes were isolated from the liver by first perfusing the liver with 10 ml of PBS via the hepatic portal vein, and then gently passing it through 200-µm wire mesh sieves into PBS containing 2% FCS (JRH Biosciences). Lymphocytes were separated from hepatocytes and cellular debris by 33% isotonic Percoll density gradient (GE Healthcare) at room temperature. Lymphocytes were harvested from spleen and thymus by gently grinding them between two frosted glass slides into PBS containing 2% FCS. Splenocytes were depleted of RBC by RBC lysis buffer (Sigma-Aldrich).
NKT cell enrichment
For thymus-derived NKT cell enrichment, thymocytes were labeled with anti-CD8 (clone 3.155) and anti-CD24 (J11D), and tagged cells were depleted using rabbit complement (C-SIX Diagnostics) in the presence of DNase (Roche Diagnostics). Viable cells were collected using a Histopaque gradient (1.083 g/ml; Sigma-Aldrich) at room temperature. Cells were then washed before being cultured or before being labeled for flow cytometric purification. NKT cells typically represented >25% of enriched thymocytes.
CFSE labeling
NKT cell-enriched thymocytes or fresh ex vivo splenocytes were washed once in 0.1% BSA in PBS before being labeled in 1 ml of 0.1% BSA in PBS with 5 µl of 1 mM CFSE (Molecular Probes) for 10 min at 37°C in the dark. The reaction was stopped with PBS containing 20% FCS before cells were washed twice in complete tissue culture medium (see below on cell culture conditions).
List of Abs and flow cytometry
All Abs used are from BD Pharmingen unless otherwise specified. Abs used include TCR-allophycocyanin (clone H57-597), NK1.1-PE/CY7 (PK-136), CD4-FITC (RM4-5), Ly49D-FITC (4E5), Ly49F-PE (HBF-719), NKG2A/C/E-FITC (20d5), NKG2D-PE (CX5), Ly49C/I-FITC (5E6), Ly49G2-allophycocyanin (LGL-1), CD94-FITC (MP-3D9), KLRG1-biotin (2F1), and mouse anti-human granzyme B-allophycocyanin (GB11; Caltag Laboratories). 7-Aminoactinomycin D (7-AAD) (Bioscientific) was added to stained samples where appropriate at a final concentration of 1 µg/ml before cells were analyzed by flow cytometry. Mouse CD1d tetramer loaded with -GalCer was produced in-house, as previously described (47), using recombinant baculovirus encoding his-tagged mouse CD1d and mouse 2-microglobulin, originally provided by Prof. M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA). Flow cytometry was performed using a FACSCalibur or LSR-II (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).
In vivo mouse stimulation
Mice were administered with 2 µg of -GalCer (provided by Kirin Brewery) prepared in 200 µl of PBS and i.p. injected as previously described (48).
Cell culture conditions
Cells were cultured in medium containing RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamax, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 15 mM HEPES buffer (Invitrogen Life Technologies), 10% FCS, and 50 µM 2-ME (Sigma-Aldrich), either in 96-well or 48-well plates (Nunc). For in vitro stimulation assays, no azide low endotoxin anti-CD3 (145-2C11; BD Pharmingen) and anti-CD28 (37.51; BD Pharmingen) were used at a concentration of 10 µg/ml to coat plates. -GalCer was used at a concentration of 100 ng/ml. Recombinant murine IL-2 (PeproTech) was used at 50 IU/ml, IL-15 (Bioscientific) at 50 ng/ml, and IL-21 (a gift from Zymogenetics) at between 1 and 300 ng/ml and usually at 100 ng/ml.
Flow cytometric purification
Following staining with -GalCer/CD1d tetramer and TCR, NKT cell populations from enriched thymus, liver, or spleen preparations were sorted using either a FACSAria (BD Biosciences) or MoFlo (DakoCytomation). A sample of sorted cells was always analyzed to assess the purity of these populations. Purified NKT cell populations were always >95% pure.
Quantitative RT-PCR
Following electronic sorting of NKT cells from livers and spleens of mice, RNA was isolated using the RNeasy kit (Qiagen), and cDNA was synthesized by using a Sensiscript kit (Qiagen) according to the manufacturer’s instructions with random hexamers (Invitrogen Life Technologies). Primers for quantitative analysis of IL-21 were designed using Primer Express software (Applied Biosystems). Quantitative RT-PCR was performed using SYBR green (Applied Biosystems) with 18 S rRNA as the normalizer. The PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min, and 95°C for 20 s, ending with a melting curve to validate product specificity. Samples were run on an Applied Biosystems ABI7700 Real-Time PCR machine. Results were analyzed using Sequence Detector software.
ELISA analysis
For IL-4 and IFN- ELISA, capture anti-IL-4 (11B11) and anti-IFN- (AN18) were grown in-house, and biotinylated detection Ab for IFN- (XMG1.2) and IL-4 (BVD6-24G2) were purchased from BD Pharmingen. IL-21 ELISA and IL-13 ELISA were performed using the DuoSet ELISA kits from R&D Systems. The Ab reagents (polyclonal) were titrated and used as follows: IL-21 kit (catalog no. DY594) capture Ab used to coat plate at 0.8 µg/ml in PBS; biotinylated detection Ab used at 0.8 µg/ml; IL-13 kit (catalog no. DY413) capture Ab used to coat plate at 8 µg/ml; and biotinylated detection Ab used at 0.2 µg/ml. Bound detection Ab was detected using streptavidin HRP with tetramethylbenzidine (Sigma-Aldrich) as the substrate and the plates were read using a LabSystems Multiskan multisoft machine.
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Results |
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IL-21 is known to have a broad range of effects on different cell types. For T and B cells, IL-21 increases their proliferative potential in vitro following stimulation via CD3 or CD40, respectively (22, 23, 24). However, cultures of NK cells expanded in IL-15 indicate that although addition of IL-21 promotes progression to a more mature NK cell phenotype, it decreases NK cell survival and proliferation (49, 50). To investigate a potential role for IL-21 in NKT cell activity, NKT cell-enriched thymocytes were first cultured over a period of 5 days in the presence of varying doses of IL-21 (1–300 ng/ml), and the viability of NKT cells in culture was monitored by flow cytometry using 7-AAD uptake to indicate dead cells (Fig. 1A). IL-21 enhanced NKT cell survival in culture over 5 days in a dose-dependent manner, with 100 ng/ml representing the maximal effective dose for NKT cell survival (Fig. 1A). However, in comparison to other c cytokines such as IL-2 and IL-15, IL-21 appeared to be less effective at promoting NKT cell survival (Fig. 1B). To determine whether IL-21 may affect the proliferation of NKT cells, NKT cell-enriched thymocytes were labeled with CFSE before culture. The presence of IL-21 alone in culture did not lead to any significant proliferation of NKT cells, in stark contrast to the effects of IL-2 or IL-15 alone (Fig. 1C). However, IL-21 enhanced NKT cell proliferation induced by IL-2 or IL-15 as illustrated by a reduction in CFSE labeling intensity at this time point (Fig. 1C). To test whether IL-21 could affect NKT cell proliferation and expansion following antigenic stimulation, IL-21 was added to CFSE-labeled whole splenocyte cultures in the presence of -GalCer. The presence of IL-21 led to increased proliferation of NKT cells, reflected by a dilution in the CFSE intensity and by the dramatically increased proportion and number of NKT cells at the end of the culture period (Fig. 1D). Similar results were observed for liver lymphocyte cultures (data not shown).
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IL-21 has been shown to increase NK cell granularity in culture (50). To test whether IL-21 also influenced NKT cell granularity, NKT cells cultured in the presence of IL-2, IL-15, or either of these cytokines in combination with IL-21 were analyzed by flow cytometry after 5–7 days of culture. NKT cells, but not conventional T cells, cultured in the presence of IL-21 were clearly more granular than NKT cells cultured in the absence of IL-21, as measured by the side scatter property of these cells (Fig. 2A). Furthermore, this increased granularity correlated with an increase in intracellular granzyme B protein (Fig. 2B). NK cells cultured in the presence of IL-21 also exhibit altered NK receptor expression, including decreased NK1.1, and increased CD94, CD154, and KLRG-1 (50). We tested whether IL-21 would have similar effects on NK receptor expression by NKT cells. After 5–7 days of culture in the presence of IL-2 and IL-21, there was a considerable increase in the proportion of NKT cells that were CD94+, NKG2A/C/E+, as well as an increase in Ly49C/I intensity but not percentage, and a decrease in the NK1.1 expression when compared with NKT cells cultured in IL-2 alone (Fig. 2C). Importantly, NKT cells cultured in IL-2 alone have a very similar NK receptor profile to fresh ex vivo NKT cells (data not shown and (51)). In contrast, when NKT cells were cultured in the presence of IL-15 and IL-21 together, NKG2A/C/E and Ly49C/I expression was no different from NKT cells cultured in IL-15 alone, although NK1.1 expression was decreased. The presence of IL-21 alone in culture did not appear to impact on NK receptor expression of NKT cells (data not shown). Although most of the experiments, including those depicted, were performed on enriched thymus-derived NKT cells that were then electronically gated using -GalCer/CD1d tetramer, very similar effects were observed when purified NKT cells were used, in terms of survival, proliferation, granularity, and NK receptor expression (data not shown).
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IL-21 is known to increase the production of both Th1 and Th2 cytokines from NK cells and IFN- from CD8 T cells (49, 50). To investigate the influence of IL-21 on NKT cell cytokine production, NKT cells from the thymus and liver were purified and separately cultured in anti-CD3/CD28-coated plates for 24 h in the presence or absence of IL-21. Most strikingly, IL-21 enhanced the production of IL-13 by both liver- and thymus-derived NKT cells (Fig. 3). IL-4 production by NKT cells was also generally increased in cultures with IL-21. However, the impact of IL-21 on NKT cell IFN- production was less clear with liver-derived NKT cells appearing to produce more IFN- in the presence of IL-21 in most experiments, but thymic NKT cells showing variable responsiveness. These effects were not observed when IL-2 was used instead of IL-21 (data not shown). Thus, IL-21 enhances NKT cell cytokine production and appears to bias thymic, but not liver NKT cell cytokine production toward a Th2 profile.
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CD4+ T cells produce IL-21 mRNA following PMA/ionomycin stimulation or CD3 and CD28 ligation (24). To determine whether NKT cells (many of which are CD4+) also produce IL-21, purified NKT cells from liver, thymus, and spleen of C57BL/6 mice were stimulated in vitro in anti-CD3/CD28-coated plates for up to 3 days, and culture supernatants were tested by ELISA. Spleen- and thymus-derived NKT cells were both capable of making IL-21 protein under these conditions and this production was delayed, detectable only on days 2 and 3 of culture (Fig. 4A). Recent findings that IL-12 could stimulate IL-21 mRNA production from T cells (52) prompted us to test whether this cytokine also boosted NKT cell IL-21 production. The addition of IL-12 to cultures of liver-derived NKT cells resulted in a dramatic increase in IL-21 production. IL-21 production by spleen- and thymus-derived NKT cells was also increased by IL-12, although to a lesser extent. Conventional CD4+ T cells purified from the spleen also produced IL-21 over the 3 days of culture, although at much lower levels than NKT cells. Importantly, the increased IL-21 production in the presence of IL-12 did not appear to be a result of increased cell survival or expansion in the presence of this cytokine over the 3 days of culture (data not shown).
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-GalCer, and 2 or 48 h later NKT cells from the livers and spleens were purified and analyzed for IL-21 mRNA production by quantitative RT-PCR. NKT cells from the liver showed IL-21 mRNA production at both time points, whereas spleen NKT cells showed detectable IL-21 mRNA primarily at the later time point (Fig. 4B). Furthermore, the results suggest that the presence of endogenous IL-12 was necessary for optimal IL-21 mRNA production in vivo. IL-21 mRNA was not detected in NKT cells derived from unstimulated mice under the assay conditions used for these experiments. Although the presence of IL-21 mRNA after short-term in vivo stimulation seems inconsistent with the in vitro results in which IL-21 was not detected until 2–3 days, in vivo stimulation with -GalCer is a very potent stimulus that also drives potent production of other cytokines such as IFN- and IL-4 within 1–2 h of stimulation at levels that we never see with in vitro CD3/CD28 stimulation based upon intracellular cytokine staining (our unpublished data). CD4+ as well as CD4– NKT cells produce IL-21 mRNA
Previous studies have shown that CD4+ T cells but not CD8+ T cells are the primary producers of IL-21 (24), which suggests that IL-21 production is limited to CD4+ T cells. Thus, we tested whether IL-21 production within the NKT cell population was also limited to the CD4+ subset. CD4+ and CD4– NKT cells from the spleens of B6 mice stimulated with -GalCer for 48 h were purified separately by flow cytometry and analyzed for IL-21 mRNA production. Both CD4+ and CD4– NKT cells were capable of IL-21 mRNA production, although CD4+ NKT cells showed slightly higher IL-21 mRNA levels upon stimulation (Fig. 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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This study is one of the first to measure IL-21 protein production by T cells, which represents a significant improvement in the field because the majority of studies thus far have been limited to quantifying IL-21 mRNA, including a very recent finding demonstrating IL-21 mRNA expression in a microarray comparison of immature and mature NKT cells in the thymus (53). We have also shown that IL-21 production is distinct between NKT cells from different organs. Intriguingly, IL-21 production by liver-derived NKT cells seemed to be dependent on the presence of IL-12, whereas thymus- and spleen-derived NKT cells were able to produce IL-21 in the absence of IL-12.
The importance of IL-21 production in NKT cell-dependent disease models remains to be tested. The ability of NKT cells to mediate potent antitumor responses is quite well characterized and relies on NKT cell IFN- production, as well as NK and CD8 T cell function (6, 12, 13, 48), although the precise mechanism by which these two cell types are induced remains unclear. The known ability of IL-21 to promote antitumor responses in a number of models by enhancing NK and CD8 T cell function (29, 30, 54, 55) suggests that NKT cells may regulate the response of these downstream effector cells at least in part by virtue of their IL-21 production.
Paradoxically, in addition to promoting tumor rejection, NKT cells are also able to mediate the suppression of autoimmune disease, allograft rejection and promotion of allergy via their production of IL-4 and IL-13 (1). In this study, we showed that IL-21 could enhance the production of these Th2 cytokines by NKT cells, suggesting a potential role for IL-21 and NKT cells in the regulation of these diseases. Supporting this theory are recent findings that NKT cells are pivotal in eliciting a potent Th2 response following helminth infection via their production of IL-13 and IL-4 (56), and also that IL-21 is crucial for optimal IL-13 and IL-4 production and eventual granuloma formation following infection with this parasite (57). Taken together, these studies are consistent with a role for NKT cell-derived IL-21 in the regulation of Th2 immune responses. Conversely, studies investigating the role of IL-21 in the NOD mouse model (35) and experimental allergic encephalomyelitis (58) suggest that IL-21 exacerbates disease via the enhanced proliferation of autoreactive CD8 T cells and enhanced NK cell function. This probably reflects the influence of the cellular microenvironment in overall disease outcome and suggests that the function of NKT cell-derived IL-21 will likely depend upon the activation of downstream cells and other cytokines present.
Although the functional importance of NKT cell-derived IL-21 remains unclear, we may hypothesize that NKT cells play a role in bridging the innate and adaptive immune responses via their production of this cytokine. This role makes sense when considering findings showing that although IL-21 can enhance NK cell function, it also increases the death of NK cells (49, 50), increases inhibitory NK receptor expression on both NK (50) and NKT cells (our study), and down-regulates costimulatory molecule expression on DC (26, 27). Conversely, because IL-21 enhances Ag-specific Ab production (25, 59), CTL responses (49, 60), and the generation of central and effector memory T cells (28, 29), the delayed production of IL-21, which we observed in vitro and also in vivo for spleen-derived NKT cells, may be timed to optimally enhance these responses. Thus, NKT cell derived IL-21 may be an important factor underlying the well-established ability of NKT cells to enhance Ag-specific Ab production and CTL responses (1, 3, 61).
In summary, the results from this study provide the first evidence of IL-21 production by thymic and peripheral NKT cells, and furthermore, that this cytokine can potently regulate NKT cell function. We hypothesize that IL-21 may play a role in a number of NKT cell-mediated immune responses by affecting the activation of downstream effector cells and also the activation of NKT cells themselves. It will therefore be important to investigate NKT cell-mediated immune responses in IL-21- and IL-21R-deficient mice to determine whether this cytokine is a critical regulator of NKT cell function in vivo.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by research grants from National Health and Medical Research Council of Australia, the National Institutes of Health, and The Association of International Cancer Research. J.M.C. is a recipient of a Cancer Research Institute Postgraduate Scholarship. D.I.G. and M.J.S. are recipients of National Health and Medical Research Council research fellowships. 
2 Address correspondence and reprint requests to Dr. Dale I. Godfrey, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail address: godfrey{at}unimelb.edu.au
3 Abbreviations used in this paper: -GalCer, -galactosylceramide; 7-AAD, 7-aminoactinomycin D; DC, dendritic cell; c, common -chain.
Received for publication September 20, 2006. Accepted for publication December 18, 2006.
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 I. D. Davis, B. Brady, R. F. Kefford, M. Millward, J. Cebon, B. K. Skrumsager, U. Mouritzen, L. T. Hansen, K. Skak, D. Lundsgaard, et al. Clinical and Biological Efficacy of Recombinant Human Interleukin-21 in Patients with Stage IV Malignant Melanoma without Prior Treatment: A Phase IIa Trial Clin. Cancer Res., March 15, 2009; 15(6): 2123 - 2129. [Abstract] [Full Text] [PDF]
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 C. S. Fernandez, A. C. Chan, K. Kyparissoudis, R. De Rose, D. I. Godfrey, and S. J. Kent Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques J. Virol., February 15, 2009; 83(4): 1617 - 1624. [Abstract] [Full Text] [PDF]
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D. Konforte, N. Simard, and C. J. Paige IL-21: An Executor of B Cell Fate J. Immunol., February 15, 2009; 182(4): 1781 - 1787. [Abstract] [Full Text] [PDF]
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 O. Dienz, S. M. Eaton, J. P. Bond, W. Neveu, D. Moquin, R. Noubade, E. M. Briso, C. Charland, W. J. Leonard, G. Ciliberto, et al. The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4+ T cells J. Exp. Med., January 16, 2009; 206(1): 69 - 78. [Abstract] [Full Text] [PDF]
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H. Yokote, S. Miyake, J. L. Croxford, S. Oki, H. Mizusawa, and T. Yamamura NKT Cell-Dependent Amelioration of a Mouse Model of Multiple Sclerosis by Altering Gut Flora Am. J. Pathol., December 1, 2008; 173(6): 1714 - 1723. [Abstract] [Full Text] [PDF]
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 R Ettinger, S Kuchen, and P E Lipsky Interleukin 21 as a target of intervention in autoimmune disease Ann Rheum Dis, December 1, 2008; 67(Suppl_3): iii83 - iii86. [Abstract] [Full Text] [PDF]
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E. Menoret, S. Maiga, G. Descamps, C. Pellat-Deceunynck, C. Fraslon, M. Cappellano, P. Moreau, R. Bataille, and M. Amiot IL-21 Stimulates Human Myeloma Cell Growth through an Autocrine IGF-1 Loop J. Immunol., November 15, 2008; 181(10): 6837 - 6842. [Abstract] [Full Text] [PDF]
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 D. T. Avery, C. S. Ma, V. L. Bryant, B. Santner-Nanan, R. Nanan, M. Wong, D. A. Fulcher, M. C. Cook, and S. G. Tangye STAT3 is required for IL-21-induced secretion of IgE from human naive B cells Blood, September 1, 2008; 112(5): 1784 - 1793. [Abstract] [Full Text] [PDF]
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J. M. Coquet, S. Chakravarti, K. Kyparissoudis, F. W. McNab, L. A. Pitt, B. S. McKenzie, S. P. Berzins, M. J. Smyth, and D. I. Godfrey Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population PNAS, August 12, 2008; 105(32): 11287 - 11292. [Abstract] [Full Text] [PDF]
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J. Gagnon, S. Ramanathan, C. Leblanc, A. Cloutier, P. P. McDonald, and S. Ilangumaran IL-6, in Synergy with IL-7 or IL-15, Stimulates TCR-Independent Proliferation and Functional Differentiation of CD8+ T Lymphocytes J. Immunol., June 15, 2008; 180(12): 7958 - 7968. [Abstract] [Full Text] [PDF]
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M. Terabe, Y. Tagaya, Q. Zhu, L. Granger, M. Roederer, T. A. Waldmann, and J. A. Berzofsky IL-15 Expands Unconventional CD8{alpha}{alpha}NK1.1+ T Cells but Not V{alpha}14J{alpha}18+ NKT Cells J. Immunol., June 1, 2008; 180(11): 7276 - 7286. [Abstract] [Full Text] [PDF]
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 M. J. Smyth, M. W.L. Teng, J. Sharkey, J. A. Westwood, N. M. Haynes, H. Yagita, K. Takeda, P. V. Sivakumar, and M. H. Kershaw Interleukin 21 Enhances Antibody-Mediated Tumor Rejection Cancer Res., April 15, 2008; 68(8): 3019 - 3025. [Abstract] [Full Text] [PDF]
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I. D. Davis, K. Skak, M. J. Smyth, P. E.G. Kristjansen, D. M. Miller, and P. V. Sivakumar Interleukin-21 Signaling: Functions in Cancer and Autoimmunity Clin. Cancer Res., December 1, 2007; 13(23): 6926 - 6932. [Abstract] [Full Text] [PDF]
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V. Ostiguy, E.-L. Allard, M. Marquis, J. Leignadier, and N. Labrecque IL-21 promotes T lymphocyte survival by activating the phosphatidylinositol-3 kinase signaling cascade J. Leukoc. Biol., September 1, 2007; 82(3): 645 - 656. [Abstract] [Full Text] [PDF]
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) represents the mean result from replicate wells (two or more) within one experiment, except for one liver experiment in which NKT cell purification yields were low and only one well was set up for each condition. The lines between symbols link samples from individual experiments.