1 RIKEN Research Center for Allergy and Immunology, Graduate School of Medicine, Chiba University, Chiba City, Chiba 260-8670, Japan 2 PRESTO, JST, Kawaguchi City, Saitama 332-0012, Japan 3 Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba City, Chiba 260-8670, Japan
Correspondence to: M. Taniguchi, RIKEN Research Center for Allergy and Immunology, Department of Molecular Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: mtaniguchi{at}faculty.chiba-u.jp
Transmitting editor: T. Watanabe
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Abstract |
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Keywords: -galactosylceramide, apoptosis, CD1d tetramer, cDNA array, cytokine production
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Introduction |
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V14 NKT cells, another type of immunoregulatory cell, possess solely an invariant V
14 antigen receptor, encoded by V
14 and J
281 gene segments, that is reactive to a glycolipid antigen,
-galactosylceramide (
-GalCer), presented by CD1d (710). V
14 NKT cells play crucial immunoregulatory roles in a variety of immune responses, including protection from autoimmune disease development, maintenance of transplantation tolerance and immunological surveillance for tumors [reviewed in (11)]. However, little is known about the molecular events operating in activated V
14 NKT cells and about the destiny of V
14 NKT cells upon stimulation. Understanding the comportment of V
14 NKT cells after activation will help to elucidate the mechanisms through which V
14 NKT cells exert regulatory functions in the immune system.
In this report we show that V14 NKT cell activation results in the rapid down-regulation of the V
14 receptor. Subsequently, V
14 NKT cells become undetectable by flow cytometry with
-GalCer/CD1d tetramer staining. Activated V
14 NKT cells remain quiescent for a while, but eventually proliferate and continue to produce cytokines. Furthermore, these cells show a resistance to apoptosis induced by TCR stimulation relative to conventional T cells. These data indicate that V
14 NKT cells neither become anergic nor are eradicated by apoptosis upon activation.
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Methods |
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Reagents and antibodies
-GalCer as previously described (10) was kindly provided by Kirin Brewery (Takasaki, Japan). The following mAb were purchased from BD PharMingen (San Diego, CA): anti-CD16/32 (2.4G2) and anti-CD3 (2C11), FITC-conjugated anti-TCRß (H57-597) and anti-NK1.1 (PK136), and biotin-conjugated anti-TCRß (H57-597), Ly-5.1 (A20) and Ly-5.2 (104). Anti-FITC microbeads were purchased from Miltenyi Biotec (Auburn, CA). Phycoerythrin-labeled
-GalCer/CD1d tetramer was prepared in our laboratory using a baculovirus expression system kindly provided by Dr M. Kronenberg (La Jolla Institute, La Jolla, CA). Cells were cultured in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 55 µM 2-mercaptoethanol (Invitrogen, Carlsbad, CA).
Preparation of dendritic cells (DC) and liver mononuclear cells
Mouse splenic DC were prepared as described previously (10). Total liver cells were suspended in a 33% Percoll solution (Pharmacia, Uppsala, Sweden) containing heparin (100 U/ml) and centrifuged at 1000 g for 15 min at room temperature. Pellets were used as liver mononuclear cells for subsequent studies after lysing red blood cells.
Stimulation with -GalCer or
-GalCer-DC
For in vivo stimulation, 100 µg/kg of -GalCer was i.p. injected into mice. For in vitro stimulation, DC were pulsed with 100 ng/ml of
-GalCer or vehicle alone for 24 h. After extensive washing, pulsed DC (10 x 103 cells/well) were added to stimulate spleen cells (0.2 x 106 cells/well) cultured in 96-well plates.
Flow cytometric analysis and cell sorting
Single-cell suspensions were prepared in PBS supplemented with 2% FCS and 0.05% sodium azide, pre-incubated with anti-CD16/CD32 mAb to prevent non-specific binding via Fc receptor interactions, and incubated with the appropriate mAb on ice for 30 min. Flow cytometric analysis and cell sorting were performed with Coulter Epics XL or Epics Elite cell sorters (Beckman Coulter, Palo Alto CA). Purity of sorted cells was estimated to be >98%.
Cell labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE)
Electronically sorted V14 NKT cells from Ly-5.2 mice liver (10 x 106) were incubated with 1 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 8 min at room temperature. Labeling was stopped by the addition of FCS at equal volume. Labeled cells were washed extensively with medium prior to addition of Ly-5.1+ spleen cells pulsed with
-GalCer or vehicle.
Cytokine concentration measurement by ELISA
Two hundred thousand fresh spleen cells, or spleen cells incubated with -GalCer-DC for the indicated time periods, were co-cultured for an additional 72 h with
-GalCer-DC or vehicle-DC. At the end of the incubation period, culture supernatants were collected and the concentration of IFN-
and IL-4 was measured using an ELISA kits (BD PharMingen).
Quantification of genomic DNA by PCR
Genomic DNA was extracted from cells using the QiaAmp DNA blood kit (Qiagen, Hilden, Germany). The amount of amplicons generated during PCR was monitored using the iCycler iQ Detection System (Bio-Rad, Hercules, CA). This method is based on the 5' to 3' nuclease activity of Taq polymerase, which allows the release of a fluorescent reporter during the PCR. The sequences of the primers and Taqman probes used in this study were as follows: V14: 5'-TGG GAGATACTCAGCAACTCTGG-3'; J
281: 5'-CAGGTATGAC AATCAGCTGAGTCC-3';TCR C
exon I forward: 5'-CAGAA CCCAGAACCTGCTGT-3'; TCR C
exon I reverse: 5'-TAGG TGGCGTTGGTCTCTTT-3'; V
14 probe FAM: 5'-FAM-CACC CTGCTGGATGACACTGCCAC-TAMRA-3'; and TCR C
exon I-probe FAM: 5'-FAM-CTCCCAAATCAATGTGCCGAAAAC CA-TAMRA-3'.
Apoptosis detection assays
Electronically purified T (TCRß+NK1.1) and -GalCer/CD1d tetramer-reactive V
14 NKT cells (TCRß+NK1.1+) from liver mononuclear cells were stimulated with plate-bound anti-CD3 mAb (10 µg/ml) for the indicated periods of time. Cells were subsequently stained with FITC-labeled Annexin-V (BD PharMingen) and analyzed with a flow cytometer. DNA fragmentation during apoptosis was monitored using the Cell Death Detection ELISA Plus kit (Roche, Mannheim, Germany).
DNA microarray analysis
Total RNA was prepared from electronically sorted hepatic T and -GalCer/CD1d tetramer-reactive V
14 NKT cells stimulated with plate-bound anti-CD3 mAb (10 µg/ml) for 2 h. cDNA was synthesized according to the manufacturers instructions and labeled antisense RNA was prepared by in vitro transcription using T7 RNA polymerase (MessageAmp aRNA kit; Ambion, Austin, TX). Mouse Apoptosis Expression Arrays (R & D Systems, Minneapolis, MN) were used to interrogate the expression of murine apoptosis-related genes. Radioactive signals were detected with FLA 8000 and the quantitative analysis was performed with Image-Gage software (Fuji Film, Tokyo, Japan).
RT-PCR
cDNA was synthesized from RNA used in DNA microarray analysis with oligo-dT primer. The following primer sets were used for the RT-PCR: NAIP, 5'-TCATGACTGTGCTTGCTTCC-3' and 5'-CCAGTGGGAACGAACAGTTT-3'; MyD118, 5'-CTC CTGGTCACGAACTGTCA-3' and 5'-GGGTAGGGTAGCCTTT GAGG-3'; IL-1ß, 5'-GCCCATCCTCTGTGACTCAT-3' and 5'-AGGCCACAGGTATTTTGTCG-3'; HPRT, 5'-AGCGTCGTGA TTAGCGATG-3' and 5'-CTTTTATGTCCCCCGTTGAC-3'. The number of PCR cycles to detect the above transcripts was as follows: 28 cycles for HPRT, 35 cycles for NAIP, and 37 cycles for MyD118 and IL-1ß. PCR products were visualized by ethidium bromide staining and subjected to DNA sequencing to verify authenticity.
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Results |
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To examine this hypothesis, we carried out a kinetic PCR analysis to rigorously measure the quantity of rearranged genomic V14J
281 upon receptor activation (Fig. 3A). When the relative numbers of V
14J
281 genomic copies were compared after an appropriate compensations had been made using C
, there was no change up to day 2, whereas the curve for the day 4 culture shifted to the left (equivalent to 3.5 PCR cycles). These results showed that there was no significant decrease in the number of V
14J
281 genomic copies during the culture periods from day 0 to 2, implying that most of the V
14 NKT cells were alive without significant cell death (Fig. 3A, left panel). The net increase in V
14 NKT cell number upon stimulation was estimated to be in the range of 11-fold over 4 days (Fig. 3A, right panel).
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It is, however, of importance to note that the Ly-5.1+ V14 NKT cells stayed alive even after
-GalCer-DC stimulation (Fig. 3B, day 04). As observed previously,
-GalCer/CD1d tetramer-reactive V
14 NKT cells reappeared on day 3 (Fig. 3B, day 3) and the number of Ly-5.1+ V
14 NKT cells subsequently expanded to account for >18% of total cultured cells (Fig. 3B, day 4). These results clearly demonstrated that Ly-5.1+ V
14 NKT cells down-regulate their invariant V
14 receptor upon
-GalCer stimulation, stay quiescent and eventually proliferate.
We also examined whether the observed cellular expansion accompanied cell division by labeling cells with CFSE. Electronically sorted liver NK1.1+TCRß+ cells from Ly-5.2 mice were labeled with CFSE and mixed with unfractionated Ly-5.1+ spleen cells. These mixtures were stimulated with -GalCer or vehicle. It was not until 57 h after stimulation that cell division could be detected only in cells activated with
-GalCer (Fig. 3C, upper panel and data not shown). After that period,
-GalCer-stimulated cells continued to divide. In marked contrast, vehicle-stimulated V
14 NKT cells showed little, if any, cell division (Fig. 3C, lower panel). These results indicated that
-GalCer promotes V
14 NKT cell proliferation and further underpinned the above hypothesis.
V14 NKT cells are relatively resistant to apoptosis induced by receptor activation
The above data, however, did not exclude the possibility that a limited number of V14 NKT cells undergo apoptosis upon activation. We thus examined, by Annexin-V staining, whether V
14 NKT cells underwent apoptosis upon stimulation. A comparison of Annexin-V staining upon anti-CD3 mAb stimulation showed less staining for V
14 NKT cells relative to conventional T cells (TCRß+NK1.1) at all time points examined (Fig. 4A, left panel), suggesting that, albeit weakly, V
14 NKT cells underwent apoptosis upon receptor-mediated activation. The number of cells that underwent apoptosis was further evaluated by measuring the amount of released DNA fragments by ELISA. As in the case of Annexin-V staining, fragmented DNA was observed in some fractions of V
14 NKT cells, but the degree of fragmentation was less than that in conventional T cells (Fig. 4A, right panel).
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These differences in expression of apoptotic genes may reflect the intrinsic nature of each cell type. We also compared the expression of apoptosis-related genes in V14 NKT cells and conventional T cells prior to receptor activation. It was found that the expression profile of these cells prior to receptor activation mirrored that of post-receptor stimulation (cf. Fig. 4B, lanes 3 and 4, and C, lanes 1 and 2). Further kinetic studies on the expression of the indicated genes in V
14 NKT cells in vivo revealed that, upon receptor activation, NAIP expression increased up to day 3, while expression thereafter gradually declined. MyD118 showed a similar expression pattern over time as NAIP, while the level of IL1-ß expression remained constant (Fig. 4C).
V14 NKT cell resistance to tolerance induction after receptor down-regulation
Since the activation of T cells often results in the loss of their function and becomes tolerant (2), the ability of V14 NKT cells to secrete IFN-
and IL-4 upon re-stimulation with
-GalCer-DC or vehicle-DC was examined at the indicated time points following the initial
-GalCer-DC stimulation. At any given time point, even after the down-regulation of the V
14 receptor, activated V
14 NKT cells produced both IFN-
and IL-4 upon re-stimulation with
-GalCer-DC (Fig. 5). In contrast, neither IFN-
nor IL-4 production could be observed following vehicle-DC re-stimulation (Fig. 5). These results indicate that the activation of V
14 NKT cells does not affect cytokine production in these cells after re-stimulation.
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Discussion |
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Our present data are somewhat contradictory to the previous reports claiming that NKT cells, defined as NK1.1+TCRßdim cells, are extremely sensitive to IL-12- and TCR-induced apoptosis (13). This is most evident in our in vitro experiments where there is no provision of exogenous V14 NKT cells or V
14 NKT cell progenitors (Fig. 2B). Under such conditions, the rapid disappearance and subsequent expansion of V
14 NKT cells upon
-GalCer challenge cannot be attributed to the immediate eradication of the cells by apoptosis. Rather, the seeming disappearance of V
14 NKT cells is better explained by the down-regulation of the V
14 receptor. This interpretation is further supported by transfer experiments using Ly-5.1+ cells and by kinetic PCR (Fig. 3). Still, more detailed studies are needed to elucidate the precise mechanism of V
14 NKT cell number reduction following receptor stimulation in vivo (Figs 1 and 2A). Apoptotic cell death may play a role in this process. However, since the expression of certain anti-apoptotic genes is up-regulated following receptor activation, it appears likely that NKT cells become less susceptible to apoptosis over time (see below, Fig. 4C).
The molecular mechanism underlying the resistance of V14 NKT cells to TCR-mediated apoptosis was further explored by DNA microarray studies combined with RT-PCR analysis (Fig. 4B). Our results indicate that the resistance to apoptosis of V
14 NKT cells relative to conventional T cells is primarily due to the preferential expression of anti-apoptotic genes, such as NAIP and MyD118. NAIP belongs to the inhibitor of apoptosis (IAP) that regulates lymphocyte sensitivity to Fas-mediated apoptosis (3,14). IAP bind activated caspases, and target their ubiquitination and degradation via the baculovirus IAP repeat (BIR) motif, which eventually allows the cells to survive upon Fas-induced signals. MyD118, also known as Gadd45ß, was first identified as a member of the Gadd45 family associated with cell-cycle control and DNA repair (15,16). MyD118/Gadd45ß is induced by an apoptotic factor, tumor necrosis factor-
, and acts as an anti-apoptotic protein by inhibiting JNK activity (17). Since continuous activation of JNK activity is tightly correlated with apoptosis (18), its inhibition results in the blocking of apoptosis. In summary, the increased expression of anti-apoptotic genes post-receptor activation together with the unaltered expression of IL-1ß in V
14 NKT cells may explain, at least in part, why these cells are more resistant to apoptosis than conventional T cells. In the light of these data, it is not surprising that V
14 NKT cells are relatively resistant to apoptosis induced by irradiation and glucocorticoid treatment (19,20). Since conventional T cells comprise naive and memory cells, a more fine comparison in the expression of anti-apoptotic genes between V
14 NKT cells and conventional T cells will provide more insight into the anti-apoptotic mechanism in V
14 NKT cells.
Another unique facet of V14 NKT cells is that its effector function, the production of cytokines, is kept intact even after activation (Fig. 5). This indicates that V
14 NKT cells do not become anergic upon receptor activation. In line with this, priming
-GalCer-pulsed DC in vivo followed by re-stimulation with
-GalCer-pulsed DC in vitro does not induce anergy in V
14 NKT cells (21). However, we have to mention that the amount of IL-4 produced by activated NKT cells was relatively constant regardless of the different NKT cell number (Figs 2A and 5). This may be explained, at least in part, by the fact that IL-4 produced from the activated NKT cells is consumed by NKT cells per se or B cells, thus culminating in the similar net production of IL-4 (22,23).
These distinct features of V14 NKT cells may be tightly linked to the role of V
14 NKT cells in controlling and suppressing autoreactive effector T cells. Collectively, it is conceivable that the nature of V
14 NKT cells to be resistance to tolerance induction as well as apoptosis after down-regulation of the invariant V
14 receptor confers on these cells the ability to mediate a long-lasting regulatory function under any given circumstances.
These findings will open the door to the elucidation of the homeostasis of immunoregulatory cells.
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Acknowledgements |
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Abbreviations |
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CFSEcarboxyfluorescein diacetate succinimidyl ester
DCdendritic cell
IAPinhibitor of apoptosis
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References |
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