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Shin-ichiro Fujii1*, Kanako Shimizu1*, Mitchell Kronenberg2 and Ralph M. Steinman1

Published online: 5 August 2002, doi:10.1038/ni827

Natural killer T (NKT) lymphocytes mediate a rapid reaction to the glycolipid drug α-galactosylceramide (αGalCer), which triggers release of large amounts of cytokines into the serumwithin 12 h, starting with interleukin 4 (IL-4). When αGalCer is administered to mice on dendriticcells (DCs) instead, the response is more prolonged (>4 days) and marked by a large expansion inIFN-γ–producing NKT cells as well as greater resistance to metastases of the B16 melanoma.Nevertheless, DCs from mice given free αGalCer are able to induce strong IFN-γ–producing NKTresponses when transferred to naïve mice, but not when transferred to αGalCer-treated recipients. Inthe latter, the NKT cells are anergized and can respond to glycolipid only in the presence ofsupplemental IL-2.Therefore, when αGalCer is selectively targeted to DCs, mice develop a stronger,more prolonged and effector type of NKT response, but this response can be blocked by theinduction of anergy after presentation of αGalCer on other cells.

1Laboratory of Cellular Physiology and Immunology,The Rockefeller University, New York, NY 10021, USA. 2La Jolla Institute for Allergy and Immunology, San Diego, CA92121, USA. *These authors contributed equally to this work. Correspondence should be addressed to R. M. S. (steinma@mail.rockefeller.edu).

Prolonged IFN-γ–producing NKT response induced with

α-galactosylceramide–loaded DCs

Natural killer T (NKT) lymphocytes1–3 are implicated in the control ofautoimmunity4–9, resistance to tumors10–18 and protection against infec-tious agents19–21. Several subsets of NKT cells have been described,defined operationally by the coexpression of T cell receptor (TCR) andNKR-P1A, but many of the regulatory properties are ascribed to theNKT cell subset that expresses a conserved TCR encoded in humans byVα24-JαQ gene segments and in mice by homologous Vα14-Jα281sequences22. Recognition by this TCR is, in turn, restricted to CD1dmolecules23–27. The drug α-galactosylceramide (αGalCer), originallyderived from mollusks, was identified because of its anti-tumor activityin mice28. This synthetic glycolipid binds tightly to CD1d29–31. The dis-covery that αGalCer is presented on CD1d to NKT cells11,12,22,23 stimu-lated this field of research enormously. Intravenous delivery of αGalCerinduces a rapid release of cytokines into the serum, including interleukin4 (IL-4), IL-12 and interferon-γ (IFN-γ). This cytokine “storm” likelyreflects a chain reaction in which the three cytokines are released suc-cessively by NKT cells, dendritic cells (DCs) and NK cells32.

The intravenous (i.v.) injection of soluble αGalCer, however, doesnot target the glycolipid to specific subsets of antigen-presenting cells.DCs efficiently present αGalCer to NKT cells in culture33–37 and, inturn, produce IL-12 in large amounts33,38. Nevertheless, other cellsexpress CD1d and may influence responses to this pharmacologicagent. We compared the ability of αGalCer-charged DCs and the freedrug to manipulate NKT numbers and function systemically in mice.We have shown that DCs elicited NKT responses distinct from thoseseen with the drug alone. The response to DC-αGalCer, as assessed bythe number of IFN-γ–secreting NKT cells after αGalCer challenge,was much stronger and more prolonged; it was also associated with

increased protection against the development of metastases with B16melanoma. These results identify qualitative and quantitative differ-ences after antigen targeting to DCs and may allow for improvedmanipulation of NKT cells in disease settings.

ResultsDC-induced prolonged IFN-γ responsesWe first compared the release of cytokines into the serum of miceinjected intravenously with free αGalCer, provided as the pharmaceu-tical compound KRN7000, or with 6 × 105 DCs pulsed with the gly-colipid ex vivo beforehand. Both forms of αGalCer induced rapidincreases in serum IFN-γ, IL-4 and IL-12 concentrations; however, theresponses to free drug were higher and more rapid, particularly in thecase of serum IL-4 (Fig. 1a). Using an enzyme-linked immunospot(ELISPOT) assay to quantify cells responsive to the synthetic αGalCer,we then looked for cytokine-producing cells 2 days after priming.Suspensions of cells from unimmunized spleen spontaneously pro-duced IL-4, but not IFN-γ, in vitro (Fig. 1b). Individually, the IL-4spots were small, which suggested relatively low amounts of IL-4 pro-duction. However, immunization with DC-αGalCer expanded the num-ber of αGalCer-responsive cytokine-producing cells, particularly cellssecreting IFN-γ, which produced larger spots (Fig. 1b). The optimaldose of glycolipid required to pulse DCs ex vivo was 100 ng/ml (Fig.1b), whereas the optimal DC dose was 6 × 105 cells/mouse (Fig. 1c).

In these dose-response studies, it was evident that the induction ofIFN-γ–producing cells was almost entirely αGalCer-dependent,requiring the presence of the drug both on the DCs and during theELISPOT assay (Fig. 1b,c). In contrast, a substantial fraction of the

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IL-4–producing cells was part of the background measurements innonimmunized spleen, that is, the cells were detected in the absence ofαGalCer either during the immunization or during the ELISPOT assay(Fig. 1b,c). Therefore DC-αGalCer induces a strong αGalCer-specif-ic, IFN-γ–producing NKT response in situ.

We then examined other tissues as well as the subcutaneous (s.c.)route of administration, as the latter is an effective route for stimulationof αβ T cells by DCs39. After intravenous (i.v.) but not s.c. injection, theinduction of αGalCer-responsive, IFN-γ–producing cells by DC-αGalCer was only observed in the spleen, not in the peripheral lymphnodes (Table 1). NKT cells, which lack CD62 ligand (CD62L) or L-selectin, may not be able to circulate into the deep cortex of lymphnodes via high endothelial venules and therefore have access to DCsinjected subcutaneously. The IFN-γ response to i.v. DC-αGalCer wasobserved in liver and bone marrow, two organs that are relatively rich inNKT cells32. The IFN-γ responsein these organs again was depen-dent upon the presence ofαGalCer, both on the injectedDCs and in the ELISPOT assay(Table 2). αGalCer-indepen-dent, IL-4–producing cells wereabundant in bone marrow ofnonimmunized mice, butαGalCer–dependent, IL-4–pro-ducing cells were expanded inliver after immunization witheither DC or DC-αGalCer

(Table 2). Thus, αGalCer-responsive, IFN-γ–producing cells in the bonemarrow, liver and spleen can be effectively expanded by DC-αGalCer.

IFN-γ–producing NKT responses with different assaysTo further compare the NKT response to αGalCer administered as afree drug or pulsed on DCs, we used three different assays: ELISPOT,enzyme-linked immunosorbent assay (ELISA) and intracellularcytokine staining. Two days after immunization, it was again apparentthat DC-αGalCer induced a stronger IFN-γ NKT response than thefree drug when αGalCer-responsive cytokine-producing cells weremeasured with an ELISPOT assay (209 versus 25 spots, Fig. 2a). Thedifferences were also substantial when total IFN-γ production wasmeasured by ELISA (394 versus 61 pg/ml, Fig. 2b). The size of theIFN-γ spots and the amounts of IFN-γ were much greater compared toIL-4 production (Fig. 2b). IFN-γ production by CD3+ cells that bind

0

100

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6x103 6x104 6x105 1x106

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IFN-γ

0

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500 IL-4

ELIS

POT

s/ 2

x10

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6x103 6x104 6x105 1x106

**

** **

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0 2 6 12 24 48 72

DCDC-αGalCerVehicleαGalCer

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0 2 6 12 24 48 720

200

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IFN

-γ (

ng/m

l)

IL-1

2 (p

g/m

l)

IL-

4 (p

g/m

l)

0 2 6 12 24 48 72

** **

60

40

20

0

IL-4 IL-4

ELIS

POT

s/ 105

spl

een

cells

60

40

20

0

DC-αGalCer (ng/ml)10 30 100 300 0.1 0.3 1 3

0

10

20

30

40IFN- IFN-γ

0

10

20

30

40

0 10 30 100 300 0.1 0.3 1 30

+αGalC–αGalCer

* *

γ

0 0

*

Time post injection (h)

αGalCer (µg/mouse)

Dose of αGalCer

spl

een

cells

Number of injected DCs

a

b c

Table 1. αGalCer-responsive IFN-γ–producing cells after different routes of administration ofDC-αGalCer

Route of DC injection: Intravenous Subcutaneous

Lymph nodes Spleen Lymph nodes Spleen

αGalCer used – + – + – + – +

DC 0 0 1±1 3±2 3±4 4±4 0 0DC-αGalCer 0 0 9±8 273±35 1±3 5±6 1±1 14±14ELISPOT assays of organs from immunized mice were done 2 days after DC injection. Data represent IFN-γ+ spots found in 2 ×105 lymph nodes and spleen with or without αGalCer restimulation in vitro. Data are the means of four mice; two mice wereused in each of two experiments.

Figure 1. Responses to αGalCer administered as a free drug or on DCs.(a) Serum concentrations of IFN-γ, IL-4 and IL-12 in mice given 6 × 105 αGalCer-loadedDCs (or unloaded DCs as control) or 2 µg of intravenous free αGalCer (or vehicle asa control) were assessed. Data are means obtained from two mice in two experiments.IL-4 concentrations after DC immunization are shown on the right y-axis. **P < 0.005.(b) Mice were immunized with DC-αGalCer loaded with different doses of glycolipidor free drug. Spleen cells (105 per well) were tested for IFN-γ or IL-4 production with-out or with the addition of αGalCer during the 16-h ELISPOT assay. *P = 0.05 for 0 ver-sus 100–300 ng/ml. (c) Two days after graded doses of DCs or DC-αGalCer (pulsedwith 100 ng/ml ex vivo) were administered intravenously, 2 × 105 spleen cells were ana-lyzed in the presence of αGalCer restimulation for cytokine-producing cells. **P < 0.005for IFN-γ in DCs versus DC-αGalCer; *P < 0.05 for IL-4 in DCs versus DC-αGalCer.(b,c) Data are means obtained from two mice per dose.

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CD1d-αGalCer tetramers was also assessed (Fig. 2c, upper row). DC-αGalCer immunization was markedly more active than free drug ininducing tetramer-binding, IFN-γ–secreting cells (Fig. 2c, lowerrows). Taken together, these assays indicated that immunization withDC-αGalCer was an effective way to induce a prolonged NKTresponse, particularly for cells producing high amounts of IFN-γ.

Turnover of IFN-γ–producing NKT cellsTwo days after immunization with αGalCer, particularly with DC-αGalCer, cytokine production—as assessed by ELISPOT—was elimi-nated by removing NKT cells with antibodies to CD3 or to NK1.1 (Fig.3a, upper panels). FACS analysis of intracellular IFN-γ productionshowed that almost all the cytokine-secreting, αGalCer-responsive cellswere CD3+ NKT cells (Fig. 3a, lower panels) and not NK cells. Thus,these data indicated that NKT cells are the major source of cytokine pro-duction in the response to DC-αGalCer. However, the NKT cells wereundergoing considerable turnover, probably due to activation-inducedcell death, which accompanies the response to soluble αGalCer31,40–42.First the total number of NKT cells per spleen dropped almost 50% in a1–2 day period after DC-αGalCer injection (Fig. 3b), although thedecrease was less rapid and severe than the one induced by the free drug.Also in the liver (where NKT cells are much more abundant than in thespleen30,31), NK1.1+ and CD3+ cells were annexin V+ and 5-bromo-deoxyuridine+ (BrdU+) (Fig. 3c). We thus concluded that DC-αGalCer

induced apoptosis of NKT cells followed by quick homeostatic regener-ation, as described for responses to free αGalCer31,40–42.

Prolonged NKT expansion and IL-2 dependenceWe prepared DCs from bone marrow progenitors using mouse serumrather than fetal calf serum (FCS) to supplement the RPMI-1640 cul-ture medium43. This was necessary for kinetic studies because wefound that DC exposure to FCS led to a large expansion of IFN-γ–secreting but αGalCer-independent cells, which presumably repre-sented αβ T cells responding to the FCS. To induce maturation of DCsas described44, we also studied DCs that were exposed to lipopolysac-charide (LPS) during the final day of a 7-day culture. LPS-inducedmaturation was not essential for the induction of IFN-γ–producingNKT cells (Fig. 4). With both immature and mature DCs, strong IFN-γ–producing αGalCer-dependent responses were evident at days 2 and4 after immunization. The response reduced to low amounts by day 7.In contrast, repeated injection of 2 µg of αGalCer as a free drug ondays 0, 4 and 8 failed to induce any IFN-γ–producing cells 10 daysafter administration (data not shown). A long-lived (peaking at day 4)boost in IL-4–producing cells was also induced by DCs (Fig. 4), butculture with αGalCer was, again, nonessential. This suggested thatDCs carry a ligand for IL-4–producing NKT cells. However, αGalCeris essential for DCs to induce a prolonged IFN-γ–producing NKT cellresponse in mice.

Figure 2. Assays for IFN-γ– and IL-4–producing NKT cells to DCs pulsed with αGalCer. Each graph shows four groups of mice immunized intravenously withαGalCer or vehicle only or with DCs that had or had not been exposed ex vivo to αGalCer.Two days later, spleens were analyzed with or without αGalCer restimulation for16 h. (a) ELISPOT-forming cells were counted. **P < 0.005 for others in IFN-γ. (b) ELISA assays were done for the culture medium. *P < 0.01 for DC-αGalCer versus others.(c) CD1d-αGalCer tetramer binding with intracellular cytokines was visualized. CD3+ T cells were selected first (upper row), then the specimens analyzed for IFN-γ–produc-ing tetramer-binding cells after 16 h of culture without (–) or with (+) αGalCer. (a,b) Data are means obtained from six mice; (c) data are representative of four mice in inde-pendent experiments.

0

100

200

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DC DC-αGalCer Vehicle αGalCer0

50

100

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250

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DC DC-αGalCer Vehicle αGalCer

ELIS

POT

s/2x

105

IFN-γ IL-4

+αGalCer–αGalCer

**

0

100

200

300

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DC DC-αGalCer Vehicle αGalCer

pg/m

l

0

10

20

30

40

50

DC DC-αGalCer Vehicle αGalCer

IFN-γ IL-4

* *

sple

en c

ells

a

b–αGalCer

IFN-γ

+αGalCer

CD1d-αGalCer

DC DC-αGalCer Vehicle αGalCer

CD3

tetramer

c

Table 2. αGalCer-responsive cytokine-producing cells after DC-αGalCer immunization

Liver Bone marrow

Restimulation DC DC-αGalCer Vehicle αGalCer DC DC-αGalCer Vehicle αGalCer

IFN-γ – 2 5.3 1.7 1.3 0.3 2 2.3 2.3IFN-γ + 1.7 99.3 5.7 2.3 3 126.3 15.7 1.7IL-4 – 8 13.3 10 14 >300 200 228.5 212.5IL-4 + 224.3 161.7 80.7 14.3 >300 >300 >300 223Four groups of mice were immunized with i.v. DCs only, DCs pulsed with αGalCer, vehicle or free αGalCer.Two days later, liver and bone marrow suspensions were restim-ulated or not with αGalCer in a 16-h ELISPOT assay that assessed either IFN-γ or IL-4. Data are ELISPOTs per 1 × 105 mononuclear cells in liver and per 3 × 105 mononu-clear cells in bone marrow, respectively.

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We evaluated the role played by IL-2 production by the DCs in theαGalCer response because mouse DCs are a recognized source of IL-245 and NKT cells are also responsive to IL-246,47. In eight wild-typemice immunized with IL-2–/– or IL-2–/+ DCs, we found that about halfthe IFN-γ–producing NKT cell response could be attributed to IL-2production by the DCs (Fig. 5). We concluded that the role played by

DCs in inducing strong IFN-γ–producing NKT cell responses is depen-dent on IL-2 production as well as other accessory functions.

αGalCer-loaded DCs protect against melanoma metastasesαGalCer was first identified as a drug capable of inducing protectionagainst metastatic tumors in mice12,28. However, in most studies, sever-

al doses of the drug were administered intravenously and—with the B16 melanoma in particular—NK cells also con-tribute to protection15,42,48. We evaluated protection against thedevelopment of B16 melanoma metastases that could beinduced by free αGalCer versus DC-αGalCer. The tumorcells and αGalCer were both administered via the i.v. route.Metastases to the lungs were evaluated 2 weeks later.Immunization with DCs pulsed with glycolipid markedlyreduced lung metastases (Fig. 6a). Immunization with DC-αGalCer reduced the number of metastases by two-thirds(Fig. 6b, upper panel), and the remaining metastases were

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spleen cells spleen cellsa BrdU Annexin V

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Figure 4. Prolonged IFN-γ–producing NKT response in mice vac-cinated with DCs. Four sets of DCs, generated in 1.5% mouse serum,were injected intravenously with or without exposure to αGalCer ex vivo.One pair of DCs was exposed to LPS to ensure optimal maturation, andthe other were not. Splenocytes from injected animals were analyzed forIFN-γ– and IL-4–producing cells without (dotted line) or with (solid line)αGalCer restimulation at each time point. Data are mean ± s.d. of threemice; the s.d. was <10%.

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Figure 3.The dynamic nature of NKT cell responses to DC-αGalCer and αGalCer. (a) Two days after mice were injected intra-venously with 6 × 105 DCs pulsed with αGalCer versus DCs only orwith 2 µg of free αGalCer versus vehicle, 2 × 105 spleen cells were ana-lyzed for IFN-γ and IL-4 production in ELISPOT assays (upper panels)with or without restimulation for 16 h with αGalCer. Spleen cells werestudied in bulk or after depletion of CD3+ or NK1.1+ cells. Data aremean±s.d. from four individual mice. Cells were also analyzed by intra-cellular cytokine staining for IFN-γ production (lower panels) to showthat the cytokine-producing cells were primarily CD3+. Data aremean±s.d. from four individual mice. (b) NKT (CD3+NK1.1+) numberswere analyzed by FACS in spleens after i.v. administration of 6 × 105

DCs with 100 ng/ml of αGalCer-loaded ex vivo or 2 µg of free drug.(c) Assessment of death and turnover of liver CD3+NK1.1+ NKT cellsby FACS, BrdU labeling and annexin V labeling. Data are means (b) orrepresentative (c) of three mice.

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much smaller in size. When the mice were depleted of NK cells byprior treatment with a polyclonal anti–asialo-GM1 (Fig. 6c), the num-ber of metastases increased relative to untreated controls; but again,the number of metastases could be reduced significantly if the NK-depleted mice were given DC-αGalCer (Fig. 6b, lower panel).Therefore, although NK cells are expanded as part of the overallresponse to αGalCer15,42,48,49, they are not the only resistance mecha-nism, presumably because NKT cells have direct anti-tumor activityagainst the B16 melanoma15. When we evaluated the frequency of

IFN-γ– and IL-4–producing αGalCer-responsive cells in these mice (2days after immunization), we found that cytokine-producing αGalCer-responsive cells were enhanced by the injection of B16 melanoma(Fig. 6d). This may reflect expansion due to endogenous glycolipid onthe tumor cells. Thus, the presentation of αGalCer to mice on DCsprovided more effective resistance to B16 melanoma metastases, andthis was independent of NK cells.

Non-DCs inhibit responses to DC-αGalCerTo further understand the distinct responses of mice injected with freeαGalCer or DC-αGalCer, αGalCer was first injected intravenouslyand, 24 h later, splenic DCs were purified with anti-CD11c–coatedmagnetic beads. When transferred into naïve mice, the DCs inducedstrong αGalCer-dependent, IFN-γ–secreting responses (Fig. 7a). Incontrast, when transferred into naïve mice, CD11c– cells were inactive(Fig. 7a, ** P < 0.005), as were positively selected CD19+ B cells (datanot shown). This indicated that DCs from mice treated with αGalCerhave the capacity to induce IFN-γ–producing NKT cells, but the DCsseemed to be inhibited in donor animals that were given free drug.When DCs from αGalCer-treated mice were adoptively transferred intorecipients that also received free drug, the DCs did not induce IFN-γ–producing NKT cells, even in recipients that had received αGalCer45 days before transfer. This indicated that αGalCer presented by non-DCs inhibited the IFN-γ NKT response to DCs (Fig. 7b).

We repeated the latter experiments using more abundant DCs derivedfrom bone marrow and pulsed with αGalCer. When bone marrow DC-αGalCer was coinjected with free αGalCer, the IFN-γ–producing NKTresponse was again inhibited, that is, the number of IFN-γ–producingcells was reduced from 320 to 100 (Fig. 7c, **P < 0.005). In mice ini-tially treated with the free drug, this inhibition of the NKT response to

Figure 5. IL-2 secretion by DCs plays a role in activating NKT cells. Bonemarrow DCs were prepared twice from IL-2–/+ and IL-2–/– mice with the use of a stan-dard culture method in FCS to increase cell yields.Two days after mice were immu-nized with αGalCer-loaded DCs, ELISPOT assays were done with spleen cells fromeight individual mice in the presence or absence of αGalCer restimulation. P < 0.005for the increased IFN-γ response to DCs able to produce IL-2.

Restim.Cell

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Figure 6. DC-αGalCer immunization pro-tects against B16 melanoma. (a) Reductionin the number of melanin-laden (black) metas-tases in the lungs of groups of eight mice, 14days after B16 melanoma cells were adminis-tered together with i.v.αGalCer, vehicle or DCswith or without ex vivo exposure to αGalCer.(b) As in a, but the number of metastases on thelung surface were counted. (Upper panel) Data from individual mice; (lower panel) the effect of NKcell depletion by treatment with anti–asialo-GM1. DC-αGalCer treatment significantly reducedmetastases. *P < 0.001, **P < 0.05. (c) Liver NK but not NKT cells were depleted from anti–asia-lo-GM1–treated mice. (Left panel) CD3–NK1.1+ NK cells (filled arrow) and CD3+NK1.1+ NKTcells (open arrow) in untreated mice.The absence of these cells in anti–asialo-GM1–treated micecontrasted with the presence of NKT cells, as assessed by the CD3+NK1.1+ phenotype (upperrow) and binding of CD1d-αGalCer tetramers (lower row). (d) Increased formation of IFN-γ–secreting cells in mice immunized with DCs pulsed with αGalCer, especially in the presence ofB16 melanoma cells. Data are from spleens sampled on day 2 and are the mean of five indepen-dent experiments. **P < 0.005 for DC-αGalCer versus others.

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Figure 7. Inhibition of DC-induced NKT responses by αGalCer on other cells. (a) CD11c+ splenic DCs, selected from mice given 2 µg of i.v. αGalCer 24 h earli-er were transferred into naïve mice intravenously. (Left panels) After 2 days, IFN-γ– and IL-4–secreting, splenic NKT cells (either CD3 or NK1.1, data not shown) were ana-lyzed with or without αGalCer restimulation. (Right panels) Responses to 5 × 105 CD11c+ and CD11c– cells were compared. **P < 0.005. (b) In parallel with a, CD11c+ DCswere transferred into mice given αGalCer 0, 2 or 45 days earlier. (c) As in a, but bone marrow–derived DCs, loaded with αGalCer, were injected together with various stim-uli. **P < 0.05 for DC-αGalcer versus others. (d) As in b, but mice were given DC-αGalCer or αGalCer and 30 days later injected with various stimuli. **P < 0.05 for DC-αGalCer versus others. (e) Four groups were primed in vivo for 2 days, then spleen cells were challenged with DC or with DC-αGalCer in culture for 16 h. Data are themean of three independent experiments. **P < 0.005 for DC-αGalCer versus others. (f) After the injection of i.v. free αGalCer or vehicle 2 days earlier, ELISPOT assays weredone with or without restimulation with αGalCer + IL-2. Data are representative of four similar experiments.

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DC-αGalCer lasted at least 30 days (Fig. 7d, **P < 0.005) and, there-fore, could not be due to a lack of NKT cells, which returned to normalwithin 2–3 days after αGalCer treatment (Fig. 3b). Inhibition was alsoseen if αGalCer was given for 2 days in vivo, and then we attempted torescue the response by restimulating the spleen cells with bone mar-row–derived DC-αGalCer in vitro (Fig. 7e, **P < 0.005). The anergyinduced by αGalCer compared to DC-αGalCer was not seen in miceprimed with DC-αGalCer (Fig. 7c–e).

To confirm that NKT cells from αGalCer–injected mice were aner-gic, we did the ELISPOT assays for IFN-γ-secreting cells in the pres-ence of graded doses of IL-2. IFN-γ–secreting αGalCer-responsiveNKT cells were regenerated from mice injected with free αGalCer for2 days but not from control mice given vehicle (Fig. 7f). Thus, theshort-lived nature of the NKT cell response to αGalCer resulted fromanergy induced by the presence of the drug on non-DCs.

DiscussionTo gain more understanding of the control of NKT cell function in vivo,we generated DCs from bone marrow progenitors, pulsed these withthe synthetic compound αGalCer and infused the ligand-bearing DCsinto adult mice. Previously, research with this active glycolipid primar-ily used free αGalCer, usually given in multiple doses12,14,15,23,42,48.However many cell types can express CD1d, so that the response to freedrug may represent a composite of effects induced by DCs and otherCD1d-expressing cells. In addition to comparing the magnitude of theNKT reaction to glycolipid delivered as a free drug versus on DCs, wehave emphasized its functional features in effector assays, that is, thecapacity to produce cytokines upon stimulation with αGalCer and toresist the B16 melanoma.

The early in vivo NKT cell response to DCs pulsed with αGalCer hasmany similarities to the well described response to free drug32. In thefirst 12 h, there is a rapid increase in several cytokines, including IL-4,IFN-γ and IL-12. With both the free drug and DC-αGalCer, there wasa rapid drop in NKT cells that was reversed within 2–3 days, due totheir regeneration; this was indicated by marked BrdU labeling. Mostlikely this rapid reduction in NKT numbers was due to antigen-inducedapoptosis, as described40–42. Therefore upon encountering αGalCer onDCs, NKT cells are capable of rapid production of cytokines and acti-vation-induced cell death.

In contrast, when we measured the number of αGalCer-responsivecytokine-producing NKT cells over longer periods, 2–7 days, theresponses in DC-vaccinated mice were different from those observed inanimals given free drug. The NKT cells behaved more like newlyprimed αβ T cells, rather than innate NK cells. DC-αGalCer, in partic-ular, induced large numbers of high IFN-γ–producing NKT cells.Therefore NKT cells not only have innate functions, but can differenti-ate to produce more IFN-γ in response to DCs. One of the mechanismsunderlying DC function is their capacity to produce IL-245. Other DC-derived cytokines, like IL-15, have not yet been tested. It also is possi-ble that different DC subsets expand different types of NKT responses36.

In trying to understand the different responses to free glycolipid ver-sus DC-αGalCer, we found that the DCs from mice given free αGalCerwere fully capable of inducing prolonged NKT cell responses uponadoptive transfer to naïve animals. However the efficacy of DCs uponadoptive transfer was blocked if the recipients were given free drug.The latter induced anergy of the IFN-γ–producing NKT cells.Therefore, the quality of the immune response is, again, affected bycells presenting glycolipid, so that non-DCs induce anergy and DCsimmunity. As a result, the usual approach of giving multiple doses offree αGalCer likely induces multiple “innate” NKT and NK responses

via DCs, but does not allow the DCs to greatly expand and prolongtheir IFN-γ component.

This finding could have broader consequences for understanding thelymphocyte anergy that is observed in mice given high doses of peptideantigens, a standard way in which this form of immune tolerance isinduced in vivo. For example, when T cells with diverse TCRαβ usageare exposed to high doses of preprocessed peptide, we suggest that thepresentation of peptide on non-DCs inhibits the response to peptidepresented on the DCs. The inhibiting cell type(s) are not yet known, butthe anergy cannot be due to simple competition of other CD1d+ cellswith DCs for αGalCer. If this were the explanation, the adoptive trans-fer of DCs from αGalCer-injected to naïve mice would not have beeneffective. The data therefore suggest an active and long-lived process inwhich CD1d+ cells other than DCs induce NKT cell anergy.

The antitumor effects of αGalCer likely require IFN-γ, as shownwith IFN-γ–deficient mice15,42. Our data, which show DCs can induceIFN-γ–producing NKT cells, complement another report, which showsrepeated stimulation of NKT cells with αGalCer induces resistance toexperimental tumor metastases15. We suspect that selective αGalCertargeting to DCs will greatly improve the efficacy with which glycol-ipid ligands for CD1 molecules can be used to analyze and manipulatethe T cell response in vivo, including tumor immunity.

MethodsReagents and antibodies. αGalCer—which is 2S, 3S, 4R-1-O(α-galactopyranosyl)-2(N-hexacosanoylamino)-1,3,4-octadecanetriol—was dissolved with 0.5% polysorbate 20(Nikko Chemical, Tokyo, Japan) in saline and subsequently diluted with this solution (vehi-cle). αGalCer was from the Pharmaceutical Research Laboratory, Kirin Brewery (Gunma,Japan). LPS was from Sigma (St. Louis, MO). Fluorescein isothiocyanate (FITC)–annexinV and the following monoclonal antibodies (mAbs) were from BD PharMingen (San Diego,CA): FITC– or allophycocyanin–anti-CD3ε (145-2C11), anti-NK1.1 (PK136) andFITC–anti-BrdU.

Mice. Pathogen-free C57BL/6, C57BL/6 IL-2–/+ and C57BL/6 IL-2–/– female mice aged 6–10weeks were from Jackson Laboratory (Bar Harbor, ME). These mice were kept in pathogen-free conditions in the animal facility of the Rockefeller University. All experiments weredone in compliance with laws and institutional guidelines of Rockefeller University.

Serum cytokines. The serum concentrations of IFN-γ, IL-4 and IL-12 were measured 2, 6,12, 24, 48 and 72 h after immunization with DCs, DC-αGalCer, vehicle or αGalCer by asandwich ELISA (Endogen, Woburn, MA).

Cells. DCs were grown as described50 in RPMI-1640 containing 5% FCS from bone mar-row progenitors with the supernatant (3% vol/vol) from J558L cells transduced with murineGM-CSF (mGM-CSF, a gift of A. Lanzavecchia). On day 6, αGalCer (100 ng/ml) wasadded to immature bone marrow DCs for 40 h. To mature the DCs, we added 100 ng/ml ofLPS on day 7 for 16 h. Mature αGalCer-pulsed DCs were collected on day 8. Alternatively,the DCs were grown from bone marrow cells cultured in 1.5% mouse serum supplementedwith recombinant mGM-CSF (rmGM-CSF) and mIL-4 (R&D Systems, Minneapolis, MN)as described45. In both FCS and mouse serum culture systems, αGalCer-pulsed immature(no LPS treatment) and mature (LPS treatment) DCs were able to induce prolonged NKTcell responses in mice. Single-cell suspensions from various organs, including lymph nodes,spleen, bone marrow and liver, were prepared from mice immunized with intravenouslyαGalCer-pulsed DCs. The livers were isolated, cut into small pieces and passed though astainless mesh. The liver cells were resuspended in Hank’s Balanced Salt Solution and cen-trifuged at 1500 rpm for 5 min at 4 °C. The cells were suspended in a 30% isotonic Percollsolution (Pharmacia, Uppsala, Sweden) that contained 100 U/ml of heparin and were cen-trifuged at 2000 rpm for 15 min at room temperature. Splenocytes were also obtained bypressing the spleen through a 70 µm strainer. Bone marrow mononuclear cells were isolat-ed on a density gradient with Lympholite-Mammal (Cederlane, Ontario, Canada). For allorgans, erythrocytes were lysed with 0.83% buffered NH4Cl, followed by two washes inRPMI-1640 containing 5% FCS.

Priming with α-galactosyl ceramide. Mice were injected once intravenously with 2 µg ofαGalCer diluted in 100 µl of PBS. Control mice were injected with an equivalent volumeof 0.5% polysorbate 20 and 0.9% NaCl diluted in PBS (vehicle). Alternatively, mice wereinjected intravenously once with αGalCer-loaded DCs; generally, 6 × 105 DCs/mouse wereused.

Identification of NKT cells. Cells were preincubated with 2.4G2 culture supernatant toblock Fcγ receptors, washed and incubated with mAb conjugates for 30 min in a total of

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100 µl of PBS containing 1% FCS. Cells were washed and analyzed on a FACSCalibur flowcytometer. NKT cells stained double-positive for FITC- and allophycocyanin-CD3ε andPE-NK1.1 mAbs. Alternatively, we used CD1d-αGalCer tetramers to identify NKT cells,which were prepared as described with PE-streptavidin31.

Apoptosis and turnover of NKT cells. Three-color staining was done to identify NKTcells that bound FITC–annexin V mAb for 15 min at room temperature (BD PharMingen).To assess the turnover of NKT cells in vivo, 1 µg of BrdU was administered intraperi-toneally to mice 1 day before immunization with αGalCer; then for 3 days, drinking waterthat contained 0.8 mg/ml of BrdU was given. Antibodies to NKT cells were added, thenlabeling with BrdU was assessed with a BrdU Flow Kit (Becton Dickinson) on fixed cells.For the latter, cells were fixed in 100 µl of Cytofix-Cytoperm buffer for 20 min on ice,washed with perm-wash buffer and incubated on ice in 100 µl of Cytoperm Plus Buffer for10 min. They were then refixed with Cytofix-Cytoperm buffer on ice for 5 min, treated with300 µg/ml of DNase for 1 h at 37 °C and then stained with anti-BrdU for 20 min at roomtemperature for flow cytometric analysis.

Assays for cytokine-producing NKT cells. ELISPOT assays for IFN-γ–secreting cells uti-lized 96-well filtration plates (Millipore, Bedford, MA) coated with rat anti–mouse IFN-γcapture antibody at 10 µg/ml (clone R4-6A2, PharMingen). Spleen cells (1 × 105–2 × 105)from immunized mice were cultured with or without αGalCer for 16 h. The plates werewashed and incubated with biotinylated anti–mouse IFN-γ detection antibody at 2 µg/ml(clone XMG 1.2, PharMingen). For detecting IL-4–producing cells, we used 11B11 at 5 µg/ml as capture antibody and BVD6 at 2 µg/ml as a detection mAb (PharMingen). Spotswere developed with avidin-peroxidase complex (Vectastain Elite Kit, Vector Labs,Burlingame, CA) and stable DAB (Research Genetics, Huntsville, AL) and were countedmicroscopically. To establish that the cytokine-producing cells were NKT cells, we verifiedthat they could be depleted with PE-conjugated antibodies to CD3ε (clone 145-2C11) orNK1.1 (clone PK136, PharMingen) and anti-PE magnetic beads (Miltenyi Biotech, Auburn,CA). To measure release of IFN-γ and IL-4 in 16 h culture supernatants, we used sandwichELISA kits from BD PharMingen. For intracellular cytokine staining by FACS, we simul-taneously stained cells with PE–CD1d-αGalCer tetramer and FITC-CD4 for 20 min at roomtemperature. Spleen cells (2 × 105) were cultured in 96-well flat-bottomed plates for 16 hwith or without 100 ng/ml of αGalCer; Golgi stop (PharMingen) was used for the last 10 hin order to accumulate cytokines intracellularly. Cells were incubated for 15 min at 4 °Cwith the 2.4G2 anti-FcγR and neutravidin (Molecular Probes) to block nonspecific staining.Cells were permeabilized in Cytofix-Cytoperm Plus (PharMingen) and stained with eitherallophycocyanin-anti–IFN-γ (XMG1.2) or anti–IL-4 (BVD4-1D11) for 30 min at 4 °C.Flow cytometry was done with FACSCalibur instrument and CellQuest software (BDBiosciences, Mountain View, CA).

Lung metastases with B16 melanoma cells. Mice were immunized with DC-αGalCer (6 × 105) or 2 µg of free drug. Soon after, B16 melanoma cells (2 × 105) suspended in 0.1ml of PBS were injected intravenously. Mice were killed 14 days after tumor inoculation,the lungs were removed and individual surface lung metastases were counted with the aidof a microscope. To deplete NK cells (monitored as NK1.1+CD3–), mice were treatedintraperitoneally with 50 µl of polyclonal antibody to asialo-GM1 (Wako Chemicals USA,Richmond, VA) 3 days before cell injection (above) and every other day until day 14.

Statistical analysis. The statistical significance of differences between the experimentalgroups were determined by the Mann-Whitney exact rank sum test.

Acknowledgments

We thank S. Sidobre for the preparation of CD1d tetramers and M. Dhodapkar for criti-cal reading of the manuscript.

Competing interests statementThe authors declare competing financial interests: see the Nature Immunology website(http://www.nature.com/natureimmunology) for details.

Received 24 April 2002; accepted 11 July 2002

1. Bendelac,A., Rivera, M. N., Park, S.-H. & Roark, J. H. Mouse CD1-specific NK1 T cells. Annu. Rev.Immunol. 15, 535–562 (1997).

2. MacDonald, H. R. CD1d-glycolipid Tetramers.A new tool to monitor natural killer T cells in healthand disease. J. Exp. Med. 192, 15–20 (2000).

3. Godfrey, D. I., Hammond, K. J., Poulton, L. D., Smyth, M. J. & Baxter,A. G. NKT cells: facts, functionsand fallacies. Immunol.Today 21, 573–583 (2000).

4. Hong, S. et al.The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes innon-obese diabetic mice. Nature Med. 7, 1052–1056 (2001).

5. Sharif, S. et al.Activation of natural killer T cells by α-galactosylceramide treatment prevents theonset and recurrence of autoimmune Type 1 diabetes. Nature Med. 7, 1057–1062 (2001).

6. Wang, B., Geng,Y. B. & Wang, C. R. CD1-restricted NKT cells protect nonobese diabetic mice fromdeveloping diabetes. J. Exp. Med. 194, 313–320 (2001).

7. Pal, E. et al. Costimulation-dependent modulation of experimental autoimmune encephalomyelitis byligand stimulation of Vα14 NK T cells. J. Immunol. 166, 662–668 (2001).

8. Jahng,A.W. et al.Activation of natural killer T cells potentiates or prevents experimental autoim-mune encephalomyelitis. J. Exp. Med. 194, 1789–1799 (2001).

9. Singh,A. K. et al. Natural killer T cell activation protects mice against experimental autoimmuneencephalomyelitis. J. Exp. Med. 194, 1801–1811 (2001).

10. Kawano,T. et al. Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activatedVα14 NKT cells. Proc. Natl. Acad. Sci. USA 95, 5690–5693 (1998).

11. Cui, J. et al. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors. Science 278,1623–1626 (1997).

12. Toura, I. et al. Inhibition of experimental tumor metastasis by dendritic cells pulsed with α-galacto-sylceramide. J. Immunol. 163, 2387–2391 (1999).

13. Shin,T. et al. Inhibition of tumor metastasis by adoptive transfer of IL-12-activated Vα14 NKT cells.Int. J. Cancer 91, 523–528 (2001).

14. Nakagawa, R. et al. Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injuryinduced by α-galactosylceramide in mice. J. Immunol. 166, 6578–6584 (2001).

15. Smyth, M. J. et al. Sequential production of interferon-γ by NK1. 1+ T cells and natural killer cells isessential for the antimetastatic effect of α- galactosylceramide. Blood 99, 1259–1266 (2002).

16. Smyth, M. J. et al. Differential tumor surveillance by natural killer (NK) and NKT cells. J. Exp. Med.191, 661–668 (2000).

17. Smyth, M. J.,Taniguchi, M. & Street, S. E.The anti-tumor activity of IL-12: mechanisms of innate immu-nity that are model and dose dependent. J. Immunol. 165, 2665–2670 (2000).

18. Takeda, K. et al. Relative contribution of NK and NKT cells to the anti-metastatic activities of IL-12.Int. Immunol. 12, 909–914 (2000).

19. Gonzalez-Aseguinolaza, G. et al. α-galactosylceramide-activated Vα14 natural killer T cells mediateprotection against murine malaria. Proc. Natl. Acad. Sci. USA 97, 8461–8466 (2000).

20. Kakimi, K., Guidotti, L. G., Koezuka,Y. & Chisari, F.V. Natural killer T cell activation inhibits hepatitis Bvirus replication in vivo. J. Exp. Med. 192, 921–930 (2000).

21. Ishikawa, H. et al. CD4+ Vα14 NKT cells play a crucial role in an early stage of protective immunityagainst infection with Leishmania major. Int. Immunol. 12, 1267–1274 (2000).

22. Grant, E. P. et al. Molecular recognition of lipid antigens by T cell receptors. J. Exp. Med. 189,195–205 (1999).

23. Kawano,T. et al. CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylce-ramides. Science 278, 1626–1629 (1997).

24. Burdin, N. et al. Selective ability of mouse CD1 to present glycolipids: α−galactosylceramide specifi-cally stimulates Vα14+ NK T lymphocytes. J. Immunol. 161, 3271–3281 (1998).

25. Brossay, L. et al. CD1d-mediated recognition of an α-galactosylceramide by natural killer T Cells ishighly conserved through mammalian evolution. J. Exp. Med. 188, 1521–1528 (1998).

26. Spada, F. M., Koezuka,Y. & Porcelli, S.A. CD1d-restricted recognition of synthetic glycolipid antigensby human natural killer T cells. J. Exp. Med. 188, 1529–1534 (1998).

27. Matsuda, J. L. & Kronenberg, M. Presentation of self and microbial lipids by CD1 molecules. Curr.Opin. Immunol. 13, 19–25 (2001).

28. Morita, M. et al. Structure-activity relationship of α-galactosylceramides against B16-bearing mice. J.Med. Chem. 38, 2176 (1995).

29. Zeng, Z.-H. et al. Crystal structure of mouse CD1: an MHC- like fold with a large hydrophobic bind-ing groove. Science 277, 339–345 (1997).

30. Benlagha, K.,Weiss,A., Beavis,A.,Teyton, L. & Bendelac,A. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191, 1895–1904 (2000).

31. Matsuda, J. L. et al.Tracking the response of natural killer T cells to a glycolipid antigen using CD1dtetramers. J. Exp. Med. 192, 741–754 (2000).

32. Tomura, M. et al.A novel function of Vα14+ CD4+ NKT cells: stimulation of IL-12 production by anti-gen-presenting cells in the innate immune system. J. Immunol. 163, 93–101 (1999).

33. Kitamura, H. et al.The natural killer T (NKT) cell ligand α-galactosylceramide demonstrates itsimmunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12receptor expression on NKT cells. J. Exp. Med. 189, 1121–1128 (1999).

34. Yang, O. O. et al. CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolyticactivity of Vα24JαQ T cells: a feedback mechanism for immune regulation. J. Immunol. 165,3756–3762 (2000).

35. Trobonjaca, Z., Leithauser, F., Moller, P., Schirmbeck, R. & Reimann, J.Activating immunity in the liver. I.liver dendritic cells (but not hepatocytes) are potent activators of IFN-γ release by liver NKT cells.J. Immunol. 167, 1413–1422 (2001).

36. Kadowaki, N. et al. Distinct cytokine profiles of neonatal natural killer T cells after expansion withsubsets of dendritic cells. J. Exp. Med. 193, 1221–1226 (2001).

37. Takahashi,T. et al.Analysis of human Vα24+ CD4+ NKT cells activated by α-glycosylceramide-pulsedmonocyte-derived dendritic cells. J. Immunol. 164, 4458–4464 (2000).

38. Nishimura,T. et al.The interface between innate and acquired immunity: glycolipid antigen presenta-tion by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specificcytotoxic T lymphocytes. Int. Immunol. 12, 987–994 (2000).

39. Inaba, K., Metlay, J. P., Crowley, M.T. & Steinman, R. M. Dendritic cells pulsed with protein antigens invitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172, 631–640 (1990).

40. Eberl, G. & MacDonald, H. R. Rapid death and regeneration of NKT cells in anti-CD3ε or IL-12-treated mice: a major role for bone marrow in NKT cell homoestasis. Immunity 9, 345–353 (1998).

41. Osman,Y. et al.Activation of hepatic NKT cells and subsequent liver injury following administrationof α-galactosylceramide. Eur. J. Immunol. 30, 1919–1928 (2000).

42. Hayakawa,Y. et al. Critical contribution of IFN-γ and NK cells, but not perforin- mediated cytotoxici-ty, to anti-metastatic effect of α-galactosylceramide. Eur. J. Immunol. 31, 1720–1727 (2001).

43. Muller, G. et al. Fetal calf serum-free generation of functionally active murine dendritic cells suitablefor in vivo therapeutic approaches. J. Invest. Dermatol. 114, 142–149 (2000).

44. Inaba, K. et al.The formation of immunogenic MHC class II- peptide ligands in lysosomal compart-ments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med. 191, 927–936 (2000).

45. Granucci, F. et al. Inducible IL-2 production by dendritic cells revealed by global gene expressionanalysis. Nature Immunol. 2, 882–888 (2001).

46. Dunne, J. et al. Selective expansion and partial activation of human NK cells and NK receptor-posi-tive T cells by IL-2 and IL-15. J. Immunol. 167, 3129–3138 (2001).

47. Wilson, S. B. & Byrne, M. C. Gene expression in NKT cells: defining a functionally distinct CD1d-restricted T cell subset. Curr. Opin. Immunol. 13, 555–561 (2001).

48. Nakui, M. et al. Potentiation of antitumor effect of NKT cell ligand, α- galactosylceramide by com-bination with IL-12 on lung metastasis of malignant melanoma cells. Clin. Exp. Metastasis 18,147–153 (2000).

49. Carnaud, C. et al. Cutting edge: Cross-talk between cells of the innate immune system: NKT cellsrapidly activate NK cells. J. Immunol. 163, 4647–4650 (1999).

50. Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow culturessupplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176,1693–1702 (1992).

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ERRATUM

www.nature.com/natureimmunology • advance online publication • nature immunology 1

Prolonged IFN-γ–producing NKT response induced with α−galactosylceramide–loaded DCsShin-ichiro Fujii, Kanako Shimizu, Mitchell Kronenberg and Ralph M. Steinman

Nature Immunology 3, doi 10.1038/ni827 (2002).

In the AOP version of this article some text was incorrect. The acknowledgments should read: We thank S. Sidobre for the preparation of CD1dtetramers and M. Dhodapkar for critical reading of the manuscript. These errors have been corrected in the HTML version and will appear cor-rectly in print. The PDF version available online has been appended.

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