Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates

Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates Invariant NKT Cell-Mediated Cross-Talk with Myeloid Dendritic Cells This information is current as of September 24, 2021. Carlos J. Montoya, Hyun-Bae Jie, Lena Al-Harthi, Candice Mulder, Pablo J. Patiño, María T. Rugeles, Arthur M. Krieg, Alan L. Landay and S. Brian Wilson J Immunol 2006; 177:1028-1039; ; doi: 10.4049/jimmunol.177.2.1028 Downloaded from http://www.jimmunol.org/content/177/2/1028

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates Invariant NKT Cell-Mediated Cross-Talk with Myeloid Dendritic Cells1

Carlos J. Montoya,*† Hyun-Bae Jie,§ Lena Al-Harthi,* Candice Mulder,* Pablo J. Patin˜o,† Marı´a T. Rugeles,† Arthur M. Krieg,‡ Alan L. Landay,2* and S. Brian Wilson2§

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-␣. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT Downloaded from cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles. The Journal of Immunology, 2006, 177: 1028–1039. http://www.jimmunol.org/ ctivation of the innate immune response is crucial to Resident DCs are among the first cells to be activated after control the early invasion of pathogens and the subse- pathogen invasion. These APCs are central to the activation of NK quent establishment of adaptive responses. Myeloid den- cells and to T lymphocyte activation (10–12). A major activation A 3 dritic cells (mDCs) capture Ags in peripheral tissues and migrate pathway by which DCs mature occurs via TLR recognition of to secondary lymphoid nodes to instruct naive T cells, while plas- PAMPs (4, 13). Importantly, there are several functional classes of macytoid DCs (pDCs) enter to the lymphoid nodes directly via the DCs, each of which expresses a different complement of TLRs (14, blood (1–4). Resting DCs that capture self-Ags in the steady state 15). This is what has given rise to the notion that DC subsets are able to induce tolerance, (5, 6) whereas in the presence of occupy special distinct niches responding to specific types of by guest on September 24, 2021 inflammation, these same cells are able to initiate adaptive immune pathogens through expression of distinct sets of TLRs (9, 16, 17). responses (7). A significant component of this discrimination is Once invading pathogens activate innate cells expressing the controlled by pattern recognition receptors (PRRs) that recognize necessary TLR, such as viruses interacting with TLR9 on pDCs, pathogen-associated molecular patterns (PAMPs) (8, 9). The TLR these cells rapidly produce IFNs, chemokines, and cytokines (8). family is the best-characterized class of PRRs. TLR recognition of After activation, these DCs undergo a complex modulation of che- PAMPs initiates inflammatory responses by orchestrating the re- mokine responsiveness, and maturation during migration into T cruitment of leukocytes and the activation of stromal and resident cell areas of secondary lymphoid organs where they activate T innate cells. cells (18–20). pDCs selectively express TLR7 and TLR9 and are specialized cells that rapidly produce massive amounts of type I IFN (IFN-I) following viral infection (21, 22). This burst of IFN-I secretion is *Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL 60612; †Group of Immunovirology, Biogenesis Corporation, University important for both pDC and mDC differentiation and migration, of Antioquia, Medellin, Colombia, South America; ‡Coley Pharmaceutical Group, and the ability of mDC to cross prime antiviral CTL (1, 7, 23–25). Wellesley, MA 02481; and §Diabetes Unit, Massachusetts General Hospital, Boston, MA 02139 In addition to specific viruses, bacterial DNA and the archetypal Received for publication October 5, 2005. Accepted for publication May 1, 2006. TLR9 agonist, CpG oligodeoxyribonucleotides (ODNs), induce The costs of publication of this article were defrayed in part by the payment of page the activation and maturation of pDCs and mDCs, stimulate mono- charges. This article must therefore be hereby marked advertisement in accordance cytes and B cells, and enhance Th1 cytokine production by acti- with 18 U.S.C. Section 1734 solely to indicate this fact. vated T and NK cells (4, 26, 27). There are three distinct classes 1 This work was supported in part by National Institutes of Health Grants of CpG ODNs which are TLR9 agonists with different patterns of 5P01AI055793 (to C.J.M., L.A-H., C.M., A.M.K., A.L.L., and S.B.W.) and 2R01AI45051 (to S.B.W.). immune activation. They are the A-, B- and C-class CpG ODNs, 2 Please address correspondence and reprint requests to Dr. S. Brian Wilson, Diabetes which differ in that the A-class ODNs activate pDC to mature and Research Unit, Massachusetts General Hospital, 65 Landsdowne Street, Room 528, to secrete high levels of IFN-␣, but do not strongly activate B cells; Cambridge, MA 02139. E-mail address: bwilson@rics. bwh. harvard or Dr. Alan L. the B-class ODNs activate B cells and mature pDCs, but only Landay, Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL 60612. E-mail address: [email protected] weakly induce pDC secretion of IFN-␣, and the C-class ODNs 3 Abbreviations used in this paper: mDC, myeloid dendritic cell; pDC, plasmacytoid exhibit the combined but attenuated effects of both the A and B DC; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pat- classes. tern; IFN-I, type I IFN; ODN, oligodeoxyribonucleotide; iNKT, invariant NK T cell; ␣-GalCer, ␣-galactosylceramide; DN, double negative; DP, double positive; IP-10, Interestingly, CD1d-restricted invariant NK T (iNKT) cells, a IFN-r-inducible protein 10; rh, recombinant human. subset of lymphocytes considered innate-like, make important

functional contributions to these very same immune processes, in- collected, filtered with a 0.22-␮m filter, and stored at Ϫ20°C until their use cluding the CpG ODN-dependent responses, despite not express- with fresh cells. ing detectable TLRs. Almost all iNKT cells recognize glycolipid Cytokine blockade Ags presented by CD1d. The regulation of mDC maturation and ϫ 6 cytokine secretion by iNKT cells is an important component of Freshly isolated cells resuspended in complete medium (1 10 /ml) were cultured, stimulated with/without A-class CpG ODN (4 ␮g/ml), rh-IFN-␣ their effector function (28–33) Recently, Steinman and coworkers (5000 U/ml), or PBMC-conditioned supernatant (dilutions: 1/20, 1/10, 1/5, (34) have argued that this interaction is a major control mechanism and 1/2). For blockade, neutralizing Abs were added 1 h before the addition for this process that is independent of TLR signaling. It is not of the stimuli: anti-IFN-␣ (10,000 U/ml), anti-IFN-␤ (3,000 U/ml), anti- known how TLR9-induced signals affect iNKT cells; however, the IFN-IR (CD118, 10 ␮g/ml), anti-TNF-␣ (10 ␮g/ml), anti-IL-6 (0.1 ␮g/ml), ␮ activation of iNKT cells in vivo clearly can lead to the subsequent and anti-IP-10 (10 g/ml). activation of mDCs, B cells, monocytes, NK cells, and T cells (30, Isolation of DC and iNKT cells 35–37) Thus, it seems reasonable to speculate that the immune BDCA-1 positive (mDC) or BDCA-2 positive (pDC) cells were magneti- responses regulated either by activated iNKT cells or TLR agonist- cally isolated from 7 ϫ 107 PBMCs per the manufacturer’s specifications activated pDCs would overlap. (BDCA-1 and BDCA-2 DC isolation kits; Miltenyi Biotec), using an Au- Despite these overlapping regulatory activities between DCs toMACS (Miltenyi Biotec) set to run the POSSELD software program. and iNKT cells, their possible interaction during the human im- Negative selection of pDCs and B cells was done with BDCA-2 and CD19 microbeads per the manufacturer’s recommendations. The number and vi- mune response has not been explored extensively. Moreover, most ability of these purified DC subgroups were determined by trypan blue of the regulatory studies in vitro have been with human DCs de- exclusion, and purity assessed by flow cytometry. rived from monocytes incubated with recombinant cytokines (GM- To isolate iNKT cells, 8 to 10 ϫ 107 PBMCs were incubated 20 min on Downloaded from CSF, IL-4, and/or TNF-␣), and it is unclear how immature DCs ice with 10 ␮g of PE labeled-anti invariant NKT cell mAb (clone 6B11; BD Pharmingen) and 100 ␮lofFcR␥-blocking reagent (Miltenyi Biotec). and iNKT cells, normally present in peripheral blood, may be ac- ϩ The 6B11 cells were then isolated using anti-PE microbeads (Miltenyi tivated by TLR agonists and interact with one another to modulate Biotec) as per manufacturers specifications and were magnetically isolated immune responses. Thus, using the model of innate immune acti- using the program Posseld on an AutoMACS instrument. Viability and vation with the TLR9 agonists CpG ODNs, we evaluated the in- purity were determined as described for DCs.

teraction between CpG ODN-activated pDCs, iNKT cells, http://www.jimmunol.org/ Coculture of purified iNKT cells, pDCs, and mDCs and mDCs. Purified iNKT cells (30K cells) were cultured alone, with either pDCs (20K cells) or mDCs (20K cells) in 0.5 ml of complete culture medium and Materials and Methods incubated either with/without A-class or control ODN; after 24 h of culture, Abs and reagents cells were analyzed by flow cytometry. Fluorochrome-labeled mAbs against human molecules 6B11, CD3, CD4, Culture of purified pDCs with A-class CpG ODN CD8, CD11c, CD19, CD25, CD38, CD40, CD69, CD86, CD45RO, CD123, CD154, HLA-DR, perforin, IFN-␥, IL-4, TNF-␣, lineage markers Freshly isolated BDCA-2-positive cells were cultured in 24-well plates (anti-CD3, CD14, CD16, CD19, CD20, and CD56) and isotype control Abs (2.5 ϫ 105 pDC/ml) and incubated with 4 ␮g/ml A-class CpG ODN during

were obtained from BD Pharmingen. 24 h at 37°C/5% CO2. Then, the pDC-conditioned supernatants were col- by guest on September 24, 2021 FITC-labeled anti-V␤11 and PE-labeled anti-V␣24 were obtained from lected centrifuged 10 min/500 ϫ g/RT, filtered with a 0.22-␮m filter, and Beckman Coulter/Immunotech. Fc␥R blocking reagent, anti-PE magnetic stored at Ϫ20°C until their use for incubation with purified iNKT cells and beads, and isolation kits for mDCs and pDCs were obtained from Miltenyi for analysis of cytokine production by ELISA. Biotec. Recombinant human (h) IFN-␣ and neutralizing mAbs against IFN-␣, Incubation of purified iNKT cells with pDC-conditioned IFN-␤, and CD118 were obtained from PBL Biomedical Laboratories. supernatant Neutralizing Abs against IL-6 and TNF-␣ were from BD Pharmingen, and 5 anti-IP-10 was obtained from R&D Systems. PMA and ionomycin were Purified iNKT cells (2.5 ϫ 10 cells/ml) were incubated for 24 h/37°C/5% obtained from Sigma-Aldrich; ␣-galactosylceramide (␣-GalCer) was from CO2 with or without pDC-conditioned supernatant (dilution 1/2 with com- Kirin Brewery. plete culture medium). Cells were washed with RPMI 1640 medium and resuspended in complete culture medium before the coculture with CpG ODNs ␣-GalCer-loaded mDCs. Synthetic endotoxin-free ODNs were provided by Coley Pharmaceutical Coculture of preactivated iNKT cells and ␣-GalCer-loaded Group (lowercase letters, phosphorothioate linkage; capital letters, phos- mDC phodiester linkage 3Ј of the base; underlined letters, CpG dinucleotides): A-class CpG ODN 2216 5Ј-ggGGGACGATCGTCgggggG-3Ј; A-class Purified mDCs were resuspended in complete medium and incubated with Ј Ј ␣ control ODN 2243 5 -ggGGGAGCATGCTGgggggG-3 ; B-class CpG or without -GalCer (10, 20, 50, or 100 ng/ml) for 24 h/37°C/5% CO2.In ODN 2006 5Ј-TCGTCGTTTTGTCGTTTTGTCGTT-3Ј; C-class CpG parallel, iNKT cells were preincubated with or without pDC-conditioned ODN 2395 5Ј-TCGTCGTTTTCGGCGCGCGCCG-3Ј; B- and C-class supernatants and then cocultured with the ␣-GalCer-loaded mDCs at a ratio control ODN 2137 5Ј-TGCTGCTTTTGTGCTTTTTGCTT-3Ј. of 25K:10K, respectively. After 24 h of incubation, 100 ␮l of culture supernatant was gently collected from each well and stored at Ϫ20oC until Isolation and culture of mononuclear cells analysis by ELISA. Then, 100 ␮l of fresh complete culture medium was added to each well, and the plate was incubated for another 24 h at Heparinized whole-blood samples were obtained from healthy adult donors 37°C/5% CO . Afterward, 1 ␮Ci per well of [3H]thymidine was added,and after obtaining informed consent for an approved protocol Institutional 2 the plate was again incubated overnight; finally, the culture was harvested Review Board ORA#0306110. PBMCs were isolated by Ficoll gradient after 24 h of culture, and incorporated cpms were counted using a 1205 (BioWhittaker). The viability of PBMCs was determined by trypan blue Beta Plate (Pharmacia). exclusion. 6 PBMCs (1 ϫ 10 /ml) were suspended in complete culture medium Flow cytometry (RPMI 1640 supplemented with 10% of heat-inactivated FBS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM L-glutamine). ODNs were Phenotypic analysis of iNKT cells, mDCs, and pDCs was performed by used at concentrations of 0.04–4 ␮g/ml; PMA at 50 ng/ml and ionomycin three- or four-color flow cytometry. For iNKT cell characterization, the ␤ ␣ ␤ at 500 ng/ml. Cultures were incubated at 37°C in 5% CO2. Culture super- following combinations of mAbs were used: V 11 FITC/V 24PE, V 11 natants for ELISA were collected and stored at Ϫ80oC until the measure- FITC/6B11 PE, or 6B11 PE/CD3 PcP. CD4-APC and 6B11-PE was used ment of cytokine levels. for iNKT cell subset analysis. Evaluation of mDC and pDC was done with Conditioned supernatants from PBMCs or cells incubated for 24 h either the combinations Lin FITC/CD11c PE/HLA-DR PcP and Lin FITC/CD123 with or without 4 ␮g/ml A-class control ODN or A-class CpG ODN were PE/HLA-DR PcP, respectively. PBMCs were labeled with CFSE per the 1030 iNKT CELLS MEDIATE PDC-MYELOID DC CROSS-TALK manufacturer’s specifications (Invitrogen Life Technologies/Molecular culture. There were no significant differences in the percentage of Probes). iNKT cells observed over the time course of activation (data not Intracellular stainings were performed following the manufacturer’s rec- ϩ ␣␣ϩ ϫ ␮ shown). Because human iNKT cell subsets are CD4 , CD8 , ommendations (BD Pharmingen). Brefeldin A solution (1 40 l per Ϫ ␣␣Ϫ well; BD Biosciences) was added during the last 6–12 h of culture. After CD4 /CD8 (double negative, DN), it was important to deter- the staining for extracellular Ags, cells were incubated with 500 ␮lof1ϫ mine whether the relative proportions and activation status of these permeabilizing solution 2 (BD Biosciences) during 10 min/room temper- subsets in response to A-class CpG ODNs stimulation were subset ature/dark. Isotype-matched control Abs were included for all experiments specific. Treatment of PBMCs with A-class CpG ODNs did not as controls for nonspecific binding. Because of the low frequency of iNKT ϩ ϩ cells and DCs in PBMCs, from 2.5 to 5 ϫ 105 total gated cells were significantly modify the proportion of CD4 , CD8 , DN and dou- analyzed for each data point. Dead cells were gated out by forward and side ble-positive (DP) iNKT cells or selectively activate any particular scatter. Flow cytometry was performed using the FACSCalibur (BD Bio- subset (Fig. 2 and data not shown). sciences) and analyzed with CellQuest software (BD Biosciences). The combination anti-V␣24/anti-V␤11 identifies a population Detection of cytokines by ELISA of T cells whose TCR coexpression for both chains is highly en- riched for iNKT cells but not specific for the invariant V␣24J␣18 Commercially available kits were used to determine cytokine concentra- TCR ␣-chain (39). To validate that the population of T cells iden- tions in culture supernatants, and assays were performed per the manufacturer’s specifications; IFN-␣, TNF-␣, IL-6, and IL-12 were obtained from tified for the donors used in this work were iNKT cells, the fre- Pierce Endogen; and IFN-␥, IP-10, IL-4, IL-7, IL-15, and IL-18 were ob- quency of iNKT cells was determined in parallel using the com- tained from R&D Systems. bination anti-V␤11 with the invariant chain CDR3 loop-specific Statistical analysis mAb 6B11. For all donors used there were no significant differences between the frequencies of V␣24/V␤11ϩ T cells and 6B11/ Downloaded from Data are shown as a mean Ϯ SD of three or more independent experiments. V␤11ϩ T cells (Fig. 3A). Hence, the frequency of iNKT cell sub- Statistical analysis for the comparison of different stimuli was performed using Student’s two-tailed t test. A value of p Ͻ 0.05 was considered sets was unaffected by activation of PBMCs with CpG ODNs. significant. To further explore the effect of CpG ODNs on iNKT cells, the expression of other T cell activation markers was evaluated (Fig. Results 3B). When compared with basal conditions or control ODNs, A- CpG ODN-stimulated PBMCs activate iNKT cells in culture class CpG ODNs significantly up-regulated the expression on http://www.jimmunol.org/ To evaluate the interaction of pDCs, mDCs, and iNKT cells during iNKT cells of the early activation markers CD38 and CD69; the the development of TLR9-dependent innate responses, PBMCs expression of CD25, CD154 and HLA-DR was unaffected (Fig. 3B were incubated with different classes of CpG ODNs or their re- and Table II). As expected, the T cell memory marker CD45RO Ͼ spective ODN controls. CpG ODNs induce the expression of ac- was expressed on 80% of iNKT cells, independent of the con- tivation markers on particular cell types (4, 27, 38) Thus, the ex- dition of cell culture. The pattern of expression of these proteins pression of CD40 on pDCs and mDCs, CD69 on iNKT cells, and after 18, 48, and 72 h of incubation with A-class control or CpG CD86 on B lymphocytes was determined. Incubation of PBMCs ODN, was identical with those at 24 h. Hence, CpG ODN treatment selectively induced iNKT cell activation markers, with A- with A-class CpG ODN induced the highest expression of CD40 by guest on September 24, 2021 on both pDCs and mDCs, while B and C classes of C CpG ODNs class CpG ODNs being the most potent. led to the highest expression of CD86 on B cells (Table I). Unex- pectedly, CpG ODN markedly stimulated CD69 expression on A-class CpG ODN-mediated activation did not stimulate iNKT iNKT cells. When compared with A- and B-class ODNs, C-class cell proliferation, cytokine secretion, or perforin expression CpG ODNs induced an intermediate level of expression of CD40 and CD69 on DCs and iNKT cells, respectively. TCR- and/or cytokine-mediated activation of iNKT cells induces Because A-class CpG ODN was the most potent ODN for in- the rapid release of cytokines (40, 41) Because incubation with ducing activation markers on DCs and iNKT cells and is thought A-class CpG ODNs induced the expression of activation markers to be pDC selective, this ODN was selected for further study. on iNKT cells, their ability to induce cytokine and perforin ex- Dose-response experiments indicated that the incubation of pression was evaluated by intracellular staining. Treatment of PBMCs with 4 ␮g/ml A-class CpG ODNs induced the highest PBMCs with CpG ODNs did not increase IFN-␥, IL-4, TNF-␣,or secretion of IFN-␣ and expression of CD40 on the positive control perforin expression by iNKT cells (Fig. 4). PBMCs were incubated pDC population and CD69 on iNKT cells (Fig. 1). Consequently, for 6 h with PMA/ionomycin as a positive control of activation, this concentration elicited equivalent maximal responses in both which significantly increased the expression of all three cytokines the iNKT and pDC populations; the A-class CpG ODN was se- and perforin by iNKT cells. Notably, A-class ODN treatment in- lected to evaluate the interaction between pDC and iNKT cells. duced CD69 expression to the same extent as did treatment with To determine whether treatment of PBMCs with A-class CpG PMA and ionomycin. To determine whether the activation with could expand the iNKT cell population, the frequency of these T CpG ODNs leads to either an early or late production of these cells in PBMCs was measured after 18, 24, 36, 48, and 72 h of cell molecules, we evaluated their expression after 12, 18, 48, and 72 h

Table I. Effect of different classes of ODNs on the expression of activation markers by immune cells (n ϭ 4)a

B and C Class ODN Cell Type No Stimulation A-Class CpG ODN A-Class Control B-Class CpG ODN Control C-Class CpG ODN

pDC CD40ϩ 1.5 Ϯ 2.2 19.7 Ϯ 10 0.7 Ϯ 1.2 3.9 Ϯ 3.9 0.8 Ϯ 0.6 7.7 Ϯ 5.9 mDC CD40ϩ 2.3 Ϯ 2.4 19.4 Ϯ 13.7 5.1 Ϯ 5.2 5.8 Ϯ 5 2.4 Ϯ 1.8 9.6 Ϯ 7.8 iNKT cells CD69ϩ 7.7 Ϯ 5.7 51.9 Ϯ 14.9 7.5 Ϯ 4.3 19.9 Ϯ 13.2 11.3 Ϯ 10.3 33.1 Ϯ 12 B cells CD86ϩ 26.9 Ϯ 4.8 48.4 Ϯ 4.4 26.4 Ϯ 4.3 63.6 Ϯ 10.7 47.9 Ϯ 2.8 65.7 Ϯ 12.1

a Expressed as the percentage (mean Ϯ SD) of positive cells for each marker, detected by ﬂow cytometry after 24 h of incubation. For pDCs and mDCs, cells analyzed were from a gate comprising all the mononuclear cells; for iNKT cells and B lymphocytes, the gate contained all the lymphocytes. The Journal of Immunology 1031 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Dose-response of innate immune cells to A-class CpG ODN. PBMCs were incubated for 24 h with different concentrations of A-class ODNs (control and CpG ODN), and the expression of activation markers and the secretion of IFN-␣ were evaluated by flow cytometry and ELISA, respectively. A, During the analysis, mononuclear cells were included in a region (R1) created in the forward vs side scatter, and used to define the lineage-negative/ HLA-DR-positive cells (R2); in the later region, the pDCs were determined as the cells bright for CD123 (R3) and mDCs as cells bright for CD11c. In the later subpopulation the expression of CD40 was analyzed (upper panel, representative dot plots for pDCs). For iNKT cells, the lymphocytes (R1) positive for both CD3 and 6B11 were defined (R2), and in this later region, the expression of CD69 was evaluated (lower panel). B, The incubation with 4 ␮g/ml A-class CpG ODN induced the highest secretion of IFN-␣ and expression of CD40 on pDC and CD69 on iNKT cells. Data are reported as means Ϯ p Ͻ 0.05 ,ء .(SD (n ϭ 4 1032 iNKT CELLS MEDIATE PDC-MYELOID DC CROSS-TALK Downloaded from http://www.jimmunol.org/

FIGURE 2. Frequency of iNKT cell subgroups in culture of PBMCs with A-class ODNs. PBMCs were incubated for 24 h with 4 ␮g/ml A-class ODNs (control and CpG ODN) and the expression of CD4 and CD8 on iNKT cells was evaluated by flow cytometry. A, iNKT cells were detected in the lymphocyte region (R1) as cells DP for V␤11 and V␣24 (R2); in this later subpopulation, the frequency of iNKT cell subgroups was defined according to the expression of the molecules CD4 and CD8 and the conditions of PBMC stimulation. B, Summary of the data showing that, in comparison to PBMCs not stimulated or incubated with A-class control ODN, the incubation with A-class CpG ODN did not significantly modify the proportion of CD4ϩ, CD8ϩ, and DN and DP iNKT cells. Data are reported as means Ϯ SD (n ϭ 5). by guest on September 24, 2021 of incubation, and there was no significant differences when com- the activation of other innate and adaptive immune cells such as NK pared with the expression observed after 24 h. cells, monocytes, and T lymphocytes (42–45). To test whether the activation of iNKT cells was dependent on soluble factors, PBMCs CpG ODN-dependent activation of iNKT cells is mediated by were incubated with CpG ODNs for 24 h, and the concentration of soluble factors cytokines that are characteristic of CpG ODN activation was deter- A-class CpG ODNs characteristically activate pDCs to produce mined (46). When compared with unstimulated PBMCs, CpG ODN massive amounts of IFN-I (␣, ␤, and ␻) and stimulate their own treatment markedly increased the secretion of IFN-␣ and IP-10, and maturation (4). This class of cytokines also is thought to be integral to the production of TNF-␣ and IL-6 was modestly elevated. There was

FIGURE 3. Flow cytometry detection of iNKT cells and impact of A-class ODNs on their expression of activation markers. A, For the analysis of iNKT cells in PB- MCs, different combinations of mAbs were used; despite that the combination of anti-V␤11 and the clone 6B11 (anti- CDR3 loop of invariant ␣chain) more specifically detects the iNKT cells, there were no significant differences regarding the use of the combination anti-V␣24/anti-V␤11. B, PBMCs were incubated for 24 h with 4 ␮g/ml A-class ODNs (control and CpG ODN) and the expression of CD25, CDCD38, CD45RO, CD69, CD154 and HLA-DR was evaluated by flow cytometry. In these representative histograms from one of five healthy subjects, it is shown that the incubation with A-class CpG ODNs up-regulated the expression of CD38 and CD69, while the expression of CD45RO and HLA-DR was not up-regulated. More than 80% of iNKT cells express CD45RO, a phenotypical marker of memory cells. The Journal of Immunology 1033

Table II. Expression of activation markers on iNKT cells incubated with A-class CpG ODN (n ϭ 5)a

Conditions CD25 CD38 CD45RO CD69 CD154 HLA-DR

Mean Ϯ SD Mean Ϯ SD Mean Ϯ SD Mean Ϯ SD Mean Ϯ SD Mean Ϯ SD Basal 1.4 Ϯ 0.7 2.4 Ϯ 1.8 84 Ϯ 11 9.3 Ϯ 3.7 0 0.1 Ϯ 0.03 No stimulation 0.8 Ϯ 0.6 3.1 Ϯ 2 86.1 Ϯ 7.5 9.8 Ϯ 5 0.03 Ϯ 0.05 0.2 Ϯ 0.1 A-Class Control ϩ ODN 0.8 Ϯ 0.7 3.6 Ϯ 3.1 83.7 Ϯ 8.7 11.5 Ϯ 6 0.2 Ϯ 0.4 0.1 Ϯ 0.1 A-Class CpG ODN 0.9 Ϯ 0.4 22.8 Ϯ 9.9** 84.7 Ϯ 7.3 75.4 Ϯ 17.8** 0 0

a Expressed as the percentage (mean Ϯ SD) of iNKT cells positive for the molecules CD69, CD38, CD25, CD45RO, CD154, or HLA-DR, detected by flow cytometry in fresh PBMCs or after 24 h of incubation with or without ODNs. iNKT cells were evaluated via the lymphocyte gate, using mAbs against V␤11 and V␣24 or against the CDR3 p Ͻ 0.05 ,ءء .(loop of the invariant ␣-chain (clone 6B11 no significant increase in the secretion of other possible activators of moderate yet equivalent inhibitory effects (Fig. 5B). Neutralization iNKT cells such as IL-7, IL-12, IL-15, IL-18, and IFN-␥ (Table III). of IP-10 and IL-6 had no effect on expression of CD69 by iNKT These results are consistent with other published findings (46). cells. Simultaneous neutralization of all four cytokines had an ad- To evaluate the potential role for IFN-I on iNKT cell activation, ditive effect but was only 40–50% effective at reducing CpG PBMCs were incubated with A-class CpG ODN with/without a ODN-induced expression of CD69. This suggests that IFN-I and mixture of mAbs to block the effect of IFN-I (neutralizing anti- TNF-␣ were important for iNKT activation but other soluble fac- Downloaded from IFN-␣, neutralizing anti-IFN-␤, and neutralizing anti-IFN-I receptor(s) contribute to this process. tor (CD118)). Although incubation with CpG ODNs alone significantly increased the expression of CD69 on iNKT cells, the A-class CpG ODN-mediated activation of purified iNKT cells coincubation with CpG ODN and IFN-I blocking mixture reduced Treatment of PBMCs with CpG ODNs activates several different the expression of CD69 ϫ 45% ( p ϭ 0.024) (Table IV). The effect subpopulations of leukocytes. To clarify the interaction mediated

of this blocking mixture was very heterogeneous in the healthy by soluble factors between CpG ODN-activated pDCs and iNKT http://www.jimmunol.org/ controls evaluated (range of CD69 reduction, 15–67%). As a pos- cells, highly purified populations of cells were isolated using itive control for these experiments, PBMCs were incubated with Miltenyi magnetic beads and an AutoMacs instrument. The puri- rh-IFN-␣ with/without the same blocking mixture. In all cases, fied pDCs were incubated for 24 h with A-class CpG ODN, and the rh-IFN-␣ up-regulated the expression of CD69 (56% Ϯ 15%), pDC-conditioned supernatants were used to treat purified iNKT while the blocking mixture was 95–100% effective at inhibiting cells (Fig. 6A). The incubation of purified iNKT cells with pDC- iNKT cell activation by rh-IFN-␣. This suggested that other cyto- conditioned supernatants resulted in the same up-regulated expres- kines/chemokines/soluble factors were potentially important for sion of CD69 (as well as CD38, data not shown), while the coin- inducing activation markers on iNKT cells. cubation with combinations of neutralizing Abs was again only The contributions of IFN-I and other soluble mediators of CpG partially effective at inhibiting this response (Fig. 6B). It is impor- by guest on September 24, 2021 ODN-induced activation of iNKT cells were confirmed using tant to note that the concentration of cytokines in supernatants PBMC-conditioned supernatants. Fresh PBMCs were incubated from CpG ODN-stimulated purified pDC is lower than in super- 24 h with/without A-class CpG ODN, rh-IFN-␣, PBMC-condi- natants from PBMCs. This is likely a consequence of using the tioned supernatant, and the anti-IFN-I mixture. The conditioned BDCA2 Ab for positive selection of pDC. Although highly selec- medium and rh-IFN-␣ consistently induced higher expression of tive for this cell subset, the Ab inhibits subsetquent in vitro re- CD69 than did optimal doses of CpG ODNs (Fig. 5A). The anti- sponses of pDC to TLR-dependent activation. However, the stim- IFN-I blocking mixture completely inhibited the expression of ulation of purified pDC with CpG still resulted in secretion of the CD69 induced by rh-IFN-␣, and only partially blocked the expres- same cytokines/chemokines as was observed with PBMC-derived sion induced by CpG ODN or PBMC-conditioned supernatant. supernatants (IFN-␣, PBMC ϭ 1,247 ϩ 477 pg/ml, pDC ϭ 449 ϩ Because A-class CpG ODN stimulation of PBMCs also induced 64 pg/ml; IP10, PBMC ϭ 1,681 ϩ 310 pg/ml, pDC ϭ 917 ϩ 466 the secretion of IP-10, IL-6, and TNF-␣ in addition to IFN-␣, pg/ml; TNF-␣, PBMC ϭ 489 ϩ 208 pg/ml pDC ϭ 263 ϩ 79 neutralizing Abs against IP-10, IL-6, and TNF-␣ were used to pg/ml; IL-6, PBMC ϭ 216 ϩ 20 pg/ml, pDC ϭ 167 ϩ 50 pg/ml). evaluate the contributions these cytokines/chemokines in the acti- Thus, the same concentration of neutralizing Abs used for the vation of iNKT cells. Only neutralization of IFN-I and TNF-␣ had PBMC experiments was used for supernatants from purified pDC;

FIGURE 4. Impact of A-class ODNs on cytokine and perforin production by iNKT cells. PBMCs were incubated for 24 h with 4 ␮g/ml A-class ODNs (control and CpG ODN) and the expression by iNKT cells of IFN-g, IL-4, TNF-␣, perforin and CD69 was evaluated by intracellular staining and ﬂow cytometry. As a control for positive activation, the PBMCs were incubated by 6 h with PMA (50 ng/ml) and ionomycin (500 ng/ ml). It is shown that, despite the stimulation with A- class CpG ODN up-regulating the expression of CD69, the incubation with this CpG ODNs did not increase the expression IFN-␥, IL-4, TNF-␣ or perforin by iNKT cells. Data are reported as means Ϯ SD (n ϭ 3). 1034 iNKT CELLS MEDIATE PDC-MYELOID DC CROSS-TALK

Table III. Concentration of cytokines in supernatants from PBMCs ODNs (control and CpG). The purity of iNKT cells was always a cultured with A-class CpG ODN Ͼ85%, while the purity of mDCs and pDCs was Ͼ90% (Fig. 7A). Consistent with the observation that iNKT cells do not express A-Class CpG ODN TLR9, treatment of purified iNKT cells with A-class CpG ODNs Cytokines No Stimuli (4 ␮g/ml) did not result in the up-regulation of CD69 expression (Fig. 7B). IFN-␣ (n ϭ 8) 62 Ϯ 79b 950 Ϯ 408b Similar results were observed when purified iNKT cells and mDCs ϭ Ϯ Ϯ IP-10 (n 8) 189 360 12,107 10,069 were cocultured and incubated without stimulation or with control TNF-␣ (n ϭ 8) 97 Ϯ 111 221 Ϯ 123 IL-6 (n ϭ 8) 67 Ϯ 82 305 Ϯ 145 and A-class CpG ODNs ( p ϭ 0.437 and p ϭ 0.382). When iNKT IL-7 (n ϭ 4) 0 0 cells were cocultured with pDCs and incubated with A-class CpG IL-12 (n ϭ 8) 33 Ϯ 44 30 Ϯ 48 ODNs, the expression of CD69 on iNKT cells significantly in- IL-15 (n ϭ 4) 0 0 IL-18 (n ϭ 4) 132 Ϯ 158 100 Ϯ 122 creased in comparison with unstimulated cells or incubated with IFN-␥ (n ϭ 4) 1 Ϯ 22Ϯ 3 control ODNs ( p ϭ 0.020 and 0.007, respectively) (Fig. 7B). Thus,

a Cytokine concentrations (pg/ml) were measured in culture supernatants after CpG ODN-mediated activation of iNKT cells is pDC dependent, 24 h of incubation with or without A-Class CpG ODNs, using commercial ELISA kits and quiescent iNKT cells fail to respond to unstimulated mDCs (detection limit, 3.5 pg/ml). when purified and cultured together in vitro. b Mean Ϯ SD. iNKT cells are strongly activated by TCR-mediated CD1d-restricted signals, particularly when mDCs are used as APCs. It also ␣ because the concentration of secreted IFN- and other cytokines is well known that ␣-GalCer is presented by CD1d and a potent Downloaded from was lower than that seen in PBMC, it is less likely that insufficient activator of iNKT cells (49, 50). To determine whether pDC-de- Abs were used. Hence, alleviating the concern that insufficient rived conditioned medium could license iNKT cells to respond to neutralization occurred. Using this reconstituted system neutral- mDCs, purified iNKT cells were treated with conditioned or con- ization by anti-IFN-I was modestly more effective when compared trol medium and then cocultured with mDCs plus increasing doses with the ability to inhibit the effect of conditioned medium pre- of ␣-GalCer. First, purified iNKT cells were incubated with/with- pared from CpG ODN-treated PBMCs. As was seen using condi- out pDC-conditioned supernatant (dilution 1/2 in complete culture http://www.jimmunol.org/ tioned medium from CpG ODN-treated PBMCs, maximal block- medium), and after 24 h of incubation, iNKT cells were washed, ade of pDC-derived conditioned medium was only 50% effective and cocultured with purified mDC with/without ␣-GalCer (0, 10, and there was no contribution by neutralization of IP-10 and IL-6. 20, and 50 ng/ml). Strikingly, only those iNKT cells that had been Thus, the induction of activation markers on iNKT cells by CpG primed with conditioned medium from CpG ODN-treated pDCs ODNs can be reproduced using highly purified pDC and iNKT cell were able to respond to ␣-GalCer-loaded mDCs. These preacti- systems suggesting that the active factors are released directly by vated iNKT cells acquired the ability to respond to ␣-GalCer in a pDCs. However, it should be noted that there might still be some ␥ contribution to this effect by rare contaminating cells (4). dose-dependent fashion by secreting IFN- , and to much lesser

extent, IL-4, and vigorously proliferating (Fig. 8). In comparison, by guest on September 24, 2021 iNKT cells activated in a pDC-dependent manner are licensed iNKT cells incubated with conditioned medium from control to interact with mDC ODN-incubated pDCs did not secrete cytokines or proliferate. Recent results indicate that mDC and iNKT cell cross-talk is im- Thus, soluble factors produced by CpG ODN-stimulated pDCs portant and that mDCs need to be activated in a TLR-dependent enhance the CD1d-restricted activation of iNKT cells and licensed fashion to present self or foreign Ag to quiescent iNKT cells (33, these T cells to interact with mDCs. 47, 48) Interestingly, human mDCs, but not pDCs express CD1d Because CpG ODN enhanced iNKT cell responses to ␣-GalCer- (4, 49) To evaluate the contribution of CD1d to CpG ODN-medi- loaded mDC and TLR4 ligands have been shown to directly en- ated iNKT cell activation, highly puriﬁed subpopulations of pDCs, hance CD1d-dependent Ag presentation to iNKT cells (33), the mDCs, and iNKT cells were isolated and cocultured with/without ability of ODNs to support expansion and survival of iNKT cells

Table IV. Role of IFN-I on A-Class CpG ODN-mediated activation of iNKT cells

Percentage of iNKT Cells Expressing CD69

Frequency of iNKT No A-Class A-Class ODN Reduction of Individual cells (%) stimulation CpG ODN and Anti-IFN-I CD69 expression

1 0.09 3.9 59.5 36.7 41.0% 2 0.10 9.5 60.1 35.9 47.8% 3 0.10 4.9 33.3 16.8 58.1% 4 0.12 3.6 41.8 19.6 58.2% 5 0.15 8.2 48.3 21.4 67.1% 6 0.20 4.6 30.4 15.2 58.9% 7 0.22 5.5 72.5 62.6 14.8% 8 0.27 2.2 27.2 21.1 24.4% 9 0.29 2 48.4 32.3 34.6% Mean (Ϯ SD) 0.17 (Ϯ 0.08) 4.9 (Ϯ 2.5) 46.8 (Ϯ 15.3) 29.1 (Ϯ 15) 44.9% (Ϯ 17.7)

The percentage of iNKT cells positive for CD69 was determined by ﬂow cytometry. The iNKT cells were evaluated via the lymphocyte gate, using mAbs against V␤11 and V␣24 or against CD3 and the invariant ␣-chain (clone 6B11). The reduction of CD69 expression by the IFN-I blocking mixture was calculated with the following formula:

(% CpG ODN ϩ anti-IFN-I) Ϫ (% No stimulation) ͩ1Ϫ ͪ ϫ 100 (% A-Class CpG ODN alone) Ϫ (% No stimulation) The Journal of Immunology 1035

FIGURE 5. Role of cytokines on the A-class CpG ODN-mediated activation of iNKT cells. A, PBMCs were incubated 24 h with A-class CpG ODN

(4 ␮g/ml), rh-IFN-␣ (5,000 U/ml) or supernatant from CpG ODN-stimulated PBMCs (dilution 1/2 with complete culture medium); the activation mediated Downloaded from by IFN-I was blocked with a combination of anti-IFN-␣ (10,000 U/ml), anti-IFN-␤ (3,000 U/ml) and anti-CD118 (10 ␮g/ml). Then, the expression of CD69 on iNKT cells was evaluated by flow cytometry, gating on the iNKT cells with the mAbs 6B11 and anti-CD3. Blocking the IFN-I partially inhibited the expression of CD69 induced by CpG ODNs or PBMC-conditioned supernatant. Data are reported as means Ϯ SD (n ϭ 3). B, PBMCs cultured with A-class CpG ODNs were coincubated with neutralizing Abs against IFN-a (10,000 U/ml), TNF-␣ (10 ␮g/ml), IL-6 (0. 1 ␮g/ml), and IP-10 (10 ␮g/ml). After 24 h, the expression of CD69 on iNKT cells was determined by flow cytometry. The neutralization of IFN-␣ and TNF-␣ significantly decreased the CpG ODN-induced CD69 expression, while there was an additive effect observed when all the neutralizing Abs were used in combination. Data are reported as means Ϯ SD (n ϭ 3). http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. Neutralization of IFN-␣ and TNF-␣ partially inhibits the expression of CD69 on purified iNKT cells incubated with supernatants from CpG ODN-stimulated purified pDCs. A, The iNKT cells and pDCs were purified from PBMCs using anti-6B11 PE plus anti-PE microbeads or anti-BDCA-2 microbeads, respectively, and the POSSELD program of AutoMACS. In these representative dot plots, the percentage of pDCs and iNKT cells is shown in the positive fraction after the magnetic isolation. B, To obtain pDC-conditioned supernatants, pDCs were purified with anti-BDCA-2 microbeads and incubated by 24 h with A-class CpG ODN; these supernatants were collected, filtered (0. 22 ␮m), and used for the coincubation with purified iNKT cells and neutralizing Abs against IFN-␣ (10000 U/ml), TNF-␣ (10 ␮g/ml), IL-6 (0.1 ␮g/ml), and/or IP-10 (10 ␮g/m). The incubation of purified iNKT cells with pDC- conditioned supernatants up-regulated the expression of CD69, which was partially blocked by the neutralization of IFN-␣ and TNF-␣. This graphic shows representative data from one of three independent experiments. 1036 iNKT CELLS MEDIATE PDC-MYELOID DC CROSS-TALK Downloaded from http://www.jimmunol.org/

FIGURE 7. Role of ODNs on the activation of purified iNKT cells cocultured with purified DCs. A, The pDCs, mDCs and iNKT cells were purified from PBMCs using anti-BDCA-2 microbeads, anti-BDCA-1 microbeads, and anti-6B11 PE and anti-PE microbeads, respectively, and POSSELD program of AutoMACS. In these representative dot plots, it is shown the percentage of these leukocyte subpopulations in whole PBMCs, the negative fraction,and by guest on September 24, 2021 in the positive fraction, before and after the magnetic isolation, respectively. B, The purified iNKT cells were cocultured 24 h with/without purified mDCs, purified pDCs and A-class ODNs (control and CpG), and the expression of CD69 on iNKT cells was determined by flow cytometry. Just the coculture of purified iNKT cells with purified pDCs and incubated with A-class CpG ODN increased significantly the expression of CD69 on iNKT cells. These dot plots are representative of one of four independent experiments.

was evaluated. PBMCs were stimulated with ␣-GalCer in the pres- of the innate and adaptive immune systems, including the induc- ence or absence of CpG ODN or LPS, with or without depletion of tion of mDC maturation (4). (23, 24). Over the next 48–72 h, in a pDC and B cells (Fig. 9). Interestingly, activation of iNKT cells in process that is dependent on autocrine stimulation by IFN-I, pDCs the presence of CpG resulted in the preferential expansion and differentiate into mature DCs capable of stimulating T cells (16). long-term survival of DN iNKT cells. After deletion of pDC and The pDC-dependent T cell effects are thought to be dependent on B cells, only CD4ϩ iNKT cells expanded in response to ␣-GalCer. IFN-I but independent of IL-12. In human DCs, the main source of And, as expected, the deletion of TLR9-responding cells had a IL-12 important for activating NK cells and inducing Th1-like T significant impact on the ability of iNKT cells to respond to ␣-Gal- cell responses is thought to be mDCs (53, 54) Consequently, as is Cer. It also should be noted that, consistent with the results of Brigl the case with TLR profiles, there appears to be clear species dif- et al. (33), LPS induced the most robust expansion of CD4ϩ and ferences between human and murine pDC and mDC effector func- DN iNKT cells (data not shown), but these cells failed to survive tion (4, 9). However, the pDC lineage is unique in the capacity to in long-term cultures (Fig. 9B). secrete massive amounts of IFN-I. The cross-talk between pDCs and mDCs and the ability of pDCs Discussion to induce adaptive T cell responses by activating mDCs have re- Freshly isolated resting pDCs express low levels of MHC class I cently been demonstrated (23, 24, 51, 55). Treatment of immature and II and CD86 and do not express detectable levels of CD80 or mDCs with IFN-I leads to their activation and enhanced produc- CD1d. In humans, these cells express CD4 and CD123 but lack the tion of IL-12, IL-15, and IL-18 (56, 57). A similar IFN-I-depen- expression of myeloid markers, such as CD11b, CD11c, CD13, dent or CpG-dependent effect has been noted for NK cell activa- and CD33, and are found in blood and secondary lymph organs tion (55, 58). (4). These cells participate in innate responses to several different Interestingly, iNKT cell activation is important for the same types of viruses, including influenza, HSV-family viruses, and mDC maturation, NK activation, and antiviral or antitumor re- HIV (24). (51, 52) pDCs rapidly produce vast amounts of IFN-I sponses (28, 29, 31, 32, 36, 59–63) Yet, iNKT cells do not express within the first 24 h of viral infection (21, 22) This burst of cyto- detectable levels of TLRs, do not respond directly to CpG ODNs kine secretion is thought to be critical for activation of other cells even in the presence of highly purified mDCs (Fig. 7), and human The Journal of Immunology 1037

FIGURE 8. Role of CpG ODN-activated pDCs on the CD1d-restricted activation of iNKT cells. A, Purified iNKT cells were incubated by 24 h with/without supernatants from CpG ODN-stimulated pDCs (dilution 1/2); after, these preincubated iNKT cells were washed and cocultured by triplicate with purified mDCs previously incubated by 24 h with/without ␣-GalCer (0, 10, 20, and 50 ng/ml). After 24 h of coculture, supernatants (100 ␮l per well) were collected and replaced with fresh complete culture medium. The concentration of IFN-␥ and IL-4 was measured in theses supernatants by ELISA; Downloaded from there was an increase in the secretion of IFN-g and IL-4 when the iNKT cells were preincubated with pDC-conditioned supernatants, and in this condition of stimulation it was observed a dose-response effect for the production of IFN-␥ regarding the concentration of ␣-GalCer used for the incubation with purified mDCs. This graphic shows representative data from one of four independent experiments. B, Twenty-four hours after the collection of supernatants, 1 ␮Ci of [3H]thymidine was added to each well and, after an incubation overnight, the cell culture plate was harvested and the proliferation of the iNKT cells was determined measuring the cpm in a beta counter. In this representative figure of four independent experiments, it is shown that, when purified iNKT cells were preincubated with pDC-conditioned supernatant, there was an increase in the proliferation of iNKT cocultured with ␣-GalCer-loaded mDCs. http://www.jimmunol.org/ pDCs and/or lymphoid DCs do not express CD1d (14, 49, 64, 65). most pronounced for the pDC-selective A-class CpG ODNs (Ta- Activation of iNKT cells by CpG ODNs has been reported to be bles I and II) (38, 68). Even though CpG ODN treatment resulted IL-12 and IL-15 dependent (60, 66, 67). However, despite the in the induction of activation markers on iNKT cells to the same significant congruent involvement of iNKT cells and pDCs in extent as PMA/ionomycin, no cell division or cytokine secretion these responses, the consequences of pDC activation on iNKT cell was noted. The activation of iNKT cells was partially dependent function has not been investigated and is poorly understood. on IFN-I and TNF-␣. However, it was noted that other soluble Thus, to determine whether pDC activation can control iNKT factors are likely to be involved. Although the conditioned medium cell function, a study of TLR9-dependent activation with CpG and Ab blockade experiments support the notion that soluble me- by guest on September 24, 2021 ODNs was undertaken. Treatment of PBMCs with CpG ODNs diator of the CpG response can preactivate iNKT cells, this does strongly induced activation markers on iNKT cells. This effect was not preclude an important role for cell-to-cell contact. It is likely

FIGURE 9. Frequency of expanded iNKT cell subgroups in culture of PBMCs activated with ␣-GalCer ODNs. A, Effect of ODNs on iNKT cells subsets after ␣-GalCer activation in the presence or absence of pDC and B cells. Whole PBMC, or PBMC where pDC and B cells were removed using anti-BDCA-2 and anti-CD19 microbeads, were labeled with CFSE and cultured for 8 days with ␣-GalCer (100 ng/ml) with or without ODNs. Cells were stained with 6B11-PE and CD4-APC to enu- merate iNKT cell subsets, and gated on 6B11ϩ cells to determine the extent of cell division by CFSE dilution. The data are representative of three separate experiments. B, Activation of iNKT cells with ␣-GalCer and ODNs results in selective long-term survival of DN iNKT cells. PBMC were activated with control IL-2 (20 U/ml), IL-2, and ␣-GalCer ϩ ODNs, or IL-2 and ␣-Gal- Cer ϩ LPS (1 ␮g/ml) and cultured for 21 days. Cells were supplemented with IL-2 and fresh medium every seventh day. The data are representative of four separate experiments. 1038 iNKT CELLS MEDIATE PDC-MYELOID DC CROSS-TALK that direct cell contact with pDC, in part mediated by OX40/ 4. Liu, Y. J. 2005. IPC: professional type 1 interferon-producing cells and plasma- OX40L, contributes to the activation of iNKT following CpG ex- cytoid dendritic cell precursors. Annu. Rev. Immunol. 23: 275–306. 5. Steinman, R. M., and M. C. Nussenzweig. 2002. Inaugural Article: Avoiding posure (69). Marschner et al. (69) examined the consequences of horror autotoxicus: the importance of dendritic cells in peripheral T cell toler- pDC and iNKT interactions, and their results are in agreement with ance. Proc. Natl. Acad. Sci. USA 99: 351–358. respect to licensing of iNKT cells, role of IFN-I, and subsequent 6. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21: 685–711. activation by mDCs. However, these authors found that cell-cell 7. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, contact, including pDCs that do not express detectable CD1d, was B. Pulendran, and K. Palucka. Immunobiology of dendritic cells. 2000. Annu. necessary for the effect. In the results presented in this study, li- Rev. Immunol. 18: 767–811. 8. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. censing of iNKT cells to recognize CD1d on mDCs did not require Rev. Immunol. 20: 197–216. cell-cell contact of iNKT cells with pDC. These differences may be 9. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive the result of different experimental protocols. Marschner et al. (69) immune responses. Nat Immunol. 5: 987–995. 10. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of admixed in semi pure iNKT cells, selected on the basis of V␣24 immunity. Nature 392: 245–252. expression, into PBMC to levels of 10% whereas either PBMC or 11. Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, and Y. J. Liu. 1999. Reciprocal control of T helper cell and reconstitution experiments were used in this study. dendritic cell differentiation [see comments]. Science 283: 1183–1186. It is unlikely that IL-6, IL-7, IL-12, IL-15, IL-18, or IFN-␥ made 12. Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, and A. any signiﬁcant contribution to the induction of activation markers O’Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose on iNKT cells, because these cytokines were absent or not induced and differential toll-like receptor ligation. J. Exp. Med. 197: 101–109. when comparing A-class CpG with control ODN treatment (Table 13. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat Rev. Immunol.

III and Figs. 5 and 6). Importantly, exposure of iNKT cells to 1: 135–145. Downloaded from ␣ 14. Liu, Y. J., H. Kanzler, V. Soumelis, and M. Gilliet. 2001. Dendritic cell lineage, IFN-I, TNF- , and soluble factors licensed these T cells to respond plasticity and cross-regulation. Nat. Immunol. 2: 585–589. to Ag-loaded mDCs (Figs. 7 and 8), and resulted in the preferential 15. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. expansion and survival of DN iNKT cells (Fig. 9). Despite the Rev. Immunol. 2: 151–161. 16. Kadowaki, N., S. Antonenko, J. Y. Lau, and Y. J. Liu. 2000. Natural interferon expression of CD1d, the in vivo activation of iNKT cells by mDCs ␣␤-producing cells link innate and adaptive immunity. J. Exp. Med. 192: presenting endogenous self-Ags does not occur unless the DCs are 219–226. 17. Kadowaki, N., S. Ho, S. Antonenko, R. W. Malefyt, R. A. Kastelein, F. Bazan, activated by either direct bacterial contact, by activation of TLRs http://www.jimmunol.org/ and Y. J. Liu. 2001. Subsets of human dendritic cell precursors express different (33, 47, 48, 70, 71). Consistent with these observations, freshly toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194: isolated human iNKT cells were refractory to TCR-dependent 863–869. stimulation. This state could be overcome by exposure to pDC- 18. Sallusto, F., A. Lanzavecchia, and C. R. Mackay. 1998. Chemokines and che- mokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. derived conditioned medium. Therefore, it seems reasonable to Today 19: 568–574. suggest that in addition to direct effects of pDC-derived cytokines 19. Sallusto, F., and A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, on mDCs, a cross-talk with mDCs mediated by iNKT cells would priming, and chronic inflammation [comment]. J. Exp. Med. 189: 611–614. 20. Randolph, G. J., S. Beaulieu, S. Lebecque, R. M. Steinman, and W. A. Muller. make significant contributions to the outcome of immune re- 1998. Differentiation of monocytes into dendritic cells in a model of transendo- sponses following pDC activation. Reciprocal activation of NK thelial trafficking [see comments]. Science 282: 480–483. cells, mDCs, and pDCs has been reported before. We have previ- 21. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, by guest on September 24, 2021 ϩ S. Ho, S. Antonenko, and Y. J. Liu. 1999. The nature of the principal type 1 ously argued that the ratio of CD4 to DN iNKT cell function has interferon-producing cells in human blood. Science 284: 1835–1837. important consequences for controlling the outcome of an immune 22. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, response and that a significant component of this regulation occurs and M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon [see comments]. Nat. Med. by controlling the effector function of mDC (30). It is now appre- 5: 919–923. ciated that CD4ϩ iNKT cells preferentially subserve immunoregu- 23. Asselin-Paturel, C., G. Brizard, K. Chemin, A. Boonstra, A. O’Garra, A. Vicari, and G. Trinchieri. 2005. Type I interferon dependence of plasmacytoid dendritic lary functions and are significant contributors to asthma and atopic cell activation and migration. J. Exp. Med. 201: 1157–1167. disorders (72–76). In contrast, DN iNKT cells are thought to be the 24. Fonteneau, J. F., M. Larsson, A. S. Beignon, K. McKenna, I. Dasilva, A. Amara, effectors of iNKT cell-dependent tumor and anti-viral responses Y. J. Liu, J. D. Lifson, D. R. Littman, and N. Bhardwaj. 2004. Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomi- (58, 77–79). Notably, iNKT cells are important for the control of tantly induces the bystander maturation of myeloid dendritic cells. J. Virol. 78: the HSV family of viruses (37, 60, 61, 80). Moreover, two separate 5223–5232. immunodeficiencies characterized by a specific loss of iNKT cells 25. Le Bon, A., N. Etchart, C. Rossmann, M. Ashton, S. Hou, D. Gewert, P. Borrow, and D. F. Tough. 2003. Cross-priming of CD8ϩ T cells stimulated by virus- with a concomitant susceptibility to HSV family infections have induced type I interferon. Nat. Immunol. 4: 1009–1015. been reported (81–84). Given that CpG are considered to be useful 26. Krug, A., A. Towarowski, S. Britsch, S. Rothenfusser, V. Hornung, R. Bals, therapeutic approaches for these diseases, it seems reasonable to T. Giese, H. Engelmann, S. Endres, A. M. Krieg, and G. Hartmann. 2001. Toll- like receptor expression reveals CpG DNA as a unique microbial stimulus for speculate that CpG regulation of iNKT cell subset function con- plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high tributes to their mechanism of action. amounts of IL-12. Eur. J. Immunol. 31: 3026–3037. 27. Hartmann, G., G. J. Weiner, and A. M. Krieg. 1999. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Disclosures Sci. USA 96: 9305–9310. A. M. Krieg is Senior Vice President, R&D CSO, and cofounder of Coley 28. Yang, O. O., F. K. Racke, P. T. Nguyen, R. Gausling, M. E. Severino, Pharmaceutical Group, which provided the CpGs used in this study. Dr. Krieg H. F. Horton, M. C. Byrne, J. L. Strominger, and S. B. Wilson. 2000. CD1d on is an active scientific collaborator on this work and National Institutes of myeloid dendritic cells stimulates cytokine secretion and cytolytic activity of V␣24J␣Q T cells: a feedback mechanism for immune regulation. J. Immunol. Health Program Project Grant PO1 AI055793, which funded this work. 165: 3756–3762. 29. Gillessen, S., Y. N. Naumov, E. E. Nieuwenhuis, M. A. Exley, F. S. Lee, References N. Mach, A. D. Luster, R. S. Blumberg, M. Taniguchi, S. P. Balk, et al. 2003. 1. Steinman, R. M., L. Bonifaz, S. Fujii, K. Liu, D. Bonnyay, S. Yamazaki, M. Pack, CD1d-restricted T cells regulate dendritic cell function and antitumor immunity D. Hawiger, T. Iyoda, K. Inaba, and M. C. Nussenzweig. 2005. The innate functions in a granulocyte-macrophage colony-stimulating factor-dependent fashion. Proc. of dendritic cells in peripheral lymphoid tissues. Adv. Exp. Med. Biol. 560: 83–97. Natl. Acad. Sci. USA 100: 8874–8879. 2. Fujii, S., K. Liu, C. Smith, A. J. Bonito, and R. M. Steinman. 2004. The linkage 30. Wilson, S. B., and T. L. Delovitch. 2003. Janus-like role of regulatory iNKT cells of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 in autoimmune disease and tumor immunity. Nat. Rev. Immunol. 3: 211–222. ligation in addition to antigen presentation and CD80/86 costimulation. J. Exp. 31. Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, and R. M. Steinman. 2003. Activation of Med. 199: 1607–1618. natural killer T cells by ␣-galactosylceramide rapidly induces the full maturation of 3. Colonna, M., G. Trinchieri, and Y. J. Liu. 2004. Plasmacytoid dendritic cells in dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 immunity. Nat. Immunol. 5: 1219–1226. T cell immunity to a coadministered protein. J. Exp. Med. 198: 267–279. The Journal of Immunology 1039

32. Smyth, M. J., N. Y. Crowe, D. G. Pellicci, K. Kyparissoudis, J. M. Kelly, dritic cells profoundly affects innate resistance functions. J. Immunol. 174: K. Takeda, H. Yagita, and D. I. Godfrey. 2002. Sequential production of inter- 727–734. feron-␥ by NK1.1ϩ T cells and natural killer cells is essential for the antimeta- 59. Vincent, M. S., D. S. Leslie, J. E. Gumperz, X. Xiong, E. P. Grant, and static effect of ␣-galactosylceramide. Blood 99: 1259–1266. M. B. Brenner. 2002. CD1-dependent dendritic cell instruction. Nat. Immunol. 3: 33. Brigl, M., L. Bry, S. C. Kent, J. E. Gumperz, and M. B. Brenner. 2003. Mech- 1163–1168. anism of CD1d-restricted natural killer T cell activation during microbial infec- 60. Gill, N., K. L. Rosenthal, and A. A. Ashkar. 2005. NK and NKT cell-independent tion. Nat. Immunol. 4: 1230–1237. contribution of interleukin-15 to innate protection against mucosal viral infection. 34. Munz, C., R. M. Steinman, and S. Fujii. 2005. Dendritic cell maturation by innate J. Virol. 79: 4470–4478. lymphocytes: coordinated stimulation of innate and adaptive immunity. J. Exp. 61. Ashkar, A. A., and K. L. Rosenthal. 2003. Interleukin-15 and natural killer and Med. 202: 203–207. NKT cells play a critical role in innate protection against genital herpes simplex 35. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, virus type 2 infection. J. Virol. 77: 10168–10171. M. Kanno, and M. Taniguchi. 1997. Requirement for V␣14 NKT cells in IL-12- 62. Hafner, M., R. Zawatzky, C. Hirtreiter, W. A. Buurman, B. Echtenacher, mediated rejection of tumors. Science 278: 1623–1626. T. Hehlgans, and D. N. Mannel. 2001. Antimetastatic effect of CpG DNA me- 36. Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, and diated by type I IFN. Cancer Res. 61: 5523–5528. A. Bendelac. 1999. Cutting edge: Cross-talk between cells of the innate immune 63. Suzuki, Y., D. Wakita, K. Chamoto, Y. Narita, T. Tsuji, T. Takeshima, H. Gyobu, system: NKT cells rapidly activate NK cells. J. Immunol. 63: 4647–4650. Y. Kawarada, S. Kondo, S. Akira, et al. 2004. Liposome-encapsulated CpG oli- 37. Skold, M., and S. M. Behar. 2003. Role of CD1d-restricted NKT cells in micro- godeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity. bial immunity. Infect. Immun. 71: 5447–5455. Cancer Res. 64: 8754–8760. 38. Ulevitch, R. J. 2004. Therapeutics targeting the innate immune system. Nat. Rev. 64. Brigl, M., and M. B. Brenner. 2004. CD1: antigen presentation and T cell func- Immunol. 4: 512–520. tion. Annu. Rev. Immunol. 22: 817–890. 39. Godfrey, D. I., K. J. Hammond, L. D. Poulton, M. J. Smyth, and A. G. Baxter. 65. Kadowaki, N., S. Antonenko, S. Ho, M. C. Rissoan, V. Soumelis, S. A. Porcelli, 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21: 573–583. L. L. Lanier, and Y. J. Liu. 2001. Distinct cytokine proﬁles of neonatal natural killer 40. Hou, R., O. Goloubeva, D. S. Neuberg, J. L. Strominger, and S. B. Wilson. 2003. t cells after expansion with subsets of dendritic cells. J. Exp. Med. 193: 1221–1226. Interleukin-12 and interleukin-2-induced invariant natural killer T-cell cytokine 66. Ashkar, A. A., and K. L. Rosenthal. 2002. Toll-like receptor 9, CpG DNA and secretion and perforin expression independent of T-cell receptor activation. Im- innate immunity. Curr Mol. Med. 2: 545–556. munology 110: 30–37. 67. Ikeda, H., K. Chamoto, T. Tsuji, Y. Suzuki, D. Wakita, T. Takeshima, and Downloaded from 41. Leite-De-Moraes, M. C., A. Hameg, A. Arnould, F. Machavoine, Y. Koezuka, T. Nishimura. 2004. The critical role of type-1 innate and acquired immunity in E. Schneider, A. Herbelin, and M. Dy. 1999. A distinct IL-18-induced pathway tumor immunotherapy. Cancer Sci. 95: 697–703. to fully activate NK T lymphocytes independently from TCR engagement. J. Im- 68. Klinman, D. M. 2004. Immunotherapeutic uses of CpG oligodeoxynucleotides. munol. 163: 5871–5876. Nat. Rev. Immunol. 4: 249–258. 42. Iho, S., T. Yamamoto, T. Takahashi, and S. Yamamoto. 1999. Oligodeoxynucle- 69. Marschner, A., S. Rothenfusser, V. Hornung, D. Prell, A. Krug, M. Kerkmann, otides containing palindrome sequences with internal 5Ј-CpG-3Ј act directly on D. Wellisch, H. Poeck, A. Greinacher, T. Giese, et al. 2005. CpG ODN enhance human NK and activated T cells to induce IFN production in vitro. J. Immunol. antigen-speciﬁc NKT cell activation via plasmacytoid dendritic cells. Eur. J. Im-

163: 3642–3652. munol. 35: 2347–2357. http://www.jimmunol.org/ 43. Sun, S., and J. Sprent. 2000. Role of type I interferons in T cell activation induced 70. Wu, D. Y., N. H. Segal, S. Sidobre, M. Kronenberg, and P. B. Chapman. 2003. by CpG DNA. Curr Top Microbiol. Immunol. 247: 107–117. Cross-presentation of disialoganglioside GD3 to natural killer T cells. J. Exp. 44. Kranzer, K., M. Bauer, G. B. Lipford, K. Heeg, H. Wagner, and R. Lang. 2000. Med. 198: 173–181. CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-␥ pro- 71. Baev, D. V., X. H. Peng, L. Song, J. R. Barnhart, G. M. Crooks, K. I. Weinberg, and duction and up-regulation of CD69 via induction of antigen-presenting cell-de- L. S. Metelitsa. 2004. Distinct homeostatic requirements of CD4ϩ and CD4Ϫ subsets rived interferon type I and interleukin-12. Immunology 99: 170–178. of V␣24-invariant natural killer T cells in humans. Blood 104: 4150–4156. 45. Rothenfusser, S., V. Hornung, A. Krug, A. Towarowski, A. M. Krieg, S. Endres, 72. Sonoda, K. H., D. E. Faunce, M. Taniguchi, M. Exley, S. Balk, and and G. Hartmann. 2001. Distinct CpG oligonucleotide sequences activate human J. Stein-Streilein. 2001. NK T cell-derived IL-10 is essential for the differentia- ␥␦ T cells via interferon-␣␤. Eur. J. Immunol. 31: 3525–3534. tion of antigen-speciﬁc T regulatory cells in systemic tolerance. J. Immunol. 166: 46. Krieg, A. M. 2002. CpG motifs in bacterial DNA and their immune effects. Annu. 42–50. Rev. Immunol. 20: 709–760. 73. Terabe, M., S. Matsui, N. Noben-Trauth, H. Chen, C. Watson, D. Donaldson,

47. Kinjo, Y., D. Wu, G. Kim, G. W. Xing, M. A. Poles, D. D. Ho, M. Tsuji, D. Carbone, W. Paul, and J. Berzofsky. 2001. NKT cell-mediated repression of by guest on September 24, 2021 K. Kawahara, C. H. Wong, and M. Kronenberg. 2005. Recognition of bacterial tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nature Im- glycosphingolipids by natural killer T cells. Nature 434: 520–525. munology 1: 515–520. 48. Mattner, J., K. L. Debord, N. Ismail, R. D. Goff, C. Cantu III, D. Zhou, 74. Gumperz, J. E., S. Miyake, T. Yamamura, and M. B. Brenner. 2002. Functionally P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, et al. 2005. Exogenous and endog- distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tet- enous glycolipid antigens activate NKT cells during microbial infections. Nature ramer staining. J. Exp. Med. 195: 625–636. 434: 525–529. 75. Akbari, O., J. L. Faul, E. G. Hoyte, G. J. Berry, J. Wahlstrom, M. Kronenberg, 49. Porcelli, S. A., and R. L. Modlin. 1999. The CD1 system: antigen-presenting R. H. DeKruyff, and D. T. Umetsu. 2006. CD4ϩ invariant T-cell-receptorϩ nat- molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol. ural killer T cells in bronchial asthma. N. Engl. J. Med. 354: 1117–1129. 17: 297–329. 76. Campos, R. A., M. Szczepanik, A. Itakura, M. Akahira-Azuma, S. Sidobre, 50. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, M. Kronenberg, and P. W. Askenase. 2003. Cutaneous immunization rapidly R. Nakagawa, H. Sato, E. Kondo, et al. 1997. CD1d-restricted and TCR-mediated activates liver invariant V␣14 NKT cells stimulating B-1 B cells to initiate T cell activation of v␣14 NKT cells by glycoceramides. Science 278: 1626–1629. recruitment for elicitation of contact sensitivity. J. Exp. Med. 198: 1785–1796. 51. Fonteneau, J. F., M. Gilliet, M. Larsson, I. Dasilva, C. Munz, Y. J. Liu, and 77. Crowe, N. Y., J. M. Coquet, S. P. Berzins, K. Kyparissoudis, R. Keating, N. Bhardwaj. 2003. Activation of influenza virus-specific CD4ϩ and CD8ϩ T D. G. Pellicci, Y. Hayakawa, D. I. Godfrey, and M. J. Smyth. 2005. Differential cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood antitumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 202: 101: 3520–3526. 1279–1288. 52. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki. 2003. Toll-like re- 78. Fleuridor, R., B. Wilson, R. Hou, A. Landay, H. Kessler, and L. Al-Harthi. 2003. ceptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid den- CD1d-restricted natural killer T cells are potent targets for human immunodefi- dritic cells. J. Exp. Med. 198: 513–520. ciency virus infection. Immunology 108: 3–9. 53. Ito, T., R. Amakawa, M. Inaba, T. Hori, M. Ota, K. Nakamura, M. Takebayashi, 79. Sandberg, J. K., N. M. Fast, E. H. Palacios, G. Fennelly, J. Dobroszycki, M. Miyaji, T. Yoshimura, K. Inaba, and S. Fukuhara. 2004. Plasmacytoid den- P. Palumbo, A. Wiznia, R. M. Grant, N. Bhardwaj, M. G. Rosenberg, and dritic cells regulate Th cell responses through OX40 ligand and type I IFNs. D. F. Nixon. 2002. Selective loss of innate CD4ϩ V␣ 24 natural killer T cells in J. Immunol. 172: 4253–4259. human immunodeficiency virus infection. J. Virol. 76: 7528–7534. 54. Ito, T., R. Amakawa, T. Kaisho, H. Hemmi, K. Tajima, K. Uehira, Y. Ozaki, 80. Grubor-Bauk, B., A. Simmons, G. Mayrhofer, and P. G. Speck. 2003. Impaired H. Tomizawa, S. Akira, and S. Fukuhara. 2002. Interferon-␣ and interleukin-12 clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells are induced differentially by Toll-like receptor 7 ligands in human blood dendritic expressing the semivariant V␣ 14-J␣ 281 TCR. J. Immunol. 170: 1430–1434. cell subsets. J. Exp. Med. 195: 1507–1512. 81. Levy, O., J. S. Orange, P. Hibberd, S. Steinberg, P. LaRussa, A. Weinberg, 55. Dalod, M., T. Hamilton, R. Salomon, T. P. Salazar-Mather, S. C. Henry, S. B. Wilson, A. Shaulov, G. Fleisher, R. S. Geha, et al. 2003. Disseminated J. D. Hamilton, and C. A. Biron. 2003. Dendritic cell responses to early murine varicella infection due to the vaccine strain of varicella-zoster virus, in a patient cytomegalovirus infection: subset functional specialization and differential reg- with a novel deficiency in natural killer T cells. J. Infect. Dis. 188: 948–953. ulation by interferon ␣␤. J. Exp. Med. 197: 885–898. 82. Pasquier, B., L. Yin, M. C. Fondaneche, F. Relouzat, C. Bloch-Queyrat, 56. Luft, T., K. C. Pang, E. Thomas, P. Hertzog, D. N. Hart, J. Trapani, and J. Cebon. N. Lambert, A. Fischer, G. de Saint-Basile, and S. Latour. 2005. Defective NKT 1998. Type I IFNs enhance the terminal differentiation of dendritic cells. J. Im- cell development in mice and humans lacking the adapter SAP, the X-linked munol. 161: 1947–1953. lymphoproliferative syndrome gene product. J. Exp. Med. 201: 695–701. 57. Nagai, T., O. Devergne, T. F. Mueller, D. L. Perkins, J. M. van Seventer, and 83. Nichols, K. E., C. S. Ma, J. L. Cannons, P. L. Schwartzberg, and S. G. Tangye. G. A. van Seventer. 2003. Timing of IFN-␤ exposure during human dendritic cell 2005. Molecular and cellular pathogenesis of X-linked lymphoproliferative dis- maturation and naive Th cell stimulation has contrasting effects on Th1 subset ease. Immunol. Rev. 203: 180–199. generation: a role for IFN-␤-mediated regulation of IL-12 family cytokines and 84. Nichols, K. E., J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons, IL-18 in naive Th cell differentiation. J. Immunol. 171: 5233–5243. S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky, and P. L. Stein. 2005. Reg- 58. Gerosa, F., A. Gobbi, P. Zorzi, S. Burg, F. Briere, G. Carra, and G. Trinchieri. ulation of NKT cell development by SAP, the protein defective in XLP. Nat. 2005. The reciprocal interaction of NK cells with plasmacytoid or myeloid den- Med. 11: 340–345.