Distinct APCs Explain the Bias of α -Galactosylceramide Variants In Vivo Li Bai, Michael G. Constantinides, Seddon Y. Thomas, Rachel Reboulet, Fanyong Meng, Frank Koentgen, Luc This information is current as Teyton, Paul B. Savage and Albert Bendelac of October 1, 2021. J Immunol 2012; 188:3053-3061; Prepublished online 5 March 2012; doi: 10.4049/jimmunol.1102414 http://www.jimmunol.org/content/188/7/3053 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2012/03/05/jimmunol.110241 Material 4.DC1

References This article cites 46 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/7/3053.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Distinct APCs Explain the Cytokine Bias of a-Galactosylceramide Variants In Vivo

Li Bai,*,†,1 Michael G. Constantinides,*,† Seddon Y. Thomas,*,† Rachel Reboulet,*,† Fanyong Meng,*,† Frank Koentgen,‡ Luc Teyton,x Paul B. Savage,{ and Albert Bendelac*,† a-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 . Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1fl/fl mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by a-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-g, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Downloaded from Th1 or Th2 cellular networks by changing APC targeting in vivo. The Journal of Immunology, 2012, 188: 3053–3061.

atural killer T cells are CD1d-restricted T cells that CD40L/CD40 interactions (7, 8), resulting in the secretion of Th1 express a conserved semi-invariant Va14(huVa24)- and Th2 cytokines and the creation of a rich proinflammatory N Ja18/Vb8,7,2(huVb11) TCR (1, 2). They constitute a milieu that can markedly enhance adaptive T and B cell responses. separate lineage of innate-like T cells characterized by the ex- Consequently, NKT ligands have often demonstrated superior pression of the signature transcription factor promyelocytic leu- adjuvant properties in vivo compared with TLR ligands (9, 10). http://www.jimmunol.org/ kemia zinc factor, which is induced during thymic development Intriguingly, extensive structure–function studies have identified and directs the acquisition of an innate effector program (3, 4). synthetic variants of aGC with a marked Th2 bias and others with This program includes the ability to produce both Th1 and Th2 a Th1 bias, raising the prospect of their use for cytokine-specific cytokines upon primary stimulation and changes in homing and adjuvant therapy as well as immunomodulation of disease (11– recirculation with permanent downregulation of the lymph node 13). On the basis of the assumption that they differentially en- homing receptor CD62L, upregulation of the adhesion/migration gaged the NKT cell TCR, the Th2 variants have been referred to receptor CD44, changes in chemokine receptor profile, and con- as “altered glycolipid ligands” in some reports (11, 14) by analogy stitutive activation of the integrin LFA-1 (5). to altered peptide ligands with similar cytokine-biasing properties. by guest on October 1, 2021 Most NKT cells recognize microbial lipids characterized by This hypothesis has received some support for one of the leading a glycuronosyl group in a-linkage to a as well as the ligands, aGC acC24:0 psC9:0 (also called OCH), which was synthetic analog a-galactosylceramide (aGC) acC26:0 psC18:0 shown to induce a conformational change of the F’ pocket of (termed aGC) (6). These agonist ligands induce the reciprocal CD1d with corresponding diminution of the binding properties of activation of NKT cells and CD1d-lipid presenting cells through the TCR (14, 15). OCH failed to induce sufficient c-rel mRNA and CD40L on NKT cells in vivo and elicited diminished IFN-g but conserved IL-4 from NKT cells (16). Whereas aGC-mediated *Committee on Immunology, Howard Hughes Medical Institute, University of Chi- cago, Chicago, IL 60637; †Department of Pathology, Howard Hughes Medical Insti- NKT stimulation indirectly induces NK cells to release a sec- tute, University of Chicago, Chicago, IL 60637; ‡Ozgene, Bentley, Western Australia ond, long-lasting wave of IFN-g secretion in vivo (17), OCH x 6983, Australia; Department of Immunology, The Scripps Research Institute, La conspicuously failed to recruit NK cells. Because the late IFN-g is Jolla, CA 92037; and {Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 dependent of CD40-mediated IL-12 production by dendritic cells 1Current address: Institute of Immunology, School of Life Sciences, University of (DCs), it was concluded that altered TCR signaling impaired both Science and Technology of China, Hefei, China. direct and indirect secretion of Th1 cytokines (18). Received for publication August 19, 2011. Accepted for publication January 30, Other well-studied Th2 variants such as aGC acC20:2 psC18 2012. (termed aGC acC20:2) (12) and aGC acC8:0 psC18 (termed aGC This work was supported by the Major State Basic Research Development Program acC8) (13) did not seem to fall into the same category, however, as of China (973 program 2012CB825806), National Natural Sciences Foundation of detailed biophysical and crystallographic studies demonstrated China Grant 31021061, National Institutes of Health Grants AI038339 and AI053725, and Digestive Disease Research Core Center Grant P30 DK42086. A.B. that their interactions with CD1d and the NKT cell TCR were is a Howard Hughes Medical Institute Investigator. indistinguishable from those of aGC (14, 19, 20). Other properties Address correspondence and reprint requests to Dr. Li Bai or Dr. Albert Bendelac, of these strong agonists might therefore account for their marked Institute of Immunology, School of Life Sciences, University of Science and Tech- Th2 bias. Recently, a unifying set of chemical and cell biological nology of China, Hefei 230027, China (L.B.) or University of Chicago, 929 East 57th Street, Chicago, IL 60637 (A.B.). E-mail addresses: [email protected] (L.B.) and properties was identified for all Th2 ligands, including aGC [email protected] (A.B.) acC20:2, aGC acC8, and OCH. Their short or unsaturated lipid The online version of this article contains supplemental material. chains conferred higher solubility in the aqueous environment, Abbreviations used in this article: B6, C57BL/6; BMDC, bone marrow-derived which allowed them to directly load CD1d at the cell surface, dendritic cell; DC, dendritic cell; aGC, a-galactosylceramide; PD-1, programmed whereas, in contrast, aGC and other Th1 ligands needed endo- death-1. somal loading by lipid transfer (20–22). Furthermore, Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 after recycling of CD1d in the lysosomal compartment, the Th2 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102414 3054 DIFFERENT APC FOR aGC VARIANTS IN VIVO variants were displaced within seconds by endogenous lipids in variants was promiscuous and mostly carried out by a variety a pH-dependent manner, whereas aGC remained stably associated of non-DC cell types that did not produce IL-12. This DC- with CD1d (22). As CD1d recycles actively between the cell independent stimulation failed to induce the IL-12/NK/IFN-g surface and the lysosome (23), cells pulsed with short and un- feedback loop of innate immunity, resulting in a dominant Th2 saturated lipids lost their ability to stimulate NKT cells in culture response. When NKT cells were forced to interact with purified faster than those pulsed with aGC (22). In addition, CD1d-aGC DCs, both aGC and the Th2 variants upregulated CD40L and complexes were shown to localize preferentially to lipid rafts at induced similar quantities of IL-12. Thus, lipid structural varia- the cell surface, whereas the “Th2” variant aGC acC20:2 was tions that do not alter TCR recognition can activate distinct Th1 largely excluded (20). These common cell biological properties of or Th2 cellular networks by changing APC targeting in vivo. the Th2 variants suggested several possible mechanisms to explain their functional differences in vivo. For example, the location of Materials and Methods CD1d-lipid complexes on membrane lipid rafts might somehow Mice alter NKT cell stimulation toward more IFN-g production or in- tm1(cre)Cgn tm1(cre)Ifo directly favor NK cell recruitment. IFN-g production might also Cd19-Cre (B6.129P2(C)-Cd19 ), Lyz2-Cre (B6.129P2-Lyz2 ), Cd4-Cre (B6 Tg(-cre)1Cwi), and C57BL/6J mice were from The be selectively impaired upon interruption of TCR signaling be- Jackson Laboratory. Cd11c-Cre (C57BL/6J-Tg(Itgax-cre,-EGFP)4097Ach/ cause of the fast dissociation of CD1d complexed with short and J) mice were obtained from Dr. A. Chervonsky at our institution. CD4p- unsaturated lipid ligands. In this study, we directly tested a third Va14-Ja18 transgenic mice (25) were maintained in the laboratory. All possibility, that the Th2 bias of the aGC variants in vivo might be mice were raised in a specific pathogen-free environment at the University the consequence of different APCs. of Chicago, and experiments were performed according to a protocol ap-

proved by the Institutional Animal Care and Use Committee. Downloaded from We generated cd1dfl/fl mice and studied the response of mice lacking CD1d selectively on DCs, macrophages, or B cells, the Generation of B6.cd1d1fl/fl mice main CD1d-expressing cell types. Although the presentation of The mouse locus includes two cd1d , cd1d1 and cd1d2, which aGC was strictly limited to DC and to a lesser extent macro- are 95% identical. Because cd1d2 is a pseudogene in the C57BL/6 (B6) phages, as previously suggested (24), the stimulation by the Th2 strain (26), we used homologous recombination to flank exons 2 and 6 of http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Characterization of cd1d1fl/fl mice. (A) The targeted C57BL/6 cd1d locus (not to scale) contains two loxP sites flanking the exons 2 and 6 of cd1d1 as well as an frt site left over after removal of the frt-neomycin-frt resistance cassette. The cd1d2 is a pseudogene in the B6 strain (26). (B) CD1d expression in splenic DC, macrophage, and B cell subsets of wild-type and D/D mice gated as indicated. Representative of two separate experiments with two to three mice per group. (C) CD1d-PBS57 tetramer+ NKT cells among CD24low thymocytes and TCRb+ spleen and liver cells of control and D/D mice as indicated. Representative of two separate experiments with two to three mice per group with mean 6 SEM of NKT cell frequency indicated in the FACS plots. (D) Cytokine released by sorted or MACS-enriched CD1d-PBS57 tetramer+ NKT cells at indicated cell numbers from thymus (left) and spleen (right) of wild-type and Cd11c D/D cocultured for 48 h with 50,000 BMDC pulsed with 200 ng/ml PBS57. Data are from three independent experiments with two to three mice pooled per group and are represented as percentage of the cytokines released by wild-type NKT cells at 30,000 cells/well. (E) Early serum IL-4 measured in individual wild-type and Cd11c D/D mice 90 min after injection of 1.25 mg anti-CD3ε. The Journal of Immunology 3055 cd1d1 with loxP sites in an ES line of B6 origin. After crossing to B6 mice produced as described previously (6, 13, 27). C-glycoside aGC was ob- expressing the Flp recombinase to remove the frt-flanked PGK-neomycin tained from National Institutes of Health Tetramer Core Facility. All the sequence used for in vitro selection of recombined ES cells, the B6.cd1d1fl/fl lipid Ags were dissolved in DMSO and stored at 220˚C at 1 mg/ml. conditional mutant line was generated. B6.cd1d1fl/fl mice were crossed to -cre, cd11c-cre and lyz2-cre to NKT cell stimulation assays obtain cd19 D/D, cd11c D/D, and lyz2 D/D mice. Littermate controls in- + + fl/+ Fresh NKT cells were purified by sorting as TCRb CD1d-PBS57 tetramer cluded Cre-negative littermates and in some cases Cre-positive cd1d1 or 2 + Cre-positive cd1d1+/+ littermates. cells (spleen) or CD24 tetramer (thymus) and cocultured with bone mar- row-derived DC (BMDC) pulsed with 200 ng/ml PBS57 for 48 h. BMDC PCR protocol for typing the cd1d1fl/fl and the cre mice were obtained after culture of bone marrow cells in medium supplemented fl/fl with 10% FCS, 10 ng/ml GM-CSF, and 10 ng/ml IL-4 for 7 d. Alternatively, B6.cd1d mice were genotyped by PCR (forward primer, 59-ATG TAT NKT cells were stimulated with 1 mM ionomycin and 20 ng/ml PMA for 48 h. CCA GAG GTT TTA CTT GGT GAA-39; reverse primer, 59-TGG GGT To obtained fresh in vivo pulsed APCs, B6 mice were i.p. injected with CCA TTC CAG ATA CAA A-39). Mice with loxP sites generated a 351-bp lipid (2 mg for aGC or 50 mg for aGC acC8), and their spleens were fragment instead of a 156-bp fragment in wild-type mice. Cre-deleter mice harvested 2 h later to sort CD11chigh DCs, CD11chigh CD8a+ lymphoid were genotyped by PCR with primers (forward primer, 59-TTA CCG GTC DCs, CD11cloCD11bloF4/80+ red pulp macrophages, and B220+ B cells, GAT GCA ACG AGT-39; reverse primer, 59-TTC CAT GAG TGA ACG prior to coculture with the CD1d-aGC–responsive NKT hybridoma DN32. AAC CTG G-39) that generated a 400-bp fragment. D3 overnight and measure of IL-2 release by Cytometric Beads Array + Flow cytometry Mouse IL-2 Flex Set. In other experiments, CD11c DCs were enriched from splenocytes by depletion with a mixture of anti-CD3, CD11b, F4/80, Splenic lymphocytes and thymus cells were isolated by mincing and pass- and B220 beads and subsequent enrichment with anti-CD11c beads using ing through a 70-mm nylon cell strainer (Falcon). Hepatic lymphocytes the autoMACS cell separator (Miltenyi Biotec). B cells were enriched by were further purified by running through Percoll gradient (Sigma- depletion with a mixture of anti-CD3, CD11b, F4/80, and CD11c beads. Aldrich). Fluorochrome-labeled mAbs against mouse CD1d (1B1), B220 For in vitro stimulation of IL-12p70 production, DCs, macrophages, and Downloaded from (RA3.6B2), F4/80 (BM8), CD11c (N418), CD11b (M1/70), CD24 (M1/69), B cells were sorted as above and cultured with fresh NKT cells sorted as CD5 (53-7.3), TCR Vb7 (TR310), TCR Vb8.1+8.2 (MR5-2), TCRb (H57- CD5+ cells from Va14 Tg spleens. In the presence of 1 mg/ml aGC or 2.5 597), CD4 (GK1.5), CD8a (53-6.7), DX5 (DX5), CD40L (MR1), IL-4 mg/ml aGC acC8 for 48 h, IL12-p70 released in supernatant was measured (11B11), IFN-g (XMG1.2), and IL-12p40 (C15.6) were purchased form with Cytometric Beads Array Mouse IL-12-p70 Flex Set. eBioscience or BioLegend. CD1d-PBS57 tetramers were obtained from the National Institute of Allergy and Infectious Diseases Tetramer Core Facility. Statistical analysis For intracellular cytokine staining, cells were stained with cell surface Different groups were compared using two-tailed t test: *p , 0.05, **p , http://www.jimmunol.org/ markers first and were fixed in Cytofix/Cytoperm buffer (BD Biosciences) 0.01, and ***p , 0.001. for 15 min and further stained with Ab against mouse IL-4, IL-12p40, and IFN-g in wash/perm buffer for 1 h on ice. Samples were analyzed on an LSR II (BD Biosciences) or sorted on a FACSAria (BD Biosciences). Results fl/fl Lipid Ags Characterization of cd1d1 mice fl/fl aGC (acC26:0 psC18:0) was purchased from Alexis Biochemicals, and its Cd1d1 mice (Fig. 1A) were produced by homologous recom- variants aGC acC8:0 psC18, aGC acC20:2 psC18, and PBS57 were bination in an ES line of B6 origin. Cd1d1fl/fl Cd4-Cre mice (cd4 by guest on October 1, 2021

FIGURE 2. Comparison of aGC with aGC acC8 in vivo. B6 mice were injected i.p. with 2 mg aGC or 5 mg aGC acC8 unless otherwise indicated. (A) Serum IL-4 at 2 h and serum IFN-g at 24 h. (B) Serum IL-12p70 at 6 h. (C) Splenic TCRb+CD1d- PBS57+ NKT cells collected at 1, 2, and 6 h as in- dicated and directly stained for intracellular IL-4 and IFN-g. Results compiled from three separate experiments. (D) Spleen cells collected at 6 h, stained with anti-DX5 and anti–IFN-g, and dis- played after gating on TCRb2B2202 cells. Left, Representative FACS plots; right, summary figures showing the frequency of IFN-g–secreting cells among NK cells and their mean fluorescence inten- sity (MFI) for IFN-g. Results compiled from three separate experiments. (E) Mice received five injec- tions (at 0, 1, 2, 3, and 5 h) of 5 mg or one injection of 25 mg aGC acC8. Sera were collected at 6 h for IL-12p70, 24 h for IFN-g, and 2 h for IL-4 and were compared with sera of mice receiving one injection of 2 mg aGC at 0 h. Results compiled from two separate experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 3056 DIFFERENT APC FOR aGC VARIANTS IN VIVO

D/D) mice showed a 90% reduction of CD1d expression on cor- PD-1 after aGC as reported by others (34) only occurred at 12 h, tical CD4+CD8+ thymocytes and a corresponding 75–86% re- beyond the time frame of this experiment. duction of NKT cell numbers in the thymus, spleen, and liver Differential APC requirements for aGC and aGC acC8 in vivo (Supplemental Fig. 1A). This result constitutes a direct demon- stration that NKT cell development is exquisitely and solely de- Cd11c D/D mice showed no detectable serum IL-4 or IFN-g after pendent on CD1d expression by DP thymocytes, as previously injection of aGC, suggesting failure to activate NKT cells (Fig. suggested by bone marrow chimeras and transgenic studies (28– 4A). This conclusion was confirmed by direct intracellular stain- 30). Furthermore, as expected in the presence of low concen- ing showing absent or massively decreased IL-4 and IFN-g in trations of CD1d ligands, the few residual NKT cells expressed splenic and liver NKT cells (Fig. 4B, 4C) and absent IFN-g in a bias toward expression of Vb7 (Supplemental Fig. 1B), which splenic and liver NK cells (Fig. 4D, 4E). Thus, despite the broad confers the highest affinity for endogenous CD1d ligands when tissue distribution of CD1d expression in mice, NKT cell stimu- paired with Va14-Ja18 (31). lation was absolutely dependent on CD1d expression by CD11c- Cd11c D/D mice lacked CD1d on splenic DCs, which are expressing cells. CD11chigh, and also on macrophage subsets that express low levels of CD11c such as the F4/80+ red pulp macrophages (Fig. 1B). Lyz2 D/D mice lacked CD1d on macrophage subsets in- cluding the F4/80+ red pulp macrophages but expressed normal levels on CD11chigh DCs. Cd19 D/D mice lacked CD1d on B cells. In contrast with the cd4 D/D mice, the cd11c D/D, lyz2 D/D,andcd19 D/D mice exhibited normal development and dis- Downloaded from tribution of NKT cells (Fig. 1C). Furthermore, the intrinsic cy- tokine secretion properties of NKT cells assessed by stimulation with PBS57-pulsed BMDCs in vitro were preserved, as was the rapid release of IL-4 upon injection of anti-CD3 in vivo, a char- acteristic properties of NKT cells (Fig. 1D, 1E) (32). Thus, the cd1d1fl/fl mouse allowed deletion of CD1d in selective APC http://www.jimmunol.org/ subsets without altering the frequencies or functional properties of NKT cells. Comparison of aGC and aGC acC8 in vivo The structure and nomenclature of aGC variants used in this study is shown in Supplemental Fig. 2. The Th2 bias of aGC variants with short acyl chain was previously defined in vitro in whole- spleen cell stimulation assays (13). A detailed comparison of by guest on October 1, 2021 their functional properties was performed in a time-course study in vivo. Unless otherwise indicated, mice were injected i.p. with doses of aGC and aGC acC8 that were previously shown to elicit similar activation of NKT cells in vivo, 2 mg for aGC and 5 mg for aGC acC8. Fig. 2A showing serum IL-4 and IFN-g at their re- spective peaks at 2 and 24 h illustrates the opposite cytokine bias of aGC and aGC acC8. The Th2 bias of aGC acC8 correlated with the lack of detectable IL-12p70 and contrasted with the abundant amount released after aGC injection (Fig. 2B). These findings are superimposable to those previously reported for the other Th2 variants aGC acC20:2 and aGC psC9 (33). Although the two lipids induced comparable intracellular IL-4 and IFN-g in splenic NKT cells at 2 h, the cytokine secretion elicited by acC8 was shifted to earlier time points for acC8 and declined, whereas the cytokines induced by aGC were still in- creasing between 2 and 6 h (Fig. 2C). Splenic NK cell production of IFN-g was markedly inferior for aGC acC8 compared with aGC both in frequency and in mean fluorescence intensity at 6 h (Fig. 2D). Repeating the injections of aGC acC8 five times over a 5-h period or increasing the dose from 5 to 25 mg resulted in a further increase in IL-4 but completely failed to reverse the defect in IL- 12 and IFN-g, indicating that the Th2 bias was independent of the dose or the duration of exposure to the lipid in vivo (Fig. 2E). FIGURE 3. Kinetics of NKT cell activation by aGC and aGC acC8 Further kinetic studies indicated that NKT cell stimulation by in vivo. (A) TCRb+CD1d-PBS57+ NKT cells in the spleen at indicated aGC was simply delayed by 2–3 h compared with aGC acC8, times after injection of lipids. Numbers represent NKT cell frequency 6 without significant alterations in the quality of this stimulation. SEM compiled from two experiments with three mice per group. (B) Ki- For example, TCR downregulation and the induction of CD25, netics of CD40L and CD25 induction on TCRb+CD1d-PBS57+ gated CD40L, and programmed death-1 (PD-1) all occurred earlier after NKT cells. (C) Kinetics of PD-1 induction by NKT cells. Results are re- injection of aGC acC8 than after aGC (Fig. 3). Full expression of presentative of two independent experiments with three mice per group. The Journal of Immunology 3057 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. APC requirement for aGC in vivo. All mice were injected with 2 mg aGC i.p. (A) Serum IL-4 at 2 h and serum IFN-g at 24 h in cd11c D/D, lyz2 D/D, and cd19 D/D mice and littermate controls. Data are normalized to the mean of littermate controls. (B) Gated TCRb+CD1d-PBS57+ NKT cells in spleens of wild-type and D/D mice were directly stained for intracellular IL-4 and IFN-g at 4 h postinjection. Top, Representative FACS plots; bottom, summary figures. (C) Same as (B) for liver NKT cells collected at 4 h. (D) DX5 and intracellular IFN-g staining on gated TCRb2B2202 spleen cells at 6 h postinjection. Left, Representative FACS plots; right, summary figure. (E) Results as in (D) for liver NK cells at 6 h. Results are compiled from two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Lyz2 D/D mice exhibited a modest but in some cases significant (Fig. 5A, 5B). Although aGC acC8 does not induce the late (NK- reduction of cytokines released in the serum or present in the dependent) IFN-g in the 24-h serum, the intracellular IFN-g cytosol of NKT and NK cells (Fig. 4). Taken together with the produced by NKT cells was similar in cd11c D/D and littermate total ablation of NKT cell activation in cd11c D/D mice, these controls. Furthermore, lyz2 D/D and cd19 D/D mice also showed results suggested that a cell type coexpressing cd11c and lyz2 the same stimulation as littermate controls. Thus, in keeping with (e.g., a macrophage or DC subset) contributed partially to the the promiscuous ability of aGC acC8 to load CD1d at the cell presentation of aGC and might be preferentially involved in the surface independently of either lysosomal recycling or lipid serum release of IL-4. transfer proteins, there was no dedicated APC-type required in Finally, no detectable changes were observed in cd19 D/D mice vivo. Furthermore, the absence of CD1d expression by DC did (Fig. 4), indicating that presentation by B cells was not signifi- not alter the pattern of IL-4 and IFN-g production by NKT cells. cantly involved in NKT or NK cell activation. a Taken together, these findings established that NKT cell stim- Other Th1 and Th2 GC variants exhibited patterns similar to a a ulation by aGC in vivo was strictly dependent on aGC presen- GC and GC acC8 tation by DCs and also, to a lesser extent, a subset of CD11c+ Other Th1 variants such as aGC C-glycoside (35) and PBS57 (27) lysM+ macrophages or DCs. exhibited a dependence of CD1d expression by DC such as aGC, In striking contrast with aGC, the Th2 variant aGC acC8 in- whereas aGC acC20:2 (12), another well characterized Th2 var- duced equivalent stimulation in controls and in cd11c D/D mice iant, followed the same pattern as aGC acC8 (Fig. 5C, 5D). These 3058 DIFFERENT APC FOR aGC VARIANTS IN VIVO

FIGURE 5. APC requirement for aGC acC8 and other variants in vivo. D/D mice and their littermate controls were injected with 5 mg aGC acC8 i.p. (A) Serum IL-4 at 2 h in cd11c D/D, lyz2 D/D, and cd19 D/D and littermate controls. Serum IFN-g at 24 h was undetectable. Data are normalized to the mean of littermate controls. (B) Wild-type and D/D splenic NKT cells gated as TCRb+CD1d-PBS57+ were stained for intracellular IL-4 and IFN-g 2h postinjection. Representative of two independent experiments with two to three mice per group. Se- rum IFN-g at 24 h (C) and serum IL-4 at 2 h (D) postinjection of 0.2 mg Th1 variant PBS57, 2 mg Th1 variant C-glycoside, or 5 mg Th2 variant aGC acC20:2. IL-4 was undetectable for C-glycoside, and IFN-g was undetectable for aGC acC20:2. *p , 0.05. Downloaded from http://www.jimmunol.org/ results indicate that the Th1 bias of NKT ligands is tightly asso- lipid for 48 h. Notably, when forced to interact exclusively with ciated with their selective presentation by DC. DCs, aGC acC8 elicited the same amount of IFN-g and IL-12 as aGC in this culture system, unambiguously demonstrating its in- a Forced interaction of NKT cells with GC acC8-pulsed DCs trinsic ability to elicit a Th1 profile (Fig. 7C). aGC acC8 also induced IL-12 tended to elicit a little more IL-4 than aGC, but this 1.5-fold in- Previous studies indicated that the CD8a++ subset of crease was not statistically significant. In this NKT-APC coculture DCs was the main cell type producing IL-12 after aGC injec- assay, highly purified B cells were able to stimulate NKT cells tion in vivo (24, 36). As IL-12 secretion by DCs required when pulsed with aGalCer acC8, but little stimulation was ob- by guest on October 1, 2021 CD40L expression by NKT cells (18, 37, 38) and the Th2 served with aGalCer, consistent with a relative defect in CD1d variants potently induced CD40L on NKT cells, one potential presentation of aGalCer by B cells. Furthermore, aGalCer acC8 explanation for the failure of the Th2 variants to induce IL-12 induced markedly more IL-4 relative to IFN-g when presented by might be that they were not presented by DCs in vivo or that B cells than by DCs, consistent with the importance of IL-12 the frequency of DC encounters was decreased because of pro- production by DCs for positive feedback on IFN-g production. miscuous presentation by other, more numerous cell types. Direct intracellular staining for IL-12 confirmed that CD8a+ Discussion DCs were the main IL-12p40 expressing cell type at 6 h postin- The generation of mice carrying a conditional allele of cd1d1 jection of aGC (Fig. 6A). However, this experiment clearly allowed a dissection of the role of different CD1d-expressing cells established that aGC acC8 also induced IL-12p40, albeit on far both in the development and in the peripheral activation of fewer DCs than aGC. NKT cells. The results directly established the essential role of Furthermore, if the Th2 bias was simply because of a DC- CD1d expression by thymocytes for NKT cell development, as intrinsic defect in presentation of aGC acC8, then injecting previously suggested by bone marrow radiation chimeras and mice with a mixture of aGC and aGC acC8 should result in strong CD1d transgenic experiments (28–30). They also demonstrated IL-12 production and NK cell activation. Fig. 6B and 6C dem- the dispensable role of CD1d expression by DCs, macrophages, or onstrated the opposite result (i.e., a strict dominance of the Th2 B cells for the development of NKT cells and the acquisition of bias). Thus, the Th2 bias likely resulted from the diversion of their cytokine profile. The most novel insight derived from these NKT cells away from DC toward interactions with other aGC studies was the in vivo demonstration of different APC require- acC8-presenting cell types. ments for aGalCer and its short and unsaturated acyl variants, The nature of the preferred APCs for aGC acC8 and aGC was which provided a simple and unifying explanation for the Th2 further investigated in vivo and in vitro. Two hours after the i.p. bias of the variants. injection of lipid in vivo, different APCs were purified and im- Whereas the presentation of aGC and other Th1 variants such mediately cocultured with the aGC-responsive IL-2–producing hy- as PBS57 and aGC C-glycoside was entirely restricted to DC/ bridoma DN32.D3 as a readout of Ag presentation. Whereas both macrophage cell types, presentation by DC/macrophages was DCs and F4/80 macrophages were effective presenters of aGC, negligible compared with other cell types after injection of the B cells were not (Fig. 7A). In marked contrast, aGC acC8 was Th2 variants aGC acC8 and aGC acC20:2 in vivo. Because only efficiently presented by B cells as well as by other cell types (Fig. DC and macrophages can produce the IL-12 that is critical for 7B). In other experiments, CD11c+ DCs, B220+ B cells, and F4/80+ amplification of Th1 responses and the recruitment of NK cells, macrophages were purified from uninjected mice and incubated these findings are sufficient to explain the Th2 bias associated with in vitro with fresh Va14 transgenic NKT cells in the presence of the main variants studied so far. The Journal of Immunology 3059

FIGURE 6. Th2 variant aGC acC8 fails to induce IL-12 in vivo. B6 mice were injected i.p. with 2 mg aGC or 5 mg aGC acC8. (A) Splenocytes were harvested 6 h later and intracellularly stained for IL-12p40. Top row, Repre- sentative FACS plots of gated CD11c+ DC cells costained with anti-CD8a. Central row, Gated CD11bloCD11clo cells costained with anti-F4/80. Bottom row, Cells in the lymphoid gate cos- tained for B220. Data representative of four experiments with two to three mice per group. (B) Mice were injected as in (A) with 2 mg aGC or 5 mg aGC acC8 and compared with mice injected with the mixture of 2 mg aGC and 5 mg aGC acC8. Top row, DC staining for IL-12p40 as in (A); bottom row, staining 2 2

of TCRb B220 splenic cells for Downloaded from DX5+ and intracellular IFN-g.(C) Se- rum IL12-p70 at 6 h, IL-4 at 2 h, and IFN-g at 24 h postinjection of 2 mg aGC, 5 mg aGC acC8, or the mixture as indicated. *p , 0.05, **p , 0.01. http://www.jimmunol.org/

The importance of aGC presentation by DC in vivo has been largely overlap with the CD8a subset and are located in the previously suggested, based on methods using a diphtheria toxin marginal zone of the spleen (36), further underscores the spe- receptor transgenically driven by a cd11c promoter (24) or by a cialized role of these IL-12 producers in the recruitment of NK langerin promoter (36) to allow DC-specific killing after admin- cells. In contrast, NKT cell production of both IL-4 and IFN-g was istration of diphtheria toxin. Notably, whereas serum IFN-g was preserved after deletion of langerin+ DCs, in keeping with the nearly absent, IL-4 was much less reduced in these experiments, ability of other DC subsets to load aGC and stimulate NKT cells. contrasting with the total ablation of both cytokines in the cd11c In the current study, the total ablation of NKT cell activation in D D cd11c D D

/ mice. The difference may reflect the role of macrophage / mice, as monitored by intracellular cytokines, unam- by guest on October 1, 2021 subsets that were reportedly spared in the diphtheria toxin receptor biguously established that DCs, and possibly a small subset of models (24) but, as a result of their intermediate expression of macrophages, constituted the exclusive cell-type mediating aGC- CD11c, had lost CD1d expression in the cd11c D/D mice. The induced stimulation in vivo. This selective presentation is at least ablation of serum IFN-g by mice lacking langerin+ DCs, which in part a consequence of two peculiar features of aGC compared

FIGURE 7. aGC acC8 can be presented by DCs to induce IL-4, IFN-g, and IL-12p70. (A) CD11chi CD8a+ DCs, B220+ B cells, and CD11clo CD11blo F4/80+ macrophages were sorted from pooled spleens from three B6 mice 2 h after injection of 2 mg aGC and cocultured at indicated numbers with the hybridoma DN32.D3 (50,000 cells/well) overnight, and IL-2 released in the supernatant was measured. Error bars represent SEM (B) CD11c+ DCs and B220+ B cells were enriched using autoMACS from the pooled spleens of three B6 mice 2 h after injection of 50 mg aGC acC8 and cocultured at indicated numbers with DN32.D3 as in (A). (C) CD11chi DCs, B220+ B cells, and CD11cloCD11blo F4/80+ macrophages were sorted from the spleens of uninjected mice and cocultured at 5000 cells/well in the presence of 1 mg/ml aGC or 2.5 mg/ml aGC acC8 for 48 h with sorted CD5+ cells (50,000/well) obtained from the spleens of Va14 transgenic mice. IL12-p70, IFN-g, and IL-4 were measured in supernatants. Three separate experiments were performed, and data were normalized to the DC + aGC group (100%) in each experiment. Error bars indicate SEM. *p , 0.05, **p , 0.01, ***p , 0.001. 3060 DIFFERENT APC FOR aGC VARIANTS IN VIVO with the Th2 variants. aGC is transported by serum lipoproteins in vivo. aGC, which selectively targeted DC and macrophages, is and its uptake by DCs and macrophages is mediated by the li- emerging as a choice adjuvant for the cross-priming of CTLs. poprotein receptor (22, 39, 40). aGC requires lysosomal lipid However, our results predict that the ability of aGC to directly transfer proteins, which are most active in DCs and macrophages, recruit NKT cell help to B cells is likely compromised by ineffi- for CD1d loading (41, 42). cient B cell presentation in vivo. By contrast, although the Th2 Although previous studies of B cell-deficient mMT mice sug- variants may be inferior at CTL priming they should provide su- gested enhanced (24) or unmodified (43) stimulation by aGC, the perior recruitment of NKT cells for cognate B cell help and Ab cd19 D/D mice demonstrated that B cell presentation was not production. In addition, similar differences are likely to govern significantly involved for systemic aGC-mediated stimulation. NKT cell responses to natural endogenous and exogenous lipid This finding was consistent with the inefficient capture and pre- ligands differing in lipid length and saturation (45, 46). The sentation of aGC by B cells when the lipid was injected alone and cd1d1fl/fl mouse provides therefore a powerful system to dissect with its efficient presentation upon BCR-mediated uptake and the complex cellular interactions involved in NKT cell-mediated endosomal delivery of Ag + lipid-coated beads (44). adjuvant effects in cellular and humoral immune responses. The uptake and presentation of the Th2 variants in vivo followed drastically different rules. The stimulation was entirely unaffected Acknowledgments in cd11c D/D mice compared with wild-type mice, demonstrating We thank members of the Bendelac laboratory for discussions and the An- that NKT cells did not require professional APC for activation. imal Resource Center, the Core Flow Cytometry Facility, the DNA Se- This finding was fully consistent with previous studies, indicating quencing Facility, and the National Institute of Allergy and Infectious that short or unsaturated aGC variants were rapidly loaded onto Diseases tetramer facility for CD1d tetramers. CD1d expressed at the cell surface and that lysosomal recycling Downloaded from was not only unnecessary but in fact was detrimental to sustained Disclosures presentation (22). Although the removal of CD1d from DCs and The authors have no financial conflicts of interest. macrophages did not impair the stimulation of NKT cells by the Th2 variants, our in vitro and in vivo experiments demonstrated that these cell types could present the Th2 variants efficiently. In References

1. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. http://www.jimmunol.org/ fact, CD8a DCs loaded with aGC acC8 or aGC acC20:2 re- Annu. Rev. Immunol. 25: 297–336. leased the same amount of IL-12 as those loaded with aGC in 2. Godfrey, D. I., S. Stankovic, and A. G. Baxter. 2010. Raising the NKT cell the presence of fresh NKT cells. Conversely, B cells stimulated family. Nat. Immunol. 11: 197–206. 3. Savage, A. K., M. G. Constantinides, J. Han, D. Picard, E. Martin, B. Li, NKT cells more effectively when loaded with the Th2 variants O. Lantz, and A. Bendelac. 2008. The transcription factor PLZF directs the than with aGC, and consistent with their inability to secrete IL-12, effector program of the NKT cell lineage. Immunity 29: 391–403. they induced relatively more IL-4 than IFN-g compared with DCs. 4. Kovalovsky, D., O. U. Uche, S. Eladad, R. M. Hobbs, W. Yi, E. Alonzo, K. Chua, M. Eidson, H. J. Kim, J. S. Im, et al. 2008. The BTB-zinc finger transcriptional Altogether, the results suggest that the overriding cause of the regulator PLZF controls the development of invariant natural killer ef- Th2 bias of the short and unsaturated variants of aGC is their wide fector functions. Nat. Immunol. 9: 1055–1064. ranging and promiscuous presentation by non–IL-12–producing 5. Thomas, S. Y., S. T. Scanlon, K. G. Griewank, M. G. Constantinides, A. K. Savage, K. A. Barr, F. Meng, A. D. Luster, and A. Bendelac. 2011. PLZF by guest on October 1, 2021 cell types, which stems from their ability to directly load CD1d at induces an intravascular surveillance program mediated by long-lived LFA-1– the cell surface. This wide ranging presentation decreases the ICAM-1 interactions. J. Exp. Med. 208: 1179–1188. frequency of NKT cell interactions with CD8a DCs and the as- 6. Savage, P. B., L. Teyton, and A. Bendelac. 2006. Glycolipids for natural killer T cells. Chem. Soc. Rev. 35: 771–779. sociated IL-12 release, whereas it increases interactions with APCs 7. Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, that are unable to enhance IFN-g production or recruit NK cells. K. Takeda, K. Okumura, L. Van Kaer, et al. 1999. The natural killer T (NKT) cell ligand a-galactosylceramide demonstrates its immunopotentiating effect by in- Our results do not rule out a contribution of other general ducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor ex- mechanisms, however, although these would appear less likely to pression on NKT cells. J. Exp. Med. 189: 1121–1128. play a prominent role in the Th2 bias of aGC variants. We did 8. Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, and R. M. Steinman. 2003. Acti- vation of natural killer T cells by a-galactosylceramide rapidly induces the full observe that the Th2 variants induced a stimulation that was maturation of dendritic cells in vivo and thereby acts as an adjuvant for com- somewhat less persistent than aGC, but the difference reflected bined CD4 and CD8 T cell immunity to a coadministered . J. Exp. Med. in part a simple time shift of 2–3 h because of the faster loading 198: 267–279. 9. Parekh, V. V., M. T. Wilson, and L. Van Kaer. 2005. iNKT-cell responses to of these lipids, and the upregulation of CD40L, CD25, PD-1, IL- glycolipids. Crit. Rev. Immunol. 25: 183–213. 4, and IFN-g by NKT cells was comparable for both types of 10. Cerundolo, V., J. D. Silk, S. H. Masri, and M. Salio. 2009. Harnessing invariant lipids. Furthermore, repeated injections of the short acyl variant NKT cells in vaccination strategies. Nat. Rev. Immunol. 9: 28–38. 11. Miyamoto, K., S. Miyake, and T. Yamamura. 2001. A synthetic glycolipid did not restore serum IL-12 or IFN-g. Although the Th2 variants prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer elicited less IL-12 production by DCs, this was mostly because T cells. Nature 413: 531–534. of a decrease in the frequency of IL-12–producing DC. It is also 12. Yu, K. O., J. S. Im, A. Molano, Y. Dutronc, P. A. Illarionov, C. Forestier, N. Fujiwara, I. Arias, S. Miyake, T. Yamamura, et al. 2005. Modulation of possible that the preferential subcellular location of CD1d-aGC CD1d-restricted NKT cell responses by using N-acyl variants of a-gal- on lipid rafts may somehow enhance the recruitment of NK actosylceramides. Proc. Natl. Acad. Sci. USA 102: 3383–3388. cells, although this effect would have to be independent of the 13. Goff, R. D., Y. Gao, J. Mattner, D. Zhou, N. Yin, C. Cantu, III, L. Teyton, A. Bendelac, and P. B. Savage. 2004. Effects of lipid chain lengths in a-gal- ability to induce CD40L, IL-4, and IFN-g in NKT cells or IL-12 actosylceramides on cytokine release by natural killer T cells. J. Am. Chem. Soc. in DCs. Furthermore, the injection of a mixture of aGC and 126: 13602–13603. 14. Wun, K. S., G. Cameron, O. Patel, S. S. Pang, D. G. Pellicci, L. C. Sullivan, aGC acC8, which would lead to preferential loading of DC by S. Keshipeddy, M. H. Young, A. P. Uldrich, M. S. Thakur, et al. 2011. A mo- aGC and of other APCs by aGC acC8, showed clear dominance lecular basis for the exquisite CD1d-restricted specificity and functional of the Th2 variant consistent with the diversion of NKT cells responses of natural killer T cells. Immunity 34: 327–339. 15. McCarthy, C., D. Shepherd, S. Fleire, V. S. Stronge, M. Koch, P. A. Illarionov, away from IL-12–producing DC. G. Bossi, M. Salio, G. Denkberg, F. Reddington, et al. 2007. The length of lipids In conclusion, the common physicochemical properties of the bound to CD1d molecules modulates the affinity of NKT cell TCR and Th2 variants, mainly their increased solubility in aqueous com- the threshold of NKT cell activation. J. Exp. Med. 204: 1131–1144. 16. Oki, S., A. Chiba, T. Yamamura, and S. Miyake. 2004. The clinical implication partment, not only underlied their cell biological properties, but and molecular mechanism of preferential IL-4 production by modified also in turn dictated their targeting to vastly different sets of APCs glycolipid-stimulated NKT cells. J. Clin. Invest. 113: 1631–1640. The Journal of Immunology 3061

17. Carnaud, C., D. Lee, O. Donnars, S.-H. Park, A. Beavis, Y. Koezuka, and 32. Yoshimoto, T., and W. E. Paul. 1994. CD4pos, NK1.1pos T cells promptly A. Bendelac. 1999. Cutting edge: cross-talk between cells of the innate immune produce in response to in vivo challenge with anti-CD3. J. Exp. system: NKT cells rapidly activate NK cells. J. Immunol. 163: 4647–4650. Med. 179: 1285–1295. 18. Oki, S., C. Tomi, T. Yamamura, and S. Miyake. 2005. Preferential T(h)2 po- 33. Venkataswamy, M. M., and S. A. Porcelli. 2010. Lipid and glycolipid of larization by OCH is supported by incompetent NKT cell induction of CD40L CD1d-restricted natural killer T cells. Semin. Immunol. 22: 68–78. and following production of inflammatory cytokines by bystander cells in vivo. 34. Parekh, V. V., S. Lalani, S. Kim, R. Halder, M. Azuma, H. Yagita, V. Kumar, Int. Immunol. 17: 1619–1629. L. Wu, and L. V. Kaer. 2009. PD-1/PD-L blockade prevents anergy induction and 19. Zajonc, D. M., C. Cantu, III, J. Mattner, D. Zhou, P. B. Savage, A. Bendelac, enhances the anti-tumor activities of glycolipid-activated invariant NKT cells. J. I. A. Wilson, and L. Teyton. 2005. Structure and function of a potent agonist for Immunol. 182: 2816–2826. the semi-invariant receptor. Nat. Immunol. 6: 810–818. 35. Schmieg, J., G. Yang, R. W. Franck, and M. Tsuji. 2003. Superior protection 20. Im, J. S., P. Arora, G. Bricard, A. Molano, M. M. Venkataswamy, I. Baine, against malaria and melanoma metastases by a C-glycoside analogue of the E. S. Jerud, M. F. Goldberg, A. Baena, K. O. Yu, et al. 2009. Kinetics and natural killer T cell ligand a-galactosylceramide. J. Exp. Med. 198: 1631–1641. cellular site of glycolipid loading control the outcome of natural killer T cell 36. Farrand, K. J., N. Dickgreber, P. Stoitzner, F. Ronchese, T. R. Petersen, and activation. Immunity 30: 888–898. I. F. Hermans. 2009. Langerin+CD8a+ dendritic cells are critical for cross- 21. Sullivan, B. A., N. A. Nagarajan, G. Wingender, J. Wang, I. Scott, M. Tsuji, priming and IL-12 production in response to systemic antigens. J. Immunol. R. W. Franck, S. A. Porcelli, D. M. Zajonc, and M. Kronenberg. 2010. Mech- 183: 7732–7742. anisms for glycolipid antigen-driven cytokine polarization by Va14i NKT cells. 37. Fujii, S., K. Liu, C. Smith, A. J. Bonito, and R. M. Steinman. 2004. The linkage J. Immunol. 184: 141–153. of innate to adaptive immunity via maturing dendritic cells in vivo requires 22. Bai, L., Y. Sagiv, Y. Liu, S. Freigang, K. O. Yu, L. Teyton, S. A. Porcelli, CD40 ligation in addition to antigen presentation and CD80/86 costimulation. J. P. B. Savage, and A. Bendelac. 2009. Lysosomal recycling terminates CD1d- Exp. Med. 199: 1607–1618. mediated presentation of short and polyunsaturated variants of the NKT cell lipid 38. Tomura, M., W. G. Yu, H. J. Ahn, M. Yamashita, Y. F. Yang, S. Ono, antigen aGalCer. Proc. Natl. Acad. Sci. USA 106: 10254–10259. T. Hamaoka, T. Kawano, M. Taniguchi, Y. Koezuka, and H. Fujiwara. 1999. A 23. Jayawardena-Wolf, J., K. Benlagha, Y. H. Chiu, R. Mehr, and A. Bendelac. 2001. novel function of Va14+CD4+NKT cells: stimulation of IL-12 production by CD1d endosomal trafficking is independently regulated by an intrinsic CD1d- antigen-presenting cells in the innate . J. Immunol. 163: 93–101. encoded tyrosine motif and by the invariant chain. Immunity 15: 897–908. 39. Freigang, S., V. Zadorozhny, M. K. McKinney, P. Krebs, R. Herro, J. Pawlak, 24. Bezbradica, J. S., A. K. Stanic, N. Matsuki, H. Bour-Jordan, J. A. Bluestone, L. Kain, N. Schrantz, K. Masuda, Y. Liu, et al. 2010. amide hydrolase J. W. Thomas, D. Unutmaz, L. Van Kaer, and S. Joyce. 2005. Distinct roles of shapes NKT cell responses by influencing the serum transport of lipid antigen in Downloaded from dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo. J. mice. J. Clin. Invest. 120: 1873–1884. Immunol. 174: 4696–4705. 40. van den Elzen, P., S. Garg, L. Leo´n, M. Brigl, E. A. Leadbetter, J. E. Gumperz, 25. Griewank, K., C. Borowski, S. Rietdijk, N. Wang, A. Julien, D. G. Wei, C. C. Dascher, T. Y. Cheng, F. M. Sacks, P. A. Illarionov, et al. 2005. A. A. Mamchak, C. Terhorst, and A. Bendelac. 2007. Homotypic interactions Apolipoprotein-mediated pathways of lipid antigen presentation. Nature 437: mediated by Slamf1 and Slamf6 receptors control NKT cell lineage develop- 906–910. ment. Immunity 27: 751–762. 41. Kang, S. J., and P. Cresswell. 2004. Saposins facilitate CD1d-restricted pre- 26. Park, S.-H., J. H. Roark, and A. Bendelac. 1998. Tissue-specific recognition of sentation of an exogenous lipid antigen to T cells. Nat. Immunol. 5: 175–181.

mouse CD1 molecules. J. Immunol. 160: 3128–3134. 42. Zhou, D., C. Cantu, III, Y. Sagiv, N. Schrantz, A. B. Kulkarni, X. Qi, http://www.jimmunol.org/ 27. Liu, Y., R. D. Goff, D. Zhou, J. Mattner, B. A. Sullivan, A. Khurana, C. Cantu, III, D. J. Mahuran, C. R. Morales, G. A. Grabowski, K. Benlagha, et al. 2004. E. V.Ravkov, C. C. Ibegbu, J. D. Altman, et al. 2006. A modified a-galactosyl ceramide Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins. for staining and stimulating natural killer T cells. J. Immunol. Methods 312: 34–39. Science 303: 523–527. 28. Wei, D. G., H. Lee, S. H. Park, L. Beaudoin, L. Teyton, A. Lehuen, and 43. Lang, G. A., T. S. Devera, and M. L. Lang. 2008. Requirement for CD1d ex- A. Bendelac. 2005. Expansion and long-range differentiation of the NKT cell pression by B cells to stimulate NKT cell-enhanced antibody production. Blood lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. 111: 2158–2162. Med. 202: 239–248. 44. Barral, P., J. Eckl-Dorna, N. E. Harwood, C. De Santo, M. Salio, P. Illarionov, 29. Coles, M. C., and D. H. Raulet. 2000. NK1.1+ T cells in the liver arise in the G. S. Besra, V. Cerundolo, and F. D. Batista. 2008. B cell receptor-mediated thymus and are selected by interactions with class I molecules on CD4+CD8+ uptake of CD1d-restricted antigen augments antibody responses by recruiting cells. J. Immunol. 164: 2412–2418. invariant NKT cell help in vivo. Proc. Natl. Acad. Sci. USA 105: 8345–8350. 30. Zimmer, M. I., A. Colmone, K. Felio, H. Xu, A. Ma, and C. R. Wang. 2006. A 45. Moody, D. B., V. Briken, T. Y. Cheng, C. Roura-Mir, M. R. Guy, D. H. Geho,

cell-type specific CD1d expression program modulates invariant NKT cell de- M. L. Tykocinski, G. S. Besra, and S. A. Porcelli. 2002. Lipid length controls by guest on October 1, 2021 velopment and function. J. Immunol. 176: 1421–1430. antigen entry into endosomal and nonendosomal pathways for CD1b presenta- 31. Wei, D. G., S. A. Curran, P. B. Savage, L. Teyton, and A. Bendelac. 2006. tion. Nat. Immunol. 3: 435–442. Mechanisms imposing the Vb bias of Va14 natural killer T cells and con- 46. Bendelac, A., L. Teyton, and P. B. Savage. 2002. Lipid presentation by CD1: the sequences for microbial glycolipid recognition. J. Exp. Med. 203: 1197–1207. short and the long lipid story. Nat. Immunol. 3: 421–422.