a Humanized Mouse Model Invariant NKT Cells in + ...

A Subset of CD8αβ+ Invariant NKT Cells in a Humanized Mouse Model This information is current as of August 7, 2015.

Xiangshu Wen, Seil Kim, Ran Xiong, Michelle Li, Agnieszka Lawrenczyk, Xue Huang, Si-Yi Chen, Ping Rao, Gurdyal S. Besra, Paolo Dellabona, Giulia Casorati, Steven A. Porcelli, Omid Akbari, Mark A. Exley and Weiming Yuan

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2015; 195:1459-1469; Prepublished online 8 July 2015; doi: 10.4049/jimmunol.1500574 http://www.jimmunol.org/content/195/4/1459

The Journal of Immunology

A Subset of CD8ab+ Invariant NKT Cells in a Humanized Mouse Model Xiangshu Wen,* Seil Kim,* Ran Xiong,* Michelle Li,* Agnieszka Lawrenczyk,*,1 Xue Huang,* Si-Yi Chen,* Ping Rao,*,2 Gurdyal S. Besra,† Paolo Dellabona,‡ Giulia Casorati,‡ Steven A. Porcelli,x Omid Akbari,* Mark A. Exley,{,‖ and Weiming Yuan*

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atural killer T cells are a group of unconventional T cells that coexpress TCR and typical surface receptors for NK cells and recognize lipid Ags presented by the MHC class I–like molecule, CD1d (1–4). Invariant NKT (iNKT) cells are *Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; †School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom; ‡Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20134 Milano, Italy; xDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; {Faculty of Medical and Human Sciences, Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester M13 9NT, United Kingdom; and ‖Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 1

Current address: BioLegend, Inc., San Diego, CA.

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Current address: UCLA Immunogenetics Center and Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA. ORCID: 0000-0002-4780-7157 (W.Y.). Received for publication March 13, 2015. Accepted for publication June 12, 2015. This work was supported by National Institutes of Health (NIH) Grants R01 AI 091987 and U01 GM 111849, a Harry Lloyd Charitable Trust grant and a Margaret Early Medical Research Trust grant (to W.Y.). G.S.B. acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick, a Royal Society Wolfson Research Merit Award, and the Medical Research Council. P.D. was supported by Associazione Italiana Ricerca sul Cancro (AIRC) Grant IG 2014 Id.15466, and G.C. was supported by AIRC Grant IG 2014 Id.15517. S.A.P. was supported by NIH Grant RO1 AI 045889. M.A.E. was supported by NIH Grant CA 170194. Address correspondence and reprint requests to Dr. Weiming Yuan, Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; BMDC, bone marrow–derived dendritic cell; DN, double-negative; Eomes, eomesodermin; a-GalCer, a-galactosylceramide; iNKT, invariant NKT; KI, knockin; KO, knockout; MFI, mean fluorescence intensity; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500574

a subset of NKT cells defined by the Va24Ja18 TCRa-chain in humans and the Va14Ja18 TCRa-chain in mice. The initial discovery of the potent antitumor function of a-galactosylceramide (a-GalCer), the prototypical ligand of iNKT cells, in mouse models stimulated great interest in the field (5–8). About 30 clinical trials using a-GalCer have been reported (8, 9). Despite continuous technical improvement, the antitumor function of a-GalCer in human clinics has been limited so far. Many factors may have contributed to this sharp contrast in a-GalCer function between human and mouse models, including a major affinity difference in the lipid-presentation properties of human versus mouse CD1d as well as the abundance, composition, and functional properties of iNKT cells in humans and mice (10–12). One major difference between human and murine iNKT cells is the composition and subsets of iNKT cells (1, 2, 11, 13, 14). Accumulating evidence has shown that iNKT cells are composed of heterogeneous populations that possess diverse function and exhibit substantially different proliferative and homeostatic properties (15–17). Therefore, differences in the composition of iNKT cells in human versus mouse may have a substantial impact on the overall immune responses to a single lipid ligand, such as a-GalCer, in vivo. There have been different approaches to categorize NKT cell subsets (18, 19). Currently, the most common classification of iNKT cell subsets has been based on the expression of conventional coreceptors, namely CD4 and CD8. Although the CD4+ and CD42CD82 (double-negative [DN]) subsets are present in both human and mice, a subset of CD8+ iNKT cells were only found in human (15, 16, 20, 21). Little is known about the development of the CD8+ iNKT cells or their contribution in diverse immune responses. We aimed to build a new mouse model to investigate the in vivo functional properties of the lipid presentation system of human


Invariant NKT (iNKT) cells are unconventional innate-like T cells demonstrating potent antitumor function in conventional mouse models. However, the iNKT cell ligands have had limited efficacy in human antitumor clinical trials, mostly due to the profound differences in the properties and compositions of iNKT cells between the two species, including the presence of a CD8+ subset of iNKT cells only in humans. To build reliable in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in. To further humanize the mouse model, we now introduced the human invariant NKT TCRa-chain (Va24Ja18) into the hCD1d-knockin mice. Similar to humans, this humanized mouse model developed a subset of CD8ab+ iNKT cells among other human-like iNKT subsets. The presence of the CD8ab+ iNKT cells in the thymus suggests that these cells developed in the thymus. In the periphery, these NKT cells showed a strong Th1-biased cytokine response and potent cytotoxicity for syngeneic tumor cells upon activation, as do human CD8ab+ iNKT cells. The low binding avidity of iNKT TCRs to the human CD1d/lipid complex and high prevalence of Vb7 TCRb among the CD8+ iNKT cells strongly point to a low avidity–based developmental program for these iNKT cells, which included the suppression of Th-POK and upregulation of eomesodermin transcriptional factors. Our establishment of this extensively humanized mouse model phenotypically and functionally reflecting the human CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell–based immunotherapies. The Journal of Immunology, 2015, 195: 1459–1469.

CD8ab+ iNKT CELLS IN HUMANIZED MICE

1460 CD1d and NKT cells and to more reliably predict the immune responses toward the glycolipid drug candidates targeting human iNKT cells in clinics. To this end, we reported the first human CD1d-knockin (hCD1d-knockin [KI]) mouse and demonstrated that the KI of human CD1d leads to the development of iNKT cells with human-like phenotypes with respect to the TCR usage, abundance, and expression pattern of CD4 coreceptor in iNKT cells (13). To further humanize the CD1d/NKT cell system, we have now introduced the invariant TCRa-chain of human iNKT cells into the hCD1d-KI mice. Interestingly, we detected a distinct group of Th1-biased iNKT cells in the thymus and periphery expressing the CD8 coreceptor and with stronger cytotoxicity in killing B16F10 tumor cells than that of DN iNKT cells, demonstrating that human CD1d/NKT lipid-presentation supports the development of functional CD8+ iNKT cells.

Mice C57BL/6 background mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred locally. C57BL/6 background CD1d2/2 mice with both CD1d1 and CD1d2 genes knocked out were generously provided by Dr. Chyung-Ru Wang from Northwestern University. hCD1d-KI/Va24transgenic (hCD1d-Va24Tg) mice were generated by crossing Va24 transgenic mice (22) and hCD1d-KI mice (13), and their genotype were confirmed as previously described (13, 22). Both Va24Tg and our hCD1d-KI mice were generated at C57BL/6 background. The Va24Tg mice were previously bred into Ja182/2 mice, which had been backcrossed into C57BL/ 6 mice for eight generations (22). The fully bred hCD1d-Va24Tg mice have the genotype of CD1dh/hJa182/2Va24Tg+. All mice were bred into homozygotes unless otherwise indicated. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Southern California.

Abs, ex vivo analysis, and flow cytometry Single-cell suspensions were prepared from thymus, spleen, and liver and incubated with anti-CD16/32 (eBioscience) for mouse and human FcR binding inhibitor (eBioscience) for human cells to block FcgRII/III, followed by staining with fluorochrome-conjugated mAbs and analysis by flow cytometry, as previously described (13). Abs were purchased from eBioscience, BD Biosciences, and BioLegend, including FITC-mCD1d (1B1), PE-mCD1d (1B1), PerCP-Cy5.5–TCRb (H57-597), allophycocyanin-eF780– TCRb (H57-597), Alexa 647–TCR Vb8.1,8.2 (KJ16-133.18), FITC–TCR Vb7 (TR310), eF450-NK1.1 (PK136), PE-Cy7–CD4 (GK1.5), allophycocyanineF780–CD8a(53-6.7), eF450-CD8a (53-6.7), FITC-CD8b (eH35-17.2), PE-Cy7–CD8b (H35-17.2), allophycocyanin-CD8b (H35-17.2), eF450CD122 (TM-b1), allophycocyanin-CD49b (DX5), eF450-mCD11a (M17-4), allophycocyanin-CXCR3 (CXCR3-173), allophycocyanin-CCR4 (2G12), allophycocyanin-CCR5 (HM-CCR5), PerCP-c5.5–CCR6 (29-2L17), allophycocyanin-CXCR4 (L276F12), eF450-CD11c (N418), Alexa 647– eomesodermin (Eomes; Dan11mag), PE–Th-POK (2POK), allophycocyanin– Annexin V, Alexa 647–Bcl2 (BCL/10C4), FITC-hTCRab (T10B9), allophycocyanin-hTCRab (T10B9), PerCP-Cy5.5–hCD4 (OKT4), allophycocyanin-Cy7–hCD8a (HIT8a), eF450-hCD8a (OKT8a), PE-Cy70– hCD8b (SIDI8BEE), PE-hCD122 (Mik-b3), allophycocyanin-hCD62L (DREG-56), allophycocyanin-CD161 (DX12), FITC-CD69 (FN50), eF450CD45RO (UCHL1), allophycocyanin-CD45RA (HI100), allophycocyaninCXCR3 (G025H7), PE–Th-POK (11H11A14), FITC-Eomes (WD1928), FITC-hCD11a (G43-25B), PerCP-Cy5.5–IFN-g (XMG1.2), PE-Cy7–IL-4 (BVD6-24G2), FITC–Ki-67 (SolA15), allophycocyanin-RORgt PerCPCy5.5–rat IgG1k isotype control (eBRG1), PE-Cy7–rat IgG1k isotype control (eBRG1), and PE-mCD1d/PBS-57 tetramer or PE-hCD1d/PBS-57 tetramer were generously provided by National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA). In anti-CD8a blocking experiments, 10 mg/ml anti-CD8a (53.6.7, eBioscience; CT-CD8a, Accurate) or isotype control (rat IgG2a) was included during stainings of iNKT cells with hCD1d/PBS-57 tetramers.

iNKT cell expansion, cell sorting, RNA isolation, reverse transcription, and quantitative real-time PCR Splenic lymphocytes were cocultured with bone marrow–derived dendritic cells (BMDCs) in the presence of 100 ng/ml a-GalCer and 50 ng/ml mouse rIL-2 in complementary RPMI 1640 containing 10% FBS,

BMDC preparation, Ag presentation assay, detection of cytokines, cytotoxicity assay, and in vivo B16F10 metastasis assay BMDC preparation, Ag presentation assay, and detection of cytokines were performed as previously described (13). For cytotoxicity assays, in vitro– expanded iNKT cells were sorted into CD8ab+ or DN iNKT cells and cocultured with CFSE-labeled B16F10 melanoma cells in a ratio of E:T cells in 1:1, 1:10, or 1:100 for 12 h. Cells were stained with Annexin V, 7-aminoactinomycin D (7-AAD), and perforin and then examined by FACS Canto II (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR) as described above. For in vivo metastatic assays, splenic iNKT cells were expanded in vitro by coculturing with BMDCs loaded with a-GalCer for 2 wk. CD42CD82 (DN) or CD8+ iNKT cells were then sorted by FACSAria III (BD Biosciences). Age/gender-matched Ja18-knockout (KO)/hCD1d-KI (6–8-wk-old) mice were administered with 0.1 million B16F10 cells, 2 mg a-GalCer, and 2 million of sorted iNKT cell subsets via tail vein. Recipient mice were euthanized after 2 wk. The mouse lungs were collected and perfused with PBS, and melanoma nodules were counted as previously described (7, 23).

Statistical analysis Descriptive statistics are expressed as the mean 6 SD values. Comparisons between groups were performed using two-tailed Student t test, and a p value ,0.05 was considered significant.

Results Development of CD8+ iNKT cells in mice with a humanized CD1d/iNKT TCR lipid presentation system In hCD1d-KI mice, we detected a reduced number of CD4+ iNKT cells and a much higher number of DN iNKT cells compared with those in wild-type (WT) mice (13), suggesting that CD1d/TCR signaling has a significant impact on coreceptor expression in iNKT cells. To further humanize the CD1d/NKT lipid presentation system in mice, we introduced a prearranged human Va24/Ja18 transgene (22) into the hCD1d-KI mice. The Va24Tg mice were in Ja182/2 mouse background (22). To eliminate the selection of endogenous mouse iNKT TCR, we also introduced the Ja182/2 allele into the hCD1d-Va24Tg mice. Because the iNKT cells in hCD1d-Va24Tg mice were developed with hCD1d as a restriction factor, we used a hCD1d tetramer loaded with a-GalCer analog, PBS57, to detect iNKT cells. We indeed detected iNKT cell populations in both thymus and periphery (Fig. 1A). To confirm that these iNKT cells are true Va24+ iNKT cells, we also costained the iNKT cells with anti-Va24 mAb 6B11 (24). All of the CD1d-tetramer+ iNKT cells are positive for Va24 expression (Supplemental Fig. 1A). The abundance (both percentage and total cell numbers) of hCD1d-tetramer+ iNKT cells in the thymi and spleens of Va24/Ja18 transgene-expressing hCD1d-KI mice (hCD1d-Va24Tg) is similar to that of Va24 transgenic (Va24Tg) mice, whereas the abundance of iNKT cells is much lower in the livers of hCD1d-Va24Tg mice than that in Va24Tg mice (Fig. 1A, right panel) (22). These results suggest that iNKT cells have different homeostatic kinetics in peripheral spleens and livers in these mice. It is important to note that the expression of hCD1d is under the endogenous mCD1d promoter in hCD1d-KI mice, and therefore, tissue-specific expression pattern of hCD1d is expected to be comparable to that of mCD1d in WT mice (13). It will be interesting to further compare the expression levels of hCD1d in different


Materials and Methods

100 U/ml penicillin, 100 mg/ml streptomycin, 5 3 1025 M 2-ME, 10 mmol HEPES, MEM (1:50), and MEM nonessential amino acids (1:100) for 14– 18 d. Cell culture media were changed every 2 to 3 d. Expanded iNKT cells or freshly isolated thymic or splenic mononuclear cells were stained and sorted as previously described (13). The sorted cells were collected in complete RPMI 1640 medium and checked for their purity (.98%). Human peripheral blood was purchased (HemaCare), and iNKT cells were enriched by AutoMACSpro sorter after staining with PE-hCD1d/PBS57 tetramers and conjugated anti-PE microbeads (Miltenyi Biotec) for further analysis. RNA isolation, reverse transcription, and quantitative real-time PCR were performed as previously described (13).


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subsets of APCs from the spleens and livers of hCD1d-Va24Tg mice to those of mCD1d in Va24Tg mice to investigate whether expression levels of hCD1d in these APCs are responsible for the differential homeostasis and/or abundance of iNKT cells in spleens versus livers. Nevertheless, the abundance of liver iNKT cells (∼3%) in hCD1d-Va24Tg mice is more similar to that in human livers (∼1%) than to that in the livers of WT C57BL/6 mice (∼20%) (11, 13, 25–27). The staining for Vb usage of the iNKT cells in hCD1d-Va24Tg mice again showed that hCD1d preferentially selects the Vb8 chain over Vb7 chain for TCRb usage, as we have observed previously in hCD1d-KI mice (13). The most abundant pop-

ulation of iNKT cells in hCD1d-Va24Tg mice use Vb8 TCRbchain (∼60% in the liver; Fig. 1B). We also compared Vb usage in total iNKT cells of hCD1d-Va24Tg mice to that of Va24Tg mice and detected a higher Vb7 usage when mCD1d was the selecting molecule (Supplemental Fig. 1B, 1C), which was consistent with previous findings that human CD1d enhances Vb8 usage (13). We then examined coreceptor expression in the iNKT cells of hCD1d-Va24Tg mice. Interestingly, in contrast to WT mice, which contain essentially no CD8+ iNKT cells, 12–20% of liver iNKT cells in hCD1d-Va24Tg mice express CD8a-chain (Fig. 1C). Further examination showed that the CD8a+ iNKT cells


FIGURE 1. Identification of CD8ab+ iNKT cells in humanized mice. (A) Development of human CD1d-restricted Va24/Ja18 iNKT cells in hCD1dVa24Tg mice. Single-cell suspensions from thymus (Thy), spleen (Sp), and liver (Liv) were stained with PBS57-loaded human CD1d tetramers and anti-TCRb Abs and analyzed for the percentage and absolute numbers of iNKT cells by flow cytometry. (B) TCR Vb usages were analyzed in iNKT (hCD1d-PBS57+TCRb+) cells from the thymus, spleen, and liver of hCD1d-Va24Tg mice. (C) Detection of CD8+ iNKT cells in hCD1d-restricted iNKT cells by staining with anti-CD4 and anti-CD8a mAbs. (D) Expression of CD8a and CD8b in CD8+ iNKT cells in thymus, spleen and liver of hCD1dVa24Tg mice. (E) Quantitation of CD8a and CD8b expression by real-time PCR in mouse thymocytes. HPRT gene was used as internal control. The data are representative of more than three independent experiments. In all of the experiments, mouse group size is n $ 3.

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CD8ab+ iNKT cells manifest a memory-like T cell phenotype and have Th1-like cytotoxic function There have been several reports for the presence of CD8ab+ iNKT cells in humans (15–17, 20, 28, 29). Due to their paucity in human samples, little is known about their phenotype and function. We enriched iNKT cells from human peripheral blood and stained for common surface markers. Similar to CD4+ and DN iNKT cells, CD8ab+ iNKT cells express CD45RO, CD161, and CD122 markers but express low to medium levels of CD69, showing a memory-like T cell phenotype (Supplemental Fig. 1D). To characterize the CD8+ iNKT cells in hCD1d-Va24Tg mice, we stained for T and NK cell markers. Similar to iNKT cells in WT mice and the CD4+ and DN subsets of iNKT cells, most CD8+ iNKT cells are CD62LloCD44hiDX5+CD122+, consistent with a memory-like T cell phenotype (Fig. 2A). Notably, in contrast to iNKT cells in WT mice, a substantial number of iNKT cells in all subsets in hCD1d-Va24Tg mice are low in NK1.1 expression, resembling the low expression of human homologous NK cell marker, CD161, in many of human iNKT cells (15, 16). The substantially lower number of iNKT cells in the livers of hCD1d-Va24Tg mice compared with that in Va24Tg mice prompted us to examine the proliferative properties of these cells. Although comparable percentages of iNKT cells stained positive for Annexin V in thymi and spleens among three mouse strains, there were substantially higher numbers of iNKT cells in the livers of hCD1d-Va24Tg mice staining positive for Annexin V (∼15%) than those of Va24Tg (Fig. 2B). We also examined the potential of expansion in the iNKT cells by stainings of Ki-67 protein, a nuclear protein associated with cell proliferation (18). Our preliminary studies did not detect a significant difference at the basal levels of potential for expansion by Ki-67 staining in splenic or hepatic iNKT cells between hCD1d-Va24Tg and Va24Tg mice (Fig. 2C). These results suggested that the higher apoptosis rate may be a major mechanism responsible for the substantial decrease in hepatic iNKT cells in hCD1d-Va24Tg mice (Fig. 1A). Interestingly, among the different subsets of iNKT cells in the liver, we detected a lower apoptosis rate in CD8+ and DN iNKT cells than that in CD4+ iNKT cells, with CD8+ iNKT cells showing the lowest apoptosis rate (Fig. 2D). Concomitantly, we detected higher Bcl2 expression in CD8+ and DN iNKT cells compared with CD4 + iNKT cells (Fig. 2E). In contrast, the proliferation capacity measured by in vitro expansion and CFSE dilution was higher in CD8+ and DN iNKT cells (Fig. 2F). This may explain why in the steady state, CD8+ and DN cells represent the

majority of iNKT cells in the livers of hCD1d-Va24Tg mice (as shown in Fig. 1C). The relatively higher proliferative capacity and lower apoptosis rate of DN iNKT cells compared to CD4+ iNKT cells in these mice are consistent with a previous report for DN and CD4+ iNKT cells in humans (17). To examine whether human CD8+ iNKT cells also have a low apoptosis rate, we also examined Annexin V staining of primary human iNKT cells in peripheral blood from healthy donors. We indeed detected low Annexin V staining in human CD8ab+ iNKT cells (Fig. 2G), suggesting that this long survival feature is an intrinsic property of CD8ab+ iNKT cells. To investigate the function of CD8ab+ iNKT cells in hCD1dVa24Tg mice, we first examined the cytokine secretion by these iNKT cells after PMA/ionomycin activation. Compared to CD4+ and DN iNKT cells, CD8+ iNKT cells had a higher percentage of cells that secrete cytokine IFN-g (Fig. 3A). Approximately 16% CD8+ iNKT cells compared with 3–6% CD4+ and DN iNKT cells secreted IFN-g. In contrast, all iNKT cells from this strain were lower in IL-4 secretion than that in WT mice (Fig. 3A, Supplemental Fig. 1E) (2, 13). Within CD8+ iNKT cells, a comparable number of CD8ab+ and CD8aa+ iNKT cells secrete IFN-g. To examine whether the CD8ab+ iNKT cells in these mice are functional in TCR-mediated cytokine responses, we sorted the splenic iNKT cells from hCD1d-Va24Tg mice and cocultured them with autologous BMDCs preloaded with a-GalCer. Whereas the CD4+ and DN iNKT cells secrete both IFN-g and IL-4, CD8ab+ iNKT cells exclusively secrete IFN-g (Fig. 3B), showing a strong Th1-like phenotype. To further characterize the functionality of CD8ab+ iNKT cells, we also examined whether these iNKT cells secret IL-17. Primary CD8ab+ or other subsets of iNKT cells were sorted from the splenocytes of hCD1d-Va24Tg mice and cocultured with BMDCs loaded with a-GalCer. Interestingly, CD8ab+ iNKT cells produced substantially higher amount of IL-17 than the other two subsets, although the difference is not significant in this assay condition (Supplemental Fig. 1F). Consistent with the higher secretion of IL-17, we detected the transcriptional factor retinoic acid–related orphan receptor gt is expressed at higher levels in CD8ab+ iNKT cells compared with that in other subsets (Supplemental Fig. 1G). These results suggest that CD8ab+ iNKT cells also have Th17like phenotype. We are very interested in further investigating whether these CD8ab+ iNKT cells play important roles in Th17type immune responses and whether they regulate the function of canonical Th17 cells as suggested previously (30, 31). To investigate the in vivo function of different iNKT subsets in response to lipid presentation in hCD1d-Va24Tg mice, we administered a-GalCer (2 mg/mouse) to the mice and examined cytokine secretion in vivo. Two hours post–lipid challenge, hepatic iNKT cells were cultured ex vivo for 12 h, and secretion of IFN-g and IL-4 was examined with intracellular staining. Again, consistent with in vitro stimulation, a higher percentage of CD8+ iNKT cells secreted IFN-g than CD4+ or DN iNKT cells, whereas few iNKT cells secreted IL-4 (Fig. 3C). We examined the kinetics of iNKT cell expansion at 3 d post–lipid treatment, and substantial expansion of iNKT cells was detected in the spleens and livers, similar to that in WT mice (Fig. 3D and data not shown). We did detect a clear expansion CD8+ iNKT cells in both the percentage and total cell numbers upon lipid challenge (Fig. 3E). Although at this time point (3 d post–lipid treatment), we did not detect a significance difference in the potential for expansion by Ki-67 staining among different subsets of hepatic or splenic iNKT cells (Fig. 3F, 3G), we have repeatedly observed a higher basal level of potential for expansion in the CD8+ iNKT cells compared with other subsets in both hepatic and splenic iNKT cells (Fig. 3F, 3G). These results may be consistent with the relatively high expression of Bcl2 staining in CD8+ iNKT cells (Fig. 2E) and the higher expansion of


are also present in the spleen and thymus (Fig. 1C). Although CD8a+ iNKT cells are also detected in Va24Tg mice expressing mCD1d, it is clear that expression of hCD1d promotes the development and/or homeostasis of CD8+ iNKT cells among the iNKT cell subsets (average ∼12–20% in hCD1d-Va24Tg mice compared with ∼3% in Va24Tg mice; Fig. 1C). CD8b costaining showed that while in the liver, there was a portion of CD8a+ CD8b2 (presumably CD8aa+) iNKT cells, .90% of splenic and virtually all of thymic CD8a+ iNKT cells are CD8ab+ iNKT cells (Fig. 1D), suggesting that the CD8ab+ iNKT cells in the periphery developed in the thymus. To further verify the coexpression of CD8a and CD8b subunits in thymic CD8ab+ iNKT cells, we sorted thymic iNKT cells from WT C57BL/6 mice or hCD1dVa24Tg mice and performed real-time PCR experiment to examine CD8a and CD8b gene expression in iNKT cells. Both CD8a and CD8b are expressed at higher levels in iNKT cells from hCD1d-Va24Tg mice (Fig. 1E). This further confirmed that authentic CD8ab+ iNKT cells are developed in the thymi of hCD1d-Va24Tg mice.



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CD8+ iNKT cells compared with other subsets upon lipid challenges (Fig. 3E). Nevertheless, a more detailed kinetic analysis of iNKT cell proliferation is needed to understand the dynamics and underlying mechanisms for different iNKT cell subsets upon lipid challenges. One intrinsic function of iNKT cells is their direct cytotoxicity against tumor cells. To investigate whether the CD8ab+ iNKT cells


FIGURE 2. CD8+ iNKT cells show a memory-like T cell phenotype and exhibit a Th1-biased cytokine profile and profound antitumor cytotoxicity. (A) iNKT cell maturation markers and NK cell markers were analyzed in CD4+, DN, and CD8+ iNKT cells in liver mononuclear cells in hCD1d-Va24Tg mice by flow cytometry. (B) The percentage of Annexin V+ iNKT cells in thymus (Thy), spleen (SP), and liver (Liv) in WT, Va24Tg, and hCD1d-Va24Tg mice. (C) Examination of the potential for expansion of splenic and hepatic iNKT cells in Va24Tg and hCD1d-Va24Tg mice by Ki-67 staining. (D) Expression of Annexin V in CD8+, CD4+, and DN iNKT cells in livers of hCD1d-Va24Tg mice. (E) Expression of antiapoptotic marker Bcl2 in CD8+ iNKT cells in hCD1d-Va24Tg mice. (F) Cell proliferation capacity by CFSE labeling in liver CD8+, CD4+, and DN iNKT cells after culturing in presence of a-GalCer (100 ng/ml) and rIL-2 (50 ng/ml). (G) In comparison with CD4+ or DN iNKT cells, relatively low expression levels of Annexin V in CD8+ iNKT cells in human PBMC. ns, not significant.

have antitumor cytotoxicity, B16F10 melanoma cells were first labeled with CFSE and then used as target cells in a cytotoxicity assay. In vitro–expanded iNKT cells were sorted into subpopulations and cocultured with CFSE-labeled B16F10 melanoma cells for 12 h. CFSE-positive B16F10 cells were analyzed for Annexin V and 7-AAD staining, and we detected a stronger cytotoxicity in killing B16F10 cells by CD8+ iNKT cells than that of

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FIGURE 3. Functional properties of iNKT cell subsets in hCD1d-Va24Tg mice in response to lipid presentation. (A) Liver mononuclear cells from hCD1d-Va24Tg mice were treated with PMA and ionomycin followed by GolgiStop. Secretion of IFN-g and IL-4 was analyzed by intracellular staining. (B) Sorted iNKT cell subsets from in vitro–expanded splenic iNKT cells of hCD1d-Va24Tg mice were cocultured with a-GalCer–loaded BMDCs for 48 h before supernatant cytokines were measured by ELISA. (C–G) Two micrograms of a-GalCer per mouse was injected to hCD1d-Va24Tg or C57BL/6 (WT) mice, and in vivo responses of iNKT cells were examined. Cytokine secretion was examined by ex vivo intracellular staining 2 h post–lipid treatment (C), and the abundance of iNKT cells was examined by hCD1d-PBS57 tetramer staining 3 d post–lipid treatment (D). The abundance (E) and potential for expansion in different hepatic (F) and splenic (G) iNKT cell subsets were examined by Ki-67 staining 3 d post–lipid treatment. ns, not significant; Veh, vehicle.


CD8 is unlikely a coreceptor molecule for CD1d lipid presentation to CD8ab+ iNKT cells In WT mice, there are virtually no CD8ab+ iNKT cells. One hypothesis put forth to explain this is that CD8 is a coreceptor for

CD1d that increases the CD1d/TCR avidity over the selection threshold to cause negative selection and deletion of CD8ab+ iNKT cells (21, 33). Consistent with this, the transgenic expression of CD8 leads to an ablation of iNKT cell development (21, 33). To examine whether CD8 molecules serve as coreceptors for the CD8ab+ iNKT cells in hCD1d-Va24Tg mice, we first performed lipid-presentation assays in the absence or presence of anti-CD8 Abs. We could not detect significant inhibition by antiCD8 Ab of CD1d presentation of lipid to CD8ab+ iNKT cells (Fig. 5A), suggesting that the CD8 molecule does not play a coreceptor role in the activation of peripheral CD8ab+ iNKT cells. To further investigate whether the CD8 molecule directly binds to CD1d in both thymi and periphery, we compared the efficiencies of CD1d binding to iNKT cell TCRs in the presence or absence of anti-CD8a Abs using the mean fluorescence intensity (MFI) of the hCD1d tetramer staining as readout (Fig. 5B, 5C). It is unknown structurally where the CD8 molecule may bind to the CD1d molecule. Epitope mapping studies demonstrated that two anti-mouse CD8a Abs 53.6.7 or CT-CD8a bind the CD8a molecule either away or in the interface of mouse CD8a/MHC class I interaction, respectively (34). Because CD1d is a MHC class I–like molecule and possesses a MHC class I–like folding in its ectodomain (35), we

FIGURE 4. Antitumor function of CD8ab+ iNKT cells. (A) CFSE-labeled B16F10 cells were cocultured with either sorted DN or CD8ab+ iNKT cells from hCD1d-Va24Tg mice in the presence of a-GalCer (100 ng/ml) in different E:T ratios for 12 h, and CFSE+ B16F10 cells were analyzed by staining with Annexin V and 7-AAD. (B) Splenic iNKT cell subsets from hCD1d-Va24Tg mice were cocultured with BMDCs cells for 48 h and analyzed for perforin expression by intracellular stainings. (C) Splenic iNKT cells from hCD1d-Va24Tg mice were expanded in vitro with BMDCs loaded with a-GalCer and coinjected with B16F10 cells and a-GalCer into Ja18KO/hCD1d-KI mice. The metastatic melanoma nodules in mouse lungs were tallied after 2 wk. Mouse group size is n $ 3. Data are representative of three repeats.


DN iNKT cells (Fig. 4A). Because DN cells are more potent in cytotoxicity than CD4+ iNKT cells (23, 32), our results suggest that CD8ab+ iNKT cells have the most potent cytotoxic function among the three subsets of iNKT cells. We then examined perforin expression levels and detected higher expression levels of perforin in CD8ab+ iNKT cells among all subsets when cocultured with a-GalCer–loaded BMDCs (Fig. 4B), consistent with results from the cytotoxicity assay (Fig. 4A). To examine whether CD8ab+ iNKT cells play antitumor function in vivo, we expanded the CD8ab+ iNKT cells from splenocytes of hCD1d-Va24Tg mice and coadministered with B16F10 melanoma cells as previously reported (7, 23). We used Ja18-KO/hCD1d-KI instead of CD1dKO mice as recipient mice so the endogenously expressed human CD1d protein can support the survival and/or activation of transferred iNKT cells. Indeed, we detected a potent antitumor function for both the DN and CD8+ iNKT cells of hCD1d-KI mice in these assays (Fig. 4C).

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examined possible effects of both anti-CD8a Abs in the binding of CD1d tetramers to iNKT TCRs. No blocking of CD1d binding to TCRs was detected in the presence of either anti-CD8a Ab (Fig. 5B, 5C), suggesting that the CD8 molecule does not bind to CD1d directly. One caveat in these experiments with hCD1d-Va24Tg mice is that human CD1d may not bind to the mouse CD8 molecule. To address this issue, we further measured the mouse CD1d binding to iNKT TCR in Va24Tg mice, which expresses mouse CD1d. Again, binding of neither of the two Abs to CD8a-chain has a detectable effect on the binding of mCD1d tetramers to iNKT TCR in CD8ab+ iNKT cells in the thymus or periphery (Fig. 5D, 5E). Although all these results do not exclude the possibility of the CD8ab molecule functioning as a coreceptor for mouse CD1d during iNKT cell development in WT mice, they suggested that CD8ab protein is unlikely a coreceptor for CD1d when human CD1d and human invariant NKT TCR are engaged during iNKT cell development. A specific transcriptional program leads to the development of CD8ab+ iNKT cells In some mouse strains, for example, in mice lacking the critical transcriptional factor for CD4/CD8 T cell lineage commitment, Th-POK, there exist CD8ab+ iNKT cells (36). The hypothesis is

that a specific gene expression program involving the suppression of Th-POK expression is the key factor for the development of CD8+ iNKT cells (36). Because Th-POK represses CD8 expression (37), the absence of CD8+ iNKT cells and lack of CD8 expression in mouse iNKT cells could be a consequence of Th-POK expression in iNKT cells of WT mice. We then examined the ThPOK expression level in CD8ab+ iNKT cells in hCD1d-Va24Tg mice. In the different subsets of iNKT cells, we found that CD4+ iNKT cells express higher levels of Th-POK protein than CD8+ and DN iNKT cells do in both thymi and periphery, with a greater difference in the periphery (Fig. 6A). We also analyzed iNKT cells in human peripheral blood. Similarly, CD4+ iNKT cells expressed a relatively higher level of Th-POK than that in CD8 and DN iNKT cells (Fig. 6B). These results suggest that the suppression of Th-POK expression may be important for the development of human CD8+ iNKT cells. In contrast, Th-POK efficiently suppresses the expression of the transcription factor Eomes (37, 38), which is critical for the development of NK cells, memory CD8+ T cells, and CD42 iNKT cells (18, 39). We then compared the expression of Eomes in different subsets of iNKT cells in both our hCD1d-Va24Tg mice and primary human iNKT cells. Indeed, CD8+ iNKT cells have the highest Eomes expression compared to CD4+ and DN iNKT cells in both human and our mouse models (Fig. 6C, 6D).


FIGURE 5. CD8 molecule is unlikely to bind hCD1d as a coreceptor in hCD1d-Va24Tg mice. (A) In vitro–expanded iNKT cells were sorted, and individual subsets were cocultured with BMDCs loaded a-GalCer for 48 h. Secretion of IFN-g by the iNKT cells was measured by ELISA. Thymocytes (Thy), splenocytes (Sp), and liver (Liv) mononuclear cells of hCD1d-Va24Tg mice were stained in the presence of anti-CD8a Ab 53.6.7 (B) or CT-CD8a (C). An isotype control Ab was used as the control. MFIs of different subsets of iNKT cells were compared. Thymocytes, splenocytes, and liver mononuclear cells of Va24Tg mice were stained in the presence of anti-CD8a Ab 53.6.7 (D) or CT-CD8a (E) as in (B) and (C). Data are representative of three repeats. ns, not significant.


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Discussion Development of CD8+ iNKT cells

FIGURE 6. Development of CD8ab+ iNKT cells is associated with downregulated expression levels of Th-POK and upregulated expression of Eomes. Expression levels of Th-POK in iNKT cell subsets in thymus (Thy; top panel), spleen (Sp; middle panel), and liver (Liv; bottom panel) in hCD1d-Va24Tg mice (A) as well as in human peripheral blood (B). Expression levels of Eomes in iNKT cell subsets were measured by flow cytometry in hCD1d-Va24Tg mice (C) and human peripheral blood (D). Thymic iNKT (hCD1d-PBS57 tet+TCRb+) cells from age/gender-matched WT (dotted line), Va24Tg (red), and hCD1d-Va24Tg (green) mice were examined for the expression of Th-POK (E) and Eomes (F) by flow cytometry. Data are representative of three repeats.

To investigate how human CD1d supports the development of more CD8+ iNKT cells than mouse CD1d does (Fig. 1C), we compared the expression levels of Th-POK and Eomes between total iNKT cells in Va24Tg (using mCD1d) versus hCD1dVa24Tg. Interestingly, we detected overall lower Th-POK and higher Eomes expression levels in the thymocytes of hCD1dVa24Tg mice compared with those of Va24Tg and WT mice (Fig. 6E, 6F). Microarray data comparing gene expression in

We are reporting in this study the first mouse model, to our knowledge, with both CD1d and the invariant TCRa-chain of iNKT cells humanized. In addition to the resembling abundance of iNKT cells to that in most humans, we also demonstrated that in this mouse model, all of the subsets of iNKT cells—CD4+, DN, CD8ab+, and CD8aa+—are present. Therefore, this extensively humanized mouse model reliably recapitulates the coreceptor expression of iNKT cells in humans. CD8ab+ iNKT cells have been reported in other mouse models including Th-POK–deficient mice and hemizygotic CD8-transgenic mice (36). Nevertheless, the developmental pathways of CD8ab+ iNKT cells in the latter two mouse models and that in the hCD1d-Va24Tg mice may be different or only partially overlapped. The expression of CD8 in CD8+ iNKT cells is likely due to the forced expression of the CD8 molecule. In Th-POK–deficient mice, the expression of CD8 coreceptor may be due to the absence of Th-POK, a suppressor of CD8 gene expression (37). For the hCD1d-Va24Tg mice, it is likely that the quality and/or quantity of hCD1d-mediated TCR signaling promote the development of the CD8ab+ iNKT cells. The lower CD1d/iNKT TCR avidity measured by relative MFI values of CD1d tetramer staining and higher prevalence of Vb7 TCRb usage in CD8ab+ iNKT cells compared to those in other subsets of iNKT cells strongly suggest that lower TCR avidity leads to a distinct transcriptional program including downregulation of Th-POK and upregulation of Eomes expression, which ultimately leads to the expression of the CD8 coreceptor. Our results suggest that in hCD1d-Va24Tg mice, more iNKT cells with Vb7 TCR developed with a CD8ab+ phenotype (Fig. 7B, 7C). The TCRs of the previously reported Vb7 iNKT cells showed lower avidity to CD1d compared with that of


thymocytes from transgenic mice expressing either human or mouse CD1d proteins also confirmed these results (X. Wen and W. Yuan, unpublished observations). These results suggest that CD1d-mediated TCR signaling has critical roles in downstream transcriptional programming and in the ensuing lineage commitments of CD4+ and CD8+ iNKT cells. To further investigate how the specific transcriptional programs arise and how CD8ab+ iNKT cells develop, we measured the avidity of CD1d/iNKT TCR interaction in different iNKT cell subsets in hCD1d-Va24Tg mice. We used the normalized MFI of CD1d tetramer staining (MFI of CD1d tetramer staining/MFI of TCRb staining) as a readout of CD1d/TCR avidity as previously reported (36). We detected the lowest relative MFI in CD8ab+ iNKT cells among all iNKT cell subsets in the thymi, suggesting that the TCRs of the CD8ab+ iNKT cells have the lowest avidity to CD1d compared with that of other iNKT cell subsets (Fig. 7A). These results are consistent with our earlier results with anti-CD8 Ab blocking (Fig. 5B–E), which suggested that CD8 does not bind human CD1d molecules as a coreceptor in the thymus. To examine how the low CD1d/TCR avidity arises for CD8ab+ iNKT cells, we compared the Vb usage in different subsets of iNKT cells in hCD1d-Va24Tg mice. Interestingly, we noticed that Vb7 was significantly overrepresented in CD8ab+ iNKT cells compared with other subsets of iNKT cells in both thymus and periphery, whereas Vb8 usage was correspondingly lower (Fig. 7B, 7C). Biochemical studies have demonstrated that the avidity of Vb7 TCR to the CD1d/lipid complex is lower than that of Vb8 TCR (40). Therefore, our results suggest that lower avidity of the CD1d/lipid complex to iNKT TCRs leads to the development of CD8ab+ iNKT cells in hCD1d-Va24Tg mice.

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Vb8 iNKT cells (40). It will be interesting to further investigate whether the differential TCR signaling ensued from different CD1d/TCR avidities leads to an instructive selection for CD8+ versus CD4+ iNKT cells in humans analogous to what was proposed for lineage commitments of conventional CD4+ and CD8+ T cells (41). In contrast, although there is no Vb7 homolog in human iNKT cells (1–3, 14), it is attractive to hypothesize that hCD1d presents specific endogenous lipid ligands with relatively low avidity (different from the lipids used to select CD4+ and DN iNKT cells) to Va24/Vb11 TCRs. This may support thymic selection of CD8ab+ iNKT cells in humans. The interaction with diverse endogenous lipid ligands can be mediated by the variable CDR3b loop of human Vb11 TCR (42). Function of CD8ab+ iNKT cells and comparison with human counterparts Our results showed that the CD8ab+ iNKT cells in hCD1dVa24Tg mice have a memory-like T cell phenotype and are particularly Th1-biased in cytokine secretion. They possess highly potent cytotoxicity and exert antitumor function when transferred to iNKT-deficient mice. Although we did not detect more potent antitumor function by these CD8ab+ iNKT cells compared with that of DN iNKT cells in our experimental settings (Fig. 4A), it is possible that these iNKT cells as well as the human counterpart CD8ab+ iNKT cells play specialized Th1-type antitumor and/or anti-infection function during immune responses. Several reports have examined the relative abundance of different iNKT cell subsets among total iNKT cells in humans (11, 15, 16, 20, 25–27). In older children (6 mo to 9 y old) or adults, the abundance of CD8+ iNKT cells in peripheral blood ranges from barely detectable to ∼20%, among which the percentage of CD8ab+ is typically ,5% in total iNKT cells (20). In contrast, ∼10% of iNKT cells in the spleens and livers of hCD1d-Va24Tg mice are CD8ab+ (Fig. 1C). This may be due to the transgenic overexpression of Va24 gene under human CD2 promoter (22). Nevertheless, our results suggested that the more humanized the CD1d/NKT cell system is, the higher percentage of CD8+ iNKT cells are developed. Our discovery of relatively low TCR avidity in CD8ab+ iNKT cells combined with the fact that the human CD1d/iNKT TCR has overall lower avidity than that in mice (40) may provide a possible explanation of why humans tend to develop CD8ab+ iNKT cells. In this regard, our mouse model

may be used to investigate the development mechanism and function of these CD8ab+ iNKT cells. Homeostasis and cell number control of iNKT cells in different organs of hCD1d-Va24Tg mice It is very interesting that a much lower number of total iNKT cells are present in the livers of hCD1d-Va24Tg mice, whereas the iNKT cells in spleen and thymi are comparable between hCD1dVa24Tg and the Va24Tg mice (Fig. 1A). Little is known about the different homeostatic requirements of iNKT cells in different organs/tissues (17, 43, 44). Our preliminary results showed that a high apoptosis rate in hepatic iNKT cells of hCD1d-KI mice (Fig. 2B) may at least partially contribute to the much lower iNKT cells in these mouse livers. Elegant studies have suggested that the peripheral survival and proliferation of mature iNKT cells are not dependent on CD1d expression but the peripheral maturation of NK1.12 to NK1.1+ iNKT cells requires CD1d expression (43, 44). It will be very interesting to further study and compare the maturation states of splenic and hepatic iNKT cells including NK1.1 expression in hCD1d-Va24Tg mice. Several groups of APCs have been reported to present exogenous lipids to iNKT cells in mouse spleens and livers (45–47). However, little is known about the type of splenic and hepatic APCs that are critical for the peripheral maturation of iNKT cells. It will be very interesting to compare the lipid presentation by mCD1d and hCD1d in these APCs in Va24Tg and hCD1d-Va24Tg mice, respectively. Both quantitative differences in CD1d expression levels and quantitative differences in the type of endogenous lipids presented by CD1d may lead to different outcomes in iNKT cell maturation and cell fate, such as apoptosis or survival. In contrast, thorough studies of the expression patterns of chemokine receptors and adhesion molecules in the iNKT cells egressed from thymus of hCD1d-Va24Tg mice may provide clues to whether potential differences in the homing properties of iNKT cells from hCD1d-Va24Tg and Va24Tg mice contribute to the sheer difference in hepatic iNKT cell numbers. In summary, we have identified a unique human-like subset of Th1-biased cytotoxic CD8ab+ iNKT cells in a new mouse model with both CD1d and iNKT TCR humanized. This model will be instrumental in further investigating the developmental mechanisms of CD8ab+ iNKT cells, as well as in delineating the exact mechanism of how these iNKT cells achieve their Th1-biased function in vivo during antitumor and anti-infection immune responses.


FIGURE 7. (A) Avidity of CD1d/iNKT TCR interaction. Normalized MFI was calculated by dividing MFIs of CD1d-tetramer staining by MFIs of corresponding TCRb staining in different subsets of iNKT cells from thymi of hCD1d-Va24Tg mice. Data are representative of three repeats. The analysis of Vb7 (B) and Vb8 (C) usage in iNKT cell subsets in hCD1d-Va24Tg mice by flow cytometry and results from three individual mice were summarized for SD. Data are representative of three repeats. Liv, liver; Sp, spleen; Thy, thymus.


Acknowledgments We thank Drs. Jae U. Jung and Shou-Jiang Gao at University of Southern California for very helpful discussions and critical readings of the manuscript. We also thank Dr. Remy Bosselut at National Institutes of Health, Dr. Mitchell Kronenberg at La Jolla Institute for Allergy Immunology, Dr. Ellen Rothenberg at California Institute of Technology, and Dr. Jonathan Kaye at Cedars-Sinai Medical Center for very helpful discussions.

Disclosures The authors have no financial conflicts of interest.

References

24. Exley, M. A., R. Hou, A. Shaulov, E. Tonti, P. Dellabona, G. Casorati, O. Akbari, H. O. Akman, E. A. Greenfield, J. E. Gumperz, et al. 2008. Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR alpha-chain CDR3 loop. Eur. J. Immunol. 38: 1756–1766. 25. Kita, H., O. V. Naidenko, M. Kronenberg, A. A. Ansari, P. Rogers, X. S. He, F. Koning, T. Mikayama, J. Van De Water, R. L. Coppel, et al. 2002. Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer. Gastroenterology 123: 1031–1043. 26. Karadimitris, A., S. Gadola, M. Altamirano, D. Brown, A. Woolfson, P. Klenerman, J. L. Chen, Y. Koezuka, I. A. Roberts, D. A. Price, et al. 2001. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl. Acad. Sci. USA 98: 3294–3298. 27. de Lalla, C., G. Galli, L. Aldrighetti, R. Romeo, M. Mariani, A. Monno, S. Nuti, M. Colombo, F. Callea, S. A. Porcelli, et al. 2004. Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis. J. Immunol. 173: 1417–1425. 28. Takahashi, T., S. Chiba, M. Nieda, T. Azuma, S. Ishihara, Y. Shibata, T. Juji, and H. Hirai. 2002. Cutting edge: analysis of human V alpha 24+CD8+ NK T cells activated by alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 168: 3140–3144. 29. Gadola, S. D., N. Dulphy, M. Salio, and V. Cerundolo. 2002. Valpha24-JalphaQindependent, CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes. J. Immunol. 168: 5514–5520. 30. Mars, L. T., L. Araujo, P. Kerschen, S. Diem, E. Bourgeois, L. P. Van, N. Carrie´, M. Dy, R. S. Liblau, and A. Herbelin. 2009. Invariant NKT cells inhibit development of the Th17 lineage. Proc. Natl. Acad. Sci. USA 106: 6238–6243. 31. Engel, I., M. Zhao, D. Kappes, I. Taniuchi, and M. Kronenberg. 2012. The transcription factor Th-POK negatively regulates Th17 differentiation in Va14i NKT cells. Blood 120: 4524–4532. 32. Liu, T. Y., Y. Uemura, M. Suzuki, Y. Narita, S. Hirata, H. Ohyama, O. Ishihara, and S. Matsushita. 2008. Distinct subsets of human invariant NKT cells differentially regulate T helper responses via dendritic cells. Eur. J. Immunol. 38: 1012–1023. 33. Lantz, O., and A. Bendelac. 1994. An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180: 1097–1106. 34. Devine, L., M. E. Hodsdon, M. A. Daniels, S. C. Jameson, and P. B. Kavathas. 2004. Location of the epitope for an anti-CD8alpha antibody 53.6.7 which enhances CD8alpha-MHC class I interaction indicates antibody stabilization of a higher affinity CD8 conformation. Immunol. Lett. 93: 123–130. 35. Zeng, Z., A. R. Castanõ, B. W. Segelke, E. A. Stura, P. A. Peterson, and I. A. Wilson. 1997. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science 277: 339–345. 36. Engel, I., K. Hammond, B. A. Sullivan, X. He, I. Taniuchi, D. Kappes, and M. Kronenberg. 2010. Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection. J. Exp. Med. 207: 1015–1029. 37. Wang, L., K. F. Wildt, E. Castro, Y. Xiong, L. Feigenbaum, L. Tessarollo, and R. Bosselut. 2008. The zinc finger transcription factor Zbtb7b represses CD8lineage gene expression in peripheral CD4+ T cells. Immunity 29: 876–887. 38. Wingender, G., and M. Kronenberg. 2008. Role of NKT cells in the digestive system. IV. The role of canonical natural killer T cells in mucosal immunity and inflammation. Am. J. Physiol. Gastrointest. Liver Physiol. 294: G1–G8. 39. Knox, J. J., G. L. Cosma, M. R. Betts, and L. M. McLane. 2014. Characterization of T-bet and eomes in peripheral human immune cells. Front. Immunol. 5: 217. 40. Pellicci, D. G., O. Patel, L. Kjer-Nielsen, S. S. Pang, L. C. Sullivan, K. Kyparissoudis, A. G. Brooks, H. H. Reid, S. Gras, I. S. Lucet, et al. 2009. Differential recognition of CD1d-alpha-galactosyl ceramide by the V beta 8.2 and V beta 7 semi-invariant NKT T cell receptors. Immunity 31: 47–59. 41. Singer, A., S. Adoro, and J. H. Park. 2008. Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Rev. Immunol. 8: 788–801. 42. Mallevaey, T., J. P. Scott-Browne, J. L. Matsuda, M. H. Young, D. G. Pellicci, O. Patel, M. Thakur, L. Kjer-Nielsen, S. K. Richardson, V. Cerundolo, et al. 2009. T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire. Immunity 31: 60–71. 43. Matsuda, J. L., L. Gapin, S. Sidobre, W. C. Kieper, J. T. Tan, R. Ceredig, C. D. Surh, and M. Kronenberg. 2002. Homeostasis of V alpha 14i NKT cells. Nat. Immunol. 3: 966–974. 44. McNab, F. W., S. P. Berzins, D. G. Pellicci, K. Kyparissoudis, K. Field, M. J. Smyth, and D. I. Godfrey. 2005. The influence of CD1d in postselection NKT cell maturation and homeostasis. J. Immunol. 175: 3762–3768. 45. Winau, F., G. Hegasy, R. Weiskirchen, S. Weber, C. Cassan, P. A. Sieling, R. L. Modlin, R. S. Liblau, A. M. Gressner, and S. H. Kaufmann. 2007. Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26: 117–129. 46. Arora, P., A. Baena, K. O. Yu, N. K. Saini, S. S. Kharkwal, M. F. Goldberg, S. Kunnath-Velayudhan, L. J. Carrenõ, M. M. Venkataswamy, J. Kim, et al. 2014. A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens. Immunity 40: 105–116. 47. Bai, L., M. G. Constantinides, S. Y. Thomas, R. Reboulet, F. Meng, F. Koentgen, L. Teyton, P. B. Savage, and A. Bendelac. 2012. Distinct APCs explain the cytokine bias of a-galactosylceramide variants in vivo. J. Immunol. 188: 3053– 3061.


1. Kronenberg, M. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23: 877–900. 2. Brigl, M., and M. B. Brenner. 2004. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22: 817–890. 3. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. Annu. Rev. Immunol. 25: 297–336. 4. Salio, M., J. D. Silk, E. Y. Jones, and V. Cerundolo. 2014. Biology of CD1- and MR1-restricted T cells. Annu. Rev. Immunol. 32: 323–366. 5. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278: 1623–1626. 6. Exley, M. A., and T. Nakayama. 2011. NKT-cell-based immunotherapies in clinical trials. Clin. Immunol. 140: 117–118. 7. Aspeslagh, S., Y. Li, E. D. Yu, N. Pauwels, M. Trappeniers, E. Girardi, T. Decruy, K. Van Beneden, K. Venken, M. Drennan, et al. 2011. Galactosemodified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis. EMBO J. 30: 2294–2305. 8. Silk, J. D., I. F. Hermans, U. Gileadi, T. W. Chong, D. Shepherd, M. Salio, B. Mathew, R. R. Schmidt, S. J. Lunt, K. J. Williams, et al. 2004. Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J. Clin. Invest. 114: 1800–1811. 9. Faveeuw, C., and F. Trottein. 2014. Optimization of natural killer T cellmediated immunotherapy in cancer using cell-based and nanovector vaccines. Cancer Res. 74: 1632–1638. 10. Lawrenczyk, A., S. Kim, X. Wen, R. Xiong, and W. Yuan. 2014. Exploring the Therapeutic Potentials of iNKT Cells for Anti-HBV Treatment. Pathogens 3: 563–576. 11. Berzins, S. P., M. J. Smyth, and A. G. Baxter. 2011. Presumed guilty: natural killer T cell defects and human disease. Nat. Rev. Immunol. 11: 131–142. 12. Moody, D. B., G. S. Besra, I. A. Wilson, and S. A. Porcelli. 1999. The molecular basis of CD1-mediated presentation of lipid antigens. Immunol. Rev. 172: 285–296. 13. Wen, X., P. Rao, L. J. Carrenõ, S. Kim, A. Lawrenczyk, S. A. Porcelli, P. Cresswell, and W. Yuan. 2013. Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells. Proc. Natl. Acad. Sci. USA 110: 2963–2968. 14. Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, and L. Van Kaer. 2004. NKT cells: what’s in a name? Nat. Rev. Immunol. 4: 231–237. 15. Gumperz, J. E., S. Miyake, T. Yamamura, and M. B. Brenner. 2002. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195: 625–636. 16. Lee, P. T., K. Benlagha, L. Teyton, and A. Bendelac. 2002. Distinct functional lineages of human V(alpha)24 natural killer T cells. J. Exp. Med. 195: 637–641. 17. Baev, D. V., X. H. Peng, L. Song, J. R. Barnhart, G. M. Crooks, K. I. Weinberg, and L. S. Metelitsa. 2004. Distinct homeostatic requirements of CD4+ and CD4subsets of Valpha24-invariant natural killer T cells in humans. Blood 104: 4150– 4156. 18. Sag, D., P. Krause, C. C. Hedrick, M. Kronenberg, and G. Wingender. 2014. IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset. J. Clin. Invest. 124: 3725–3740. 19. Dao, T., D. Guo, A. Ploss, A. Stolzer, C. Saylor, T. E. Boursalian, J. S. Im, and D. B. Sant’Angelo. 2004. Development of CD1d-restricted NKT cells in the mouse thymus. Eur. J. Immunol. 34: 3542–3552. 20. Berzins, S. P., A. D. Cochrane, D. G. Pellicci, M. J. Smyth, and D. I. Godfrey. 2005. Limited correlation between human thymus and blood NKT cell content revealed by an ontogeny study of paired tissue samples. Eur. J. Immunol. 35: 1399–1407. 21. Bendelac, A., N. Killeen, D. R. Littman, and R. H. Schwartz. 1994. A subset of CD4+ thymocytes selected by MHC class I molecules. Science 263: 1774–1778. 22. Capone, M., D. Cantarella, J. Sch€umann, O. V. Naidenko, C. Garavaglia, F. Beermann, M. Kronenberg, P. Dellabona, H. R. MacDonald, and G. Casorati. 2003. Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice. J. Immunol. 170: 2390–2398. 23. Crowe, N. Y., J. M. Coquet, S. P. Berzins, K. Kyparissoudis, R. Keating, D. G. Pellicci, Y. Hayakawa, D. I. Godfrey, and M. J. Smyth. 2005. Differential antitumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 202: 1279–1288.

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