Type I NKT cells protect (and type II NKT cells suppress) the host’s innate antitumor immune response to a B cell lymphoma

doi:10.1182/blood-2007-05-092866Prepublished online April 16, 2008;2008 111: 5637-5645

and Randy R. BrutkiewiczGourapura J. Renukaradhya, Masood A. Khan, Marcus Vieira, Wenjun Du, Jacquelyn Gervay-Hague innate antitumor immune response to a B-cell lymphomaType I NKT cells protect (and type II NKT cells suppress) the host's

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IMMUNOBIOLOGY

Type I NKT cells protect (and type II NKT cells suppress) the host’s innateantitumor immune response to a B-cell lymphomaGourapura J. Renukaradhya,1-3 Masood A. Khan,1-3 Marcus Vieira,1-3 Wenjun Du,4 Jacquelyn Gervay-Hague,4 andRandy R. Brutkiewicz1-3

1Department of Microbiology and Immunology, Indiana University School of Medicine, 2The Walther Oncology Center, and 3The Walther Cancer Institute,Indianapolis, IN; and 4Department of Chemistry, University of California, Davis

Natural killer T (NKT) cells are a T-cellsubpopulation known to possess immu-noregulatory functions and recognizeCD1d molecules. The majority of NKTcells express an invariant T-cell receptor(TCR) � chain rearrangement (V�14J�18in mice; V�24J�18 in humans) and arecalled type I NKT cells; all other NKT cellsare type II. In the current study, we haveanalyzed the roles for these NKT-cellsubsets in the host’s innate antitumorresponse against a murine B-cell lym-

phoma model in vivo. In tumor-bearingmice, we found that type I NKT cellsconferred protection in a CD1d-dependent manner, whereas type II NKTcells exhibited inhibitory activity. Pro- andanti-inflammatory cytokines secreted bysplenocytes from tumor-bearing mice cor-related with tumor progression. Myeloidcells (CD11b�Gr1�) were present in largenumbers at the tumor site and in thespleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor

immunosurveillance was inhibited byCD11b�Gr1� cells. Overall, these datasuggest that there are distinct roles forNKT-cell subsets in response to a B-celllymphoma in vivo, pointing to potentialnovel targets to be exploited in immuno-therapeutic approaches against bloodcancers. (Blood. 2008;111:5637-5645)

© 2008 by The American Society of Hematology

Introduction

Tumors must escape from the host’s immune surveillance tosurvive and grow. Lymphoma is a general term for cancers thatdevelop in the lymphatic system and it is the third most commoncancer in children and adolescents.1 To improve the outcome inpatients with hematopoietic tumors, a better understanding of theimmunobiology of lymphoma is essential. A novel lymphocytepopulation that has been identified as a key player in both innateand acquired immune responses, including as antitumor effectorcells, is the natural killer T (NKT) cell.2-4 NKT cells recognize lipidantigens presented by the MHC class I-like CD1d molecule andexpress cell surface markers shared with NK cells.3 The vastmajority of these T cells are canonical or invariant NKT (type INKT) cells that possess a specific T-cell receptor (TCR) �–chainrearrangement (V�14J�18 in mice; V�24J�18 in humans), associ-ated with V� chains of limited diversity. All other NKT cells thatare CD1d-restricted and do not express this invariant TCR arecalled type II NKT cells.5,6 Unlike type I NKT cells, little is knownabout type II NKT cells, but there is some evidence that type IINKT cells are a functionally important T-cell subset.5 As CD1dmolecules are vital for NKT-cell development,7 mice lacking theCD1d1 gene (CD1KO mice) are deficient in all CD1d-restrictedNKT cells (both type I and type II).8 Mice lacking the J�18 gene(J�18KO mice) are deficient only in type I NKT cells. As NKTcells are capable of secreting both Th1 and Th2 cytokines, this hasmade it difficult to predict the consequences of their activation invivo but has nonetheless created much speculation that they play acentral role in immunoregulation.2

NKT cells are directly cytotoxic and their activation can alsoresult in “adjuvant effects” during antitumor immune responses by

activating other cytotoxic lymphocytes, mainly through a Th1cytokine cascade.4 However, there are reports demonstrating asuppressive antitumor role for CD4� NKT cells in some murinetumor models,9 and type II NKT cells were shown to be capable ofdown-regulating tumor immunosurveillance.10 The role of NKTcells in the evasion of hematopoietic tumors from the host’s innateantitumor immune response in vivo has only recently begun to beinvestigated, and we have reported that type I NKT cells play aninhibitory role in a murine T-cell lymphoma model.11 Severalhuman hematopoietic cell types express CD1d on their surface,12

but the overall role of CD1d in antitumor immunity is not wellunderstood. We have previously demonstrated that certain hemato-poietic tumors shed glycolipids that mask CD1d-mediated antigenpresentation to both type I and II NKT cells.13 Recently, in vitrokilling of EL-4 T-cell lymphoblastic lymphoma cells by type I NKTcells and their in vivo eradication in a CD1d-dependent manner hasbeen reported.14

CD11b and Gr-1 are the most common markers found onmyeloid derived suppressor cells (MDSCs) and these cells aredistinct from T lymphocytes and NK cells.15 Accumulating evi-dence since the 1980s has demonstrated that MDSCs of themyeloid macrophage/DC lineage, are significantly increased in thespleen and bone marrow of animals bearing large tumors and underconditions associated with impaired immune reactivity.16 TheMDSC population is heterogenous, including mature granulocytes,immature macrophages, immature DCs, immature myeloid cells,monocytes, and a broad representation of immature cells of themyelomonocytic lineage.15 They express abnormally low levels ofMHC class II molecules and low to undetectable levels of

Submitted May 28, 2007; accepted March 30, 2008. Prepublished online asBlood First Edition paper, April 16, 2008; DOI 10.1182/blood-2007-05-092866.

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© 2008 by The American Society of Hematology

5637BLOOD, 15 JUNE 2008 � VOLUME 111, NUMBER 12




costimulatory molecules. They are unable to process and presentantigens, and therefore do not induce effective antitumor cellularimmune responses.16 Recently, Taniguchi and colleagues havereported that an NKT cell–mediated antitumor immune responsecould be enhanced by inhibiting MDSCs in B16 melanoma and3LL Lewis lung cancer models.17

In the current report, we have studied the role(s) of both typeI and type II NKT cells in the control of a B-cell lymphoma invivo. We have found that type I NKT cells are protective,whereas type II NKT cells are suppressive in the antitumorimmune response to this tumor.

Methods

Mice

Inbred BALB/c wildtype mice were obtained from The JacksonLaboratory (Bar Harbor, ME). CD1d1-deficient (CD1KO) mice8 wereobtained from L. Van Kaer (Vanderbilt University, Nashville, TN).J�18KO mice18 were kindly provided by Dr R. Singh (University ofCincinnati, OH) with permission from Dr M. Taniguchi (Chiba Univer-sity, Chiba, Japan). All gene-targeted mice were backcrossed onto theBALB/c background for 10 generations. All mice were age- andsex-matched and used between 6 and 12 weeks of age. All animalprocedures were approved by the Indiana University School of MedicineInstitutional Animal Care and Use Committee.

Antibodies

The mouse CD1d-specific 1H6 mAb has been previously described.19 1H6ascites and the isotype control mAb HB158 hybridoma supernatant werepurified on a protein A column for in vivo use. Phycoerythrin (PE)– orfluorescein isothiocyanate (FITC)–conjugated antibodies specific for mouseCD4, CD8, TCR�, B220, Gr-1, CD11b, CD44, and DX5 were purchasedfrom BD-PharMingen (San Diego, CA). PE-conjugated rabbit anti–mouseIg antiserum was purchased from Dako (Carpinteria, CA). Purified andbiotinylated antibodies specific for mouse transforming growth factor�(TGF�) and IL-13 were purchased from R&D Systems (Minneapolis, MN),whereas those specific for mouse IL-2, IL-12, IFN-�, and GM-CSF werepurchased from BD-PharMingen. All the cytokine standards used in theenzyme-linked immunosorbent assays (ELISA) were purchased fromPeproTech (Rocky Hill, NJ).

Tumor cell lines and murine type I NKT cells

The B-cell lymphoma cell line NS0 was kindly provided by S. Joyce (VanderbiltUniversity). NS0 cells were transfected with the pSR�-neo vector alone (NS0-V)or the vector with a cd1d1 cDNA insert (NS0-CD1) by standard electroporationtechniques. Isolation of murine type I NKT cells was performed as previouslydescribed.20 Briefly, isolated liver mononuclear cells (LMNC),21 were blockedwith anti-FcR�II (2.4G2, ATCC, Manassas, VA) and treated with recombinantsoluble dimeric mouse CD1d-Ig fusion proteins (DimerXI; BD Biosciences)loaded with a 40 M excess of �-galactosylceramide (�-GalCer) and labeled witha Zenon R-PE mouse IgG1 (Invitrogen, Carlsbad, CA) as described previously.20

In some experiments, PBS-57-loaded allophycocyanin-conjugated CD1d tetram-ers (National Institutes of Health [NIH] National Institute of Allergy andInfectious Diseases [NIAID] Tetramer Facility at the Emory University VaccineCenter,Atlanta, GA) were used. Unloaded CD1d1 dimers conjugated with ZenonR-PE mouse IgG1 and unloaded allophycocyanin-conjugated CD1d tetramerswere used as negative controls. Dimer or tetramer-positive cells were electroni-cally sorted using a BD FACSAria cell sorter.

In vivo tumor assays

Single cell suspensions of tumor cells were prepared in Iscove modifiedEagle medium (IMDM) containing 5% fetal bovine serum (FBS) andinjected intraperitoneally. Liver-derived NKT cells from BALB/c mice

were adoptively transferred on the indicated day’s posttumor inoculation toJ�18KO mice, intravenously. To block CD1d in vivo, 50 �g purified 1H6 orisotype control mAb in PBS was injected on the indicated day’s posttumorinoculation to BALB/c mice, intraperitoneally.

Ex vivo cytokine analyses, flow cytometry, and NKT-cellfunctional assays

Ex vivo cytokine analysis from splenocytes of tumor-bearing mice wasperformed as previously described.11 Briefly, single cell suspensions ofsplenocytes from NS0-V- or NS0-CD1-inoculated BALB/c wildtype,CD1KO, and J�18KO mice were prepared from tumor-bearing mice on day25 and 50 after tumor inoculation. The cells were cultured at a density of3 � 106 cells/well in a 24-well plate along with irradiated (5000 rads)NS0-V- or NS0-CD1 cells (7.5 � 105 tumor cells/well) at a 4:1 ratio. Twodays after the coculture, supernatants were harvested and stored at �20°Cuntil used for cytokine measurement by ELISA. All samples were analyzedin triplicate. Supernatants from NS0-V- or NS0-CD1 cells, and splenocytesof tumor and non–tumor-bearing mice cultured alone were included ascontrols in the ELISA assays. The background cytokine levels weresubtracted from the experimental values. Single cell suspensions ofsplenocytes harvested from NS0-V– or NS0-CD1–bearing mice for FACSanalyses of the MDSC population was performed as previously described.11

The murine NKT-cell hybridoma assay was performed as described.19

Briefly, NS0-V- or NS0-CD1 cells were cocultured with murine NKT-cellhybridoma DN32.D322,23 at 1:1 ratio for 48h, supernatants harvested andIL-2 production was measured by ELISA as previously described.19 Thecytotoxic activity of murine NKT cells against NS0-V or NS0-CD1 cellswas performed as previously described.24

Statistical analysis

For the statistical analysis of tumor growth in mice, the log-rank test wasperformed using GraphPad PRISM software (version 4.00 for Windows;GraphPad, San Diego, CA). A nonparametric statistical analysis using SAS,version 9 (SAS Institute, Cary, NC) was performed for comparing the levelsof cytokines secreted by different tumor-bearing mouse strains. Twoexperiments (n � 6) were combined (P .05, Wilcoxon test) for thestatistical analysis of those data. An overall test of difference betweenstrains was performed using the Kruskal-Wallis test. Pairwise comparisonsbetween strains were performed using Wilcoxon tests with a Bonferroniadjustment for multiple comparisons.

Results

Type I NKT cells are essential for the survival of mice against aB-cell lymphoma

CD1d molecules are expressed on B cells, macrophages, dendriticcells, thymocytes and some hematopoietic tumor cells.12,14,25 Therole of CD1d on tumor cells in the host’s antitumor immuneresponse has not been well established. The B-cell lymphoma NS0naturally expresses a very low level of CD1d on the cell surface(Figure 1A). Upon expression of mouse CD1d1 in NS0 cells(NS0-CD1), these cells were able to stimulate cytokine productionby NKT cells and were susceptible to NKT cell–mediated lysis invitro (Figure 1B,C). Vector control NS0 (NS0-V) cells failed toactivate NKT cells and were not killed by NKT cells in vitro. Theseresults are consistent with a previous report showing that thesensitivity of leukemia cells to NKT cell–mediated lysis iscorrelated with CD1d expression.12 Some glycolipid fractions fromthe tumor cell membrane are known to be presented by CD1d andto be recognized by NKT cells,4 although we have reported in atleast one case that tumor-derived glycolipids can be inhibitory.13

Our in vitro experiments using NS0 cells suggested that tumor-derived lipid antigens are capable of being presented by CD1dmolecules and activating the lytic effector program of NKT cells.

5638 RENUKARADHYA et al BLOOD, 15 JUNE 2008 � VOLUME 111, NUMBER 12




The in vivo role for NKT cells in the control of B-celllymphomas and the importance of CD1d in the antitumor immuneresponse against these tumors are unknown. To address thesequestions, syngenic BALB/c wildtype, CD1KO, and J�18KO micewere inoculated intraperitoneally with NS0-V or NS0-CD1 cells.Ascites tumor development was detected as early as day 25 aftertumor inoculation and 5 to 10 days later, the mice exhibited severemorbidity and rapid mortality. More than 70% of the J�18KO miceinoculated with NS0-V tumors died within 100 days, with a meansurvival time (MST) of 61 days, whereas all of the NS0-CD1–bearing J�18KO mice died within 100 days (MST � 45 days;Figure 2A). Less than 30% of the wildtype and CD1KO micesuccumbed to either tumor; these mice suffered less and survivedlonger compared with J�18KO mice (Figure 2A and Table 1). Thepercentage of J�18KO mice bearing NS0-CD1 tumors was higherthan those inoculated with the control tumor (P � .0153) and this

may have been due to tumor cell CD1d-mediated activation of typeII NKT cells in the absence of protective type I NKT cells. Thus,these results suggested that type I NKT cells were protective andtype II NKT cells were suppressive in this tumor model. Thisobservation was confirmed by the enhanced survival after adoptivetransfer of electronically sorted liver type I NKT cells fromwildtype BALB/c mice to tumor-bearing J�18KO mice (P � .0163;Figure 2B). Notably, this protection depended upon CD1d beingexpressed by the tumor. Therefore, these results confirmed that typeI NKT cells are responsible for protecting mice from rapid death bya B-cell lymphoma, and this protective effect is CD1d-dependent.Because both type I and type II NKT cells have oppositefunctions,2,3,5 the similar growth of NS0-V and NS0-CD1 tumors in

Figure 1. The parental murine B-cell lymphoma NS0 is deficient for cell surfaceCD1d expression and does not stimulate NKT cells. (A) NS0-V and NS0-CD1cells were stained for mouse CD1d (or Kd) and analyzed by flow cytometry. Openhistogram: isotype control; filled histogram: anti-mouse CD1d or Kd. (B) NKT cells killonly CD1d-expressing NS0 cells. Splenic NKT cells were used as effectors against51Cr-labeled NS0-V (f) or NS0-CD1 (�) target cells in a 24-hour 51Cr release assay.(C) NKT cells are activated only by CD1d-expressing NS0 cells. NS0-V or NS0-CD1cells were cocultured with the NKT-cell hybridoma DN32.D3 for 48 hours. Thesupernatant was tested for IL-2 production as an indicator of NKT-cell activation.Each bar represents triplicate determinations, and the data are shown as mean plusSD. The data shown are representative of 3 independent experiments.

Figure 2. Protective antitumor immune response mediated by type I NKT cellsto a murine B-cell lymphoma. (A) BALB/c wildtype ( ), CD1KO (�), and J�18KO(�) mice were inoculated with 107 NS0-V or NS0-CD1 cells. The results consist of thepooled data from 3 independent experiments (n � 20). The log-rank test was used todetermine P values. Tumor incidence in mice inoculated with NS0-V cells comparedwith NS0-CD1 cells: wildtype, P � .537; in CD1KO, P � .6560; and in J�18KO mice,P � .015. (B) BALB/c J�18KO mice were inoculated with tumors as above and ondays 0, 10, and 20, the mice received 2.5 � 105 electronically-sorted type I NKT cellsby adoptive transfer or sham, intravenously. The results shown are pooled data(4 mice/experiment; n � 8). The log-rank test was used to determine P values fortumor-bearing mice receiving sham treatment (�) or NKT cells (�) and NS0-V(P � .6130) or NS0-CD1 cells (P � .0163).

Table 1. Statistical analysis of tumor growth in wildtype, CD1KO,and J�18KO mice inoculated with NS0-V or NS0-CD1 cells (n � 20mice/group)*

Mice with tumors

Tumor and mouse group† P Significance‡

NS0-V with wildtype and CD1KO .993 NS§

NS0-V with wildtype and J�18KO .003 S¶

NS0-V with CD1KO and J�18 KO .003 S

NS0-CD1 with wildtype and CD1KO .792 NS

NS0-CD1 with wildtype and J�18 KO .001 S

NS0-CD1 with CD1KO and J�18KO .001 S

Wildtype with NS0-V and NS0-CD1 .537 NS

CD1KO with NS0-V and NS0-CD1 .656 NS

J�18KO with NS0-V and NS0-CD1 .015 S

*Mice were inoculated with 107 tumor cells/mouse, intraperitoneally.†Comparisons were made between the different combinations of groups in order

to determine the statistical significance of the analyses.‡The P values are based on the log-rank test comparing either two groups of

mice with one cell line or two cell lines with one group of mice as indicated.§Not significant.¶Significant (entries in bold).

NKT CELLS PROTECT AGAINST A B-CELL LYMPHOMA 5639BLOOD, 15 JUNE 2008 � VOLUME 111, NUMBER 12




wildtype or CD1d-deficient mice (the latter of which has neithertype I nor type II NKT cells) was predicted. Furthermore, theadoptive transfer of type I NKT cells reduced the tumor incidencein NS0-CD1–bearing J�18KO mice to the levels observed inwildtype mice (Figure 2B right panel), also consistent with ourhypothesis. However, why was there no change in the tumorincidence in J�18KO mice bearing NS0-V control tumors that wereadoptively-transferred with NKT cells (Figure 2A left panel)? It isknown that upon activation, NKT cells can stimulate NK cells,26,27

and these 2 lymphocyte populations can work together or indepen-dently in various tumor models.28 With hematopoietic tumors inparticular, there is an inverse correlation between MHC class Iexpression on the cells and their susceptibility to NK cell–mediatedlysis.29 However, with the very high level of surface MHCclass I (eg, H2-Kd) on the NS0-V cells (Figure 1A), NKactivation likely would effectively be irrelevant. Hence, no differ-ence in NS0-V growth in the presence or absence of type I NKTcells in J�18KO mice was observed (Figure 2B left panel). Thus,overall, the data strongly suggest that type I NKT cells serve asantitumor effector cells, whereas type II NKT cells act opposite totheir type I counterparts.

Liver NKT cells are considered to be functional representativesof all invariant NKT cells in mice.30 In an earlier study, it was foundthat 2.5 � 105 liver NKT cells injected once to tumor-bearingC57BL/6 J�18KO mice were sufficient to confer substantialprotection of mice from tumor burden.30 Thus, additionally, ourdata suggest that a similar number of BALB/c liver NKT cellsinjected 3 times is also significantly protective in a B-celllymphoma (and CD1d�) model.

Enhanced mortality of tumor-bearing J�18-deficient mice isdue to type II NKT cells

The function of type II NKT cells in vivo can be assessed bycomparing results from experiments using J�18- and CD1d-deficient mice.4,10 J�18KO mice only lack type I NKT cells,whereas CD1KO mice are deficient in both type I and type II.Therefore, considering the reduced survival in J�18KO com-pared with CD1KO mice (Figure 2A), we speculated that type IINKT cells were playing an immunosuppressive role. Therecruitment and activation of type I NKT cells is an importantconsideration during the immunotherapy of early stage tu-mors.31 In our B-cell lymphoma model, a similar recruitmentand activation of type II NKT cells appeared to be responsiblefor inhibiting the host’s innate antitumor immunosurveillance.To address this question, tumor-bearing mice were injected witha mouse CD1d-specific mAb (1H6) that blocks CD1d-dependentNKT-cell activation in vitro19 or an isotype control mAb. It wasfound that the 1H6 mAb completely restored the level ofsurvival in tumor-bearing J�18KO mice to that observed inwildtype mice (Figure 3; Table 2). Furthermore, 1H6 cansubstantially impair cytokine production by NKT cells afterinjection of �-GalCer in vivo (data not shown). These data aresupported by an earlier study showing that the in vivo functionof NKT cells could be blocked using an anti-CD1d antibody.10

These results further confirmed a possible suppressive functionfor type II NKT cells against a B-cell lymphoma. The antibody-mediated CD1d block in wildtype mice also did not alter tumorincidence, suggesting that inhibiting both NKT-cell subsets hasno effect on survival in this tumor model.

Pro- and anti-inflammatory cytokine profiles reflect tumorprogression

In other murine tumor model systems, it has been reported thatCD4� NKT cells exert an inhibitory effect on antitumor immuno-surveillance mediated through the actions of anti-inflammatorycytokines like IL-13.9 Similarly, we have reported in a murineT-cell lymphoma model that J�18KO and CD1KO mice producelower levels of Th2 cytokines and survive longer than wildtypemice.11 In the current study, to explore how type I and II NKT-cellsubsets mediated the divergent antitumor immune response to aB-cell lymphoma, the pro- and anti-inflammatory cytokine profilesin tumor-bearing wildtype and mutant mice at various stages oftumor growth were assessed. Our in vivo experiments indicatedthat the maximum ascites tumor development was detectablebetween days 25 and 60 after inoculation. Therefore, spleens fromwildtype, CD1KO, and J�18KO mice bearing NS0-V and NS0-CD1 tumors were harvested on day 25 or 50, splenocytes werecocultured with the respective irradiated tumor cells, and theproduction of IFN-�, GM-CSF, IL-12, TGF�, and IL-13 into thesupernatant was measured (Figure 4). Secretion of the pro-inflammatory cytokines IFN-� and IL-12 were significantly higherin wildtype and CD1KO mice compared with J�18KO mice(P .05; Figure 4A,B). In contrast, the levels of the anti-inflammatory cytokines TGF� and IL-13 were significantly higherin J�18KO mice and nearly undetectable in wildtype and CD1KOmice (Figure 4A,B). TGF� is a cytokine that has been shown toblock cytotoxic T lymphocyte–mediated tumor immunosurveil-lance.32 We detected high levels of serum TGF� in J�18KO mice atvarious stages of tumor growth compared with wildtype andCD1KO mice (Figure 4C). We did not detect any cytokine byintracellular staining and FACS analysis in splenocytes fromtumor-bearing mice (data not shown).

Figure 3. Inhibition of the antitumor immune response by type II NKT cells intumor-bearing mice. NS0-V or NS0-CD1 cells (107) were injected intraperitoneallyinto BALB/c wildtype ( ), CD1KO (�) and J�18KO (�) mice. On days �1, 5, 10, 20,30, 40, and 50, the mice were injected intraperitoneally with 50 �g of isotype controlantibody (A) or the anti-mouse CD1d antibody 1H6 (B). Tumor growth was monitoredfor 100 days. The results shown are pooled data from 2 independent experiments(4 mice/experiment; n � 8).





IL-12 has been shown to possess potent antitumor activity in awide variety of murine tumor models via NK and/or NKT cells.33

As NKT cells are activated by the IL-12 produced during atumor-induced inflammatory process, NKT cells might be able torespond to any type of tumor.4 We also detected significantly higherlevels of IL-12 in wildtype mice (Figure 4A,B), correlating with areduced tumor incidence and is supportive of a possible protectivefunction mediated by type I NKT cells.

Cytokine production from splenocytes of tumor-bearing J�18KOmice adoptively transferred with type I NKT cells was analyzed aswell. The results obtained support the survival data (Figure 2),whereby the pro-inflammatory cytokines IFN-�, IL-12, and GM-CSF were detected more in J�18KO mice bearing NS0-CD1 andadoptively transferred with NKT cells as opposed to those that didnot (Figure 5). A similar but reduced trend in cytokine productionwas detected in NS0-V–bearing J�18KO mice receiving NKT cells(Figure 5). The anti-inflammatory cytokine TGF� was detected inmore sham-treated NS0-CD1–bearing mice than in those receivingNKT cells. It is important to note that the reduced tumor incidencein NS0-CD1–bearing J�18KO mice after NKT cell transfer was notcomplete, although it was significant. This suggests that theadoptively-transferred type I NKT cells induced a prevalence ofpro-inflammatory cytokine production in tumor-bearing mice.FACS analyses of splenocytes from NS0-CD1–bearing J�18KOmice receiving NKT cells showed a marginal rescue in CD4�

and CD8� T-cell numbers and with a concomitant reductionin CD11b�Gr1� cells compared with the control group (datanot shown).

Tumor-bearing type I NKT cell–deficient mice have elevatedlevels of myeloid suppressor cells

Necropsy analyses of tumor-bearing mice revealed metastasesin the liver, spleen (3-10 times larger than normal), kidney,lymph nodes, intestines, and peritoneum. The host’s response toprevent tumor progression is reflected by the predominanttumor-infiltrating leukocytes and other cells, which participatein the neoplastic process by fostering tumor cell proliferation,survival, and metastasis.34 To analyze the tumor microenviron-ment in the current study, ascites tumor cells from wildtype,CD1KO, and J�18KO mice inoculated with NS0-V or NS0-CD1cells were stained with various mAb to permit the identification

of the individual leukocyte populations present. Interestingly,most of the ascites tumors had 2 or 3 distinct cell populationsand tumor cells from each mouse had various levels of B220,DX5, TCR��CD4�, TCR��CD8�, and CD11b�Gr1� cells(Figure 6A). Interestingly, we found lower numbers of DX5�

and B220� cells, but substantially higher numbers of myeloidsuppressor cells (MDSC; CD11b�Gr1�) in the ascites fromtumor-bearing J�18- (type I NKT cell)–deficient mice (Figure6A). Furthermore, elevated levels of MDSC were detected in thespleens of these mice (Figure 6B).

Discussion

Unlike murine NKT cells, the number of type I NKT cells inhumans is highly variable among individuals and they representonly a small portion of total NKT cells, whereas type II NKT cellsare the major population.35-40 Therefore, it is very essential todelineate the independent roles of NKT cell subsets in antitumorimmunity to design effective immunotherapeutic strategies. Ourprevious studies have identified a suppressive antitumor immuneresponse mediated by type I NKT cells in a murine T-celllymphoma model.11 Interestingly, our in vivo results in the currentstudy demonstrate opposing roles for type I and type II NKT cells.Differences in the immune response between J�18KO and CD1KOmice have been observed in a protozoan disease model (Trypano-soma cruzi), with J�18KO mice more susceptible to disease.41

Recently, opposite immunoregulatory functions by type I and typeII NKT cells during the acute phase of schistosomiaisis has beenreported.42 Enhanced tumor incidence in wildtype and J�18KOcompared with CD1KO mice has been observed by Berzofsky andcolleagues in models of fibrosarcoma (15-12RM), colon carcinoma(CT26), and mammary carcinoma (4T1), suggesting a suppressiverole for type II NKT cells, but a role for type I NKT cells inwildtype mice in those models was not demonstrated.10

We attempted to address the role of both CD1d molecules andNKT cells in a single tumor model system. Although B cellsnormally express CD1d on their surface, we found that the NS0B-cell lymphoma has low/undetectable levels of CD1d. Thus, wehypothesized that down-regulation of CD1d is one means by whicha B-cell lymphoma could evade the host’s innate antitumor

Table 2. Statistical analysis of tumor growth in BALB/c wildtype, CD1KO, and J�18KO mice inoculated with NS0-V or NS0-CD1 cells andtreated with an isotype control or anti-CD1d mAb (n � 8 mice/group)*

In vivo CD1d block

Tumor and mouse group†

Isotype control Anti-mouse CD1d

P Significance‡ P Significance

NS0-V with wildtype and CD1KO .144 NS§ .843 NS

NS0-V with wildtype and J�18KO .021 S¶ .779 NS

NS0-V with CD1KO and J�18 KO .001 S .612 NS

NS0-CD1 with wildtype and CD1KO .283 NS .846 NS

NS0-CD1 with wildtype and J�18 KO .040 S .926 NS

NS0-CD1 with CD1KO and J�18KO .006 S .943 NS

Wildtype with NS0-V and NS0-CD1 .609 NS .665 NS

CD1KO with NS0-V and NS0-CD1 .317 NS .690 NS

J�18KO with NS0-V and NS0-CD1 .364 NS .943 NS

*Mice were inoculated with 107 tumor cells/mouse and 50 �g of isotype or anti-mouse CD1d mAb on days �1, 5, 10, 20, 30, 40, and 50 intraperitoneally.†Comparisons were made between the different combinations of groups in order to determine the statistical significance of the analyses.‡The P values are based on the log-rank test comparing either 2 groups of mice with 1 cell line or 2 cell lines with 1 group of mice as indicated.§Not significant.¶Significant (entries in bold).





immune response. Because of this, we overexpressed CD1d in NS0cells and compared it to the same tumor without CD1d overexpres-sion. The role of CD1d expressed on NS0 tumors was evident intumor-bearing J�18KO mice, as NS0-CD1 tumors killed moremice with a shorter mean survival time than those inoculated withNS0-V tumors. The adoptive transfer of type I NKT cells signifi-cantly protected only NS0-CD1–bearing J�18KO mice. The ab-sence of CD1d on NS0-V cells is the likely reason why adoptively

transferred type I NKT cells failed to protect NS0-V-bearingJ�18KO mice; the cytokine profile data correlated with thisobservation. However, it cannot be ruled out that the adoptivetransfer of more type I NKT cells would reduce the tumor incidencein NS0-V–bearing J�18KO mice. That being said, other lympho-cyte populations do have the capacity to participate in antitumorimmune responses. In particular, NK cells are well-known antitu-mor effector cells and are highly lytic against hematopoietic tumorsboth in vivo and in vitro.29,43,44 The function of NK cells can beaffected by activated NKT cells.26,27 Furthermore, it is known thatNK cells can work with (or can function independently of) NKTcells in a variety of tumor cell models.28,45,46 Thus, one might havepredicted that the adoptive transfer of type I NKT cells could havehad an effect on NS0-V tumor growth in J�18KO mice through theaction of NK cells. As indicated above, no difference in NS0-Vtumor incidence was observed in J�18KO mice regardless ofwhether type I NKT cells were adoptively transferred or not.Considering the high levels of MHC class I molecules on NS0 cells(Figure 1A), and the well-known inverse correlation between MHCclass I levels and susceptibility to lysis by NK cells,29 this resultmay not be that surprising.

Recently, it has been demonstrated that interactions betweentype II NKT cells and hepatic DCs result in the regulation of type INKT-cell activity in concanavalin A–induced hepatitis47; it isimportant to note that BALB/c mice are mainly Th2 cytokine-biased.48 Some human tumor cells express CD1d and are able topresent glycolipids to NKT cells, eliciting IFN-� release andcytolysis.49,50 GD3, a ganglioside expressed on the CD1d-negativehuman melanoma cell line SK-MEL-28, can be cross-presented bymurine APC in a CD1d-dependent manner in vivo and this is aputative mechanism for NKT recognition of tumor glycolipids inCD1d� tumors.51 Therefore, cross-presentation of tumor-derivedglycolipids by professional antigen presenting cells such as den-dritic cells to NKT cells can be very important for the developmentof antitumor immunity.51,52 Were cross-presentation to be the majormechanism operative in our NS0 system, we would expect there tobe a comparable reduction in NS0-V and NS0-CD1 tumor growthin J�18KO mice receiving type I NKT cells (dendritic cells in these

Figure 4. Levels of pro- and anti-inflammatory cytokines are indicators ofmurine B cell lymphoma growth in vivo. Splenocytes from BALB/c wildtype,CD1KO, and J�18KO mice inoculated with NS0-V or NS0-CD1 cells were collected25 (A) or 50 (B) days later and cocultured with irradiated tumor cells. The culturesupernatants were tested for the indicated cytokines by ELISA. Each bar representsthe cytokine production from individual mice (n � 6 from 2 independent experi-ments). No bar in a group indicates that the cytokine was below the level of detection.In the day 50 NS0-CD1 tumor-bearing J�18KO mouse group; 3 mice were dead byday 50 (#) and hence, cytokine production could not be measured. (C) BALB/c micewere inoculated with the tumors as above, and on days 0, 5, 10, 20, 30, 40, and 50,mice were bled and the serum assayed for TGF� by ELISA. Error bars represent SD.In panels A and B, the asterisk indicates a significant P value ( .025) as determinedby the Kruskal-Wallis test for the comparisons made between the indicated groups.

Figure 5. Pro- and anti-inflammatory cytokine production correlates withsurvival of tumor-bearing J�18KO mice adoptively transferred with type I NKTcells. BALB/c J�18KO mice were inoculated with 107 NS0-V or NS0-CD1 tumors andon days 0, 10, and 20 received 2.5 � 105 electronically sorted type I NKT cells orsham intravenously. Splenocytes were collected 25 days later and cocultured withirradiated tumor cells for 2 days. The culture supernatants were tested for theindicated cytokines by ELISA. Each bar represents the cytokine production fromindividual mice (n � 6 from 2 independent experiments). No bar indicates that thecytokine was not detectable. The asterisk indicates a significant P value ( .025) asdetermined by the Kruskal-Wallis test for the comparisons made between theindicated groups.





animals are CD1d�). As there was only a decrease in NS0-CD1tumor growth upon the adoptive transfer of type I NKT cells inJ�18KO mice, it is likely that cross-presentation plays a moreimportant role when the tumors are CD1d�.

We demonstrated the suppressive role for type II NKT cells to aB-cell lymphoma NS0 as indicated by increased tumor incidence inJ�18KO mice bearing both NS0-V or NS0-CD1 tumors and whichis supported by significantly reduced tumor incidence in CD1KOmice. These results correlate with recent findings using CD1d�

tumors as well10 and therefore potentially suggest an even moresignificant immunoregulatory (albeit negative) role played by typeII NKT cells in the host’s innate antitumor immune response. Theabsence of type II NKT cells in CD1KO mice would therefore notprohibit other immune cells (eg, NK cells and tumor-specificcytotoxic T lymphocytes [CTL]) from clearing the tumor burdenand resulting in a significantly reduced tumor incidence. Thus, our

work has revealed an important protective antitumor effect medi-ated by type I NKT cells against a B-cell lymphoma. This is the firstunique (and CD1d�) tumor model study demonstrating distinct,independent and contrasting antitumor functions for the individualNKT cell subsets, providing useful information that can potentiallybe applied to NKT cell–based antitumor immunotherapy.

The progressive lethal tumor growth in 30% of the wildtypemice bearing either NS0-V or NS0-CD1 observed in the currentstudy is similar to that found with the BW-Sp3 lymphoma insyngenic AKR mice, where a marked increase in MDSC wasdetected in those mice as well.15 The local effects of inhibitorycytokines in both lymphoid organs and at the tumor site by myeloidcells can thus target the generation of antitumor CTL with tumorprogression occurring as a result.15 Studies using the BW-Sp3lymphoma line, MZ1851RC and MZ1851LN renal carcinomas,and B16 melanoma have shown that Th2 cytokines dominate thehost’s response during the progressive growth stage of the tumor.15

Our in vivo analyses in a B-cell lymphoma model demonstrate howthe effectiveness of NKT cell–dependent antitumor immunosurveil-lance can be predicted based on the cytokines produced.

It is known that mice bearing various types of tumors (either byinoculation or carcinogen-induced) progressively accumulate MDSCin the spleen and blood, and they are profoundly impaired in theirimmune response to various tumor-associated antigens.15 It hasbeen reported that MDSC from J�18KO mice in a pancreatic islettransplant model failed to produce IFN-�; the production of IFN-�in that model was dependent on the activation status of type I NKTcells.53 IL-13 produced from NKT cells activates CD11b�Gr1�

cells to produce TGF� which directly suppresses tumor-specificCD8� CTLs.32 The results from our study suggest that theCD11b�Gr1� cells found in significant numbers in the ascites oftumor-bearing J�18KO mice probably produced higher levelsof TGF�, (rather than IFN-�) which resulted in the impairment ofantitumor immunosurveillance.

We have detected very high expression of CD44 in all theascites tumor cells ( 90%); this molecule is often used as amarker of a tumor’s metastatic potential.54 Hematopoiesis in atumor-bearing host is generally altered, manifested by an increasein CD11b�Gr1� cells in both spleen and ascites; these cells directlypromote neoplastic growth by decreasing tumor cell apoptosis andnecrosis.16 This correlates very well with our observations in thecurrent study, where we have observed a similar increase in theMDSC population (especially in type I NKT cell–deficient mice)by 4- to 6-fold in the spleen and in the ascites tumors. Thus, ourfindings suggest that there is active immunosurveillance at thetumor site. Depending on the final outcome, there is a shift in theimmune cell populations that will either favor survival or death inthe tumor-bearing host. Furthermore, the effects of the NKT-cellsubsets in vivo may be regulated by the levels of Th1 and Th2cytokines, and ultimately through MDSCs. Conventional treat-ments of hematologic malignancies using chemotherapeutic agentsare often unsuccessful due to severe side effects and incompletelong term remission. Therefore, the availability of a novel immune-based therapeutic option would be of great interest. B-cell malignan-cies are considered to be the most responsive types of all humancancers in an immunotherapeutic setting.55 Our study has clearlydemonstrated the importance of NKT-cell subsets in the host’sinnate antitumor immunity against a B-cell lymphoma. Thisknowledge will be beneficial in the design of NKT cell–basedimmunotherapeutic strategies against these tumors.

Figure 6. Ascites from tumor-bearing Type I NKT cell–deficient mice haveelevated levels of myeloid suppressor (CD11b�Gr1�) cells. (A) NS0-V orNS0-CD1 tumor-bearing BALB/c wildtype, CD1KO, and J�18KO mice were eutha-nized, and ascites tumor cells were harvested and stained for surface expression ofCD44, B220, DX5, TCR� and CD4, TCR� and CD8, or CD11b and Gr1 and analyzedby flow cytometry. Each bar corresponds to the mean percentage of positive cells fora specific antigen obtained from 3 individual mice in each group (� SD).(B) Splenocytes harvested from the above groups of mice were stained for Gr1 andCD11b. A representative FACS analysis of 3 individual mice is shown. Numbers onthe plots indicate the percentage of total cells in that quadrant.





Acknowledgments

We thank Drs L. Van Kaer, R. Singh, and M. Taniguchi for mice. DrV. Sriram provided help during type I NKT-cell isolation. K.Gillett-Heacock, C. Willard, J. Eltz, and B. Champ provided experttechnical assistance. Dr H. Twigg and P. Smith provided invaluablehelp in the CBA cytokine analyses. Drs M. Kaplan, D. Godfrey, J.Berzofsky, and M. Terabe provided valuable suggestions. Ms B. E.Juliar performed the statistical analysis of our data. The allophycocyanin-labeled CD1d tetramer loaded with the �-galactosylceramide analogPBS-57 was provided by the NIH Tetramer Facility (Emory UniversityVaccine Center, Atlanta, GA). We thank the members of theFlow Cytometry Resource Facility, Indiana University School ofMedicine for their invaluable assistance in the electronic sorting of NKTcells for this study.

This work was supported by funding from NIH grants (R01CA89026 and AI46455) and the Walther Cancer Institute to R.R.B.,

and NSF CHE-0194682 from the National Science Foundation toJ.G.H. G.J.R. was supported by NIH training grant T32 DK007519.R.R.B. is a Scholar of the Leukemia & Lymphoma Society.

Authorship

Contribution: G.J.R. performed research, analyzed, and interpreteddata, and wrote the paper; M.A.K. and M.V. assisted with animalstudies; W.D. and J.G.-H. contributed resources; R.R.B. conceptu-alized the work, analyzed and interpreted the data, and reviewedthe manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Randy R. Brutkiewicz, PhD, Department ofMicrobiology and Immunology, Indiana University School ofMedicine, Building R2, Room 302, 950 W Walnut St, Indianapolis,IN 46202-5181; e-mail: [email protected].

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Documents

Type I NKT cells protect (and type II NKT cells suppress) the host’s innate antitumor immune response to a B cell lymphoma