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-Chain-Dependent Non-T Non-B Cells to IL-12-Mediated Rejection of Tumors 1
* Department of Molecular Biology, Princeton University, Princeton, NJ 08540;
Graduate School of Biotechnology, Korea University, Seoul, South Korea; and
Institut National de la Santé et de la Recherche Medicale Unité 25 and E0209, Paris, France
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Abstract |
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14-J18 NKT cell was the essential cell type recruited in most, if not all tumor rejection models, including the B16 melanoma. In this study, we have examined and compared the role of NKT cells, T cells, NK cells, and other non-T non-B cells in the rejection of B16 melanoma cells after exogenous administration of IL-12. Surprisingly, our results failed to confirm a necessary role for NKT cells in this model. Instead, we found that NK cells mediated the rejection of liver metastases, whereas other 
c-dependent non-T non-B cells, possibly lymphoid dendritic cells, were required for rejection of skin tumors. These findings challenge the view that NKT cells are systematically required for IL-12-mediated rejection of tumors, and instead reveal that a variety of effector pathways can be recruited depending on the tumor microenvironment. |
Introduction |
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14-J18 NKT cells, a conserved population of CD1d-autoreactive CD4+ and double-negative T cells, and suggested its involvement in the regulation of a number of autoimmune, infectious, and cancer conditions (reviewed in Refs 1 and 2). V14 NKT cells have long been confused with NK cells because they share the expression of various receptors encoded by the NK gene complex, such as NK1.1, and are sensitive to the same type of Ab-mediated depletion experiments that have been used to determine the functions of NK cells in vivo. NKT and NK cells also share functional similarities and intricacies in vivo because both respond to IL-12 by rapidly releasing IFN-, and because NKT cell activation by its cognate glycolipid Ag -galactosylceramide rapidly transactivates NK cells in vivo (3, 4).
The mechanisms underlying the antitumor activity of IL-12 illustrate some of the difficulties recently encountered in dissecting the specific contribution of NK and NKT cells. The beneficial effects of exogenously administered IL-12 in antitumor therapy were first ascribed to the recruitment and activation of CD8+ CTL or conventional NK cells (reviewed in Ref.5). Indeed, depending on the model studied, the abrogation of one or the other subset significantly reduced the therapeutic benefit of IL-12. More recently, however, using J18-deficient mice lacking NKT cells, as well as RAG0.V14-J18/V
8 TCR transgenic mice lacking NK cells, Cui et al. (6) suggested that V14 NKT cells were not only necessary, but also sufficient for tumor rejection. Their findings in three of three tumor models investigated suggested that V14 NKT cells functioned as the obligate mediator of all the antitumor effects of IL-12. This requirement of NKT cells has not been systematically confirmed by others, because, based on the study of RAG0 mice treated with anti-NK1.1 Ab, NK cells rather than NKT cells were suggested to induce protection against B16 melanoma cells (7). However, in support of the importance of NKT cells, Smyth et al. (8) found a correlation between the NKT dependence and the IL-12 dependence of the rejection of some tumors.
Similar confusion exists in the understanding of the cellular mechanisms underlying the generalized Shwartzman reaction, a model of IL-12-mediated septic shock induced by repeated exposure to LPS. Although NK cells were thought to be the essential cell type in the sequence of events leading to the lethal shock (9), this was not confirmed in a genetic model of NK deficiency in mice (10). In contrast, another study reported that both J180 and CD1d0 mice lacking NKT cells, but otherwise normal with respect to NK cells and conventional T cells, resisted more efficiently to LPS-induced death than control wild-type mice, suggesting a prominent role of NKT cells in the IL-12-induced shock (11).
To reconcile these discrepancies, several authors have proposed a threshold model whereby high doses of IL-12, in the case of tumor therapy, or high doses of LPS in the Shwartzman reaction recruited both NKT and NK cells, each one being able to mediate an efficient response, whereas infraoptimal doses of the same stimuli selectively recruited NKT cells (11, 12). The model was supported by IL-12 titration experiments in the Shwartzman reaction. Because the physiological role of NK and NKT cells needed to be further clarified in view of their putative contribution to a large variety of biological responses, we have tested experimentally the threshold hypothesis in the IL-12-mediated tumor rejection model. Our data failed to confirm the hypothesis that NKT cells become the critical effector cell type when the IL-12 stimulus reaches infraoptimal zones. In contrast, we report that the site of implantation of the tumor critically affects the nature of the cell types recruited by IL-12, which include NK cells as well as other c-dependent non-T non-B cells.
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Materials and Methods |
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C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). RAG20 and RAG20.c0 (RAG.c) mice in a C57BL/6 background were a kind gift of J. DiSanto (Institut Pasteur, Paris, France). CD1-deficient mice lacking both CD1d1 and CD1d2 and their heterozygous littermates were in a C57BL/6 background (13). Mice were treated according to institutional animal care and use guidelines.
Tumor cell injection and IL-12 treatment
B16.F10 melanoma cells were obtained from N. P. Restifo (National Institutes of Health, Bethesda, MD) and cultured in RPMI enriched with 10% FCS (Biofluids, Rockville, MD) and antibiotics. The cell line tested negative for mycoplasma and other transmissible infectious agents. A total of 0.4–1 x 106 cells resuspended in PBS were injected into the s.c. tissue of the flank, or into the portal system through intrasplenic injection of 0.1 ml cell suspension, immediately followed by splenectomy (day 0). Mouse rIL-12 (2.6 x 106 U/mg; a gift from Genetics Institute, Cambridge, MA) was injected i.p. at days 1, 3, 5, and 9. Tumor growth was assessed by measuring two perpendicular diameters of the skin tumor, or by weighing the whole liver for liver metastases.
Ab treatment
Mice were injected on days -5 and -2 with 200 µg purified PK136 anti-NK1.1 mAb or control isotype-matched 14.4.4S anti-I-E mAb. Depletion was monitored by staining with anti-CD3 and DX5 mAb.
Flow cytometry
Liver lymphocytes were collected and stained using APC-conjugated CD1d-GalCer tetramers, anti-NK1.1 PE, annexin V FITC, or anti-5-bromo-2'-deoxyuridine (BrdU)3 FITC, as previously described (14). Continuous BrdU exposure in drinking water and BrdU staining were as recommended by BD PharMingen (San Diego, CA).
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Results |
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The B16 melanoma line has been largely used in IL-12 antitumor therapy studies and is at the center of the controversy regarding the specific involvement of NK vs NKT cells (6). Although the tumor line does not metastasize spontaneously, one can mimic the metastatic process by inoculating tumor cells i.v. for the lung metastasis or intrasplenically for liver metastasis. In contrast, when injected s.c. or in the muscle, B16 cells expand as a single solid tumor mass. We have examined the two modes of expansion, local upon inoculation in the flank and metastatic following embolization in the liver through intrasplenic injection. No differences in tumor growth between wild-type and NKT-deficient mice could be observed in the absence of IL-12. When repeatedly injected i.p. with 500 ng of IL-12, both CD1+/- and CD1-/- slowed the growth of B16 in the s.c. tissue to a similar extent (Fig. 1A). Similarly, CD1d expression had no influence on the liver metastastic process (Fig. 1B). These results showed that, irrespective of the site of inoculation, therapeutic doses of IL-12 were as effective in normal mice as in NKT-deficient mice. Thus, using one of the same tumor types and the same IL-12 treatment regimen as Cui et al. (6), our data did not support the notion that NKT cells were systematically required for the IL-12-mediated rejection of tumors.
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To directly test the threshold hypothesis, we performed additional experiments to ask whether suboptimal doses of IL-12 might reveal an essential role of NKT cells. Two independent experiments were performed, and their pooled results are presented in Fig. 2. Cohorts of CD1-/- and heterozygous (+/-) littermates were inoculated in the spleen with B16 cells and subsequently treated with varying amounts of IL-12 ranging from 200 to 2 ng repeated four times at days 1, 3, 5, and 9. There was a clear dose effect because, whereas protection was almost complete at 100 ng, it decreased at 50 ng, and the difference with the PBS-treated group was marginal at 10 ng. The most important point demonstrated by these experiments, however, was that mice deficient in NKT cells behave like heterozygous littermates at every dose tested. Thus, there was no indication that NKT-deficient mice were less sensitive than littermate controls to low doses of IL-12.
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c-dependent non-T non-B cells depending on the mode of inoculationThe contribution of NK1.1+ cells to IL-12 therapy was further examined in experiments in which RAG20 mice, naturally lacking both NKT and mainstream T cells, were further depleted of NK cells by PK136 anti-NK1.1 Abs.
We investigated the two modes of inoculation, the flank for local tumor growth (Fig. 3A) and the spleen for visceral metastatic dissemination (Fig. 3B). In both models, RAG20 mice rejected the tumor as well as wild-type mice in response to IL-12, suggesting the lack of requirement for components of adaptive immunity in this system (compare Figs. 1A and 3A and Figs. 1B and3B). However, the two models revealed different requirements. Thus, the depletion of NK1.1+ NK cells did not alter the therapeutic effect of IL-12 against s.c. tumor growth (Fig. 3A). In contrast, it abrogated protection against liver metastasis (Fig. 3B). Thus, the presence of CD3-NK1.1+ cells, but not NKT cells, seemed to be clearly required to mediate the antitumor effects of IL-12 in the liver, whereas in the s.c. tissue, none of these cell types appeared to be necessary.
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secretion and tumor rejection in the skin model, we tested RAG.c doubly deficient mice, which are severely impaired in all lymphoid lineages, including B, T, NKT, NK, and lymphoid dendritic cells (DC). As shown in Fig. 4, these mice lost the antitumor response to IL-12 when inoculated with B16 s.c. This result contrasted with the indifference of RAG20 mice to PK136-mediated ablation of NK cells, and suggested that a distinct population of c-dependent non-T non-B non-NK cells was required for rejection of s.c. tumors.
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The above results suggesting that NKT cells may not be, as initially reported, essential for IL-12-mediated tumor rejection prompted us to re-evaluate the claim that NKT cells were highly responsive to IL-12 in vivo (15). In mice injected with IL-12, NKT cells underwent rapid activation-induced cell death. We found a prompt down-modulation of NK1.1 associated with a gain of annexin V staining, suggesting apoptosis (Fig. 5A). In contrast, NK1.1+CD1d-GalCer- cells (mainly NK cells) did not down-modulate NK1.1 and appeared conserved. At day 2, NKT cells were virtually absent from the liver, whereas NK cells were conserved (not shown). By day 5 after a single injection of IL-12, NKT cells were back to subnormal levels (Fig. 5B). Interestingly, most of these NKT cells were BrdU positive, indicating that they were the progeny of dividing cells. Altogether, these results are consistent with a previously published study (15) reporting drastic effects of IL-12 on NKT cells in vivo, with rapid activation-induced cell death followed by renewal from a pool of dividing cells. The present study demonstrates that these striking effects did not appear to be necessary for the rejection of B16 tumors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release from various cellular sources, exerting significant antitumor effects both in natural as well as in experimental tumorigenesis. Thus, for example, IFN- enhances tumor Ag presentation to CTL, exerts antiangiogenetic effects through the induction of IFN--inducible protein-10 and monokine induced by IFN-, and may also directly prevent tumorigenesis by activating IFN regulatory factor-1 (18).
Independently from microbial challenges, NKT cell activation by their cognate glycolipid ligand can also elicit a cascade including IL-12 secretion by DC through cognate NKT-DC interaction (19), and subsequent activation of both IFN- secretion and cytolytic properties of NK cells (3, 4). Importantly, NKT cells have been reported to be recruited in the natural surveillance of fibrosarcomas induced by methylcholanthrene (MCA) injection as well as in the rejection of transplants of MCA-induced tumors (8, 20). In this system, NKT cell activation appeared to be mediated by the recognition of CD1d complexed with natural ligands expressed on nontumor cells, possibly DC, rather than on tumor cells that did not express CD1d.
Thus, IL-12 appears to be at the center of a number of intricated and overlapping mechanisms leading to the natural or therapeutic rejection of many tumors. Although many cellular targets of IL-12 can be induced to secrete IFN-, recent reports have suggested a hierarchy based on their relative contribution. Taniguchi and colleagues (6) suggested that V14 NKT cells, but not T cells or NK cells, were a necessary component of the immune network, leading to tumor rejection in three of three tumor systems tested, including B16 melanoma, LLC lung carcinoma, and FBL3 erythroleukemia. Others reported that NKT cells expressed higher levels of the IL-12R1 and 2 mRNA (21), and were more sensitive to low doses of IL-12 than NK cells (11). Because many tumors, including B16, do not express surface CD1d, the antitumor mechanism elicited by NKT cells might be a direct, NK-like killing of tumor cells by activated NKT cells themselves or by recruited NK cells, or, alternatively, noncytolytic mechanisms induced by the IFN- released from NKT cells. However, other studies challenged these results. For example, lymphoid DC were suggested to be the major source of IFN- after IL-12 injection (22). In addition, IL-12 induced the same pattern of rejection of B16 lung metastasis in RAG0 and in wild-type mice (7). Interestingly, the controversy over the cellular targets of IL-12 extends to the Schwartzman reaction, in which the respective contribution of NKT cells, NK cells, and other cell types has not been fully resolved (11, 23).
In this study, we have re-examined the effect of IL-12 on one of the three tumor types studied by Taniguchi and colleagues (6). We confirmed that, concomitantly with the extensive activation, cell division, and death of NKT cells, IL-12 induced the rejection of the tumor inoculated intrasplenically or s.c. However, we failed to observe a requirement of these NKT cells for tumor clearance over a broad range of doses administered i.p. at days 1, 3, 5, and 9, according to the protocol used by Taniguchi and colleagues. The basis for these discrepancies is unclear at present, and may involve different tumor sublines or differences in mouse colonies. Furthermore, it is possible that the CD1d-deficient mice used in our study differed significantly from the J18-deficient mice used in the previous study, although it is expected that the loss-of-function phenotype should be conserved. For example, one might envision a more complex scenario whereby two opposing subsets contributed to the IL-12 effect, the V14-J18 cells being protective, while another hypothetical subset regulated by CD1d was aggravating. Our results, however, are important in that they directly challenge the prevailing notion that NKT cells are systematically required for rejection of tumors mediated by high or low doses of IL-12. Recent studies of the Schwartzman reaction have also challenged the notion that NKT cells were required to observe the lethal effects of LPS injection (23) (our data not shown). Altogether, these results suggest that a reassessment of the conditions under which NKT cells may be necessary is warranted. In this respect, a recent study indicated that, while NK cells dominated the early rejection of an MCA-induced sarcoma following cyclophosphamide and IL-12 treatment, a role for CD1d-restricted NKT cells could be suggested by the late tumor recurrence in CD1d-deficient mice (24).
Interestingly, our comparison of the patterns of tumor growth in different tissue environments of immunological mutant mice has revealed unexpected differences in the effector mechanisms leading to tumor rejection under IL-12 treatment. Thus, rejection of liver metastasis was conserved in RAG0 mice, but disappeared after treatment of these RAG0 mice with anti-NK1.1 mAb, suggesting a prominent role of NK cells in this location. This conclusion is consistent with the high frequency of NK cells in the liver and their demonstrated functions in other systems. In contrast, the same anti-NK1.1 treatment did not alter the rejection of skin tumors, suggesting that NK cells were not operating in this location. However, our finding that RAG.c doubly mutant mice failed to reject the skin tumors suggested the involvement of yet another cell type, which can be operationally defined as c-dependent non-T non-B non-NK cell. One attractive candidate is the lymphoid DC, which is c dependent and has been reported to be the main source of IFN- following IL-12 treatment (22). A definitive test of this hypothesis awaits the development of a methodology to selectively deplete lymphoid DC in vivo. It is conceivable that solid skin tumors are refractory to NK cell attack, but are most effectively controlled by the antiangiogenetic factors elicited by IFN-. It should be noted that the same cell type could also be essential in the rejection of liver metastasis, which might depend on the combined activation of NK cells locally and the presence of high IFN- levels systematically.
In conclusion, our study directly challenges the model suggesting that all antitumor effects of IL-12 are mediated by NKT cells, and further suggests that the notion that NKT cells are the most sensitive targets of IL-12 needs to be experimentally re-examined in vivo. Our results instead reveal a complexity of mechanisms depending on specific microenvironments as well as tumor cell types, which will be important to dissect for further understanding on the physiological as well as therapeutic roles of IL-12.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Albert Bendelac, Department of Pathology, University of Chicago, Chicago, IL 60637. E-mail address: abendela{at}bsd.uchicago.edu
3 Abbreviations used in this paper: BrdU, 5-bromo-2'-deoxyuridine; DC, dendritic cell; MCA, methylcholanthrene.
Received for publication August 28, 2002. Accepted for publication November 20, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623. production in vivo. J. Immunol. 164:1643./(1) T cell receptor /(1) intestinal intraepithelial lymphocytes in the absence of both classical major histocompatibility complex class I and nonclassical CD1 molecules. J. Exp. Med. 190:885.
- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis. Immunity 9:345.[Medline]
. Annu. Rev. Immunol. 15:749.[Medline]
-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189:1121. production by CD8+ lymphoid dendritic cells. J. Exp. Med. 189:1981.This article has been cited by other articles:
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  N. A. Nagarajan and M. Kronenberg Invariant NKT Cells Amplify the Innate Immune Response to Lipopolysaccharide J. Immunol., March 1, 2007; 178(5): 2706 - 2713. [Abstract] [Full Text] [PDF]
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 M. Emoto, I. Yoshizawa, Y. Emoto, M. Miamoto, R. Hurwitz, and S. H. E. Kaufmann Rapid Development of a Gamma Interferon-Secreting Glycolipid/CD1d-Specific V{alpha}14+ NK1.1- T-Cell Subset after Bacterial Infection. Infect. Immun., October 1, 2006; 74(10): 5903 - 5913. [Abstract] [Full Text] [PDF]
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L. Broderick, S. J. Yokota, J. Reineke, E. Mathiowitz, C. C. Stewart, M. Barcos, R. J. Kelleher Jr., and R. B. Bankert Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-{gamma}, and Eradicate Tumor Cells J. Immunol., January 15, 2005; 174(2): 898 - 906. [Abstract] [Full Text] [PDF]
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 F. Granucci, I. Zanoni, N. Pavelka, S. L.H. van Dommelen, C. E. Andoniou, F. Belardelli, M. A. Degli Esposti, and P. Ricciardi-Castagnoli A Contribution of Mouse Dendritic Cell-Derived IL-2 for NK Cell Activation J. Exp. Med., August 2, 2004; 200(3): 287 - 295. [Abstract] [Full Text] [PDF]
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M. Falcone, F. Facciotti, N. Ghidoli, P. Monti, S. Olivieri, L. Zaccagnino, E. Bonifacio, G. Casorati, F. Sanvito, and N. Sarvetnick Up-Regulation of CD1d Expression Restores the Immunoregulatory Function of NKT Cells and Prevents Autoimmune Diabetes in Nonobese Diabetic Mice J. Immunol., May 15, 2004; 172(10): 5908 - 5916. [Abstract] [Full Text] [PDF]
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M. L. Salem, A. N. Kadima, Y. Zhou, C. L. Nguyen, M. P. Rubinstein, M. Demcheva, J. N. Vournakis, D. J. Cole, and W. E. Gillanders Paracrine Release of IL-12 Stimulates IFN-{gamma} Production and Dramatically Enhances the Antigen-Specific T Cell Response after Vaccination with a Novel Peptide-Based Cancer Vaccine J. Immunol., May 1, 2004; 172(9): 5159 - 5167. [Abstract] [Full Text] [PDF]
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 D. Pelloso, K. Cyran, L. Timmons, B. T. Williams, and M. J. Robertson Immunological Consequences of Interleukin 12 Administration after Autologous Stem Cell Transplantation Clin. Cancer Res., March 15, 2004; 10(6): 1935 - 1942. [Abstract] [Full Text] [PDF]
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