NK cells and NKT cells collaborate in host protection from methylcholanthrene-induced fibrosarcoma

Mark J. Smyth, Nadine Y. Crowe1, and Dale I. Godfrey1,

Cancer Immunology, Peter MacCallum Cancer Institute, St Andrews Place, East Melbourne, Victoria 8006, Australia
1 Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria 3181, Australia

Correspondence to: M. Smyth, Cancer Immunology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett Street, Victoria, Australia


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
NK1.1+ V{alpha}14J{alpha}281+ (NKT) cells can be induced by IL-12 therapy to mediate tumor rejection; however, methylcholanthrene (MCA)-induced fibrosarcoma is the only tumor model described where NKT cells play a natural role in controlling tumor initiation. From our previous study in C57BL/6 mice it remained unclear whether NK cells were also involved in this natural response. Herein, to discriminate the function of NK and NKT cells, we have evaluated fibrosarcoma development in mice deficient in NKT cells, but not NK cells, and mice deficient in NK cells, but not NKT cells. The results indicate that both NK cells and NKT cells are essential and collaborate in natural host immunity against MCA-induced sarcoma. In contrast, sarcoma incidence and growth rate were reduced using IL-12 therapy, this effect was mediated in the absence of T cells (including NKT cells), but not NK cells.

Keywords: immunotherapy, in vivo animal model, NK cell, NKT cell, tumor immunity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Chemical induction of tumors in inbred mice, particularly sarcomas induced by methylcholanthrene (MCA), has been a tumor model often used by immunologists to investigate immune surveillance (1). Treatment of mice with the powerful T and NK cell-stimulatory cytokine, IL-12, was shown to delay and reduce tumor appearance in MCA-inoculated mice (2). This study, and other studies using perforin (pfp)-deficient mice (3) and IFN-{gamma} receptor-deficient mice (4), first defined the importance of immunological control, at the molecular level, on tumor induction by MCA.

Despite these advances, studies in nu/nu mice by Stutman (5) had demonstrated a similar tumor incidence compared with wild-type mice and thus controversy persisted concerning what cellular mechanisms regulated sarcoma induction by MCA. However, our recent study using TCRJ{alpha}281–/– mice specifically deficient for NKT cells defined that natural surveillance of MCA-induced sarcomas was controlled by these cells (6). This is the only tumor model described where NKT cells naturally regulate tumor initiation or growth, despite several studies that define the involvement of NKT cells in IL-12- or {alpha}-galactosylceramide-mediated anti-metastatic activity (7,8). A difficulty with the aforementioned MCA study (6) was discriminating whether NK cells were additionally important in host protection, since anti-NK1.1 mAb depletes both NK and NKT cells. This is an important issue since it has remained unclear as to whether NKT cells can act alone as anti-tumor effector cells. Herein, we have taken advantage of the ability of anti-asialo-GM1 antibody to deplete NK cells, but not NKT cells, to demonstrate that indeed both NK and NKT cell subsets collaborate to constitute protective natural anti-tumor immunity.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Mice
Inbred C57BL/6 (B6), BALB/c and BALB/c.scid mice were purchased from the Walter and Eliza Hall Institute of Medical Research (WEHI; Melbourne, Australia). BALB/c.B6Cmv1r (NK1.1+) were obtained from the Animal Resources Centre (Perth, Australia). The following gene-targeted mice were bred at the Austin Research Institute Biological Research Laboratories (ARI-BRL): C57BL/6.RAG-1-deficient (B6.RAG-1–/–) mice (from Dr Lynn Corcoran, WEHI) (9) and C57BL/6 TCRJ{alpha}281-deficient (B6.J{alpha}281–/–) mice (from Dr Masaru Taniguchi, Chiba University School of Medicine, Chiba, Japan) (6). Mice aged 6–12 weeks old were used in all experiments that were performed according to animal experimental ethics committee guidelines.

NK/NKT cell subset depletion
The protocols for depletion of NK1.1+ (NK and NKT) cells in B6 mice using anti-NK1.1 mAb (PK136) were as previously described (6,10). Groups of BALB/c and BALB/c.B6Cmv1r (NK1.1+) mice were depleted of asialo-GM1+ cells using 20 µl of the rabbit anti-asialo-GM1 antibody (prepared as recommended; Wako Chemicals, Richmond, VA) on days –1, 0 (the day of MCA inoculation) and weekly thereafter. The BALB/c.B6Cmv1r (NK1.1+) mice were used to show that this depletion was effective, since DX5 and other surrogate markers are not entirely suitable for detecting NKT cells (11). Anti-asialo-GM1 antibody significantly depleted NK1.1+ TCR{alpha}ß NK cells, but not NKT cells in both C57BL/6 and BALB/c.B6Cmv1r mice (Fig. 1Go). Assessment of depletion was performed following preparation of cell suspensions from spleen and liver as described (11). Flow cytometry of cells was performed with allophycocyanin-conjugated anti-{alpha}ßTCR (clone H57-597), phycoerythrin-conjugated anti-NK1.1 (clone PK-136), FITC-conjugated anti-CD8a (clone 53.6.7) and biotin-conjugated anti-CD4 (clone CT-CD4; Caltag, Burlingame, CA). Streptavidin-conjugated PerCP was used to detect biotin-conjugated anti-CD4. Asialo-GM1 expression was revealed using sheep anti-rabbit IgFITC. All flow cytometry reagents were purchased from PharMingen (San Diego, CA), unless otherwise indicated. Cells were isolated, gated and analyzed as previously described (11). To assess the maintenance of NKT cell function in anti-asialo-GM1 antibody-treated mice, mice were treated i.v. with 1.5 µg of anti-mouse CD3 mAb (145-2C11) and serum collected 8 h later. Serum IFN-{gamma} was measured by a mouse IFN-{gamma}-specific ELISA according to the manufacturer's protocol (PharMingen).



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1. NK cells are preferentially depleted by anti-asialo-GM1 treatment. (A and B) Liver lymphocytes were labeled with anti-NK1.1, -{alpha}ßTCR and -asialo-GM1 (AGM1). AGM1 expression was revealed using sheep anti-rabbit Ig–FITC, and is shown on T (dashed line), NKT (solid line) and NK (shaded histogram) cells of the liver. Background staining by non-specific rabbit anti-sera is shown for whole liver lymphocytes (dotted line). (C and F). Groups of B6 and BALB/c.B6Cmv1r mice were injected with anti-asialo-GM1 or PBS and the spleen and liver harvested at days 1 and 6. Isolated lymphocytes were labeled with anti-NK1.1 and -{alpha}ßTCR. The percentages of NK (C and E) and NKT (D and F) cells in the spleen (C and D) and liver (E and F) are shown. Results represent the mean ± SE, n = 3 unless otherwise indicated. (*P < 0.05, **P < 0.005, Mann–Whitney U-test).

 
Tumor surveillance in vivo
Effector function was examined in gene-targeted mice or mice depleted of lymphocyte subsets by inducing fibrosarcoma in mice using MCA as described (6). Anti-asialo-GM1 or anti-NK1.1 mAb was administered on day –1, 0 (the day of MCA inoculation) and weekly thereafter. Mouse IL-12 was kindly provided by Genetics Institute (Cambridge, MA). The preparation of IL-12 was diluted in PBS immediately before use. Mice received 250 U of IL-12 i.p. 5 days a week for 20 weeks, in a schedule of 3 weeks on and 1 week off. Mice were weighed periodically during the first 120 days to determine any potential toxicity of IL-12. Relative sarcoma growth rate was calculated from the gradient of individual growth curves [plotted as s.c. tumor size – the product of two perpendicular diameters (cm2)]. The mean gradient of each group was standardized with the group of BALB/c mice receiving 100 µg MCA alone = 1.0.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Anti-asialo-GM1 antibody depletes NK cells, but not NKT cells
Anti-asialo-GM1 antibody is an effective means of depleting NK cells from a variety of mouse strains (12,13). Analysis of the specificity and effectiveness of this depletion has been difficult in BALB/c mice that lack the NK1.1 marker for suitable discrimination between NK cells and other T cell subsets. Since anti-NK1.1 mAb is the preferred reagent for staining NK and NKT cells, we have made use of the congenic BALB/c.B6.Cmv1r strain (14) that expresses the NK1.1 allele. Asialo-GM1 was shown to be expressed at significantly higher levels on liver NK1.1+TCR{alpha}ß cells than on NK1.1+TCR{alpha}ß+ or other TCR{alpha}ß+ cells (Fig. 1Go). A protocol of weekly injection of anti-asialo-GM1 antibody was employed to treat BALBc and B6 mice inoculated with MCA. This same protocol was shown to maintain significant depletion of NK1.1+TCR{alpha}ß cells, but not NK1.1+TCR{alpha}ß+ or other TCR{alpha}ß+ cells, in both B6 mice and BALB/c.B6.Cmv1r mice (Fig. 1Go). A lack of effect of anti-asialo-GM1 antibody treatment on the function of NKT cells was confirmed by the elevated serum IFN-{gamma} levels in these mice responding to anti-CD3 antibody (Fig. 2Go). Previous studies have indicated that NKT cells were the major T cell population responsible for IFN-{gamma} production in response to TCR–CD3 ligation (15,16). Interestingly, our data also suggested that NK cells are not required for NKT cell-mediated IFN-{gamma} production.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Anti-asialo-GM1 antibody does not reduce IFN-{gamma} production by anti-CD3 mAb-activated NKT cells. Untreated or anti-asialo-GM1 antibody-treated (day –6 or –1) B6 mice and B6.J{alpha}281–/– mice were treated i.v. with 100 µl of PBS or 1.5 µg of anti-mouse CD3 mAb (145-2C11) and serum collected 8 h later. Serum IFN-{gamma} was measured by a mouse IFN-{gamma}-specific ELISA. Serum of all PBS-treated mice did not contain detectable IFN-{gamma} (<50 pg/ml). The results are recorded as the mean serum IFN-{gamma} (pg/ml) ± SE for two mice performed in triplicate.

 
NK and NKT cells collaborate in host protection from MCA-induced fibrosarcoma
A clear picture of which host immune cells control sarcoma induction by MCA has been lacking. Our recent analysis using TCRJ{alpha}281–/– mice identified NKT cells to be critical for natural immunity against MCA-induced fibrosarcoma in the C57BL/6 mouse (6). However, previous depletion of NK1.1+ cells in B6 mice using the anti-NK1.1 mAb did not discriminate whether NK cells also played a role since both NK and NKT cells were eliminated by the protocol employed (6). Therefore, herein we compared the effect on MCA-induced sarcoma incidence in B6 mice when specifically depleting NK cells using anti-asialo-GM1 antibody (Fig. 3Go). At either dose of MCA inoculated (100 or 25 µg), depletion of NK cells alone was sufficient to cause B6 mice to develop significantly more sarcomas. Susceptibility to sarcoma was equivalent in mice specifically lacking NKT cells (B6.J{alpha}281–/–) or NK cells (B6 + anti-asialo-GM1), suggesting that both NK and NKT cells were indispensable for innate protection from MCA-induced sarcoma. No further increase in susceptibility has been observed in B6.J{alpha}281–/– mice depleted using anti-NK1.1 mAb (6). Increased susceptibility of B6.RAG-1–/– mice (Fig. 3AGo) and B6.ß2microglobulin–/– mice (data not shown) was also consistent with the lack of NKT cells in these two strains (1519).




View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3. NK and NK cells collaborate in host protection from MCA-induced fibrosarcoma. Groups of (A) B6, B6.RAG-1–/– and B6.J{alpha}281–/– or (B) BALB/c and BALB/c.scid mice were injected s.c. in the hind flank as indicated with 100, 25 or 5 µg MCA diluted in 0.1 ml corn oil. Some groups of B6 or BALB/c mice were also depleted of NK cells (+ anti-asGM1) or NK1.1+ cells (B6 + anti-NK1.1) in vivo. Mice were observed weekly for tumor development over the course of 50–180 days. Tumors >5 mm in diameter and demonstrating progressive growth over 3 weeks were counted as positive. Sarcoma development was recorded at 180 days as a percentage of the mice in each group (in parentheses). The number of mice in each group is shown in parentheses. Significant differences from (A) B6 mice or (B) BALB/c mice were determined by Fisher's exact test (*P < 0.05, ** P < 0.01).

 
We next examined groups of BALB/c, BALB/c.scid and BALB/c mice depleted of NK cells with anti-asialo-GM1 antibody. Mice were injected s.c. with 100, 25 or 5 µg of MCA and fibrosarcoma development was monitored for a period of 180 days (Fig. 3BGo). BALB/c.scid mice and BALB/c mice depleted of NK cells developed sarcomas more frequently than BALB/c control mice regardless of the dose of MCA administered. These data supported those in the C57BL/6 strain indicating that both NK cells and NKT cells contribute to natural host protective immunity in this model.

IL-12 reduces sarcoma incidence and growth rate in a NK cell-dependent fashion
Given that Noguchi et al. (2) previously demonstrated that IL-12 prolonged the latent period for tumor induction by MCA, we next evaluated the incidence and growth rates of sarcoma in the same groups of BALB/c mice (Fig. 3BGo) treated with IL-12 and 100 µg MCA. Because of the latency of these tumors, and the long period of IL-12 treatment, we could not readily test various IL-12 regimes and therefore employed a similar IL-12 treatment schedule to Noguchi et al. (2) (see Methods). Recombinant mouse IL-12 administered over a 20-week course was shown to significantly (P < 0.05) reduce the incidence of sarcoma in wild-type mice receiving MCA (Fig. 4AGo). Mice receiving IL-12 appeared healthy and gained weight (data not shown). A reduction in sarcoma incidence was also observed in BALB/c.scid mice receiving IL-12, suggesting that T cells, in particular NKT cells, were not essential for IL-12-mediated anti-tumor activity. By contrast, IL-12 had no effect on sarcoma incidence in mice depleted of NK cells. Individual sarcomas arising in these groups were monitored for growth weekly and their rate of growth was compared with tumors arising in BALB/c mice receiving MCA alone (Fig. 4BGo). In concert with IL-12-mediated reductions in sarcoma incidence, treatment also significantly reduced sarcoma growth rate. Notably, the suppression of tumor growth was dependent upon NK cells, but not T cells, including NKT cells. Interestingly, IL-12 treatment of NK cell-depleted mice led to an increase in mean tumor growth rate. It may be that the altered cytokine environment induced by IL-12 in the absence of NK cells promotes tumor growth.




View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4. IL-12 reduces sarcoma incidence and growth rate in a NK cell-dependent fashion. Similar groups of BALB/c and BALB/c.scid mice injected s.c. in the hind flank with 100 µg MCA were additionally treated with 250 U of mouse IL-12 i.p. 5 days a week for 20 weeks, in a schedule of 3 weeks on and 1 week off. One group of BALB/c mice was also depleted of NK cells (+ anti-asGM1). Sarcoma development was recorded (A) at 180 days as a percentage of the mice in each group (in parentheses). Significant differences from groups of mice not receiving IL-12 were determined by Fisher's exact test (*P < 0.05, ** P < 0.01). (B) Individual tumor growth was measured weekly with a caliper square as the product of two diameters. Growth rates were calculated from individual growth curves and the mean growth rate of each group was compared with BALB/c mice receiving 100 µg MCA alone (standardized to 1.0). The significance of IL-12 treatment was determined by the Mann–Whitney test (*P < 0.05). Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma Innate immune control of MCA-induced sarcoma

 

    Concluding remarks
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
This study has significantly enhanced our understanding of what effector cells contribute to host immune protection from MCA-induced sarcoma. Previously, and herein, we have illustrated very specifically that NKT cells are critical for host resistance to sarcoma formation. The use of a specific regime of anti-asialo-GM1 antibody that depletes NK cells, but leaves NKT cells intact, has enabled us to demonstrate that NK cells also are critical for an effective immune reaction to MCA-induced sarcoma. These data compare well with the recent observations that specific activation of CD1d-restricted NKT cells by exogenous stimuli such as IL-12 or {alpha}-Gal-Cer can result in the potent activation of NK cell cytokine production, proliferation and cytotoxicity (15,20). The activation of NK cell effector functions by NKT cell IFN-{gamma} production and antigen-presenting cell IL-12 production (20) is also consistent with an important role for endogenous IL-12 (6) and IFN-{gamma} (Smyth, unpublished data) in host natural immunity to MCA-induced sarcoma.

Clearly IL-12 treatment also suppressed the initiation and growth of MCA-induced sarcomas; however, the anti-tumor activity of IL-12 was not strictly dependent on the presence of V{alpha}14J{alpha}281 NKT cells. While the IL-12 treatment experiments further supported an important role for effector NK cells in controlling sarcoma initiation and growth, it must be noted that in other tumor models we have demonstrated that the relative dose of IL-12 administered dictates the relative role of NK and NKT cells (21). NKT cells have higher basal levels of IL-12 receptor expression than NK cells (22) and thus it is probable that at lower doses of IL-12, perhaps more akin to those naturally triggered by MCA-induced tumors, NKT cell function is an important amplifier of an effective anti-tumor response. Recent evidence supports the ability of TCR stimulated NKT cells to rapidly secrete IFN-{gamma} that promotes secondary activation of NK cell proliferation and effector functions (15). It now remains to be formally demonstrated by adoptive transfer experiments exactly how NKT cells induce NK cell activity against MCA-induced tumors.

Sarcoma is a well-studied experimental tumor in mice, but is relatively infrequent in humans. The use of adoptive transfer experiments and the recent exciting development of CD1d tetramers that can detect NKT cells (23,24), should enable better dissection of how NK cells and NKT cells collaborate to control immune rejection of MCA-induced sarcoma, and whether other highly immunogenic tumors are innately controlled by both NK and NKT cells.


    Acknowledgments
 
M. J. S. is currently supported by a National Health and Medical Research Council of Australia (NH & MRC) Principal Research Fellowship. D. I. G. is supported by the NH & MRC and AdCorp-Diabetes Australia. This work was supported by a NH & MRC Project Grant. We thank the staff of the PMCI animal facility and ARI-BRL for their maintenance and care of the mice in this project.


    Abbreviations
 
MCA methylcholanthrene
pfp perforin

    Notes
 
Transmitting editor: A. Kelso

Received 25 September 2000, accepted 18 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 

  1. Basombrio, M. A. 1970. Search for common antigenicities among twenty-five sarcomas induced by methylcholanthrene. Cancer Res. 30:2458.[ISI][Medline]
  2. Noguchi, Y., Jungbluth, A., Richards, E. C. and Old, L. J. 1996. Effect of interleukin 12 on tumor induction by 3-methylcholanthrene. Proc. Natl Acad. Sci. USA 93:11798.[Abstract/Free Full Text]
  3. van den Broek, M. E., Kagi, D., Ossendorp, F., Toes, R., Vamvakas, S., Lutz, W. K., Melief, C. J., Zinkernagel, R. M. and Hengartner, H. 1996. Decreased tumor surveillance in perforin-deficient mice. J. Exp. Med. 184:1781.[Abstract]
  4. Kaplan, D. H., Shankaran, V., Dighe, A. S., Stockert, E., Aguet, M., Old, L. J. and Schreiber, R. D. 1998. Demonstration of an interferon {gamma}-dependent tumor surveillance system in immunocompetent mice. Proc. Natl Acad. Sci. USA 95:7556.[Abstract/Free Full Text]
  5. Stutman, O. 1975. Immunodepression and malignancy. Adv. Cancer Res. 22:261.[Medline]
  6. Smyth, M. J., Thia, K. Y. T., Street, S. E. A., Cretney, E., Trapani, J. A., Taniguchi, M., Kawano, T., Pelikan, S. B., Crowe, N. Y. and Godfrey, D. I. 2000. Differential tumor surveillance by NK and NKT cells. J. Exp. Med. 191:661.[Abstract/Free Full Text]
  7. Takeda, K., Hayakawa, Y., Atsuta, M., Hong, S., Van Kaer, L., Kobayashi, K., Ito, M., Yagita, H. and Okumura, K. 2000. Relative contribution of NK and NKT cells to the anti-metastatic activities of IL-12. Int. Immunol. 12:909.[Abstract/Free Full Text]
  8. Kawano, T., Cui, J., Koezuka, Y., Toura, I., Kaneko, Y., Sato, H., Kondo, E., Harada, M., Koseki, H., Nakayama, T., Tanaka, Y. and Taniguchi, M. 1998. Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated V{alpha}14 NKT cells. Proc. Natl Acad. Sci. USA 95:5690.[Abstract/Free Full Text]
  9. Spanopoulou, E., Roman, C. A., Corcoran, L. M., Schlissel, M. S., Silver, D. P., Nemazee, D., Nussenzweig, M. C., Shinton, S. A., Hardy, R. R. and Baltimore, D. 1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8:1030.[Abstract]
  10. Smyth, M. J., Thia, K. Y., Cretney, E., Kelly, J. M., Snook, M. B., Forbes, C. A. and Scalzo, A. A. 1999. Perforin is a major contributor to NK cell control of tumor metastasis. J. Immunol. 162:6658.[Abstract/Free Full Text]
  11. Hammond, K. J. L., Pelikan, S. B., Crowe, N. Y., Hooi, H. L. C., Randle-Barrett, E., Smyth, M. J., Baxter, A. G., van Driel, I. R., Scollay, R. and Godfrey, D. I. 1999. NKT cells are a phenotypically and functionally diverse population. Eur. J. Immunol. 29:3768.[ISI][Medline]
  12. Habu, S., Fukui, H., Shimamura, K., Kasai, M., Nagai, Y., Okumura, K. and Tamaoki, N. 1981. In vivo effects of anti-asialo-GM1. I. Reduction of NK activity and enhancement of transplanted tumor growth in nude mice. J. Immunol. 127:34.[Abstract/Free Full Text]
  13. Kasai, M., Iwamori, M., Nagai, Y., Okumura, K. and Tada, T. 1980. A glycolipid on the surface of mouse natural killer cells. Eur. J. Immunol. 10:175.[Medline]
  14. Scalzo, A. A., Lyons, P. A., Fitzgerald, N. A., Forbes, C. A. and Shellam, G. R. 1995. The BALB.B6-Cmv1r mouse: a strain congenic for Cmv1 and the NK gene complex. Immunogenetics 41:148.[ISI][Medline]
  15. Carnaud, C., Lee, D., Donnars, O., Park, S-H., Beavis, A., Koezuka, Y. and Bendelac, A. 1999. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163:4647.[Abstract/Free Full Text]
  16. Bendelac, A., Rivera, M. N., Park, S. H. and Roark, J. H. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.[ISI][Medline]
  17. Bix, M., Coles, M. and Raulet, D. 1993. Positive selection of Vß8+ CD48 thymocytes by class I molecules expressed by hematopoietic cells. J. Exp. Med. 178:901.[Abstract]
  18. Bendelac, A., Killeen, N., Littman, D. R. and Schwartz, R. H. 1994. A subset of CD4+ thymocytes selected by MHC class I molecules. Science 263:1774.[ISI][Medline]
  19. Ohteki, T. and MacDonald, H. R. 1994. Major histocompatibility complex class I related molecules control the development of CD4+8 and CD48 subsets of natural killer 1.1+ T cell receptor-{alpha}+ cells in the liver of mice. J. Exp Med. 180:699.[Abstract]
  20. Eberl, G. and MacDonald, H. R. 2000. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol. 30:985.[ISI][Medline]
  21. Smyth, M. J., Taniguchi, M. and Street, S. E. A. 2000. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J. Immunol. 165:2665.[Abstract/Free Full Text]
  22. Kawamura, T., Takeda, K., Mendiratta, S. K., Kawamura, H., Van Kaer, L., Yagita, H., Abo, T. and Okumura, K. 1998. Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J. Immunol. 160:16.[Abstract/Free Full Text]
  23. Benlagha, K., Weiss, A., Beavis, A., Teyton, L. and Bendelac, A. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191:1895.[Abstract/Free Full Text]
  24. Matsuda, J. L., Naidenko, O. V., Gapin, L., Nakayama, T., Taniguchi, M., Wang, C. R., Koezuka, Y. and Kronenberg, M. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192:741.[Abstract/Free Full Text]