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The Journal of Immunology, 2004, 172: 6550-6557.
Copyright © 2004 by The American Association of Immunologists

Essential Role of Bystander Cytotoxic CD122+CD8+ T Cells for the Antitumor Immunity Induced in the Liver of Mice by {alpha}-Galactosylceramide

Ryusuke Nakagawa*, Takuo Inui{ddagger}, Ikuko Nagafune§, Yoshiko Tazunoki§, Kazuhiro Motoki§, Akira Yamauchi*, Mitsuomi Hirashima{dagger}, Yoshiko Habu{ddagger}, Hiroyuki Nakashima{ddagger} and Shuhji Seki1,{ddagger}

Departments of * Cell Regulation and {dagger} Immunology and Immunopathology, Kagawa Medical University, Kagawa, Japan; {ddagger} Department of Microbiology, National Defense Medical College, Tokorozawa, Japan; and § Pharmaceutical Research Laboratory, Kirin Brewery, Takasaki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently reported that NK cells and CD8+ T cells contribute to the antimetastatic effect in the liver induced by {alpha}-galactosylceramide ({alpha}-GalCer). In the present study, we further investigated how CD8+ T cells contribute to the antimetastatic effect induced by {alpha}-GalCer. The injection of anti-CD8 Ab into mice 3 days before {alpha}-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-{gamma} production nor did it reduce the NK activity of liver mononuclear cells after {alpha}-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after {alpha}-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8+ T cells did. CD122+CD8+ T cells in the liver increased after {alpha}-GalCer injection, and antitumor cytotoxicity of CD8+ T cells in the liver gradually increased until day 6. These CD8+ T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122+CD8+ T cells. The critical, but bystander role of CD122+CD8+ T cells was further confirmed by adoptive transfer experiments into CD8+ T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/{alpha}-GalCer injection for the mice to generate CD8+ T cells that can reject s.c. rechallenged B16 cells. These findings suggest that {alpha}-GalCer activates bystander antitumor CD122+CD8+ T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8+ CTLs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mouse NK1.1 Ag+ T cells (NKT cells) are abundant in the liver (1, 2, 3, 4, 5). These cells use V{alpha}14J{alpha}281 gene product combined with V{beta}8 and V{beta}7 gene products for their TCR (6, 7), and their development is dependent on the MHC class I-like molecule, CD1d (8). A glycolipid Ag, {alpha}-galactosylceramide ({alpha}-GalCer),2 is a synthetic ligand of NKT cells and induces NKT cells to produce IFN-{gamma} and IL-4 (9). Liver NKT cells of mice acquired a potent antitumor cytotoxicity after IL-12 stimulation in vivo and inhibit hematogenous tumor metastases in the liver, lung, and kidney (2, 4, 10, 11, 12, 13, 14). However, liver NKT cells in mice injected with {alpha}-GalCer have been reported to rapidly disappear (15, 16, 17), while, in addition, it was recently shown that NKT cells do not disappear, but transiently down-regulate their TCR and NK1.1 Ag (18, 19). We recently demonstrated that NK cells that are activated by IFN-{gamma} produced by {alpha}-GalCer-activated NKT cells are the main and direct antimetastatic effector cells in the liver and reject the liver metastases of intrasplenically (i.s.) injected tumors, while CD8+ T cells are also required for the antimetastatic effect in the liver and mouse survival, whereas activated NKT cells caused the hepatocyte injury (20, 21). The mice that rejected liver metastases of i.s. injected tumors are already resistant to the same s.c. rechallenged tumor cells, while they could not reject s.c. inoculated other tumors (22, 23). Furthermore, the inhibitory effect of {alpha}-GalCer on s.c. rechallenged tumors was mainly mediated by CD8+ T cells (22, 23). These findings suggested that NK and NKT cells might be important for innate immunity against tumor metastasis, while tumor-specific memory CD8+ T cells may play an important role in the inhibition of s.c. inoculated tumor growth.

{alpha}-GalCer stimulation reportedly induces an Ag-independent proliferation of memory phenotype T cells (bystander proliferation) (24). An extensive T cell proliferation occurs during viral infections in mice (25), and in mice injected with LPS (26), CpG DNA (27), or poly(I:C) (25), and for the most part, these proliferative responses are restricted to memory phenotype CD8+ T cells (28). The proliferations of these CD8+T cells stimulated with poly(I:C) occurred in MHC class I-deficient hosts (using bone marrow chimeras). As a result, although the proliferative response of the memory phenotype CD8+ T cells represented a MHC-independent bystander response, it may be an important immune response to protect the hosts against infections (25).

In the present study, we show that {alpha}-GalCer induces the bystander proliferation of memory phenotype CD122+CD8+ T cells with an antitumor function in the liver. These CD122+CD8+ T cells are tumor-nonspecific CTLs; however, they become more critical antitumor effectors than NK cells as early as 2–3 days after {alpha}-GalCer injection. As a result, {alpha}-GalCer-induced antitumor immunity of NK cells triggered by IFN-{gamma} produced by NKT cells is rapidly taken over by CD122+CD8+ T cells, and thereafter, adaptive antitumor immunity may further be induced by tumor-specific memory CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College (Tokorozawa, Japan).

Mice and preparation of hepatic mononuclear cells (MNCs)

Male C57BL/6 (B6) mice at 6 wk of age were obtained from Nippon SLC (Hamamatsu, Japan). Mice were maintained and fed under standard laboratory conditions. Hepatic MNCs were prepared essentially as described (14). In brief, the liver was passed through a stainless steel mesh, and the resulting dissociated cells were suspended in HBSS, washed, resuspended in an isotonic 33% Percoll solution (Amersham Biosciences, Arlington Heights, IL) containing heparin (100 U/ml (Sigma-Aldrich, St. Louis, MO)), and centrifuged at 2000 rpm (500 x g) for 15 min at room temperature. The resulting pellet was resuspended in RBC lysis solution and then was washed twice in RPMI 1640 medium supplemented with 5% FCS.

Reagents

{alpha}-GalCer, or (2S,3S,4R)-1-O-({alpha}-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,2,4-octadecanetriol (KRN7000), was synthesized in our laboratory (29). The original solution of {alpha}-GalCer (220 µg/ml) was prepared with 0.5% polysorbate 20 (Nikko Chemicals, Tokyo, Japan) in saline, and then was subsequently diluted with this solution (vehicle) or with saline before i.v. injection at a dose of 100 µg/kg body mass.

Flow cytometric analysis

The surface phenotypes of liver MNCs were characterized by a four-color flow cytometric analysis. FITC-conjugated anti-TCR{alpha}{beta} mAb (H57-597, hamster IgG), FITC-conjugated anti-CD8 mAb (53-6.7, rat IgG2a), PE-conjugated anti-NK1.1 mAb (PK136, mouse IgG2a), PE-conjugated anti-CD122 (IL-2R{beta}) mAb (TM-{beta}1, rat IgG2b), allophycocyanin-conjugated anti-CD44 mAb (IM7, rat IgG2b), and allophycocyanin-conjugated anti-CD3 mAb (145-2C11, hamster IgG) were purchased from BD PharMingen (San Diego, CA). Before staining with Abs, the MNCs were incubated for 10 min with Fc blocker (2.4 G2; BD PharMingen) to prevent any nonspecific binding. Flow cytometry was performed with the FACSCalibur device (BD Biosciences, San Jose, CA).

In vivo and in vitro cell depletion

Anti-CD4 mAb (L3T4), anti-CD8 mAb (Lyt-2.2), anti-NK1.1 mAb, and anti-CD122 (IL-2R{beta}) mAb were derived from GK1.5, 2.43, PK136, and TM-{beta}1 hybridoma cells, respectively, and each Ab was prepared from mice ascites fluid (IBL, Gunma, Japan). We previously showed that a single i.v. injection of an optimal dose of anti-NK1.1 Ab depleted both NK and NKT cells, for at least 5 days. Mice were injected i.v. with 500 µg of anti-CD4 mAb, anti-CD8 mAb, or anti-CD122 mAb, or 200 µg of anti-NK1.1 mAb. For the in vitro depletion of CD8+CD122+ T cells, whole liver MNCs were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated anti-CD122 mAb, while the CD8+CD122+ T cells were sorted out using the Epics Altra (Coulter Pharmaceutical, Miami, FL) and the purity of the sorted cells was checked by Epics XL (Coulter Pharmaceutical).

Cytotoxicity assay

NK cell-sensitive YAC-1 lymphoma cells, EL-4 lymphoma cells, and B16 melanoma cells (both of B6 origin) were used as target cells. Target cells (3 x 106) were labeled for 60 min at 37°C with 100 µCi of Na251CrO4 in 500 µl of RPMI 1640 supplemented with 10% FCS. They were then washed three times with medium alone and subjected to a cytotoxicity assay. Labeled targets (2 x 103 cells/well) were incubated for 4 h at 37°C in 96-well round-bottom microtiter plates containing RPMI 1640 (total volume of 100 µl) with liver MNCs obtained from mice injected 24 h previously with {alpha}-GalCer (100 µg/kg). The plates were then centrifuged, and the resulting supernatants were harvested and their radioactivity was determined with a gamma counter. Cytotoxicity was calculated as the percentage of released radioactivity after correcting for spontaneous release, which was <15% of maximal release. In some experiments, liver MNCs or sorted liver CD8+ T cells were obtained from mice injected with {alpha}-GalCer 6 days before, and cells were incubated with target cells for 16 h and determine the role of CD8+ T cells in the cytotoxicity of liver MNCs.

Proliferation assay

To determine the proliferation of the MNCs (2 x 105 cells/200 µl) stimulated with {alpha}-GalCer, the cells were pulsed with 0.5 µCi/well [3H]thymidine ([3H]TdR) 12 h before the cells were harvested. The radioactivities of the harvested cells at the indicated culture time points were assessed by the liquid scintillation counting method.

Measurement of serum IFN-{gamma}

The peripheral blood of individual mice was collected at the indicated time points from the retro-orbital sinus. The serum concentration of IFN-{gamma} was measured by using a cytokine-specific ELISA kit (Endogen, Boston, MA).

B16 model of hepatic metastasis

Hepatic metastases of B16 tumor cells were produced, as previously described (22). In brief, the spleen of anesthetized mice was exposed to allow the direct i.s. injection of 3 x 106 B16 cells in 0.1 ml of medium. The spleen was then removed after clamping of the artery and vein, and the abdomen and skin were surgically sutured. Using this method, B16 tumor cells almost exclusively metastasized to the liver, but not to other organs.

Adoptive transfer of purified CD8+ T cells

Liver CD8+ T cells were positively purified by magnetic cell sorting (MACS system; Miltenyi Biotec, Bergisch Gladbach, Germany) from {alpha}-GalCer-injected mice with or without anti-CD122 mAb pretreatment. Briefly, liver MNCs were stained with magnetic bead-conjugated anti-CD8 mAb for 20 min at 4°C, and then were washed twice in the medium. Next, they were resuspended in 500 µl of the medium, and transferred onto a separation column for positive sorting, which was attached to a Midi MACS separation unit (Miltenyi Biotec). Any adherent CD8+ MNCs in the column were washed with 500 µl of the medium and collected using a plunger when the column was removed from the unit. The collected cells were resuspended in PBS, and 2 x 106 CD8+ cells (200 µl)/mouse were injected i.v. into B16-inoculated mice that had been injected with anti-CD8 Ab.

Statistical analysis

All data are expressed as the means ± SD, and differences among groups were analyzed by the Mann-Whitney U test using Stat View software. Then mouse survival rates were analyzed by the log-rank test. A p value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Depletion of CD8+ T cells or CD4+ T cells affects neither cytotoxic activity of liver NK cells nor production of IFN-{gamma} in mice injected with {alpha}-GalCer, whereas depletion of CD8+ T cells markedly decreased the proliferative response of liver MNC

We and other researchers recently reported that the antitumor cytotoxicity of liver NK cells and serum IFN-{gamma} levels remarkably increased after an {alpha}-GalCer injection (21, 30, 31). The pretreatment of mice with anti-CD4 or anti-CD8 Ab did not reduce either the cytotoxicity of the liver MNCs or serum IFN-{gamma} levels in {alpha}-GalCer-injected mice (Fig. 1A, a and b). Although an injection of either anti-CD8 Ab or anti-CD4 Ab depleted CD8+ T cells and CD4+ T cells, respectively (Fig. 1B, right panels), anti-CD4 Ab injection unexpectedly increased the proportion of NK1.1+ cells to 43.1% (Fig. 1B, left middle panel), and the proportion of NKT cells was not substantially affected (or even slightly increased) (Fig. 1B, middle central panel). CD4+ NKT cells presumably down-regulated surface expression of CD4 molecule after anti-CD4 Ab injection (Fig. 1B, left middle panel, indicated by an arrow). Although ~60% of NKT cells are CD4+, they have been shown to express CD4 only at a low level (10, 32). These findings may explain why the cytotoxic activity, IFN-{gamma} production, and proportion of NKT cells of liver MNC did not decrease by CD4 Ab injection.



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  		FIGURE 1. Effect of the depletion of either CD8+ T cells or CD4+ T cells in mice on the {alpha}-GalCer-induced effect. Aa, Mice were injected with anti-CD4 Ab or anti-CD8 Ab 2 days previously, and 18 h after {alpha}-GalCer (100 µg/kg, i.v.) injection, liver MNCs were isolated and the cytotoxicities were determined. Ab, Serum specimens were collected from mice from retro-orbital plexus at the indicated time points, and the amounts of IFN-{gamma} were determined by ELISA. Ac, Mice were injected with anti-CD4 Ab or anti-CD8 Ab 2 days previously. MNCs were isolated from the liver and spleen, and cultured with or without {alpha}-GalCer (100 ng/ml) in a 96-well round-bottom plate. The proliferations of MNCs were examined after 2-day culture. Data are from an experiment that was repeated three times with similar results. d, MNCs were isolated from the liver and stained with anti-CD8 and anti-CD122. CD8+CD122+ T cells were depleted by sorting. Sorted MNCs and whole MNC were cultured with {alpha}-GalCer (100 ng/ml) in a 96-well round-bottom plate. The proliferations were examined after 2-day culture. B, Mice were injected with anti-CD4 Ab or anti-CD8 Ab 2 days before MNC preparation. MNCs were isolated from the liver and stained with anti-CD4, anti-CD3, anti-CD8, and anti-NK1.1. An arrow shows the population of presumably CD4 down-regulated NKT cells.

 
However, the proliferation of liver MNCs of anti-CD8 Ab-pretreated mice stimulated with {alpha}-GalCer in vitro decreased significantly (Fig. 1Ac). Sorting experiments revealed that depletion of CD8+CD122+ cells from whole liver MNCs also greatly decreased the proliferation of liver MNCs stimulated with {alpha}-GalCer, while depletion of CD8+CD122– cells did not (Fig. 1Ad), thus suggesting that mainly CD8+CD122+ cells proliferated, while CD8+CD122– cells did not. Consistently, no remarkable change was seen in the proliferation of spleen MNCs from anti-CD8 Ab-pretreated mice (Fig. 1Ac) presumably because spleen MNCs contain a much smaller population of CD8+CD122+ cells than liver MNC (5).

Treatment of mice with anti-CD8 Ab decreases an antimetastatic effect induced by {alpha}-GalCer after B16 inoculation

To examine when NK cells or CD8+ T cells are crucial to reject tumors, we injected each Ab at various time points, as indicated in Fig. 2A. The pretreatment of mice with either anti-CD8 Ab or anti-NK1.1 Ab-reduced {alpha}-GalCer induced a prolongation of survival time of B16-inoculated mice. The treatment of mice with anti-CD8 Ab on day 3 also decreased the survival time (Fig. 2B), whereas the treatment of anti-NK1.1 Ab on day 3 hardly inhibit the {alpha}-GalCer-induced effect (Fig. 2C). Furthermore, the treatment of anti-CD8 Ab on day 8 slightly reduced the survival rate of the mice. These findings suggest that liver CD8+ T cells are more important antitumor effectors than NK cells in the liver beyond 3 days after {alpha}-GalCer injection.



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  		FIGURE 2. Effect of the treatment with either anti-NK1.1 Ab or anti-CD8 Ab (PBS as a control) on the {alpha}-GalCer-induced prolongation of mouse survival rate. A, The mice were i.s. inoculated with B16 cells and injected with {alpha}-GalCer (100 µg/kg, i.v.) or vehicle and received the Ab injection at the indicated time points before/after tumor inoculation. B, Survival of anti-CD8 Ab-treated mice was evaluated at the indicated days after injection of tumor cells. *, p < 0.05 vs day 3 and day 2 Ab-treated groups. C, The survival of anti-NK1.1 Ab-treated mice was evaluated at the indicated days after injection of tumor cells. All experimental mice groups in B and C included five to eight mice. *, p < 0.05 vs day 2 Ab-treated group.

 
CD8+ T cells acquired antitumor cytotoxicity against B16 cells with or without B16 inoculation

Although anti-CD8 Ab pretreatment did not decrease the cytotoxicity of liver MNCs 1 day after the {alpha}-GalCer injection (Fig. 1A), the liver MNCs prepared from anti-CD8 Ab-treated mice (3 days after {alpha}-GalCer injection) 6 days after {alpha}-GalCer injection exhibited a decreased antitumor cytotoxicity compared with that of liver MNCs prepared from mice without Ab treatment (Fig. 3A). However, because the treatment of mice with both anti-CD8 Ab and anti-NK1.1 Ab completely inhibited the cytotoxicity toward B16 (Fig. 3A) NK cells may still demonstrate a cytotoxicity toward B16 at this time point. Furthermore, isolated CD8+ T cells in the liver MNCs of mice with or without B16 inoculation at 6 days after {alpha}-GalCer injection exhibited similar up-regulated antitumor cytotoxicities toward B16 (Fig. 3B), thus suggesting that the killing activity of these CD8+ T cells may not be B16 specific.



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  		FIGURE 3. The antitumor cytotoxicity of liver CD8+ T cells. A, Normal mice were i.s. inoculated with B16 cells and then were injected with {alpha}-GalCer (100 µg/kg, i.v.) 24 h after tumor injection. Three days after {alpha}-GalCer injection, some mice were injected with anti-CD8 Ab or both CD8 Ab and NK1.1 Ab. Six days after {alpha}-GalCer injection, liver MNCs were isolated and incubated with 51Cr-labeled B16 cells for 16 h, and cytotoxic activities were determined. The data are the means ± SD of values. B, Mice were i.s. inoculated with B16 cells and then were injected with {alpha}-GalCer (100 µg/kg, i.v.) or vehicle 24 h after tumor injection. At the same time, normal mice were injected with {alpha}-GalCer (100 µg/kg, i.v.) or vehicle. Six days after {alpha}-GalCer injection, liver MNCs were isolated from the mice and CD8+ T cells were purified by the magnet sorting. The purities of sorted cells were >95%, and the cytotoxic activity of sorted liver CD8+ T cells was determined. The data are the means ± SD of values from five mice. All data are from experiments that were repeated three times with similar results.

 
Time course analysis of the activation of CD8+ T cells

The cytotoxicity of the liver CD8+ T cells prepared from {alpha}-GalCer-injected mice (without B16 inoculation) gradually increased and exhibited a maximum cytotoxicity toward B16 as well as YAC-1 cells at 6 days after {alpha}-GalCer injection (Fig. 4A). In addition, the population of liver CD8+ T cells increased (Fig. 4B) and continuously maintained an activating profile like IL-2R{beta} (CD122) and CD69 expression after {alpha}-GalCer injection (Fig. 4C). CD122+CD8+ T cells at every time point showed lower TCR/CD3 intensities than those of CD122–CD8+ T cells (data not shown).



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  		FIGURE 4. The time course analysis of the activation of liver CD8+ T cells after {alpha}-GalCer injection. A, Cytotoxic activities of liver CD8+ T cells against B16 cells at the indicated time points after {alpha}-GalCer injection. Normal mice were injected with {alpha}-GalCer (100 µg/kg, i.v.) or vehicle. At the indicated time points after {alpha}-GalCer injection, liver MNCs were isolated and the liver CD8+ T cells were sorted. The purities of the sorted cells were >95%, and the cytotoxic activity of sorted liver CD8+ T cells was determined. B, The percentage of CD8+ T cells in the liver MNCs. Normal mice were injected with {alpha}-GalCer (100 µg/kg, i.v.) or vehicle. At the indicated time points after {alpha}-GalCer injection, liver MNCs were isolated and stained with anti-CD3 and anti-CD8. C, CD122 and CD69 expressions of liver CD8+ T cells were examined at indicated time points after {alpha}-GalCer injection. The data are from experiments that were repeated three times with similar results.

 
Depletion of CD122+CD8+ T cells reduced the antitumor cytotoxicity of liver CD8+ T cells

The administration of anti-CD122 Ab has been reported to deplete CD122+ cells, such as NK, NKT, and CD122+CD8+ T cells (5, 33). To further confirm such depletion, we analyzed the expression of CD44 because most of CD122+CD8+ T cells are also known to have a high expression of CD44 (34, 35). CD44high cells in liver CD8+ T cells of {alpha}-GalCer- or vehicle-injected mice (without B16 inoculation) remarkably decreased by the subsequent injection of anti-CD122 Ab (Fig. 5A). Liver CD8+ T cells prepared from mice at 6 days after {alpha}-GalCer injection (without B16 inoculation) exhibited the antitumor cytotoxicity against either B16 or EL-4 cells; however, their cytotoxicity was suppressed by the depletion of the CD122+ fraction by anti-CD122 Ab (Fig. 5B). Hence, CD122+CD8+ T cells, but not CD122–CD8+ T cells, prepared from {alpha}-GalCer-injected mice 6 days previously exhibited cytotoxicity in an Ag-independent bystander-activated manner.



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  		FIGURE 5. Effect of the injection of anti-CD122 Ab {alpha}-GalCer on the CD44 expression (A) and cytotoxic activity of liver MNCs (B). Normal mice were injected with {alpha}-GalCer or vehicle, and then anti-CD122 Ab was injected 3 days after {alpha}-GalCer injection. After an additional 3 days, liver MNCs were isolated from the mice and the liver CD8+ T cells were sorted. The purities of sorted cells were >95%; A, FACS analysis of sorted liver CD8+ T cells were conducted. B, The cytotoxicity of liver CD8+ T cells was determined in vitro with B16 cells (left panel) or EL-4 cells (right panel). The data are from experiments that were repeated three times with similar results.

 
Anti-CD122 Ab treatment inhibited {alpha}-GalCer-induced antimetastatic effect, and adoptive transfer of {alpha}-GalCer-activated CD8+ T cells without CD122+ fraction did not exhibit an antimetastatic effect

The depletion of NK and NKT cells with anti-NK1.1 Ab 3 days after {alpha}-GalCer injection (4 days after B16 injection) did not affect the survival time of mice inoculated with B16 (Figs. 2B and 6A). However, the survival time of B16-inoculated mice greatly reduced either by anti-CD122 Ab or anti-CD8 Ab injection at 3 days after {alpha}-GalCer injection (Fig. 6A). As a result, although NK cells may have cytotoxicity against B16 cells, as suggested by the results in Fig. 3A, CD122+CD8+ T cells rather than NK cells contributed to the inhibition of B16 metastases beyond 3 days after {alpha}-GalCer injection. Furthermore, to confirm the bystander antimetastatic activity of CD122+CD8+ T cells, we conducted the adoptive transfer of {alpha}-GalCer-activated CD8+ T cells with or without CD122+CD8+ T fraction (Fig. 6B). The adoptive transfer of CD8+ T cells of donor type 1 mice that were treated with {alpha}-GalCer (6 days before) (without B16 inoculation) rescued B16-inoculated recipient mice that were already pretreated with anti-CD8 Ab and {alpha}-GalCer. However, the adoptive transfer of CD8+ T cells prepared from donor type 2 mice that were depleted of the CD122+ fraction only slightly prolonged the survival time of B16-inoculated mice (Fig. 6C). These findings revealed that the transferred CD122+CD8+ T cells activated by {alpha}-GalCer contributed to the rejection of B16 cells without the prior sensitization by B16 cells (bystander antimetastatic activity), and that CD122–CD8+ T cells could not transfer the antimetastatic effect. These findings also suggest that CD122–CD8+ T cells could not substitute CD122+CD8+ T cells, and they are therefore essentially different populations.



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  		FIGURE 6. A, Effect of the treatment with various Abs on the survival rate of mice inoculated with B16 cells. Three days after {alpha}-GalCer injection (4 days after B16 inoculation), B6 mice were injected (i.p.) with anti-NK1.1 Ab, anti-CD8 Ab, or anti-CD122 Ab (200 µg) (PBS as a control). B, Schedule of the adoptive transfer of CD8+ T cells with/without CD122+ fraction. Recipient mice were treated with anti-CD8 Ab before B16 and {alpha}-GalCer injection at the indicated time point. Donors were normal mice that were not injected with B16 cells. CD8+ T cells prepared from donor type 1 mice contained CD122+ fraction, while CD8+ T cells prepared from donor type 2 mice lacked CD122+ fraction (indicated by the flow cytometric profiles). These CD8+ T cells were adoptively transferred to recipient mice twice at indicated time points (day 1 (3 h after {alpha}-GalCer injection) and day 5). C, Effect of adoptive transfer of {alpha}-GalCer-activated CD8+ T cells with (donor type 1) or without CD122+ fraction (donor type 2). Survival was evaluated at the indicated time points after B16 inoculation. All experimental mice groups in A and C included five to eight mice. *$, p < 0.05 vs group transferred from donor type 2 mice.

 
Tumor-specific CD8+CTL exhibited their function 14 days after tumor inoculation and {alpha}-GalCer injection

We previously showed that {alpha}-GalCer injection induced tumor-specific memory immunity (22, 23). B16 i.s. inoculated mice could reject liver-metastasized B16 cells by {alpha}-GalCer treatment, and these survived mice could also reject s.c. rechallenged B16 cells, but could not reject s.c. challenged EL-4 cells (36). Mice that survived i.s. inoculated with B16 cells were selected and pretreated with various Abs and were thereafter s.c. inoculated with B16. The results showed that the tumor-specific memory immunity was largely due to the CD8+ T cells, but the NK1.1+ cells (NK and NKT cells) contributed very little (Fig. 7A). To confirm when memory CTL generate and to reject specific tumor cells, we s.c. inoculated B16 cells to mice at various time points that were already i.s. inoculated with B16 cells and injected with {alpha}-GalCer (Fig. 7B). The {alpha}-GalCer injection did not cause a regression of the s.c. inoculated B16 cells early after first B16 i.s. inoculation (days 1–7), while the most mice could reject s.c. inoculated B16 cells 14 days after first B16 i.s. inoculation (Fig. 7C). However, all mice (n = 5) that had received the i.s. B16 injection 14 days before could not reject the s.c. inoculated EL-4 cells (data not shown). These results suggest that it takes 14 days for tumor-specific CD8+CTLs to be generated, and that the CD122+CD8+ T cells that expanded within 1 wk after {alpha}-GalCer injection in the liver are not tumor-specific CTLs.



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  		FIGURE 7. A, Effects of various Ab treatment on the survival of mice s.c. rechallenged with B16 tumor cells. Mice that had already rejected i.s. inoculated B16 cells were s.c. rechallenged with B16 cells. Various Ab were treated weekly from 2 days before tumor rechallenge, and survival was evaluated at the indicated times after B16 inoculation. All experimental mice groups included five to eight mice. *, p < 0.05 vs anti-CD8 Ab-treated group. B, The schedule of s.c. inoculation of B16 cells to mice that had received an i.s. inoculation of B16 and {alpha}-GalCer treatment. The B16 (i.s.)- and {alpha}-GalCer-injected mice were s.c. rechallenged with B16 cells at the indicated days after first performing the i.s. inoculation of B16 cells (day 0). C, The growth of s.c. rechallenged B16 cells in mice. The mice were s.c. rechallenged with B16 cells, as shown in B. The control group (without previous i.s. B16 inoculation) included six mice, while the other groups included five mice each.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The depletion of NK and NKT cells 2–3 days after {alpha}-GalCer injection no longer inhibited antimetastatic effect of {alpha}-GalCer against B16 tumor cells in the liver, whereas the depletion of CD8+ T cells at this time point significantly reduced the {alpha}-GalCer-induced antimetastatic effect and mouse survival time. CD8+CD122+ T cells were found to be critical for the proliferation of liver MNCs after {alpha}-GalCer injection. Adoptive transfer experiments also showed CD8+CD122+ T cells, but not CD8+CD122– T cells, to be responsible for the antitumor function in the liver, suggesting that CD8+CD122+ T cells and CD8+CD122– T cells are essentially different populations. These results suggest that although NK cells in the liver are important antitumor effectors in the early phase after {alpha}-GalCer injection, CD8+CD122+ T cells thereafter are more critical antitumor effectors. However, these CD8+CD122+ T cells expanded within 1 wk appeared to be distinct from tumor-specific memory CTLs and are bystander CTLs. It took 14 days for {alpha}-GalCer to generate/differentiate tumor-specific memory CD8+ T cells.

Although we recently showed that liver NK cells proportionally increased until 5 days after {alpha}-GalCer injection (21), the present study revealed that NK cells no longer play an essential role in the antimetastatic effect in the liver as early as 2–3 days after {alpha}-GalCer injection, even though they still have antitumor cytotoxicity. The antimetastatic role in the liver MNCs was taken from NK cells by CD122+CD8+ T cells at this time point. As a result, coordination of antimetastatic function of the liver MNCs rapidly occurs among NKT, NK, and CD122+CD8+ T cells.

Antitumor cytotoxicity of CD122+CD8+ T cells expanded within 1 wk after B16 and {alpha}-GalCer injection was not B16 specific because their cytotoxicity was also exhibited toward EL-4 cells, and their cytotoxicity against B16 cells could not be blocked by the treatment of B16 cells with anti-H-2D and anti-H-2K Ab (data not shown). In addition, the adoptive transfer of CD8+ T cells from mice injected with {alpha}-GalCer 6 days before (but without B16 injection) similarly inhibited liver metastasis of B16 cells and prolonged the mouse survival time. Moreover, the mice that received the i.s. B16 tumor injection and {alpha}-GalCer treatment could not reject rechallenged s.c. inoculated B16 tumors until 10–14 days after the first i.s. B16 injection. The CD122+CD8+ T cells that thus expanded within 1 wk by {alpha}-GalCer stimulation are not likely B16-specific CD8+ T cells that can inhibit s.c. rechallenged B16 tumors. It is generally accepted that it takes 2 wk after an Ag challenge for the development of either Ag-specific Ab or Ag-specific CTL, and their manner of recognition by Ab or by TCR is known to be highly restricted. From the view of kinetics and the recognition of targets, CD122+CD8+ T cells induced by {alpha}-GalCer injection within 1 wk are thus different from tumor-specific memory CTLs and are bystander CTLs.

{alpha}-GalCer injection was recently reported to induce the expansion of CD122+CD8+ T cells, and this phenomenon is called bystander proliferation because of their Ag independency (24). Viral infections and IFN-{alpha}{beta} (25) or IL-15 (34) also reportedly increase these bystander CD122+CD8+ T cells, and these cells have been called memory phenotype CD8+ T cells as distinguished from memory CD8+ T cells (34, 37, 38). LPS (26) and poly(I:C), both of which are activators of NK cells, also reportedly induce the proliferation of CD122+CD8+ T cells in vivo in mice (34). We previously reported that CD122+CD8+ T cells secreted a much larger amount of IFN-{gamma} and acquired a more potent antitumor cytotoxicity by anti-CD3 stimulation than did CD122–CD8+ T cells (39). CD122+CD8+ T cells were abundant in the liver, and {alpha}-GalCer injection further expanded these cells. As a result, bystander CD122+CD8+ T cells may be important as a Th1 effector population in both antitumor and anti-infection immunity and may create a bridge from innate immunity to adaptive immunity.

It is unlikely that conventional CD122–CD8+ T cells are activated by {alpha}-GalCer and became CD122+CD8+ T cells because depletion of CD122+CD8+ T cells from liver MNCs greatly cause a reduction in the proliferation of liver MNCs. In addition, the depletion of CD8+ T cells did not significantly reduce {alpha}-GalCer-induced proliferation of the spleen MNCs in which CD122–CD8+ T cells are predominant and there were fewer CD122+CD8+ T cells than in the liver MNCs (3, 5). Furthermore, the finding that CD122–CD8+ T cells could not inhibit tumor metastasis, as evidenced by adoptive transfer experiments, supports that activating cells are CD122+CD8+ T cells, but not CD122–CD8+ T cells, and these cells are essentially different populations.

It has been proposed that CD122+CD8+ T cells may develop independently of the thymus in the liver and also other sites because these cells increase age dependently in parallel with the thymus involution in normal mice as well as in athymic nude mice (5, 40, 41, 42, 43). In addition, CD122+CD8+ T cells emerged early in the livers of mice before thymus reconstitution in radiation bone marrow chimera mice (44). The report that CD122+CD8+ T cells were positively selected by H-Y Ag outside of the thymus in H-Y Ag transgenic mice suggests that some CD122+CD8+ T cells are dependent on autologous Ags for their development and expansion (40, 41) and may be autoreactive. These findings together with present results suggest that thymus-independent T cells are included in bystander CD122+CD8+ T cells and expand against infections and tumors.

Finally, it should be noted that although ligand-activated NKT cells induce severe hepatocyte damage in aged mice through Fas/Fas-ligand system (21), we have recently found that anti-TNF-{alpha} Ab pretreatment of mice completely inhibited liver injury without attenuating antitumor and antimetastatic effect of {alpha}-GalCer. 3

In conclusion, {alpha}-GalCer induces the activation of bystander CD122+CD8+ T cells following the activation of NKT and NK cells, and finally, tumor-specific memory CD8+ T cells develop ~14 days after tumor/{alpha}-GalCer injection.


   Acknowledgments
 
We thank Hitomi Tomura for her help in experiments.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Shuhji Seki, Department of Microbiology, National Defense Medical College, Tokorozawa 359-8513, Japan. E-mail address: btraums{at}res.ndmc.ac.jp Back

2 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galactosylceramide; i.s., intrasplenic; MNC, mononuclear cell. Back

3 T. Inui, H. Nakashima, Y. Habu, R. Nakagawa, M. Fukasawa, M. Kinoshita, and S. Seki. Neutralization of TNF{alpha} abrogates hepatic failure induced by {alpha}-galactosylceramide without attenuating its antitumor effect in aged mice. Submitted for publication. Back

Received for publication October 20, 2003. Accepted for publication March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ohteki, T., H. R. MacDonald. 1994. Major histocompatibility complex class I related molecules control the development of CD4+8– and CD4–8– subsets of natural killer 1.1+ T cell receptor-{alpha}/{beta}+ cells in the liver of mice. J. Exp. Med. 180:699.[Abstract/Free Full Text]
  2. Hashimoto, W., K. Takeda, R. Anzai, K. Ogasawara, H. Sakihara, K. Sugiura, S. Seki, K. Kumagai. 1995. Cytotoxic NK1.1 Ag+ {alpha}{beta} T cells with intermediate TCR induced in the liver of mice by IL-12. J. Immunol. 154:4333.[Abstract]
  3. Watanabe, H., C. Miyaji, Y. Kawachi, T. Iiai, K. Ohtsuka, T. Iwanage, H. Takahashi-Iwanaga, T. Abo. 1995. Relationships between intermediate TCR cells and NK1.1+ T cells in various immune organs: NK1.1+ T cells are present within a population of intermediate TCR cells. J. Immunol. 155:2972.[Abstract]
  4. Seki, S., Y. Habu, T. Kawamura, K. Takeda, H. Dobashi, T. Ohkawa, H. Hiraide. 2000. The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol. Rev. 174:35.[Medline]
  5. Tsukahara, A., S. Seki, T. Iiai, T. Moroda, H. Watanabe, S. Suzuki, T. Tada, H. Hiraide, K. Hatakeyama, T. Abo. 1997. Mouse liver T cells: their change with aging and in comparison with peripheral T cells. Hepatology 26:301.[Medline]
  6. Makino, Y., H. Koseki, Y. Adachi, T. Akasaka, K. Tsuchida, M. Taniguchi. 1994. Extrathymic differentiation of a T cell bearing invariant V{alpha}14J{alpha}281 TCR. Int. Rev. Immunol. 11:31.[Medline]
  7. Lantz, O., 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.[Abstract/Free Full Text]
  8. Bendelac, A.. 1995. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J. Exp. Med. 182:2091.[Abstract/Free Full Text]
  9. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, et al 1997. CD1d-restricted and TCR-mediated activation of V{alpha}14 NKT cells by glycosylceramides. Science 278:1626.[Abstract/Free Full Text]
  10. Takeda, K., S. Seki, K. Ogasawara, R. Anzai, W. Hashimoto, K. Sugiura, M. Takahashi, M. Satoh, K. Kumagai. 1996. Liver NK1.1+ CD4+ {alpha}{beta} T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. J. Immunol. 156:3366.[Abstract]
  11. Seki, S., W. Hashimoto, K. Ogasawara, M. Satoh, H. Watanabe, Y. Habu, H. Hiraide, K. Takeda. 1997. Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice. Immunology 92:561.[Medline]
  12. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, M. Taniguchi. 1997. Requirement for V{alpha}14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623.[Abstract/Free Full Text]
  13. Kawamura, T., K. Takeda, S. K. Mendiratta, H. Kawamura, L. Van Kaer, H. Yagita, T. Abo, K. Okumura. 1998. Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J. Immunol. 160:16.[Abstract/Free Full Text]
  14. Ogasawara, K., K. Takeda, W. Hashimoto, M. Satoh, R. Okuyama, N. Yanai, M. Obinata, K. Kumagai, H. Takada, H. Hiraide, S. Seki. 1998. Involvement of NK1+ T cells and their IFN-{gamma} production in the generalized Shwartzman reaction. J. Immunol. 160:3522.[Abstract/Free Full Text]
  15. Osman, Y., T. Kawamura, T. Naito, K. Takeda, L. Van Kaer, K. Okumura, T. Abo. 2000. Activation of hepatic NKT cells and subsequent liver injury following administration of {alpha}-galactosylceramide. Eur. J. Immunol. 30:1919.[Medline]
  16. Leite-de-Moraes, M. C., A. Herbelin, C. Gouarin, Y. Koezuka, E. Schneider, M. Dy. 2000. Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes. J. Immunol. 165:4367.[Abstract/Free Full Text]
  17. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C. R. Wang, Y. Koezuka, M. Kronenberg. 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]
  18. Crowe, N. Y., A. P. Uldrich, K. Kyparissoudis, K. J. Hammond, Y. Hayakawa, S. Sidobre, R. Keating, M. Kronenberg, M. J. Smyth, D. I. Godfrey. 2003. Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells. J. Immunol. 171:4020.[Abstract/Free Full Text]
  19. Wilson, M. T., C. Johansson, D. Olivares-Villagomez, A. K. Singh, A. K. Stanic, C. R. Wang, S. Joyce, M. J. Wick, L. Van Kaer. 2003. The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion. Proc. Natl. Acad. Sci. USA 100:10913.[Abstract/Free Full Text]
  20. Inui, T., R. Nakagawa, S. Ohkura, Y. Habu, Y. Koike, K. Motoki, N. Kuranaga, M. Fukasawa, N. Shinomiya, S. Seki. 2002. Age-associated augmentation of the synthetic ligand-mediated function of mouse NK1.1 Ag+ T cells: their cytokine production and hepatotoxicity in vivo and in vitro. J. Immunol. 169:6127.[Abstract/Free Full Text]
  21. Nakagawa, R., I. Nagafune, Y. Tazunoki, H. Ehara, H. Tomura, R. Iijima, K. Motoki, M. Kamishohara, S. Seki. 2001. Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by {alpha}-galactosylceramide in mice. J. Immunol. 166:6578.[Abstract/Free Full Text]
  22. Nakagawa, R., K. Motoki, H. Nakamura, H. Ueno, R. Iijima, A. Yamauchi, S. Tsuyuki, T. Inamoto, Y. Koezuka. 1998. Antitumor activity of {alpha}-galactosylceramide, KRN7000, in mice with EL-4 hepatic metastasis and its cytokine production. Oncol. Res. 10:561.[Medline]
  23. Nakagawa, R., K. Motoki, H. Ueno, R. Iijima, H. Nakamura, E. Kobayashi, A. Shimosaka, Y. Koezuka. 1998. Treatment of hepatic metastasis of the colon26 adenocarcinoma with an {alpha}-galactosylceramide, KRN7000. Cancer Res. 58:1202.[Abstract/Free Full Text]
  24. Eberl, G., P. Brawand, H. R. MacDonald. 2000. Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation. J. Immunol. 165:4305.[Abstract/Free Full Text]
  25. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947.[Abstract]
  26. Tough, D. F., S. Sun, J. Sprent. 1997. T cell stimulation in vivo by lipopolysaccharide (LPS). J. Exp. Med. 185:2089.[Abstract/Free Full Text]
  27. Sprent, J., X. Zhang, S. Sun, D. Tough. 2000. T-cell proliferation in vivo and the role of cytokines. Philos. Trans. R. Soc. London B Biol. Sci. 355:317.[Medline]
  28. Sprent, J.. 2003. Turnover of memory-phenotype CD8+ T cells. Microbes Infect. 5:227.[Medline]
  29. Kobayashi, E., K. Motoki, T. Uchida, H. Fukushima, Y. Koezuka. 1995. KRN7000, a novel immunomodulator, and its antitumor activities. Oncol. Res. 7:529.[Medline]
  30. Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, A. Bendelac. 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]
  31. Eberl, G., H. R. MacDonald. 2000. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol. 30:985.[Medline]
  32. Seki, S., D. H. Kono, R. S. Balderas, A. N. Theofilopoulos. 1994. V{beta} repertoire of murine hepatic T cells: implication for selection of double negative {alpha}{beta}+ T cells. J. Immunol. 153:637.[Abstract]
  33. Toyabe, S., S. Seki, T. Iiai, K. Takeda, K. Shirai, H. Watanabe, H. Hiraide, M. Uchiyama, T. Abo. 1997. Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice. J. Immunol. 159:1537.[Abstract]
  34. Zhang, X., S. Sun, I. Hwang, D. F. Tough, J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8:591.[Medline]
  35. Ohteki, T., R. Okuyama, S. Seki, T. Abo, K. Sugiura, A. Kusumi, T. Ohmori, H. Watanabe, K. Kumagai. 1992. Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice. J. Immunol. 149:1562.[Abstract]
  36. Nakagawa, R., I. Serizawa, K. Motoki, M. Sato, H. Ueno, R. Iijima, H. Nakamura, A. Shimosaka, Y. Koezuka. 2000. Antitumor activity of {alpha}-galactosylceramide, KRN7000, in mice with the melanoma B16 hepatic metastasis and immunohistological study of tumor infiltrating cells. Oncol. Res. 12:51.[Medline]
  37. Nishimura, H., T. Yajima, Y. Naiki, H. Tsunobuchi, M. Umemura, K. Itano, T. Matsuguchi, M. Suzuki, P. S. Ohashi, Y. Yoshikai. 2000. Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo. J. Exp. Med. 191:157.[Abstract/Free Full Text]
  38. Judge, A. D., X. Zhang, H. Fujii, C. D. Surh, J. Sprent. 2002. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8+ T cells. J. Exp. Med. 196:935.[Abstract/Free Full Text]
  39. Takayama, E., S. Seki, T. Ohkawa, K. Ami, Y. Habu, T. Yamaguchi, T. Tadakuma, H. Hiraide. 2000. Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-{gamma} production. J. Immunol. 164:5652.[Abstract/Free Full Text]
  40. Yamada, H., T. Ninomiya, A. Hashimoto, K. Tamada, H. Takimoto, K. Nomoto. 1998. Positive selection of extrathymically developed T cells by self-antigens. J. Exp. Med. 188:779.[Abstract/Free Full Text]
  41. Yamada, H., G. Matsuzaki, Q. Chen, Y. Iwamoto, K. Nomoto. 2001. Reevaluation of the origin of CD44high "memory phenotype" CD8 T cells: comparison between memory CD8 T cells and thymus-independent CD8 T cells. Eur. J. Immunol. 31:1917.[Medline]
  42. Terra, R., N. Labrecque, C. Perreault. 2002. Thymic and extrathymic T cell development pathways follow different rules. J. Immunol. 169:684.[Abstract/Free Full Text]
  43. Clegg, C. H., J. T. Rulffes, P. M. Wallace, H. S. Haugen. 1996. Regulation of an extrathymic T-cell development pathway by oncostatin M. Nature 384:261.[Medline]
  44. Watanabe, H., C. Miyaji, S. Seki, T. Abo. 1996. c-kit+ stem cells and thymocyte precursors in the livers of adult mice. J. Exp. Med. 184:687.[Abstract/Free Full Text]



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