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International Immunology Advance Access originally published online on August 16, 2006
International Immunology 2006 18(9):1397-1404; doi:10.1093/intimm/dxl073
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8+ T cells in a manner dependent on IL-4

Naoko Ueda1, Hiroko Kuki1, Daisuke Kamimura1,2, Shinichiro Sawa1, Kenichiro Seino3, Takuya Tashiro3, Ken-ichi Fushuku3, Masaru Taniguchi3, Toshio Hirano1,2 and Masaaki Murakami1

1 Laboratory of Developmental Immunology, Graduate School of Medicine and Graduate School of Frontier Biosciences, Osaka University, 565-0871 Osaka, Japan
2 Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology (RCAI), 230-0045 Yokohama, Japan
3 Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology (RCAI), 230-0045 Yokohama, Japan

Correspondence to: M. Murakami; E-mail: murakami{at}molonc.med.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide ({alpha}-GalCer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that {alpha}-GalCer enhanced homeostatic proliferation of CD8+ but not CD4+ T cells and this effect of {alpha}-GalCer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8+ T cell homeostatic proliferation. Importantly, the expression of IL-4R{alpha} and STAT6 in CD8+ T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8+ T cells. Consistent with this, the level of IL-4R{alpha} expression on memory phenotype CD8+ T cells was higher than that on naive phenotype one and CD4+ T cells. Thus, these results showed the ‘involvement’ of IL-4 that is produced from activated NKT cells for CD8+ T cell homeostatic proliferation in vivo.

Keywords: NKT cells, homeostatic proliferation, CD8+ T cells, IL-4


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
NKT cell is a unique subset of T lymphocytes having TCRs and NK cell markers and functional properties shared with conventional T cells and NK cells (1–4). NKT cells have restricted TCR expression (V{alpha}14-J{alpha}18/Vß8.2 in mice and V{alpha}24-J{alpha}18/Vß11 in humans) and recognize glycolipid antigens such as endogenous isoglobotrihexosylceramide and exogenous alpha-galactosylceramide ({alpha}-GalCer) presented by the MHC class I-like protein CD1d (5, 6). The most remarkable property of NKT cells is their capacity to rapidly produce large amounts of various kinds of cytokines, including IFN{gamma} and IL-4, in response to their TCR engagement (1). These cytokines from NKT cells are known to be important for suppressing autoimmunity and graft rejection, enabling resistance to infection and promoting tumor immunity through regulating other types of cells including conventional T cells (7). It was also reported that activation of NKT cells induced selective bystander proliferation of memory phenotype but not naive phenotype CD4+ and CD8+ T cells (8). However, a role of NKT cells and cytokines produced by NKT cells for homeostatic proliferation of T cells has not yet been investigated.

Recovery of normal T cell numbers in a lymphopenic condition involves the proliferation of naive T cells (9). It is known that T cell lymphopenic situations physiologically occurred at neonatal period when T cells firstly move from the thymus to periphery, at elder period when thymus function reduced followed by the decrease of T cell input in periphery or when some infectious agents, in particular viruses, that reduced T cell numbers in periphery (10–12). It is also reported that slower proliferation is occurred for a whole period of life in a body to maintain memory T cells (13, 14). This process, called homeostatic proliferation, requires TCR recognition of self-peptide/MHC ligands (15, 16). Much evidence has been accumulated that cytokines play a critical role in driving homeostatic proliferation (16, 17). It was reported that IL-7, IL-12 and IL-15 play a critical role for CD8+ T cell homeostatic proliferation (18, 19), while IL-7 and thymic stromal lymphopoietin are important for that of CD4+ T cells (20, 21). These results suggested that IL-2 receptor family cytokines play a critical role for T cell homeostatic proliferation. However, role of IL-4 on homeostatic proliferation of T cells has not completely been evaluated.

IL-4 plays a major role in the induction of certain protective CD8+ T cell responses. It was reported that IL-4 was necessary to activate CD8+ T cells in vitro (22) and to establish in vivo anti-tumor protective immunity mediated through activation of CD8+ T cells (23). Recently, it was reported that IL-4 directly influences CD8+ T cell function against malaria parasites (24). All these data suggested that IL-4 plays a role for homeostasis of CD8+ T cells in vivo. However, a source of IL-4 for activation of CD8+ T cell and a role of induced IL-4 for T cell homeostatic proliferation have not been identified, although a basal level of IL-4 did not affect T cell homeostatic proliferation (18).

Here we showed that {alpha}-GalCer-mediated activation of NKT cells enhanced CD8+ T cell but not CD4+ T cell homeostatic proliferation. {alpha}-GalCer-mediated enhancement of CD8+ T cell homeostatic proliferation was dependent on IL-4 produced by NKT cells. Using several knockout (KO) mice and in vivo over-expression of cytokines, we showed that IL-4 directly acts on CD8+ T cells through IL-4R{alpha}-STAT6 signaling to induce their homeostatic proliferation. Thus, these results showed that IL-4 from activated NKT cells plays a role for CD8+ T cell homeostatic proliferation in vivo.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
C57BL/6 mice and Balb/c mice were obtained from Japan SLC, Inc. C57BL/6 background CD1dKO, TNF{alpha}KO, IL-4KO, IL-10KO, IL-12KO, IL-15KO, C57BL/6/SJL (CD45.1+) and C57BL/6/PL (CD90.1+) mice were purchased from Jackson Laboratory. C57BL/6 background J{alpha}18KO was established by us (25). Balb/C background IL-4R{alpha}KO and STAT6KO mice were provided by Kubo (RIKEN, Research Center for Allergy and Immunology). C57BL/6 background IL-6KO and IFN{gamma}KO mice were obtained from Iwakura (University of Tokyo, Japan). All mice were maintained under SPF conditions according to the instructions of Osaka University Medical School.

Antibody and reagents
Antibodies against FITC-conjugated anti-mouse CD19, NK1.1, MHC class II, PE-conjugated anti-mouse CD4, CD5, CD8, Cychrome-conjugated anti-mouse CD4, CD8, CD44, streptavidin, allophycoerythrin (APC)-conjugated anti-mouse CD4, CD45.1, CD90.1, streptavidin and biotin-conjugated TCRß, CD19, CD45.2 were purchased from e-Bioscience. FITC-conjugated anti-mouse I-Ab and H-2Kb/H-2Dd were purchased from Biolegend. PE-conjugated anti-mouse IL-4R{alpha} (CD124), APC-conjugated anti-mouse CD11c, anti-IL-12 and purified anti-mouse CD3{varepsilon} were from BD PharMingen (Tokyo, Japan). Cytometric bead array kits (mouse Th1/2 cytokine kit and inflammation kit) were purchased from BD (Tokyo, Japan). Anti-IL-7 was provided by Marrack (Howard Hughes Medical Institute, National Jewish Medical and Research Center). ELISA kit for mouse IL-13 and IL-4 was obtained from R&D. Recombinant mouse IL-4 and IL-13 were purchased from Peprotech. {alpha}-GalCer was mainly synthesized in our laboratory and some was provided by KIRIN. Both {alpha}-GalCer from our laboratory and KIRIN had the same activity (data not shown).

Cell preparation and cell sorting
The lymph nodes (inguinal, axillaries, cervical and mesenteric) and spleen from C57BL/6/SJL or C57BL/6/PL mice were harvested, passed through a 100-µm cell strainer (BD Falcon, Tokyo, Japan) with RPMI-1640 and washed. Erythrocytes were eliminated with 0.165 M NH4Cl. A T cell-enriched sample was then prepared using a nylon wool column, and the CD4+CD44low and CD8+CD44low T cells were purified by a Moflo high-performance cell sorter (DakoCytomation, Kyoto, Japan). For NKT cell sorting, a MHC class II negative fraction was purified by a magnetic sorting system (BD) and CD5+NK1.1+ cells were sorted by Moflo. The purities of the T cells and NKT cells were routinely >98%.

Homeostatic proliferation assay
The purified T cell populations were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen Life Technologies, Grand Island, NY, USA) by a standard method (26). The 5-Gray-irradiated syngeneic Thy-1.2 mice were given the CFSE-labeled donor T cells [1–2 x 106 cells per mouse, intravenously (i.v.)] with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. Seven or eight days after transfer, the recipient mice were sacrificed, and the lymph nodes and spleen were harvested, passed through a 100-µm cell strainer (BD Falcon) with RPMI-1640 and washed. Erythrocytes were eliminated with 0.165 M NH4Cl. A T cell-enriched sample was then prepared using a nylon wool column. CFSE levels and CD8, CD45.1 (or CD90.1) levels of the T cell fraction were examined by FACSCalibur (BD) or Cyan (DakoCytomation). The division of donor T cells was detected by the dilution of CFSE.

RNA extraction and reverse transcription–PCR
Total RNA of CD44lowCD8+, CD44highCD8+, CD44lowCD4+ or CD44highCD4+ T cells was extracted by using RNeasy kit (Qiagen, Germany). Reverse transcriptase reaction was performed using Rever Tra Ace (TOYOBO). The following primers were used to determine the level of specific mRNA: IL-13R{alpha}1, sense, 5'-gcacgataatatggacgtgg-3' and anti-sense, 5'-ttgagcacttttctccaggc-3' and IL-13R{alpha}2, sense, 5'-atggcttttgtgcatatcagatgct-3' and anti-sense, 5'-gacaaatgcgtacgtatctt-3'. DNA was amplified by a Gene Amp PCR system 9700 under following conditions: IL-13R{alpha}1, 30 cycles of 1 min at 95°C, 30 s at 62°C and 1 min at 72°C; IL-13R{alpha}2, 30 cycles of 1 min at 95°C, 30 s at 54°C and 1 min at 72°C.

Preparation and injection of IL-4 and IL-13 plasmid DNAs
The full-length cDNA for mouse IL-4 and mouse IL-13 was obtained by PCR using cDNA from splenocytes of the C57BL/6 mice 2 h after 2 µg of {alpha}-GalCer treatment. The cDNAs were cloned into an expression vector: the pCAGGS vector (27). The controls were the pCAGGS vectors without insert DNA. These plasmid DNAs were purified with the EndoFree plasmid Mega kit (Qiagen). For IL-4 or IL-13 protein expression in vivo, mice were given a bolus injection of 1 ml lactate Ringer's solution containing the IL-4 or IL-13 plasmid DNA. The serum level of IL-4 and IL-13 were detected by a sandwich ELISA systems (BD and R&D).

In vitro proliferation assay
CD44lowCD8+, CD44highCD8+, CD44lowCD4+ or CD44highCD4+ T cells from C57BL6 mice were sorted and cultured with or without IL-4 or IL-13 in the presence or absence of anti-CD3 mAb (BD). Anti-CD3 mAb (1 µg ml–1) was coated at 37°C for 3 h and the resulted plates were washed with PBS twice. The resulted cells were plated in anti-CD3-coated 96-well plates at a concentration of 5 x 105 cells per well in RPMI-1640 medium supplemented with 10% FCS and added mouse recombinant IL-4 or IL-13 in some wells. Two days after, 20 µl of 5 mg ml–1 MTT in PBS were added to each well. After 4 h at 37°C, cell proliferation was measured by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

Statistical analysis
All the data were statistically analyzed with a student's t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
{alpha}-GalCer-mediated activation of CD1d-restricted NKT cells enhanced CD8+ but not CD4+ T cell homeostatic proliferation
To investigate whether activation of CD1d-restricted NKT cells regulates homeostatic proliferation of T cells, we treated mice with {alpha}-GalCer and analyzed homeostatic proliferation of CD4+ and CD8+ T cells in irradiated hosts. {alpha}-GalCer treatment significantly enhanced homeostatic proliferation of CD8+ T cells but rather suppressive for CD4+ T cells (Fig. 1A). We next confirmed that CD1d-restricted NKT cells are critical for the enhancement of CD8+ T cell homeostatic proliferation after {alpha}-GalCer treatment. We employed two types of mutant mice, J{alpha}18KO and CD1dKO mice. We showed that {alpha}-GalCer-mediated enhancement of CD8+ T cell homeostatic proliferation was dependent on CD1d-restricted NKT cells (Fig. 1B). On the other hand, without {alpha}-GalCer stimulation, the homeostatic proliferation of CD8+ T cells in J{alpha}18KO and CD1dKO mice was almost the same level to that in control mice (Fig. 1B), suggesting that NKT cells have an impact on CD8+ T cell homeostasis only upon their activation. Thus, these results demonstrated that {alpha}-GalCer-mediated CD1d-restricted NKT cell activation is critical for the enhancement of CD8+ T cell homeostatic proliferation.


Figure 1
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  		Fig. 1 {alpha}-GalCer-mediated activation of NKT cells increased CD8+ but not CD4+ T cell homeostatic proliferation. (A) CD44lowCD4+ T cells and CD44lowCD8+ T cells from mice expressing Thy-1.1 molecules were sorted and labeled with CFSE. The 5-Gray-irradiated syngeneic Thy-1.2 mice were given the CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the Thy-1.1-expressing donor population. (B) CD44lowCD8+ T cells from mice expressing Thy-1.1 molecules were sorted and labeled with CFSE. The 5-Gray-irradiated syngeneic Thy-1.2+ J{alpha}18KO or CD1dKO mice were given the CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the Thy-1.1-expressing donor population.

 
IL-4 was essential and sufficient for the activated NKT cell-mediated enhancement of CD8+ T cell homeostatic proliferation
Because activated NKT cells produce a variety of cytokines, we hypothesized that {alpha}-GalCer-mediated cytokine production by NKT cells plays a role for the enhancement of CD8+ T cell homeostatic proliferation. To check this, we investigated serum cytokine concentrations after {alpha}-GalCer injection. All cytokines investigated here, tumor necrosis factor {alpha} (TNF{alpha}), IL-4, IL-6, IL-10, IL-12, IL-13 and IFN{gamma}, increased short time after the treatment with {alpha}-GalCer (within 4 h after the treatment) (Fig. 2A). We employed several KO mice and antibodies for these cytokines to examine their roles for {alpha}-GalCer-mediated enhancement of CD8+ T cell homeostatic proliferation. In IL-4KO, {alpha}-GalCer-mediated enhancement of CD8+ T cell homeostatic proliferation was lower as compared with that in control mice (Fig. 2B). However, other cytokine KO mice nor mice treated with antibodies against cytokines showed the similar level of {alpha}-GalCer-mediated increase of CD8+ T cell homeostatic proliferation compared with control animals (Fig. S1A, Fig. S1B, available at International Immunology Online, and data not shown). Consistent with a previous report (18), in normal condition without {alpha}-GalCer stimulation, the similar level of CD8+ T cell homeostatic proliferation was observed in IL-4KO mice as compared with controls (Fig. 2B). In addition, IL-4 in sera did not increase after irradiation without {alpha}-GalCer treatment. Therefore, we concluded that IL-4 plays a role for activated NKT cell-mediated enhancement of CD8+ T cell homeostatic proliferation.


Figure 2
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  		Fig. 2 IL-4 increased CD8+ T cell homeostatic proliferation after {alpha}-GalCer treatment. (A) The serum cytokine levels following the i.v. injection of {alpha}-GalCer in C57BL6 mice, measured by cytometric bead array (BD). Data give the mean ± SEM (n = 4–5). (B) CD44lowCD8+ T cells from mice expressing Thy-1.1 molecules were sorted and labeled with CFSE. The 5-Gray-irradiated syngeneic Thy-1.2+ IL-4KO mice were given the CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the Thy-1.1-expressing donor population.

 
Because {alpha}-GalCer treatment in vivo induces IL-4 production in NKT cells (1) and we showed that IL-4 in serum increased at 2 h after NKT cell activation in vivo (Fig. 2A), we hypothesized that IL-4 produced by activated NKT cells plays a role for enhancing homeostatic proliferation of CD8+ T cells. To check this hypothesis, we transferred NKT cells from wild-type mice to IL-4KO recipients and investigated {alpha}-GalCer-mediated enhancement of CD8+ T cell homeostatic proliferation. We showed that {alpha}-GalCer treatment enhanced homeostatic proliferation of CD8+ T cells even in IL-4KO mice after transferring wild-type NKT cells (Fig. 3). Thus, NKT cells produced enough amount of IL-4 for the enhancement of CD8+ T cell homeostatic proliferation after {alpha}-GalCer treatment, although this does not exclude the possible involvement of IL-4 produced by other cells. Since activated NKT cells produce many cytokines and may express several cell surface molecules which may affect homeostatic proliferation of CD8+ T cells, we next asked a question whether IL-4 produced by NKT cells is sufficient for the enhancement of CD8+ T cell homeostatic proliferation. To check this, we expressed IL-4 by using an expression plasmid in vivo. We used IL-13 as a control, because both IL-4 and IL-13 have very similar in vivo activities via the same signaling components (28). We showed a dose-dependent increase of serum IL-4 and IL-13 levels after injection of the expression plasmid (Fig. 4A and data not shown). Importantly, expression of IL-4 but not IL-13 significantly enhanced homeostatic proliferation of CD8+ T cells (Fig. 4B). All these results showed that IL-4 was essential and sufficient for NKT cell-mediated enhancement of CD8+ T cell homeostatic proliferation in vivo.


Figure 3
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  		Fig. 3 IL-4 produced by activated NKT cell was essential for the enhancement of CD8+ T cell homeostatic proliferation. CD44lowCD8+ T cells from mice expressing Thy-1.1 molecules were sorted and labeled with CFSE. The CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) were transferred into the 5-Gray-irradiated syngeneic IL-4KO mice on day 0. NKT cells (3 x 105 cells per mouse, i.v.) from wild-type C57BL/6 mice were also given i.v. on day 0 in the presence or absence of {alpha}-GalCer treatment. CFSE levels were examined on day 7. Histograms shown are gated on the Thy-1.1-expressing donor population.

 

Figure 4
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  		Fig. 4 IL-4 produced by activated NKT cell was sufficient for the enhancement of CD8+ T cell homeostatic proliferation. (A) The serum cytokine levels following the i.v. injection of a plasmid carrying IL-4, IL-13 or a mock plasmid in non-irradiated C57BL6 mice, measured by ELISA. Data give the mean ± SEM (n = 4–5). (B) CD44lowCD8+ T cells from mice expressing Thy-1.1 molecules were sorted and labeled with CFSE. The CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) were transferred into the 5-Gray-irradiated syngeneic C57BL6 mice on day 0. A 10 µg of plasmid carrying IL-4, a 1 µg of plasmid carrying IL-13 or a mock plasmid was also given i.v. on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the Thy-1.1-expressing donor population.

 
IL-4 directly acted on CD8+ T cells to enhance their homeostatic proliferation
Then we asked a question whether IL-4 directly acts on CD8+ T cells. For this, we first examined whether either IL-4 or IL-13 induces proliferation of CD8+ T cells in the presence of weak TCR-mediated signaling in vitro. We prepared memory and naive phenotype of CD4+ and CD8+ T cells and cultured them with the cytokines plus anti-CD3 mAb. Because we coated a low dose of anti-CD3 mAb to mimic MHC/self-peptide–TCR interaction, there was almost no T cell proliferation under this condition (Fig. 5A). IL-4 alone showed a low-level proliferation of T cells (Fig. 5A). Combination of TCR stimulation and IL-4 increased proliferation of CD8+ T cells especially memory phenotype ones (Fig. 5A and B), but that of TCR plus IL-13 stimuli hardly induced proliferation of any T cells (Fig. 5D). We hardly observed proliferation of CD4+ T cells after stimulation of TCR plus IL-4 (Fig. 5A and C). Then we examined whether IL-4 directly acts on CD8+ T cells in vivo homeostatic proliferation. We isolated CD8+ T cells from either STAT6KO or IL-4R{alpha}KO mice and analyzed their homeostatic proliferation in wild-type recipients after treatment of {alpha}-GalCer. These mutant CD8+ T cells showed almost comparable basal level of homeostatic proliferation as compared with those from wild-type controls in the absence of {alpha}-GalCer treatment (Fig. 6A and B). However, {alpha}-GalCer failed to enhance the homeostatic proliferation of either STAT6- or IL-4R{alpha}-deficient CD8+ T cells (Fig. 6A and B). These results demonstrated that the activation of IL-4R{alpha}–STAT6 signaling in CD8+ T cells was essential for NKT cell-mediated enhancement of CD8+ T cell homeostatic proliferation, in consistent with the notion that IL-4 directly acts on CD8+ T cells in vivo.


Figure 5
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  		Fig. 5 IL-4 but not IL-13 promotes proliferation of CD8+ T cells in vitro. CD44lowCD8+, CD44highCD8+, CD44lowCD4+ or CD44highCD4+ T cells from C57BL6 mice were sorted and cultured with or without IL-4 (A, B and C) or IL-13 (D) in the presence or absence of anti-CD3 mAb. Proliferation of the T cell populations was measured by MTT assay on day 2. Data give the mean ± SEM (n = 4–5).

 

Figure 6
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  		Fig. 6 STAT6 and IL-4R{alpha} were critical for {alpha}-GalCer-mediated increase of CD8+ T cell homeostatic proliferation. (A) CD44lowCD8+ T cells from Balb/c STAT6KO mice were sorted and labeled with CFSE. The 5-Gray-irradiated Balb/c mice were given the CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the CD8-expressing donor population. (B) CD44lowCD8+ T cells from Balb/c IL-4R{alpha}KO mice were sorted and labeled with CFSE. The 5-Gray-irradiated Balb/c mice were given the CFSE-labeled donor T cells (1–2 x 106 cells per mouse, i.v.) with or without {alpha}-GalCer (2 µg per mouse, i.v.) on day 0. CFSE levels were examined on day 7. Histograms shown are gated on the CD8-expressing donor population.

 
This was further supported by the following results. Memory phenotype CD8+ T cells showed higher level of IL-4R{alpha} expression compared with naive phenotype CD4+ and CD8+ T cells and memory phenotype CD4+ T cells either before or after {alpha}-GalCer stimulation (Fig. 7). After induction of homeostatic proliferation, the expression level of IL-4R{alpha} was significantly increased on only memory phenotype CD8+ T cells (Fig. 7). On the other hand, IL-13R{alpha} expression in all T cells decreased after {alpha}-GalCer treatment in lymphopenic condition (data not shown), in consistent with no effect of IL-13 on homeostatic proliferation of CD8+ T cells.


Figure 7
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  		Fig. 7 {alpha}-GalCer treatment increased IL-4R{alpha} level on CD8+ T cells especially memory phenotype in irradiated mice. The 5-Gray-irradiated C57BL6 mice were injected 2 µg of {alpha}-GalCer. IL-4R{alpha} level on CD44lowCD8+, CD44highCD8+, CD44lowCD4+ or CD44highCD4+ T cells in spleen was measured by FACS. Data give the mean ± SEM (n = 2).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
MacDonald's group demonstrated that NKT cell activation via {alpha}-GalCer increases memory phenotype of T cells in normal condition (not in lymphopenic condition) (8). In addition, homeostatic proliferation always induces memory phenotype of T cells from naive ones (10). These results suggested that activated NKT cells might affect homeostatic proliferation of T cells. We here demonstrated that activation of NKT cells by {alpha}-GalCer in lymphopenic condition enhanced homeostatic proliferation of CD8+ but not CD4+ T cells in a manner dependent on IL-4.

We showed that in vivo {alpha}-GalCer treatment enhanced homeostatic proliferation of CD8+ T cells (Fig. 1A). Moreover, this enhancing effect of {alpha}-GalCer was observed in neither J{alpha}18- nor CD1d-deficient mice (Fig. 1B), indicating that the importance of activated NKT cells for CD8+ T cell homeostatic proliferation. However, almost the same level of CD8+ T cell homeostatic proliferation was observed in either J{alpha}18- or CD1d-deficient mice compared with controls under lymphopenic condition, when we did not treat the mice with {alpha}-GalCer. These results suggested that NKT cells are not involved in homeostatic proliferation in lymphopenic condition of the steady state where activation stimuli, such as {alpha}-GalCer, are absent (Fig. 1B). In physiological conditions, NKT cells are strongly activated by exogenous glycolipid antigens derived from pathogens (6, 29–32). Furthermore, infection of pathogens especially viruses induced lymphopenic condition in vivo (10). In other words, infection of pathogens simultaneously induces both strong activation of NKT cells and lymphopenic condition in vivo. This change of condition of immune system may facilitate the defense mechanisms against pathogen through increased homeostatic proliferation of CD8+ T cells.

{alpha}-GalCer injection induced significant level of various kinds of cytokines in sera (including IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF{alpha}, monocyte chemoattractant protein-1 and IFN{gamma}) (Fig. 2A and data not shown) and activated NKT cells produced these cytokines (data not shown). IL-7, IL-12 and IL-15 have been reported as stimulators of CD8+ T cell homeostatic proliferation (18, 19), suggesting that NKT cells enhance homeostatic proliferation of CD8+ T cells through IL-7, IL-12 and IL-15. However, we showed that {alpha}-GalCer significantly increased CD8+ T cell homeostatic proliferation even in IL-15KO mice and mice that were treated with anti-IL-7 or anti-IL-12 mAb, although depletion of IL-7 or IL-15 in vivo decreased a basal level of homeostatic proliferation of CD8+ T cells (see {alpha}-GalCer untreated results in Fig. S1B). These results suggested that activated NKT cells might produce an unknown stimulator of CD8+ T cell homeostatic proliferation in vivo. It was reported that a basal level of IL-4 does not affect CD8+ T cell homeostatic proliferation (18) (see Fig. 2B without {alpha}-GalCer), but several groups demonstrated that IL-4 stimulates CD8+ T cells in vivo (23, 24). This led us to a hypothesis that IL-4 derived from activated NKT cells might be an enhancer of homeostatic proliferation of CD8+ T cells. Three lines of in vivo experiments directly supported that this hypothesis is physiologically relevance: (i) {alpha}-GalCer effect was abrogated in IL-4KO (Fig. 2B), (ii) transferring of wild-type NKT cells at least partially induced the enhanced homeostatic proliferation of CD8+ T cells in IL-4KO mice (Fig. 3) and (iii) expression of IL-4 alone could enhance CD8+ T cell homeostatic proliferation in vivo (Fig. 4B). Therefore, we concluded that IL-4 was essential and sufficient for the enhancement of CD8+ T cell homeostatic proliferation by activated NKT cells.

To demonstrate the importance of NKT cell-derived IL-4 in homeostatic proliferation of CD8+ T cells, we transferred activated NKT cells into IL-4KO mice and observed slightly enhanced homeostatic proliferation of CD8+ T cells, which remained much reduced compared with that occurred in {alpha}-GalCer-injected wild-type mice. There might be two possible explanations for this phenomenon. First, the number of transferred NKT cells was not sufficient enough. Second, {alpha}-GalCer injection induced IL-4 production by the cell type other than NKT cells.

Anyway, all results indicated that IL-4 produced by activated NKT cells plays a role in homeostatic proliferation of CD8+ T cells in vivo. The question we asked next was whether IL-4 directly acts on CD8+ T cells or IL-4 acts on the other cells that then affect CD8+ T cell homeostatic proliferation. We clearly showed that IL-4 but not IL-13 directly induces proliferation of memory/activated phenotype CD8+ T cells in the presence of weak stimulation of TCR in vitro. Furthermore, {alpha}-GalCer could not enhance CD8+ T cell homeostatic proliferation when we used either STAT6- or IL-4R{alpha}-deficient CD8+ T cells, clearly indicated the direct action of IL-4 on CD8+ T cells. Consistent with these results, only memory phenotype CD8+ T cells expressed higher level of IL-4R{alpha} than naive phenotype CD8+ and CD4+ T cells (Fig. 7). Thus, all the results presented here demonstrated the ‘involvement’ of IL-4 from activated NKT cells for CD8+ T cell homeostatic proliferation in vivo.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary figure is available at International Immunology Online.


   Acknowledgements
 
We thank M. Kubo (RIKEN, Research Center for Allergy and Immunology) for providing IL-4R{alpha}KO and STAT6KO mice. We also thank KIRIN for {alpha}-GalCer. We appreciate Ms E. Iketani and Ms T. Hayashi for their excellent technical assistance and thank Ms R. Masuda and Ms M. Shimura for their excellent secretarial assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan; the Uehara Foundation and the Osaka Foundation for the Promotion of Clinical Immunology.


   Abbreviations
 
{alpha}-GalCer, alpha-galactosylceramide
APC, allophycoerythrin
CFSE, carboxyfluorescein diacetate succinimidyl ester
i.v., intravenously
KO, knockout
TNF{alpha}, tumor necrosis factor {alpha}

   Notes
 
Transmitting editor: S. Koyasu

Received 19 June 2006, accepted 6 July 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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