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International Immunology Advance Access originally published online on January 17, 2007
International Immunology 2007 19(3):249-256; doi:10.1093/intimm/dxl140
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

AsialoGM1+CD8+ central memory-type T cells in unimmunized mice as novel immunomodulator of IFN-{gamma}-dependent type 1 immunity

Akemi Kosaka1,2, Daiko Wakita1, Naoki Matsubara3, Yuji Togashi1, Shin-Ichiro Nishimura2, Hidemitsu Kitamura1 and Takashi Nishimura1,3

1 Division of Immunoregulation, Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
2 Division of Biological Sciences, Graduate School of Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Sapporo 001-0021, Japan
3 Division of ROYCE' Health Bioscience, Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan

Correspondence to: T. Nishimura; E-mail: tak24{at}igm.hokudai.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In unimmunized specific pathogen-free mice, there are unique memory-type CD8+ T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (TCMT) judging from their expression profile of CD44, IL-2Rß, CD62L and CCR7 cell-surface molecules. Among CD44highCD8+ so-called memory CD8+ T cell population, ASGM1+CD44highCD8+ TCMT, but not ASGM1–CD44highCD8+ memory T cells, produced IFN-{gamma} by stimulation with anti-CD3 mAb. The physiological significance of ASGM1+CD8+ TCMT as early source of IFN-{gamma} was also demonstrated in vivo. Namely, intravenous injection of anti-CD3 mAb (2 µg) resulted in early activation of IFN-{gamma}-producing ASGM1+CD8+ TCMT cells as well as NKT and NK cells. Unexpectedly, however, few IFN-{gamma}-producing CD4+ T cells were detected until 4 h after anti-CD3 mAb administration. Thus, ASGM1+CD8+ TCMT were demonstrated to be early IFN-{gamma} producer, which may be crucial for Th1-dependent cellular immunity. Indeed, co-culture of naive CD4+ T cells with ASGM1+CD8+ TCMT but not ASGM1–CD8+ T cells caused a great acceleration of IFN-{gamma}-producing Th1 cells in vitro. Finally, we found that Th1-prone C57BL/6 mice possessed higher percentage (10%) of ASGM1+CD8+ TCMT in CD8+ T cells compared with that (3%) of Th2-prone BALB/c mice. Moreover, ASGM1+CD8+ TCMT derived from C57BL/6 mice produced higher levels of IFN-{gamma} compared with those from BALB/c mice. Thus, ASGM1+CD8+ TCMT, whose differentiation in vivo is genetically controlled, appear to play a critical role in the control of type 1 immunity, which is essential for therapy of tumors and infectious diseases.

Keywords: genetic differences, IFN-{gamma}-producing CTLs, immunoregulatory cells, Th1 commitment


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
It has been demonstrated that immune balance is controlled by cytokines derived from two distinct Th subsets, Th1 and Th2 (1–3). Th1 produce IL-2 and IFN-{gamma}, while Th2 produce IL-4, IL-5 and IL-6. The former plays a critical role in cellular immunity, while the latter is involved in humoral immunity. It has also been demonstrated that CD8+ T cell populations are subdivided into IFN-{gamma}-producing Tc1 and IL-4-producing Tc2 cells such as Th subsets (4–9). More confusingly, innate effector cells such as dendritic cells (DC), NK, NKT and {gamma}{delta} T cells produce various cytokines including Th1 cytokines and Th2 cytokines (2, 10–12). Type 1 immunity is regulated by DC, macrophages (M{phi}), NK, NKT, {gamma}{delta} T, Th1 and Tc1 cells which produce Th1-inducing cytokines (e.g. IL-12, IL-18 and IFN-{gamma}), whereas type 2 immunity is regulated by DC, M{phi}, NKT, {gamma}{delta} T, Th2 and Tc2 cells producing Th2-inducing cytokines (e.g. IL-4, IL-10 and transforming growth factor-ß). Thus, the immune balance is controlled by many immunoregulatory cells in addition to Th1 and Th2.

As reported previously, type 1 acquired immunity regulated by IFN-{gamma}-producing Th1 and Tc1 cells is crucial for inducing protective immunity against infectious and malignant diseases, though too much acceleration is a causative of autoimmune diseases (13–16). Therefore, it is of great importance to define the early IFN-{gamma} source of immunoregulatory cells, which is beneficial for inducing Tc1- and Th1-mediated type 1 immunity.

In the previous paper (17), we found that freshly isolated CD8+ T cells from unimmunized mice contained minor asialoGM1+ (ASGM1+) CD8+ T cell subsets, which exhibited augmented immune responses to IL-12 in the presence of IL-2. In this paper, we further investigated the phenotypic and functional characteristics of ASGM1+CD8+ T cell subsets and demonstrated the following results: (i) ASGM1+CD8+ T cells were central memory-type T cells (TCMT) expressing CD44, IL-2Rß, CD62L and CCR7 cell-surface markers, (ii) ASGM1+CD8+ TCMT exhibited the highest responsiveness to immobilized anti-CD3 mAb stimulation among CD44+CD8+ memory T cell population, (iii) ASGM1+CD8+ TCMT as well as NKT and NK cells were early IFN-{gamma} source in anti-CD3 mAb-administered mice, (iv) ASGM1+CD8+ TCMT accelerated the generation of IFN-{gamma}-producing Th1 from naive CD4+ T cells and (v) Th1-prone C57BL/6 mice possessed higher frequency of IFN-{gamma}-producing ASGM1+CD8+ TCMT compared with Th2-prone BALB/c mice. Thus, the present data indicate the critical role of genetically controlled ASGM1+CD8+ TCMT in the regulation of type 1 immunity, which is crucial for inducing protective immunity against infectious and malignant diseases.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
Wild-type C57BL/6 and BALB/c female mice, 5–6 weeks of age, were purchased from Charles River Japan Inc. (Kanagawa, Japan). IFN-{gamma}-deficient C57BL/6 mice were kindly gifted by Y. Iwakura (Institute of Medical Science, University of Tokyo, Tokyo, Japan). All animals were housed under specific pathogen-free (SPF) conditions.

Flow cytometry
Phenotypic characterization of ASGM1+CD8+ T cells was analyzed by FACSCalibur (BD Biosciences, San Jose, CA, USA) using the following mAbs: PE–Cy5-anti-CD8{alpha}, PE–anti-CD44, PE–anti-CD122 (IL-2Rß), PE–anti-CD62L, PE–anti-NK1.1, PE–anti-TCRß, PE–anti-CD3{epsilon}, PE–anti-CD8ß.2, biotin–anti-CD44 and allophycocyanin (APC)–streptavidin. All mAbs were purchased from BD Biosciences PharMingen (San Jose, CA, USA), except PE–anti-CCR7 mAb (eBioscience, San Diego, CA, USA) and anti-ASGM1 antibody plus FITC–anti-rabbit antibody (WAKO Pure Chemical Industries, Ltd, Osaka, Japan). Samples were analyzed by CellQuest software (BD Biosciences).

Cell purification
ASGM1+CD8+ T cells, ASGM1–CD8+ T cells and the other CD8+ T cell populations were separated from nylon–wool passaged spleen cells using MACS system (Miltenyi Biotec, Bergisch Glabach, Germany) and FACSVantage (BD Biosciences). For purification of naive (CD45RB+) CD4+ T cells, selected CD4+ T cells by MACS system were stained with PE–anti-CD4 and FITC–anti-CD45RB (BD Biosciences PharMingen) and then isolated using FACSVantage. The sorted cells were >95% pure.

In vitro stimulation and detection of IFN-{gamma}
The purified CD8+ T cell subsets were stimulated with immobilized anti-CD3 mAb or IL-2 plus IL-12 under standard culture conditions (37°C, 5% CO2) on a flat-bottomed 96-well plate. Anti-CD3 mAb was purchased from BD Biosciences PharMingen. Recombinant IL-12 and IL-2 were kindly donated by Wyeth Research (Cambridge, MA, USA) and Takuko Sawada (Shionogi Pharmaceutical Institute Co. Ltd, Osaka, Japan), respectively. IFN-{gamma} and IL-4 levels in culture supernatant were measured using BD OptEIATM ELISA kits (BD Biosciences PharMingen). IL-13 concentrations in culture supernatant were measured using Quantikine ELISA kit (R&D System, Minneapolis, MN, USA). The amount of cytokine production was calculated from the extrapolated curve of a standard graph.

Intracellular cytokine staining
For in vivo experiments, spleens were harvested at various indicated time points after anti-CD3 mAb injection and then single-cell suspensions were prepared. For in vitro experiments, CD4+ T cells were harvested at 42 h after co-culturing with each CD8+ T population. The cells were stained with the indicated cell-surface mAbs for 15 min, washed with PBS/0.05% NaN3/0.5% BSA, fixed with 4% PFA for 15 min at 4°C and treated with permeabilizing solution (50 mM NaCl, 5 mM EDTA, 0.02% NaN3 and 0.5% Triton X-100, pH 7.5) at 4°C followed one wash. After 10 min, these cells were washed and stained with APC-conjugated anti-IFN-{gamma} mAb (BD Biosciences PharMingen) for 45 min at 4°C. The percentage of cells producing cytoplasmic IFN-{gamma} was determined using FACSCalibur.

Statistical analyses
All experiments were repeated at least three times. Mean values and standard errors were calculated for data from three independent experiments and are shown in the figures. Significant differences in the results were determined by using the two-sided Student's t-test. P < 0.05 was considered significant in the present experiments.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
ASGM1+CD8+ T cells are TCMT expressing CD62L and CCR7 antigens
The phenotypic characteristics of ASGM1+CD8+ T cells existed in unimmunized C57BL/6 mouse was analyzed by flow cytometry. As shown in Fig. 1(A), ASGM1+CD8+ T cells occupied ~10% of unfractionated CD8+ T cells. Although CD8+ T cells are divided into CD44low naive T cells and CD44high memory T cells, almost all ASGM1+CD8+ T cells expressed CD44 memory marker and ~35% of CD44highCD8+ memory T cells expressed ASGM1 (Fig. 1B). Moreover, it was demonstrated that ASGM1+CD8+ T cells also expressed CD122 (IL-2Rß), CD62L and CCR7 cell-surface molecules, and there was no significant levels of NK1.1 expression on these cells (Fig. 1C). Thus, consistent with our previous report (18), these results suggested that ASGM1+CD8+ T cells were defined as TCMT. Because we isolated ASGM1+CD8+ T cells from unimmunized naive C57BL/6 mice and the number of them greatly reduced in Rag2–/– ovalbumin peptide-specific TCR-transgenic mice (OT-1 Rag2–/– mice) (18), these ASGM1+CD8+ TCMT were probably induced by homeostatic expansion mechanisms in peripheral lymphoid organs, involving expansion of T cells recognizing self-MHC/peptide or intestinal flora antigen.


Figure 1
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  		Fig. 1. Phenotypic features of ASGM1+CD8+ T cells. (A) Spleen cells from C57BL/6 mice were stained with anti-CD8 mAb and anti-ASGM1 antibody, and then analyzed by flow cytometry. ASGM1 expression levels on CD8+ T cells were evaluated. (B) CD8+ T cells were gated from total spleen cells and expression levels of CD44 and ASGM1 were evaluated. (C) Expression levels of CD122, CD62L, CCR7 or NK1.1 together with ASGM1 were evaluated for the gated CD8+ T cells. The number in the FACS profiles represents the percentage of each population. Similar results were obtained in three independent experiments and representative data were shown in the figures.

 
ASGM1+CD8+ TCMT are responsible for IFN-{gamma} production among CD44highCD8+ memory T cell population
CD8+ T cells, which were isolated from C57BL/6 mouse spleen T cells, were further separated into CD44lowCD8+ naive CD8+ T cells, CD44highCD8+ memory T cells, ASGM1–CD44highCD8+ major (~65%) memory T cells and ASGM1+CD44highCD8+ minor (~35%) memory T cells using FACSVantage. Then, each separated CD8+ T cell populations was cultured with immobilized anti-CD3 mAb for 48 h and IFN-{gamma} and IL-4 activity in culture supernatants were measured by ELISA. There was no IL-4 production, which was crucial for Th2 immunity, from each population (data not shown). In Fig. 2, IFN-{gamma} producers in CD8+ T cells were enriched in CD44high memory T cells. Moreover, it was demonstrated that ASGM1+CD44highCD8+ TCMT, but not ASGM1–CD44highCD8+ memory T cells, were responsible cells for IFN-{gamma} production among CD44high memory CD8+ T cells.


Figure 2
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  		Fig. 2. ASGM1+CD8+ TCMT were major producers of IFN-{gamma} among CD44highCD8+ memory T cell population. (A) Whole CD8+, ASGM1+CD8+ or ASGM1–CD8+ T cells were freshly isolated from C57BL/6 mice. Each population was stimulated with or without immobilized anti-CD3 mAb (5 µg ml–1) for 48 h. IFN-{gamma} levels produced in the supernatant were measured by ELISA. (B) Whole CD8+, CD44highCD8+ or CD44lowCD8+ T cells were isolated, and CD44highCD8+ T cells were further separated by ASGM1 expression levels. Each population was stimulated with or without immobilized anti-CD3 mAb (5 µg ml–1) for 48 h. IFN-{gamma} levels produced in the supernatant were measured by ELISA. The data represent mean and standard error of three independent experiments.

 
We further characterized the surface phenotype of ASGM1+CD8+ TCMT. It was shown that ASGM1+CD44highCD8+ TCMT expressed intermediate TCRß and CD3{epsilon} as compared with naive (CD44low) CD8+ T cells and ASGM1–CD44highCD8+ T cells (Fig. 3). There was no significant difference in the expression levels of CD8ß, indicating that ASGM1+CD8+ TCMT expressed the CD8{alpha}ß heterodimer.


Figure 3
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  		Fig. 3. Comparison of surface expression levels on CD8+CD44low, ASGM1+CD44highCD8+ and ASGM1–CD44highCD8+ T cells. CD8+ T cells were purified from spleen of C57BL/6 mice and stained with anti-TCRß, anti-CD3{epsilon} or anti-CD8ß mAbs together with anti-ASGM1 antibody and anti-CD44 mAb. The expression levels of TCRß, CD3{epsilon} or CD8ß on each population were analyzed by flow cytometry. The data shown in the figure are representative of two independent experiments.

 
ASGM1+CD8+ TCMT are early IFN-{gamma} producers as well as NKT and NK cells in anti-CD3 mAb-administered mice
To evaluate physiological significance of ASGM1+CD8+ TCMT, C57BL/6 mice were intravenously treated with anti-CD3 mAb (2 µg) and early IFN-{gamma} producer was determined various times after the treatment. The induction of IFN-{gamma}-producing cells in NK, NKT, CD8+ T and CD4+ T cells was determined by flow cytometry after cytoplasmic staining of IFN-{gamma}. As shown in Fig. 4, NKT cells are the most rapid IFN-{gamma} producer in vivo after stimulation with anti-CD3 mAb. IFN-{gamma} production of NKT cells became detectable 30 min after anti-CD3 mAb injection and reached peak at 1 h. Interestingly, ASGM1+CD8+ TCMT were also rapid responders to anti-CD3 mAb stimulation in vivo and produced high levels of IFN-{gamma} in the same kinetics as NKT cells. NK cells were also demonstrated to be early IFN-{gamma} source after anti-CD3 mAb injection. Judging from the fact that NK cells do not express CD3 antigen and the kinetics of IFN-{gamma} production was delayed compared with that of NKT cells and ASGM1+CD8+ TCMT, IFN-{gamma} production by NK cells might be indirectly triggered by anti-CD3 mAb via some cytokines derived from anti-CD3-activated NKT cells or ASGM1+CD8+ TCMT. In contrast to NKT, ASGM1+CD8+ TCMT and NK cells, CD4+ T cells produced little levels of IFN-{gamma} at early phase of anti-CD3 mAb stimulation in vivo.


Figure 4
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  		Fig. 4. Kinetics of IFN-{gamma}-producing cells in the spleen cells of mice injected with anti-CD3 mAb. (A) C57BL/6 mice were intravenously injected with 2 µg of anti-CD3 mAb. At different time points (0.6, 1, 2 and 4 h) after the treatment, spleen cells were harvested and intracellular IFN-{gamma} production of NK, NKT, ASGM1+CD8+ T and CD4+ T cells were analyzed by flow cytometry. The percentages of the population of IFN-{gamma}-producing cells were calculated for each population and plotted at the time after anti-CD3 mAb injection. The data represent mean and standard error of three independent experiments. (B) Intracellular IFN-{gamma} levels were detected for the same population as (A) at 1 h post-anti-CD3 mAb injection. The number shown in each FACS profile represents the percentage of IFN-{gamma}+ cells in each population. Similar results were obtained in three independent experiments and representative data were shown in the figures.

 
The critical role of ASGM1+CD8+ TCMT in the induction of IFN-{gamma}-producing Th1 from naive Th
ASGM1+CD8+ TCMT from wild-type or IFN-{gamma}–/– mice were co-cultured with naive CD4+ T cells from wild-type mice for 42 h and the generation of IFN-{gamma}-producing Th1 was determined by FACSCalibur. As shown in Fig. 5, co-culture of naive Th with wild-type ASGM1–CD8+ T cells caused the induction of small percentages (1.7%) of IFN-{gamma}-producing Th1. In contrast, co-culture of naive Th with ASGM1+CD8+ TCMT resulted in a great enhancement (22.1%) of IFN-{gamma}-producing Th1 generation. Moreover, very little IFN-{gamma} production from Th co-cultured with IFN-{gamma}–/– ASGM1+CD8+ TCMT was observed (1.2%). Thus, it was demonstrated that IFN-{gamma}-producing ASGM1+CD8+ TCMT could accelerate the induction of Th1 from naive Th.


Figure 5
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  		Fig. 5. ASGM1+CD8+ TCMT induced differentiation of IFN-{gamma}-producing CD4+ T cells. Naive CD4+ T cells purified from wild-type mice were co-cultured with ASGM1+CD8+ or ASGM1–CD8+ T cells from wild-type or IFN-{gamma}–/– mice in the presence of immobilized anti-CD3 mAb (5 µg ml–1). After 42 h, these cells were stained with anti-CD4 and anti-IFN-{gamma} mAbs. IFN-{gamma}-producing cells were evaluated for the CD4+ T cells by flow cytometry. Similar results were obtained in three independent experiments and representative FACS profiles were shown in the figures (left panel). The number in the FACS profiles represents the percentage of IFN-{gamma}+ cells against total CD4+ T cells (right panel). The mean and standard error were calculated from the results of three independent experiments and indicated in the figure.

 
Quantitative and qualitative differences of ASGM1+CD8+ TCMT between Th1-prone C57BL/6 and Th2-prone BALB/c mice
During investigating the frequency of ASGM1+CD8+ TCMT, we found that C57BL/6 mice possessed higher frequency of ASGM1+CD8+ TCMT in CD8+ T cells compared with that of BABL/c mice. As shown in Fig. 6(A), C57BL/6 mouse CD8+ T cells contained ~10% of ASGM1+CD8+ TCMT, while BALB/c mouse CD8+ T cells included just 3% of ASGM1+CD8+ TCMT. ASGM1+CD8+ TCMT obtained from C57BL/6 mice exhibited high response to IL-2 plus IL-12 (Fig. 6B) or anti-CD3 mAb stimulation (Fig. 6C). However, BALB/c-derived ASGM1+CD8+ TCMT enriched by sorting showed very low IFN-{gamma} production in response to IL-2 plus IL-12 or anti-CD3 mAb stimulation. Here, even in the case of ASGM1+CD8+ TCMT from Th2-prone BALB/c mice, there was no IL-4 and IL-13 production, which may interfere IFN-{gamma} production from Th in the same experiments (Fig. 6C and data not shown). Therefore, the differentiation of ASGM1+CD8+ TCMT in vivo appeared to be controlled by some unknown genetic factors.


Figure 6
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  		Fig. 6. Genetically controlled differences in ASGM1+CD8+ TCMT between C57BL/6 and BALB/c mice. (A) ASGM1 expression levels were analyzed for CD8+ T cells of the spleen from C57BL/6 and BALB/c mice (upper panel). Similar results were obtained in three independent experiments and representative data were shown in the figures. The number in the FACS profiles represents the percentage of ASGM1+ cells in CD8+ cells (lower panel). The mean and standard error were calculated from the results of three independent experiments and indicated in the figure. (B and C) ASGM1+CD8+ TCMT were freshly isolated from spleen of C57BL/6 or BALB/c mice and stimulated with IL-2 plus IL-12 (20 U ml–1 each) (B) or immobilized anti-CD3 mAb (5 µg ml–1) (C), and then IFN-{gamma} and IL-4 levels in the culture supernatant were measured by ELISA at 48 h after the stimulation. The data represent mean and standard error of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we showed that ASGM1+CD8+ T cells, a unique population of freshly isolated CD8+ T cells, are classified into central memory-type T cells (TCMT) expressing CD44, CD122 (IL-2Rß), CD62L and CCR7 cell-surface markers, and early source of IFN-{gamma} upon TCR stimulation. Interestingly, in spite of a memory phenotype, ASGM1–CD44highCD8+ T cells were poor IFN-{gamma} producers, whereas ASGM1+CD8+ TCMT produced a large amount of IFN-{gamma} among CD44high memory CD8+ T cells. Moreover, the finding that in vivo administration of anti-CD3 mAb caused the early production of IFN-{gamma} by ASGM1+CD8+ TCMT as well as NK and NKT cells showed the physiological significance of ASGM1+CD8+ TCMT as immunoregulatory cells.

Generally, memory CD8+ T cells were differentiated from naive T cells by antigen immunization. To acquire immunological memory against pathogenic antigens, naive T cells first differentiated into CD44highCD62L–CCR7– effector–memory T cells (TEM) and finally differentiated into CD44highCD62L+CCR7+ central memory T cells (TCM) which exhibited strong immune responses by recall antigen stimulations (19, 20, 25). However, in the present work, we used unimmunized C57BL/6 mice. As reported previously, even in unimmunized naive mice maintained under SPF conditions, there are small percentage of memory T cells, which may be derived from thymus-derived naive T cells by peripheral homeostatic expansion mechanisms receiving unknown self-antigen stimulation, and these memory-type CD8+ T cells convert to phenotypic, functional and gene expression characteristics similar to those of ‘true’ memory T cells (21–25). Although it has been recently reported that CD8+NKT cells have the potential of immediate antigen-specific effector functions, ASGM1+CD8+ TCMT seemed to be distinct from CD8+NKT cells in terms of their cell surface phenotype (26) and freshly isolated ASGM1+CD8+ TCMT showed <2% cytotoxicity against YAC-1 cells at 20:1 effector-target (E/T) ratio (data not shown).

In the previous paper (27), we demonstrated that CD45RB–CD62L–CD4+ memory T cells induced by homeostatic expansion produced higher levels of IL-4 and IL-10 to regulate Th1–Th2 immune responses. In this paper, we defined novel ASGM1+CD8+ TCMT, which might be involved in regulation of type 1–type 2 immune balance without IL-4 and IL-13 production. During the differentiation of naive CD4+ T cells into a Th1 or a Th2 phenotype, many factors influence the Th lineage commitment, including cytokines, transcription factors, dose of antigens and co-stimulatory pathways (3, 28, 29). However, the cytokine environment is generally considered to be the most potent inducer of Th1–Th2 fate, and the critical factors are IL-12 to promote Th1 and IL-4 to induce Th2. Moreover, IFN-{gamma} has been shown to enhance the differentiation of CD4+ T cells into Th1 phenotype (30–32). Although it had been well known that NK and NKT cells were the major source of early IFN-{gamma} upon activation, we demonstrated that ASGM1+CD8+ TCMT also produced early IFN-{gamma} (Figs 2 and 4). Furthermore, when naive CD4+ Th were co-cultured with wild-type ASGM1+CD8+ TCMT under TCR stimulation, they differentiated into IFN-{gamma}-producing Th (Fig. 5), though they had no differentiation in the presence of IFN-{gamma}-deficient ASGM1+CD8+ TCMT. These results suggest that IFN-{gamma}-producing ASGM1+CD8+ TCMT have the ability to promote Th1 polarization and contribute to immune responses.

Th phenotype development is controlled not only by cytokines but also by the other factors including genetic background and classical examples of mice strains are Th1-prone C57BL/6 and Th2-prone BALB/c mice. Our results revealed that both the proportion and the functional property of ASGM1+CD8+ TCMT were genetically controlled between C57BL/6 and BALB/c mice. In both strains, ASGM1+CD8+ TCMT had similar surface phenotype in the expression of CD44, CD122, CD62L and CCR7, indicative of a central memory phenotype (data not shown). However, the proportion of ASGM1+ subset in CD8+ T cells was ~3-fold higher in C57BL/6 mice in comparison to BALB/c mice (Fig. 6A). In addition, ASGM1+CD8+ TCMT from C57BL/6 mice produced a large amount of IFN-{gamma} than that from BALB/c mice in response to IL-2 plus IL-12 stimulation (Fig. 6B) or immobilized anti-CD3 mAb (Fig. 6C). In both strains of mice, there was no IL-4 and IL-13 production by ASGM1+CD8+ TCMT following anti-CD3 stimulation (Fig. 6C and data not shown). We have also identified two suggestive quantitative trait loci that influence the proportion of ASGM1+CD8+ T cells: IL-1{alpha} [logarithm of odds (LOD) 2.2, P = 0.0016] and D2Mit62 (LOD 2.1, P = 0.0021) on chromosome 2 at 73 and 65 cM, respectively, using the 13 CXB recombinant inbred mouse strains derived from BALB/c and C57BL/6 progenitors and linkage analysis by Map Manager QTb29 software [(33) and data not shown]. These results suggest that the differences of ASGM1+CD8+ TCMT in not only the frequency but also the capacity to produce IFN-{gamma} may, at least in part, account for the differences in immune response between Th1-type C57BL/6 and Th2-type BALB/c mice.

Recent works have suggested that CD8+ T cells produce IFN-{gamma} rapidly in response to cytokines such as IL-12 or IL-18 from antigen-stimulated DC or M{phi} (34–38), indicating that IFN-{gamma} production of CD8+ T cells is important for the initiation of early immunoregulation. Moreover, it was shown that TCM were superior to TEM in conferring protective immunity against viral or bacterial challenge, and established tumor (19, 25, 39–41). The evidence that ASGM1 antigen is selectively expressed on IL-12-responding pre-killer T cells producing IFN-{gamma} (17) and early IFN-{gamma} producers upon TCR stimulation indicates that ASGM1 antigen combined with CD62L and CCR7 may become a good marker to isolate great precursor cells for IFN-{gamma}-producing Tc1 cells with cytotoxic activity.

We have already demonstrated that ASGM1+CD8+ TCMT were also differentiated into IFN-{gamma}-high-producing CTLs in response to type 1 IFNs in addition to anti-CD3 mAb or IL-12 stimulation (D. Wakita et al., unpublished data). Therefore, it is possible to speculate that ASGM1+CD8+ TCMT directly differentiated into IFN-{gamma}-producing CTLs in response to IL-12 and/or type 1 IFNs produced by innate effector cells such as DC and M{phi} activated with pathogenic antigens. Alternatively, ASGM1+CD8+ TCMT may indirectly play a critical role in the regulation of Th1 immunity as early IFN-{gamma}-producing CD8+ T cells. In addition, we have also shown that allospecific CTLs were efficiently generated from ASGM1+CD8+ TCMT but not from ASGM1–CD8+ T cells (data not shown). Moreover, in CpG-based tumor vaccine therapy model, we previously demonstrated that depletion of ASGM1+CD8+ TCMT caused the inhibition of the generation of tetramer-positive tumor-specific CD8+ CTLs and anti-tumor activity (D. Wakita et al., unpublished data). However, such inhibition was not observed when NK and NKT cells were depleted by treatment with anti-NK1.1 mAb (42). Taken together, these observations suggest that ASGM1+CD8+ T cells appear to be a precursor of antigen-specific CTLs. We are now investigating the precise role of ASGM1+CD8+ TCMT in the integration between innate and acquired T cell immunity during regulation of type 1 immune balance, which is crucial for inducing anti-tumor immunity.


   Abbreviations
 
APC, allophycocyanin
ASGM1, asialoGM1
SPF, specific pathogen free
TCM, central memory T cells
TCMT, central memory-type T cells
TEM, effector memory T cells

   Notes
 
Transmitting editor: T. Hamaoka

Received 14 October 2006, accepted 13 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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