Th1/Th2 cell differentiation of developing CD4 single-positive thymocytes
Emiko Kikkawa1,3,
Masakatsu Yamashita1,4,
Motoko Kimura1,
Miyuki Omori1,
Kaoru Sugaya4,
Chiori Shimizu1,
Takuo Katsumoto1,
Masahiko Ikekita6,
Masaru Taniguchi1,5 and
Toshinori Nakayama1,2
Departments of 1 Molecular Immunology and 2 Medical Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 3 Life Science Group, Hitachi Ltd, Kawagoe, Saitama 350-1165, Japan 4 PRESTO, Japan Science and Technology Corporation (JST), 5 Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, and 6 Department of Applied Biology, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Chiba 278-8510, Japan
The first two authors contributed equally to this work
Correspondence to: T. Nakayama, Department of Medical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan. E-mail: nakayama{at}med.m.chiba-u.ac.jp
Transmitting editor: A. Singer
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Abstract
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In this study we investigate the stage at which developing T cells in the thymus acquire the ability to differentiate into Th1 and Th2 cells. We addressed this question by using sorted heat-stable antigen (HSA)+ and HSA CD4 single-positive (SP) thymocytes prepared from ovalbumin-specific TCR
ß transgenic mice and an in vitro Th1/Th2 differentiation culture system. HSA CD4 SP thymocytes show nearly full functional capacity to differentiate into either Th1 or Th2 cells. A dramatic difference was observed, however, between HSA+ and HSA CD4 SP thymocytes in the efficiency for Th1 cell differentiation. TCR function of HSA+ CD4 SP thymocytes appeared to be fully developed because antigen-induced proliferation and IL-2 production were essentially equivalent to that of HSA CD4 SP thymocytes. However, the levels in IL-12 receptor (IL-12R) ß2 chain expression following anti-TCR stimulation were dramatically low in the HSA+ CD4 SP thymocytes. Decreased IL-12-induced STAT4 phosphorylation was also observed. Moreover, IL-12-dependent transcriptional up-regulation of T-bet and STAT4 was deficient in the HSA+ CD4 SP thymocytes. Thus, the poor capacity of HSA+ CD4 SP thymocytes to proceed to Th1 cell differentiation appears to be at least partly due to underdeveloped capacity in IL-12R expression and function.
Keywords: CD4 single-positive thymocyte, IL-12 receptor, STAT4, Th1/Th2
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Introduction
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The development of Th1 and Th2 cells is a central issue for understanding the diversity of T cell-dependent immune responses in infectious, allergic and autoimmune diseases. Both Th1 and Th2 cells are thought to differentiate from a common precursor (naive peripheral CD4 T cells), depending on the cytokines presented during the primary antigenic stimulation of naive T cells (15). Differentiation of Th1 cells is induced by IL-12 (69). In mice deficient for the p40, a subunit of IL-12 receptor (IL-12R) (10), and also in mice deficient for STAT4, a downstream signaling molecule of the IL-12 receptor pathway (11,12), Th1 differentiation does not occur. On the other hand, IL-4 is required for the differentiation of naive T cells into Th2 effector cells (1316). No significant Th2 response is observed in IL-4-deficient mice (17,18) or STAT6-deficient mice (1921).
In addition to the cytokines mentioned above, TCR-mediated signaling events also influence the generation of Th1 and Th2 cells (3). We recently reported that inefficient activation of Ras/mitogen-activated protein kinase (MAPK) cascade or calcineurin activation results in diminished Th2 cell differentiation (22,23). In contrast, Th1 cell differentiation and Th1 cytokine production are dependent on other MAPK pathways, such as c-Jun N-terminal kinase and p38MAPK respectively (2426).
Developing thymocytes are most clearly defined by the surface expression of CD4 and CD8 co-receptor, i.e. the most immature are CD4CD8 [double-negative (DN)], immature are CD4+ CD8+ [double positive (DP)] and mature are either CD4 CD8+ [CD8 single positive (SP)] or CD4+ CD8 (CD4 SP) cells [reviewed in (27)]. The CD4 SP thymocytes can be divided further into two subpopulations based on their surface expression of heat-stable antigen (HSA) and Qa-2 (2831). HSA Qa-2+ CD4 SP thymocytes are reported to be fully matured cells in terms of anti-TCR-induced proliferative responses. HSA+ Qa-2 CD4 SP thymocytes, however, are less mature and poorly responsive to stimulation by immobilized anti-TCR mAb even in the presence of IL-2 or syngeneic antigen-presenting cells (APC) (31). Both HSA+ and HSA CD4 SP thymocytes differentiate from immature CD4+CD8+ thymocytes (32), and express high levels of TCRCD3 complex on the cell surface (31). Similarly, only a portion of human CD4 SP thymocytes is reported to be immunocompetent in terms of cytokine production (33,34). What remains to be clarified is the reason for the difference in the responsiveness between these two CD4 SP thymocyte subpopulations. In addition, the immunocompetence of these phenotypically distinct CD4 SP thymocytes in terms of antigen-induced T cell functions, such as the differentiation into Th1/Th2 cells, has not been formally addressed.
Here, we have employed CD4 SP thymocytes from ovalbumin (OVA)-specific TCR Tg mice that were fractionated into HSA+ and HSA cells, to investigate their capacity to differentiate into Th1 and Th2 cells after antigenic stimulation. HSA CD4 SP thymocytes were found to differentiate into either Th1 or Th2 cells in a manner similar to that of naive splenic CD4 T cells. We found that the maturation of the capacity for Th1 cell differentiation slightly lags that for Th2 differentiation during the transition from HSA+ CD4 SP thymocytes to HSA CD4 SP thymocytes. This lag may be due to the developmental programming of the IL-12R functions.
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Methods
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Mice
C57BL/6 (B6) and BALB/c mice were purchased from Clea (Tokyo, Japan). Anti-OVA TCR
ß transgenic (DO.11.10 Tg) mice (68 weeks old) (35) on a (B6 x BALB/c)F1 background were used. All mice used in this study were maintained under specific pathogen-free conditions.
Reagents
The reagents used in this study were as follows: FITC-conjugated anti-CD4 mAb (GK1.5FITC), anti-HSAFITC (J11dFITC), phycoerythrin (PE)-conjugated anti-CD4 mAb (GK1.5PE), anti-CD44PE (IM7PE), anti-Thy-1 mAb (30H.12), anti-IFN-
FITC (XMG1.2FITC) and anti-IL-4PE (11B11PE) were purchased from BD PharMingen (San Diego, CA). Anti-FcR
II and III mAb (2.4G2), unconjugated anti-IL-4 mAb (11B11), anti-NK1.1 mAb (PK136), and an IgM class anti-CD8 mAb (83-12-5) were used as culture supernatants. Recombinant mouse IL-12 was purchased from Genzyme (Cambridge, MA) and recombinant mouse IL-4 was from Toyobo (Osaka, Japan). The OVA peptide (residues 323339; ISQAVHAAHAEINEAGR) was synthesized by BEX (Tokyo, Japan).
Cell purification
Thymocytes from DO.11.10 Tg mice with (B6 x BALB/c)F1 background were incubated on ice with culture supernatants of both anti-NK1.1 (PK136, 20% v/v) and anti-CD8 (83-12-5, 20% v/v) mAb followed by treatment with rabbit complement (Cedarlane, Hornby, Ontario, Canada; 1:14) at 37°C for 30 min. (B6 x BALB/c)F1 background DO.11.10 mice were used because NK T cell depletion can be done with anti-NK1.1 mAb. Thymic NK T cells (3638) were eliminated by complement-dependent cytotoxic reactions (39) and the residual NK1.1+ cells in the CD4 thymocyte subpopulation were <0.5%. After washing extensively, the treated cells were stained with anti-CD4, anti-CD8 (53.6-72) and anti-HSA mAb, and then subjected to a FACS Vantage cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA). The anti-CD8 staining by 53.-72 mAb was not affected by the binding of another anti-CD8 mAb, 83-12-5 mAb (data not shown). HSA+ and HSA CD4 SP thymocytes were obtained. Splenic CD4 T cells with naive phenotype (CD44low/) were isolated also from DO.11.10 Tg mice with (B6 x BALB/c)F1 background by cell sorting as described (22). APC were prepared from BALB/c (H-2d) splenocytes by cytotoxic killing with anti-Thy1 mAb and irradiated (3000 rad).
T cell stimulation culture
Proliferative responses of sorted HSA+ or HSA CD4 SP thymocytes and naive splenic CD4+ T cells were determined by measuring [3H]thymidine uptake as described (22). The amounts of a specific cytokine produced in the supernatant of 24 h stimulation cultures were determined by appropriate standard ELISA assays as described (40).
In vitro Th1/Th2 cell differentiation culture and intracellular staining
In vitro Th1/Th2 cell differentiation cultures with DO.11.10 Tg T cells were performed as described (22). In brief, 1 x 104 sorted naive CD4+ T cells were stimulated with a various concentrations of antigenic OVA peptide (0.3.0 µM) and 2.5 x 105 irradiated T-depleted APC in the presence or absence of selected concentrations of IL-4 or IL-12 for 5 days. For IL-12-induced Th1 cell differentiation culture, anti-IL-4 mAb (11B11, 10% culture supernatant) was added. No exogenous IL-2 was added in the culture. Intracellular staining of IL-4 and IFN-
was performed as described (22). Briefly, the cultured T cells were re-stimulated with anti-TCR mAb (H57-597; 30 µg/ml) for 6 h in the presence of 2 µM monensin, which inhibited the secretion of newly produced protein. Then, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton X-100 (in 50 mM NaCl, 5mM EDTA and 0.02% NaN3, pH7.5) for 10 min. on ice. After blocking with 3% BSA in PBS for 15 min, cells were incubated on ice for 45 min with anti-IFN-
FITC (XMG1.2FITC) and anti-IL-4PE (11B11PE) which were purchased from PharMingen. Flow cytometry analysis was performed on FACSort and results were analyzed using CellQuest software (Becton Dickinson).
RT-PCR
RT-PCR was done by a standard method as described (41). Total RNA was prepared using Trizol (Gibco/BRL, Rockville, MD). Reverse transcription was done using Superscript II (Gibco/BRL). The primers used were as follows IL-12Rß2, 5'-ACATCCAATAAGCAGCCTACAGCC-3' and 5'-GGCCATGC CATCAGGAGATTATCC-3'; ß-actin, 5'-GTGGGGGCTATTTT ATTGATG-3' and 5'-GAGAGGGAAATCGTGCGTGA-3'; ST AT4, 5'-AGCCAACATGCCTATCCAGGGACC-3' and 5'-CTC CTGTAGTTTCTCCAGTTGCTG-3'; T-bet, 5'-TGAAGCCCAC ACTCCTACCC-3' and 5'-GCGGCATTTTCTCAGTTGGG-3'; GATA-3, 5'-GGAGGCATCCAGACCCGAAAC-3' and 5'-ACC ATGGCGGTGACCATGC-3'.
Immunoprecipitation and immunoblotting
Tyrosine phosphorylation of STAT4 was detected by immunoprecipitation with anti-STAT4 antiserum (R & D Systems, Minneapolis, MN) and subsequent immunoblotting with anti-phosphotyrosine mAb (RC20; Transduction Laboratories, Lexington, KY) as described previously (23).
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Results
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Antigen-induced proliferative responses and IL-2 production of HSA+ and HSA CD4 SP thymocytes
Our study aims to elucidate the stage at which CD4 SP thymocytes acquire the ability to differentiate into Th1 or Th2 cells after antigenic stimulation. We analyzed the antigen-induced proliferative responses and IL-2 production of HSA+ and HSA CD4 SP thymocyte subpopulations using OVA-specific TCR
ß (DO.11.10) Tg thymocytes with (B6 x BALB/c) F1 background. Thymocytes were treated with anti-CD8 and anti-NK1.1 mAb and complement. The resulting CD8 NK1.1 thymocytes were subjected to cell sorting after staining with anti-CD4, anti-CD8 and anti-HSA mAb (Fig. 1A). Residual NK1.1+ cells in the CD4+ thymocyte population were <0.1% (Fig. 1A, right panel). The sorted NK1.1 HSA+ CD4 SP and NK1.1 HSA CD4 SP thymocytes were cultured with graded doses of antigenic peptide and H-2d BALB/c splenic APC. The proliferative responses of HSA+ CD4 SP thymocytes were equivalent to or slightly higher than that of the HSA CD4 SP thymocytes or naive splenic CD4 T cells. Thus, the capacity for antigen-induced proliferation appears to have been acquired at the HSA+ CD4 SP thymocyte stage (Fig. 1B). Concurrently, proliferative responses induced by immobilized anti-TCR mAb were measured in parallel and significantly decreased responses were observed in HSA+ CD4 SP thymocytes (Fig. 1C). Next, IL-2 production of the sorted HSA+ and HSA CD4 SP thymocytes was assessed after stimulation with antigenic peptides and APC. As can be seen in Fig. 1(D), the production of IL-2 in HSA+ CD4 SP thymocytes was not decreased, but significantly enhanced. This result is consistent with observations previously reported (42). Our experiments with mature CD4 naive T cells suggest that in the anti-TCR stimulation culture, mature T cells do not consume a large amount of IL-2 up to 48 h (data not shown). This suggests that the decreased IL-2 production is not due to the high level consumption of produced IL-2. The production of IL-4 from HSA+ and HSA CD4 SP thymocytes was detectable, but at low levels, and it was lower in HSA+ CD4 SP thymocytes (data not shown). The early intracellular signaling events, such as anti-TCR mAb-induced calcium mobilization, are induced equivalently in HSA+ CD4 SP thymocytes compared to freshly prepared CD4 T cells (data not shown). Thus, both HSA+ and HSA CD4 SP thymocytes show similar patterns of responsiveness by these parameters.

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Fig. 1. Proliferative responses and IL-2 production of HSA+ and HSA CD4 SP thymocytes. (A) Purification of HSA+ and HSA CD4+ thymocytes. Thymocytes were first treated with anti-CD8 mAb (83-12-5) and anti-NK1.1 mAb (PK136) followed by complement. The treated cells were stained with anti-CD4, anti-CD8 (53.6-72) and anti-HSA mAb. CD4/CD8 profiles and CD4/HSA profiles are shown. The gates for cell sorting (HSA+CD4+ and HSACD4+) are depicted. Re-analyses CD4/HSA profiles of the sorted cells are also shown with the original gates for sorting. In addition, NK1.1 staining profiles of CD4 SP thymocytes after anti-CD8 treatment (left) and anti-CD8 + anti-NK1.1 treatment (right) are also shown. Indirect staining was performed. Background control staining represents second antibody (anti-MIgFITC) alone. (B) Flow cytometry-sorted naive CD4 splenic T cells and HSA+ and HSA CD4 SP thymocytes (1 x 105) were stimulated with various doses of antigenic peptides and 2.5 x 105 irradiated BALB/c APC. The mean values + SD of [3H]thymidine uptake of triplicate cultures are shown. The results are representative of three independent experiments. (C) Flow cytometry-sorted naive CD4 splenic T cells and HSA+ and HSA CD4 SP thymocytes (1 x 105) were stimulated with 10 µg/ml of antigenic peptides and 2.5 x 105 irradiated BALB/c APC or with immobilized anti-TCR ß mAb. The mean values + SD of [3H]thymidine uptake of triplicate cultures are shown. (D) The amounts of IL-2 in the culture supernatant of stimulation cultures as (A) measured by ELISA are shown.
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Antigen-induced Th1/Th2 cell differentiation of HSA+ and HSA CD4 SP thymocytes
Next, the ability of HSA+ and HSA CD4 SP thymocytes to differentiate into Th1 or Th2 subpopulations was assessed using a well-documented in vitro OVA peptide-induced Th1/Th2 cell differentiation system (22,43). The sorted NK1.1 HSA+ CD4 SP and NK1.1 HSA CD4 SP thymocytes from DO.11.10 Tg thymocytes with (B6 x BALB/c)F1 background were cultured with graded doses of antigenic peptide, and T cell-depleted and irradiated BALB/c APC. No exogenous cytokine was added. As shown in Fig. 2(A), the efficiency in the generation of Th2 cells from HSA+ CD4 SP cells was significantly lower than that of naive splenic CD4 T cells, particularly in the groups stimulated with lower peptide concentrations (compare upper and middle panels). However, the levels of Th2 cell generation from HSA CD4 SP thymocytes were almost equivalent (compare upper and bottom panels). These results suggest that HSA CD4 SP thymocytes have already acquired functional maturation for Th2 cell differentiation that naive splenic CD4 T cells demonstrate.
The efficiency for Th2 cell differentiation was further examined in the presence of exogenous IL-4 (Fig. 2B). The levels in Th2 cell differentiation of HSA+ CD4 SP thymocytes were significantly lower than those of the other two groups at only 0.3 µM peptide concentration, but equivalent at higher doses (Fig. 2B). The decreased generation of Th2 cells at 0.3 µM peptide stimulation was not rescued by the presence of 100 U/ml of IL-4 (data not shown), suggesting there exist some intrinsic deficiency in HSA+ CD4 SP thymocytes. Taken together with the results in Fig. 2(A), these results also suggest that the Th2 cell differentiation of HSA+ CD4 SP thymocytes is partly augmented by the presence of exogenous IL-4. As can be seen in the bottom panels in Fig. 2(B), the levels of Th2 cell differentiation in HSA CD4 SP thymocytes were equivalent or even slightly greater than those in naive splenic CD4 T cells.
Concurrently, the efficiency for Th1 cell differentiation was examined. Significant levels of Th1 cell generation were induced in the DO.11.10 Tg/OVA stimulation culture system when recombinant IL-12 and anti-IL-4 mAb were added in the culture (22) (Fig. 2C, top panels). Under these conditions, the levels in IL-12-induced Th1 cell differentiation of the HSA+ CD4 SP thymocytes were examined. As shown in Fig. 2(C, middle panels), the generation of Th1 cells was very poor at all doses of the antigenic peptide. Similar results were seen in the presence of 100 U/ml of IL-12 (data not shown). In contrast, HSA CD4 SP thymocytes differentiated into Th1 cells at almost equivalent levels to those of naive splenic CD4 T cells, although a decrease was detected at 0.3 µM peptide stimulation. The decreased generation of Th1 and Th2 cells appeared not to be due to the increased cell death of the developing Th1 or Th2 cells (Fig. 2D). Taken collectively, these results indicate that the ability to differentiate into Th1 and Th2 cells is fully acquired at the HSA CD4 SP thymocyte stage.
Expression levels of IL-12Rß2 in HSA+ and HSA CD4 SP thymocytes and naive splenic CD4 T cells
Since we had observed a dramatic difference in the capacity of HSA+ and HSA CD4 SP thymocytes to differentiate into Th1 cells, we sought to clarify the molecular basis that could account for the difference. IL-12R expression is critical for Th1 cell differentiation and, therefore, we assessed the levels of IL-12R expression by RT-PCR with specific primers for IL-12Rß2. As shown in Fig. 3(A), the expression levels of IL-12Rß2 in the freshly prepared HSA+ and HSA CD4 SP thymocytes as well as naive T cells were marginal. The sorted HSA+ and HSA CD4 SP thymocytes were stimulated with anti-TCR mAb and IL-2 for 48 h, and the IL-12Rß2 expression was determined by RT-PCR. The levels in IL-12Rß2 expression in HSA+ CD4 SP thymocytes were very low; the difference in the levels of expression between HSA+ and HSA CD4 SP thymocytes was >5-fold (Fig. 3B). Thus, the induction of IL-12Rß2 was significantly weaker in HSA+ CD4 SP thymocytes. In naive splenic CD4 T cells, the induction of IL-12Rß2 mRNA was higher than that in HSA CD4 SP thymocytes.

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Fig. 3. IL-12Rß2 expression and IL-12-induced STAT4 phosphorylation in HSA+ and HSA CD4 SP thymocytes. Flow cytometry-sorted HSA+ and HSA CD4 SP thymocytes and naive splenic CD4 T cells (naive T) were stimulated with anti-TCR mAb and IL-2 for 48 h. Total RNA was extracted from freshly prepared cells (A) or the stimulated cells (B). The mRNA levels of IL-12Rß2 and ß-actin were determined by RT-PCR. Three-fold serial dilutions of template cDNA were performed. The ratios of the band intensities at the highest cDNA concentration (IL-12Rß2/ß-actin) were calculated by a densitometer, and were 0.18, 0.24 and 0.04 (A, left to right), and 0.14, 1.00 and 1.74 (B, left to right). (C) Flow cytometry-sorted HSA+ and HSA CD4 SP thymocytes were stimulated with anti-TCR mAb and IL-2 for 2 days. The cultured cells were stimulated with IL-12 for 5 min, and phosphorylated STAT4 molecules were detected by immunoprecipitation with anti-STAT4 mAb and anti-phosphotyrosine immunoblotting. Arbitrary densitometric units are shown under each band.
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IL-12-induced tyrosine phosphorylation of STAT4 in HSA+ and HSA CD4 SP thymocytes
Next, we assessed the IL-12R function in the HSA+ CD4 SP thymocytes. Sorted HSA+ and HSA CD4 SP thymocytes (1 x 106 cells) were stimulated with anti-TCR mAb and IL-2 for 2 days, and then IL-12-induced phosphorylation of STAT4 in the cultured cells was determined by immunoprecipitation with anti-STAT4 mAb and anti-phosphotyrosine immunoblotting. IL-12-induced tyrosine phosphorylation of STAT4 was detected in HSA CD4 SP thymocytes, but it was significantly lower in HSA+ CD4 SP thymocytes (Fig. 3C). These results suggest that the IL-12-induced STAT4 activation, which is known to be required for Th1 cell differentiation, is inefficiently triggered in the HSA+ CD4 SP thymocytes, probably as a consequence of the deficiency in IL-12R expression.
Transcriptional up-regulation of T-bet and STAT4 after TCR stimulation with IL-12 in HSA+ and HSA CD4 SP thymocytes
Finally, we compared the levels of transcription of STAT4, T-bet and GATA3 in the HSA+ and HSA CD4 SP thymocytes after stimulation with anti-TCR, IL-2 and IL-12. Total RNA was prepared from fresh and stimulated HSA+ and HSA CD4 SP thymocytes, and subjected to semiquantitative RT-PCR analysis (Fig. 4A and B). A 2-fold up-regulation of STAT4 transcription was detected in naive splenic CD4 T cells; however, STAT4 up-regulation was not detected in the HSA+ CD4 SP thymocytes (Fig. 4C). The induction of T-bet mRNA was
2-fold in both naive splenic CD4 T cells and HSA CD4 SP thymocytes, whereas the induction was <50% in HSA+ CD4 SP thymocytes as compared to the former. The transcription of GATA-3 is reported to be down-regulated in developing Th cells under the Th1-skewed culture conditions (44). Interestingly, down-regulation of GATA-3 mRNA was detected in all three subpopulations. These results suggest that the capacity of IL-12-induced up-regulation of STAT4 and T-bet is not fully developed in HSA+ CD4 SP thymocytes.

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Fig. 4. Transcriptional levels of STAT4, T-bet and GATA-3 in HSA+ and HSA CD4 SP thymocytes stimulated with anti-TCR mAb, IL-2 and IL-12. Flow cytometry-sorted HSA+ and HSA CD4 SP thymocytes were cultured with immobilized anti-TCR mAb, IL-2 (25 U/ml) and IL-12 (100 U/ml) for 2 days. Total RNA was extracted from freshly prepared and stimulated cells. The mRNA levels of STAT4, T-bet, GATA3 and ß-actin were determined by a semiquantitative RT-PCR analysis with 3-fold serial dilution of template cDNA (A and B). (C) The ratios of the band intensities at the highest cDNA concentration (STAT4/ß-actin, T-bet/ß-actin and GATA-3/ß-actin) were calculated by a densitometer and arbitrary densitometric units are depicted with SE bars. Three independent experiments were performed with similar results.
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Discussion
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In this report, we addressed the functional maturation of developing CD4 SP thymocytes by investigating the ability of these cells to differentiate into Th1/Th2 cells after antigenic stimulation. We show that functional maturation for differentiating into either Th1 or Th2 cells is acquired at the HSA CD4 SP thymocyte stage. In addition, the Th1 cell differentiation of HSA+ CD4 SP thymocytes occurs poorly, and this appears to be due to underdeveloped IL-12R expression and function.
In CD4 SP thymocytes, at least two distinct subpopulations have been identified, i.e. less mature HSA+ Qa-2 and mature HSA Qa-2+ cells (2831). Reduced proliferative capacity was noted in HSA+ Qa-2 cells when immobilized anti-TCR mAb was used for the stimulation of such cells (31,42). The response was not augmented by the addition of exogenous IL-2, but it could be partially restored by stimulation with allogenic cells or anti-TCR mAb on the surface of syngeneic APC, suggesting additional co-stimulatory signals may be required for full proliferation of HSA+ Qa-2 cells. In addition, using immobilized anti-TCR mAb for stimulation, HSA+ Qa-2 cells were reported to produce considerable amounts of IL-2, but not IL-4 or IFN-
, whereas HSA cells produce large amounts of IL-4 and IFN-
as well (42). In this report, these issues were re-evaluated using a peptide stimulation system, i.e. thymocytes were prepared from OVA-specific DO.11.10 TCR
ß Tg mice, and the specific antigenic peptide and mature splenic APC were used for stimulation. In contrast to the results obtained using immobilized anti-TCR mAb stimulation, proliferation of HSA+ CD4 SP thymocytes induced by peptide stimulation was not impaired (Fig. 1B). Our results, however, are not contradictory to the idea that the co-receptor dependency is different in these two CD4 SP subpopulations (31), although no direct evidence supporting this idea has been reported to date. The IL-2 production by HSA+ CD4 SP thymocytes was not impaired (Fig. 1D). These results are consistent with the results of studies with anti-TCR stimulation (42).
Another line of experiments performed by Nikolic-Zugic et al. (30,45,46) used anti-CD8 mAb for panning and identified two subsets of TCRhighCD4high SP cells (CD8 and CD8ow). The immature CD8lowCD4high cells were TCR highCD24 intCD44 high and did not produce significant amounts of IL-2 or IL-4 (30). This population appeared to be distinct from HAS+ CD4 SP population identified by Bendelac et al. (42), which produces a large amount of IL-2. More mature CD8CD4high cells were also TCR highCD24 intCD44 high and produced both IL-2 or IL-4 (45). Our HSA (CD24)+ cells were prepared by a similar method as employed by Bendelac et al. (42) and produced high levels of IL-2 (Fig. 1D). Both the HSA+ and HSA CD4 SP thymocytes express high and equivalent levels of
ß TCR on their surface (unpublished observation). Thus, our HSA CD4 SP cells may contain some of the CD8lowCD4high cells and mature CD8CD4high cells reported by Nikolic-Zugic et al. (45). However, it appears to be clear that the most mature immunocompetent cells belong to the HSA CD4 SP cell population and CD8low CD4high cell population, because HSA CD4 SP cells were fully competent in Th1/Th2 cell differentiation (Fig. 2) and the CD8 CD4high cells demonstrated mature T cell function such as the induction of lethal graft versus host disease (45).
More interestingly, a dramatic difference between HSA+ and HSA CD4 SP thymocytes in their capacity to initiate IL-12-induced Th1 cell differentiation was observed (Fig. 2C). An important consequence of T cell antigen recognition, the differentiation into Th1/Th2 cells, requires both TCR-mediated signals (3,22) and specific cytokine receptor-mediated signals, such as IL-12R-mediated STAT4 activation for Th1 and IL-4R-induced STAT6 activation for Th2 cell differentiation (1,2,4). In the HSA+ CD4 SP thymocytes, the early TCR-mediated signaling events, such as anti-TCR mAb-induced calcium influx, are fully programmed (unpublished observation). The peptide-induced proliferative responses and IL-2 production were not reduced (Fig. 1). Thus, it is most reasonable to ascribe the reduced capacity of HSA+ CD4 SP thymocytes to undergo Th1 cell differentiation to the immaturity of IL-12 cytokine receptor function. We show here that the expression of IL-12R and IL-12-mediated STAT4 phosphorylation was significantly low in the HSA+ CD4 SP thymocytes (Fig. 3). IL-12-induced transcriptional up-regulation of STAT4 and T-bet was not very efficient in HSA+ CD4 SP thymocytes (Fig. 4). Thus, the weak IL-12R-mediated signal transduction may explain in part the poor Th1 cell differentiation capacity. The reduced capacity in STAT4 up-regulation was observed in both HSA+ and HSA thymocytes, but the reduced up-regulation of T-bet is only prominent in the HSA+ population. The expression of STATs is generally ubiquitous and, while its up-regulation is important, it may not be indispensable for Th1 cell generation. The induction of T-bet, however, may be more critical for the Th1 cell differentiation. Thus, our results suggest that the reduced capacity in up-regulation of T-bet is a critical factor for the decreased Th1 cell generation in HSA+ CD4+ thymocytes. On the other hand, differentiation of Th2 cells from HSA+ CD4 SP thymocytes seemed to be more matured as compared to the Th1 cells (Fig. 2A and B). Taken together, the ability to differentiate into Th2 cells is acquired during the HSA+ CD4 SP thymocyte stage, whereas the process is slower in the case of Th1.
We detected significant GATA3 transcription by RT-PCR analysis in both HSA+ and HSA CD4 SP thymocytes as well as naive CD4 T cells (47) (Fig. 4). Interestingly, significant GATA3 down-regulation was detected in the HSA+ CD4 SP thymocytes after stimulation with anti-TCR, IL-2 and IL-12 (see Fig. 4C, bottom panel). The down-regulation of GATA3 in developing Th1 cells is IL-12 mediated and STAT4 dependent (44). Here, a significant decrease in the IL-12-induced STAT4 phosphorylation was observed in the HSA+ CD4 SP thymocytes (Fig. 3C). It is possible that GATA3 down-regulation is very sensitive to the activation of IL-12-mediated signaling and occurs efficiently even with a low level of activation. Alternatively, the molecular requirements for the down-regulation of GATA3 transcription may be distinct between HSA+ CD4 SP thymocytes and naive CD4 T cells.
In summary, the results demonstrated in this report suggest that one of the most important functions of immunocompetent T cells, the ability to differentiate into Th1/Th2 cells, is almost fully acquired at the HSA CD4SP thymocyte stage. In addition, the maturation of specific cytokine receptor signaling programs that help direct differentiation into Th1 and Th2 cells proceeds to completion in the transition from the HSA+ to the HSA CD4 SP thymocyte stages.
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Acknowledgements
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The authors are grateful for Drs Ralph Kubo and Steven Ziegler for helpful comments and constructive criticisms in the preparation of the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grant-in-Aid for Scientific Research, Priority Areas Research #13218016 and #12051203, Scientific Research C #12670293, and Special Coordination Funds for Promoting Science and Technology), the Ministry of Health, Labor and Welfare (Japan), and Human Frontier Science Program Research Grant (RG00168/2000-M206).
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Abbreviations
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APCantigen-presenting cell
B6C57BL/6
DNdouble negative
DO.11.10 TgOVA-specific
ßTCR transgenic
DPdouble positive
HSAheat-stable antigen
IL-12RIL-12 receptor
MAPKmitogen-activated protein kinase
OVAovalbumin
PEphycoerythrin
SPsingle positive
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References
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