Immunologic immaturity, but high IL-4 productivity, of murine neonatal thymic CD4 single-positive T cells in the last stage of maturation

Madoka Koyanagi1, Ken’ichi Imanishi1, Yutaka Arimura1, Hidehito Kato1, Junji Yagi1 and Takehiko Uchiyama1

1 Department of Microbiology and Immunology, School of Medicine, Tokyo Women’s Medical University,8-1 Kawada-Cho, Shinjuku-Ku, Tokyo 162-8666, Japan

Correspondence to: T. Uchiyama; E-mail: tuchi{at}research.twmu.ac.jp
Transmitting editor: S. Koyasu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To determine the levels of maturation and differentiation of murine CD4 single-positive (SP) T cells, we compared the secondary responses of staphylococcal enterotoxin A (SEA)-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts prepared from whole or heat-stable antigenlow CD4 SP T cells. Proliferative responses upon re-stimulation with SEA were strong in adult splenic CD4 SP T cell blasts, but quite weak in neonatal thymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2 production was weaker in neonatal thymic blasts than in the adult splenic CD4 SP T cell blasts. In contrast, SEA-induced IL-4 production was high in neonatal thymic CD4 SP T cell blasts, and low in adult splenic and thymic CD4 SP T cell blasts. Expression of GATA-3, that directs production of IL-4 in T cells, examined at protein and mRNA levels, was higher in neonatal thymic cells than in adult thymic and splenic cells. These results suggest that neonatal and adult thymic CD4 SP T cells in the final stage of maturation are relatively immature compared with adult splenic CD4 SP T cells. The cytokine production profile of neonatal thymic CD4 SP T cells suggests that they are inclined towards a Th2 response.

Keywords: anergy, cytokine, neonate, Th1/Th2 cell, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During the process of T cell maturation, progenitor T cells derived from the fetal liver or bone marrow enter into the thymus as immunologically incompetent CD4CD8 double-negative T cells and differentiate there into immunologically competent CD4 or CD8 single-positive (SP) T cells (1,2). Murine thymic SP T cells spend an average of 14 days in the medulla (3), during which they down-regulate the heat-stable antigen (HSA), and up-regulate Qa-2 and CD45RB (4). Two million cells leave the thymus each day (5,6). Most of these recent thymic emigrants are CD69 and CD62Lhigh. In 1991, Bendelac and Schwartz reported a functional difference between thymic CD4 SP T cells and peripheral CD4 SP T cells in mice (7). They showed that thymic CD4 SP T cells secrete high levels of a variety of lymphokines including IL-4, IL-5, IL-10 and IFN-{gamma} upon primary stimulation as compared with peripheral CD4 SP T cells. Later, several lines of evidence demonstrated that this high inducibility of lymphokines in thymic CD4 SP T cells is largely attributable to a specialized population of TCR {alpha}ß+ T cells, NK T cells which express NK1.1 and a highly restricted TCR with polyclonal Vß2+, Vß7+ or Vß8+ ß chains and invariant {alpha} chain composed of V{alpha}14 and J{alpha}281 (810). However, recently, Chen et al. reported that NK1.1 thymic CD4 SP T cells produce high levels of IL-4, suggesting that conventional thymic CD4 SP T cells may also function differently from peripheral CD4 SP T cells (11).

Human thymic CD4 SP T cells consist of two populations—CD1a+ and CD1a fractions. The latter are thought to be in a final stage of maturation in the thymus (12). Thus, human CD1a CD4 SP T cells supposedly correspond to murine HSAlow, Qa-2high and CD45RBhigh CD4 SP T cells in their level of maturity. We previously found that human thymic CD1a CD4 SP T cells and cord blood CD4 SP T cells, a large part of which are presumed to be recent thymic emigrants, were susceptible to anergy induction by the superantigen (SAG) toxic shock syndrome toxin-1 (TSST-1) (13,14). In contrast, adult peripheral blood CD4 SP T cells exhibited enhanced responses. Thus, CD1a CD4 SP T cells and cord blood CD4 SP T cells are still immunologically immature compared with adult peripheral blood CD4 SP T cells, suggesting the existence of a post-thymic maturation process in human T cells (13). Moreover, we hypothesize that the immunological immaturity seen in human thymic CD4 SP T cells is a common feature of thymic CD4 SP T cells, irrespective of the animal species.

In this study, to clarify further the functional differences between maturational stages and to prove our hypothesis, we analyzed mainly the secondary responses of CD4 SP T cell blasts, obtained by stimulating thymic and splenic HSAlow CD4 SP T cells of C57BL/6 mice of various ages with the SAG, staphylococcal enterotoxin A (SEA). SEA stimulates T cells bearing Vß3+ and Vß11+ ß chains, but not Vß2+, Vß7+ and Vß8+ ß chains, which are related to NK T cells (810). SEA-reactive T cells thus do not induce NK T cells and are suitable for the analysis of conventional T cells. We found that SEA-induced proliferative responses were much lower in neonatal and adult thymic CD4 SP T cell blasts than in adult splenic CD4 SP T cell blasts. Notably, however, IL-4 production and expression of GATA-3 were much higher in neonatal thymic CD4 SP T cell blasts than adult splenic CD4 SP T cell blasts. These findings indicate that murine thymic CD4 SP T cells in the final stage of maturation, irrespective of the age of the mice, are immature compared with adult splenic CD4 SP T cells and that thymic CD4 SP T cells seem to decrease the ratio of Th2 cells from neonatal to adult stages.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
C57BL/6 mice were bred in the Animal Care Unit of the Department of Microbiology and Immunology, Tokyo Women’s Medical University, School of Medicine. Mice of various ages ranging from 0 days to 8 weeks were used as donors of thymic and splenic CD4 SP T cells.

Reagents and antibodies
SEA was purchased from Toxin Technology (Sarasota, FL). RPMI 1640 culture medium contained 100 µg/ml streptomycin, 100 U/ml penicillin, 10% FCS and 5 x 10–5 M 2-mercaptoethanol. Human rIL-2 was kindly provided by Dainippon Pharmaceutical (Osaka, Japan). Phorbol myristate acetate (PMA) was obtained from Nacalai Tesque (Kyoto, Japan), and the calcium ionophore A23187 was purchased from Sigma (St Louis, MO).

The mAb to CD8 (83.12.5), Thy1.2 (HO13), I-Ab/d (28-16-8s), CD25 (7D4), Vß3 (KJ25), Vß6 (RR4-7) and Vß11 (RR3-15) have been described (15). Anti-CD25, -Vß3, -Vß6 and -Vß11 mAb were conjugated with biotin. Antibodies to IL-4 (11B11), CD44 (IM7), TCR ß (H57-597) and NK1.1 (PK136), biotinylated antibodies to CD3 (145-2C11), CD24 (HSA) (30-F1), CD45RB (16A) and IL-4 (BVD6-24G2), FITC-conjugated antibodies to CD3 (145-2C11), CD8 (Lyt-2), CD44 (1M7) and IFN-{gamma} (XMG1.2), phycoerythrin (PE)-conjugated antibodies to CD4 (RM4-5) and IL-4 (11B11), and streptavidin–CyChrome were purchased from BD PharMingen (San Diego, CA). Biotinylated antibodies to CD24 (J11d) and CD62L (MEL-14) were purchased from Beckman Coulter (Miami, FL). FITC-conjugated goat anti-mouse IgG was obtained from Biosource (Camarillo, CA), PE-conjugated streptavidin was purchased from Becton Dickinson (Mountain View, CA), antibodies to GATA-3 and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and horseradish peroxidase-conjugated secondary antibody was purchased from Tago Immunologicals (Camarillo, CA).

Cells
The preparation of splenic lymphoid cells has been described elsewhere (16,17). CD4 SP T cells were obtained by treating C57BL/6 thymic or splenic cells (2 x 107/ml) with a mixture of 83.12.5, 28-16-8s, and guinea pig complement. In indicated experiments, CD44low HSAlow T cells were isolated from these cells by cell sorting using Epics Altra (Beckman Coulter), yielding >99 % purity. T cell-depleted spleen cells used as accessory cells (AC) were obtained by treating spleen cells (2 x 107/ml) with mAb HO 13 and guinea pig complement, followed by irradiation at 3500 rad with an X-ray irradiator. At the final step, viable cells were recovered at the bottom of tubes by Percoll gradient (density 1.055) centrifugation of mAb-treated cells.

SEA-induced CD4 SP T lymphoblasts were prepared as described elsewhere (16,18). Briefly, thymic and splenic CD4 SP T cells (2 x 106/well) were stimulated with 10 ng/ml SEA in the presence of irradiated AC (2 x 106/well) for 3 days and the recovered cells were applied to Percoll density gradients (1.055 and 1.075). The large lymphoblasts obtained at the interface of 1.055 and 1.075 were expanded by incubating them in the presence of rIL-2 (100 U/ml) for 2 days in two cycles. CD4 SP T cell blasts were recovered from the interface of 1.055 and 1.075 after Percoll gradient centrifugation.

Characterization of cells by flow cytometry
All procedures for cell staining were performed on ice. TCR Vß elements and CD molecules were analyzed by two-color flow cytometry as described elsewhere (15,17). For analysis of CD3 expression versus Vß3, Vß6, Vß11, CD25 or CD44 expression, the cells were stained with biotinylated antibodies to TCR Vß elements, CD25 or CD44, and then with a combination of FITC-conjugated anti-CD3 and PE-conjugated streptavidin. For analysis of CD44 expression versus CD45RB or CD62L in CD4 SP T cells, the cells were stained with biotinylated antibodies to CD45RB or CD62L, and then with a combination of FITC-conjugated anti-CD44, PE-conjugated CD4 and CyChrome-conjugated streptavidin. For analysis of TCRß expression versus NK1.1 expression, the cells were stained with a combination of anti-NK1.1 and FITC-conjugated anti-mouse IgG, incubated with an excess of normal murine serum to block unbound binding sites on FITC-conjugated anti-IgG, washed, and incubated with a combination of biotinylated anti-TCRß mAb and PE-conjugated streptavidin. Samples containing >10,000 viable cells were analyzed with an Epics XL flow cytometer (Beckman Coulter).

T cell proliferation assays
[3H]Thymidine uptake was assayed as described elsewhere (16). CD4 SP T cells and SEA-induced CD4 SP T cell blasts (unprimed cells, 1 x 105/well; T cell blasts, 6 x 104/well) were stimulated with SEA in the presence of AC in a total volume of 0.2 ml in each well of a round-bottom 96-well microplate (Corning Glass Works, Corning, NY). The cultures were pulsed with 0.5 µCi (18.5 kBq) of [3H]thymidine for the last 6 or 16 h of culture. Data were presented as the average c.p.m. (+ SD) of triplicate samples.

CD4 SP T cell blasts were labeled with CFSE as described elsewhere (15,19). Briefly, SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts were stained with CFSE (10 µM) for 10 min at 37°C and incubated on ice for 5 min to terminate the reaction. The cells (2 x 106/culture) were cultured for 3 days with SEA in the presence of irradiated AC and stained with biotinylated anti-Vß3 or anti-Vß11 mAb, followed by a combination of PE-conjugated anti-CD4 and streptavidin–CyChrome. After gating for Vß3+ and Vß11+ CD4 SP T cells, cell divisions were analyzed by flow cytometry.

Cytokine assays
Cytokine secretion was assayed by incubating T cells (5 x 105/well) with various concentrations of SEA, in the presence of AC, in a total volume of 1 ml in each well of a 48-well culture plate (Corning Glass Works) and collecting the culture supernatants at various times. IL-2 activity in these supernatants was determined using IL-2-dependent CTLL-2 cells, as described elsewhere (20); data presented as units of IL-2/ml. Secreted IL-4 was measured by ELISA (BD PharMingen) (15), with anti-mouse IL-4 (11B11) and biotinylated anti-mouse IL-4 (BVD6-24G2) serving as coating and detecting mAb respectively, and using peroxidase-conjugated streptavidin (Vector, Burlingame, CA) and substrate solution (TMB; Dako, Glostrup, Denmark) for color development. Standard curves were generated using mouse rIL-4 (BD PharMingen). IFN-{gamma} was measured with an Opt EIA Mouse IFN-{gamma} set (BD PharMingen) according to the manufacturer’s instructions.

Intracellular cytokine staining was assayed by flow cytometry (15). CD4 SP T cell blasts (2 x 106/well) were stimulated for 16 h with 10 ng/ml SEA in the presence of AC (2 x 106/well), and re-stimulated for 5 h with 10 ng/ml PMA and 0.4 µM calcium ionophore A23187 in the presence of GolgiStop (BD PharMingen). The harvested cells were stained with a combination of biotinylated anti-Vß3 or -Vß11 and CyChrome-conjugated streptavidin. The cells were fixed and permeabilized using Cytofix/Cytoperm (BD PharMingen), stained with FITC-conjugated anti-IFN-{gamma} and PE-conjugated anti-IL-4, and analyzed with an Epics XL flow cytometer.

Analysis of GATA-3 expression
For western blot analysis, 2 x 106 cells were solubilized in 30–50 µl Leammli’s sample buffer containing 4% SDS, boiled for 5 min and sonicated for 5 min to shear high-mol.-wt DNA. The cell lysates (20 µg protein/sample) were separated by 10% SDS–PAGE, transferred to nitrocellulose membranes and reacted with anti-GATA-3 or anti-actin antibodies. After reaction with the horseradish peroxidase-coupled secondary antibody, the blots were visualized with a chemiluminescence substrate (Santa Cruz Biotechnology) according to the manufacturer’s instructions.

For RT-PCR, total RNA was extracted from cells as described (21), and cDNA was synthesized by incubating these RNAs with RAV-2 reverse transcriptase (Takara Biomedicals, Osaka, Japan) and random primers for 2 h at 42°C, and terminating the reactions by heating at 65°C for 10 min. Competitive PCR was performed using specific primer pairs for GATA-3 and ß-actin; the competitors against the PCR reaction for GATA-3 and ß-actin were designed to yield different-sized PCR products and were prepared using reagents from the Competitive DNA Construction Kit (Takara Biomedicals). Using a series of 10-fold dilutions of the DNA competitors, their optimal concentrations for the measurement of GATA-3 and ß-actin expression levels were determined. For each PCR reaction, cDNA was amplified in the presence of 104 copies of competitor for GATA-3 or 106 copies of competitor for ß-actin, using a protocol consisting of 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, followed by a final extension of 72°C for 10 min. The GATA-3-specific primers were 5'-CACGCGGCCGCGATCCAGCACAGAAGGCAGG-3' (sense) and 5'-CGATCTAGACTAACCCATGGCGGTGACCA-3' (antisense), while the ß-actin-specific primers were 5'-TGA AGCTGTGCTATGTTGCT-3' (sense) and 5'-TCAGTAACAG TCCGCCTAGA-3' (antisense).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Primary responses of thymic and splenic CD4 SP T cells from neonatal and adult mice to in vitro stimulation with SEA
Neonatal thymic, adult thymic and adult splenic CD4 SP T cell populations were stimulated with SEA in the presence of AC for 72 h, and examined for [3H]thymidine uptake response. Adult thymic CD4 SP T cells exhibited a substantial response at 100 pg/ml of SEA. The other two T cell preparations needed 30- to 100-fold higher SEA doses to exhibit similar levels of response (Fig. 1). Production of IL-2 and IFN-{gamma} was quite low in neonatal thymic CD4 SP T cells over the doses examined compared with adult splenic CD4 SP T (Table 1), while it was intermediate in adult thymic CD4 SP T cells. IL-4 production was too low to evaluate in these three T cell populations (data not shown).



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Fig. 1. [3H]Thymidine uptake in neonatal thymic, adult thymic and splenic CD4 SP T cells stimulated with SEA. Neonatal thymic (circle), adult thymic (diamond) and adult splenic (square) CD4 SP T cells (1 x 105/well) were stimulated in vitro with increasing doses of SEA in the presence of AC (1 x 105/well). Cell proliferation was determined by incorporating 0.5 µCi (18.5 kBq) of [3H]thymidine during the last 16 h of the 72-h culture. Three independent experiments were performed with similar results.

 

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Table 1. Cytokine production by CD4 SP T cell populations in response to stimulation with SEA
 
Immunologic phenotypes of SEA-induced CD4 SP T cell blasts
We next addressed the secondary response of the SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts to re-stimulation with SEA. If murine thymic CD4 SP T cells have immature traits, SEA-induced thymic CD4 SP T cells would exhibit an anergic response to re-stimulation with SEA.

The percentages of TCR Vß3+ and Vß11+ T cells, which constitute the major SEA-reactive T cell fractions (15,22) in the three SEA-induced CD4 SP T cell blasts, from around 4–5% in the unstimulated cells were around 33 and 20% respectively (Fig. 2A). The percentages of TCR Vß6+ T cells, which do not react with SEA, were around 1% in all of them. All three CD4 SP T cell blast populations expressed equally high levels of CD25 and CD44. In addition, NK1.1+ T cells, which have been reported to produce large quantities of IL-4 (8), constituted <1.2% of each of these three preparations (Fig. 2B). Collectively, these results indicate that potentially SEA-reactive CD4 SP T cell fractions are equally enriched in the three T cell blast preparations.



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Fig. 2. The immunologic phenotypes of SEA-induced CD4 SP T cell blasts derived from neonatal thymic, adult thymic and adult splenic CD4 SP T cells. Neonatal thymic, adult thymic and adult splenic CD4 SP T cells were stimulated with SEA (10 ng/ml) for 3 days, and expanded with rIL-2 for 4 days. (A) CD4 SP T cell blasts were stained with several combinations of biotinylated anti-Vß3, -Vß6, -Vß11, -CD25 or -CD44 mAb, and then stained with PE-conjugated streptavidin and FITC-conjugated anti-CD3 mAb. Numbers indicate the percentage of T cells expressing a particular Vß element. (B) CD4 SP T cell blasts were stained with a combination of anti-NK1.1 mAb/FITC-anti-rat IgG and biotinylated anti-TCRß/avidin–PE. Numbers indicate the percentage of NK1.1+ T cells, calculated by subtracting background staining. Three independent experiments were performed with similar results.

 
Proliferative response of CD4 SP T cell blast populations to re-stimulation with SEA
First, SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts were re-stimulated with SEA, and examined for [3H]thymidine uptake response. Overall, higher magnitudes of response were obtained in the secondary responses with much lower SEA doses than in the primary responses. Adult splenic CD4 SP T cell blasts exhibited a substantial response at 1 pg/ml of SEA. Neonatal and adult thymic CD4 SP T cell blasts needed a 30–100 times higher SEA dose to exhibit similar levels of response respectively (Fig. 3). When compared with adult splenic CD4 SP T cells, it is noteworthy that the proliferative capacity of adult thymic CD4 SP T cells decreased by 103–104 times after their encounter with antigenic stimulation compared with before.



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Fig. 3. [3H]Thymidine uptake of SEA-induced neonatal thymic, adult thymic and splenic CD4 SP T cell blasts upon re-stimulation with SEA. SEA-induced neonatal thymic (circle), adult thymic (diamond) and adult splenic (square) CD4 SP T cell blasts, obtained as in Fig. 2 (6 x 104/well), were stimulated in vitro with increasing doses of SEA in the presence of AC (6 x 104/well). Cell proliferation was determined by incorporating 0.5 µCi (18.5 kBq) of [3H]thymidine during the last 6 h of the 72-h culture. Three independent experiments were performed with similar results.

 
Second, to directly analyze cell division in these T cell populations, the three CD4 SP T cell blasts stained with CFSE were stimulated with 1 pg/ml of SEA for 3 days, and analyzed for the intensity of CFSE fluorescence in the Vß3+ and Vß11+ cell fractions (Fig. 4). The intensity of the CFSE fluorescence decreases by one-half in one cell division, permitting us to obtain an accurate count of the number of cell divisions. In the adult splenic T cell blasts, >60% of the Vß3+ T cell fraction underwent two or more cell divisions, while no cell division took place in 15% of these cells. In the neonatal and adult thymic T cell blasts, about 40% of the Vß3+ T cell fraction had undergone two or more divisions, while no cell division occurred in 30% of the cells in these preparations. Vß11+ T cells also exhibited a similar response pattern. It is noteworthy that cell division was greater in Vß3+ T cells than in Vß11+ T cells in all three CD4 SP T cell blasts.



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Fig. 4. In vitro cell division of SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts upon re-stimulation with SEA. SEA-induced neonatal thymic, adult thymic and splenic CD4 SP T cell blasts, prepared as described in Fig. 2, were stained with CFSE and re-stimulated in vitro with 10 pg/ml SEA in the presence of AC for 3 days. (A) Histogram of cell division of Vß3+ and Vß11+ CD4 SP T cells. (B) The percentages of Vß3+ and Vß11+CD4 SP T cells expressed as the function of cycles of cell division in neonatal thymic (circle), adult thymic (diamond) and adult splenic (square) CD4 SP T cell blasts. Three independent experiments were performed with similar results.

 
Taken together, the results shown in Figs 3 and 4 indicate that both SEA-induced neonatal and adult thymic CD4 SP T cell blasts are quite low in the proliferating activity to re-stimulation with SEA compared with SEA-induced adult splenic CD4 SP T cell blasts. We think that the lower two T cell blasts were in an anergic state, although not complete. As the proliferative response is weaker in the neonatal thymic T cell blasts than in the adult thymic T cell blasts, it seems likely that the level of anergy was slightly deeper in the former than the latter.

Preferential high IL-4 production of SEA-induced neonatal thymic CD4 SP T cell blasts
Following stimulation with a specific antigen, unprimed CD4 SP T cells are known to differentiate into two functional subsets, Th1 and Th2. Th1 cells preferentially produce IL-2 and IFN-{gamma}, whereas Th2 cells preferentially produce IL-4, IL-5, IL-10 and IL-13 (23,24). First, SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts were stimulated with varying doses of SEA for 8 h, and supernatants of the cultures were assayed for amounts of IL-2, IFN-{gamma} and IL-4 (Table 2). It was found that adult thymic and adult splenic T cell blasts produced substantial amounts of IL-2 at >=0.1 ng/ml SEA, whereas neonatal thymic cells produced low amounts of this cytokine over the doses examined (Table 2). No difference was observed in IFN-{gamma} production among these three populations (data not shown). Surprisingly, it was found that neonatal thymic T cell blasts produced >=10 times the amount of IL-4 than adult splenic T cell blasts over the SEA doses examined, with adult thymic cells producing an intermediate response. Several preparations of SEA-induced thymic CD4 SP T cell blasts were also prepared from mice of different ages and examined for production of IL-4 upon re-stimulation with SEA. It was found that production of IL-4 was maximal in day 0–2 neonatal thymic CD4 SP T cell blasts and thereafter decreased with the age (data not shown).


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Table 2. Cytokine production by SEA-induced CD4 SP T cell blast populations in response to re-stimulation with SEA
 
Secondly, the three preparations of CD4 SP T cell blasts were stimulated with 10 ng/ml of SEA for 18 h, and then PMA and A23187 in the presence of monensin for 5 h. Cells harvested were examined for production of IL-4 and IFN-{gamma} at the cell level of Vß3+ and Vß11+ T cells. The percentage of cells producing IL-4, but not IFN-{gamma}, was much higher in neonatal thymic CD4 SP T cell blasts than in adult splenic T cell blasts. Adult thymic T cell blasts gave an intermediate response (Fig. 5). The percentage of cells producing IFN-{gamma}, but not IL-4, was comparable in the three T cell preparations.



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Fig. 5. Intracellular staining of IL-4 and IFN-{gamma} produced in the SEA-induced neonatal thymic, adult thymic and splenic CD4 SP T cell blasts re-stimulated with SEA. Neonatal thymic, adult thymic and splenic CD4 SP T cell blasts (2 x 106/ml), prepared as described in Fig. 2, were re-stimulated with 10 ng/ml SEA in the presence of AC (2 x 106/ml) for 16 h, and then stimulated with 10 ng/ml PMA and 0.4 µM calcium ionophore A23187 in the presence of GolgiStop for 5 h. Harvested cells were stained with combinations of biotinylated anti-Vß3 or -Vß11/CyChrome-conjugated streptavidin and FITC-conjugated anti-IFN-{gamma} and PE-conjugated anti-IL-4 mAb. Analysis gates were set on Vß3+ or Vß11+.

 
Low proliferative response and high IL-4 production of neonatal thymic CD4 SP T cell blasts prepared from CD44low HSAlow CD4 SP T cells
The HSAlow fraction, which is thought to be a more mature fraction, constituted ~40% of the neonatal thymic CD4 SP T cells, 45% of the adult thymic CD4 SP T cells and 95% of the adult splenic CD4 SP T cells (data not shown). Since HSAhigh (Qa-2low) thymic CD4 SP T cells, which are thought to be a less immature fraction, showed quite a low proliferative response (25) and a high susceptibility to apoptotic death (26) after stimulation through TCR, we think that thymic SEA-induced CD4 SP T cell blasts were derived mainly from the HSAlow fractions.

However, it is possible that HSAhigh cells and/or memory-type CD4 T cells contained in each CD4 SP T cell preparations may contribute the above results. In order to address this question, we tried to perform the same analysis as above using SEA-induced CD4 T cell blasts prepared from naive HSAlow CD4 SP T cells. CD45RBhighCD62LhighCD44low is accepted as the phenotype of peripheral naive CD4 T cells (27). However, this appeared not applicable to thymic preparations, since the expression of CD45RB was much lower in thymic CD4 SP T cells than in adult splenic CD4 SP T cells and the majority of neonatal thymic CD4 SP T cells expressed low amounts of CD62L (Fig. 6A). Therefore, we isolated CD44low HSAlow cells from three CD4 SP T cell preparations (Fig.6B). The intensity of CD45RB and CD62L did not show any difference between whole CD4 SP T cells and CD44low HSAlow CD4 SP T cells in three populations. We induced CD4 SP T cell blasts with SEA from these three populations and compared their secondary responses.



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Fig. 6. Expression of activation/memory markers on three CD4 SP T cell preparations and low proliferative responses of thymic CD4 SP T cell prepared from CD44low HSAlow CD4 SP T cells. (A) Neonatal thymic, adult thymic and splenic CD4 SP T cells were stained with biotinylated anti-CD45RB or CD62L mAb, and then stained with CyChrome-conjugated streptavidin, PE-conjugated anti-CD4 mAb and FITC-conjugated anti-CD44 mAb. Profiles of CD44 versus CD45RB or CD62L expression on gated CD4+ T cells are shown. Numbers indicate the percentage of positive cells in each quadrant. (B) CD44low HSAlow CD4 T cells were isolated from whole CD4 SP T cells by cell sorting. Profiles of CD44 versus HSA, CD45RB and CD62L expression on gated CD4+ T cells are shown. Numbers indicate the percentage of cells in each quadrant. (C) SEA-induced neonatal thymic (circle), adult thymic (diamond) and adult splenic (square) CD4 SP T cell blasts (6 x 104/well), prepared from CD44low HSAlow CD4 SP T cells as described in Fig. 2, were stimulated in vitro with increasing doses of SEA in the presence of AC (6 x 104/well). Cell proliferation was determined by incorporating 0.5 µCi (18.5 kBq) of [3H]thymidine during the last 4 h of the 72-h culture. Three independent experiments were performed with similar results.

 
Adult splenic CD4 SP T cell blasts showed a maximal proliferative response at 10 pg/ml of SEA. In contrast, adult thymic and neonatal thymic CD4 SP T cell blasts exhibited only marginal responses at that dose, although the former proliferated a little more than the latter (Fig. 6C). Analysis for cytokine production revealed that adult splenic CD4 SP T cell blasts produced substantial amounts of IL-2 at >=0.1 ng/ml SEA, whereas neonatal and adult thymic CD4 SP T cell blasts produced low amounts of IL-2 over the doses examined (Table 3). Neonatal thymic CD4 SP T cell blasts again produced >=10-fold the amount of IL-4 than adult splenic and adult thymic CD4 SP T cell blasts over the SEA doses examined. Thus, although the reason why cytokine production from adult thymic CD4 SP T cell blasts decreased is not clarified, the results indicate low proliferative capacity of thymic CD4 SP T cell blasts and a high level of IL-4 production in neonatal thymic CD4 SP T cell blasts to be characteristic of the most mature naive (CD44low HSAlow) thymic population.


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Table 3. Cytokine production by SEA-induced CD4 SP T cell blasts populations prepared from CD44low HSAlow CD4 SP T cells in response to re-stimulation with SEA
 
Expression of GATA-3 in CD4 SP T cells
To clarify the molecular mechanism underlying the preferentially high production of IL-4 by SEA-induced neonatal thymic CD4 SP T cell blasts, several preparations of CD4 SP T cells were examined for expression of GATA-3 protein that directs the IL-4 production (28,29) in two experimental systems. In the western blot analysis (Fig. 7A), unprimed neonatal thymic T cells expressed GATA-3 at a higher level than unprimed adult thymic T cells. It was not detected in adult splenic T cells. Expression of GATA-3 was enhanced in either preparation of SEA-induced CD4 SP T cell blasts. The level of GATA-3 expression was much higher in neonatal thymic T cell blasts than in adult thymic and adult splenic T cell blasts, and the levels in the latter two blasts were almost equal. In the analysis using RT-PCR, the level of GATA-3 mRNA in unstimulated CD4 SP T cells was higher in neonatal thymic cells than in adult thymic T cells (Fig. 7B), irrespective of the presence of competitor. Expression of the GATA-3 message was not detected in adult splenic T cells. A marked enhancement of the GATA-3 message was observed in SEA-induced CD4 SP T cell blasts. The level of GATA-3 mRNA was in the order neonatal thymic T cell blasts, adult thymic cells and adult splenic cell blasts. Thus, the results showed that neonatal thymic CD4 SP T cells express high levels of GATA-3 and that this expression decreases with age.



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Fig. 7. Higher expression of GATA-3 in neonatal thymic CD4 SP T cells than in adult thymic and splenic counterparts. (A) The total cell lysates were prepared from neonatal thymic, adult thymic and adult splenic CD4 SP T cells, and corresponding SEA-induced CD4 SP T cell blasts, prepared as described in Fig.2. All lysates (20 µg total protein/sample) were subjected to western blot analysis for GATA-3 expression. (B) Total RNA was prepared from neonatal thymic, adult thymic and adult splenic CD4 SP T cell, and corresponding SEA-induced CD4 SP T cell blasts populations, which were re-stimulated with SEA (10 ng/ml) for 6 h in the presence of AC. Two micrograms of total RNA was reverse transcribed, and subjected to competitive PCR using cDNA and specific competitors as templates. Three independent experiments were performed with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The results reported here partially support our hypothesis that immunological immaturity is a common feature of thymic HSAlow CD4 SP T cells in the final stage of maturation and is not species specific. In addition, we found that murine neonatal thymic HSAlow CD4 SP T cells are in a Th2-dominant state, whose trait decreases with time after birth.

Proliferative capacity is essential for the expansion of T cell clones activated by antigens. It has been thought that immature T cell clones lose the capacity to proliferate after undergoing strong antigenic stimulation and, rather than expanding, are deleted. In contrast, mature T cells are thought to expand their clonotypes upon strong antigenic stimulation. Our findings that the proliferative capacity of SEA-induced thymic CD4 SP T cell blasts was reduced upon re-stimulation with SEA compared with SEA-induced adult splenic CD4 SP T cell blasts (Figs 1, 3 and 6) suggest that thymic CD4 SP T cells in the final stage of maturation are still in an immunologically immature state. Since the proliferation was lower in the neonatal thymic CD4 SP T cell blasts than the adult thymic CD4 SP T cell blasts, neonatal thymic CD4 SP T cells may be more immature than the adult counterpart. It is likely, however, that mouse thymic CD4 SP T cells are not as immature as the human counterpart. In the present study, we found that adult thymic CD4 SP T cell blasts responded well to SEA at 100 pg/ml or higher concentrations and at levels similar to the response of adult splenic CD4 SP T cells. In contrast, TSST-1-induced human thymic CD4 SP T cell blasts exhibited markedly decreased proliferative responses to both TSST-1 (unpublished data) and IL-2 (13).

It was observed that the proliferative capacity is higher in Vß3+ SEA-induced CD4 SP T cell blasts than the Vß11+ counterpart, irrespective of thymic or splenic origin (Fig. 4). We recently showed that Vß3+ CD4+ T cells exhibited a higher level of protracted expansion in mice implanted with an osmotic pump filled with SEA than did Vß11+ CD4+ T cells (15). These findings strongly suggest that Vß3+ CD4+ T cells are high responders to SEA, whereas Vß11+ CD4+ T cells are moderate responders. We think that this difference is due to a greater binding affinity of SEA to Vß3+ TCR than to Vß11+ TCR, although both affinities are sufficient to trigger responses in the two fractions of cells.

IL-4 production was quite different among SEA-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts. It was quite higher in the neonatal thymic T cell blasts. The thymic production of IL-4 decreased with time after birth. IL-4 production with adult splenic T cell blasts was quite low. Since NK T cells are known to be high IL-4 producers (8), one possibility is that the percentage of NK T cells is high in the neonatal thymic T cell blasts. We found, however, that neonatal thymic, adult thymic and adult splenic CD4 SP T cell blast preparations contained equally low percentages of NK T cells (Fig.2). Moreover, NK T cells express a highly restricted TCR composed of an invariant {alpha} chain (V{alpha}14–Jß281) and a ß chain with restricted Vß elements (Vß2, Vß7 or Vß8), and heterogeneous Dß and Jß elements (9,10), indicating that they are unreactive to SEA. Supporting this, depletion of NK1.1 + T cells using anti-NK1.1 antibody and magnetic beads from whole neonatal thymic CD4 SP T cells did not affect the level of IL-4 production from SEA-induced CD4 SP T cell blasts (data not shown). Thus, these findings remove the possibility that NK T cells are involved in the high IL-4 production in neonatal thymic T cell blasts.

In addition to NK T cells, it is reported that CD25+ CD4 regulatory T cells have a role in Th2 development (30), therefore CD25+ CD4 regulatory T cells may affect the results in this study. We examined whether a large number of regulatory T cells are present in the neonatal thymic CD4 SP T cells. The percentage of CD25+ T cells in adult splenic, adult thymic and neonatal (day 0) thymic CD4 SP T cells was 2.8, 3.3 and 3.2% respectively (data not shown). Although it is not clear whether they preferentially affect neonatal thymic CD4 SP T cell responses, equal percentages in three CD4 SP T cell preparations suggested that CD25+ CD4 regulatory T cells do not have a major role in the present study.

Murine thymic CD4+ T cells have been reported to produce higher quantities of IL-4 upon primary stimulation than splenic CD4+ T cells (11). Since IL-4 produced by naive CD4+ T cells has an important role in the generation of IL-4-producing cells (23,24), primary stimulation of thymic CD4+ T cells was thought to account for the high IL-4 production by neonatal thymic cells. We found, however, that IL-4 production was below the level of detection upon primary stimulation of neonatal thymic CD4 SP T cells. Thus, an inherent property of the cells, rather than microenvironmental factors such as IL-4 concentration, seems to be responsible for the high IL-4 production by neonatal thymic CD4 SP T cell blasts. Lymph node T cells from postnatal day 4 mice have been reported to produce copious amounts of IL-4 in response to primary stimulation with anti-CD3 antibody (3135), but this capacity disappears by postnatal day 6 (31). It seems likely therefore that splenic CD4 SP T cells of mice aged 4 days or less preserve the capacity to produce IL-4 carried by neonatal thymic CD4 SP T cells. These phenotypes of newborn mice appear to contribute to neonatal tolerance to alloantigens, which may be achieved by the suppressive effect of IL-4 on Th1 cell-mediated immunity (3638), although the physiological role of IL-4 in the neonate or indeed the neonatal thymus remains to be defined. In IL-4-deficient mice, there was no apparent alteration in thymic T cell development (39).

IL-4 productions of thymic T cells were higher in the Vß11+ CD4 SP T cell blasts than in the Vß3+ CD4 SP T cell blasts (Fig. 5). It has been previously demonstrated that altering the affinity of antigen peptide for MHC or TCR molecules or changing the dose of antigen in priming resulted in a different pattern of functional T cell responses (40). Peptides of low MHC- or TCR-binding affinity or low doses of peptide favor the generation of Th2 responses, whereas high-affinity peptides or high doses of peptide lead to Th1 response. If the above conception is applied to the present study using SEA, the difference in binding affinity of SEA to TCR between Vß3+ and Vß11+ CD4 SP T cell blasts may be attributable to the difference in IL-4 production. However, this assumption is not applicable to adult splenic CD4 SP T cell blasts (Fig. 5). Alternatively, it is possible that IL-4-producing Vß11+ T cells may be a specific thymocyte population. Detailed studies on these issues are now under way.

As shown in experiments using GATA-3 knockout mice and lacZ knockin mice at the GATA-3 gene locus, this transcription factor is essential for the development of thymic T cells (41,42). In mature T cells, GATA-3 is involved in the polarization of Th0-type T cells into Th2-type cells by chromatin remodeling of loci, including the gene encoding IL-4 (28,29). Our finding showed that the level of GATA-3 expression was much higher in neonatal thymic CD4 SP T cell blasts as compared with those in adult thymic and adult splenic CD4 SP T cell blasts (Fig. 7). This result is in agreement with the finding that the level of IL-4 production in the three populations (Tables 2 and 3). The significance of high GATA-3 expression in the neonatal thymic CD4 SP T cell blasts remains unknown. Other members of the GATA family such as GATA-2 and -6 have been reported to promote cell proliferation while inhibiting differentiation in non-lymphoid organs (43,44). Moreover, Hendriks et al. have suggested the association of GATA-3 with thymic autonomous proliferation and thus its down-regulation in the periphery might allow T cells to proceed to terminal differentiation (42).

Signal transduction via TCR stimulation involves at least two pathways: the activation of phospholipase C{gamma}1 and the activation of the RAS–mitogen-activated protein kinase pathway. Both pathways require the activation of Src family kinases such as Lck and Fyn. We found that the TSST-1-induced human thymic and peripheral CD4+ T cell blasts are heavily phosphorylated in Lck at the negative regulatory site of Tyr505 Lck. Its dephosphorylation occurs in the peripheral CD4 SP T cell blasts, but not in the thymic CD4+ T cell blasts (14). It is our current consideration that the protein tyrosine phosphatase CD45 is involved in this dephosphorylation of Lck. Whether or not a similar mechanism is involved in the mouse system would be important to determine.


    Acknowledgements
 
We thank H. Yagi and M. Maruyama for their technical assistance, and N. Wakisaka and M. Yoshikawa for taking care of the animals used in this study. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labor and Welfare of Japan, and the research fund contributed by Professor Emeritus Dr Kiyoko Nakanishi.


    Abbreviations
 
AC—accessory cell

PE—phycoerythrin

PMA—phorbol myristate acetate

SAG—superantigen

SEA—staphylococcal enterotoxin A

SP—single positive

TSST-1—toxic shock syndrome toxin-1


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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