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The Journal of Immunology, 2005, 174: 1188-1195.
Copyright © 2005 by The American Association of Immunologists

Polarized Development of Memory Cell-Like IFN-{gamma}-Producing Cells in the Absence of TCR {zeta}-Chain

Ludmila Krymskaya, Wen-Hui Lee, Lingwen Zhong and Chih-Pin Liu1

Division of Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR/CD3 complex-mediated signals play critical roles in regulating CD4+ Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule {zeta}-chain in regulating the differentiation of Th cells. We have studied T cells from {zeta}-chain-deficient mice ({zeta}KO mice), {zeta}-chain-bearing mice ({zeta}+ mice), and from {zeta}KO mice expressing a FcR{gamma} chain transgene (FcR{gamma}TG, {zeta}KO mice). Our results demonstrated that, compared with those of control mice, CD4+ T cells and not CD8+ T cells from {zeta}KO mice were polarized into IFN-{gamma}-producing cells. Some of these IFN-{gamma}-producing cells could also secrete IL-10. Interestingly, {zeta}KO mouse T cells produced IFN-{gamma} even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-{gamma} production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from {zeta}KO mice. Further studies showed that noncultured {zeta}KO mice CD4+ T cells and thymocytes bore a unique memory cell-like CD44high, CD62Llow/neg phenotype. Altogether, these results suggest that, in the absence of the {zeta}-chain, CD4+ T cells develop as polarized IFN-{gamma}-producing cells that bear a memory cell-like phenotype. The {zeta}-chain-bearing T cells may produce a large amount of IFN-{gamma} only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of {zeta}-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ Th1 cells produce cytokines such as IFN-{gamma} and are involved in the development of autoimmune diseases, whereas Th2 cells produce cytokines including IL-4 and participate in allergic immune responses (1, 2, 3). The differentiation of T cells into Th1 cells or Th2 cells can be regulated by several factors such as the local cytokine environment, the type of APCs, the type of Ags, and the nature/strength of the TCR-mediated signals (4, 5, 6, 7). The influence of the local cytokine environment on Th1 and Th2 cell differentiation is well established. Upon TCR engagement, naive T cells can become Th1 cells if they are cultured in medium containing IL-12, or they can become Th2 cells if the medium contains IL-4 (2, 6, 7, 8). Culturing cells with a combination of different cytokines, such as IL-12 and IL-18, may also lead to IFN-{gamma} production independent of TCR stimulation (6, 7, 9). Furthermore, the expression and activation of different signaling and transcription factors can regulate the differentiation of Th1 and Th2 cells. For example, activation of STAT1 or STAT6 signaling molecules can regulate the expression and activation of transcription factors T-bet or GATA-3, respectively (7, 10). Activation of T-bet then drives IFN-{gamma} gene transcription leading to the differentiation of Th1 cell, and activation of GATA-3 drives the transcription of IL-4 gene leading to the differentiation of Th2 cells (11, 12). These two different transcription factors can exert a negative effect on the expression of cytokines by the opposing Th cell subsets.

In addition to cytokines, the signals mediated through TCR/CD3 complexes play critical roles during the differentiation of both Th1 and Th2 cells. Previous studies have demonstrated that the strength of the signals mediated through TCRs can influence Th cell lineage commitment (5, 13, 14, 15, 16). It is possible that activation of T cells via TCRs may polarize the cells toward being Th1 or Th2 cells, and these cells further differentiate and proliferate in response to IL-12 or IL-4, respectively (17). Moreover, the strength of TCR signals and the up-regulation of T-bet expression may play more important roles in Th1 cell commitment than do IL-12-mediated signals (17). Following these interesting findings, further studies are necessary to investigate the role of TCR complexes and how the signals delivered by TCRs may influence the in vivo differentiation of Th1 and Th2 cells. The TCR complex is composed of several peptides including the {alpha}/{beta} heterodimer associated with the CD3 chains and the {zeta}-chain homodimer (18, 19). The results obtained from our previous studies and those of others in using gene knockout mice have demonstrated that the {zeta}-chain homodimer plays critical roles in regulating TCR-mediated signals and development of T cells (20, 21, 22, 23). Interestingly, T cells from patients bearing tumors, autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, or chronic infectious diseases down-regulate the expression of {zeta}-chain (24, 25, 26, 27, 28, 29). Furthermore, sustained bacterial infection induces IFN-{gamma}-dependent {zeta}-chain down-regulation and defective T cell function (30). Altogether, these studies suggest that regulation of {zeta}-chain expression in T cells is associated closely with the development of immunity and autoimmunity in animals. Interestingly, some of the {zeta}-chain-deficient T cells from animals or humans may instead express a homologous molecule, the {gamma}-chain of the high affinity IgE receptor (FcR{gamma} chain), which can substitute for the {zeta}-chain (20, 31, 32, 33). Our studies and those of others have demonstrated that expression of a FcR{gamma} chain transgene can restore T cell development in {zeta}-chain-deficient animals (34, 35).

Because the differentiation of Th1 vs Th2 cells plays critical roles in defining the nature of an immune response and in the pathogenesis of various diseases, it is important to determine whether and how the in vivo expression of {zeta}-chain influences such processes. We report here that T cells from {zeta}-chain-deficient animals display a memory cell-like phenotype, up-regulate STAT4 and T-bet expression, and are polarized IFN-{gamma}-producing cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CD3{zeta}–/– ({zeta}KO mice) and FcR{gamma} chain transgenic {zeta}KO mice have been described in previous studies (20, 34). Mice expressing {zeta}-chain were used as the {zeta}+ mice control and are heterozygous ({zeta}+/–) rather than homozygous ({zeta}+/+) mice. The {zeta}+ mice and the {zeta}KO mice used in our study are littermates. The {zeta}KO mice, generated from a 129 ES cell line, have been backcrossed onto C57BL/6 mice for eight generations. Despite this extensive backcross, we cannot exclude the possibility that the genetic background differences may still exist between the {zeta}KO mice and the {zeta}+ mouse littermates. It is possible that such genetic differences may contribute to the observed differences of IFN-{gamma} production between the {zeta}KO mice and the {zeta}+ mouse littermates. Mice were housed under specific pathogen-free conditions at the animal facility of the Beckman Research Institute of City of Hope (Duarte, CA). The animals used in the experiments were 8- to 10-wk of age unless otherwise stated.

Abs and reagents

All Abs used in FACS analyses were purchased from BD PharMingen. Ionomycin and PMA were purchased from Sigma-Aldrich. Monensin was purchased from Calbiochem-Novabiochem.

Cell preparation and phenotypic characterization by flow cytometry

Single-cell suspensions of splenic cells were isolated from individual mice or pooled from two to three mice of each strain. For cell surface phenotype analysis, freshly isolated cell populations were stained with the indicated Abs. A total of 0.5–1 x 106 cells were incubated with Abs for 30 min, washed, and acquired using FACSCalibur (BD Biosciences). The data from at least 25,000 events were analyzed using the CellQuest program (BD Biosciences). Isotype-matched Abs were used as negative controls.

Cell purification and culture

Splenic T cells were isolated using magnetic beads (Miltenyi Biotech) according to the manufacturer’s instructions. The purity of cells was routinely >98%. T cells (1 x 106 cell/ml) were cultured in RPMI 1640 medium supplemented with 5% FBS in 12-well plates coated with anti-TCR Ab H57 (50 µg/ml) plus anti-CD28 (2 µg/ml). Th1 condition medium contains anti-mouse IL-4 (5 µg/ml), rIL-12 (5 ng/ml, R&D Systems), and rIL-2 (10 U/ml). Th2 condition medium contains IL-4 (50 ng/ml), anti-IL-12 (2 µg/ml), and anti-IFN-{gamma} (3 µg/ml). In Th2 cultures, cells were collected 2 days after culture, washed, and reincubated for 3 days with IL-4, anti-IL-12, and anti-IFN-{gamma}. On day 3 for Th1-conditioned cultures and on day 5 for Th2-conditioned cultures, T cells were harvested for analyses.

Intracellular cytokine staining and ELISA

T cells (1 x 106/ml) were stimulated with PMA (5 ng/ml) plus ionomycin (500 ng/ml) and monensin (3 µM) in RPMI 1640 medium for 4 h, stained with Abs, fixed with 4% formaldehyde, and permeabilized by 0.5% saponin (Sigma-Aldrich). Permeabilized T cells were incubated with FITC-anti-IFN-{gamma} plus PE-anti-IL-4 or PE-anti-IL-10 (BD PharMingen). Isotype Abs were used as negative controls.

Primed CD4+ T cells (5 x 105/ml) were stimulated with immobilized H57 and anti-CD28 in 96-well plates. After 24 and 48 h, supernatants were collected and assayed for cytokines by ELISA using Opt EIA mouse cytokine kits (BD PharMingen).

Western blot analysis

Freshly isolated CD4+ T cells were lysed in buffer containing 20 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 5 mM iodoacetamide, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, and small peptidase inhibitors. Cell lysates were separated on 10% SDS-PAGE gels, and proteins were transferred onto polyvinylidene difluoride membrane (Millipore). The primary Abs include: anti-T-bet, anti-GATA-3 (Santa Cruz Biotechnology); anti-phospho-STAT4, anti-STAT4, anti-phospho-STAT1, anti-STAT1 (Zymed Laboratories); anti-p38 MAPK or phospho-p38 MAPK (Cell Signaling Technology). The secondary Abs include: HRP-conjugated anti-rabbit IgG (Pierce) or anti-mouse IgG (DakoCytomation). Target proteins were visualized using an enhanced West Pico chemifluorescence substrate (Pierce). To normalize for protein content, the membranes were stripped and reprobed with anti-{beta}-actin.

Statistical analysis

In addition to using the Student’s t test for statistical analyses, we also have performed one-way ANOVA and Tukey-Kramer Honestly Significant Difference test using the JMP statistical discovery software (SAS Institute) for statistical analyses to calculate the statistical significance for differences between different groups. The p value of <0.05 was considered to be statistically significant. All data are expressed as the mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biased differentiation of IFN-{gamma}-producing CD4+ T cells lacking the TCR {zeta}-chain

The TCR {zeta}-chains play an important role in regulating the signals mediated through TCRs and in regulating T cell developments (17, 18, 19, 20, 21, 22). However, the effect of {zeta}-chain expression on the differentiation of CD4+ IFN-{gamma}-producing Th1 cells and IL-4-producing Th2 cells remains unclear. To address this question, we have studied T cells isolated from mice expressing varied TCR complex structures. These animals include mice expressing the TCR {zeta}-chain ({zeta}+ mice), mice deficient in the TCR {zeta}-chain expression ({zeta}KO mice), and {zeta}KO mice expressing a FcR{gamma} transgene (FcR{gamma}TG, {zeta}KO mice). The splenic T cells were activated through their TCRs using an anti-TCR Ab, H57, plus an anti-CD28 Ab in a Th1 culture condition that favored the differentiation of IFN-{gamma}-producing T cells (IFN-{gamma}+ cells)(see Materials and Methods). For the initial studies, we analyzed both CD4+ and CD8+ T cells that were cultured under such a Th1 condition for 3 days. These cells were then restimulated for 3 h with PMA/ionomycin before being used for intracellular cytokine staining. Our previous studies have shown that {zeta}KO mouse T cells were hyporeactive to stimuli and produced less IL-2 than did T cells from {zeta}+ mice (20, 34). Therefore, one would expect that T cells from {zeta}KO mice have the least number of IFN-{gamma}+ T cells. Consistent with this notion, we observed that CD8+ T cells from {zeta}KO mice contained significantly less IFN-{gamma}+ T cells (66.3 ± 13.9%) than did the cells from {zeta}+ mice (77.3 ± 9.9%) and FcR{gamma}TG, {zeta}KO mice (82.6 ± 8.9%)(Fig. 1, A and C). The percentage of IFN-{gamma}+CD8+ T cells from {zeta}+ mice and FcR{gamma}TG, {zeta}KO mice were not significantly different from each other. However, {zeta}KO mouse CD4+ T cells contained the highest percentage of IFN-{gamma}+ cells (65.3 ± 9.9%), contrary to that of CD8+ T cells and more than the CD4+ T cells from the other two mouse strains (Fig. 1, B and C). In addition, more CD4+ T cells from FcR{gamma}TG, {zeta}KO mice (39.5 ± 14.6%, p < 0.05) than from {zeta}+ mice (25.7 ± 10%) produced IFN-{gamma}.



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  		FIGURE 1. IFN-{gamma} production by CD4+ and CD8+ T cells. Splenic T cells from {zeta}+, FcR{gamma}TG {zeta}KO, and {zeta}KO mice were cultured in a Th1 condition (see Materials and Methods). After being cultured for 3 days, cells were restimulated with PMA/ionomycin for 4 h, stained for surface CD4 and CD8, and then stained for intracellular IFN-{gamma}. CD8+ (A) and CD4+ (B) cell populations capable of IFN-{gamma} production were assessed using FACS. The numbers represent the percentage of cells in each quadrant. The results shown are the representative of nine independent experiments. C, The average percentage of IFN-{gamma}+ cells within CD4+ and CD8+ cell subsets. The percentage of IFN-{gamma}+ cells was calculated as the number of CD4+IFN-{gamma}+ or CD8+IFN-{gamma}+ cells/the total number of CD4+ or CD8+ T cells x 100%. Results are the mean ± SD. * and **, Statistically significant difference when compared with cells from {zeta}+ and FcR{gamma}TG, {zeta}KO mice, respectively.

 
To exclude the possibility that cytokines produced by CD8+ T cells may affect the differentiation of CD4+ T cells, we examined purified CD4+ T cells cultured in the same Th1 condition. Consistent with the data shown in Fig. 1, the {zeta}KO mouse CD4+ T cells contained the highest percentage of IFN-{gamma}+ cells (59.8 ± 6.3%), which was significantly more than that of FcR{gamma}TG, {zeta}KO mice (37.3 ± 7.1%)(Fig. 2, A and B). Similar results were obtained from ELISA of cytokines released in the supernatants of stimulated T cells (Fig. 2C). In addition, essentially no T cells from these mice produced IL-4 when cells were cultured in Th1 condition. These results demonstrated that, compared with {zeta}-chain-bearing CD4+ T cells, {zeta}-chain-deficient CD4+ T cells appeared to be more biased toward becoming IFN-{gamma}-producing T cells. Expression of a {zeta}-chain homologous molecule FcR{gamma} chain in {zeta}KO mice also reduced the frequency of IFN-{gamma}+CD4+ T cells. Altogether, these results show that the expression of {zeta}-chain and, to a lesser degree, FcR{gamma} chain influences the differentiation of IFN-{gamma}-producing CD4+ T cells.



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  		FIGURE 2. IFN-{gamma} production by purified CD4+ T cells cultured in a Th1 condition. A, CD4+ T cells were activated with H57 and anti-CD28 in a Th1 condition for 3 days. The cultured cells were then stimulated with PMA/ionomycin for 4 h, stained for surface CD4, and for intracellular IFN-{gamma} and IL-4. The dot plots shown are the representatives of ten independent experiments. B, The bar chart represents the average percentage of IFN-{gamma}+CD4+ T cells. C, ELISA of IFN-{gamma} production by CD4+ T cells cultured in a Th1 condition for 3 days and restimulated with H57 plus anti-CD28 for 24 and 48 h. The results represent the mean ± SD of ELISA results. These cells did not produce a significant amount of IL-4. * and **, Statistically significant difference when compared with cells from {zeta}+ or FcR{gamma}TG, {zeta}KO mice, respectively.

 
Production of IL-10 by IFN-{gamma}-producing {zeta}KO mice T cells cultured in Th1 and Th2 condition

We then determined whether CD4+ T cells isolated from these three mouse strains were varied in their differentiation toward IL-4-producing (IL-4+) CD4+ Th2 cells. Contrary to the results of IFN-{gamma}+ cells shown above, {zeta}KO mice CD4+ T cells contained the least percentage of IL-4+ T cells (10.8 ± 5.2%), significantly less than that of {zeta}+ mice (25 ± 8.7%) and of FcR{gamma}TG, {zeta}KO mice (17.6 ± 8.8%)(Fig. 3A). The ELISA results also showed that {zeta}KO mouse T cells secreted the least amount of IL-4 (Fig. 3B). Therefore, {zeta}-chain-deficiency results in defective differentiation of IL-4+CD4+ T cells cultured under a Th2 condition.



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  		FIGURE 3. Decreased IL-4 but increased IL-10 production by CD4+ T cells from {zeta}KO mice. Purified CD4+ T cells were cultured in a Th2 condition (see Material and Methods). After being cultured for 5 days, cells were resimulated with PMA/ionomycin for 4 h, and stained for surface CD4 and intracellular IL-4 (A) or IL-10 (C). The results represent the average percentage of IL-4+ or IL-10+ cells from four independent experiments. The results represent the mean ± SD. Secretion of IL-4 (B) and IL-10 (D) by CD4+ T cells were determined using ELISA. The cells were cultured for 5 days in a Th2 condition, and restimulated with H57 plus anti-CD28 for 24 and 48 h. The results represent the mean ± SD. * and **, Statistically significant difference when compared with cells from {zeta}+ or FcR{gamma}TG, {zeta}KO mice, respectively.

 
Surprisingly, in contrast to the IL-4 production results, {zeta}KO mice CD4+ T cells contained the highest percentage of IL-10+ T cells (22.8 ± 3.4%), significantly more than that of {zeta}+ mice (8.1 ± 6.7%) and FcR{gamma}TG, {zeta}KO mice (10.2 ± 4.7%)(Fig. 3C). Similar results were obtained from ELISA and {zeta}KO mice CD4+ T cells secreted significantly more IL-10 than did T cells from the other two strains of mice (Fig. 3D). These data show that production of IL-10 by CD4+ T cells cultured in a Th2 condition increased in the absence of {zeta}-chain and, to a lesser extent, FcR{gamma} chain expression.

We then investigated whether Th1-conditioned {zeta}KO mice CD4+ T cells could also produce IL-10. Interestingly, a significant percentage of {zeta}KO mice T cells produced IL-10 (35.5 ± 7.9%), significantly more than the cells from {zeta}+ mice (8.4 ± 2.1%) and FcR{gamma}TG, {zeta}KO mice (27.2 ± 3.4%)(p < 0.05, Fig. 4A). The ELISA results confirmed the staining results (Fig. 4B). These results show that {zeta}-chain-deficient T cells, but not T cells bearing {zeta}-chain or FcR{gamma} chain, were more biased toward becoming IL-10-producing cells not only when they were cultured in a Th2 condition but also in a Th1 condition.



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  		FIGURE 4. {zeta}KO mouse CD4+ T cells are polarized IFN-{gamma}-producing cells that can coproduce IL-10. A, CD4+ T cells were cultured in a Th1 condition for 3 days. The cells were then restimulated with PMA and ionomycin for 4 h and stained for surface CD4 and intracellular IL-10. The results represent the average percentage (mean ± SD) of IL-10+ cells from 10 experiments. B, ELISA of IL-10 secreted by CD4+ T cells restimulated with PMA/ionomycin for 24 and 48 h. The results represent the mean ± SD. C, A majority of IL-10-producing CD4+ T cells from {zeta}KO and FcR{gamma}TG, {zeta}KO mice also coproduce IFN-{gamma}. CD4+ T cells were stained for intracellular expression of IL-10 and IFN-{gamma}. The dot plots are representative of seven independent experiments. D, Bar chart analyses of IFN-{gamma}+/IL-10–, IFN-{gamma}+/IL-10+, and IFN-{gamma}–/IL-10+ cells from seven experiments. The results shown are mean ± SD. * and **, statistically significant differences compared with cells from {zeta}+ or FcR{gamma}TG, {zeta}KO mice, respectively.

 
Our previous studies and those of others have demonstrated that a unique population of CD4+ T cells can coproduce both IFN-{gamma} and IL-10 (36, 37). Therefore, we determined whether Th1-conditioned {zeta}KO mice T cells contained IFN-{gamma}+/IL-10+ cells that produced both cytokines. These studies should also tell us whether the expression of {zeta}-chain in TCR complex has an effect on the differentiation and expansion of the IFN-{gamma}+/IL-10+ cells in vitro. The results showed that {zeta}KO mice CD4+ T cells contained the highest percentage of not only IFN-{gamma}+/IL-10– cells (31.7 ± 5.6%) but also IFN-{gamma}+/IL-10+ cells (30.4 ± 6.2%)(Fig. 4, C and D). In comparison, both {zeta}+ mice and FcR{gamma}TG, {zeta}KO mice had significantly less IFN-{gamma}+/IL-10– cells (14.7 ± 5.6% and 19.3 ± 6.2%, respectively) and IFN-{gamma}+/IL-10+ cells (3.6 ± 0.4% and 20.7 ± 4%, respectively) than did {zeta}KO mice. These studies demonstrate that {zeta}KO mice CD4+ T cells contained not only more IFN-{gamma}+/IL-10– cells but also more IFN-{gamma}+/IL-10+ cells than did CD4+ T cells from FcR{gamma}TG, {zeta}KO, and {zeta}+ mice. The presence of more IFN-{gamma}-producing cells in FcR{gamma}TG, {zeta}KO mice than in {zeta}+ mice was due to an increased population of IFN-{gamma}+/IL-10+ cells.

{zeta}-chain deficient CD4+ T cells can produce IFN-{gamma} when cultured in a Th2 condition

Our results suggest that the expression of {zeta}-chain and, to a lesser extent, FcR{gamma} chain negatively regulates the differentiation of IFN-{gamma}+CD4+ T cells. We hypothesized that {zeta}-chain-deficient T cells are thus intrinsically polarized toward IFN-{gamma}-producing cells whether these cells are cultured in a Th1 condition or not. To test this hypothesis, we performed the following experiments. First, we examined whether freshly isolated {zeta}-chain-deficient CD4+ T cells are able to produce IFN-{gamma} without the need to be cultured in a Th1 condition. The cells were stimulated in vitro with PMA/ionomycin for 1–4 h and stained for intracellular IFN-{gamma} and IL-10. The results demonstrated that a significant percentage of {zeta}KO mice T cells produced IFN-{gamma} within 1 h after simulation (~32%), and that the percentage of those cells increased further at 4 h after stimulation (~73%)(Fig. 5). A small percentage of IFN-{gamma}+/IL-10+ cells (~4%) can be detected at 2 h after stimulation and the percentage increased up to ~11% at 4 h. In comparison, T cells obtained from the other two strains of mice showed not only a delayed but also a significantly weaker response in IFN-{gamma} production. Moreover, essentially no IFN-{gamma}+ T cells from these two strains of mice produced IL-10.



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  		FIGURE 5. A large number of freshly isolated {zeta}KO mice CD4+ T cells were able to produce IFN-{gamma} shortly after activation. Freshly isolated CD4+ T cells were stimulated with PMA/ionomycin for 1–4 h, and stained for intracellular IFN-{gamma} and IL-10. The numbers represent the percentages of cells in each quadrant. The results are the representative of three experiments.

 
We then examined whether {zeta}KO mouse T cells still retained their biased IFN-{gamma}-producing capacity and became IFN-{gamma}+/IL-10– cells and IFN-{gamma}+/IL-10+ cells when cultured in a Th2 condition. The results showed that a significant percentage of CD4+ T cells from {zeta}KO mice were either the IFN-{gamma}+/IL-10– cells (38.5 ± 3.9%) or the IFN-{gamma}+/IL-10+ cells (15.8 ± 3.9%). In comparison, very few CD4+ T cells from both {zeta}+ mice and FcR{gamma}TG, {zeta}KO mice contained the IFN-{gamma}+/IL-10– cells (1.45 ± 0.76% and 4.1 ± 1.8%, respectively) and the IFN-{gamma}+/IL-10+ cells (1.0 ± 0.5% and 3.6 ± 1.3%, respectively) (Fig. 6, A and B). The ELISA results were consistent with the staining results (Fig. 6C), although Th2-conditioned {zeta}KO mice T cells produced an ~4- to 5-fold less amount of IFN-{gamma} than did Th1-conditioned T cells.



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  		FIGURE 6. Production of IFN-{gamma} by {zeta}KO mice CD4+ T cells cultured in a Th2 condition. A, CD4+ T cells were cultured in a Th2 condition (see Material and Methods). After 5 days, T cells were restimulated with PMA/ionomycin, and stained for intraceullular IL-10 and IFN-{gamma}. The dot plots are representative of four experiments. B, The results represent the average percentage of CD4+ IFN-{gamma}+/IL-10–, IFN-{gamma}+/IL-10+, or IFN-{gamma}–/IL-10+ cells from four experiments. C, ELISA of IFN-{gamma} production by these CD4+ T cells. The results represent the mean ± SD. *, statistically significant difference when compared with cells from {zeta}+ mice.

 
Taken together, these results are consistent with the idea that {zeta}KO mice T cells are intrinsically polarized or predisposed toward IFN-{gamma}-producing cells, and that these cells can produce IFN-{gamma} even after being cultured in a Th2 condition. These results also suggest that expression of {zeta}-chain and, to a lesser degree, the FcR{gamma} chain can prevent CD4+ T cells from becoming IFN-{gamma}-producing cells unless these cells are cultured under a Th1 condition.

{zeta}KO mouse CD4+ T cells express up-regulated level of STAT4 and T-bet

We then investigated the molecular basis that would account for the polarized IFN-{gamma}-producing cells from {zeta}KO mice. Previous studies have demonstrated that TCR-mediated signals cooperate with other signals mediated through cell surface cytokine receptors such as IFN-{gamma} receptors and IL-12 receptors (7, 10). These signals then result in an up-regulation and activation of downstream signaling molecules leading to Th1 cell differentiation and IFN-{gamma} production. These molecules include STAT1, STAT4, p38 MAPK, and transcriptional factors such as T-bet. Up-regulation of different molecules, such as STAT6 and GATA-3, may inhibit Th1 and facilitate Th2 cell differentiation and IL-4 production (7, 8, 10).

To assess the expression level of STAT1, STAT4, p38, T-bet, and GATA-3 molecules, we conducted Western blot analysis using freshly isolated CD4+ T cells. The results demonstrated that the expression level of STAT4 and T-bet, but not STAT1, GATA-3, and p38 MAPK, were up-regulated in {zeta}KO mouse T cells when compared with T cells from {zeta}+ or FcR{gamma}TG, {zeta}KO mice (Fig. 7). The expression level of the phosphorylated form of STAT4, but not STAT1 and p38, was also increased in {zeta}KO mouse T cells. These results suggest that up-regulation of STAT4 and T-bet in T cells from {zeta}KO mice may be involved in the polarization of these T cells and may lead to their quick IFN-{gamma} production response.



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  		FIGURE 7. Up-regulation of T-bet and STAT4 expression in freshly isolated noncultured CD4+ T cells from {zeta}KO mice. Total cell lysates were obtained from freshly isolated CD4+ T cells. Equal amounts of proteins (40 µg) from each cell lysate were resolved in 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with Abs against total or phosphorylated STAT4, STAT1, p38, and with Abs against T-bet and GATA-3. Ab against {beta}-actin was used as a control. Results are the representative of three experiments.

 
Memory cell-like phenotype of CD4+ T cells from {zeta}KO mice

The rapid IFN-{gamma}-production by {zeta}KO mouse T cells bears interesting similarity to that of CD44highCD62Llow memory T cells. It has been known that memory T cells respond more easily than naive T cells to Ag stimulation and can rapidly secrete cytokines (38, 39, 40). Therefore, we performed experiments to determine whether {zeta}KO mouse T cells exhibited a memory cell-like phenotype. The results showed that freshly isolated T cells from all three strains of mice did not increase the expression of the activation markers CD25 and CD69 (Fig. 8A). However, unlike T cells from {zeta}+ and FcR{gamma}TG, {zeta}KO mice, essentially all {zeta}KO mice CD4+ T cells bore a memory cell-like CD44highCD62Llow/neg phenotype (Fig. 8, A and B). Additionally, T cells from 3-day-old {zeta}KO mice, the youngest age of mice studied, have already displayed the same CD44highCD62low/neg phenotype as that observed in older (3 wk and 6 wk) mice (Fig. 8B). These {zeta}KO mice CD4+ T cells did not up-regulate CD25 and CD69 expression even when isolated from newborn 3-day-old mice. Therefore, it is unlikely that these cells acquired this phenotype during peripheral expansion while the mice age or due to activation in vivo.



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  		FIGURE 8. {zeta}KO mouse CD4+ T cells and thymocytes bear a memory cell-like phenotype. A, Histogram analyses of activation and memory cell markers. CD4+ T cells from 8-wk-old {zeta}+; FcR{gamma}TG, {zeta}KO; and {zeta}KO mice were stained for the expression of CD44 and CD62L as markers for memory cell-like phenotype, and for the expression of CD25 and CD69 as activation markers. The dark areas represent cells stained with the indicated Abs and the solid lines represent cells stained with negative control Abs. B, Phenotypic study of CD4+ T cells from {zeta}+ mice and {zeta}KO mice at different ages (3-day-, 3-wk-, and 6-wk-old mice). Splenocytes were stained for surface expression of CD4, CD44, and CD62L. Electronically gated CD4+ T cells were analyzed for the expression of CD44 and CD62L. C, Analyses of CD4+CD8– single-positive thymocytes. Freshly isolated thymocytes from 6-wk-old {zeta}+ mice and {zeta}KO mice were stained for surface expression of CD4, CD8, and CD44 or CD62L. Thymocytes were also stained for intracellular IFN-{gamma} after being stimulated with PMA/ionomycin for 4 h. The results represent analyses of electronically gated CD4+CD8– thymocytes. The numbers are the percentage of cells in each gated area.

 
We then sought to examine whether {zeta}KO mice thymus select for CD4+ thymocytes bearing such a memory cell-like phenotype. Phenotypic studies demonstrated that some CD4+ thymocytes from {zeta}KO mice, and not from {zeta}+ mice, also bore the CD44highCD62low/neg phenotype (Fig. 8C). Furthermore, only freshly isolated {zeta}KO mice CD4+ thymocytes contained a significant percentage of IFN-{gamma}-producing cells after being stimulated with PMA/ionomycin for 3 h (Fig. 8C). Altogether, these results argue in favor for the idea that {zeta}KO mice select for a population of memory cell-like CD4+ T cells that are polarized into producing IFN-{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Production of IFN-{gamma} by Th1 cells is considered a critical step in promoting resistance to many pathogens, in the generation of immunity against tumors, and in causing autoimmune diseases (2, 3). Therefore, it is important to understand how the differentiation of IFN-{gamma}-producing T cells is regulated by TCR-mediated signals. In this study, we provide evidence that the expression of TCR {zeta}-chain plays an important regulatory role during the development of CD4+ IFN-{gamma}-producing T cells. In the absence of {zeta}-chain expression, CD4+ T cells are polarized toward becoming IFN-{gamma}-producing cells. These {zeta}-chain-deficient CD4+ T cells retain their IFN-{gamma}-production capability even when they are cultured in a Th2 condition.

Several possibilities exist that would account for the biased IFN-{gamma} production by {zeta}KO mouse T cells. First, it is possible that the biased differentiation occurs in peripheral lymphoid organs when T cells from {zeta}KO mice, but not from the other two strains of mice, encounter Ags and are activated in vivo. The following evidences exclude such a possibility: the {zeta}+ littermates and the FcR{gamma}TG, {zeta}KO mice were raised under the same condition but their T cells did not display the same polarized IFN-{gamma} production. Additionally, only CD4+ T cells but not CD8+ T cells of {zeta}KO mice were polarized into becoming IFN-{gamma}-producing cells. Moreover, freshly isolated T cells from {zeta}KO mice of various ages, like the other two strains of mice, did not display the activation markers CD25 and CD69. Second, it is also possible that the polarized differentiation of {zeta}KO mouse T cells is due to a weaker signal mediated through their very low TCR expression levels. However, {zeta}KO mouse CD8+ T cells, which also express lower level of TCRs, are not polarized IFN-{gamma}-producing cells, suggesting that lower TCR expression level alone is not the reason for the polarized CD4+ T cells. Third, the {zeta}KO mice, which were originally generated from a 129 strain ES cell line, were backcrossed onto C57BL/6 mice for eight generations before the pups were generated from brother and sister mating. Despite this extensive backcross, it remains possible that the genetic background of the {zeta}KO mice and the {zeta}+ mice were not identical. It is likely that such a difference in their genetic background may contribute to the observed differences in IFN-{gamma} production by their T cells. In particular, multiple, additional genetic differences on the {zeta} gene locus chromosome may come from the other strain and may contribute to the differences of IFN-{gamma} production by T cells isolated from the {zeta}KO mice and the {zeta}+ mice. To demonstrate that the observed differences are due to the {zeta}-chain deletion rather than to unrelated differences in background genes, further studies would be necessary to study T cells from {zeta}KO mice reintroduced with {zeta}-chain or to introduce the {zeta}-chain into the cells tested in vitro. Fourth, phenotypic studies have shown that {zeta}KO mouse T cells, unlike {zeta}+ mouse T cells, are almost all CD44highCD62low/neg cells. These results, together with their rapid IFN-{gamma} production in response to activation, suggest that {zeta}-chain-deficient T cells bear a memory cell-like phenotype. Our additional studies demonstrated that T cells from as early as 3-day-old {zeta}KO mice have already had the CD44highCD62Llow/neg phenotype, suggesting that these cells did not acquire this phenotype as the mice aged.

Based on these results, it appears more likely that {zeta}KO mice select for a polarized T cell population during thymocyte selection. Our results and those of others have previously shown that thymocyte development was altered in {zeta}KO mice (20, 21, 22, 23). Interestingly, the selected T cells from {zeta}KO mice bear high affinity TCRs for self-Ags (34, 41). It is thus possible that the selected thymocytes acquire their memory cell-like phenotype during the altered thymocyte selection in {zeta}KO mice. This possibility is further supported by our additional studies on thymocytes. The results from these studies demonstrate that a significant percentage of CD4+ thymocytes from {zeta}KO mice (~35–40%) and not from {zeta}+ mice bore the CD44highCD62low/neg phenotype. In addition, the fact that the activation of freshly isolated {zeta}KO mouse and not {zeta}+ mouse thymocytes could produce IFN-{gamma} is consistent with this idea.

Our further analyses of the role of {zeta}-chain on IFN-{gamma}-producing cells have shown that {zeta}-chain expression can also regulate the differentiation of IFN-{gamma}+/IL-10+ coproducing cells. The observation that {zeta}-chain-deficiency resulted in the differentiation of a significant population of IFN-{gamma}+/IL-10+ coproducing cells was unexpected because IL-10 is usually considered as a Th2 cytokine. Our recent studies and those of others have demonstrated that a unique T cell subset, termed regulatory type 1 T cells, can secrete both IFN-{gamma} and IL-10 (36, 37). Although we demonstrate in this report that a large percentage of IL-10-producing IFN-{gamma}+/IL-10+ cells can be obtained from culturing {zeta}KO mouse T cells, we do not know whether these cells function as regulatory T cells because we were not able to purify these cells. We could partially enrich for IL-10+ cells using magnetic beads. However, these partially enriched IFN-{gamma}+/IL-10+ T cells (up to ~50% of the recovered cells) died within 3 days after being activated in vitro. Furthermore, these cells also contained a significant population of IFN-{gamma}+/IL-10– cells that could secrete a large amount of IFN-{gamma}. Therefore, it is likely that, if some of the IFN-{gamma}+/IL-10+ cells were indeed IL-10-producing regulatory cells, their regulatory function would be counteracted by the presence of secreted IFN-{gamma}.

In addition to studying {zeta}-chain, we also examined the role of FcR{gamma} chain during the IFN-{gamma} production. The results demonstrated that the expression of a FcR{gamma} chain in {zeta}KO mice significantly decreased the percentage of IFN-{gamma}+/IL-10– T cells to the same level as that of T cells from {zeta}+ mice. Furthermore, both intracellular cytokine staining and ELISA studies demonstrated that the expression of FcR{gamma} also resulted in a significant decrease in the percentage of IFN-{gamma}+/IL-10+ T cells when compared with that of {zeta}KO mice, although the percentage of such cells is still higher than that of T cells from {zeta}+ mice. Taken together, these results suggest that the FcR{gamma} chain-mediated signals, like those mediated by {zeta}-chain, can regulate the differentiation of both IFN-{gamma}+/IL-10– and IFN-{gamma}+/IL-10+ T cells. We have reported in a separate study that development of T cells bearing FcR{gamma}, like {zeta}-chain-bearing T cells, requires both ZAP-70 and lck kinases (42). Therefore, it is likely that activation of similar signaling pathways mediated through {zeta}-chain and FcR{gamma} chain that involve ZAP-70 and lck kinases can result in a decrease in the number of IFN-{gamma}-producing T cells.

We have studied the potential molecular mechanisms underlying the unexpected selection of polarized IFN-{gamma}-producing cells in {zeta}KO mice. It has been demonstrated that expression of STAT1 and STAT4 associate with IFN-{gamma} production, and they play critical roles during the differentiation of Th1 cells (7, 43, 44, 45). During in vitro Th1 cell differentiation, the induction of T-bet expression by IFN-{gamma} could be dependent on activation of STAT1. In addition, the expression and activation of T-bet may precede IL-12-dependent signaling events in regulating Th1 cell differentiation (45, 46). Interestingly, our studies have shown that freshly isolated CD4+ T cells from {zeta}KO mice, and not from the other two strains of mice, up-regulated the expression and phosphorylation level of STAT4 but not STAT1. Furthermore, only {zeta}KO mouse T cells demonstrated an increased level of T-bet. A recent study has shown that activation of T-bet-transfected T cells can still produce a significant amount of IFN-{gamma} when they are cultured in a Th2 condition (45). Our results are consistent with these studies and suggest that up-regulation of T-bet may lead to the polarized production of IFN-{gamma} by {zeta}KO mouse T cells, even after they are cultured in a Th2 condition. It is currently unclear how the signals mediated through the {zeta}-chain lead to the up-regulation of T-bet and STAT4. Further investigations, such as studies to determine tyrosine residues in the ITAM of the {zeta}-chain, would be helpful to further understand the role of {zeta}-chain in the development of these T cells. Altogether, these results support the idea that a signaling pathway, which is downstream or independent of STAT1, is activated in {zeta}KO mouse T cells leading to the activation of STAT4 but not STAT1. This novel pathway also induces the up-regulation and activation of T-bet and results in the polarized production of IFN-{gamma}. In addition, our results suggest that the signals mediated through {zeta}-chain or, to a lesser extent, FcR{gamma} chain may suppress such a pathway, allowing for the production of a large amount of IFN-{gamma} by {zeta}- or FcR{gamma}-bearing T cells only after these cells have been cultured in a condition favoring Th1 cell differentiation.

In vivo regulation of the development of IFN-{gamma}-producing CD4+ T cells by {zeta}-chain expression may play important roles in several physiological and pathological conditions in animals and humans. Expression of {zeta}-chain may ensure for proper regulation of the differentiation of IFN-{gamma}-producing cells and the development of autoimmune disease-inducing pathogenic Th1 cells. For example, our findings bear interesting similarities to those that show the loss of {zeta}-chain in T cells associated with rheumatoid arthritis and lupus (24, 26). An imbalanced activation and differentiation of Th1 cells may play important roles in the pathogenesis of several autoimmune diseases (47, 48). Based on our results, one may speculate that the loss of {zeta}-chain in TCR complexes of T cells found in autoimmune disease patients may predispose the patients to a proinflammatory condition and facilitate the disease progress. In addition, sustained bacterial infection induces IFN-{gamma}-dependent {zeta}-chain down-regulation and defective T cell function (30). Moreover, it has also been shown that human effector CD4+ T cells generated in vitro by activation through their TCRs lose their {zeta}-chain expression (49). Interestingly, activation of these effector CD4+ T cells can produce a large quantity of IFN-{gamma}. Therefore, the results from our studies and those of others support the idea that down-regulation of {zeta}-chain and rapid IFN-{gamma} production play important roles during the selection of CD4+ T cells that perform more potent effector functions in protecting individuals from infections.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Chih-Pin Liu, Division of Immunology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3000. E-mail address: cliu{at}coh.org Back

Received for publication July 9, 2004. Accepted for publication October 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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