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Regulation of Apoptosis in Mature ß⁺CD4^-CD8^- Antigen-Specific Suppressor T Cell Clones¹

Qasim Khan^*, Josef M. Penninger, Liming Yang^*, Luciano E. Marra^*, Ivona Kozieradzki and Li Zhang^2,*

^* Department of Laboratory Medicine and Pathobiology, and Multi Organ Transplant Program, Toronto Hospital Research Institute, University of Toronto, Toronto, Ontario, Canada; and The Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada

	Abstract

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The regulation of apoptosis in mature CD4⁺ or CD8⁺ ß⁺ T cells has been well studied. How the survival and death is regulated in peripheral CD4^-CD8^- (double negative, DN) ß⁺ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4⁺ or CD8⁺ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of ß⁺ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for L^d and permanently accepted L^d+ skin allografts after pretransplant infusion of L^d+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-x_L, but not Bcl-2; 4) both Bcl-x_L and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-x_L and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.

	Introduction

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The majority of ß T cells in the periphery of normal mice express the CD4 or CD8 molecule (1). About 1–5% of peripheral T cells express neither CD8 nor CD4, i.e., they are CD4 and CD8 double negative (DN)³ ß⁺ T cells (2). To date, extensive studies have been done on CD4⁺ and CD8⁺ T cells to investigate the mechanisms involved in the apoptosis and homeostasis of mature lymphocytes (3, 4). For instance, it is well known that the Fas/Fas ligand (FasL) and TNF-/TNFR interactions are important pathways for mediating apoptosis in mature CD4⁺ and CD8⁺ T lymphocytes (5, 6, 7, 8, 9, 10, 11, 12, 13). Resistance or susceptibility to apoptosis is influenced by intrinsic or extrinsic survival signals received by the T cell. Among them, Bcl-2 and Bcl-x_L have been shown to be potent repressors of apoptosis in CD4⁺ or CD8⁺ T cells (14, 15, 16, 17). In contrast to CD4⁺ or CD8⁺ cells, the mechanisms that regulate survival and death of mature DN T cells have never been studied.

Recent studies have suggested that DN T cells possess an immunoregulatory/suppressive function and can prevent graft-versus-host disease and autoimmunity (18, 19, 20). Other studies suggest that DN T cells may be a source of IL-4 and may initiate a Th2 response (21, 22). Using 2C anti-L^d TCR transgenic mice (the transgenic ß-TCR can be detected by the mAb 1B2) as a model, we have recently demonstrated that encountering the alloantigen L^d in vivo leads to activation-induced cell death (AICD) in the majority of 1B2⁺CD8⁺ T cells in peripheral lymphatic organs. Interestingly, no AICD was observed in the peripheral DN T cells that express the same TCR (1B2⁺CD8^-CD4^-), suggesting that these 1B2⁺DN T cells may be more resistant to AICD in vivo than 1B2⁺CD8⁺ cells (23). Furthermore, when the 1B2⁺DN T cells were purified from the spleen of the 2C mice made tolerant to L^d+ skin allografts by pretransplant infusion of L^d+ cells and tested in vitro, these cells were able to inhibit proliferation of naive anti-L^d T cells. Moreover, 1B2⁺DN T cell clones generated from the 2C mice that become tolerant to L^d+ skin allografts inhibit the proliferation of naive syngeneic T cells in a dose-dependent manner in vitro. These DN T cell clones can also specifically prevent L^d+ skin allograft rejection when adoptively transferred to naive syngeneic animals.⁴ These findings, together with the reports by other groups (18, 19, 20), provide evidence that DN cells may function as Ag-specific suppressor cells in vitro and in vivo. Therefore, it is important to understand the mechanisms regulating their survival and death.

In the present study, we used the 1B2⁺DN Ag-specific suppressor T cell clones in an in vitro TCR cross-linking-induced apoptosis model to investigate the molecules and pathways involved in regulating apoptosis in mature DN T cells. It was found that when cultured in the presence of exogenous IL-4, the 1B2⁺DN T-suppressor clones were highly resistant to TCR cross-linking-induced apoptosis. In contrast to apoptosis-sensitive CD4⁺ or CD8⁺ T cells, neither Fas/FasL pathway nor TNF-/TNFR interactions seemed to play a significant role in regulating apoptosis in 1B2⁺DN T cells. IL-4 stimulation induced up-regulation of the death-repressing molecule Bcl-x_L but had no effect on Bcl-2 expression. Moreover, IL-4 stimulation triggered induction of the AP-1 transcription factor c-Jun and activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Together, these results provide a better picture of how the survival of DN suppressor T cells is regulated and suggest that IL-4 plays a pivotal role in determining the fate of DN suppressor T cells.

	Materials and Methods

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Mice

C57BL/6 (B6, H-2^b), (B6 x BALB/c)F₁ (H-2^b/d L^d+), and BALB/c H-2-dm2 (dm2, a BALB/c L^d loss mutant, H-2 D^d+, K^d+, L^d+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding stock of 2C transgenic mice was kindly provided by Dr. Dennis Y. Loh (24). A large fraction of T cells in the periphery of the 2C mice express a transgenic ß-TCR reactive against the L^d (class I MHC). These T cells can be detected by the clonotypic mAb 1B2 (24, 25). B62C mice (H-2^b/b) were bred with dm2 mice to generate 1B2⁺ 2C F₁ mice (H-2^b/d, L^d-, anti-L^d TCR⁺). All animals were maintained in the animal facility at the Ontario Cancer Institute.

Generation and maintenance of 1B2⁺CD8^-CD4^- T cell clones

Spleen cells were collected from 2C F₁ mice that permanently accepted L^d+ skin allografts from (B6 x BALB/c)F₁ donors for the generation of T cell clones using standard cloning and subcloning procedures. To maintain the T cell clones, 5 x 10⁴ clone cells were cultured in a 24-well plate containing 5 x 10⁵ irradiated L^d+ cells in an -MEM supplemented with 10% FCS and 30 U/ml of rIL-2 and 50 U/ml of rIL-4. The cells were incubated at 37°C with 5% CO₂. The T cell clones were stimulated in the above manner every 3–5 days. From the initial 28 clones generated, 7 1B2⁺DN clones were grown successfully. All the DN clones express an equivalent level of ß-TCR as seen in activated 1B2⁺CD8⁺ T cells, and are CD3⁺, CD8^-, CD4^-, NK1.1^-, CD25⁺, CD69⁺ (data not shown). In addition, all the 7 1B2⁺DN T cell clones display Ag-specific suppressive function.⁴

Mixed lymphocyte reaction (MLR)

Splenocytes from naive 2C F₁ were collected and used as responder cells in a MLR. A total of 7.5 x 10⁵ cells/ml were cocultured with irradiated (20 Gy) sex-matched splenocytes (7.5 x 10⁵ cells/ml) from (B6 x BALB/c)F₁ in -MEM supplemented with 10% FCS and 30 U/ml of rIL-2 and 50 U/ml of rIL-4 as a source of growth factor in 24-well plates. After a 3.5 day incubation at 37°C and 5% C0₂, the activated 2C F₁ lymphocytes were collected using Lympholyte M (Cedarlane, Ontario, Canada) for further analysis.

Flow cytometric analysis for Fas and TNFR expression

Cells were stained with PE-conjugated anti-Fas Ab (PharMingen, San Diego, CA) to examine surface Fas receptor expression. Hamster anti-mouse TNFR-I (p55) (T55-593.4) and TNFR-II (p75) (T75-54.7.14) Abs (26) followed by anti-hamster FITC-conjugated Ab were used to examine surface expression of TNFRs. Data were acquired and analyzed on an Epics XL-MCL flow cytometry machine (Coulter, Miami, FL).

Purification of 1B2⁺CD8⁺ and 1B2⁺DN T cells

Spleen cells were collected from 2C F₁ mice and stained with FITC-labeled clonotypic mAb 1B2, PE-conjugated anti-CD4 (GK1.5) and Cy-chrome conjugated anti-CD8 (53-6.7) as described previously (27). 1B2⁺CD8⁺ and 1B2⁺CD4^-CD8^- T cells were sorted by using a cell sorter (Coulter, Epics V). The purity and viability of the cells after sorting were >95%.

Induction of apoptosis by TCR cross-linking

The 1B2 mAb was diluted in PBS at a final concentration of 65 µg/ml and incubated in a 24-well plate at 4°C overnight. Nonspecific binding sites were blocked with 10% FCS/PBS. Following activation of the clones and naive 2C F₁ lymphocytes, 1.25 x 10⁵ cells were plated in each well either precoated with or without 1B2 mAb. Numbers of viable and dead cells were measured using eosin exclusion at 21, 44, 72, and 96 h after cross-linking. Apoptotic cell death after TCR cross-linking was confirmed by DNA gel electrophoresis (28) and the TUNEL assay (29).

Detection of Fas/FasL mRNA levels by RT-PCR

Total RNA was extracted from the L12 clone (5 x 10⁶ cells) with TriZol reagent (Life Technologies, Grand Island, NY). cDNA was prepared from RNA with 0.5 mg/ml of pd(N)6 Random Hexamer Primer (Pharmacia Biotech, Uppsala, Sweden) and 300 U of murine leukemia virus (MLV) reverse transcriptase (Life Technologies); 2 µl of the cDNA mixture was used in a PCR with 10 pmol of forward and reverse primers, as described elsewhere for Fas and FasL (27), and 2.5 U of Taq DNA polymerase (Life Technologies). The sequences of the specific sense and antisense oligonucleotide primer pairs, 5' and 3' for GAPDH are as follows (30): sense, 5'-TGATGACATCAAGAAGGTGGTGAA-3'; and antisense, 5'-TCCTTGGAGGCCATGTAGGCCAT-3'. Samples were amplified through 30 (Fas and FasL) or 35 (GAPDH) cycles at an annealing temperature of 59°C in a PCR Thermal Cycler (MJ Research, Watertown, MA). The amplified products were separated on a 1.5% agarose gel stained with ethidium bromide. GAPDH was used as an internal control for RNA integrity.

Western blot analysis

L12 cells were collected after treatment with or without 1B2 mAb and IL-4, washed with PBS, pelleted by centrifugation, and lysed using RIPA buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM NaF, 2 mM EDTA, 1 mM sodium orthovanadate, and 0.05% NaN₃) supplemented with 0.1% aprotinin, 0.1% leupeptin, and 1 mM PMSF. Samples were incubated on ice for 15 min and then centrifuged at 13,000 x g to remove cellular debris. Protein concentration of the supernatant was determined using the colorimetric BCA assay (Bio-Rad, Richmond, CA). A total of 10 µg of total protein for each sample was denatured in SDS sample loading buffer and separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and blocked with 5% milk and 0.5% Tween-20 in Tris-buffered saline (T-TBS). After blocking, blots were incubated with anti-Bcl-x (1:5000) (kindly provided by Dr. Craig Thompson, University of Chicago), anti-Bcl-2 (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-IRF-4 (gift of T. W. Mak, Amgen Institute, Toronto), anti-ß-actin (Sigma, St. Louis, MO) anti-c-Jun, anti-MAPK, SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase; all from New England Biolabs, Beverly, MA) Abs in T-TBS. Activation of MAPK and PKB/Akt was detected using phospho-MAPK and phospho-PKB/Akt-specific Abs indicative of active MAPK/ERK and PKB/Akt (New England Biolabs). Blots were washed in T-TBS and subsequently incubated with HRP-conjugated anti-rabbit (1:10000) (Sigma) and anti-mouse (1:10000) (Bio-Rad) Abs. Western blots were developed using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL).

	Results

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Resistance to TCR cross-linking-induced apoptosis in 1B2⁺DN T cell clones

Cross-linking the TCR of activated T cells using mAb, either anti-TCR or anti-CD3, in vitro causes the cell to die by a process known as TCR cross-linking-induced apoptosis (31, 32, 33, 34, 35). To study the response of the 1B2⁺DN T cell clones to TCR cross-linking-induced apoptosis, the 1B2 mAb, which specifically recognizes the transgenic TCR - and ß-chains expressed on the DN clones (36), was used. First, the 1B2 mAb was titrated at 10, 25, 50, 65, 75, and 100 µg/ml to find the optimal concentration for TCR cross-linking-induced cell death. A total of 65 µg/ml 1B2 mAb was chosen because this is the lowest dose that can induce maximal cell death after cross-linking (data not shown). Two 1B2⁺DN T cell clones (L2 and L12) that possessed immunosuppressive function were stimulated with L^d+ cells for 3.5 days. Viable cells were purified and plated in 24-well tissue culture plates that were precoated with 1B2 mAb. At various time points (21, 44, 72, and 96 h) after cross-linking, the cells were harvested and cell viability was measured by eosin exclusion. The fresh splenic 1B2⁺CD8⁺ and 1B2⁺DN T cells syngeneic to the DN T cell clones were purified using FACS and treated in the same way as controls. Fig. 1 shows that the activated apoptosis-sensitive 1B2⁺CD8⁺ cells exhibited dramatic cell death (about 60%) within the first 21 h after cross-linking. By 96 h, only 25% of the apoptosis-sensitive 1B2⁺CD8⁺ cells were viable. Cell death was due to apoptosis as assessed by DNA gel electrophoresis and the TUNEL assay (data not shown). In contrast, both of the DN T cell clones as well as primary activated 1B2⁺DN T cells were highly resistant to cross-linking-induced cell death (Fig. 1). These data demonstrate that both primary activated DN T cells and DN T cell clones generated from mice that permanently accepted an L^d+ skin allograft are resistant to TCR cross-linking-induced cell death. In a separate study, we have demonstrated that both fresh isolated 1B2⁺DN T cells and 1B2⁺DN T cell clones were able to down-regulate the anti-L^d responses in vitro and in vivo.⁴ These findings suggest that the 1B2⁺DN T cells and clones (L2 and L12) have similar phenotypes and function. Therefore, the L12 clone was used for further analysis of the molecules and pathways that are involved in the resistance to TCR cross-linking-induced cell death for the remainder of the study.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Resistance to TCR cross-linking-induced apoptosis in 1B2+DN T cells and clones. IB2+DN L2 clone (filled bars), 1B2+DN L12 clone (open bars), fresh purified 1B2+CD8+ (hatched bars), and 1B2+DN (dotted bars) T lymphocytes were stimulated by Ld+ cells in an MLR. After 3.5 days, viable cells were collected and plated in wells precoated with or without 1B2 mAb. Cells were collected at various time points (21, 44, 72, and 96 h), and cell viability was measured using eosin exclusion. Data are shown as percent viability, i.e., number of viable cells in 1B2-treated wells as a percent of number of viable cells in non-1B2-treated wells. Data are the mean ± SEM of at least three independent experiments for T cell clones and two independent experiments for purified T cells.

The Fas surface molecule and the low (p55) and high (p75) m.w. TNFRs, TNFR-I and TNFR-II, are known to be potent inducers of cell death in apoptosis-sensitive CD4⁺ and CD8⁺ T cells. Numerous studies have shown that activated CD4⁺ and CD8⁺ T cells undergo AICD when the Fas molecule binds with its ligand, FasL (5, 6, 7, 8, 9). Furthermore, the deficiency of Fas/FasL seen in MLR-lpr/gld mice results in an accumulation of autoreactive T cells in the periphery (37, 38). Similarly, recent studies have shown that when TNF- binds to the TNFR-I and/or TNFR-II expressed on the surface of CD4⁺ or CD8⁺ T cells (11, 39), the cells will die by apoptosis (10, 11, 12, 13). Because our DN clones were highly resistant to TCR cross-linking-induced apoptosis, we first wanted to see if this resistance was due to an impairment of the Fas/FasL pathway. Apoptosis-resistant 1B2⁺DN T cells were collected 3.5 days after activation, and their expression of Fas and FasL was compared with activated apoptosis-sensitive 1B2⁺CD8⁺ T cells. As shown in Fig. 2a, the level of FasL mRNA was similar for apoptosis-resistant 1B2⁺DN and apoptosis-sensitive 1B2⁺CD8⁺ T cells. However, the level of Fas mRNA was lower in apoptosis-resistant 1B2⁺DN L12 cells compared with apoptosis-sensitive 1B2⁺CD8⁺ T cells. The same result was found when Fas protein expression was measured using flow cytometry (Fig. 2b). When both apoptosis-sensitive and -resistant cells were incubated with soluble anti-Fas mAb, there was no significant increase in the number of dead cells by 96 h after cross-linking in the apoptosis-resistant 1B2⁺DN cells, whereas the addition of anti-Fas mAb to apoptosis-sensitive 1B2⁺CD8⁺ T cells did induce more cell death when compared with TCR cross-linking alone (Fig. 2c). These results demonstrate that apoptosis-resistant 1B2⁺DN cells express a lower level of Fas, but not FasL, than apoptosis-sensitive 1B2⁺CD8⁺ T cells. Further, addition of anti-Fas mAb does not cause additional cell death in the apoptosis-resistant 1B2⁺DN T cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Expression of Fas and FasL on DN T cells and 1B2+CD8+ T cells, and the addition of anti-Fas Ab to DN T cells and 1B2+CD8+ T cells. a, Fas and FasL expression were detected by RT-PCR. Total RNA was extracted from the following cells: 1B2+CD8+ cells 3 days after activation (lane 1), 1B2+CD8-CD4- L12 cells 3 days after activation (lane 2), L12 cells without 1B2 cross-linking (lane 3), and L12 cells 96 h after 1B2 cross-linking (lane 4). GAPDH was used as a positive internal control for RNA integrity. Data shown are representative of three independent experiments. b, FACS analysis for Fas surface receptor expression. Activated apoptosis-sensitive 1B2+CD8+ cells and apoptosis-resistant 1B2+DN L12 cells were collected 96 h after IB2 cross-linking and stained with PE-conjugated anti-Fas Ab. Surface expression was compared with unstained controls. Data represent staining of three independent experiments. c, Addition of soluble anti-Fas mAb does not cause additional apoptosis in 1B2+DN T cells. Apoptosis-resistant DN T cells and apoptosis-sensitive 1B2+CD8+ T cells were collected after 96 h of cross-linking in the presence (open bars) or absence (filled bars) of anti-Fas mAb. Cell viability as percent of controls (no 1B2 cross-linking) is shown. Data represent the mean of at least two experiments.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Apoptosis-resistant and sensitive 1B2+ T cells express similar levels of TNFR. Apoptosis-resistant 1B2+DN L12 cells and apoptosis-sensitive 1B2+CD8+ were collected at 96 h after TCR cross-linking and stained with hamster anti-mouse TNFR-II mAb (T75-54.7.14) followed by goat anti-hamster FITC. Cells stained with goat anti-hamster FITC alone were used as control. Data were analyzed by flow cytometry. Data are representative of two independent experiments.

Based on studies from apoptosis-sensitive CD4⁺ or CD8⁺ cells, the fate of a cell during apoptosis is not solely dependent on the death pathways. It is also influenced by survival signals received by the T cell. The Bcl-2 family of gene products is well known to play a dominant role in regulating apoptosis. Among them, Bcl-2 and Bcl-x_L have been shown to be potent repressors of apoptosis in CD4⁺ or CD8⁺ T cells, whereas Bcl-x_S has been shown to be an inducer of apoptosis (14, 15, 16, 17). To understand the role of Bcl-2 and Bcl-x_L in the maintenance of survival of DN T cells, we investigated whether there was an increase in Bcl-2 and/or Bcl-x_L expression in the DN L12 cells that could account for the resistance of DN T cells to apoptosis. Using Western blot analysis, it was found that unlike what is seen in resting and activated apoptosis-sensitive CD4⁺ or CD8⁺ T cells (40), there was no Bcl-2 expression in activated apoptosis-resistant 1B2⁺DN L12 cells (Fig. 4, time 0). However, the level of Bcl-2 protein was increased at all time points after 1B2 cross-linking compared with the non-cross-linked cells (Fig. 4, time 21, 44, 96 h; X-linking). When the level of Bcl-x_L expression was studied, a significantly higher level of Bcl-x_L was detected in L12 cells compared with that of apoptosis-sensitive 1B2⁺CD8⁺ T cells (Fig. 4, Bcl-x_L time 0 vs 1B2⁺CD8⁺). At 44 and 96 h after TCR cross-linking, both Bcl-x_L and Bcl-2 expression were increased compared with non-cross-linked L12 cells. In contrast, no difference in the level of Bcl-x_S, which is the protein product of a smaller, alternatively spliced mRNA of the bcl-x gene and potent apoptosis inducer (15), was detected between 1B2⁺DN T cells and 1B2⁺CD8⁺ T cells (Bcl-x_S; Fig. 4). These results indicate that while Bcl-2 may play a major role in the maintenance of the survival of apoptosis-sensitive 1B2⁺CD8⁺ T cells, Bcl-x_L may be the principle molecule involved in maintaining the survival of apoptosis-resistant 1B2⁺DN T cells. TCR mediated up-regulation of both Bcl-2 and Bcl-x_L may contribute to the high resistance of these cells to TCR cross-linking-induced apoptosis. In contrast, Bcl-x_S was either playing no part in cross-linking-induced apoptosis in DN L12 cells, or the ratio of Bcl-x_S to Bcl-2 and Bcl-x_L favored the death-preventing molecules.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Expression of Bcl-2, Bcl-xL, and Bcl-xS in apoptosis-resistant 1B2+DN T cells. 1B2+DN L12 cells were collected before cross-linking (time 0) and 21, 44, and 96 h after 1B2 (X-linking) or no 1B2 (no X-linking) treatment and lysed with Nonidet P-40. The expression of Bcl-2, Bcl-xL, Bcl-xS, and ß-actin as an internal control for protein integrity was examined by Western blotting. Activated apoptosis-sensitive 1B2+CD8+ T cells were used as a control. Data are representative of at least two independent experiments.

Numerous studies have shown that IL-4 protects against a variety of apoptotic stimuli, particularly in B cells (41, 42, 43, 44). However, its role in cross-linking-induced T cell death is not known. Because the L12 clone requires the presence of exogenous IL-4 to grow and proliferate, and our previous results indicated that apoptosis-resistant T cells expressed a high level of IL-4 mRNA (27), it was possible that IL-4 may protect 1B2⁺DN T cells from AICD. To test this possibility, DN L12 cells were stimulated and grown in the absence of IL-4 for either 6 (Fig. 5, top) or 30 days (Fig. 5, bottom). The cells were then harvested and their TCR cross-linked with 1B2 mAb in the presence of IL-4 (-IL4,+IL4) or absence of IL-4 (-IL4,-IL4). DN L12 cells activated and cross-linked in the presence of IL-4 were used as controls (+IL4,+IL4). As early as 21 h after TCR cross-linking, there was a significant increase in the number of dead cells among cells activated and cross-linked without IL-4 (-IL4,-IL4) compared with cells activated and cross-linked in the presence of IL-4 (+IL4,+IL4). By 96 h, there was significantly increased cell death in the -IL4,-IL4 cells compared with the control cells and -IL4,+IL4 cells. Together, these results demonstrate that IL-4 plays a protective role against cross-linking-induced apoptosis in DN L12 cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. IL-4 protects against IB2 cross-linking-induced apoptosis in DN L12 cells. L12 cells were activated in the absence of IL-4 for 6 days (top) or 30 days (bottom), and then cross-linked using the IB2 Ab in the presence (-IL4,+IL4, ) or absence of IL-4 (-IL4,-IL4, ). Cells were collected at the indicated time points, and cell death was measured using eosin exclusion. Dead cells are shown as a difference between the number of dead cells in the 1B2-treated wells and non-1B2-treated wells. L12 cells activated and cross-linked in the presence of IL-4 were used as a control (+IL4,+IL4, ). Data are an average of three independent experiments.

Apoptosis-resistant 1B2⁺DN T cells, which were cultured in the presence of IL-4, express a significantly lower level of Fas upon activation compared with apoptosis-sensitive 1B2⁺CD8⁺ T cells (Fig. 2). To investigate the mechanisms of how IL-4 exerts its role on preventing apoptosis in DN T cells, we first examined whether this protection is through down-regulation of Fas expression on DN cells. The 1B2⁺CD8^-CD4^- L12 cells were cultured in the presence or absence of exogenous IL-4 for 6 days, and the expression of the Fas molecule on the cell surface was analyzed using flow cytometry. As shown in Fig. 6, there was no down-regulation of Fas expression on the L12 cells that were cultured in the presence of IL-4 compared with those cultured in the absence of IL-4. In fact, the expression of Fas is slightly higher in the L12 cells cultured in the presence of IL-4. The higher expression of Fas in the L12 cells became more pronounced when compared with L12 cells that were cultured in the absence of IL-4 for a longer time period (data not shown). These data indicate that IL-4 does not down-regulate Fas expression in DN L12 cells, suggesting that IL-4 may protect DN cells from TCR cross-linking-induced cell death through other mechanisms.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. IL-4 up-regulates Fas expression in DN L12 cells. L12 cells were stimulated in the presence or absence of IL-4 for 6 days. Cells were collected and stained with PE-conjugated anti-Fas Ab to determine the level of Fas expression. Surface expression was compared with unstained controls. Data represent staining of at least two independent experiments.

Next, we investigated whether IL-4 plays any role in the regulation of the death-repressing molecules Bcl-2 and Bcl-x_L. L12 cells were stimulated and cross-linked in the presence or absence of IL-4 as described in the previous section. Because the greatest difference in cell death was seen at 96 h after TCR cross-linking, the cells were collected at this time point, and the protein was extracted for Western blot analysis. When L12 cells cultured in the presence of IL-4 were compared with those in the absence of IL-4, no significant difference in the expression of Bcl-2 was found (Fig. 7, +IL4,+IL4 vs -IL4,-IL4). In fact Bcl-2 expression is highest in the absence of exogenous IL-4. However, when Bcl-x_L was studied, the L12 cells cultured in the presence of IL-4 had a significantly higher expression of Bcl-x_L (+IL4,+IL4) compared with the cells that did not have IL-4 present at any time (-IL4,-IL4) (Fig. 7). Furthermore, when IL-4 was added back during cross-linking to cells not activated with IL-4, the level of Bcl-x_L increased to almost the same level as the cells that were activated and cross-linked in the presence of IL-4 (Fig. 7, +IL4,+IL4 vs -IL4,+IL4). Because the only difference between the cells was the presence or absence of IL-4, these results demonstrate that IL-4 up-regulates the expression of the death suppressor Bcl-x_L, but IL-4 does not induce Bcl-2 expression.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. IL-4 up-regulates Bcl-xL, but not Bcl-2. L12 cells stimulated and cross-linked in the presence of IL-4 (+IL4,+IL4) and L12 cells not stimulated with IL-4, but cross-linked in the presence of IL-4 (-IL4,+IL4) or absence of IL-4 (-IL4,-IL4) were collected after 96 h and lysed as before. The levels of Bcl-2 and Bcl-xL were examined by standard Western blot analysis, and ß-actin was used as a positive internal control. Data are representative of at least two independent experiments.

Previously it has been shown that MAPK/ERK, SAPK/JNK, and PKB/Akt activation have an important role in regulating the cellular resistance and susceptibility to apoptosis (45, 46, 47, 48, 49, 50). Moreover, PKB/Akt and SAPK/JNK signaling cascades protect T cells from apoptosis (51, 52, 53, 54). Interestingly, culture of L12 cells in the presence of IL-4 led to the induction of MAPK phosphorylation and transactivation of the c-Jun transcription factor (Fig. 8). c-Jun transactivation and MAPK phosphorylation were dependent on IL-4 but not on TCR cross-linking. In contrast, transactivation of the transcription factor IRF-4, which is strongly induced by TCR stimulation and regulates T cell proliferation in vivo (55), was dependent on TCR cross-linking but not on the presence of IL-4, suggesting that the IL-4 effects might be specific for MAPK and c-Jun activation (Fig. 8). Moreover, we failed to detect activation of PKB/Akt in the presence of IL-4 and/or TCR cross-linking in DN L12 cells (data not shown). These results imply that IL-4 mediated protection from cell death in mature DN T cells might be mediated through activation of the MAPK and c-Jun signaling cascades.

View larger version (39K): [in this window] [in a new window]   FIGURE 8. IL-4 induces MAPK phosphorylation and up-regulation of c-Jun. DN L12 cells were cross-linked in the presence of IL-4 (+IL4,+IB2) or absence of IL-4 (-IL4,+IB2) or cultured without cross-linking in the presence of IL-4 (+IL4) or absence of IL-4 (-IL4). After 96 h of culture, the levels of MAPK, phospho-MAPK (P-MAPK), c-Jun, and IRF-4 were examined by Western blot analysis. ß-Actin was used as a positive internal control. Data are representative of at least two independent experiments.

	Discussion

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In this study, we report that the apoptosis-resistant 1B2⁺DN T cell clones that possess suppressive function in vitro and in vivo differ from apoptosis-sensitive CD8⁺ and CD4⁺ T cells in the following aspects. 1) Most CD8⁺ or CD4⁺ T cells are known to be susceptible to TCR-cross-linking induced cell death (31, 32, 33, 34, 35). However, the DN T cell clones are highly resistant to TCR cross-linking-induced apoptosis. This finding is consistent with our previous observation that 1B2⁺DN T cells are more resistant to AICD in vivo than 1B2⁺CD8⁺ cells (23). 2) Unlike what is seen in apoptosis-sensitive CD8⁺ or CD4⁺ T cells, neither the Fas/FasL nor TNF-/TNFR pathway plays a critical role in DN T cells. Although 1B2⁺DN T cells expressed lower levels of Fas compared with 1B2⁺CD8⁺ cells (Fig. 2), it is unlikely that this low expression of Fas is responsible for the resistance to apoptosis in 1B2⁺DN T cells for the following reasons. First, because the apoptosis-resistant 1B2⁺DN T cells cultured in the absence of IL-4 had an even lower expression of Fas (Fig. 6), yet were more susceptible to cross-linking-induced cell death (Fig. 5). Second, addition of anti-Fas mAb did not have any influence on apoptosis in these DN T cells (Fig. 2c). DN L12 T cells express equivalent levels of FasL and TNFRs. They also produce high levels of TNF- upon activation (data not shown). Addition of either anti-TNFR Abs or exogenous TNF- did not have any effect on the number of apoptotic L12 cells, suggesting that these death pathways may not exert a dominant effect on regulating apoptosis in these DN T cells. 3) Compared with apoptosis-sensitive 1B2⁺CD8⁺ T cells, Bcl-2 is barely detectable whereas Bcl-x_L is highly up-regulated in activated apoptosis-resistant 1B2⁺DN T cells (Fig. 4).

The results from our studies support the notion that Bcl-x_L may be the major molecule responsible for the resistance to apoptosis in DN T cells. This hypothesis is based on the following findings. First, constitutively high expression of Bcl-x_L, but barely detectable Bcl-2 expression is observed in apoptosis-resistant 1B2⁺DN T cells. Second, upon TCR cross-linking, Bcl-x_L expression is further up-regulated. Third, IL-4, which protects DN T cells from apoptosis, can also up-regulate Bcl-x_L, but not Bcl-2 or Bcl-x_S. Together, these data indicate that Bcl-x_L may play an essential role in protecting the DN T cells from apoptosis. Because there is also an up-regulation of Bcl-2 during TCR cross-linking, it is possible that Bcl-x_L may be acting synergistically with Bcl-2 to confer greater protection against a death signal. Over-expression of Bcl-x_L has been shown to be able to prevent Fas-mediated cell death in B cells and CD8⁺ T cells (56, 57). The constitutive high expression of Bcl-x_L may prevent DN T cells from undergoing cross-linking-induced apoptosis. It could be exerting its effect by overriding the death signals induced by the Fas/FasL pathway or by distinct apoptosis signaling pathways. The molecular mechanisms of how Bcl-x_L exerts its role in protecting lymphocytes from apoptosis needs to be elucidated.

Our previous data showed that apoptosis-resistant 1B2⁺CD8⁺ T cells expressed a high level of IL-4 mRNA (27), and sera from tolerant mice, which had a substantial number of DN T cells in the periphery, expressed high levels of IL-4 (23). Moreover, our DN T cell clones required IL-4 for maximal proliferation and suppression. IL-4 has been previously shown to protect B-cells from Fas-mediated death (43). Studies from T cells have shown that Th2 cells are preferentially protected from apoptosis using the Fas/FasL pathway as opposed to Th1 cells (58, 59). A recent study has also shown that IL-4 rescued resting CD4⁺ T cells from apoptosis by prolonging the expression of Bcl-2 and Bcl-x_L (60). However, no work had been done to see if IL-4 played a similar role in activated DN T cells. In this study, we demonstrate that IL-4 protects peripheral DN T cells from TCR cross-linking-induced cell death. In the absence of IL-4, Bcl-x_L expression is significantly down-regulated, whereas both Bcl-2 and Fas are similarly expressed compared with cells that have IL-4, but the cells are still sensitive to cross-linking-induced apoptosis. These data suggest that the protective role of IL-4 is due to the up-regulation of Bcl-x_L rather than Bcl-2 or down-regulation of Fas in DN T cells. Whether IL-4 has any effect on modulation of Fas signaling needs to be elucidated. Taken together, these data indicate that IL-4 plays an important part in preventing cross-linking-mediated cell death, and this protection may be conferred by the up-regulation of Bcl-x_L, but not by the down-regulation of Fas.

Furthermore, we demonstrate the IL-4-induced MAPK phosphorylation and c-Jun expression in DN L12 cells, which indicate the role of IL-4 in activation of MAPK and the c-Jun, and possibly SAPK/JNK signaling cascades in DN T cells. It has been shown recently that the SAPK/JNK signaling cascade can protect thymocytes from Fas and anti-CD3 mediated apoptosis (50) and SAPK/JNK activation prevents Ag receptor-induced cell death of activated peripheral CD4⁺ and CD8⁺ T cells (54). Moreover, it has been suggested that SAPK/JNK links TCR signaling to the induction of Bcl-x_L (54). Together, these findings suggest the possibility that IL-4 might exert its death suppressive effect via activation of the MAPK and c-Jun signal transduction pathways to up-regulate Bcl-x_L expression.

How does this translate to the survival of DN T cells in vivo? Previously, we have shown that pretransplant infusion of donor lymphocytes can induce permanent donor-specific skin allograft survival in the host (23). An interesting finding from that study showed a large population of DN T cells that remained in the periphery of the host, being resistant to apoptosis in vivo. As well, the serum from the long-term skin allograft tolerant mice showed an elevated level of IL-4 (23). The source of this IL-4 was not known; however, some studies have shown that the DN T cells themselves may produce it (21, 22). It is possible that these cells are able to avoid undergoing apoptosis due to the protective effect of IL-4. IL-4 probably protects DN T cells from apoptosis via an up-regulation of the death-repressing molecule Bcl-x_L. Because DN T cells have been shown to possess a suppressor function in vitro and in vivo (Refs. 18–20; footnote 4), it is possible that mature DN T cells are able to regulate the function of CD4⁺ or CD8⁺ T cells, and this regulatory role is made possible by their prolonged survival in vivo. Studies done on DN T cells isolated directly from the periphery of the skin graft tolerant mice will provide further evidence on the regulation of apoptosis in these cells. Whether IL-4 is a survival factor for the homeostasis and persistence of immunosuppressive DN T cells in vivo needs to be determined.

	Acknowledgments

 
We thank Drs. Craig Thompson and T. W. Mak for kindly providing anti-Bcl-x and IRF-4 Abs, respectively; D. Y. Loh for providing the breeding stock of 2C transgenic mice; and Dr. Jinyi Zhang and Ms. Barb DuTemple for technical help.

	Footnotes

 
¹ This work is supported by the Medical Research Council of Canada Grant No. MT 14431 (to L. Zhang), and Q. Khan is partially supported by University of Toronto Open Fellowship.

² Address correspondence and reprint requests to Dr. Li Zhang, CCRW 2-809, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. E-mail address:

³ Abbreviations used in this paper: DN, double negative; AICD, activation-induced cell death; FasL, Fas ligand; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; IRF-4, IFN regulatory factor-4; PKB, protein kinase B.

⁴ L. M. Yang, Z. X. Zhang, K. Young, B. DuTemple, and L. Zhang. Identification and characterization of a novel antigen-specific regulatory T cell. Submitted for publication.

Received for publication September 11, 1998. Accepted for publication March 1, 1999.

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