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The Journal of Immunology, 2004, 172: 1437-1443.
Copyright © 2004 by The American Association of Immunologists

Lck Is Required for Activation-Induced T Cell Death after TCR Ligation with Partial Agonists1

Xue-Zhong Yu2,*,{ddagger}, Steven D. Levin3,{dagger}, Joaquin Madrenas§ and Claudio Anasetti*,{ddagger}

* Human Immunogenetics Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; Departments of {dagger} Immunology and {ddagger} Medicine, University of Washington, Seattle, WA 98195; and § Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, and the John P. Robarts Institute, London, Ontario, Canada.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR engagement can induce either T cell proliferation and differentiation or activation-induced T cell death (AICD) through apoptosis. The intracellular signaling pathways that dictate such a disparate fate after TCR engagement have only been partially elucidated. Non-FcR-binding anti-CD3 mAbs induce a partial agonist TCR signaling pattern and cause AICD on Ag-activated, cycling T cells. In this study, we examined TCR signaling during the induction of AICD by anti-CD3 fos, a non-FcR-binding anti-CD3 mAb. This mAb activates Fyn, Lck, and extracellular signal-regulated kinase, and induces phosphorylation of Src-like adapter protein, despite the inability to cause calcium mobilization or TCR polarization. Anti-CD3 fos also fails to effectively activate {zeta}-associated protein of 70 kDa or NF-{kappa}B. Using Ag-specific T cells deficient for Fyn or Lck, we provide compelling evidence that activation of Lck is required for the induction of AICD. Our data indicate that a selective and distinct TCR signaling pattern is required for AICD by TCR partial agonist ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Naive T cells typically respond to TCR ligation by proliferating and differentiating, but premature restimulation through the TCRs leads to apoptosis of Ag-activated, cycling T cells. Full activation of naive T cells requires multivalent engagement and aggregation of the TCR/CD3 complex. Ag can be mimicked by anti-TCR/CD3 mAbs that mediate cross-linking of the TCR through their interaction with FcR-bearing cells. This TCR cross-linking leads to a cascade of intracellular biochemical events that promote cell division and cytokine production. The need for multivalent TCR aggregation is demonstrated by the fact that bivalent, non-FcR-binding anti-CD3 mAbs fail to induce activation and proliferation of naive T cells (1, 2). However, these engineered anti-CD3 mAbs can trigger apoptosis on Ag-activated, cycling T cells in vitro (3, 4, 5), and can effectively deplete alloreactive T cells and prevent graft-vs-host disease in mice (6). Moreover, such reagents have also been shown to be effective in blunting new onset type-1 diabetes (7) in humans. Thus, non-FcR-binding anti-CD3 mAbs show considerable promise as an immunosuppressive agent with the potential for treating graft-vs-host disease, graft rejection, and autoimmune diseases.

Stimulation of TCR initiates a complex signaling cascade (8) that is critically dependent on the expression of nonreceptor tyrosine kinases. In particular, members of the Src family of tyrosine kinases play a critical role during the initial steps of TCR signaling. T cells express primarily two members of the Src family of tyrosine kinases, Lck and Fyn (9), and both kinases are essential for early TCR-mediated signal transduction (10). Although assignment of specific functions to Lck and Fyn is incomplete, available evidence indicates that these kinases play some overlapping roles, but that they also have distinct functions (11, 12, 13).

Previous studies in resting T cells have shown that non-FcR-binding anti-CD3 mAbs induce a TCR signaling pattern (1, 2, 14, 15) similar to that observed in T cells activated with altered peptide ligands (APL)4 (16, 17). This partial agonist signaling pattern was characterized by incomplete phosphorylation of TCR{zeta} and CD3{varepsilon}, activation of Fyn, recruitment of {zeta}-associated protein of 70 kDa (ZAP-70) without phosphorylation and activation of this kinase, attenuated intracellular calcium mobilization, and transient activation of mitogen-activated protein kinases (MAPKs). Partial TCR signaling induces anergy in noncycling T cells (16, 18, 19) and activation-induced cell death (AICD) through apoptosis in Ag-activated, cycling T cells (3, 5, 20). This AICD requires a TCR signal and expression of one or more death receptor ligands. Engagement of death receptors like CD95 (Fas) with their ligands induces apoptotic cell death in appropriately sensitized T cells. This induction of sensitivity to AICD by a partial TCR signal in activated cells has been termed a "competence to die" signal (20, 21). Although it is clear that AICD represents an important mechanism for curbing the expansion of activated T cells, the molecular events whereby TCR restimulation evokes death rather than proliferation are not well defined.

In this study, we examined the effects of a non-FcR-binding anti-CD3 mAb on TCR polarization and signal transduction, and correlated these effects with the induction of AICD. Non-FcR-binding anti-CD3 mAb induce phosphorylation of Fyn, Lck, extracellular signal-regulated kinase (ERK)-1/2 and the Fyn-substrate Src-like adapter protein (SLAP-130). However, using peripheral T cells from mice deficient in either Fyn or Lck, we found that AICD depends on Lck but not Fyn. We found no evidence for activation of ZAP-70, induction of Ca2+ mobilization, and TCR polarization among the early events in AICD. Together, our data indicate that the induction of AICD by TCR partial agonists is the result of a very restricted and distinct pattern of early TCR-mediated signaling.


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

C57/BL6 (B6) mice and B6 lpr mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The Bcl-xL transgenic mouse line was provided by C. Thompson (University of Pennsylvania, Philadelphia, PA). Founders for the DO11.10 strain were provided by D. Y. Loh (Nippon Roche Research Center, Kamakura-shi, Japan). DO11.10 TCR transgenic mice were constructed originally from a BALB/c line (H-2d), and the TCR is specific for OVA323–339 peptide presented by I-Ad. Fyn-/- and Lck-/- mice had been bred more than 10 generations to B6 background. LGF Lck-/- mice, which express Lck in thymocytes but not in peripheral T cells, have been previously described (22, 23). Fyn-/- and LGF Lck-/- mice were then crossed with OT-I TCR transgenic mice. Similarly, Fyn-/- mice were crossed with OT-II TCR transgenic mice to generate Fyn-deficient class II-restricted TCR population. However, the relatively high level of expression of thymic Lck in the LGF+Lck-/- mice resulted in negative selection of the OT-II class II-restricted TCR. Hence, to get around this, another Lck transgenic mouse was made using a wild-type Lck transgene as described (24). A single line was generated and designated LGY7998. This line expressed about one-third the normal levels of Lck and was subsequently bred 10 generations onto the B6 background and crossed to Lck-/- mice that were also on the B6 background to generate animals that expressed Lck in the thymus, but lacked expression in peripheral T cells. The resultant animals were subsequently bred to OT-II mice to generate Lck-deficient peripheral T cells that expressed a class II-restricted TCR. Because these mice express relatively low levels of a wild-type Lck transgene in the thymus, the OT-II TCR+ T cells were not negatively selected, and could be readily detected in the periphery (our unpublished data). Both OT-I and OT-II transgenic mice were constructed originally from a B6 line (H-2b). The OT-I TCR is specific for OVA257–264 peptide presented by H-2Kb, while the OT-II TCR is specific for OVA323–339 peptide presented by I-Ab. All the mice used in this study were maintained under specific pathogen-free conditions in the animal facility at the University of Washington or the Fred Hutchinson Cancer Research Center (Seattle, WA) in accord with institutional policies.

Peptides and Abs

The antigenic peptide for OT-I cells is OVA257–264, SIINFEKL, and the antigenic peptide for DO11.10 or OT-II cells is OVA323–339, ISQAVHAAHAEINEASGR. Both peptides were synthesized by United Biochemical Research (Seattle, WA). Anti-CD3 fos and anti-CD3-CD4 fos-jun chimeric Abs were kindly provided by Dr. J. Tso (Protein Design Lab, Fremont, CA) and have been previously described (2, 3). These chimeric Abs combine a Fab against CD3 or CD4 linked to fos or jun, respectively. Fos homodimers will lead to bivalent engagement of CD3 upon stimulation with anti-CD3 fos, and fos-jun heterodimers will lead to CD3 and CD4 coengagement upon stimulation with anti-CD3-CD4 fos-jun. KJ1-26, a clonotypic mAb specific for DO11.10 TCR-V{alpha} (3), was produced and biotinylated in our laboratory. Anti-phosphotyrosine mAb, 4G10, was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal Abs against Fyn (FYN3), Lck (2102) and ZAP-70 (LR) and mouse mAbs against Fyn (15) and Lck (3A5) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep polyclonal Ab against SLAP-130 was kindly provided by Dr. G. Koretzky (University of Pennsylvania, Philadelphia, PA). HRP-conjugated anti-mouse, rabbit, or sheep IgG secondary Abs were purchased from Amersham Life Science (Piscataway, NJ). Phospho-ERK (Thr202/Tyr204) mAb was purchased from Cell Signaling (Beverly, MA) and anti-ERK mAb (c-14) was from Santa Cruz Biotechnology. Polyclonal Abs specific for phospho-I{kappa}B or total I{kappa}B were purchased from Cell Signaling.

Cell cultures and apoptosis assay

Cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 5 x 105 M 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. To activate T cells, DO11.10, OT-II or OT-I transgenic spleen cells at 106/ml were stimulated for 3 days with 0.5 µM OVA323–339, 2 µM OVA323–339, or 10 nM OVA257–264, respectively. After primary stimulation, viable cells were isolated and expanded with 50 U/ml human IL-2 for 2 additional days. Apoptosis of preactivated T cells was then induced by incubating these cells with 200 ng/ml anti-CD3{varepsilon} mAb for various times in the presence of human IL-2 at 50 U/ml. Cell death was measured by propidium iodide (PI) uptake or cell cycle analysis, as previously described (3, 4). FACS analysis was performed using a FACSCan instrument and CellQuest software (BD Biosciences, San Jose, CA).

TCR capping assay

Preactivated DO11.10 cells were incubated with either 5 µg/ml anti-CD3 fos or no Ab at 37°C for 30 min, washed twice with cold PBS containing 0.2% NaN3. TCR expression was measured by staining cells with biotinylated KJ1-26 at 4°C for 30 min, followed by streptavidin-Texas Red (Molecular Probes, Eugene, OR) at 4°C for 30 min. To cross-link CD3, cells were incubated with biotinylated anti-CD3 mAb at 4°C for 30 min, followed by streptavidin-Texas Red at 37°C for 30 min. Cells were allowed to adhere to cover slips, fixed, and analyzed by an Axiovert 100 TV microscope (Zeiss, Germany). Data were acquired using a Photometrix PXL camera and Delta Vision 2.10 software (Applied Precision, Issaquah, WA).

T cell stimulation and cell lysate preparation

Primed T cells were stimulated with no Ab, anti-CD3 fos, or anti-CD3-CD4 fos-jun for different time periods at 37°C on a Thermomixer. Cells were washed in cold PBS containing 0.4 mM Na3VO4 and 0.4 mM EDTA and lysed with 1x lysis buffer on ice for 30 min. The lysis buffer contains 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µM p-nitrophenyl-p-guanidino-benzoate. Cell lysates were obtained after removal of cell debris by centrifugation at 13,500 rpm for 10 min at 4°C.

Immunoprecipitation, Western blotting, and kinase assays

Immunoprecipitation and Western blotting were performed as previously described (14, 15). Cell lysates were immunoprecipitated for target molecules with specific Ab conjugated on protein A-Sepharose beads at 4°C overnight. The next day, beads were pelleted, washed with lysis buffer, and resuspended in sample buffer. Proteins were separated in 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with appropriate Abs. Signal detection was performed by chemiluminescence (Roche Diagnostics, Indianapolis, IN).

In vitro Fyn and Lck kinase assays were performed as previously described by Huang et al. (25). Briefly, the immunoprecipitates were washed twice with lysis buffer and once in kinase buffer (20 nM HEPES, pH 7.2, 5 mM MgCl2, and 5 mM MnCl2). The samples were then resuspended in 30 µl of kinase buffer containing 10 µCi [32P]ATP (NEN Life Science, Boston, MA) and incubated at room temperature for 15 min. Reactions were terminated by adding 10 µl of 4x SDS sample buffer followed by heating at 95°C for 5 min. Proteins were separated in 8% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. The membranes were treated with 1 M potassium hydroxide 95°C for 1 h to remove alkalilabile phosphate groups from serine- and threonine-phosphorylated proteins. Radiolabeled tyrosine-phosphorylated proteins were detected by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Soluble anti-CD3 fos induces apoptosis of Ag-activated T cells through Fas ligand (FasL)/Fas death pathway

We have tested the effect of anti-CD3 fos, a non-FcR-binding anti-CD3 mAb, on Ag-activated T cells. DO11.10 cells were stimulated with specific OVA323–339 peptide, and then viable, activated T cells were restimulated with soluble anti-CD3 fos. Exogenous IL-2 was added in the secondary culture to keep cells cycling and facilitate apoptosis (26, 27). Although non-FcR-binding anti-CD3 mAbs generate a partial agonist TCR signaling pattern and do not induce proliferation of naive or resting T cells (1, 2, 14, 15), the soluble anti-CD3 fos was readily capable of inducing apoptotic death on previously Ag-activated, cycling DO11.10 cells (Fig. 1, and Refs. 3 and 4). This AICD was largely Fas-dependent, because T cells from lpr mice exhibited markedly reduced levels of apoptosis after stimulation. However, overexpression of the Bcl-xL transgene could not protect these activated T cells from apoptosis induced by anti-CD3 fos (Fig. 1C). These results demonstrate that the T cell apoptosis under the condition tested requires Fas expression, and cannot be prevented by Bcl-xL, consistent with the results in other systems studying AICD (20, 28).



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  		FIGURE 1. The effects of anti-CD3 fos on apoptotic cell death of activated, cycling T cells. A, DO11.10 splenocytes were stimulated with OVA323–339 for 3 days. Viable cells after primary stimulation were isolated and expanded with IL-2 for an additional 2 days. Apoptosis of preactivated T cells was then induced by restimulating these cells with IL-2 alone or IL-2 plus anti-CD3 fos. Cells were harvested 18 h after restimulation and cell death was measured by PI uptake. Numbers represent the percentage of live cells. B, The same experimental settings as above, but cells were harvested 3 h after restimulation, and the cell cycle was measured by nuclear DNA staining with PI and apoptosis was measured by TUNEL assay. Numbers represent the percentage of TUNEL-positive cells. C, Splenocytes from B6 wild-type, lpr, or Bcl-xL mice were stimulated with immobilized anti-CD3 mAb for 3 days. Activated T cells were isolated and cultured in medium containing IL-2 for an additional 2 days. These T cells were then restimulated with soluble anti-CD3 fos mAb in the presence of IL-2. After overnight incubation, cell death was measured by PI uptake with flow cytometry. Data are presented as averages of triplicate wells with error bars indicating 1 SD.

 
Activation of Src family kinases is required for AICD induced by anti-CD3 fos

Protein tyrosine kinases such as Fyn, Lck, and ZAP-70 play an essential role in TCR signaling, and therefore it seemed reasonable that they may play an important role in T cell apoptosis after TCR engagement (29, 30, 31, 32). To examine whether activation of Src family kinases was required for AICD induced by anti-CD3 fos, we took advantage of pyrazolopyrimidine (PP)1, a selective inhibitor of Src family kinases (33). We tested whether PP1 could block AICD induced by anti-CD3 fos in preactivated, cycling cells. PP1 by itself did not affect cell viability over the course of the assay, but inhibited T cell apoptosis induced by anti-CD3 fos in a concentration-dependent manner (Fig. 2). In contrast, the related compound PP3, which does not inhibit Src family kinases (34), had no effect on AICD induced by anti-CD3 fos. These results suggest that activation of Src kinases is required for T cell apoptosis in this system.



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  		FIGURE 2. Requirement of Src kinase activation for AICD by anti-CD3 fos. Preactivated DO.11.10 cells were restimulated with IL-2 and: 1) PP1 alone (src inhibitor); 2) anti-CD3 fos plus PP1; 3) PP3 alone (negative control for PP1); or anti-CD3 fos plus PP3. Cells were harvested 18 h after restimulation and cell death was measured by PI uptake with flow cytometry. Data represent the mean of percent cell death in triplicates, and the results are representative of three similar experiments.

 
Anti-CD3 fos activates Fyn and Lck on Ag-activated T cells

Because activation of Src family kinases was required for AICD (Fig. 2), we next examined whether anti-CD3 fos could activate Fyn and Lck in primed T cells. For this experiment, we used Ag-primed, noncycling T cells, because there was a high level of basal activity for Fyn and Lck in cycling T cells. Anti-CD3 fos induced significant phosphorylation of Fyn as compared to unstimulated controls, consistent with the findings that TCR partial agonists preferentially induce phosphorylation of Fyn (19, 25). In fact, the activity of Fyn was higher after stimulation with anti-CD3 fos than with anti-CD3-CD4 fos-jun (Fig. 3A). In addition to inducing the autophosphorylation of Fyn, a protein of around 130 kDa was also phosphorylated after anti-CD3 fos treatment. Immunoblot analysis revealed that this protein was SLAP-130 (Fig. 3B), a well-characterized substrate of Fyn. Anti-CD3 fos also induced activation of Lck, but in this case the activity of Lck was induced to much higher levels after stimulation with anti-CD3 fos plus anti-CD4 jun compared with anti-CD3 fos alone (Fig. 3C), consistent with what has been previously reported (14). Although anti-CD3 fos induced phosphorylation of Lck, it was unable to induce phosphorylation of ZAP-70, a proposed substrate of Lck (Fig. 3D). These results indicate that anti-CD3 fos strongly activates Fyn and induces phosphorylation of the Fyn substrate SLAP-130. Anti-CD3 fos also induces Lck phosphorylation but does not induce phosphorylation of the Lck substrate ZAP-70.



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  		FIGURE 3. Activation of Src kinase and their substrates after stimulation with anti-CD3 fos. DO11.10 cells were activated with Ag for 3 days and cultured with IL-2 for 7–10 days. These rested cells were then restimulated with medium alone, anti-CD3 fos, or anti-CD3-CD4 fos-jun for 1 min (A and B) or 5 min (C and D). Whole cell lysates were prepared and immunoloprecipitated with polyclonal Abs specific for Fyn (A and B), Lck (C), or ZAP-70 (D). Anti-Fyn or Lck immunoprecipitates were subjected to in vitro kinase assays and analyzed by SDS-PAGE. Proteins were transferred to membranes and the phosphorylated proteins were analyzed by autoradiography (A–C). Anti-ZAP-70 immunoprecipitates were separated by SDS-PAGE, transferred to membranes, and immunoblotted with anti-phosphotyrosine Ab (D). The same membranes were subsequently stripped and immunoblotted with anti-Fyn (A), SLAP-130 (B), Lck (C), or ZAP-70 (D). Results are representative of three similar experiments.

 
Activation of Lck is responsible for AICD induced by anti-CD3 fos

To pinpoint which Src kinase is required for AICD induced by anti-CD3 fos in Ag-activated T cells, we examined AICD in OT-II TCR transgenic mice lacking each of these kinases in peripheral T cells. Because Lck-/- mice fail to develop significant numbers of T cells, we used Lck-/- that expressed a Lck transgene selectively in the thymus, but not in peripheral T cells (LGY+Lck-/-). This strategy allowed development of significant numbers of mature CD4+ cells bearing the OT-II TCR transgene. TCR transgenic cells from OT-II Fyn+/-, Fyn-/-, Lck+/-, or Lck-/- mice were activated with peptide and then restimulated with anti-CD3 fos. In this case, we analyzed DNA content to evaluate apoptosis (sub-G1 peak) and cell cycle progression (S/G2 phases) at the same time. We noted that Fyn-/- T cells were just as susceptible to apoptosis as Fyn+/- cells, suggesting that Fyn does not play a significant role in the induction of AICD (Fig. 4A). However, we found that Lck-/- cells were essentially resistant to apoptosis as compared to Lck+/- cells (Fig. 4A). The resistance of Lck-/- cells to apoptosis was not due to a reduced level of cells in cycle, as the percentage cells at S/G2 phases were similar for Lck+/- and Lck-/- cells (Fig. 4A).



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  		FIGURE 4. Requirements for Fyn or Lck for induction of cell death and cell cycle progression. Splenocytes from Fyn+/-, Fyn-/-, LGY Lck+/-, or LGY Lck-/- OT-II (A) or Fyn+/-, Fyn-/-, LGF Lck+/- or LGF Lck-/- OT-I (B) mice were stimulated with specific peptide. Three days after stimulation, peptide was washed away and cells were cultured with IL-2 for an additional 2 days. After expansion, viable cells were isolated and restimulated with IL-2 alone or IL-2 plus anti-CD3 fos for 18 h. After restimulation, DNA content was measured by nuclear DNA staining with PI. Data represent the mean percentage of cells with a subdiploid DNA content (left numbers) and the percentage of cells at S/G2 phases (right numbers) in duplicate cultures. The results are representative of two similar experiments.

 
Because CD8+ T cells are also sensitive to AICD induced by non-FcR-binding anti-CD3 mAbs (5, 6) we examined the necessity for Lck and Fyn to this process in these cells. This was done by using CD8+ T cells with the OT-I transgenic TCR on the Fyn-/- and LGF+Lck-/- background. The sensitivity of these cells to AICD was assessed as described above and the level of AICD was compared to relevant controls. Fig. 4B illustrates that virtually the same results were obtained looking at CD8+ cells, in that a lack of Fyn had no effect on AICD, whereas Lck-deficient T cells were less sensitive than controls. Collectively, our data indicate that Lck plays a critical role in the induction of AICD by anti-CD3 fos in both CD4+ and CD8+ T cells, even though at least one of its critical substrates (ZAP-70) is not being phosphorylated.

Resistance of Lck-deficient T cells to anti-CD3-induced apoptosis is not due to lack of FasL expression

Because the apoptosis induced by anti-CD3 fos requires the interaction of FasL and Fas, it is conceivable that the resistance of Lck-deficient T cells to apoptosis is due to the failure of FasL up-regulation after activation. To test this hypothesis, FasL expression was compared on DO11.10 Lck+/+, OT-II Lck+/-, or OT-II Lck-/- T cells after stimulation with OVA peptides (Fig. 5). We found that FasL was expressed on Lck-/- T cells, although at relatively lower levels than that on Lck+/+ or Lck+/- cells, at the point when these cells were tested for anti-CD3-induced apoptosis (Fig. 4). These results suggest that the resistance of Lck-/- T cells to apoptosis was unlikely due to a defect in FasL expression.



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  		FIGURE 5. The effect of Lck on FasL expression. Splenocytes from DO11.10 Lck+/+, OT-II Lck+/-, or OT-II Lck-/- mice were stimulated with OVA323–339 for 3 days. Viable cells were isolated and strained with FITC-conjugated anti-mouse CD4 and biotin-conjugated anti-mouse FasL or isotype control IgG followed by streptavidin-PE (all Abs from BD PharMingen). The histograms show the staining of CD4-gated T cells with anti-FasL (thick lines) or control IgG (thin lines). The results are representative of two similar experiments.

 
Ca2+ mobilization and TCR polarization are not required for AICD

Ca2+ mobilization and TCR polarization are essential for efficient T cell activation and proliferation (35, 36, 37, 38). Non-FcR-binding anti-CD3 mAbs fail to mobilize Ca2+ or aggregate TCR and are unable to induce proliferation of naive T cells (1). As anti-CD3 fos is able to trigger apoptosis on Ag-activated, cycling T cells, we tested whether anti-CD3 fos could induce Ca2+ flux or TCR polarization on these cells under the conditions in which AICD was efficiently induced. Analysis of Ca2+ mobilization using cells loaded with the Ca2+-sensitive dye indo-1 showed that anti-CD3 fos was unable to induce a Ca2+ flux, while cross-linking CD3 and CD4 readily elicited this response (Fig. 6A). We next tested whether TCR polarization could be induced by anti-CD3 fos in T cells preactivated with Ag, and found no detectable TCR aggregation under conditions that effectively induced T cell apoptosis. In contrast, TCR aggregation was induced after cross-linking CD3 molecules (Fig. 6B). These results suggest that Ca2+ mobilization and TCR polarization are not required for the induction of AICD on Ag-activated T cells.



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  		FIGURE 6. The effects of anti-CD3 fos on Ca2+ flux, TCR aggregation, and phosphorylation of ERK or I{kappa}B. A, Ag-activated, cycling DO11.10 cells were loaded with Indo-1 and then incubated with anti-CD3 fos, biotinylated anti-CD4 mAb, or biotinylated anti-CD3 fos plus biotinylated anti-CD4 mAb, each at 5 µg/ml. Baseline Ca2+ levels were established for 10 s, and then streptavidin was added into each sample. B, Ag-activated, cycling DO11.10 cells were incubated with medium alone or 5 µg/ml anti-CD3 fos at 37°C for 30 min, washed twice with cold PBS containing 0.2% NaN3. TCR expression was measured by staining cells with biotinylated KJ1-26 at 4°C for 30 min, followed by streptavidin-Texas Red at 4°C for 30 min. To cross-link CD3, cells were incubated with biotinylated anti-CD3 mAb at 4°C for 30 min, followed by streptavidin-Texas Red at 37°C for 30 min. Cells were allowed to adhere to coverslips, fixed, and analyzed by an Axiovert 100 TV microscope. Visual data were acquired using a Photometrix PXL camera and Delta Vision 2.10 software. C, Ag-activated cells were stimulated with 10 µg/ml Ab indicated at 37°C for 5 min. After stimulation, whole cell lysates were prepared and immunoblotted for doubly phosphorylated ERK (upper bands). The same membrane was stripped and reblotted for total ERK (lower bands). D, Ag-activated cells were stimulated with 10 µg/ml Ab indicated at 37°C for 0, 5, 15, 30, and 60 min. After stimulation, whole cell lysates were prepared and immunoblotted for phosphorylated I{kappa}B (upper bands). The same membrane was stripped and reblotted for total I{kappa}B (lower bands).

 
Apoptosis induced by anti-CD3 fos is associated with increased activation of ERK, but not NF-{kappa}B

Partial agonists can selectively activate the Ras/MAPK pathway, although the response is generally less robust than what is seen with full agonists (14, 15, 18). Despite the failure of anti-CD3 fos to induce a Ca2+ flux and TCR polarization, we expected that anti-CD3 fos would activate ERK-1 and ERK-2. Thus, we assessed activation of the Ras/MAPK pathway by measuring the levels of phosphorylated ERK-1 and ERK-2. The results showed that anti-CD3 fos activated ERK-1/2 nearly as well as anti-CD3-CD4 fos-jun on Ag-activated, cycling T cells (Fig. 6C), consistent with previous observations from us and others that ERK activation plays an important role in the induction of AICD (39, 40).

Considerable evidence indicates that NF-{kappa}B is a major regulator of T cell survival (41). Recently, Wan and DeGregori (42) found that activation of NF-{kappa}B promotes survival of activated T cells by antagonizing up-regulation of p73, a common mediator of TCR-induced apoptosis (43). Furthermore, activation of NF-{kappa}B along with activation of ERK protects Jurkat cells from Fas-mediated apoptosis (44). Hence, it seemed possible that the AICD induced by anti-CD3 fos was a consequence of activation of ERK in the absence of NF-{kappa}B activation. To test this possibility, we determined whether anti-CD3 fos could activate NF-{kappa}B on Ag-activated T cells with anti-CD3-CD4 fos-jun as a positive control. Because activation of NF-{kappa}B involves the phosphorylation and degradation of its inhibitor I{kappa}B, we analyzed the phosphorylation state of I{kappa}B as an indicator of NF-{kappa}B activation (45). Although stimulation with anti-CD3-CD4 fos-jun induced a strong and sustained phosphorylation of I{kappa}B, stimulation with anti-CD3 fos induced a weak and transient phosphorylation of I{kappa}B (Fig. 6D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have showed that AICD can be induced by soluble non-FcR-binding anti-CD3 fos (Fig. 1) (3, 4). Anti-CD3 fos activates Fyn, Lck, and ERK, but does not cause Ca2+ flux, TCR aggregation, or activation of NF-{kappa}B under conditions in which AICD is effectively induced. We conclude that partial agonist signals delivered by non-FcR-binding anti-CD3 mAbs are sufficient for the induction of AICD on Ag-activated, cycling T cells. Furthermore, because NF-{kappa}B activation has been correlated with "prosurvival" signals, the lack of NF-{kappa}B activation may contribute to T cell apoptosis under these experimental conditions.

Recent studies on AICD have suggested that some of the TCR proximal signals leading to death of activated T cells are similar to those leading to proliferation and differentiation. For instance, protein tyrosine kinases (i.e., Fyn, Lck, and ZAP-70) (29, 30, 31, 32) and tyrosine protein phosphatase (i.e., Src homology protein-1) (46) appear to play a role in T cell proliferation as well as in T cell death following TCR stimulation. Using transformed Lck-deficient Jurkat cell lines, previous studies suggested that Lck plays a key role in transducing signals leading to apoptotic cell death (30, 31). However, Al-Ramadi et al. (47) reported that Lck deficiency causes cell cycle arrest and hypersusceptibility to apoptosis in a nontransformed T cell line. In the current study, we have found that anti-CD3 fos induces low but reproducible levels of Lck kinase activation and a specific inhibitor of Src family kinase blocks AICD. Furthermore, by using Ag-specific, Lck-deficient T cells from mutant mice, we observed that Lck deficiency renders Ag-activated T cells resistant to AICD, providing compelling evidence that Lck is required for AICD.

Because the interaction between FasL and Fas is essential for T cell apoptosis induced by anti-CD3 fos, one possibility for the resistance of Lck-/- T cells to apoptosis is that these cells could not up-regulate FasL expression. However, we have shown that Lck-/- T cells can up-regulate FasL expression as long as they can get into cell cycle after Ag activation with IL-2, and hence FasL expression does not fully account for T cells insensitive to AICD. It has been reported that TCR signals induce AICD by sensitizing activated T cells to Fas-mediated apoptosis (4, 48), we therefore reason that Lck may transduce the TCR signal that enhances the susceptibility to apoptosis.

Anti-CD3 fos also activates Fyn and induces phosphorylation of SLAP-130, consistent with published data that TCR partial agonists or TCR antagonists induce activation of Fyn (19, 25). However, Fyn activation does not seem to play an essential role in AICD under the conditions tested, as T cells with or without Fyn had a comparable susceptibility to apoptosis induced by anti-CD3 fos (Fig. 4). In contrast, Fyn activation has been shown to be important in the induction of T cell anergy (19, 49). Thus, Fyn and Lck activation might be differentially involved in determining T cell anergy vs apoptosis, respectively.

Our data and those of others indicate that anti-CD3 fos induces Fyn activation as TCR partial agonists or TCR antagonists do, but anti-CD3 fos does not cause TCR polarization while TCR partial agonists or TCR antagonists do (17, 25). It is possible that under the conditions where APL was used for stimulation, costimulatory signals delivered by APCs together with a partial TCR signal contribute to TCR polarization, while the partial TCR signal elicited by anti-CD3 fos alone is unable to do so. As anti-CD3 fos readily induces AICD on Ag-activated, cycling T cells without TCR polarization, it appears that TCR polarization is not required for the induction of AICD.

As reported by others (1, 2, 14, 15), we found that anti-CD3 fos was unable to cause ZAP-70 phosphorylation, suggesting that activation of ZAP-70 is not necessary for AICD. Eischen et al. (32) reported that ZAP-70 is required for up-regulation of FasL in AICD in Jurkat cells. However, the data from Combadiere et al. (17) indicate that APL can trigger a high level of apoptosis without ZAP-70 activation in nontransformed cycling T cells. We and Combadiere et al. (17) studied apoptosis in Ag-activated, cycling T cells on which FasL was already expressed (3, 4, 17, 21), whereas Jurkat cells do not express FasL but it is induced upon TCR triggering and thereby promotes apoptosis (32). Thus, it is possible that ZAP-70 activation might be required for the up-regulation of FasL, but not required for transducing the "competence to die" signal to T cells once FasL is already expressed.

An intriguing question is why anti-CD3 fos induces Lck activation but fails to induce phosphorylation of ZAP-70. The level of Lck activation after stimulation with anti-CD3 fos was much lower than that with anti-CD3-CD4 fos-jun. We speculate that the low level of Lck activation might account for the poor phosphorylation of ZAP-70. Despite the failure to detect ZAP-70 phosphorylation, our data indicate that the low level of Lck activation triggered by anti-CD3 fos is necessary and sufficient for the induction of AICD. By using T cell transfectants expressing kinase-active and Src homology (SH) 2- or SH3-impaired Lck mutants, Miceli and colleagues (51) found that these impaired mutants disrupt costimulation-dependent raft recruitment, sustained TCR phosphorylation and IL-2 production, but not TCR-mediated apoptosis (50, 51). They concluded that TCR-induced IL-2 production requires SH3 activity resulting in CD28 recruitment to the TCR cap while TCR-induced apoptosis does not require TCR capping. Our data further substantiate this conclusion.

We have previously reported that ERK activation is required for AICD induced by anti-CD3 fos (39), and here we demonstrate that Lck is also essential for this process. One possible explanation for this is that Lck is required for the activation of ERK after TCR engagement. In fact such a conclusion is supported by previous studies in Lck-deficient Jurkat cells (52). The phosphorylation of Lck induced by partial TCR agonist does not lead to activation of ZAP-70, Ca2+ mobilization or cytoskeleton reorganization, yet we propose that it is sufficient to transduce a competence to die signal through the ERK MAPK pathway to preactivated T cells. Because NF-{kappa}B is a major antiapoptotic transcription factor (41), we expected that stimulation with anti-CD3 fos would not activate NF-{kappa}B. Indeed, strong and sustained phosphorylation of I{kappa}B was induced by anti-CD3-CD4 fos-jun, but not by anti-CD3 fos. Under the same culture condition, stimulation with anti-CD3-CD4 fos-jun induced apoptosis of Ag-activated T cells at much lower levels than that induced with anti-CD3 fos (our unpublished observation). Therefore, we postulate that the failure of NF-{kappa}B activation may contribute to T cell apoptosis mediated by anti-CD3 fos.


   Acknowledgments
 
We thank L. A. Chau, C. Moore, S. Bidwell, and M. Castor for their technical assistance, and Dr. J. Tso for providing anti-CD3 fos and anti-CD3-CD4 fos-jun mAbs.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA 84132 (to X.-Z.Y.), and CA 18029 and AI 51693 (to C.A.). Back

2 Address correspondence and reprint requests to Dr. Xue-Zhong Yu, Human Immunogenetics Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Mail Box: D2-100, Fairview Avenue North, Seattle, WA 98109. E-mail address: xyu{at}fhcrc.org Back

3 Current address: Department of Cytokine Biology, ZymoGenetics, 1201 Eastlake Ave East, Seattle, WA 98102. Back

4 Abbreviations used in this paper: APL, altered peptide ligand; ZAP-70, {zeta}-associated protein of 70 kDa; MAPK, mitogen-activated protein kinase; AICD, activation-induced cell death; ERK, extracellular signal-regulated kinase; SLAP, Src-like adapter protein; PI, propidium iodide; FasL, Fas ligand; PP, pyrazolopyrimidine; SH, Src homology. Back

Received for publication May 19, 2003. Accepted for publication November 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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