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The Journal of Immunology, 2000, 164: 1193-1199.
Copyright © 2000 by The American Association of Immunologists

Blocking the Common {gamma}-Chain of Cytokine Receptors Induces T Cell Apoptosis and Long-Term Islet Allograft Survival1

Xian Chang Li2,*, Azine Ima*, Yongsheng Li*, Xin Xiao Zheng*, Thomas R. Malek{dagger} and Terry B. Strom*

* Department of Medicine, Division of Immunology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215; and {dagger} Department of Microbiology and Immunology, University of Miami, Miami, FL 33101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The common {gamma}c-chain is an essential signaling component shared by all known T cell growth factor (TCGF) receptors (i.e., IL-2, IL-4, IL-7, IL-9, and IL-15). In the present study, we have studied the effect of {gamma}c-chain blockade on T cell activation and allograft rejection. Treatment of B6AF1 (H-2b/d.k) recipient mice with anti-{gamma}c mAbs induced long-term survival of DBA/2 (H-2d) islet allografts (>150 days, n = 8), whereas control Ab-treated mice rejected the islet allografts within 17 days (n = 6). The state of engraftment induced by the anti-{gamma}c mAbs was remarkably stable, as recipient mice bearing the primary islet allografts accepted a second DBA/2 islet allograft without further immunosuppression and systemic administration of high doses of IL-2Ig fusion protein failed to provoke rejection. Blocking the {gamma}c-chain inhibited T cell proliferation and induced T cell apoptosis by repressing expression of Bcl-2. Our data suggest that one means of inducing T cell apoptosis and stable allograft survival can be achieved via {gamma}c-chain blockade.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Unique to the allograft response across full MHC barriers, by comparison to immune responses to nominal Ags, is the direct recognition of intact donor MHC Ags by recipient T cells (direct Ag presentation) and activation of an unusually large mass of alloreactive T cells (1). Thus, acquisition of peripheral allograft tolerance, a true tolerant state established in the absence of immune system ablation, is a challenging task in transplantation. Central to the events of T cell activation and acute allograft rejection is the production of T cell growth factors (TCGFs)3 that drive the proliferation, clonal expansion, and functional maturation of alloreactive T cells. Indeed, in the absence of TCGFs during a critical stage of T cell activation, activated T cells become anergic or are committed to apoptotic cell death (2).

IL-2, IL-4, IL-7, IL-9, and IL-15, all TCGFs, utilize a common receptor element, the IL-2R {gamma}c-chain, also known as the common {gamma}c-chain, as an essential signaling component in their multichain receptor complexes (3, 4). The importance of the {gamma}c-chain in immune activation is further highlighted by the finding that mutations of this protein in mice or humans result in X-linked severe combined immunodeficiency characterized by impaired development of T cells, B cells, and NK cells, and a total absence of peripheral lymph nodes and gut-associated lymphoid tissue (5, 6, 7).

The primary role of the {gamma}c-chain is to polymerize with other receptor subunits (i.e., the {alpha}-chain for IL-4, IL-7, IL-9 receptors or the {alpha}- and ß-chains for IL-2, IL-15 receptors) to form functional receptor complex upon ligand binding. One of the key signaling events conferred by the cytoplasmic domain of the common {gamma}c-chain is the recruitment and activation of Jak3 kinase (8). This is supported by the finding that the phenotype of Jak3 knockout mice is remarkably similar to that of {gamma}c knockout mice (9). Activation of Jak3 kinase by the {gamma}c, in concert with signals from other receptor subunits ({alpha} or ß subunit), triggers a variety of downstream signaling events through activation of Src family tyrosine kinases, STAT proteins, and phosphatidylinositol kinase 3 (PI-3) kinases (8). Thus, the {gamma}c signals play a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T cells.

In the present study, we tested the hypothesis that targeting the common {gamma}c of TCGF receptors may lead to a reduction in the mass of alloreactive T cells via T cell apoptosis and/or prevention of clonal expansion, thus permitting the induction of stable allograft tolerance. We now report that blocking the common {gamma}c-chain of TCGF receptors using noncytolytic mAbs induced rapid apoptotic cell death of responding T cells and produced long-term allograft survival in a murine islet transplantation model.


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

Male DBA/2 (H-2d), B6AF1 (H-2b/d.k), and BALB/c (H-2d) mice (8- to 10-wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME).

Islet cell transplantation

Islet transplantation was performed as described previously (10). Briefly, crude islets were isolated from donor DBA/2 pancreata through collagenase digestion and the Ficoll gradient centrifugation method. Islets (300–400 islets) were transplanted under the renal capsule into B6AF1 recipients rendered diabetic by a single i.p. injection of streptozocin (225 mg/kg; Sigma, St. Louis, MO). Allograft function was monitored by sequential blood glucose measurements. Primary graft function was defined as blood glucose levels under 200 mg/dl on day 3 after transplantation, and graft rejection was defined as a rise in blood glucose levels exceeding 300 mg/dl following a period of primary graft function.

Reagents

Recombinant human IL-2 and IL-15 were obtained from PharMingen (San Diego, CA). Two blocking mAbs (4G3/3E12 and rat IgG2a) that bind to different epitopes on the common {gamma}c-chain of IL-2R were used as previously reported (11, 12). Rat IgG2a (Sigma) was used as an isotype control. Biotinylated mouse anti-human IL-2 (B33-2), biotinylated mouse anti-human IL-15 mAb (G243-886), biotinylated anti-mouse CD4 (GK1.5), biotinylated anti-mouse CD8a (53-6.7), hamster anti-mouse CD3 (145-2C11), rabbit anti-Bcl-2, PE-conjugated annexin V, PE-conjugated anti-mouse Fas (Jo-2), streptavidin-PE, and streptavidin-CyChrome were purchased from PharMingen.

The IL-2Ig fusion protein was constructed, expressed, and tested in our laboratory as reported previously (13, 14, 15).

BAF-B03 cells, an IL-3-dependent hematopoietic cell line selected for high expression of IL-2R {alpha}-chain was kindly provided by Dr. T. Taniguchi (Osaka University, Osaka, Japan). BAF-B03 cells were transfected with full-length cDNA encoding human IL-2R ß-chain as described previously (16). Since parent BAF-B03 cells constitutively express the common {gamma}c and the IL-15R {alpha}-chain, the transfected BAF-B03 cells are fully responsive to either IL-2 or IL-15 (17). Cells were routinely maintained in IL-3-conditioned RPMI 1640 medium supplemented with 10% FCS and 1% penicillin and streptomycin (BioWhittaker, Walkersville, MD).

Cell proliferation assay

BAF-B03 cells were starved from IL-3 for at least 6 h before each experiments. Cells (2 x 10 5/ml) were resuspended in RPMI 1640 medium with 10% FCS and 1% penicillin and streptomycin, and stimulated with IL-2 (100 U/ml) or IL-15 (10 ng/ml) in the presence or absence of anti-{gamma}c mAbs for 48 h. Cells were pulsed with 1 µCi [3H]TdR (Amersham, Boston, MA) for 6 h and [3H]TdR uptake was determined by scintillation counting (Beckman Coulter, Columbia, MD).

Preparation of T lymphoblasts

Splenic leukocytes were prepared from BALB/c mice. Cells were resuspended in RPMI 1640 medium with 10% FCS and 1% penicillin/streptomycin at 2 x 106/ml and stimulated with anti-CD3 (145-2C11; PharMingen) for 3 days. Cells were harvested and viable T lymphoblasts were prepared with Lympholyte-M separation medium (Cedarlane Laboratories, Ontario, Canada).

Flow cytometry

For analysis of apoptotic cell death, BAF-B03 cells were stimulated with IL-2 (100 U/ml) or IL-15 (10 ng/ml) in the presence or absence of anti-{gamma}c mAbs for overnight. Cells were harvested and stained with PE-conjugated annexin V (PharMingen) at 4°C for 15 min, washed in labeling buffer, and analyzed by FACS (Becton Dickinson, Mountain View, CA). For detection of cell surface Fas expression, BAF-B03 cells were stained with PE-conjugated anti-mouse Fas mAb (Jo-2; PharMingen) on ice for 20 min, and cells were washed twice in PBS-0.5% BSA before FACS analysis. For intracellular Bcl-2 staining, BAF-B03 cells were fixed and permeabilized with Cytofix-Cytoperm solution for 20 min and then stained with a rabbit anti-Bcl-2 Ab (PharMingen) at 4°C for 10 min. Cells incubated with normal rabbit IgG were included as a control. The cells were washed in washing buffer, further stained with PE-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA), and analyzed by FACS.

Cytokine-binding assay

BAF-B03 cells (1 x 106) were incubated with recombinant human IL-2 (0.5 µg) and IL-15 (0.5 µg) on ice for 20 min in the presence or absence of a saturating amount of anti-{gamma}c mAbs (100 µg/ml) or anti-CD25 mAb (100 µg/ml), washed twice in PBS-0.5%BSA, and further stained with biotinylated mouse anti-human IL-2 or biotinylated mouse anti-human IL-15 mAbs. Cells were washed again in PBS-0.5% BSA, followed by staining with PE-streptavidin. Cells stained with isotype control mAb were included as a control. Binding of IL-2 and IL-15 to the BAF-B03 cells were then analyzed by FACS.

RT-PCR

Cellular RNA was extracted using a Qiagen RNA isolation kit (Qiagen, Chatsworth, CA) and reverse transcribed into cDNA. A total of 1 µl of cDNA was amplified in a 50-µl reaction mix containing 10 mM dNTPs, 100 ng of sense and antisense primers, and 0.25 U of Taq polymerase (Promega, Madison, WI). The specific primers for murine Fas ligand and GAPDH were used as previously reported (18). The PCR amplification schema consisted of the following elements: denature at 94°C for 30 s, anneal at 55°C for 30 s, and extension at 72°C for 45 s for each cycle in a GeneAmp thermocycler (Perkin-Elmer/Cetus, Norwalk, CT) for a total of 40 cycles. PCR products were analyzed in ethidium bromide-stained 1% agarose gel.

5-Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and in vivo quantitation

Labeling of lymphocytes with a tracking fluorochrome CFSE, whose per cell fluorescent intensity halves with each round of cell division, was performed as reported previously (19). Briefly, spleen and lymph nodes from B6AF1 mice were harvested and single-cell suspensions were prepared in HBSS. RBC were lysed by hypotonic shock. Lymphocytes (107/ml) were then labeled with CFSE (Molecular Probes, Portland, OR) at a final concentration of 5 µM in HBSS for 5 min. Cell labeling was terminated by addition of one-tenth the volume of FCS. Cells were then washed twice in HBSS before i.v. injection.

DBA/2 mice were lethally irradiated (1000 rad) with a Gammacell 40 Exactor (Kanata, Ontario, Canada). Each mouse then received 4–6 x 107 CFSE-labeled cells via the penile vein. Mice were killed 2 days after i.v. injection of labeled lymphocytes, host spleens were harvested, and single-cell suspensions were prepared. Cells were stained with biotinylated anti-mouse CD4 (GK1.5) or CD8a (53-6.7) mAb (PharMingen) on ice for 20 min, followed by staining with streptavidin-CyChrome and PE-conjugated annexin V (PharMingen) on ice for 15 min. Proliferation and apoptosis of CFSE-labeled CD4+ T cells and CD8+ T cells in each distinct cell division cycle was analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the impact of common {gamma}c blockade on T cell activation and the allograft response, crude islets from DBA/2 (H-2d) donor mice were transplanted into B6AF1 (H-2b/d.k) recipients treated with anti-{gamma}c mAbs or an isotype control Ab. As shown in Fig. 1Go, control Ab-treated mice rejected the islet allografts with a mean survival time of 17 days (17 ± 3, n = 6). In contrast, treatment of recipient mice with anti-{gamma}c mAbs (0.5 mg i.p on posttransplant days 0, 1, 3, 5, and 7) markedly prolonged the islet allograft survival. In fact, six of eight recipient mice survived indefinitely (mean survival time, >150 days after transplantation) whereas the other grafts were rejected on posttransplant days 28 and 35, respectively.



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  		FIGURE 1. Anti-{gamma}c mAbs induced long-term islet allograft survival. Crude islets from DBA/2 mice (300–400 islets) were transplanted into B6AF1 recipients rendered diabetic by a single i.p. injection of streptozocin. Recipient mice were treated with anti-{gamma}-chain mAbs or an isotype control mAb i.p. at 0.5 mg/day on posttransplant days 0, 1, 3, 5, and 7.

 
Nephrectomy of the left kidney (the islet allografts were placed under the left renal capsule) on three recipients 150 days after transplantation led to a sharp rise of blood glucose levels, demonstrating that euglycemia was maintained by the islet allografts. Each of the three nephrectomized mice accepted a second DBA/2 islet allograft without further immunosuppression (Fig. 2Go). To probe whether the long-term allograft survival was due to T cell anergy, we then injected IL-2-Ig fusion protein into three recipient mice 150 days after primary islet transplantation (2500 U i.p. daily for 8 days). The IL-2-Ig fusion protein is divalent in avidity and has a remarkable long circulatory half-life (half-life >24 h as compared with few minutes for native IL-2), and its efficacy in immune activation is well characterized in our previous studies (13, 14, 15). As shown in Fig. 3Go, systemic IL-2-Ig failed to provoke islet allograft rejection, suggesting a state of stable engraftment is achieved following {gamma}c blockade.



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  		FIGURE 2. Anti-{gamma}c mAbs induced stable engraftment of islet allografts. Removal of the primary islet allografts from three recipient mice 150 days after transplantation led to rapid hyperglycemia, and each of the three mice accepted a second donor-specific islet allograft without any immunosuppression.

 


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  		FIGURE 3. Systemic IL-2Ig failed to provoke islet allograft rejection. IL-2Ig was injected i.p. into three recipient mice at 2500 U/day for 8 days started on posttransplant day 150.

 
Clearly, blocking the common {gamma}c of TCGF receptors produces a state of permanent engraftment that can withstand rechallenge with the donor-specific allografts and with administration of exogenous TCGF, i.e., IL-2. To probe the mechanisms by which anti-{gamma}c mAbs enable the induction of long-term allograft survival, we first analyzed the effect of the anti-{gamma}c mAbs on proliferation and apoptosis of BAF-B03 cells in vitro. BAF-B03 cells proliferated vigorously to either IL-2 (100 U/ml) or IL-15 (10 ng/ml), and such proliferation can be completely inhibited by provision of the anti-{gamma}c mAbs in the culture. In contrast, proliferation of BAF-B03 cells stimulated with IL-3, whose receptor does not contain the {gamma}c-chain, was not affected by the anti-{gamma}c mAbs (Fig. 4Go).



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  		FIGURE 4. Anti-{gamma}c mAbs inhibited IL-2- and IL-15-stimulated cell proliferation. BAF-B03 cells were cultured in IL-3-conditioned medium or stimulated with IL-2 (100 U/ml) or IL-15 (10 ng/ml) in the presence or absence of anti-{gamma}c mAbs for 48 h. Cell proliferation was determined by [3H]TdR uptake. Results are presented as means ± SD of triplicate assays.

 
To analyze the fate of BAF-B03 cells following {gamma}c blockade, cells were stimulated with IL-2 or IL-15 in the presence of anti-{gamma}c mAbs and examined for apoptotic cell death by annexin V staining (20). Annexin V binds to cell membrane-associated phosphatidylserine, which is restricted to the interior side of the cell membrane in living cells and is rapidly exposed to the exterior side in the early stage of apoptotic cell death. As shown in Fig. 5GoA, the anti-{gamma}c mAbs did not affect the survival of BAF-B03 cells cultured in IL-3-conditioned medium (annexin V+ cells, <7%). In striking contrast, there was a marked increase in apoptotic cell death (>40%) of BAF-B03 cells cultured in IL-2 or IL-15 in the presence of anti-{gamma}c mAbs. Apoptotic cell death of BAF-B03 cells induced by the anti-{gamma}c mAbs was further confirmed by DNA fragmentation analysis (data not shown). Similarly, anti-{gamma}c mAbs also induced apoptosis of anti-CD3-activated primary T lymphoblasts in vitro (Fig. 5GoB).



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  		FIGURE 5. A, Blocking the common {gamma}c induced apoptotic cell death. BAF-B03 cells were cultured in IL-3-conditioned medium or stimulated with IL-2 (100 U/ml) or IL-15 (10 ng/ml) in the presence of anti-{gamma}c mAbs (100 µg/ml) for 14 h. Apoptotic cell death was analyzed by annexin V staining. Cells cultured with IL-2 or IL-15 and rat IgG2a were used as a control. B, Anti-{gamma}c mAbs induced apoptotic cell death of anti-CD3-activated T lymphoblasts. Splenic leukocytes were activated with anti-CD3 (2 µg/ml) for 3 days. Viable cells were isolated with Lympholyte-M and stimulated with IL-15 (5 ng/ml) in the presence or absence of anti-{gamma}c mAbs (100 µg/ml) for 14 h. Cell death was analyzed by annexin V staining.

 
To more precisely probe the effect of {gamma}c blockade on proliferation and apoptosis of alloreactive T cells in vivo, lymphocytes from B6AF1 mice (H-2b/d.k) were labeled with CFSE, whose per cell fluorescent intensity halves with each round of cell division (19, 21), and adoptively transferred into lethally irradiated DBA/2 (H-2d) hosts and treated with the anti-{gamma}c mAbs. As shown in Fig. 6Go, 2 days after passive cell transfer, a population of CFSE-labeled CD4+ T cells proliferated in vivo. Although the magnitude of T cell proliferation appeared to be similar in either control or anti-{gamma}c-treated recipients at this time point, there was a striking difference regarding apoptotic cell death. When cells were gated and analyzed for annexin V staining, >34% of CFSE-labeled CD4+ T cells stained positive for annexin V in anti-{gamma}c-treated mice. In contrast, only 6% in untreated controls were annexin V positive (Fig. 6Go). CFSE-labeled CD8+ T cells recovered from anti-{gamma}c-treated hosts exhibited similar patterns of proliferation and apoptotic cell death (data not shown). Thus, blocking the common {gamma}c in the early stage of T cell activation rapidly programs T cells for apoptotic cell death.



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  		FIGURE 6. Blocking the common {gamma}c promoted T cell apoptosis in vivo. CFSE-labeled B6AF1 lymphocytes were injected i.v. into lethally irradiated DBA/2 hosts treated with anti-{gamma}c mAbs (0.5 mg/day for 2 days) or control rat IgG2a. Two days after passive cell transfer, cells were recovered from the host spleen and stained with CyChrome-anti-CD4 and PE-conjugated annexin V. Proliferation and apoptosis of CFSE-labeled CD4+ T cells was determined by FACS.

 
The apoptotic cell death induced by the anti-{gamma}c mAbs (in vivo and in vitro) might be attributed to blockade of growth factor binding to its receptor, mimicking a situation of growth factor deprivation or to perturbation of {gamma}c-triggered signaling events without interfering with growth factor binding. To gain some insights into these possibilities, we examined IL-2 and IL-15 binding to BAF-B03 cells, which constitutively express high-affinity IL-2 and IL-15 receptors, in the presence or absence of a saturating amount of anti-{gamma}c mAbs (100 µg/ml). As shown in Fig. 7Go, binding of IL-2 to BAF-B03 cells was consistently demonstrated by FACS and this binding was completely inhibited by anti-IL-2R {alpha}-chain mAb (anti-CD25), which is known to block IL-2 binding to its high-affinity receptor (22). In contrast, binding of IL-2 or IL-15 to BAF-B03 cells was not blocked by the anti-{gamma}c mAbs, although cell proliferation was completely inhibited (Fig. 4Go). Since the IL-2R {alpha}- and the ß-chain can form pseudo-high-affinity receptor for IL-2 (22) and the IL-15R {alpha} monomeric structure displays high affinity for IL-15 (23), our data suggest that the anti-{gamma}c mAbs most likely interfere with the {gamma}c-imparted signaling events rather than blocking growth factor binding.



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  		FIGURE 7. Anti-{gamma}c mAbs failed to block IL-2 and IL-15 binding to BAF-B03 cells. BAF-B03 cells were incubated with IL-2 and IL-15 on ice for 30 min in the presence or absence of anti-{gamma}c mAbs (100 µg/ml) or anti-CD25 (100 µg/ml), followed by staining with biotinylated mouse anti-human IL-2 or IL-15 mAb. Cells were washed and further stained with PE-streptavidin. Binding of IL-2 and IL-15 to BAF-B03 cells was analyzed by FACS. A, Effect of anti-CD25 on IL-2 binding. B, Effect of anti-{gamma}c mAbs on IL-2 binding. C, Effect of anti-{gamma}c mAbs on IL-15 binding. Bold line, negative control; thin line, IL-2 and IL-15 binding in the presence of isotype control Ab; dotted line, IL-2 and IL-15 binding in the presence of anti-CD25 (A) or anti-{gamma}c mAbs (B and C).

 
To determine the pathways by which anti-{gamma}c mAbs induce apoptotic cell death, we first analyzed Fas and Fas ligand expression by BAF-B03 cells. BAF-B03 cells constitutively express low levels of Fas on the cell surface, but Fas ligand expression was not detected regardless of IL-2 or IL-15 stimulation (Fig. 8Go), suggesting that apoptosis of BAF-B03 cells upon {gamma}c blockade is not mediated by the Fas-Fas ligand interactions. Interestingly, there was a consistent decline of anti-apoptotic molecule Bcl-2 expression in BAF-B03 cells following {gamma}c blockade as compared with control Ab-treated cells (Fig. 9Go), indicating that apoptotic cell death following {gamma}c blockade may be associated with down-regulation of Bcl-2.



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  		FIGURE 8. BAF-B03 cells express low levels of Fas determined by FACS but do not express Fas ligand analyzed by RT-PCR.

 


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  		FIGURE 9. Bcl-2 expression was inhibited by anti-{gamma}c mAbs. BAF-B03 cells stimulated with IL-2 (100 U/ml) in the presence or absence of anti-{gamma}c mAbs (100 µg/ml) for 14 h. Cells were fixed and permeabilized, and expression of Bcl-2 protein was determined by FACS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TCGFs are of central importance in the clonal expansion of activated T cells and execution of acute allograft rejection. Thus, targeting TCGFs remains one of the key aspects of organ transplantation. However, TCGFs exhibit high degree of redundancy and pleiotropism, targeting individual TCGFs and/or its private receptor component has generally failed to produce stable engraftment of allotransplants. Interestingly, all known TCGFs utilize the same IL-2R {gamma}c-chain as a critical signaling component in their receptor complex (3). One major clinical corollary about sharing the common {gamma}c by all TCGFs is that therapies directed toward the common {gamma}c may be far more efficacious than blocking each growth factor alone. In the present study, we have demonstrated, by using an islet transplantation model, that blocking the common {gamma}c with noncytolytic mAbs produces long-term islet allograft survival. In this model, the state of islet engraftment induced by the common {gamma}c blockade is remarkably stable, it can withstand rechallenge with the donor allografts and with high doses of exogenous TCGF, i.e., IL-2Ig, suggesting that the long-term engraftment is unlikely maintained by an IL-2-sensitive anergic process. The mechanism by which the anti-{gamma}c mAbs induces long-term islet allograft survival appears to be linked to the rapid apoptosis of activated T cells, since blocking the common {gamma}c readily induced apoptotic cell death of BAF-B03 cells and primary anti-CD3 activated T lymphoblasts in vitro and alloreactive T cells in vivo (Figs. 5Go and 6Go). Our data suggest that apoptosis of alloreactive T cells may be necessary, albeit not sufficient, to the induction of stable peripheral allograft tolerance. The unusually large mass of cytopathic T cells in alloimmune response, in contrast to immune responses to nominal Ags, may invoke a requirement for apoptotic cell death to reduce the mass of alloreactive T cell clones to achieve stable peripheral tolerance. Our data also suggest that one means of selectively depleting alloreactive T cells and facilitating tolerance induction can be achieved via {gamma}c blockade.

The anti-{gamma}c mAbs used in the present study appear to inhibit {gamma}c function with little, if any, effect on cytokine binding to its receptor complex, as the anti-{gamma}c mAbs failed to block the binding of IL-2 and IL-15 to BAF-B03 cells (Fig. 7Go) despite complete inhibition of IL-2- and IL-15-driven cell proliferation in vitro (Fig. 4Go). The functional high-affinity IL-2R is a heterotrimeric structure consisting of {alpha}, ß, and {gamma}c three units (3). Although the IL-2R {alpha}-chain displays a very low binding affinity for IL-2 (Ka = 10-8 M), the {alpha}-chain can associate with the ß-chain to form a high-affinity binding structure for IL-2 (Ka > 10-10 M). Because the {gamma}c is an essential signaling component, the {alpha}ß complex can bind IL-2 but cannot initiate signaling events, and, therefore, this structure is often regarded as a pseudo-high-affinity IL-2R (3). It is likely that IL-2 binding to the BAF-B03 cells in the presence of a saturating amount of anti-{gamma}c mAbs is due to the formation of pseudo-high-affinity IL-2R. The receptor for IL-15 utilizes the same IL-2R ß- and the {gamma}c-chain with an exception of a private {alpha}-chain. In contrast to the IL-2R {alpha}-chain, the IL-15R {alpha}-chain alone displays a remarkably high affinity for IL-15 (Ka = 10-11 M) (24). We have previously reported that targeting the IL-15R {alpha}-chain with a mutant IL-15Ig fusion protein, which precludes the recruitment of the {gamma}c-chain, can effectively block IL-15 function and immune activation (17). The unusually high affinity of IL-15R {alpha}-chain for IL-15 may also explain the failure of anti-{gamma}c mAbs to block the binding of IL-15 to the BAF-B03 cells (Fig. 7Go). Furthermore, the remarkable similarity of {gamma}c knockout mice with the chimeric mice created by lethal irradiation and bone marrow reconstitution treated with our anti-{gamma}c mAbs suggests strongly that the effect anti-{gamma}c mAbs is most likely to block is {gamma}c function rather than blocking growth factor binding (12).

Engagement of IL-2R has been shown to deliver mitotic signals as well as survival or antiapoptotic signals via activation of multiple signaling pathways (16). There is compelling evidence that activation of PI-3 kinase is essential for prevention of apoptosis in a number of cell types, and this appears to be mediated by PI-3 kinase-dependent activation of Akt, a serine-threonine protein kinase (25, 26, 27), since expression of a constitutive form of Akt is sufficient to protect cells from apoptosis caused by growth factor withdrawn, whereas the dominant negative form of Akt can cause apoptosis (28, 29). It is important to emphasize that the {gamma}c-triggered Jak3 kinase activation is instrumental for the activation of the PI-3 kinase-Akt pathway (8). The precise nature of PI-3-Akt pathway in conferring the antiapoptotic effects upon growth factor stimulation remains to be clearly defined. There is some evidence to suggest that Akt can phosphorylate Bad, which normally associates with Bcl-2 and prevents its antiapoptotic effect; the phosphorylated Bad then dissociates from Bcl-2 and releases the active form of Bcl-2 (30). Akt has also been shown to phosphorylate caspase 9 and prevent its proteolytic activation (31). More recently, Akt has been shown to prevent apoptosis by phosphorylating forkhead transcription factors (32). Phosphorylation of these transcription factors causes them to be exported from the nucleus and prevents them from inducing expression of proapoptotic genes. Our study in the BAF-B03 cells is consistent with the finding that the {gamma}c-imparted survival signal is mediated at least in part through up-regulation of Bcl-2, since {gamma}c blockade markedly decreased the level of Bcl-2 expression. More recently, apoptosis of CD8+ T cells induced by common {gamma}c blockade is also associated with Bcl-2 down-regulation (33).

Clearly, targeting the common {gamma}c using noncytolytic mAbs may have important clinical implications in some T cell-dependent cytopathic conditions such as allograft rejection. Because the binding affinity of TCGFs to their receptors is remarkably high, it is possible that there may be a competitive action between growth factors and the anti-{gamma}c mAbs; therefore, the effects of the anti-{gamma}c mAbs may be critically dependent on the levels of growth factors produced during immune activation by the hosts and the amount of blocking Abs available. It seems that perturbation of {gamma}c function is a key to the blocking effects of our anti-{gamma}c mAbs. We anticipate that targeting the {gamma}c-triggered signaling events, ca. Jak3 activation, may produce similar inhibitory effects in T cell activation. Certainly, this notion needs to be vigorously tested.


   Footnotes
 
1 This work was supported by the Juvenile Diabetes Foundation International Grant 1-1999-16 (to X.C.L.) and National Institutes of Health Grants RO1-AI40114 (to T.R.M.), RO1-AI37798 (to T.B.S.), and P01-AIGF4152 (to T.B.S.). Back

2 Address correspondence and reprint requests to Dr. Xian C. Li, Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, P.O. Box 15707, RN389, Boston, MA 02215. E-mail address: Back

3 Abbreviations used in this paper: TCGF, T cell growth factor; PI-3, phosphophatidylinositol kinase 3; CFSE, 5-carboxyfluorescein diacetate succinimidyl ester. Back

Received for publication September 22, 1999. Accepted for publication November 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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