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The Journal of Immunology, 2002, 168: 4382-4390.
Copyright © 2002 by The American Association of Immunologists

On CD28/CD40 Ligand Costimulation, Common {gamma}-Chain Signals, and the Alloimmune Response1

Gülçin Demirci*, Wenda Gao*, Xin Xiao Zheng*, Thomas R. Malek{dagger}, Terry B. Strom* and Xian Chang Li2,*

* Department of Medicine, Harvard Medical School, Division of Immunology, 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
 
Activation and robust expansion of naive T cells often require T cell costimulatory signals and T cell growth factors. However, the precise growth and costimulation requirements for activation and expansion of CD4+ and CD8+ T cells in vivo in allograft response are still not clearly defined. In the present study, we critically examined the role of CD28/CD40 ligand (CD40L) costimulation and the common {gamma}-chain ({gamma}c) signals, a shared signaling component by receptors for all known T cell growth factors (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21), in activation and expansion of CD4+ and CD8+ T cells in the allogeneic hosts. We found that CD28/CD40L costimulation and the {gamma}c signals are differentially involved in proliferation and clonal expansion of CD4+ and CD8+ T cells in response to alloantigen stimulation. CD8+ T cells are highly dependent on the {gamma}c signals for survival, expansion, and functional maturation, whereas in vivo expansion of alloreactive CD4+ T cells is largely {gamma}c independent. T cell costimulation via CD28 and CD40L, however, is necessary and sufficient for activation and expansion of CD4+ T cells in vivo. In a skin transplant model, blocking both CD28/CD40L and the {gamma}c pathways induced prolonged skin allograft survival. Our study provides critical insights that the CD4 and CD8 compartments are most likely governed by distinct mechanisms in vivo, and targeting both costimulatory and {gamma}c signals may be highly effective in certain cytopathic conditions involving activation of both CD4+ and CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells are of central importance in the execution of allograft rejection. Thus, blocking T cell activation remains a key component in the induction of transplantation tolerance. However, acquisition of peripheral allograft tolerance by immune competent hosts also requires active interactions with donor Ags by the host T cells. In this regard, preservation of TCR signaling (signal 1) is critically important, as the TCR signals are often required to program activated T cells for apoptosis and for regulatory properties, two key components in the induction and maintenance of transplant tolerance (1, 2). Indeed, protocols that completely block TCR signaling often lead to generalized immunosuppression, and induction of stable allograft tolerance is precluded under such circumstances, albeit graft survival can beprolonged (3, 4). Thus, a detailed understanding of T cell costimulation (signal 2) and T cell growth factors (TCGFs)3 (signal 3) in regulating T cell expansion and T cell apoptosis after alloantigen stimulation is essential in transplantation.

T cell costimulatory molecules, especially CD28/CD40 ligand (CD40L) molecules, are clearly important in T cell activation and allograft rejection, as blocking CD28 and CD40L can dramatically prolong allograft survival (5, 6). However, the effect of costimulation blockade in blunting allograft rejection is not universal, and graft survival is often dictated by the animal species, animal strains, and animal models used as well as the activation status of responding T cells (7, 8). Also, definitively proven tolerance by costimulation blockade is rare, and induction of tolerance often requires application of other reagents such as rapamycin or bone marrow cells to the costimulation blockade protocol (3, 9). The nature of this striking variation remains unclear. There is evidence to suggest that a subset of CD8+ T cells may play a key role in supporting costimulation blockade-resistant allograft rejection (10, 11, 12). Nonetheless, under certain conditions, costimulatory signals are absolutely critical to CD8+ T cell activation (13, 14, 15). Furthermore, depletion of asialo+CD8+ T cells in addition to costimulation blockade delayed, but certainly did not prevent skin allograft rejection (10). Hence, the precise role of costimulatory signals in regulating alloimmunity and the identity of factors that support costimulation blockade-resistant rejection remain to be clearly defined.

TCGFs play a key role in supporting expansion and effector function of activated T cells. Of particular interest is that the receptors for all known TCGFs (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) share the same IL-2R {gamma}-chain, also known as the common {gamma}-chain ({gamma}c), as a critical signaling element (16, 17). TCGFs are expressed by a variety of different cell types, and their expression is regulated by distinct mechanisms. For example, IL-2 is produced primarily by activated T cells, especially activated CD4+ T cells, and its expression is critically dependent on costimulatory signals (18). In contrast, IL-7 and IL-15 are produced primarily by nonlymphoid cells such as stromal cells, fibroblasts, endothelial cells, epithelial cells, and dendritic cells, and their expression is not directly affected by T cell costimulatory signals (19), and, therefore, their impact on T cell activation is likely to be distinct. Moreover, rejection of MHC-mismatched allografts often involves activation of both CD4+ and CD8+ T cells, and interactions between both T cell subsets may define the nature of the rejection process and probably also the strategy of tolerance induction. Nonetheless, a clear understanding of costimulatory signals and the {gamma}c signals in activation of CD4+ and CD8+ T cells in vivo is still lacking, and their impact on allograft rejection and tolerance induction remains uncertain.

In the present study, we critically examined CD28/CD40L costimulation and the {gamma}c signals in activation and expansion of CD4+ and CD8+ T cells in vivo in the allogeneic hosts. We found that in vivo expansion of CD4+ and CD8+ T cells in response to alloantigen stimulation exhibited distinct sensitivity to blockade of costimulatory signals and the {gamma}c. Selective blockade of CD28/CD40L costimulation and {gamma}c signals differentially affected the alloreactive CD4 and the CD8 compartments, and targeting both T cell costimulatory and {gamma}c signals may be required to block cytopathic conditions involving activation of both CD4+ and CD8+ T cells.


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

Male B6AF1 (H-2b/a), DBA/2 (H-2d), and C57BL/6 (H-2b) mice, 8–10 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Male CD4-deficient and CD8-deficient mice on the C57BL/6 background were purchased from Taconic Farms (Germantown, NY). All animals were housed in the animal facility at the Beth Israel Deaconess Medical Center (Boston, MA). Animal use was conformed to the guidelines established by the Animal Care Committee in our institution.

Reagents

The following staining Abs were obtained from BD PharMingen (San Diego, CA): CyChrome anti-mouse CD4 (GK1.5), CyChrome anti-mouse CD8{alpha} (clone 53-6.7), PE anti-mouse CD44 (clone IM7), PE anti-mouse CD62L (L-selectin, clone MEL-14), PE anti-mouse CD25 (clone PC61), PE anti-mouse IL-2 (clone JES6-5H4), PE anti-mouse TNF-{alpha} (clone MP6-XT22), PE anti-mouse IFN-{gamma} (clone XMG 1.2), and PE isotype control Abs.

Murine CTLA-4Ig was constructed, expressed, and tested in our laboratory, as previously described (20). Hybridoma cell lines secreting a hamster anti-mouse CD40L mAb (MR1, IgG) and a rat anti-mouse CD25 mAb (TIB 222, IgG1) were obtained from American Type Culture Collection (Manassas, VA). The hybridoma cells were grown in serum-free UltraCulture medium (BioWhittaker, Walkersville, MD), and mAbs were purified from the culture supernatant with protein G columns. Rat anti-mouse {gamma}c mAbs (4G3/3E12) were produced and used, as we previously reported (21, 22). A mutant IL-15/Fc fusion protein that acts as an IL-15R-specific antagonist was constructed, tested, and produced in our laboratory and used as described elsewhere (23).

CFSE labeling

Spleen and peripheral lymph nodes were harvested from donor mice, and single-cell suspension was prepared in HBSS. RBCs were lysed by hypotonic shock. Lymphocytes were resuspended in HBSS at 1 x 107 cells/ml for labeling with fluorochrome CFSE (Molecular Probes, Portland, OR), as previously described (24). Briefly, cells were incubated with CFSE at a final concentration of 5 µM in HBSS at room temperature for 6 min. The labeling was then terminated by the addition of FCS (10% of the total volume). Cells were washed twice in HBSS before i.v. injection.

Adoptive transfer of CFSE-labeled cells into allogeneic hosts

Host DBA/2 mice were lethally irradiated (1000 rad) with a Gammacell Exactor (Kanata, Ontario, Canada). Each mouse then received ~6 x 107 CFSE-labeled donor cells in 0.5 ml HBSS via the tail vein shortly after irradiation. Three days later, the host mice were sacrificed, spleens and peripheral lymph nodes were harvested, and single-cell suspension was prepared for cell surface staining and intracellular cytokine staining. The large number of cells transferred did not cause homeostatic expansion, and cell proliferation in vivo is solely driven by alloantigens in this model (3).

Treatment of irradiated host mice

Treatment with anti-{gamma}c mAbs consisted of 1 mg daily for 3 consecutive days starting at i.v. injection of CFSE-labeled cells. Treatment with costimulation blockade consisted of 0.3 mg MR1 (anti-CD40L mAb) and 0.3 mg CTLA-4Ig i.p. daily for 3 days starting at cell transfer. Host mice were also treated with anti-mouse CD25 mAb at 1 mg daily for 3 days or anti-CD25 (1 mg/day for 3 days) plus mutant IL-15/Fc (10 µg/day for 3 days) starting at i.v. injection of CFSE-labeled cells. In some experiments, host mice were treated with combined CTLA-4Ig, anti-CD40L, and anti-{gamma}c mAbs for 3 days with the same doses as described above. Animals treated with rat IgG and hamster IgG (Sigma-Aldrich, St, Louis, MO) were included as controls.

Cell staining and flow cytometry

For cell surface staining, CFSE-labeled cells were recovered from the host spleen. Cells were resuspended in PBS/0.5% BSA (2 x 106/ml) and stained with CyChrome-conjugated anti-mouse CD4 and CyChrome anti-mouse CD8, respectively, on ice for 30 min, washed in PBS/BSA, and fixed in 1% formaldehyde before analysis. In some experiments, cells were stained with CyChrome anti-mouse CD4 or CyChrome anti-mouse CD8 in combination with PE anti-mouse CD25, PE anti-mouse CD44, and PE anti-mouse CD62L to analyze the activation status of either T cell subsets. PE-conjugated isotype control Ab was included in the staining as a control. Cells were then washed in PBS/0.5% BSA and fixed before analysis.

For intracellular cytokine staining, CFSE-labeled cells harvested from the host mice were resuspended in complete RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin at 5 x 106/ml. Cells were restimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) at 37°C for 4 h. In the last 2 h of restimulation, GolgiStop (BD PharMingen) was added at a concentration of 1 µg/ml into the culture. Cells were harvested following the in vitro culture and stained with CyChrome anti-mouse CD4 and CyChrome anti-mouse CD8, respectively, fixed, and cell membrane permeabilized in Cytofix/Cytoperm solution (BD PharMingen) at 4°C for 10 min, followed by washing in Perm/Wash solution (BD PharMingen). Cells were then resuspended in Perm/Wash solution (1 x 106) and stained with PE-conjugated Abs against mouse IL-2, TNF-{alpha}, and IFN-{gamma} on ice for 30 min. Cells were washed twice in Perm/Wash solution and resuspended in PBS/0.5% BSA. Isotype-matched control Abs were included in the staining as negative controls for FACS analysis.

For annexin V staining, CFSE-labeled cells were recovered from the host mice and briefly stained with CyChrome anti-CD4 or CyChrome anti-CD8, as described above, along with PE-annexin V in a calcium-rich annexin-binding buffer (BD PharMingen). Cells were washed twice after the staining before FACS analysis. Cell division history and apoptotic cell death in individual rounds of cell divisions were analyzed by FACS, calculated, and compared.

All samples were analyzed using a FACSort equipped with CellQuest software (BD Biosciences, Mountain View, CA). Data were collected and analyzed by gating on the CFSE-positive population. At least 100,000 events were collected for each sample.

Calculation of responder frequency and clonal expansion

The frequency of CD4+ and CD8+ T cells proliferating in vivo in the allogeneic hosts was calculated, as previously reported (24). Briefly, distinct rounds of cell divisions were identified by their CFSE profiles. The absolute number of cells in each cell division was counted using the FACS acquisition software CellQuest; the number of precursors that proliferated and gave rise to the absolute number of daughter cells was extrapolated using the formula: y/2n (y = absolute number of cells in each cell cycle; n = number of cell divisions). For example, 16 daughter cells in the third cell division are the progeny of two precursors, each of which have divided three times (16/23 = 2). The frequency of proliferating T cells in the responder population was then calculated by dividing the total number of precursors by the sum of total CFSE-labeled cells collected.

Skin grafting and treatment protocol

Full-thickness tail skin graft (~1 cm2) from DBA/2 mice (H-2d) was transplanted onto the thoracic wall of B6AF1 recipients (H-2b/a), and the skin graft was secured with an adhesive bandage for the initial 5 days. Graft survival was then followed by daily visual inspection. Rejection was defined as the complete necrosis and loss of viable skin tissue.

Treatment of skin graft recipients with costimulation blockade consisted of 0.5 mg CTLA-4Ig and 0.5 mg anti-CD40L i.p. on days 0, 1, and 3 after transplantation. Anti-{gamma}c mAbs were given at 1 mg i.p. on days 0, 1, 3, 5, and 7 after transplantation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Quantitative analysis of CD4+ and CD8+ T cell expansion in vivo

To precisely analyze the cellular basis of T cell activation in vivo, lymphocytes from B6AF1 mice (H-2b/a) were labeled with the tracking dye CFSE and injected into lethally irradiated DBA/2 hosts (H-2d); cell division history and the magnitude of clonal expansion of CD4+ and CD8+ T cell subsets were determined. As shown in Fig. 1Go, both CD4+ and CD8+ T cells proliferated vigorously in vivo. Responder frequency calculation revealed that ~22% of CD4+ T cells and as high as 42% of CD8+ T cells recovered from the host mice entered the cell cycle 3 days after adoptive transfer, and the responding T cell clones expanded over 10 times at this time point. Acquisition of primed phenotype was closely linked to cell divisions for both T cell subsets. At least five cell divisions were required for dividing T cells to shed the L-selectin and to up-regulate CD25 and CD44 expression on the cell surface (Fig. 2Go, A and B), the hallmarks of Ag-activated T cells. The acquisition of primed phenotype was also closely associated with their functional maturation, as determined by intracellular staining for effector cytokines IFN-{gamma} and TNF-{alpha}. As shown in Fig. 3Go, dividing CD4+ and CD8+ T cells that displayed primed phenotype also expressed IFN-{gamma} and TNF-{alpha}. Thus, rapid cell cycle progression is critical for both CD4+ and CD8+ T cells to acquire effector functions in vivo.



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  		FIGURE 1. Proliferation of allogeneic lymphocytes in irradiated hosts. CFSE-labeled lymphocytes from B6AF1 mice were injected into lethally irradiated DBA/2 mice (6 x 107 cells/mouse). Proliferation of CFSE-labeled CD4+ and CD8+ T cells in the host spleen was examined 3 days later by FACS. Representative data of one of five individual experiments are shown.

 


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  		FIGURE 2. Acquisition of primed phenotype by dividing CD4+ and CD8+ T cells in vivo. A, CFSE-labeled B6AF1 cells were recovered from irradiated DBA/2 hosts 3 days after adoptive transfer. Cells were stained with CyChrome anti-mouse CD4 and anti-CD8 as well as PE-conjugated mAbs against CD25, CD44, and CD62L. Expression of the activation markers was analyzed by gating onto CFSE+CD4+ or CFSE+CD8+ cells. Cells stained with isotype control Abs were used as a control to set up the quadrants for analysis. B, Expression of CD25, CD44, and CD62L in each cell division was analyzed, and the mean fluorescence intensity (MFI) was calculated and plotted against the number of cell divisions. Representative data of five experiments shown.

 


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  		FIGURE 3. Expression of IFN-{gamma} and TNF-{alpha} by proliferating CD4+ and CD8+ T cells in vivo. A, CFSE-labeled cells were activated in vivo in the allogeneic hosts for 3 days. Cells were recovered and briefly restimulated in vitro with PMA and ionomycin. Cells were then fixed, permeabilized, and stained for IFN-{gamma} and TNF-{alpha}. Cells stained with PE-conjugated isotype control Ab were used as a control. B, IFN-{gamma} and TNF-{alpha} staining in each cell division was analyzed, and the mean fluorescence intensity (MFI) was calculated and plotted against the number of cell divisions. Representative data of five experiments shown.

 
Blocking the {gamma}c on proliferation of CD4+ and CD8+ T cells in vivo

T cell proliferation is believed to require growth factors, and all known TCGFs (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) utilize the {gamma}c as a critical signaling component in their receptor complexes (16, 17). We therefore examined the effect of blocking the {gamma}c with anti-{gamma}c mAbs on proliferation of CD4+ and CD8+ T cells in the allogeneic hosts. As shown in Fig. 4GoA, treatment of host mice with saturating doses of anti-{gamma}c mAbs (1 mg i.p. daily for 3 days) virtually abolished the in vivo expansion of CD8+ T cells. Surprisingly, such treatment had little effect on blocking the in vivo proliferation of CD4+ T cells in the same animals examined. CFSE-labeled CD4+ T cells recovered from the anti-{gamma}c-treated mice had a division profile that was similar to control Ab-treated mice (Fig. 4GoA). Furthermore, recovery of CFSE-labeled CD8+ T cells from the allogeneic hosts was consistently reduced by ~40% in the anti-{gamma}c-treated mice as compared with the controls, presumably reflecting inhibition of cell expansion and increased cell death (25), whereas recovery of CFSE-labeled CD4+ T cells was comparable between anti-{gamma}c-treated and control Ab-treated mice (Fig. 4GoB).



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  		FIGURE 4. A, Effect of anti-{gamma}c treatment on expansion of CD4+ and CD8+ T cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with anti-{gamma}c mAbs or rat IgG as a control. Cells were recovered 3 days later, and cell division was analyzed by gating onto the CFSE+CD4+ and CFSE+CD8+ population, respectively. B, Recovery of CFSE-labeled CD4+ and CD8+ T cells in anti-{gamma}c-treated hosts. Data are presented as percentage of CFSE+CD4+ and CFSE+CD8+ T cells in the total CFSE+ cells recovered from the host mice. Results are shown as mean ± SD of three experiments.

 
To further study the expansion of CD4+ and CD8+ T cells in vivo and the role of {gamma}c-sensitive signals, we injected CFSE-labeled cells obtained from CD4 knockout (KO) mice (primarily CD8+ T cells) or CD8KO mice (primarily CD4+ T cells) into lethally irradiated DBA/2 hosts with or without anti-{gamma}c treatment, and cell proliferation in vivo was examined 3 days later. As shown in Fig. 5Go, the division profile of CFSE-labeled CD8KO cells in vivo in anti-{gamma}c-treated hosts was remarkably similar to the control Ab-treated mice. In stark contrast, proliferation of CD4KO cells in vivo was markedly inhibited in the anti-{gamma}c-treated mice.



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  		FIGURE 5. Effect of anti-{gamma}c on in vivo proliferation of cells from CD4KO mice or CD8KO mice. Lymphocytes were prepared from CD4KO and CD8KO mice, labeled with CFSE, and injected into lethally irradiated DBA/2 hosts. The host mice were treated with anti-{gamma}c mAbs or control IgG, and cell proliferation in the host spleen was analyzed 3 days later. Representative data of two experiments shown.

 
CD4+ T cells that entered the cell cycle could differentiate into effector cells despite the {gamma}c blockade, as determined by staining for intracellular IFN-{gamma} expression (Fig. 6Go). Interestingly, blocking the {gamma}c induced increased apoptotic cell death of proliferating CD4+ T cells, as determined by annexin V staining, as compared with the control Ab-treated mice (Fig. 7Go), suggesting that {gamma}c-dependentsignals may be required for sustained survival of activated and actively dividing CD4+ T cells, but proliferation of CD4+ T cells in vivo may be resistant to the {gamma}c blockade.



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  		FIGURE 6. IFN-{gamma} expression by CD4+ T cells from anti-{gamma}c-treated mice. CFSE-labeled cells were recovered from the anti-{gamma}c-treated allogeneic hosts, briefly restimulated in vitro with PMA and ionomycin, and stained for intracellular IFN-{gamma} expression. Analysis was gated onto the CFSE+ and CD4+ cells. Representative data of three experiments shown.

 


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  		FIGURE 7. Effect of anti-{gamma}c treatment on apoptosis of dividing CD4+ cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with the anti-{gamma}c mAbs. Host spleen cells were harvested 3 days later and stained with CyChrome anti-mouse CD4 and PE-annexin V. Staining for annexin V was analyzed by gating CFSE+CD4+ cells. Control Ab-treated mice were included as a control. Data are presented as percentage of annexin V-positive cells in each cell division. Representative data of three experiments shown.

 
CD28/CD40L costimulation and in vivo expansion of CD4+ and CD8+ T cells

The apparent resistance of in vivo expansion of CD4+ T cells, but not CD8+ T cells, to {gamma}c blockade treatment in the allogeneic hosts suggests that TCR stimulation plus T cell costimulation may be necessary and sufficient for proliferation of CD4+ T cells. Also, it remains unclear whether CD4+ T cell-dependent activation of CD8+ T cells requires CD28/CD40L costimulation to gain responsiveness to {gamma}c-dependent cytokines. To probe these possibilities, we treated host mice with CTLA-4Ig and anti-CD40L mAb (MR1) to block both CD28/B7 and CD40L/CD40, two critical costimulatory pathways in T cell activation (26), and proliferation of CD4+ and CD8+ T cells in vivo was determined. As shown in Fig. 8Go, blocking both costimulatory pathways markedly inhibited the division of adoptively transferred CD4+ T cells in the allogeneic hosts, and the responder frequency was reduced by ~2-fold (~10%) when compared with the control mice (~21%). A small subset of CD4+ T cells still entered the cell cycle, despite the costimulation blockade treatment, and divided for multiple times. However, their proliferative capacity (the number of daughter cells generated by a given precursor) was strongly inhibited by blocking both CD28 and CD40L pathways (Fig. 8Go), suggesting that CD28/CD40L signals are required for activated CD4+ T cells to enter the proliferating pool and for sustained proliferation.



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  		FIGURE 8. Effect of CTLA-4Ig and anti-CD40L treatment on divisions of CD4+ and CD8+ T cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with CTLA-4Ig and anti-CD40L or control IgG. Cells were recovered 3 days later, and cell division in vivo was analyzed by gating onto the CFSE+CD4+ and CFSE+CD8+ population, respectively. A, Histogram presentation of CD4+ and CD8+ T cell division in vivo. B, Each cell division was individually gated; the percentage of cells in each cell division among the total number of CFSE+ cells gated was calculated and plotted against the number of cell divisions. Representative data of three experiments shown.

 
The impact of costimulation blockade on in vivo expansion of CD8+ T cells exhibited distinct patterns. Cell cycle entry and initial expansion of CD8+ T cells are clearly CD28/CD40L independent, as CD8+ T cells could enter the proliferative pool and divide more than three times in the allogeneic hosts treated with CTLA-4Ig and anti-CD40L (Fig. 8Go, right panel), suggesting that recruitment of activated CD8+ T cells into the proliferating pool is not dependent on CD28/CD40L costimulation. Nonetheless, continued expansion of activated CD8+ T cells beyond the fifth cell divisions was completely blocked in CTLA-4Ig- and anti-CD40L-treated mice (Fig. 8Go).

Role of IL-2 and IL-15 in proliferation of CD8+ T cells in vivo

As blocking the {gamma}c virtually blocked the in vivo expansion of alloreactive CD8+ T cells (Figs. 4GoA and 5), whereas costimulation blockade inhibited only the late, but not the early phase, of CD8+ T cell expansion (Fig. 8Go), we reasoned that once activated CD8+ T cells are recruited into the proliferating pool, continued expansion of CD8+ T cells may depend on CD4+ T cells, possibly via the production of T cell-derived growth factors (e.g., IL-2), which is a costimulation-dependent process (18). Staining for cytosolic IL-2 revealed that CD8+ T cells in this model did not produce IL-2, whereas IL-2 was highly expressed by CD4+ T cells only after five cell divisions (Fig. 9Go). Production of IL-2 by activated CD4+ T cells was closely associated with the high expression of CD25 on dividing CD8+ T cells (Fig. 2GoA, right panel). Treatment of host mice with saturating doses of anti-CD25 to block the IL-2R, similar to costimulation blockade, inhibited the late phase of CD8+ T cell expansion, while cell cycle entry and initial expansion of CD8+ T cells were intact (Fig. 10Go). Treatment with both anti-CD25 and mutant IL-15/Fc, a fusion protein that acts as an IL-15R-specific antagonist (23, 27), inhibited both early and late phase of CD8+ T cell expansion (Fig. 10Go), and such a division profile was similar to anti-{gamma}c-treated hosts (Fig. 4GoA). These data suggest that recruitment and initial expansion of alloreactive CD8+ T cells appear to be supported by non-T cell-derived growth factors, especially IL-15, and its expression is not directly affected by T cell costimulation (28), and continued expansion of dividing CD8+ T cells may rely on activated CD4+ T cells for IL-2 production in this model.



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  		FIGURE 9. Comparison of IL-2 expression by CD4+ and CD8+ T cells in vivo. CFSE-labeled cells were activated in vivo in allogeneic hosts for 3 days. Cells were recovered and briefly restimulated in vitro with PMA and ionomycin. Cells were then fixed, permeabilized, and stained for intracellular IL-2. Cells stained with PE-conjugated isotype control Ab were used as a control to set up the quadrants for analysis. Representative data of three experiments shown.

 


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  		FIGURE 10. Effect of anti-CD25 and mutant IL-15/Fc on expansion of CD8+ T cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with anti-CD25 or anti-CD25 plus mutant IL-15/Fc. Cells were recovered 3 days later from the host spleen, and cell division in vivo was analyzed by gating onto the CFSE+CD8+ population. Representative data of three experiments shown.

 
Blocking both CD28/CD40L and the {gamma}c pathways on T cell expansion and allograft rejection

Clearly, selective targeting of costimulatory pathway or the {gamma}c pathway preferentially affects CD4+ and CD8+ T cells in vivo. As the allograft response often involves activation of both CD4+ and CD8+ T cells (Fig. 1Go), and either T cell subset is capable of mediating graft destruction (29), targeting both T cell costimulatory and the {gamma}c pathways may be required in blocking activation of both CD4+ and CD8+ T cells in vivo. To probe this possibility, we treated the irradiated allogeneic hosts with CTLA-4Ig, anti-CD40L, and the anti-{gamma}c mAbs following adoptive transfer of CFSE-labeled cells, and cell expansion in vivo was examined 3 days later. As shown in Fig. 11Go, proliferation of both CD4+ and CD8+ T cells in vivo in the allogenic hosts was simultaneously blocked with concurrent blockade of CD28/CD40L and the {gamma}c when compared with the control Ab-treated mice or mice treated with CD28/CD40L blockade alone or the {gamma}c blockade alone (Figs. 4GoA and 8).



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  		FIGURE 11. Effect of CTLA-4Ig + anti-CD40L + anti-{gamma}c treatment on expansion of CD4+ and CD8+ T cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with CTLA-4Ig + anti-CD40L + anti-{gamma}c mAbs or control IgG. Cells were recovered 3 days later, and cell division in vivo was analyzed by gating onto the CFSE+CD4+ and CFSE+CD8+ population, respectively. Representative data of two experiments shown.

 
To further determine the impact of costimulation blockade and {gamma}c blockade on allograft rejection, we used a stringent skin allograft model. As shown in Fig. 12Go, skin grafts from DBA/2 donors (H-2d) were uniformly rejected by the B6AF1 recipients (H-2b/a) with a mean survival time of 10 days (n = 8). Treatment of recipient mice with CTLA-4Ig and anti-CD40L (n = 4) or anti-{gamma}c mAbs (n = 5) failed to prolong skin allograft survival, and all of the skin grafts were rejected at about day 12 after transplantation. In contrast, treatment with CTLA-4Ig and anti-CD40L in combination with the anti-{gamma}c mAbs induced prolonged allograft survival (n = 7), and three of seven skin allograft recipients survived for over 100 days.



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  		FIGURE 12. Skin allograft survival in mice treated with costimulation blockade and {gamma}c blockade.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the remarkable features of allograft response is the multiplicity of cell types involved and the complexity of their interactions and regulations. It is generally believed that robust activation of naive T cells requires costimulatory signals (30), and the fate of activated T cells to become armed effector cells, to undergo activation-induced cell death, or to evolve as immune regulatory cells, is probably dictated by the availability and accessibility of TCGFs (31). Our data demonstrate, by using an in vivo model, that expansion of alloreactive T cells in vivo is clearly not a uniform process, and different T cell subsets exhibit distinct sensitivity to costimulatory signals and growth factor signals for activation and expansion. In keeping with previous reports (12, 32), CD28- and CD40L-imparted costimulatory signals are clearly essential for CD4+ T cells to enter the dividing pool and to expand efficiently since blocking both CD28/B7 and CD40L/CD40 costimulatory pathways using CTLA-4Ig and anti-CD40L mAb markedly inhibited the responder frequency and the responder capacity of activated CD4+ T cells (Fig. 8Go). Surprisingly, signals transduced from the {gamma}c, a critical signaling element used by receptors for all known TCGFs (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) (16, 17), appear to be dispensable in this process, as blocking the {gamma}c had little effect in blocking alloantigen-driven proliferation of CD4+ T cells in vivo (Fig. 4Go). The lack of effects of anti-{gamma}c on the CD4+ T cells is unlikely due to inadequate amount of blocking mAbs given, since proliferation of CD8+ T cells in the same animals examined was nearly abolished (Fig. 4Go). CD4+ T cells in this model express high levels of IL-2 upon activation in vivo (Fig. 9Go); they certainly have the competitive advantage over anti-{gamma}c mAbs to bind to the IL-2R via the autocrine fashion. However, only a fraction of dividing CD4+ T cells in vivo expresses CD25 (Fig. 2GoA, left panel), the private {alpha}-chain of the IL-2R that is required for high-affinity IL-2 binding (16). Furthermore, CD4+ T cells from IL-2KO mice can expand with similar kinetics in the allogeneic hosts regardless of anti-{gamma}c treatment (our unpublished observation). Nonetheless, activated CD4+ T cells are clearly sensitive to {gamma}c-dependent cytokines, as they proliferate vigorously in vitro in response to IL-2, IL-4, IL-7, or IL-15 (33). It is not clear why {gamma}c signals are dispensable for in vivo proliferation of CD4+ T cells. Alternatively, proliferation of activated CD4+ T cells in vivo may be supported by other {gamma}c-independent cytokines, such as IL-5, IL-6, IL-12, IL-18, IFN-{gamma}, etc. However, detailed analysis showed that neither of such cytokines, alone or in combinations, can stimulate T cell proliferation in vitro (34).

Thus, it seems likely that TCR stimulation plus costimulatory signals may be necessary and sufficient to sustain the proliferative activity of CD4+ T cells in vivo. This notion is supported by a recent study that CD4+ T cells carrying a TCR transgene, but lacking the expression of {gamma}c, can mount a proliferative response to the cognate Ag in vivo, albeit such CD4+ T cells exhibit an increased apoptotic index (35), which is consistent with our finding using the in vivo CFSE model. However, the role of costimulatory molecules in proliferation and expansion of such CD4+ T cells was not examined in that study (35). Furthermore, CD4+ T cells, but not CD8+ T cells, selectively expand in the periphery of {gamma}c mutant mice over time (36). Such CD4+ T cells are functional, as they can synthesize IFN-{gamma} and control the early phase of Toxoplasma gondii infection (37). In a novel transgenic system, Malek et al. (38) have convincingly demonstrated that signaling through the TCR and CD28/CD40L costimulatory molecules can drive T cell proliferation that is independent of IL-2 and other {gamma}c cytokines. In fact, some of the signaling pathways required for cell cycle progression (e.g., activation of mitogen-activated protein kinase and phosphatidylinositol-3 kinase) are directly linked to CD28 stimulation (39). Thus, a detailed study of costimulatory signals and cell cycle regulators may help further delineate this issue.

Another key finding is that CD8+ T cells, in stark contrast to CD4+ T cells, are exquisitely dependent on {gamma}c signals for survival, expansion, and functional maturation (Figs. 4Go and 5Go). However, individual {gamma}c cytokines appear to play a distinct role in this process. Clearly, recruitment of activated CD8+ T cells into the dividing pool in vivo in the allogeneic hosts requires non-T cell-derived growth factors, especially IL-15, and sustained expansion of activated CD8+ T cells requires the production of IL-2, a T cell-produced growth factor and expression of which often relies on activation and expansion of CD4+ T cells. Indeed, blocking the IL-15/IL-15R pathway using an antagonist IL-15/Fc or the IL-2/IL-2R pathway using anti-CD25 mAb can partially contain CD8+ T cell expansion in vivo and facilitate costimulation blockade-induced allograft survival (27, 40). However, the dependency on CD4+ T cells for continued expansion of CD8+ T cells in vivo seems to be conditional, as T cells from CD4KO mice (primarily CD8+ T cells) proliferated vigorously in the allogeneic hosts with a similar kinetics to the wild-type controls, and such expansion is also extremely sensitive to the {gamma}c blockade (Fig. 5Go), suggesting that other {gamma}c cytokines may support robust CD8+ T cell expansion in the complete absence of CD4+ T cells, or activated CD8+ T cells may produce IL-2 under such conditions. Our finding provides clear explanation for some of the conflicting observations reported in the literature (14, 41). It appears that CD28/CD40L costimulation regulates CD8+ T cell expansion indirectly via activation of CD4+ T cells and production of IL-2 under certain conditions, but activation of CD8+ T cells per se is less dependent on CD28/CD40L costimulatory signals (11, 42). However, multiple alternative costimulatory pathways (e.g., 4-1BB/4-1BBL, 2B4/CD48) have been described previously (43, 44), and such alternative pathways may play an important role in regulating CD8+ T cell function. The question as to whether CD8+ T cells require alternative costimulatory signals to gain optimal responsiveness to {gamma}c-dependent cytokines warrants further study.

Clearly, activation and interaction of CD4+ and CD8+ T cells in allograft response is not a uniform process. Selective targeting of CD28/CD40L costimulation or the {gamma}c signals may preferentially affect CD4+ or CD8+ T cell activation, and such interventions may be effective in blocking a CD4- or CD8-dominated rejection process. Under certain conditions in which both CD4+ and CD8+ T cells are involved and either subset can mediate graft destruction, targeting either T cell costimulation or {gamma}c signals may be necessary, but not sufficient for stable allograft survival, and blocking both pathways may be critically important in this regard. Our study also suggests that the intrinsic features of the grafts that affect the expression of nonconventional growth factors (i.e., IL-7, IL-15) may have a significant impact on the final outcome of the grafts (45). For example, renal tubular epithelial cells are a rich source of IL-15, and ligation of CD40 can enhance IL-15 expression (46, 47). In the skin, keratinocytes constitutively express IL-15, and its expression is enhanced in inflammation, which stimulates not only CD8+ T cells, but also dendritic cell maturation (48); such process may also be important in chronic graft destruction (45). Given the fact that TCGFs are extremely redundant in supporting T cell activation and allograft rejection (49), and the sharing of the {gamma}c by all known TCGFs and the {gamma}c blockade can inhibit CD8+ T cell expansion, targeting the {gamma}c may be a critical component to the costimulation blockade protocol in blocking acute/chronic rejection and in tolerance induction under stringent conditions.


   Footnotes
 
1 This work was supported by grants from the Juvenile Diabetes Foundation International, 1-1999-16 and 1-2000-47 (to X.C.L.), and the National Institutes of Health, RO1 AI42298 (to T.B.S.) and PO AI/GF 41521 (to T.B.S.). G.D. was supported by a grant from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Xian C. Li, Department of Medicine, Harvard Medical School, Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215. E-mail address: xli{at}caregroup.harvard.edu Back

3 Abbreviations used in this paper: TCGF, T cell growth factor; CD40L, CD40 ligand; KO, knockout; {gamma}c, common {gamma}-chain. Back

Received for publication December 5, 2001. Accepted for publication February 19, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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