Selective Blockade of IL-15 by Soluble IL-15 Receptor α-Chain Enhances Cardiac Allograft Survival1

IL-15 is a T cell growth factor that shares many functional similarities with IL-2 and has recently been shown to be present in tissue and organ allografts, leading to speculation that IL-15 may contribute to graft rejection. Here, we report on the in vivo use of an IL-15 antagonist, a soluble fragment of the murine IL-15R α-chain, to investigate the contribution of IL-15 to the rejection of fully vascularized cardiac allografts in a mouse experimental model. Administration of soluble fragment of the murine IL-15R α-chain (sIL-15Rα) to CBA/Ca (H-2k) recipients for 10 days completely prevented rejection of minor histocompatibility complex-mismatched B10.BR (H-2k) heart grafts (median survival time (MST) of >100 days vs MST of 10 days for control recipients) and led to a state of donor-specific immunologic tolerance. Treatment of CBA/Ca recipients with sIL-15Rα alone had only a modest effect on the survival of fully MHC-mismatched BALB/c (H-2d) heart grafts. However, administration of sIL-15Rα together with a single dose of a nondepleting anti-CD4 mAb (YTS 177.9) delayed mononuclear cell infiltration of the grafts and markedly prolonged graft survival (MST of 60 days vs MST of 20 days for treatment with anti-CD4 alone). Prolonged graft survival was accompanied in vitro by reduced proliferation and IFN-γ production by spleen cells, whereas CTL and alloantibody levels were similar to those in animals given anti-CD4 mAb alone. These findings demonstrate that IL-15 plays an important role in the rejection of a vascularized organ allograft and that antagonists to IL-15 may be of therapeutic value in preventing allograft rejection.

C ytokines play a key role in regulating and amplifying the T cell-dependent immune response to an organ allograft. T cell growth factors such as IL-2, IL-4, IL-7, IL-9, and IL-15 promote differentiation and clonal expansion of activated alloreactive T cells, whereas proinflammatory cytokines such as IFN-␥ and TNF-␣ contribute significantly to the effector mechanisms responsible for graft destruction (1,2). The T cell-derived cytokine IL-2 was the first major T cell growth factor identified, and therapeutic Abs directed against the ␣-chain of the IL-2R (CD25) have recently been shown, when combined with conventional immunosuppressive agents, to reduce the incidence of acute rejection after human organ transplantation (3,4). However, allograft rejection still occurs in the presence of IL-2/IL-2R blockade (3)(4)(5). Moreover, IL-2 gene knockout mice and IL-2/IL-4 doubleknockout mice are still able to reject allografts, highlighting the redundancy in the cytokine network and suggesting a potentially important role for other immunoregulatory cytokines, such as IL-7 and IL-15, in the graft rejection process (6,7).
IL-15 is a recently identified cytokine that is similar in structure to IL-2 and shares with it a number of biological activities, including its ability to stimulate the proliferation and differentiation of activated T cells (8,9). However, unlike IL-2, IL-15 mRNA is expressed in a wide range of different cell types, including acti-vated macrophages, activated vascular endothelial cells, fibroblasts, muscle cells, epithelial cells, and T cells (10,11). Moreover, although IL-15 and IL-2 share the same IL-2R␤ and common ␥-chain receptor subunits, IL-15 uses a unique ␣-chain, IL-15R␣, which is expressed on a wide variety of cell types, including activated T cells, B cells, NK cells, macrophages, and some types of nonimmune cells (10 -12). The biological effects of IL-15 are diverse. In addition to acting as a T cell growth factor, IL-15 stimulates the differentiation of NK cells, induces T cell chemotaxis, promotes B cell activation and isotype switching, and increases the production of proinflammatory cytokines by macrophages (10,11). It also promotes the growth and activation of mast cells and influences activity in some types of nonimmune cell (10,11).
The role of IL-15 in the allograft rejection response is not clearly defined, but an important role for this cytokine has been suggested in other types of immunopathology, such as inflammatory arthritis, inflammatory bowel disease, sarcoidosis, and multiple sclerosis (13)(14)(15)(16)(17). Several recent studies have observed an increase in the expression of intragraft IL-15 mRNA transcripts and IL-15-expressing cells after transplantation and suggested that increased IL-15 expression correlates with acute rejection (18 -22). Therefore, blockade of the IL-15/IL-15R pathway may be of value in modifying the immune response to an organ allograft. In this paper we report that administration of a soluble fragment of the murine IL-15R ␣-chain (sIL-15R␣) 3 to neutralize IL-15 prevents rejection of fully vascularized, minor histocompatibility Ag-mismatched cardiac allografts. Furthermore, sIL-15R␣, in combination with a single dose of a nondepleting anti-CD4 mAb, markedly prolongs the survival of fully allogeneic heart allografts.

Heart transplantation
Vascularized cervical heterotopic heart transplantation was performed using standard microsurgical techniques as previously described (23). The innominate artery of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient, and similarly, the donor pulmonary artery was anastomosed to the recipient external jugular vein. Cold ischemic times were Ͻ30 min. Graft function was assessed by daily palpation of the heart, and rejection was defined as the complete cessation of myocardial contraction.

Skin grafting
Tail skin grafts from adult donor mice were transplanted to the flanks of heart graft recipients, essentially as described previously (24). Dressings were removed after 7 days, and skin graft rejection was defined as complete destruction of the graft.

Histology
Heart grafts were excised, formalin fixed, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin.

sIL-15R␣
A soluble fragment of IL-15R ␣-chain lacking transmembrane and cytoplasmic domains was generated as described previously (14). The histidine-tagged recombinant protein was purified using nickel-agarose and dialyzed into PBS for in vivo use. The sIL-15R␣ fragment used has been shown previously to inhibit rIL-15-mediated in vitro proliferation of CTLL and D10 cells, but does not bind to IL-2 or inhibit IL-2-dependent CTLL proliferation (14). Heart graft recipients received 60 g of sIL-15R␣ i.p. on the day of transplantation and daily for 9 days thereafter.
This schedule was chosen on the basis that daily i.p. administration of 40 g of sIL-15R␣ for 10 days profoundly suppressed the development of murine collagen-induced arthritis (14), although it is not known whether the amount of sIL-15R␣ given was sufficient to completely neutralize IL-15 for the treatment duration.

CTL activity
The CTL activity of splenocytes was determined as described previously (26). Recipient splenocytes were cocultured with irradiated donor splenocytes (20 Gy) for 5 days to generate CTL, then incubated with [ 3 H]thymidine-labeled donor strain Con A blast target cells for 4 h to determine their cytotoxic activity. Reduced scintillation counts indicated enhanced cytotoxic activity.

Lymphocyte proliferation and cytokine production
Lymphocyte proliferation assays were performed essentially as described previously (27). Responder splenocytes (2 ϫ 10 5 /well) were cultured with irradiated (20 Gy) stimulator splenocytes (2 ϫ 10 5 /well) for 72, 96, and 120 h in 96-well plates at 37°C in 5% CO 2 . Proliferation was assessed by uptake of [ 3 H]thymidine for 6 h at the end of the culture period. Supernatant was harvested from cell culture plates and was assayed by ELISA for production of IFN-␥ and IL-4 using capture and detection Abs (PharMingen) according to the manufacturer's instructions.

Statistical analysis
Differences between groups were compared by nonparametric analysis using the Mann-Whitney U test. Values of p (two-tailed) Ͻ 0.05 were considered significant.

Effect of sIL-15R␣ on cardiac allograft survival
The effect of disrupting the IL-15/IL-15R pathway on cardiac allograft survival was examined by administering sIL-15R␣ to recipients of either minor (mHC) or MHC Ag-mismatched heart grafts. CBA/Ca (H-2 k ) recipients of mHC-disparate B10BR (H-2 k ) heart grafts were treated daily with an i.p. injection of 60 g of sIL-15R␣ for 10 days from the day of transplant. This treatment schedule was based on our experience of using sIL-15R␣ to control murine collagen-induced arthritis (14). Control recipients rejected their heart grafts promptly, with a median survival time (MST) of 10 days, whereas treatment with sIL-15R␣ extended heart graft survival well beyond the 10-day treatment schedule in all recipients, and in 70% of recipients led to long term (Ͼ100 days; p Ͻ 0.01) graft survival (Fig. 1). Histologic examination of rejected heart grafts in control recipients showed characteristic features of acute rejection, with a heavy mononuclear cell infiltrate and widespread tissue destruction ( Fig. 2A). Nonrejecting hearts in sIL-15R␣-treated recipients examined at Ͼ100 days were still beating strongly and showed no histologic evidence of graft rejection (Fig. 2B).
To determine whether sIL-15R␣ treatment had induced donorspecific tolerance, three sIL-15R␣-treated CBA/Ca recipients with long surviving B10BR heart allografts (Ͼ100 days) were grafted with both donor strain (B10BR, H-2 k ) and third-party (AKR/J, H-2 k ) skin. Third-party AKR/J skin grafts were rapidly rejected (MST, 14 days) by CBA/Ca recipients, whereas donor strain B10BR grafts survived for Ͼ100 days, confirming the development of donor-specific immunologic tolerance.
Administration of sIL-15R␣ to CBA/Ca recipients bearing fully allogeneic BALB/c (H-2 d ) heart grafts produced only a modest prolongation of graft survival compared with MHC-mismatched controls (MST of 11 days vs MST of 7 days, respectively; Fig. 3a).
Because sIL-15R␣ alone was relatively ineffective in this fully MHC-disparate mouse strain combination, we chose in subsequent FIGURE 1. Effect of sIL-15R␣ on mHC-mismatched heart graft survival. CBA/Ca (H-2 k ) recipients of B10BR (H-2 k ) heart grafts were treated i.p. with 60 g of sIL-15R␣ or human serum albumin (HSA) daily for 10 days. Treatment with sIL-15R␣ induced indefinite survival in 70% of mHC-disparate recipients (p Ͻ 0.01 vs control group). Long-surviving recipients subsequently accepted B10BR donor strain skin grafts indefinitely (MST, Ͼ100 days; n ϭ 3), but rejected third-party, mHC-mismatched AKR/J (H-2 k ) skin grafts (MST, 14 days; n ϭ 3). experiments to combine it with an additional immunomodulatory agent in the form of a nondepleting anti-CD4 mAb. YTS 177.9 is a rat anti-mouse CD4 mAb of the IgG2a subclass (25) that after in vivo administration induces rapid and profound modulation of CD4 from residual CD4 T cells, resulting in transient interruption of the alloimmune response, but only limited prolongation of cardiac allograft survival (28). CBA/Ca recipients of BALB/c hearts received 1 mg of YTS 177.9 given i.p. at the time of heart transplantation together with a 10-day course of sIL-15R␣. Phenotypic analysis of spleen cells from transplanted animals showed that the anti-CD4 mAb treatment schedule led to a modest reduction in CD4 T cell numbers, and this reduction was most apparent in recipients given a combination of sIL-15R␣ and YTS 177.9 (Fig.  4). Cardiac allograft recipients given anti-CD4 mAb alone rejected their grafts with an MST of 15 days (Fig. 3b). In recipients treated with a combination of sIL-15R␣ and anti-CD4 mAb, cardiac allograft survival was markedly prolonged (MST of 60 days vs MST of 20 days for controls; Fig. 3b). Histologic examination of heart grafts excised on day 15 from control recipients given anti-CD4 mAb showed a heavy mononuclear cell infiltrate and widespread tissue destruction (Fig. 5A). In contrast, examination of hearts excised on day 15 from sIL-15R␣ plus anti-CD4-treated recipients showed a moderate mononuclear cell infiltrate, but minimal graft damage (Fig. 5B).

CTL activity
Spleen cells were obtained from CBA/Ca recipients of BALB/c hearts at 10 and 15 days after transplantation, and cytotoxic T cell activity against donor target cells was determined. CTL activity against BALB/c targets was higher on day 10 than on day 15 after transplantation, but at neither time point was there any significant difference between experimental groups (Fig. 6).

FIGURE 2.
Effect of sIL-15R␣ treatment on the histopathologic appearance of mHC-mismatched B10BR heart grafts. Heart grafts were formalinfixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. A, Rejecting heart removed on day 10 from control CBA/Ca recipient with heavy mononuclear cell infiltration and graft destruction. B, Nonrejecting heart graft removed at Ͼ100 days from sIL-15R␣-treated recipient. Long-surviving grafts contained minimal cellular infiltration and no evidence of chronic allograft rejection (photomicrographs, ϫ200 magnification).

Alloantibody levels
Anti-BALB/c Ab was readily detected by flow cytometry in serum obtained from anti-CD4-treated CBA/Ca recipients of BALB/c heart grafts and was tested using the F0313 rabbit anti-mouse Ig that detects both IgG and IgM. Additional treatment with sIL-15R␣ had no discernible effect on the Ab titers observed (Fig.  7). Three of five recipients treated with sIL15R␣ plus anti-CD4 plus showed low titers (Ͻ1/27) of the Th1-type IgG2a alloantibody and no detectable IgG3 or Th2-type IgG1 alloantibody. Control recipients given anti-CD4 mAb alone had marginally higher titers of circulating IgG alloantibody, with four of six animals having detectable IgG2a alloantibody with titers of up to 1/81, no detectable IgG3, and IgG1 titers of Ͻ1/27 (data not shown).

Lymphocyte proliferation and cytokine production
Spleen cells obtained on day 15 from CBA/Ca recipients of BALB/c heart allografts were assessed for their ability to proliferate in vitro and to produce IFN-␥ in response to irradiated allogeneic stimulators. Lymphocytes from recipients treated with a combination of sIL-15R␣ and anti-CD4 mAb showed a marked reduction in proliferation to donor-specific (BALB/c) stimulator cells compared with naive responder cells from normal CBA/Ca mice ( p Ͻ 0.05). The level of proliferation observed was signif-icantly lower ( p Ͻ 0.02) than that shown by cells obtained from control recipients treated with anti-CD4 mAb alone (Fig. 8a). Spleen cells from animals treated with a combination of sIL-15R␣  Generation of CTL in sIL-15R␣-treated heart allograft recipients. Splenocytes were prepared on day 10 (a) or on day 15 (b) posttransplant from MHC-mismatched heart graft recipients treated with sIL-15R␣ (or human serum albumin (HSA)) plus anti-CD4. Cells were cocultured with donor-strain BALB/c splenocytes to generate CTL, and their donor-directed lytic activity was compared with that of cocultured splenocytes taken on day 10 from an untreated CBA/Ca recipient rejecting a BALB/c heart. Results are the mean Ϯ SD of three to five mice per group.

FIGURE 7.
Alloantibody production in sIL-15R␣-treated heart allograft recipients. Serum was taken on day 15 post-transplant from BALB/c heart-grafted CBA/Ca recipients treated with sIL-15R␣ (or human serum albumin (HSA)) plus anti-CD4. Serum was diluted sequentially and incubated with donor strain splenocytes, and bound alloantibody was analyzed by flow cytometry using class-and isotype-specific mAbs. and anti-CD4 mAb also showed lower levels of spontaneous proliferation than cells obtained from control recipients given anti-CD4 mAb alone ( p Ͻ 0.02). Lymphocytes from recipients treated with sIL-15R␣ plus anti-CD4 mAb gave a lower proliferative response to third-party (C57BL/10) stimulators compared with naive control cells, but the residual levels of proliferation observed were not significantly different from those shown by spleen cells from control recipients given anti-CD4 mAb alone.
Spleen cells from CBA/Ca recipients treated with sIL-15R␣ and anti-CD4 mAb produced levels of IFN-␥ comparable to those produced by naive splenocytes from normal CBA/Ca mice and significantly less IFN-␥ after 96 and 120 h of coculture than splenocytes from control recipients treated with anti-CD4 mAb alone ( p Ͻ 0.05; Fig. 8b). IL-4 was not detected in any of the coculture supernatants examined in these experiments.

Discussion
IL-15 is a member of the 4␣-helix bundle family of cytokines, initially identified through its ability to stimulate T cell growth and differentiation, but with wide ranging effects on both lymphoid and nonlymphoid cells (8,9,29). Many cell types constitutively ex-press IL-15 mRNA, although production of IL-15 protein is subject to considerable post-transcriptional regulation (10,11). Recent studies have shown that expression of IL-15 mRNA is increased in some types of immune-mediated tissue injury, and there is considerable interest in the potential contribution of this cytokine to the pathogenesis of tissue destruction in these situations (13,(15)(16)(17)(18)(19)(20)(21)(22). Following organ transplantation, expression of IL-15 mRNA is readily detectable in the graft, and increased levels of expression have been shown to correlate with the presence of acute rejection, leading to speculation that IL-15 may contribute to the rejection process (18 -22).
The results of the present study provide clear in vivo evidence that IL-15 plays a role in the rejection of fully vascularized heart allografts. In the case of multiple mHC-mismatched cardiac allografts, the contribution of IL-15 to acute rejection appears critical, because administration of a sIL-15R␣ not only prevented the rejection of mHC-mismatched cardiac allografts, but also led to the development of operational donor-specific tolerance, as shown by rejection of third-party, but not donor strain, skin grafts. It might have been expected, in view of the considerable functional overlap between cytokines of the T cell growth factor family, that there would be sufficient redundancy in the cytokine network to allow rejection of mHC-mismatched heart grafts in the presence of IL-15 antagonism (1,30). IL-15, produced mainly by monocytes, and IL-2, produced by T cells, have considerable functional overlap, and both cytokines support T cell proliferation and differentiation, augment B cell and NK cell activities, and are chemoattractant for T cells. The extent of this functional overlap is explained in part by sharing of common receptor subunits (30). Both IL-15 and IL-2 use the IL-2R ␤-chain and common ␥-chain, but each has a distinct ␣-chain. Interestingly the IL-15R ␣-chain has such a high affinity for IL-15 that it binds with a K a of 10 11 M, which is 1000-fold higher than that for IL-2R␣ for IL-2 (12). In view of the functional similarities between IL-15 and IL-2, it is important to note that the sIL-15R␣ fragment used in the present study does not bind to IL-2 in vitro and does not inhibit IL-2-dependent CTLL proliferation (14). This argues strongly against cross-reactivity of the sIL-15R␣ with IL-2 as an explanation for our findings and points instead to the importance of IL-15 in mediating allograft rejection.
IL-15 has a number of biological functions not shared with IL-2, and these may arise because of the site of synthesis and regulation of IL-15 expression, the wide tissue distribution of the IL-15R, and the existence of another unique IL-15R (IL-15RX) (10,11). Production of IL-15 by dendritic cells may, for example, strongly influence T cell activation. In addition, IL-15 stimulates the proliferation of mast cells and bone marrow cells and plays a key role in the differentiation and development of NK cells (10,11). Neither the cell type and anatomic location of IL-15 production nor the nature of the target cells through which IL-15 exerts its effects on cardiac allograft rejection were directly addressed in the present study. Many cell types express IL-15 mRNA, but there is considerable post-transcriptional control of protein production as well as regulation at the level of intracellular trafficking of IL-15 protein (11). Activated monocytes and macrophages are one of the major sources of IL-15 protein and large numbers of macrophages expressing IL-15 can be identified in human cardiac allografts (19). APCs, recipient lymphocytes, monocytes, NK cells, and donor endothelial cells may all express receptors for IL-15 and therefore are potential target cells for IL-15 during an alloimmune response (11). Further studies are now needed to dissect in detail the role of IL-15 in allograft rejection.
The rejection response to MHC-mismatched heart allografts is considerably more powerful than that to grafts differing only in mHC, and therefore it is not surprising that administration of FIGURE 8. In vitro proliferative response and cytokine production in splenocytes from sIL-15R␣-treated heart allograft recipients. Splenic lymphocytes were harvested from naive CBA/Ca mice or from CBA/Ca recipients of BALB/c heart allografts on day 15 following transplant and treatment with sIL-15R␣ (or human serum albumin (HSA)) plus anti-CD4. Cells were cocultured with irradiated BALB/c (donor strain) or C57BL/10 (third-party) splenocytes for 96 h. Proliferation (a) was assessed by uptake of [ 3 H]thymidine for 6 h at the end of the culture period. Results are the mean Ϯ SD of three to five mice per group. ‫,ء‬ p Ͻ 0.02 vs anti-CD4 plus HSA controls; †, p Ͻ 0.05 vs naive controls; ‡, p Ͻ 0.02 vs naive controls. IFN-␥ levels (b) in supernatants were assayed by ELISA (mean Ϯ SD of three to five mice per group). ‫,ء‬ p Ͻ 0.05 vs HSA plus anti-CD4; †, p Ͻ 0.01 vs naive controls. sIL-15R␣ alone induced only a modest prolongation of graft survival beyond that of control recipients. Therefore, we chose in subsequent experiments to combine sIL-15R␣ treatment with a single dose of the nondepleting anti-CD4 mAb YTS 177.9. A combination of sIL-15R␣ and anti-CD4 mAb prolonged survival of fully MHC-mismatched heart grafts far more effectively than anti-CD4 mAb alone, thereby demonstrating the contribution of IL-15 to rejection of MHC disparate allografts. The choice of a nondepleting anti-CD4 mAb as an additional immunomodulatory agent for these studies arose because of our long-standing interest in anti-CD4 mAbs as agents for preventing allograft rejection. However, it seems reasonable to hypothesize that combinations of sIL-15R and various other immunosuppressive agents might also have a synergistic effect on allograft survival.
Analysis of the effector mechanisms responsible for allograft rejection reveals that delayed-type hypersensitivity, cytotoxic T cells and alloantibody-dependent effector mechanisms may all contribute to graft destruction. In the present study administration of sIL-15R␣ did not reduce the level of anti-donor CTL in the spleens of anti-CD4-treated heart graft recipients, nor did it further reduce the total serum Ig alloantibody levels below those seen in control animals given anti-CD4 mAb alone, although experimental data suggested that the anti-CD4-mediated reduction in IgG alloantibody was enhanced by additional sIL-15R␣. However, spleen cells from recipients given sIL-15R␣ showed a reduction in proliferation and produced less of the proinflammatory cytokine IFN-␥ in response to in vitro restimulation by donor alloantigen.
The finding that IL-15 contributes to allograft rejection is consistent with the emerging role of IL-15 in other types of T celldependent immunopathology, such as rheumatoid arthritis (13). Targeting the IL-15R with an antagonist IL-15 mutant/Fc␥2a fusion protein has recently been shown to attenuate the ability of mice to mount an Ag-specific delayed-type hypersensitivity response (31). Similarly, injection of sIL-15R␣ delays the development of collagen-induced arthritis and suppresses the accompanying collagen-specific T cell response (14). The recent availability of mice genetically deficient in IL-15 will further clarify the role of IL-15 in these and other immunopathologies (32) T cell growth factors play an essential role in allograft rejection, as highlighted by the recent demonstration that blockade of the common ␥-chain, a key signaling component shared by IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, prevents rejection in a murine islet transplant model (33). Further studies are now needed to define more clearly the contributions of individual growth factors in the graft rejection process, and in the case of IL-15 these will be facilitated by the availability of sIL-15R␣. In view of the increasing use of anti-CD25 mAb in clinical transplantation it will be of interest to combine sIL-15R␣ with anti-CD25 mAb in experimental studies. Combinations of sIL-15R␣ and calcineurin blockade will also be of particular interest in view of the resistance of in vitro IL-15 production to treatment with cyclosporin (34).
In conclusion, the results of this study provide in vivo evidence that IL-15 plays an important role in the rejection of a vascularized organ allograft and suggest that targeting IL-15/IL-15R interaction may, when combined with other chemical or biological immunosuppressive agents, be of therapeutic value in preventing allograft rejection.