Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is a widely performed therapy for many hematologic malignancies. Effective use of this powerful therapeutic modality has been hindered by its significant toxicity, acute graft-versus-host disease (GVHD). However, the graft-versus-leukemia (GVL) effect provided by allogeneic BMT represents a very potent form of immune therapy against malignancy.1 Unfortunately, GVHD and GVL are tightly linked as demonstrated by the inverse correlation between leukemia relapse rates and the severity of GVHD.2-4 Prevention of GVHD by T-cell depletion or nonspecific immune suppression is associated with increased risk for leukemia relapse after allogeneic BMT.5-7 Thus, approaches that can reduce the toxicity of GVHD while preserving the beneficial GVL effects are necessary to better harness this very effective therapeutic modality against hematologic malignancies.

Interleukin 18 (IL-18) is a recently discovered cytokine that is structurally related to IL-1 but functionally similar to IL-12.8 It is produced by a wide variety of cells such as macrophages, dendritic cells, keratinocytes, T cells, osteoblasts, and Kupffer cells and is a potent inducer of Th1 response in concert with IL-12.8,9 We have recently demonstrated that when administered early after allogeneic BMT it can significantly attenuate acute GVHD.10 Because IL-18 enhances the cytolytic activity of T cells and has been shown to provide antitumor immunity,8,11,12 we examined whether IL-18 could preserve the GVL effect conferred by donor T cells after allogeneic BMT.

Female C57BL/6 (B6 Ly5.2, H-2^b, CD45.1⁺), B6D2F1 (H-2^b/d , CD45.2⁺) and perforin-deficient C57BL/6 (H-2^b, pfp^−/−) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed and fed as previously described.10 

All the methods were done as described previously.10,13-16 

Mice underwent transplantation according to a standard protocol described previously.13 Briefly, on day 0 mice received 13 Gy total body irradiation (TBI; ¹³⁷Cs source) split into 2 doses. After TBI, 5 × 10⁶ T-cell–depleted (TCD) bone marrow (BM) and 2 × 10⁶ nylon wool purified (NWP) splenic donor T cells were injected intravenously into recipients. Survival was monitored daily; recipient body weight and GVHD clinical score were measured weekly.10,13 

Recombinant murine IL-18 (RD, Flanders, NJ) was reconstituted per the manufacturer's recommendations in sterile distilled H₂O and injected intraperitoneally (10 μg/mouse/d) from day −2 to +2. Mice from the control groups received phosphate-buffered saline (PBS).

Briefly, 2000 P815 (H-2^d) cells (a mastocytoma derived from a DBA/2 mouse) were injected intravenously into B6D2F1 recipients on day 0 along with the BM transplant inoculum. Survival was monitored daily. P815-induced leukemic death was defined by the occurrence of either macroscopic tumor nodules in liver or spleen or hindleg paralysis.15 GVHD death was defined by the absence of leukemia and the presence of clinical signs of GVHD.13,14 Animals surviving beyond day 50 after BMT were killed and the liver and spleen were harvested for flow cytometric evaluation (has sensitivity of 0.5%15).

Fluorescein isothiocyanate–conjugated monoclonal antibodies to mouse CD45.1⁺, CD45.2⁺, and H-2^d+, phycoerythrin-conjugated CD4⁺, CD8⁺, and H-2^b+ were purchased from Pharmingen (San Diego, CA). Cells were analyzed by 2-color flow cytometry on a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).13,15 

Briefly, splenocytes were harvested from animals 14 days after transplantation and 3 spleens combined from each group. Donor (CD45.1⁺CD3⁺) T cells in the spleens were determined and normalized between groups. Donor T-cell engraftment was 94% ± 4% in the controls and 86% ± 8% in IL-18 recipients (P = NS) on day +14 after BMT. These cells were then plated in 96-well flat-bottomed plates (Falcon, Lincoln Park, NJ) at a concentration of 2 × 10⁵ T cells (CD45.1⁺CD3⁺)/well with 2 × 10⁵irradiated (2000 rad) splenocytes harvested from naive B6D2F1 (allogeneic) or B6 (syngeneic) animals. At 48 hours, supernatants were collected for cytokine analysis and the cultures were pulsed with³[H]-thymidine (1 μCi/well; 0.037 MBq) and proliferation was determined 24 hours later on a 1205 Betaplate reader (Packard, Downers Grove, IL).

Antibodies used in the interferon γ (IFN-γ) and IL-2 assays were purchased from Pharmingen. All assays were performed according to the manufacturer's protocol in a 1:5 dilution. Plates were read at 450 nm using a microplate reader (Bio-Rad Labs, Hercules, CA). Recombinant murine IFN-γ and IL-2 (Pharmingen) were used as standards for enzyme-linked immunosorbent assays (ELISAs). Samples and standards were run in duplicate and the sensitivity of the assays was 0.063 U/mL for IFN, and less than 0.13 U/mL for IL-2.

Briefly, splenocytes were removed from B6D2F1 recipients 14 days after BMT, and 3 spleens were combined from each group. Donor CD8⁺ cells in each group were determined and the counts were normalized. They were added at varying effector to target ratios and incubated for 4 hours with either allogeneic P815 (H-2^d) or syngeneic EL-4 (H-2^b) targets (2 × 10⁶ cells), labeled with 100 μCi (3.7 MBq) ⁵¹Cr. ⁵¹Cr activity in supernatants was determined in an autogamma counter (Packard Instrument, Meriden, CT). The percentage of specific lysis was calculated as follows: 100 × (sample count − background count)/(maximal count − background count).15 

Survival curves were plotted using Kaplan-Meier estimates. The Mantel-Cox log-rank test was used to analyze survival data. Statistical significance was set at P < .05.

We have previously demonstrated that administration of IL-18 early in the time course of allogeneic BMT attenuated early in vivo donor T-cell proliferation by enhancing activation induced Fas-mediated apoptosis and resulted in reduced GVHD.10 We now determined whether the effects of IL-18 on acute GVHD severity were also associated with a decrease in donor responses to host antigens after BMT. B6D2F1 mice received 13 Gy of TBI followed by infusion of BM and T cells from either allogeneic B6 Ly5.2 or syngeneic donors as described in “Study design.” Donor splenic T cells were harvested from allogeneic BM transplant recipients on day +14 and then restimulated in vitro with B6D2F1 stimulators in standard mixed lymphocyte reaction (MLR) cultures. As shown in Figure1A, splenic T cells from both IL-18–treated or control allogeneic recipients displayed similar proliferative responses to host antigens. There was also no difference in secretion of either IFN-γ or IL-2 between T cells from IL-18–treated or control mice (Figure 1B,C). We next tested the cytotoxic capability of donor CD8⁺ T cells harvested from recipient spleens on day +14 against host type P815 (H2^d) tumor targets, which was also equivalent between the 2 groups (Figure1D). Taken together with our previous study,10 these data demonstrate that administration of IL-18 to BM transplant recipients attenuates donor T-cell proliferation by enhancing Fas-mediated increasing activation induced cell death early after BMT but does not alter donor T-cell responses to host antigens measured 2 weeks after BMT.

The preservation host-specific responses in donor cells suggested that GVL effects might be preserved. We next determined the ability of IL-18 treatment to promote leukemia-free survival after allogeneic BMT in a well-established mouse GVL model described in “Study design.”15,16 As expected all recipients of syngeneic BM transplants receiving P815 tumor cells died with evidence of massive hepatosplenomegaly. Although IL-18 by itself has been shown to possess antitumor effects,8,17 all IL-18–treated syngeneic mice that received P815 cells also died from leukemia by day 18, thus ruling out any direct antitumor effect of IL-18 in this system (Figure2A). All allogeneic BM transplant recipients treated with control died by day 40. By contrast, 50% of IL-18–treated allogeneic animals survived the entire observation period (P < .03). Clinical GVHD scores were also more severe in controls than in IL-18–treated animals (5.6 ± 0.7 versus 3.5 ± 0.4, P < .05) consistent with our previous observation.10 In each case, allogeneic recipients effectively rejected their leukemia with no evidence of tumor at autopsy. In additional experiments with a lower dose (1 × 10⁶) of allogeneic T cells, 25% of the control compared to 70% of IL-18–treated recipients survived (P < .04) and had less clinical GVHD. No animals showed morphologic evidence of leukemia at the end of the observation period, demonstrating that IL-18 preserved GVL effect at both higher and lower T-cell doses. We tested for minimal residual leukemia by killing all the surviving animals on day 50 and analyzing peripheral blood and splenocytes by FACS. No cells with the P815 phenotype (CD45.2⁺, H2^d+/H2^b−) were detected. Residual leukemia was evaluated by injecting 10 × 10⁶ TCD splenocytes from these mice into lethally irradiated secondary F1 hosts. No secondary hosts (0 of 6) died from leukemia, confirming the eradication of P815 cells by adoptive transfer (ie, < 2000 tumor cells in 10 × 10⁶ cells, sensitivity of 0.02%). These results indicate that IL-18 enhances leukemia-free survival after allogeneic BMT by attenuating acute GVHD and maintaining GVL activity.

Earlier studies have demonstrated that CD8⁺ T cells are more potent effectors of GVL than are CD4⁺ cells in several models.15,18,19 CD8⁺ cells mediate cytotoxicity, which is critical for GVL effect, either by the perforin-granzyme granule exocytosis pathway or the Fas pathway.20 Several recent studies have demonstrated that perforin rather than Fas ligand (FasL) is important for GVL activity.19-21 We therefore evaluated the cytotoxic mechanisms responsible for GVL by using perforin (pfp)–deficient B6 (H2^b) mice as donors and undergoing transplantation as above. Consistent with previous studies, most of the recipients of allogeneic perforin-deficient splenic T cells treated with control diluent died from GVHD (Figure 2B).22,23 In contrast, IL-18 treatment conferred a significant survival advantage to allogeneic BM transplant recipients (88% versus 12%; Figure 2B). When F1 recipients were injected with P815 at the time of BMT all the recipients died from leukemia regardless of treatment if they received perforin-deficient donor T cells (Figure 2C). These data indicate that the perforin-dependent cytotoxicity of donors is critical for preservation of GVL activity in this model.

Interleukin 18 did not impede engraftment of donor marrow in this parent into F1 model.10 It should be noted, however, that the effects of IL-18 in other models where rejection might be mediated by T cells as well as natural killer cells have not yet been evaluated. Immune reconstitution after IL-18 treatment was also better than controls in this model (data not shown) and IL-18 has been shown to be critical for defense against cytomegalovirus24,25 andAspergillus.26,27 Thus IL-18 treatment may reduce the risk of opportunistic infections after allogeneic BMT, but detailed evaluation of this effect of IL-18 on posttransplantation immune responses against infections remains to be determined.

In conclusion, when taken together with our previous study,10 these data demonstrate that brief administration of IL-18 to recipients early after allogeneic BMT may represent a novel strategy to attenuate acute GVHD without compromising GVL activity.