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Ferrara JLM: An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation. I. The roles of minor H antigens and endotoxin.
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Prepublished online July 5, 2002; doi:10.1182/blood-2002-04-1252

Interleukin-18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation Pavan Reddy, Takanori Teshima, Gerhard Hildebrandt, Ulrich Duffner, Yoshinobu Maeda, Kenneth R Cooke and James L M Ferrara

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Interleukin-18 Preserves a Perforin Dependent Graft-versus-Leukemia Effect After Allogeneic Bone Marrow Transplantation Pavan Reddy1, Takanori Teshima1, Gerhard Hildebrandt1, Yoshinobu Maeda1, Kenneth R. Cooke1, and James L. M. Ferrara1* 1

Blood and Marrow Transplantation Program, Division of Hematology-Oncology, Departments of Internal Medicine and Pediatrics, University of Michigan Comprehensive Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0942. Scientific Heading: Brief Report (Transplantation) Running Title: IL-18 preserves GVL Words (Abstract/ Text): 178 / 1578

Research Grant: Supported by NIH grants CA 49542 to JLMF and 2KIZHD28820 to KRC P.R. is the recipient of an ASCO Young Investigator Award K.R.C is a scholar of NMDP Amy Strelzer- Manasevit Scholarship Program, a Fellow of the Robert Wood Johnson Minority Medical Faculty Development Program

*To whom correspondence should be addressed: University of Michigan Cancer Center 1500 East Medical Center Drive Ann Arbor, MI 48109-0942 Phone; (734) 615-1340 Fax; (734) 647-9271 Email; [email protected]

1 Copyright 2002 American Society of Hematology

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Abstract We have recently shown that early administration of IL-18 after bone marrow transplantation (BMT) attenuates acute graft-versus-host disease (GVHD) in a lethally irradiated Parent into F1 (B6 B6D2F1) BMT model 1. In this study, we investigated whether IL-18 can maintain graft-versus-leukemia (GVL) effect in this context. B6D2F1 mice were transplanted with T cell–depleted (TCD) bone marrow (BM) and splenic T b/d

cells from either syngeneic (H2

b

) or allogeneic B6 (H2 ) donors. Recipient mice were

treated with rmIL-18 or the control diluent. Initial studies demonstrated that IL-18 treatment did not affect the proliferative responses or the cytolytic effector functions of T cells after BMT. In subsequent experiments, animals also received host-type P815 mastocytoma cells at the time of BMT. All syngeneic BMT recipients died from leukemia by day 18. The allogeneic BMT recipients effectively rejected their leukemia regardless of treatment and IL-18 significantly reduced GVHD-related mortality. Examination of the cytotoxic mechanisms with perforin-deficient donor T cells demonstrated that perforin is critical for the GVL effect. Taken together these data demonstrate that IL-18 can attenuate acute GVHD without impairing the in vitro cytolytic function or the in vivo GVL activity after allogeneic BMT.

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Introduction

Allogeneic bone marrow transplantation (BMT) is a widely performed therapy for many hematological 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 effect provided by allogeneic BMT represents a very potent form of immune therapy against malignancy 2. Unfortunately, GVHD and GVL are tightly linked as demonstrated by the inverse correlation between leukemia relapse rates and the severity of GVHD 3-5. Prevention of GVHD by T cell depletion or nonspecific immune suppression is associated with increased risk in leukemia relapse after allogeneic BMT 6-8. 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 hematological malignancies. Interleukin 18 (IL-18) is a recently discovered cytokine that is structurally related to IL-1 but functionally similar to IL-12 9. 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 9,10. We have recently demonstrated that when administered early after allogeneic BMT it can significantly attenuate acute GVHD 1. Because IL-18 enhances the cytolytic activity of T cells and has been shown to provide antitumor immunity 9,11,12 we examined whether IL-18 could preserve the GVL effect conferred by donor T cells after allogeneic BMT.

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Methods b + b/d + Mice: Female C57BL/6 (B6 Ly5.2, H-2 , CD45.1 ), B6D2F1 (H-2 , CD45.2 ) and b perforin-deficient C57BL/6 (H-2 , pfp–/–) mice were purchased from The Jackson

Laboratory (Bar Harbor, Maine, USA). Mice were housed and fed as previously described (1). All the methods were done as described previously (1, 13 16). Bone Marrow Transplantation: Mice were transplanted according to a standard protocol described previously 13. Briefly, on day 0 mice received 13 Gy total body 6

irradiation (TBI; 137Cs source) split into 2 doses. After TBI, 5 x 10 TCD BM and 2 x 6

10 NWP splenic donor T cells were injected intravenously into recipients. Survival was monitored daily, recipient body weight and GVHD clinical score were measured weekly (1, 13). IL-18 treatment: Recombinant murine IL-18 (RDInc., Flanders, NJ, USA) was reconstituted per manufacturers recommendations in sterile DI H2O and injected intraperitoneally (10 µg/mouse/day) from day -2 to +2. Mice from the control groups received PBS. d Leukemia induction: Briefly, 2000 P815 (H-2 ) cells (a mastocytoma derived from a

DBA/2 mouse) were injected intravenously into B6D2F1 recipients on day 0 along with the BMT inoculum. Survival was monitored daily. P815-induced leukemic death was defined by the occurrence of either macroscopic tumor nodules in liver and/or spleen, or

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hindleg paralysis (15). GVHD death was defined by the absence of leukemia and the presence of clinical signs of GVHD 14 13. Animals surviving beyond day 50 of BMT were killed and the liver and spleen were harvested for flow cytometric evaluation (has sensitivity of 0.5% 15). +

FACS analysis: FITC-conjugated mAbs to mouse CD45.1 , CD45.2+, and H-2

d+ , PE-

+ + b+ conjugated CD4 , CD8 and H-2 were purchased from PharMingen San Diego, USA.

Cells were analyzed by two color flow cytometry on a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, USA) 13,15. Cell cultures: Briefly, splenocytes were harvested from animals 14 days after transplant + + and three 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-flat bottomed plates (Falcon, Lincoln Park, NJ) at a 5 + + 5 concentration of 2 × 10 T cells (CD45.1 CD3 )/well with 2 x 10 irradiated (2,000

Rad) splenocytes harvested from naive B6D2F1 (allogeneic) or B6 (syngeneic) animals. At 48 h, supernatants were collected for cytokine analysis and the cultures were pulsed with 3[H] thymidine (1 µCi per well) and proliferation was determined 24 h later on a 1205 Betaplate reader (Wallac, Finland). Cytokine ELISAs: Antibodies used in the IFN- and IL-2 assays were purchased from PharMingen (San Diego, CA). 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

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Labs, Hercules, CA). Recombinant murine IFN- and IL-2 (PharMingen) were used as standards for ELISA assays. Samples and standards were run in duplicate and the sensitivity of the assays was 0.063 U/ml for IFN, and < 0.13 U/ml for IL-2. 51 Cr release assays. 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 was determined and the counts were normalized. They were added at varying d effector/target ratios and incubated for 4 hours with either allogeneic P815 (H-2 ) or b 6 51 syngeneic EL-4 (H-2 ) targets (2 x 10 cells), labeled with 100 µCi of 51Cr. Cr

activity in supernatants was determined in an auto-gamma counter (Packard Instrument Company, Meriden, Connecticut, USA). The percentage of specific lysis was calculated as follows: 100 x (sample count – background count)/(maximal count – background count)

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.

Statistical analysis. 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 < 0.05.

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Results and Discussion

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 1. 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 13Gy of TBI followed by infusion of BM and T cells from either allogeneic B6 Ly5.2 or syngeneic donors as described in Methods. Donor splenic T cells were harvested from allogeneic BMT recipients on day +14 and then restimulated in vitro with B6D2F1 stimulators in standard MLR cultures. As shown in Fig. 1a, splenic T cells from both IL18 or control treated allogeneic recipients displayed similar proliferative responses to host antigens. There was also no difference in secretion of either IFN- or IL-2 between T cells from IL-18 or control treated mice (Fig. 1b and 1c). We next tested the cytotoxic capability of donor CD8+ T cells harvested from recipient spleens on day +14 against d host type P815 (H-2 ) tumor targets, which was also equivalent between the two groups

(Fig. 1d). Taken together with our previous study 1, these data demonstrate that administration of IL-18 to BMT recipients attenuates donor T cell proliferation by enhancing Fas mediated increasing AICD early after BMT but does not alter donor T cells responses to host antigens measured two 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

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survival after allogeneic BMT in a well-established mouse GVL model described in Methods 15 16. As expected all recipients of syngeneic BMT receiving P815 tumor cells died with evidence of massive hepatosplenomegaly. Although IL-18 by itself has been shown to possess anti-tumor effects 9,17, all IL-18 treated syngeneic mice that received P815 cells also died from leukemia by day 18, thus ruling out any direct anti-tumor effect of IL-18 in this system (Fig 2a). All allogeneic BMT recipients treated with control died by day 40. By contrast, 50% of IL-18 treated allogeneic animals survived the entire observation period (P < 0.03). Clinical GVHD scores were also more severe in controls than in IL-18–treated animals (5.6± 0.7 v 3.5± 0.4, P < 0.05), consistent with our previous observation 1. In each case, allogeneic recipients effectively rejected their leukemia with no evidence of tumor at postmortem. In additional experiments with a 6 lower dose (1 X 10 ) of allogeneic T cells, 25% of the control compared to 70% of IL-18

treated recipients survived (P < 0.04) and had less clinical GVHD. No animals showed morphological evidence of leukemia at the end of 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 + d+ blood and splenocytes by FACS. No cells with the P815 phenotype (CD45.2 , H-2 /H2b– 6 ) were detected. Residual leukemia was evaluated by injecting 10 x 10 TCD

splenocytes from these mice into lethally irradiated secondary F1 hosts. No secondary hosts (0/6) died from leukemia, confirming the eradication of P815 cells by adoptive 6 transfer (i.e. less than 2000 tumor cells in 10 x10 cells, sensitivity of 0.02%). These

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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 FasL is important for GVL activity 19-21. We therefore evaluated the cytotoxic b mechanisms responsible for GVL by utilizing perforin (pfp) deficient B6 (H2 ) mice as

donors and transplanted as above. Consistent with previous studies, most of the recipients of allogeneic perforin deficient splenic T cells treated with control diluent died from GVHD (Fig. 2b) 22,23. In contrast, IL-18 treatment conferred a significant survival advantage to allogeneic BMT recipients (88% v 12%, Fig 2b). When F1 recipients were injected with P815 at the time of BMT all the BMT recipients died from leukemia regardless of treatment if they were transplanted with perforin deficient donor T cells (Fig. 2c). These data indicate that the perforin dependent cytotoxicity of donors is critical for preservation of GVL activity in this model.

IL-18 did not impede engraftment of donor marrow in this parent into F1 model 1. 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 NK cells, has not yet been evaluated.

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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 CMV 24,25and Aspergillus 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 post transplant immune responses against infections remains to be determined.

In conclusion, when taken together with our previous study 1 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.

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References 1.

Reddy P, Teshima T, Kukuruga M, Ordemann R, Liu C, Lowler K,

Ferrara JL: Interleukin-18 Regulates Acute Graft-Versus-Host Disease by Enhancing Fas-mediated Donor T Cell Apoptosis. J Exp Med 194:1433, 2001 2.

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Rimm AA, Ringden O, Rozman C, Speck B, Truitt RL, Zwaan FE, Bortin MM: Graftverus-leukemia reactions after bone marrow transplantation. Blood 75:555, 1990 4.

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understanding and

using the alloimmune response to treat haematological malignancies. Brit. J. Haematol. 93:754, 1996 5.

Truitt RL, Johnson BD, McCabe C, Weiler MB: Graft versus leukemia, in

Ferrara JLM, Deeg HJ, Burakoff SJ (eds): Graft-vs-Host Disease (ed 2nd). New York, Marcel Dekker, Inc., 1997, p 385 6.

Butturini A, Gale RP: T cell depletion in bone marrow transplantation for

leukemia: current results and future directions. Bone Marrow Transplant 3:185, 1988 7.

Butturini A, Gale RP: Graft versus leukemia. Immunol Res 11:24, 1992

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15:248, 1997 9.

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regulates both th1 and th2 responses. Annu Rev Immunol 19:423, 2001

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response. J Exp Med 194:F11, 2001 11.

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MT, Tahara H: IFN-gamma-inducing factor/IL-18 administration mediates IFN-gammaand IL-12-independent antitumor effects. J Immunol 160:1742, 1998 12.

Heuer JG, Tucker-McClung C, Hock RA: Neuroblastoma cells expressing

mature IL-18, but not proIL-18, induce a strong and immediate antitumor immune response. J Immunother 22:324, 1999 13.

Hill GR, Crawford JM, Cooke KJ, Brinson YS, Pan L, Ferrara JLM: Total

body irradiation and acute graft versus host disease. The role of gastrointestinal damage and inflammatory cytokines. Blood 90:3204, 1997 14.

Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J, Crawford JM,

Ferrara JLM: An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation. I. The roles of minor H antigens and endotoxin. Blood 88:3230, 1996 15.

Teshima T, Hill GR, Pan L, Brinson YS, van den Brink MRM, Cooke KR,

Ferrara JLM: IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation. J. Clin. Invest. 104:317, 1999 16.

Pan L, Teshima T, Hill GR, Bungard D, Brinson YS, Reddy VS, Cooke

KR, Ferrara JLM: G-CSF mobilizes allogeneic stem cell transplantation maintains GVL effects through a perforin dependent pathway while preventing GVHD. Blood , 1999 17.

Belardelli F, Ferrantini M: Cytokines as a link between innate and

adaptive antitumor immunity. Trends Immunol 23:201, 2002

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12 preserves the graft-versus-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-versus-host disease in mice. Blood 90:4651, 1997 19.

Schmaltz C, Alpdogan O, Horndasch KJ, Muriglan SJ, Kappel BJ,

Teshima T, Ferrara JL, Burakoff SJ, van den Brink MR: Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graftversus-leukemia effect. Blood 97:2886, 2001 20.

Russell JH, Ley TJ: Lymphocyte-mediated cytotoxicity. Annu Rev

Immunol 20:323, 2002 21.

Tsukada N, Kobata T, Aizawa Y, Yagita H, Okumura K: Graft-versus-

leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood 93:2738, 1999 22.

Blazar BR, Taylor PA, Vallera DA: CD4+ and CD8+ T cells each can

utilize a perforin-dependent pathway to mediate lethal graft-versus-host disease in major histocompatibility complex-disparate recipients. Transplantation 64:571, 1997 23.

Baker MB, Altman NH, Podack ER, Levy RB: The role of cell-mediated

cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice. J Exp Med 183:2645, 1996 24.

Pien GC, Satoskar AR, Takeda K, Akira S, Biron CA: Cutting edge:

selective IL-18 requirements for induction of compartmental IFN-gamma responses during viral infection. J Immunol 165:4787, 2000

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Peterson PA, Fung-Leung WP: Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J Exp Med 189:1129, 1999 26.

Brieland JK, Jackson C, Menzel F, Loebenberg D, Cacciapuoti A, Halpern

J, Hurst S, Muchamuel T, Debets R, Kastelein R, Churakova T, Abrams J, Hare R, O'Garra A: Cytokine networking in lungs of immunocompetent mice in response to inhaled Aspergillus fumigatus. Infect Immun 69:1554, 2001 27.

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asthma in a murine model; putative involvement of Toll-like receptor-2. Inflamm Res 50:552, 2001

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Figure Legend

Figure 1: Donor T cell cytokine and cytolytic functions after BMT B6D2F1 animals were injected with control diluent ( , solid bar) or IL-18 ( , dotted bar) 6 from day –2 to +2, received 13 Gy TBI and were transplanted with 5 x 10 TCD BM and 6

2 x 10 splenic T cells from B6 Ly5.2 (CD45.1+) donor mice. Splenocytes from the recipients (n = 3/group) were harvested on day 14 after BMT, combined and normalized + + for donor T cells (CD45.1 and CD3 ); and restimulated in quadruplicate with irradiated

naive host (B6D2F1) splenocytes in MLR cultures. Supernatants were collected after 48 hours of culture and proliferation was determined by pulsing with 3H-thymidine (1 µCi/well) for an additional 20 hours. T cell proliferation (a), IFN- secretion (b), IL-2 production (c), all were similar (

v

, P = NS). Results from one of three similar

experiments are shown. (d) Splenocytes harvested from allogeneic animals on day 14 + post-BMT were pooled (n=3/group), and normalized for donor CD8 cells and used in a 51 Cr release assay. CTL activity against allogeneic P815 in control ( , filled triangle) and

IL-18 ( , filled circle) groups was similar; there was no significant lysis of syngeneic targets by either group, (IL-18, open square

; control, filled square, ).

Figure 2: IL-18 retains a perforin-dependent GVL effect (a) B6D2F1 mice were injected with IL-18 or diluent, given 13Gy TBI and transplanted with allogeneic or syngeneic BM and T cells as in Figure 1. All animals were also 15

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injected intravenously with 2000 P815 tumor cells on day 0. (Allogeneic: control treated, , n = 8 and IL-18 treated, O, n = 8) or (syngeneic: control treated, treated, , n = 8) donors. P < 0.03 for O,allo IL-18 v

, allo

, n = 8 and IL-18

control. Data from one of two

similar experiments are shown. (b) Lethally irradiated (13 Gy) B6D2F1 mice were transplanted with 5 x 106 TCD BM b 6 b –/– from wt B6 (H2 ) and 2 x 10 splenic T cells from allogeneic pfp B6 (H2 ) donors.

Survival in control (closed circles, , n = 8) and IL-18–treated (open circles, O, n=8) –/–

recipients of T cells from pfp

donors was significantly different.

P < 0.01, O v .

(c) B6D2F1 mice were transplanted as above and injected intravenously with two thousand P815 cells on day 0. All syngeneic ( , n=10), allogeneic control ( , n= 10), –/–

allogeneic IL-18 treated pfp

T cells (O, n= 10) and allogeneic IL-18 treated wt T cells

( , n= 10) survived. P = NS, O v ; and P < 0.04,

v O.

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Figure 1 Reddy et al

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