BIOLOGY

IL-2–Targeted Therapy Ameliorates the Severity of Graft-versus-Host Disease: Ex Vivo Selective Depletion of Host-Reactive T Cells and In Vivo Therapy

Shai Yarkoni,1 Tatyana B. Prigozhina,2 Shimon Slavin,3 Nadir Askenasy4

T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2- cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical trans- 1 plants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3 ; 1 1 50%) and CD25 FoxP3 T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro 1 2 1 1 apoptosis in both CD4 CD25 and CD4 CD25 subsets of from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2- cas–pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of 1 CD25highFoxP3 T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2–targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploident- ical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transi- tion to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion. Biol Blood Marrow Transplant 18: 523-535 (2012) Ó 2012 American Society for Blood and Marrow Transplantation KEY WORDS: transplantation, Interleukin-2, Caspase-3, Fusion protein, Apoptosis, Regulatory T cell

INTRODUCTION due to propagation of cytokine cascades superposed on injury caused by pretransplantation conditioning, T cells have dual beneficial function in bone mar- resulting in severe tissue damage and often a fatal out- row transplantation: support of hematopoietic progen- come [1-3]. Most clinical approaches use extensive itor engraftment and . For effective depletion of T cells and sometimes B lymphocytes implementation of T cell-mediated activities, it is es- from donor inoculum [4-6], particularly when cells sential to reduce and/or abrogate the potentially lethal are collected from mobilized peripheral blood and in graft-versus-host disease (GVHD). The activation and haploidentical transplants. More recent approaches proliferation of effector T cells are self-perpetuating use selective depletion of host-reactive donor T cells ex vivo to eliminate clones endowed with GVHD potential. Repeated TCR engagement induces IL-2– From the 1GASR Biotechnology, Kfar-Saba, ; 2Department of dependent cell proliferation and concomitant sensiti- Bone Marrow Transplantation, Hadassah University Hospital, zation to apoptosis [7]. Based on the physiological , Israel; 3International Center for Cell Therapy and process of T cell activation, selective depletion has 4 , Tel Aviv, Israel; and Frankel Labora- been attained targeting Fas [8-10], the IL-2 receptor tory, Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel. [11-15], and proliferating cells [16]. Identification of Financial disclosure: See Acknowledgments on page 533. the high-affinity a-chain of the IL-2 receptor Correspondence and reprint requests: Shai Yarkoni, MD, PhD, (CD25) as a reliable target in alloreactive T cells [17] GASR Biotechnology, 14 Kaplan Street, Kfar-Saba 44395, led to implementation of immunomagnetic separation Israel (e-mail: [email protected]). of activated T cells based on phenotype [11,12], along Received April 2, 2011; accepted November 8, 2011 Ó 2012 American Society for Blood and Marrow Transplantation with a number of fusion proteins that target the IL-2 1083-8791/$36.00 receptor to deliver toxic moieties for selective killing doi:10.1016/j.bbmt.2011.11.016 of potential GVHD effectors. Anti-CD25 antibodies

523 524 S. Yarkoni et al. Biol Blood Marrow Transplant 18:523-535, 2012 conjugated to Pseudomonas exotoxin [12] or deglycosy- MATERIALS AND METHODS lated ricin toxin-A chain [12,14,15] have shown superior efficacy in preventing GVHD compared with Animal Preparation and Disease Model 1 diphtheria toxin fused to IL-2 in depleting CD25 BALB/c (H2Kd), C57BL/6J (B6, H2b, CD45.2), reactive T cells [12]. In addition to several promising B6.SJL-Ptprca Pepcb/BoyJ (H2Kb, CD45.1), and clinical trials using selective depletion of host-reactive C57BL/6-TgN(ACTbEGFP)1Osb (GFP, H2kb, T cells ex vivo by targeting the IL-2 receptor [18-21], CD45.2) mice were purchased from Jackson Laborato- anti-CD25 monoclonal antibodies have been applied ries (Bar Harbor, ME) and housed in a barrier facility. for GVHD prophylaxis in allogeneic and haploidentical F1 chimeras (H2Kb/d, CD45.2) were inbred from transplantations [22-24]. C57BL/6 and BALB/c mice. The Institutional Animal In view of the significant role of IL-2 in immune Care Committee of the Beilinson Campus approved all reconstitution after transplantation [25], with particu- procedures. Recipients were conditioned by sublethal lar efficacy in anticancer immunotherapy [26], thera- doses of total body irradiation (550-700 rad) using an peutic approaches to GVHD based on neutralization X-ray irradiator (RadSource 2000; Rad Source Tech- of the effects of IL-2 might impede transplantation nologies, Suwanee, GA) at a rate of 106 rad/min. Irra- outcomes and exacerbate GVHD [27]. Furthermore, diation was routinely performed 18-24 hours before approaches based on specific targeting of the IL-2 re- transplantation. (Of note, X-ray irradiation has a lower ceptor raise questions regarding the effects on regula- myeloablative dose and greater toxicity compared with tory T cell (Treg) homeostasis. Naturally occurring g irradiation.) For transplantation, cells suspended in Tregs are characterized by high-level expression of 0.2 mL of PBS were infused into the lateral tail vein. CD25 and the associated transcription factor X-linked forkhead/winged helix (FoxP3), making this subset ex- Agents tremely susceptible to IL-2– and IL-2– receptor- targeted therapy. On the one hand, adoptive transfer IL-2 conjugated to truncated diphtheria toxin (de- of donor Tregs protects from lethal GVHD without nileukin diftitox; Ontak) was purchased from Seragen impairing graft-versus-tumor (GVT) reactivity (San Diego, CA). IL2-cas is composed of full-frame [28-33]. Consistently, IL-2–dependent expansion of IL-2 conjugated head to tail to full-frame caspase-3 host Tregs suppresses GVHD while retaining GVT (without the leading procaspase segment) [38,39]. activity [34] and ameliorates host-versus-graft (HVG) The sequence was inserted into a PET-28 plasmid un- resistance [35]. On the other hand, adoptive transfer der control of the T7 promoter, and protein retrieved of naturally occurring Tregs negatively affects the en- from the inclusion bodies of Escherichia coli was purified graftment of hematopoietic progenitors [36]. It re- under denatured conditions and refolded to a biologi- mains to be determined whether, similar to different cally active molecule. subsets of na€ıve/effector T cells [37], selective protec- tion from GVHD can be dissociated from suppression Cell Preparation of engraftment based on phenotypic and/or functional Whole bone marrow cells (BMCs) were harvested characteristics of Tregs. from femurs and tibia in PBS (Beit Haemek, Israel) un- We have tested a chimeric molecule composed of der aseptic conditions [41]. T cell depletion was per- IL-2 as a targeting moiety and caspase-3 to activate formed immunomagnetically by incubating the cells the intrinsic apoptotic pathway (IL2-cas). This conju- for 45 minutes at 4C with saturating amounts of rat gate was demonstrated effective in blunting the patho- anti-mouse mAbs specific for CD4, CD5, and CD8 genesis of 2 model disorders characterized by relative (harvested from hybridoma cell lines). The antibody- resistance of pathogenic cells to apoptosis: inflamma- coated cells were washed twice with PBS containing tory bowel disease [38] and autoimmune insulitis 1% FCS (Biological Industries, Kibutz Haemek, [39]. Although targeting the IL-2 receptor has partic- Israel) and incubated with sheep anti-rat IgG conju- ular affinity for Treg depletion, an interesting immu- gated to M-450 magnetic beads (Dynal, Lake Success, nomodulatory feature of the chimeric protein is NY), with 4 beads per cell. T cell–depeleted (TCD) 1 1 a marked elevation in CD4 FoxP3 T cells, a hetero- BMCs were collected by exposure to a magnetic field, geneous population that includes precursors of both and the efficiency of separation was reassessed by flow effector and regulatory subsets [40]. In this study, cytometry using FITC-labeled mAbs against these IL2-cas was used in 2 GVHD models: posttransplanta- markers (eBioscience, San Diego, CA and BD Phar- tion therapy and prevention by pretransplantation mingen, Erembodegem, Belgium). To achieve a higher ex vivo deletion of host-sensitized donor lymphocytes. degree of purity (.95%), the immunomagnetic sepa- In both models, IL2-cas proved effective in preventing ration was repeated in some cases. lethal GVHD, and, moreover, the particular mode of Spleens and lymph nodes were harvested and action of this chimeric protein resulted in expansion gently minced on a 40-mM nylon mesh in HBSS of suppressor subsets. (Beit Haemek) to prepare single-cell suspensions Biol Blood Marrow Transplant 18:523-535, 2012 IL-2–Targeted Therapy in GVHD 525

[38,39]. The cells were aspirated with an 18G needle to Measurements were performed with a Vantage SE obtain single-cell suspensions. For flow cytometry, the flow cytometer (BD, Franklin Lakes, NJ). Positive red cells were removed by ammonium chloride lysis staining was determined on a log scale, normalized for 4 minutes at room temperature. The reaction was with control cells stained with isotype control anti- arrested with excess ice-cold solution, and cells were bodies. washed. For proliferation assays, the lymphocytes were centrifuged over 1.5 mL of separa- In Vitro Apoptosis tion media (Cedarlane, Ontario, Canada). Cell death In this procedure, 2 Â 106 cells/mL were incubated was assayed by the trypan blue exclusion test to show in DMEM supplemented with 2 mM L-glutamine, . 90% viability in all samples. 1 mM sodium pyruvate, 13.6 mM folic acid, 270 mM L-asparagine, 548 mM L-arginine HCL, 10 mM GVHD Model HEPES, 50 mM2b-mercaptoethanol, 100 mg/mL F1 chimeras (H2Kb/d, CD45.2) were sublethally streptomycin, 100 U/mL penicillin, and 5% heat- irradiated and transplanted after 18 hours (day 0) inactivated FBS (MLR medium) [44]. All ingredients with semiallogeneic BMCs (H2KbCD45.1) and sple- were purchased from Beit Haemek and Sigma- 1 nocytes (H2KbGFP ). A calibration curve for lethal Aldrich. Apoptotic challenge was applied by the addi- GVHD was calculated to determine the number of tion of 2 mg/mL of IL2-cas fusion protein. donor haploidentical splenocytes required to induce lethal GVHD under our experimental conditions. Dis- Proliferation Assay ease severity was scored using weight loss and a semi- Cells were plated in plastic dishes (5 Â 107), and af- quantitative clinical 0-4 scale including posture (ie, ter 45 minutes, the nonadherent lymphocytes were hyperkeratosis of the foot pads), diffuse erythema (par- collected and washed [38,39]. The lymphocytes were ticularly of the ear) or dermatitis (including hair loss), incubated at room temperature for 7 minutes with diarrhea, and activity [42,43]. At the experimental 10 mM 5-(and 6-)-carboxyfluorescein diacetate endpoint, ear edge biopsy specimens and livers succinimidyl ester (CFSE; Molecular Probes, were harvested and fixed in 4% paraformaldehyde. Eugene, OR), followed by arresting of labeling by the Cryosections were stained with H & E and inspected addition of 50% FCS and washing with PBS. Stained using a Zeiss Axioplan 2 microscope (Carl Zeiss, cells were cultured at a density of 106/mL in MLR  Oberkochen, Germany). Histological scoring was done medium in a humidified 5% CO2 atmosphere at 37 C using the following scale: 0, no infiltration; 1, scarce for 3 days, then stimulated with 5 mM concanavalin A infiltrates; 2, patchy infiltration; 3, diffuse infiltration; (ConA, Sigma-Aldrich). All proliferation assays were 4, deterioration of tissue infrastructure [43]. performed in triplicate and related to unstimulated cells. CFSE dilution was analyzed by flow cytometry Flow Cytometry with gating on the live lymphocytes, and proliferation Cells were labeled by incubation for 45 minutes at was quantified using ModFit software (Verity Software 4C with the appropriate antibodies conjugated to House, Topsham, ME). FITC, PE, allophycocyanin, and peridinin chlorophyll Statistical Analysis a-protein (BD Pharmingen): CD4 (clone RM 4-5), CD8 (clone 53-6.7), and CD25 (clone BC96) [41]. Data are presented as mean 6 standard deviation FoxP3 was measured after permeabilization and intra- for each experimental protocol. Differences between cellular staining with a PE-labeled antibody (Foxp3 the experimental results were estimated with a post staining buffer set NRRF-30; eBioscience) [38,39]. hoc Scheffe t test, and survival curves were assessed Antibodies were purchased from BD Pharmingen the using Mantel-Cox test, with a threshold signifi- and eBioscience. Donor chimerism in syngeneic cance of P \ .05. Statistical processing of survival transplants was determined from the percentage of was performed using BMDP software. donor and host peripheral blood lymphocytes using monoclonal antibodies against minor antigens CD45.1 (clone A20; eBioscience) and CD45.2 (clone RESULTS 104; eBioscience), respectively. Donor chimerism in allogeneic transplants was determined using PE-anti- IL-2–Targeted Therapy Protects Against GVHD H2Kb (clone CTKb; Caltag, Burlingame, CA) and In the first set of experiments, we assessed the ther- FITC anti-H2Kd mAb (clone SF-1.1; BD Pharmin- apeutic efficacy of IL2-cas in a haploidentical model gen). Cell death and apoptosis were determined in cells (H2Kb/H2Kb/d) of lethal GVHD induced by infu- incubated with 5 mg/mL of 7-aminoactinomycin-D (7- sion of 107 BMCs and 3 Â 107 parent lymphocytes AAD; Sigma-Aldrich, St. Louis, MO) and Annexin-V into sublethally irradiated (700 rad total body irradia- (IQ Products, Groningen, the Netherlands). tion [TBI]) F1 recipients (Figure 1A). The recipients 526 S. Yarkoni et al. Biol Blood Marrow Transplant 18:523-535, 2012

Figure 1. IL2-cas ameliorates the course of GVHD in vivo. (A) A haploidentical murine model of GVHD was established by transplantation of 107 BMCs and 3 Â 107 splenocytes (day 0) into sublethally irradiated (700 rad) F1 recipients (H2Kb/H2Kb/d). IL2-cas was administered at daily doses of 1 mg/g for 3 weeks and then 2 mg/g at 2-day intervals for 4 weeks, starting on day 0 (protocol 1A) or day 16 (protocol 1B). (B) Impact of early (day 0) therapy with IL2-cas (n 5 11) and 2 mg/g anti-CD25 antibodies (n 5 10) and late IL2-cas therapy (day 16; n 5 11) compared with untreated mice (GVHD; n 5 12). Biol Blood Marrow Transplant 18:523-535, 2012 IL-2–Targeted Therapy in GVHD 527 displayed signs of severe GVHD, with a mean survival [29,30]. To evaluate whether IL2-cas augments hema- time of 17.3 6 2 days, which was insignificantly atten- topoietic cell engraftment, it was compared with the uated by daily administration of 2 mg/g of anti-CD25 immunosuppressive activity of cyclophosphamide antibodies (mean survival, 21 6 6.5 days; Figure 1B). [42,48] in a modified model adopted from clinical In contrast, initiation of therapy with 1 mg/g of IL2- studies [23,24]. Sublethally irradiated BALB/c mice cas at the time of transplantation resulted in superior (550 rad TBI) were engrafted with 5 Â 106 TCD survival (protocol 1A; P 5 .017 versus GVHD con- BMCs from B6 donors (H2Kb/H2Kd), and 2 doses trol), which was less pronounced when IL2-cas therapy of adjuvant were administered on days was initiated in mice with established disease (protocol 0 and 12(Figure 2A). Administration of 2 doses of 1B; P 5 .038 versus GVHD control). Consistent vari- 2 mg/g IL2-cas on days 0 and 12 had an insignificant ations in body weight (Figure 1C) indicate that pro- effect on donor chimerism, whereas cyclophospha- phylactic therapy with IL2-cas is more effective than mide promoted engraftment to full donor chimerism treatment of established disease. by 3 weeks posttransplantation (Figure 2B). All mice One of the consequences of severe GVHD is sup- survived the transplantation procedure, and both con- pression of donor cell engraftment and decreased levels trol and IL2-cas–treated mice progressed to full donor of chimerism [45,46]. Mice that survived for 7 weeks chimerism at 16 weeks posttransplantation. These showed reduced levels of donor cells in peripheral results indicate that IL2-cas neither improves nor blood (P \ .01; Figure 1D), followed by an increase impairs hematopoietic cell engraftment and durable at 11 weeks posttransplantation. Of note, there were chimerism, suggesting that the decreased chimerism no significant differences in the donor and host mye- at 7 weeks posttransplantation (Figure 1D) was related loid/lymphoid ratio in peripheral blood of mice among mainly to GVHD, not to IL2-cas therapy. the various experimental groups. Further analysis of We next assessed the activity of IL2-cas on GVHD the peripheral lymphoid organs at the end of the ex- effectors in vivo, in the absence of bone marrow- periment revealed insignificant variations in the num- derived host-tolerant lymphoid progeny that compete bers of T cells in the spleen (Figure 1E), which was with the grafted cells [43]. Administration of 2 doses of quantitatively reconstituted at this time point (94 6 4 mg/g IL2-cas (protocol 2) to sublethally irradiated 11 Â 106 cells, compared with 104 6 106 cells in na€ıve (650 rad) F1 hosts grafted with 5 Â 106 haploidentical 1 1 mice). In contrast, the CD4 /CD8 ratio was inverted splenocytes (H2Kb/H2Kb/d) improved survival from in the lymph nodes of long-term survivors in both 38% in untreated mice (n 5 8) to 70% (n 5 10) treatment protocols (Figure 1E). Another common (Figure 2C), and also improved posttransplantion re- feature of all treated mice was a significant increase in covery of body weight (P \ .001; Figure 2D). Taken 1 2 1 the number of CD4 CD25 FoxP3 T cells in the together, these data indicate that the grafted GVHD spleen (P \ .005 versus na€ıve mice), and marked frac- effectors are not outcompeted by host-tolerant prog- 1 1 tional enrichment in CD4 FoxP3 T cells, including eny of the bone marrow graft in the process of 2 1 both CD25 and CD25 subsets in the mesenteric lymphopenia-induced proliferation. The remarkable lymph nodes (P \ .001 versus na€ıve mice) effect on the histological GVHD score (Figure 2E) (Figure 1F). Similar profiles of increased fractions of links IL2-cas with reduced lymphocytic infiltration 1 1 1 CD4 CD25 FoxP3 T cells in peripheral lymphoid in the affected organs, indicating direct targeting of organs were also observed in a GVHD model of alloge- the GVHD effectors in vivo. neic bone marrow transplantation (H2Kb/H2Kd). To examine the role of the enriched fractions of 2 1 CD25 FoxP3 T cells observed after IL2-cas therapy (Figure 1F), we assessed the suppressive function of this Mechanism of IL-2–Targeted Therapy subset in an in vitro proliferation assay. Na€ıve mice Protection from lethal GVHD by IL2-cas therapy given daily doses of 2 mg/g IL2-cas for 10 days demon- 1 2 1 and the extensive variations in immune profiles ques- strated elevated fractions of CD4 CD25 FoxP3 T 1 tioned the mechanism of action of this fusion protein. cells (Figure 2F). The FoxP3 subsets were sorted IL2-cas might enhance engraftment, generating a large from the spleens and mesenteric lymph nodes of these pool of T cells tolerant to the host that compete with mice in reference to CD25 expression. Coincubation of 2 1 the GVHD effectors [43,47], target the GVHD both sorted subsets of CD25 FoxP3 and 1 1 effectors [12-15], or amplify suppressor subsets CD25 FoxP3 T cells suppressed the proliferation = The data summarize 2 experiments, each including all experimental groups. (C) Percent changes in body weight of the corresponding experimental groups. (D) Donor chimerism in peripheral blood at 7 weeks (end of therapy) and 11.5 weeks (experimental endpoint) posttransplantation of BMCs and T cells in recipients of early (n 5 5) and late (n 5 4) therapy. Chimerism was compared with that in mice grafted with BMCs alone (n 5 6). 1 (E and F) Immune profiles of survivors of early (n 5 5) and late (n 5 2) therapy compared with na€ıve F1 mice (n 5 6). (E) Distribution of CD4 1 1 and CD8 T cells in the spleen and mesenteric lymph nodes presented as percentage of total cells. (F) Total numbers of CD4 T cells expressing 1 CD25 and FoxP3 in the spleen (left), representative plots of CD25 and FoxP3 expression in gated CD4 subsets (middle), and fractional CD25 and 1 FoxP3 expression in CD4 T cells in the mesenteric lymph nodes (right). 528 S. Yarkoni et al. Biol Blood Marrow Transplant 18:523-535, 2012 Biol Blood Marrow Transplant 18:523-535, 2012 IL-2–Targeted Therapy in GVHD 529

1 2 of immunomagnetically isolated CD4 CD25 T cells differences in reference to the depleting agent: domi- 2 1 (at a 1:1 ratio; Figure 2F). Thus, along with direct tar- nant CD25 FoxP3 subset in recipients of medium- geting of GVHD effectors, IL2-cas has an additional incubated splenocytes (P \ .01) and dominant 1 1 immunomodulatory activity related to increased frac- CD25 FoxP3 subset after IL2-cas depletion (P \ 2 1 1 1 tions of CD25 FoxP3 and CD25 FoxP3 suppres- .005) (Figure 3F). Further analysis revealed that 1 1 sor subsets. CD4 FoxP3 T cells commonly expressed low levels of CD25 after Ontak pretreatment (P \ .005; Figure 3F), and only recipients of IL2-cas–pretreated Pretransplantation Depletion of Activated Cells splenocytes were characterized by high CD25 levels Protects Against Lethal GVHD (P \ .005; Figure 3G). Taken together, these data im- Various approaches to selective depletion of host- ply that selective depletion of presensitized splenocytes reactive T cells ex vivo have targeted the IL-2 receptor improves survival and induces sustained functional dif- as an activation marker [11-20], including an IL-2 con- ferences in response to stimulation in vivo, such as 1 jugate of diphtheria toxin (Ontak) [12,19]. Reduced blunted peripheral migration of CD4 T cells. activity of GVHD effectors in vivo suggests that the Compared with depletion with Ontak, depletion with fusion proteins might be particularly effective in IL2-cas results in increased fractions of cells with depleting host-presensitized GVHD effectors. We re- Treg phenotypes in the survivors. capitulated ex vivo depletion of host-sensitized T cells [18-21] from donors immunized against the host (Figure 3A) to attain physiological immune stimula- Differential Apoptosis of T Cells Ex Vivo 1 tion [49]. Adoptive transfer of 1.5 Â 106 H2Kd-sensi- The consistent enrichment in FoxP3 T cells in- tized H2Kb splenocytes into sublethally irradiated duced by IL2-cas in both models of ex vivo depletion (650 rad) haploidentical recipients (H2Kb/d) resulted of GVHD effectors and in vivo treatment of estab- in acute GVHD and death of 40% of the mice within lished disease might be caused by survival of this subset 1 week, whereas ex vivo exposure to IL2-cas and Ontak and/or increased proliferation rates. In the first stage decreased mortality to 10% (Figure 3B). Recipients of of our analysis, we assessed the apoptotic activity of T cells in all experimental groups displayed a similar IL2-cas during extended incubation for 3 days, which 1 fractional distribution of CD4 T cells in peripheral is generally required for ex vivo depletion of sensitized lymphoid organs, and only recipients of cells pre- lymphocytes [50]. Unlike in shorter incubation periods 1 2 treated with Ontak exhibited lower fractional expres- [38], apoptosis of CD4 CD25 /low cells induced by sion of CD25 and FoxP3 (Figure 3C). Thus, both extended exposure to IL2-cas exceeded that of 1 IL-2 conjugates were effective in ameliorating CD4 CD25high cells (Figure 4A), consistent with in- GVHD, with varying consequences on immune recon- ternalization of the fusion protein by other compo- stitution. nents of the IL-2 receptor [51]. A lipopolysaccharide (LPS) challenge applied on In the second stage, our analysis focused on FoxP3 day 17 polarized the survival curves of the experimen- expression, to test whether cells positive for this tal groups; uniform lethality was triggered in recipients transcription factor are less sensitive to apoptosis in- of cells incubated in medium, whereas 50%-60% of re- duced by IL2-cas [38]. The fusion protein had no 1 cipients of cells preincubated with IL2-cas and Ontak significant effect on apoptosis of the CD4 CD25low survived the challenge (P \ .001; Figure 3D). Assess- subset; however, it induced apoptosis preferentially 1 ment of the immune phenotypes on day 12 after in CD4 CD25high cells lacking FoxP3 expression 1 LPS administration revealed several characteristics (P \ .005; Figure 4B). Superior survival of CD4 1 associated with acute immune stimulation. LPS- CD25highFoxP3 T cells appears to be the basis for mediated stimulation induced an apparent redistribu- selective enrichment of this subset in treated mice 1 tion of CD4 T cells from the spleen the lymph nodes, (Figure 1F) and recipients of cells pretreated with which was blunted in recipients of selectively depleted IL2-cas (Figure 3F-G). 1 splenocytes (Figure 3E). A reactive increase in CD4 In the third stage, we evaluated whether the fusion subsets expressing FoxP3 in the mesenteric lymph protein induces proliferation, given that its compo- nodes of survivors showed marked phenotypic nents IL-2 and caspase-3 are both known to promote = Figure 2. Mechanisms of IL2-cas amelioration of GVHD. (A) Sublethally irradiated mice (550-650 rad) were grafted with BMCs or splenocytes, and 2 doses of adjuvant therapy were administered on days 0 and 12. (B) Sublethally irradiated (550 rad) BALB/c mice were engrafted with 5 Â 106 allogeneic (H2Kb/H2Kd) TCD BMCs (control, n 5 7) and treated with 2 doses of 100 mg/g cyclophosphamide (n 5 10) or 2 mg/g of IL2-cas (n 5 14) were ad- ministered on days 0 and 12. Hematopoietic chimerism was measured in peripheral blood at 3 weeks posttransplantation. (C-E) Sublethally irradiated (650 rad) F1 recipients were engrafted with 5 Â 106 haploidentical (H2Kb/H2Kb/d) splenocytes (GVHD; n 5 8) and treated with 2 doses of 4 mg/g of IL2-cas (n 5 10). Survival (C) and changes in body weight (D) in the posttransplantation period, and histology (E) on day 21 in liver and ear pinna sections 2 1 (representative images). (F) B6 mice treated for 10 days with daily doses of 2 mg/g IL2-cas displayed a significant increase in CD25 FoxP3 subset (left). 2 1 1 1 2 The CD25 FoxP3 and CD25 FoxP3 subsets were sorted and coincubated with CFSE-loaded CD25 responders at a 1:1 ratio (right). Proliferation assessed from CFSE dilution was quantified with the ModFit software. Data are representative of 3 independent experiments. 530 S. Yarkoni et al. Biol Blood Marrow Transplant 18:523-535, 2012

Figure 3. Ex vivo exposure of sensitized splenocytes to IL2-cas protects against lethal GVHD. (A) H2Kb mice were immunized with 7 Â 106 H2Kd splenocytes, and after 1 week spleens were harvested and incubated for 72 hours in medium and with 0.3 mg/mL of Ontak and 2 mg/mL of IL2-cas. Then 1.5 Â 106 viable splenocytes from the immunized donors were infused into sublethally irradiated (650 rad) F1 (imH2Kb/H2Kb/d) recipients. (B) Survival of recipients of presensitized splenocytes incubated in medium (GVHD; n 5 17), with Ontak (n 5 12), and with IL2-cas (n 5 14). (C) 1 Immunophenotype of CD4 T cells in the mesenteric lymph nodes of mice in the corresponding experimental groups (n 5 4). (D) Survival of mice after i.v. administration of 10 mg of LPS on day 17 after adoptive transfer of cells preincubated in medium (GVHD; n 5 15), with Ontak (n 5 13), and with IL2-cas (n 5 14). *Mice sacrificed for analysis. (E-G) Immunophenotypes of the spleen and mesenteric lymph nodes at 2 days after LPS administration to survivors of infusion of cells incubated in medium (n 5 3), with Ontak (n 5 5), and with IL2-cas (n 5 6). (F) Fractional CD25 and 1 1 1 FoxP3 expression within the CD4 subset. (G) Fractions of CD4 FoxP3 T cells with low and high CD25 expression.

1 cell cycling [52-54]. The fusion protein synergized tor and suppressor cells, but is less toxic to CD4 T with the mitogenic activity of ConA, resulting in cells expressing FoxP3 and high levels of CD25. faster proliferation rates of splenocytes in vitro (Figure 4C). All assays presented here for splenocytes DISCUSSION yielded similar results in cultures of lymph node cells. Taken together, these data indicate that IL2-cas in- IL2-cas is emerging as an alternative therapeutic duces both apoptosis and proliferation in na€ıve/effec- approach to GVHD, both for pretransplantation Biol Blood Marrow Transplant 18:523-535, 2012 IL-2–Targeted Therapy in GVHD 531

Figure 4. Differential apoptosis of T cell subsets ex vivo. C57BL/6 mice (H2Kb) were immunized with 7 Â 106 BALB/c (H2Kd) splenocytes. After 1 week, splenocytes were incubated for 3 days in medium (control) and with 2 mg/mL of IL2-cas for determination of apoptosis from Annexin-V uptake. Data 1 represent 5 individual incubations. Apoptosis was determined by gating on CD4 T cells in reference to CD25 expression (A) and in reference to FoxP3 expression (B). The left panel represents fractional distribution of FoxP3 in the viable CD25low and CD25high subsets. (C) Proliferation index as determined from CFSE dilution in splenocytes incubated for 3 days in the presence of 5 mg/mL of ConA in medium and with 2 mg/mL of IL2-cas. Data are representative of 4 independent incubations. CFSE dilution was quantified with the ModFit simulation software compared with the parent signal (right peak). selective depletion of host-sensitized donor T cells and attributes have been shown to ameliorate the course as posttransplantation therapy. The chimeric molecule of GVHD both by apoptotic targeting of effectors targets cells expressing the IL-2 receptor, a ubiquitous and by triggering the expansion of suppressor subsets. characteristic of activated T cells, and uses a terminal IL2-cas receptor has been successfully imple- caspase to simulate the physiological process of mented in the treatment of disorders characterized apoptosis. This agent does not induce significant lym- by relative insensitivity of effector cells to apoptosis, phopenia under steady-state and disease conditions such as autoimmune insulitis and colitis [38,39]. [38], and does not perturb the kinetics of hematopoi- Direct internalization of caspase-3 by the IL-2 recep- etic cell engraftment and the consequent immune tor bypasses and overcomes pathways of pathogenic reconstitution. However, its immunomodulatory cell resistance to apoptosis and negative regulation, 532 S. Yarkoni et al. Biol Blood Marrow Transplant 18:523-535, 2012 such as reduced caspase-3 activity in colitogenic cells T cell subsets. Reciprocal to suppression of graft func- [55-57]. Similarly, in NOD mice, direct elimination tion and immune reconstitution by GVHD [45,46], of diabetogenic cells protects against exacerbation of prompt repopulation of the lymphoid compartment destructive insulitis by cyclophosphamide, an agent has the capacity to counteract GVHD, and early that selectively depletes Treg subsets [39]. In principle, reinstitution of regulatory mechanisms prevents a therapeutic approach that targets the IL-2 receptor uncontrolled expansion of pathogenic effector clones as a marker of T cell activation has the capacity to sup- [68] and induces a state of tolerance [69]. press both GVHD and HVG resistance mediated by Given the nature of the chimeric protein that tar- residual host immune cells after nonmyeloablative gets of the IL-2 receptor, naturally occurring Tregs conditioning. We have demonstrated that the fusion characterized by high-level CD25 expression are protein reduces the severity of established GVHD expected to be prime targets of deletion, leading to ex- with superior prophylactic efficacy compared with acerbation of the immune reaction. Accordingly, apo- anti-CD25 antibodies, possibly because antibodies ptosis is effectively induced in significant fractions of 1 1 do not always deplete, but sometimes only inhibit, ef- CD25 cells, including naturally occurring FoxP3 fector cell function [58]. Furthermore, the fusion pro- Tregs. Although host Tregs are dominant in the early tein was safely administered over extended periods, posttransplantation period [70], the stable levels of do- and its immunomodulatory consequences persisted nor chimerism indicate that HVG was not amplified after discontinuation of therapy. The fusion protein by eliminating suppressor subsets to mediate rejection. is also effective in ex vivo selective depletion of host- Reciprocally, GVH reactivity was not exacerbated by 1 presensitized GVHD effectors in stringent haploi- CD25 T cell depletion, but rather was suppressed. dentical transplantation models. Both phenomena are explained based on the dual Direct cytotoxic activity of IL2-cas in na€ıve/effec- mode of action of the chimeric protein, which results 2 tor (CD25 /low) T cells depends on internalization of in expansion and relative dominance of Treg pheno- the receptor–ligand complex, which occurs primarily types in vitro and in vivo. In essence, both constituents through the g chain [51]. This component of the of the chimeric protein are involved in the prolifera- IL-2 receptor is shared by a number of receptors, in- tion of effector and suppressor T cells [52-54]. Our cluding IL-4, IL-7, IL-9, and IL-15 [59]. Deletion of data suggest that cells with the highest capacity for host-reactive cells in vitro requires 2-3 days of culture IL-2 uptake are induced to proliferate in vitro, the for the sufficient sensitization of lymphocytes [50] and likely mechanism of the dominant expansion of the for accumulation of intracellular concentrations of the Treg phenotype in vivo. However, preferential enrich- toxic moiety that causes dose-dependent apoptosis ment in CD25high T cells also might originate from the [39]. Even if the deletion process is not completed IL-2–mediated signaling through heterodimers of the ex vivo, caspase-loaded cells will become apoptotic IL-2 receptor that include CD25, without internaliza- and further contribute to Treg expansion and protec- tion of caspase-3 [71]. These differences between IL-2 tion against GVHD [60]. Direct internalization of signaling versus internalization of the fusion protein toxic moieties, including caspase-3 and diphtheria underlie selective induction of apoptosis in effector 2 toxin, also prevents the accumulation of primed/ cells (including CD25 subsets) and expansion of sup- memory T cells [61], which are relatively insensitive pressor cells. In contrast, IL2-cas has proven effective to apoptosis because of low levels of caspase-3 [62]. in the killing of malignant cell lines expressing the IL-2 1 Both CD8 [63] and effector/memory CD4 subsets receptor without inducing proliferation (S. Yarkoni, [64] are less potent mediators of acute GVHD than unpublished data). Current experimental evidence in- na€ıve T cells; however, these cells are resistant to dicates a retained antileukemia effect of T cells in the receptor-mediated apoptotic signaling and contribute presence of IL2-cas in vitro and preserved GVT activ- to chronic GVHD [65,66]. Preventing the transition ity of selectively depleted T cells in vivo. to memory phenotypes ex vivo and in vivo explains Robust expansion under lymphopenic conditions the continued suppression of GVH activity after generates dominant fractions of 50%-60% 1 1 discontinuation of IL2-cas therapy [67]. CD4 FoxP3 T cells in the lymph nodes of long- The lower cytotoxic activity of caspase-3 com- term survivors. Expansion of this subset is mediated pared with diphtheria toxin [38] is a significant advan- by several mechanisms. First, IL2-cas spares 1 1 tage in the transplantation setting. On the one hand, CD4 FoxP3 T cells during short [38] and extended IL2-cas does not cause lymphopenia and thus does periods of incubation. Second, the heterogeneous 2 1 not promote engraftment, and on the other hand, CD25 FoxP3 subset is endowed with suppressive administration of therapeutic doses does not impair activity, as demonstrated by inhibition of T cell prolif- hematopoietic cell engraftment. This feature is signif- eration in vitro, and includes Treg progenitors that ex- icant in the reinstitution of immune homeostasis of the pand in the periphery without obligatory thymic reconstituted lymphoid compartment; Ontak blunted priming [40]. Third, TCR engagement is essential to and IL2-cas promoted the recovery of regulatory initiate Treg proliferation but is not required to Biol Blood Marrow Transplant 18:523-535, 2012 IL-2–Targeted Therapy in GVHD 533 maintain Tregs’ suppressive function, whereas IL-2 is Shai Yarkoni serves as the Chief Executive Officer of mandatory to sustain suppression and may promote GASR Biotechnology and has significant equity in cycling [71-73]. Sustained engagement of host this company. The authors have no conflicts of interest antigens provides a strong proliferative stimulus, and to disclose. consequently Treg clones with antigen-specificity have an expansion advantage [74]. Fourth, cytokine environments created by radiation-induced lympho- REFERENCES penia and LPS-mediated activation foster homeostatic and spontaneous expansion [68]. Rebound of 1. Shlomchik WD. Graft-versus-host disease. Nat Rev Immunol. 1 1 1 2007;7:340-352. CD4 CD25 FoxP3 Tregs accounting for 35% of 1 2. 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