Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-γ and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

The ␣4␤7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the ␤7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive ␤7 ؊/؊ T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of ␤7 ؊/؊ donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versustumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of ␤7 ؊/؊ donor T cells. In conclusion,

Interleukin-15 (IL-15 IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) is an important therapy for a variety of malignant and nonmalignant diseases. Immune deficiency after allogeneic HSCT is one of the major causes of early and late posttransplantation morbidity and mortality. Strategies to enhance posttransplantation immune reconstitution could decrease the incidence of fatal infectious complications, enhance graft-versus-tumor activity, and significantly increase the overall survival after allogeneic HSCT. Interleukin-7 (IL-7) and IL-15 are critical cytokines in lymphocyte development and homeostasis. Administration of IL-7, which plays an important role in the homeostasis of B and T lymphocytes, can improve immune reconstitution after syngeneic and allogeneic HSCT. [1][2][3][4][5] The effect on immune reconstitution of administration of IL-15 to HSCT recipients has not been determined.IL-15 is a pleiotropic cytokine that plays a role in natural killer (NK) development, innate and adaptive immune homeostasis, and the activation and homing of immune effector cells including NK cells, NK T-cells, and cytotoxic T lymphocytes. 6 It binds to a receptor complex that consists of IL-15R␣, IL-2R␤, and the ␥-common chain. 7-9 IL-15 is produced by antigen-presenting cells (APCs), 10 bone marrow stroma, 7-9 thymic epithelium, 11 and epithelial cells in the kidney, 8 [7][8][9]17 and has a proliferative and survival effect on CD8 ϩ memory T-cells. 18 IL-15 is required for optimal proliferation of CD8 ϩ T-cells, and IL-15 (Ϫ/Ϫ) mice cannot support homeostatic proliferation of CD8 ϩ memory T-cells. 19,20 Recently, IL-15 has been shown to have a role in the survival and antigen-independent expansion in vitro of naive CD8 ϩ T-cells. 21,22 Until now, few studies have evaluated the effects of IL-15 administration in vivo. Sprent and colleagues showed that the proliferation of CD8 ϩ memory T-cells increased after a single dose of IL-15 in normal mice. However, IL-15 did not affect CD4 ϩ memory T-cells, which could be due to their low expression of IL-2R␤. 18 Administration of IL-15 to mice also enhanced the antitumor activity after syngeneic bone marrow transplantation (BMT) and antigen-specific primary CD8 ϩ T-cell responses following vaccination with peptide-pulsed dendritic cells. 23,24 In this study, we used murine models to investigate the effects of IL-15 administration to recipients of an allogeneic BMT on immune reconstitution, graft-versus-host disease (GVHD), and graft-versus-leukemia (GVL) activity. (Germantown, NY). Mice used in BMT experiments were between 8 and 10 weeks or 10 months (CBA/J) of age. BMT protocols were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. Bone marrow (BM) cells were removed aseptically from femurs and tibias. Donor BM was T-cell depleted (TCD) by incubation with anti-Thy-1.2 for 30 minutes at 4°C followed by incubation with Low-TOX-M rabbit complement (Cedarlane Laboratories, Hornby, ON, Canada) for 40 minutes at 37°C...

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT).Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8 ؉ T cells is relevant for the control of CD8 ؉ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2 ؊/؊ ) CD8 ؉ T cells developed less damage of gut and liver than recipients of wild-type CD8 ؉ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8 ؉ T-cell target organ infiltration revealed that CCR2 ؊/؊ CD8 ؉ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-␥ production and cytotoxicity of CCR2 ؊/؊ CD8 ؉ T cells were intact. Interestingly, the graft-versustumor effect mediated by CCR2 ؊/؊ CD8 ؉ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity. ( IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapy for a variety of malignant and nonmalignant disorders of the hematopoietic system and for certain solid tumors. 1 A major complication limiting the success and wider application of allogeneic HSCT is the occurrence of acute graft-versus-host disease (GVHD), which is a rapidly progressive illness with immunosuppression, cachexia and specific target organ damage of liver, intestines, skin, and lung. 2 Donor-derived alloreactive T cells play a major role in the pathogenesis of GVHD and depletion of T cells from the donor cell inoculum remains the most effective approach to prevent the development of disease. 3 However, alloreactive donor T cells also display graft-versus-tumor (GVT) activity, which is increasingly being recognized as an important component of the overall antitumor effect of an allogeneic HSCT. 4 Recent murine bone marrow transplantation studies suggest that specifically interfering with T-cell migration represents an attractive therapeutic approach toward the goal of amelioration of GVHD without reducing GVT activity. 5,6 It is known that 3 families of migration molecules (selectins, chemokines, integrins, and their respective ligands and receptors) control T-cell migration in homeostasis and inflammation 7 and members of all 3 families have been identified as important players during GVHD. 5 CC chemokine receptor 2 (CCR2) and its main ligand chemokine ligand 2 (CCL2) are among the chemokine receptor-ligand pairs that control leukocyte migration during inflammatory processes. 8,9 CCL2 (originally termed monocyte chemoattractant protein-1 [MCP-1]) belongs to the family of inflammatory CC chemokines and was one...

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494 Several randomised pediatric trials have demonstrated that intensification of Asparaginase (ASP) treatment in ALL can contribute to improved outcome. In adult ALL few data are availabe and optimal ASP preparation, schedule and intensity with respect to efficacy and tolerability have to be defined. The optimisation of ASP treatment is therefore an essential aim of the GMALL. Treatment: Induction treatment of the ongoing study 07/2003 consists of dexamethasone, vincristine, daunorubicine, pegylated asparaginase (PEG-ASP) (phase I), mercaptopurine, cyclophosphamide and cytarabine (phase II) as previously described (Brueggemann et al, Blood 2006: 107; 1116). During the study the dose for PEG-ASP was increased from 1000 to 2000 U/m2 in induction and from 500 to 2000 U/m2 in consolidation (combined with HDMTX and MP) for pts aged between 15 and 55 years. 1 application for high risk and 7 applications for standard risk (SR) were scheduled during the first year and the aim was improvement of overall survival (OS) and remission duration (RD). Patients: From more than 100 centers in Germany 1226 pts with a median age of 35 (15-55) yrs were evaluable. 826 pts were treated with 1000 U/m2 (cohort 1) and 400 pts with 2000 U/m2 (cohort 2) and both groups were comparable regarding major entry criteria. The analysis was restricted to pts who received one of the scheduled PEG-ASP doses during induction. Outcome: CR rate after induction was 91% vs 91% in cohort 1 and 2 resp., with comparable rates for early death (4% vs 5%) and failure (5% vs 4%). Data on molecular response (MRD below 10−4) after induction are available in a subset and showed no difference between both cohorts after induction (79% vs 82%). OS after 3 years was improved in cohort 2 (60% vs 67%; p>.05). The positive effect was specifically evident in SR patients (N=407 vs 190) with respect to OS (68% vs 80%; p=.02) and RD (61% vs 74%; p=.02). It was demonstrated in younger pts (15-45 yrs) (71% vs 82%; p=.02) and older pt (45-55 yrs) (56% vs 74%; p>.05). Excellent results were achieved in young adults (15-25 years) with respect to OS (77% vs 86%; p>.05) and RD (60% vs 78%; p>.05). Toxicity: The analysis of toxicity was focused on grade III-IV events during induction with potential correlation to PEG-ASP (764/382 pts in cohort 1/cohort 2)). Incidences are as follows: GOT or GPT (30%/30%), bilirubine (10%/16%), thrombosis (5%/5%) and hypersensitivity (<1%/<1%). In a subset of pts additional AEs were assessed as amylase (5%/13%), lipase (23%/15%) and glucose (10%/12%). Significantly less toxicity was observed during consolidation cycles. Bilirubine °III/IV occurred median 16d after PEG-ASP during phase II of induction. In univariate analysis it was correlated to dose (10% vs 16%; p=.004), age <> 45 yrs (11% vs 17%; p=.005), BMI <> 30 (12% vs 18%; p=.04) and rituximab application (11% vs 18%; p=.009). Hepatomegaly, infections or imatinib application had no significant effect. In multivariate analysis dose and age remained independent significant prognostic factors. Bilirubine increase during induction was associated with treatment delays and inferior prognosis. Conclusions: This is the largest cohort of adult ALL treated with PEG-ASP. Due to prolonged activity fewer applications are required which is a pre-requisite for realisation of ASP intensification in the context of an intensive multidrug chemotherapy for adult ALL. Although CR rate and molecular CR were not significantly improved PEG-ASP intensification was associated with an improved OS and RD. The improvement was specifically evident in SR pts treated with up to 7 doses of PEG-ASP. Overall intensified PEG-ASP was feasible. The rate of grade III-IV bilirubine elevation increased after dose escalation and led to treatment delays in individual pts which were prognostically relevant. It would be an important goal to identify parameters to predict severe ASP related toxicity. Further intensification of ASP by additional applications would be of interest. Supported by Deutsche Krebshilfe 70–2657-Ho2 and partly BMBF 01GI 9971 and Medac GmbH. Disclosures: Goekbuget: Medac: Consultancy, Research Funding, Speakers Bureau. Hoelzer: Medac: Speakers Bureau.

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is IntroductionAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematologic malignancies and certain benign hematopoietic disorders. Apart from the antitumor effect of the conditioning regimen, graft versus leukemia (GVL) activity by alloreactive donor T cells plays an important role in the prevention of posttransplantation tumor relapse. However, alloreactive donor T cells are also responsible for graft versus host disease (GVHD), a major cause of posttransplantation morbidity and mortality. 1 Thus, a major challenge for allo-HSCT is to inhibit GVHD while maintaining the beneficial effects of T-cell-mediated GVL activity.T cells require 2 signals for an effective immune response. The first signal is generated by the interaction of the T-cell receptor (TCR) with a cognate peptide: major histocompatibility complex (MHC) on the surface of an antigen-presenting cell (APC). 2 T cells that are stimulated via the TCR alone fail to produce the appropriate cytokines, are unable to sustain proliferation, and will often undergo apoptosis. 3 The second signal is a costimulatory signal required to prevent apoptosis and induce full activation and differentiation into effector and memory T cells. 4 Because full T-cell activation requires 2 signals, blocking T-cell costimulatory molecules is a strategy utilized to induce tolerance in solid organ transplantation 5 and may be useful in allo-HSCT to inhibit GVHD. Costimulatory blockade has been studied in allo-HSCT models to induce immune deviation or suppression after transplantation. 6 Inducible costimulator (ICOS) is a relatively new member of B7 family involved in the costimulation of T cells. [7][8][9][10] This costimulatory family also includes CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1). 3 The most studied receptor is CD28, which is constitutively expressed on naive T cells and regulates the threshold for T-cell activation and interleukin-2 (IL-2) production. 11 ICOS is expressed upon T-cell activation and contributes to the production of effector cytokines including interferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), IL-4, IL-5, and IL-10 but may have little impact on IL-2 production. 12-14 ICOS shares 28% amino acid identity and 39% similarity with CD28 7 and contains several highly conserved motifs that are found in CD28. 15 ICOS expression is restricted to activated T cells present in lymphoid organs such as spleen, lymph nodes, and Peyer patches. 7,10,16 Mice genetically deficient for ICOS have normal thymi and normal peripheral T-cell development. 12-14 However, because ICOS signaling induces CD40L expression on T cells, ICOS Ϫ/Ϫ mice are deficient in germinal center formation and immunoglobulin (Ig) class switching. This phenotype can be rescued by in vivo administration of an antibody that stimulates CD40 signaling. 13 The ICOS ligand (ICOSL) is constitutively expressed on unstimulated B cells, sple...