Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

Multiple myeloma (MM) has been suggested to be associated with different neoplasms. Of 589 consecutive patients with MM, 59 (10%) had different neoplasms: solid tumors in 78% and hematological neoplasms in 22%. Different neoplasms were separated into those emerging prior or synchronously (p/s; n = 41) versus subsequently after the MM (n = 18). The rate of different neoplasms at the time of MM diagnosis was estimated as 6.6%, and estimated different neoplasm rates at 2, 5, and 10 years were 7.8%, 10.3%, and 11.6%, respectively. Patients with MM with p/s different neoplasms showed a hazard ratio (HR) for impaired overall survival of 1.2 (95% CI 0.8-2.0), whereas in those with subsequent neoplasms the HR was 2.5 (95% CI 1.4-4.4). This demonstrates that (1) p/s are more frequent compared with subsequent different neoplasms, and (2) the prognosis is more impaired with subsequent different neoplasms. Age ≥60 years was a confounding covariable with a HR of 2.021 (95% CI 1.6-2.6).

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1336 Poster Board I-358 Conditioning prior allogeneic hematopoietic cell transplantation causes local tissue injury. Cellular molecules that are released upon cell stress, injury or cell death can act as potent danger associated molecular patterns (DAMP). Nucleotides like ATP are ideal candidates to serve as such indicators of cell damage, since they are present in the extracellular environment in the nanomolar range under physiological conditions but are released upon unphysiological cell death which causes activation of purinergic receptors. The role of purinergic signaling in alloantigen driven immune responses such as graft-versus-host disease (GvHD) is unclear. We found that ATP levels in the peritoneal fluid increased when GvHD was present in human and mice. Detection of extracellular ATP with a luciferase based in vivo detection system demonstrated early ATP release from the gastrointestinal tract. Blockade of multiple purinergic receptors on T cells or ATP neutralization during GvHD development reduced disease severity and proinflammatory cytokine production significantly. Gene expression analysis of different P2R in GvHD target organs identified the P2X7(R)receptor to be increasingly expressed. P2X7R−/− recipients developed less severe GvHD as compared to wildtype mice. This was paralleled by the induction of STAT5 signaling with increased numbers of Foxp3 positive regulatory T cells while STAT1 phosphorylation and IFN-γ production was reduced. Despite P2X7R blocking, T cells were still able to reconstitute the lymphoid compartment with a polyclonal intact TCR repertoire analyzed by spectratyping DNA sequence analyses of the CDR3 region and to reject established B-cell lymphoma. Therefore, DAMPs such as endogenous nucleotides may not be required for anti-tumor activity but dominantly trigger acute GvHD. Blockade or genetic deficiency of P2X7R in BMT recipients conferred GvHD protection without loss of GvL effects, which has important clinical implications given the availability of P2X7R inhibitors. Disclosures No relevant conflicts of interest to declare.

4642 Next to the control of malignant disease, tolerance induction of the grafted cells remain to be a critical issue for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are believed to be involved in the process of tolerance induction to solid organ grafts and in the regulation of alloreactivity, e.g. graft versus host disease (GvHD) and graft versus leukemia effect (GvL), after allogeneic HCT. Since GvHD causes substantial morbidity, medication with calcineurin inhibitors (CI) like cyclosporine are established prophylactic measures for the prevention of GvHD after HCT. Next to the substantial renal toxicity of CI, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. In contrast, data from in vitro and animal experiments suggest that inhibition of the mammalian target of rapamycin (mTOR) has not only an antiproliferative effect on many malignant cell lines but also results in an inhibition of proliferation of alloreactive T cells with sustained Treg function in a murine HCT model. Therefore we initiated a phase I/II, monocenter trial using everolimus and mycophenolate sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after conditioning with fludarabine, melphalan, and BCNU (FBM). No additional T cell depleting agents were used for conditioning/GvHD prophylaxis. Enrolment was started in april 2008, and up to august 2009 10 patients were included (median age: 50.7 years, range: 26-64). The diagnoses included de novo AML (n=3), sAML (n=4), RAEB II (n=1), CML (n=1), T-PLL (n=1). 6/10 patients were regarded as high risk (not in CR1) for early relapse. PBSC grafts were obtained from unrelated (n=5) and related (n=5) HLA-matched donors. With no graft failures, engraftment kinetics for myeloid cells were normal, and reconstitution of the T cell compartment reached median cell counts of 251 CD4+ cells/μl and 163 CD8+ cells/μl at day +30. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow-up of 6 month two patients have died. The causes were acute GvHD, refractory to several lines of treatment, in a patient with CML, and severe pulmonary toxicity/BOOP in a patient with sAML. Out of 9 patients reaching CR after HCT, only one high risk patient relapsed after 6 month. In total 6 patients are alive and show complete donor chimerism for time periods of 1-14 months post transplant. The observed early recovery of T cell immunity correlated in 8/10 patients with an early brief period of acute GvHD, with 4 patients experiencing grade III/IV severities. Most of the cases could be controlled with steroids alone. Chronic GvHD could be observed in 6/7 patients, with mild to moderate forms in 5 cases, mainly involving skin, mucosa and liver. Interestingly, while early tapering of MMF-Na did not cause any problems, reduction of everolimus earlier as 6 month after HCT resulted in an induction of GvHD symptoms. Although viral reactivation (CMV, HHV6) did occur in patients receiving additional immunosuppression with steroids, no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of mild to moderate chronic GvHD. Since this sustained mild alloreactivity might reduce the risk of relapse, this GvHD prophylaxis could well be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures: Marks: Novartis: Research Funding. Off Label Use: Everolimus for prophylaxis of GvHD. Finke:Novartis: Research Funding.