Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

The interferon-g (IFN-g)-induced enzyme indoleamine 2,3-dioxygenase (IDO) is discussed to play an important role in the induction of allogeneic immune tolerance in pregnant women. IDO is expressed in human placenta and in certain macrophages that affects the suppression of T-cell activity by catabolizing the essential amino acid L-tryptophan [1]. Indeed, during pregnancy, the systemic plasma tryptophan concentration falls significantly [2]. Moreover, T-cell proliferation could be inhibited by macrophage tryptophan catabolism as demonstrated by Munn et al. [3] But it is still not clear how allogeneic T-cell tolerance in pregnancy may be achieved by this mechanism.In a previous study, we have shown a spontaneous IDO expression in monocytes of healthy pregnant women by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in contrast to nonpregnant healthy volunteers [4]. Up to date, it is not clear why pregnant women have this spontaneous IDO expression. An increased expression of IFN-g does not seem to induce IDO, because IFN-g is one of the cytokines that are significantly decreased in the decidua of healthy pregnant women [5].Here we analysed the most upregulated hormones in pregnancy for their ability to induce IDO. The IDO expression induced by the hormones human choriongonadotropine (b-HCG), oestrogen and progesterone in monocytes derived of the peripheral blood of six healthy nonpregnant women was assessed by a quantitative real-time RT-PCR and compared to the spontaneous and IFN-g-induced IDO expression. The median age of the examined healthy nonpregnant women was at 35 years (range 24-43 years), two of the six women had previous pregnancies. The monocytes isolated from whole blood were incubated for 24 h (37 C, 5% CO 2 ) with 1500 IU b-HCG, 1.4-mg oestrogen and 20-mg progesterone.Real-time RT-PCR was performed using the Lightcycler (Roche Diagnostics, Mannheim, Germany) with the forward primer 5 0 -ACAGACCACAAGTCACAGCG and the reverse primer 5 0 -AACTGAGCAGCATGTCCTCC and the hybridization probes GCAGTGCAGGCCAAAG-CAGCGTCTTTCAGTGCTTT-X and LC Red640-ACGTCCTTGCTGGGCATCCA. The expression of IDO transcript was determined and normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-housekeeping gene as an endogenous reference with the primers 5 0 -TTCACCACCATGGAGAAGGCT and 5 0 -ATGGCATGGACTGTGGTCATG and the probes ATCATCAGCAATGCCTCCTGCACCACCAACTGCT-X and LC Red640-AGCACCCCTGGCCAAGGTCATC-CAT-ph. All analyses were performed in duplicate.Here we describe for the first time that IDO was expressed in the monocytes of all the six nonpregnant healthy volunteers after the induction of the above hormones. Interestingly, the b-HCG-induced IDO expression was the highest with 117% at median (range 27.9-609.7%), which was quit comparable to the IFN-g-induced IDO expression with a median at 109.7% (range 35.7-301.4%). Further, we also found an IDO expression after stimulation with oestrogen and progesterone, which was lower as the IFN-g-and b-HCG-induced IDO expression. The median of the oestrogen-...

Background Chronic graft-versus-host disease (GVHD) is a major complication following allogeneic hematopoietic stem cell transplantation and is associated with a substantial morbidity and mortality. It is a systemic inflammatory disorder that reflects the lack of immune tolerance between donor-derived immune competent cells and host organs. Human chorionic gonadotropin hormone (hCG) is a natural occurring hormone during pregnancy secreted by syncytiotrophoblasts of the placenta. We had previously observed (Koldehoff et al; J Leukoc Biol 2011) that the rejection of transplanted skin was significantly delayed by hCG in a mouse skin transplant model and had also demonstrated that tryptophan-catabolizing enzyme, indoleamine-2,3-dioxygenase(IDO), interleukin-10 (IL 10) and T-regulatory cells (Tregs) increased significantly in females treated with hCG as preconditioning therapy for in-vitro-fertilization. Since all these factors are known to induce tolerance and given the low rate of adverse effects, we off-label used low dose of hCG to treat 20 patients as forth- or fifth-line therapy with steroid-refractory or intolerant severe-grade chronic GVHD. Patients Because all of these factors are known to induce tolerance and given the low rate of adverse effects in preconditioning therapy, we off-label used low dose of hCG (187 IU) to treat 8 male and 12 female patients (median age 48, r. 28-68) with moderate or severe grade of chronic GVHD according to the NIH criteria; all patients had been informed of the experimental state of this treatment and provided written consent. Results The median number of sites of chronic GVHD involvement per patient was 3 (range, 1-6). hCG therapy was started as 4 or 5th line-therapy together with preexisting medication with prednisone and a calcineurin inhibitor. Twelve of 20 patients (60%) had an objective partial response during 8 weeks of hCG treatment with at least 50% improvement according to the TSS score. Responses included softened skin and subcutaneous tissue; decreased erythema and extent of sclerodermatous, hidebound skin; improved joint mobility and gait; gastrointestinal improvements; and resolution of neuropathy. Nine patients had stable disease (6 with minor responses). Only one patient with previous ATG treatment showed progression of her liver GVHD (histologically proven) and died from GHVD. All other patients were well and alive. Daily low-dose hCG was well tolerated. Adverse events that were possibly related to hCG included reversible and asymptomatic CTCAE grade 4 hypertriglyceridemia (n=1), grade 2 constitutional symptoms (fever, malaise, fatigue; flush, breast enlargement). IDO expression increased up to 8 times and IL10-serum level up to 2 times after 3 weeks of hCG therapy (p<0.003 and p<0.04). T-regulatory cell expansion was documented in 3 patients. Conclusion This successful use of hCG in an immune disorder warrants further studies to assess its role as an immunosuppressant in GVHD and potentially other autoimmune disorders. Disclosures: Off Label Use: The off-label use of HCG will be presented here for the first tiem for treatment of chronic GVHD and will clearly marked as off-label use.

5503 Introduction Early detection of inapparent replicative human cytomegalovirus (HCMV) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening HCMV disease after allogeneic hematopoietic stem cell transplants (alloSCT). A new aspect of HCMV reactivation and pretransplant HCMV serostatus has recently emerged by an earlier retrospectively performed report from us showing that the occurrence of a HCMV-reactivation after transplant reduces the risk for relapse in patients with AML and NHL. This idea was supported by a study by Scheper and co-worker (Leukemia, 2013) reporting recently, that gamma-delta T cells elicited by HCMV reactivation after alloSCT cross-recognize HCMV and leukemia. Here we evaluate the potential impact of early HCMV replication in a prospectively performed observational study about the occurrence of a HCMV- reactivation after T cell repleted alloSCT on the risk for leukemic relapse in patients with AML (Registration Trial DRKS00004300). Patients and Method Between January 2012 and March 2013 we enrolled in this trial 83 patients with AML who were consecutively transplanted at the University Hospital of Essen. 48 of 83 patients received a myeloablative (TBI based conditioning n=27, chemotherapy based conditioning n=23) and 35 patients a RIC regimen. Patients were transplanted in 1.CR (n=40), 2.CR (n=23) or more progressive disease stages (n=20) from HLA-identical sibling donor (n=17) or HLA-identical unrelated donor (URD) (n=42) or mismatched unrelated donor (n=24). The median age of patients was 53 years (range 18-72) and that of the donors 38 years (range 12-61). GVHD prophylaxis was performed with MTX and CSA, or CSA and MMF with or without ATG (n=64) (30-60mg total dose). The incidence of acute GVHD grade 2-4 was statistically not different in both groups (78% versus 85%). Results HCMV status of recipient (R) or donors (D) were in 29% R-/D-, 8% R-/ D+; 34% D+/R- and 29% R+/D+. Patients with a documented HCMV-reactivation (HCMV-R) had an estimated relapse incidence (CIR) at 1-year after transplant of only 8% compared to 43% in patients without a HCMV-R (p=0.03). Patients in more progressive disease phase of AML (N=43) benefit more from a HCMV-R in regard of CIR than patients in 1.CR of AML (0% versus 55% estimate for relapse at 1-year after transplant for patients with HCMV-R compared to patients without HCMV –R, p=0.028). One-year overall survival was statistically not different in both groups. Non relapse mortality was greater in patients with HCMV reactivation 37.8% versus 12.5%, p=0.1) Conclusion The first result of this prospective study confirms an independent advantageous effect of early HCMV replication on the leukemic relapse risk in patients with AML after transplant, which was more pronounced in patients in  progressive disease phase of AML than patients in 1.CR of AML. Disclosures: Off Label Use: The off-label use of HCG will be presented here for the first tiem for treatment of chronic GVHD and will clearly marked as off-label use.