Abstract: The intestine is preferentially damaged in acute graft-versus-graft disease (aGVHD). Patients with intestinal GVHD are usually associated with drug-resistant diarrhea and microflora disturbance. Recent studies suggest that toll-like receptor (TLR) signaling can protect the intestinal epithelial barrier and confer commensal tolerance in health. But less is known about how functional versus dysfunctional TLR pathway opposes or favours the intestinal GVHD. Methods In the current study, BALB/c mice were transplanted whole spleen and T cell deleted (TCD) bone marrow cells from C57BL/6 mice as GVHD group, and transplanted TCD bone marrow cells as control group. The jejunum, ileum, colon and rectum epithelium were harvested and total RNA of the intestinal epithelium were extracted in two groups. The mRNA expression of classical TLR pathway TLRx/MYD88/IRAK4 signaling molecules (TLR2, TLR4, MYD88, IRAK4 and Tollip) and cytokines (IFN-γ, TNF-α and TGF-β) were detected by RT-PCR. Results The intestine of aGVHD recipients showed severe mucosal edema and erythema with histologic changes of apoptotic epithelial cells and crypt cell dropout, while the intestine of recipients in the control group did not show any intestinal GVHD evidence. TLR2 expression was markedly down-regulated and little TLR4 expression was observed in GVHD intestinal epithelium in comparison to control group. MYD88 and IRAK4 expression were lower in the entire intestinal epithelium of GVHD group but only significant in colon and rectum epithelium between the two groups. Tollip, a TLR signaling inhibitor by interfering IRAK, was found much higher in the GVHD group. For cytokines, both of IFN-γ and TNF-α expression were markedly up-regulated from proximal to distal intestine in GVHD group as compared to control group. There was no difference in TGF-β expression between the two groups. Conclusions We propose TLR signaling in the intestinal epithelium, especially in colon and rectum, presents disruption in intestinal graft-versus-host disease. IFN-γ and TNF-α might contribute to accelerate TLR pathway alteration. Disclosures: Liu: It was supported by 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Wu:It was supported by 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Zhao:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Wu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhang:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yin:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zheng:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yi:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

Abstract: Noninfectious lung injury caused by graft-versus-graft disease (GVHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplant (allo-HSCT). Epithelial injury is a central event in the pathogenesis of noninfectious lung injury. Recent studies have shown that alveolar epithelial cells are able to self-renew and reestablish a functional alveolar epithelium. In the inflammatory and fibrotic lung diseases, differentiated epithelia also can acquire a myofibroblast phenotype in the process termed epithelial to mesenchymal transition (EMT), which contributes to aberrant healing and fibrosis. Many factors such as inflammatory cytokine TGF-β have been suggested to induce EMT. However, the role of EMT in the remodeling of acute GVHD (aGVHD) induced lung injury is unclear. Methods BALB/c mice were lethally irradiated and transplanted T cell-deleted (TCD) bone marrow plus whole spleen cells from C57BL/6 mice as aGVHD group, and only transplanted TCD bone marrow cells as control group. Alveolar epithelial cells were isolated from mice of two groups and Ep-CAM expression was measured by flow cytometry. The mRNA expression of cytokines including IFN-γ, TNF-α and TGF-β was detected by RT-PCR. The mRNA and protein expressions of specific markers, including E-cadherin, vimentin, Snail and surfactant proteins (SP)-C in lung tissue, were detected by RT-PCR and western blot. Results All mice in the aGVHD group showed diffuse periluminal infiltrates and parenchymal pneumonitis by histopathology, while the mice in the control group did not show any lung injury evidence. IFN-γ, TNF-α, TGF-β mRNA expressions were markedly up-regulated in the lung injury group as compared to the control group (P=0.045, P=0.032, P=0.025). Alveolar epithelial cells of injured lung expressed higher Ep-CAM (P=0.017) and lower SP-C (P=0.023). RT-PCR and western blot analyses revealed a significant decrease in epithelial marker E-cadherin (P=0.029) and increase in mesenchymal marker vimentin (P=0.026) in the GVHD damaged lung. Snail, a key EMT related transcription factor, was significantly elevated at mRNA and protein level in comparison to control group (P=0.015). Conclusion EMT is involving in the remodeling of lung injury induced by aGVHD. TGF-β is demonstrated to induce EMT. Whether up-regulation of IFN-γ and TNF-α contributes to EMT is deserved to be further explored. Disclosures: Wu: 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhao:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Wu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhang:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yin:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zheng:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yi:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

Abstract: Abstract 4069 Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cells, derived from bone marrow progenitor cells and immature myeloid cells. In tumor development and progression, MDSCs play a role of immunosuppressive effect. Recently, researchers found that granulocyte colony-stimulating factor (G-CSF) could mobilize MDSCs from bone marrow (BM) to peripheral blood (PB), but there is unknown about the effect of G-CSF to MDSCs in the human body and the relationship between MDSCs and graft-verse-host disease (GVHD). So we investigated the association between MDSCs and acute graft-verse-host disease (aGVHD). The expression of MDSCs and adhesion molecules in the BM, PB and peripheral blood stem cell grafts in 30 related donors, were detected by flow cytometry before and after recombined human G-CSF (rhG-CSF) mobilization. The expression of arginase (ARG) and inducible nitric oxide synthase (iNOS) were detected by enzyme-linked immunosorbent assay. According to the median number of MDSCs in peripheral blood stem cell harvests (9.60×106/kg), 30 leukemia patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were divided into two groups, high MDSCs group and low MDSCs group. The hematopoietic/immune reconstitution, incidence of aGVHD, relapse, and survival rate were compared between two groups. MDSCs could be detected in the blood and BM in healthy donors, the proportion in nuclear cells in the blood and BM was 0.30% ± 0.09% and 0.53% ± 0.16%, respectively. MDSCs in BM was higher than that in PB (P 〈 0.001). After 5 days of rhG-CSF mobilization, the proportion in the blood and BM was 0.66% ± 0.28% and 0.72% ± 0.13%, respectively. The difference between proportion in the blood before and after mobilization was significant (P =0.017). Adhesion molecules on MDSCs surface after rhG-CSF mobilization were significant lower than that before rhG-CSF mobilization, both in blood and BM (P 〈 0.001). The expression of ARG and iNOS after mobilization were significantly higher than that before mobilization (P 〈 0.001), but not in BM (P =0.695, 0.073). There was a significant negative correlation between the number of transplanted MDSCs cells and the grading of aGVHD (correlation coefficient rs =0.445, P =0.014). The difference between the incidence of aGVHD in high MDSCs group and low MDSCs group (20.00% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(315\) \end{document} vs 66.67% \batchmode \documentclass[fleqn,10pt,legalpaper] {article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(1015\) \end{document}, P =0.011), there was no significant difference between the hematopoietic/immune reconstitution, relapse rate (26.67% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(415\) \end{document} vs 20.00% \batchmode \documentclass[fleqn,10pt,legalpaper] {article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(315\) \end{document}, P =0.671) and disease-free survival rate (46.7% ±14.2% vs 38.9%±15.8%, P =0.833) in two groups. In conclusion, in the human body, rhG-CSF could transfer more MDSCs from BM into the blood. RhG-CSF mobilization could increase the number of MDSCs in graft, which may be related to low incidence of aGVHD after allo-HSCT. The mechanism of rhG-CSF mobilized MDSCs to blood may be related to adhesion factor on MDSCs surface. The incidence of aGVHD in patients who accepted high MDSCs cells was lower; there was a significantly negative correlation between MDSCs transplant value and the grading of aGVHD. MDSCs cell infusion did not affect the function of immune reconstitution and the effect of graft versus leukemia. Disclosures: Liu: National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.

Abstract: We aimed to investigate outcomes of different post-remission treatment (PRT) choices based on dynamic measurable residual disease (MRD) by multiparameter flow cytometry in favorable-risk AML (FR-AML). Four hundred and three younger patients with FR-AML in first complete remission (CR1) were enrolled in this registry-based cohort study, including 173 who received chemotherapy (CMT), 92 autologous stem cell transplantation (auto-SCT), and 138 allogeneic SCT (allo-SCT). The primary endpoint was the 5-year overall survival (OS). Subgroup analyses were performed based on dynamic MRD after the 1st, 2nd, and 3rd courses of chemotherapy. In subgroups of patients with negative MRD after 1 or 2 course of chemotherapy, comparable OS was observed among the CMT, auto-SCT, and allo-SCT groups ( p  = 0.340; p  = 0.627, respectively). But CMT and auto-SCT had better graft-versus-host-disease-free, relapse-free survival (GRFS) than allo-SCT in both subgroups. For patients with negative MRD after three courses of chemotherapy, allo-SCT had better disease-free-survival than CMT ( p  = 0.009). However, OS was comparable among the three groups ( p  = 0.656). For patients with persistently positive MRD after 3 courses of chemotherapy or recurrent MRD, allo-SCT had better OS than CMT and auto-SCT ( p  = 0.011; p  = 0.029, respectively). Dynamic MRD might improve therapy stratification and optimize PRT selection for FR-AML in CR1.

Abstract: Backgroud: Although the introduction of rituximab as a first-line treatment has improved outcome of post-transplant lymphoproliferative disorder (PTLD), PTLD with central nervous system involvement (CNS-PTLD) still has a dismal prognosis because of low penetrance across the blood-brain barrier. In this prospective study, we reported intrathecal rituximab was efficacy and safety in the patients with CNS-PTLD who had failed to respond to the intravenous rituximab-based treatments. Methods: From June 2009 to November 2013, 32 cases of EBV-associated PTLD were recorded in the Southern Medical University Institute of Hematology. Fourteen patients diagnosed with CNS-PTLD were enrolled in this prospective study. For the patients who failed to response to the initial intravenous rituximab-based treatments, sequential dose-escalation schedule of intrathecal rituximab (initial dose of 20mg, increased by 10mg each week and maximum dose of 50 mg) was administrated weekly. Results: Three patients were responsive and 11 were unresponsive to the initial treatments within one week after the treatments. For the 11 patients who failed to respond to the initial treatments, 9 patients received intrathecal rituximab within 7-11 days and 2 patients refused the treatment. After two cycles of rituximab-based treatments, 10 patients achieved complete response (CR), 2 patients were partial response and 2 patients were non-response. With a median follow up of 664 (range 18 to 1545) days after the diagnosis of CNS-PTLD, 7 patients survived and 7 died. The causes of death included PTLD progressing (n=3), PTLD relapse (n=1), GVHD (n=1), CMV pneumonia (n=1) and pseudomonas aeruginosa sepsis (n=1). The 3-year probability of OS was 45.7% ±14.7% in CNS-PTLD, which was no significant difference as compared to PTLD without CNS involvement(55.6% ±11.7%, P=0.706). Conclusion: Intrathecal rituximab might be an effective and safe method for CNS-PTLD in the allo-HSCT recipients who were unresponsive to the intravenous rituximab-based treatments. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 1959 Background. Epstein-Barr virus (EBV) -associated post-transplant lymphoproliferative disorder (PTLD) with central nervous system (CNS) involvement is a rare, but life-threatening complication in the recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The management of systemic PTLD, including EBV molecular monitoring, preemptive therapy, and treatment with reduction of immunosuppression, chemotherapy, rituximab and cellular therapy is reported, but there is no uniform approach to the management of PTLD with CNS involvement. To date, limited data exist for treatment in patients with CNS-PTLD. Methods. Eight CNS-PTLD patients from 198 patients undergoing allo-HSCT between April 2009 and March 2012 in our single institution were enrolled in this prospective study. Diagnosis of CNS-PTLD was based on clinical manifestations, EBV-DNA in blood and cerebrospinal fluid (CSF), immunophenotypic analysis of CSF cells and neuroimaging. The treatment of patient with CNS-PTLD included reduction of immunosuppression, antiviral agents, intravenous rituximab-based treatment, cellular therapy as well as intrathecal rituximab. Results. The median time of diseases onset was 63 (range 49 to 186) days post-transplants. Six patients had systemic PTLD with CNS involvement, while two patients revealed isolated CNS-PTLD. All patients were EBV-DNA positive of blood and CSF, except one patient was EBV-DNA positive of CSF but negative of blood. EBV-DNA-emia appeared 7 (range 2–13) days before clinical manifestations. The EBV-DNA levels of CSF were significantly higher than that of blood (201321 ± 202053 copies/ml vs. 28083 ± 35706 copies/ml, P=0.032). The immunophenotypic analysis of CSF cells revealed that 7 cases had CD19+CD20+ B cell monoclonality and 1 case had CD3+ T cells, CD19+ B cells and CD14+ monocytes polyclonality. Magnetic resonance imaging (MRI) of CNS on the early stage of diseases onset showed normal (n=1), inflammatory changes (n=2) and space-occupying lesions (n=5).With the development of diseases, 2 cases who initially presented as normal and encephalitis MRI, had space-occupying lesions on CNS imaging. Immunosuppressive agents were withdrawn in all patients (ie, total dose reduced by 20%/week). Two patients were only treated with antiviral agents, 5 received rituximab-based treatments and 1 abandoned. The two patients underwent antiviral agents were unresponsive and died of PTLD. Of the 5 patients underwent intravenous rituximab-based treatments, 2 cases obtained complete remission (CR) and 3 did not. Three patients who had failed to respond to intravenous rituximab-based treatments all obtained CR after the administration of intrathecal rituximab in dose 10–30 mg. Five patients who were CR all received donor lymphocyte infusion after CR. With a median follow up of 183 (range 7 to 918) days, four cases were alive and 4 died. The causes of death included progression of CNS-PTLD (n=3) and cytomegalovirus pneumonia (n=1). Conclusions. EBV-DNA detection of CSF is an important indicator for early diagnosis and therapeutic evaluation of CNS-PTLD. The immunophenotypic analysis of CSF cells is helpful in diagnosis of EBV-associated CNS-PTLD. The administration of intrathecal rituximab is an effective method for patients who had failed to respond to intravenous rituximab-based treatments. Cellular therapy may be beneficial for long term survival. Disclosures: No relevant conflicts of interest to declare.

Abstract: Mesenchymal stem cells (MSCs) have been considered as a promising strategy for the prevention and treatment of acute graft-versus-host disease (aGVHD), but the mechanism of MSCs ameliorating GVHD is still not fully understood. A few studies suggested that MSCs ameliorated GVHD via protecting and repairing the function of thymus. Methods Twenty refractory aGVHD patients receiving MSCs treatment were enrolled in this study, and twenty grade II to IV aGVHD patients treated without MSCs were matched as non-MSCs group. MSCs were given at a median dose of 1×106 cells/kg once weekly until aGVHD got complete response (CR) or MSCs were infused for a total of 8 doses. Lymphocyte subsets of T-cell, B-cell, and NK-cell in peripheral blood were analyzed by flow cytometry before and 30, 60, 90, 180 and 360 days after the MSC infusion in MSC group and the corresponding period in the control group. Results A total of the 33 patients who survived more than 30 days after the study treatments were evaluated for the alterations in cellular immune compartment and cGVHD, including 15 cases in MSCs group and 18 in non-MSCs group. In MSCs group, patients had a significantly higher CD4+/CD8+ T-cell ratio at 60, 90 and 180 day after MSCs treatments, which subsequently equalized at 360 day compared with non-MSCs group. The MSC group presented higher percentages of CD4+CD25+ regulatory T cells (Treg) comparing with non-MSCs group, especially at 90 and 180 day, and approached at 360 day. The proportion of CD4+CD45RO+ and CD4+CD45RA+ naïve T-cells in MSC group were also higher than those in non-MSCs group at 60, 90 and 180 day, but not different at 360 day. The proportion of CD19+ and CD16+CD56+ was not detected striking difference between the two groups. Four patients (26.7%) experienced cGVHD in MSCs group, compared with twelve (66.7%) in non-MSCs group (p=0.02). Conclusions In conclusion, our data indicate that MSCs might ameliorate aGVHD and induce longer-lasting immune tolerance by altering cellular immune compartments in peripheral blood. The alteration of the cellular immune compartments is associated with the protecting and repairing role of MSCs to thymus. Disclosures: Liu: 863 Program (No. 2011AA020105): Research Funding; National Public Health Grand Research Foundation (Grant No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647): Research Funding; Science and Technology Program of Guangzhou of China (11A72121174) : Research Funding.