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Objective To investigate the effectiveness and safety of rituximab in treating autoimmune haemolytic anaemia (AIHA) after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Methods Patients with refractory AIHA following allo-HSCT were treated once-weekly with rituximab 375 mg/m2 for a total of four doses. In an animal study, recipient CB6F1 mice were conditioned with busulfan/fludarabine and transplanted with splenocytes and T-cell-depleted bone marrow from C57Bl/6 mice. In this animal model, anti-CD20 monoclonal antibody (mAb) was evaluated to see if it could prevent graft versus host disease (GVHD). GVHD was monitored by body weight loss, GVHD clinical scores and the survival of each group of mice. Histopathological analyses of the skin, intestine, liver and lung were used to analyse the severity of GVHD. Results After rituximab therapy, refractory AIHA was resolved in all four patients as shown by increased haemoglobin levels. B-cell proportions were reduced with a relative increase of the proportions of T-cells following rituximab treatment. None of the four patients experienced chronic GVHD. In the animal model, anti-CD20 mAb treatment reduced GVHD. Conclusions Rituximab therapy deserves consideration for the treatment of post-HSCT patients with refractory AIHA. Further studies are needed to define the therapeutic role of this anti-CD20 mAb.

Objective To investigate the cytotoxic effects of suberanilohydroxamic acid (vorinostat) in combination with arsenic trioxide (ATO) on the human NB4 cell line in vitro. Methods The rates of cell proliferation following treatment with vorinostat with or without ATO were measured. Flow cytometry of Annexin-V/propidium iodide double-stained cells was used to measure apoptosis. Acridine Orange and ethidium bromide staining was used to observe morphological changes characteristic of apoptosis. Western blot analysis was used to measure protein levels. Results Vorinostat and ATO, alone and in combination, inhibited the proliferation of NB4 cells in a time- and dose-dependent manner and the effect was additive. NB4 cells treated with vorinostat + ATO demonstrated greater levels of apoptosis compared with cells treated with either drug alone. Both vorinostat and ATO alone and in combination resulted in lower levels of promyelocytic leukaemia/retinoic acid receptor alpha fusion protein and increased levels of acetyl-histone H3 and acetyl-histone H4 proteins compared with controls. Vorinostat + ATO resulted in lower levels of Akt protein compared with either drug alone. Conclusion The combination of vorinostat and ATO inhibited cell proliferation, induced apoptosis, and enhanced the chemosensitivity of NB4 cells. The mechanism might be associated with increasing histone acetylation levels as well as downregulation of the Akt signalling pathway.

Objective:To assess the relevance between minimal residual disease (MRD) levels and disease recurrence and prognosis in patients with acute leukemia before and after hematopoietic stem cell transplantation (HSCT), and further to explore potential benefit based on pre-transplant MRD stratification. Methods: A total of 113 patients with newly diagnosed acute leukemia in Fujian Union hospital from April 2013 to April 2019 were retrospectively analyzed. 94 patients were complete remission (CR)( 50 cases of AML and 44 cases of ALL). 19 patients didn't achieve CR( 10 patients with AML and 9 patients with ALL). The median age was 26(1-56) years. 67 patients were male and 46 patients were female. The sources of hematopoietic stem cell include bone marrow, cord blood and peripheral blood stem cell. All patients underwent myeloablative conditioning before transplantation. Standard graft-versus-host disease (GVHD) prophylaxis was applied in all patients. MRD was measured by flow cytometry and real-time quantitative polymerase chain reaction(PCR). MRD values >10-4 or BCR-ABL were considered positive for minimal residual disease and compared with bone marrow morphological findings. Median follow time was 15.2 (2.2 to 70.5) months. Results: Among patients in CR, achieving pre-MRD negativity was associated with longer 2-year relapse-free survival (2-year RFS; 81.4% vs 49.1%; P=0.003) and 2-year overall survival (2-year OS; 84% vs 50.6%; P=0.012). Compared to pre-MRDneg patients, pre-MRDpos patients had a higher incidence of relapse (31.2% vs. 5.1%, P=0.001). There was no significant difference between OS and MRD level after transplantation (OS; post-MRDneg 82.9% vs. post-MRDpos 66.7%, P =0.468). In the second set of analyses, CR patients were classified into the MRDpos /MRDpos group, the MRDpos /MRDneg group, the MRDneg /MRDpos group, and the MRDneg /MRDneg group according to MRD dynamics. Compared to the other three groups, patients from the MRDpos /MRDpos group had higher cumulative incidences of relapse (MRDpos /MRDpos, 48.3%; MRDpos /MRDneg, 22.2%, MRDneg /MRDpos,40.0%; MRDneg /MRDneg, 2.7%; P Conclusion: MRD can be used as a sensitive indicator to evaluate disease prognosis. Patients who are in morphologic remission and have no evidence of MRD before the HSCT come to encouraging leukemia-free survival. Continuous regular dynamic observation is important for guiding disease recurrence and prognosis. Disclosures No relevant conflicts of interest to declare.

Objective: Invasive fungal disease (IFD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). However, limited data is available on its clinical features after cord blood transplantation (CBT) for patients with hematological malignancies. Method: We retrospectively reviewed 63 patients who underwent myeloablative CBT at our institution between July 2013 and June 2019 with a median follow-up of 14 months. Micafungin, Voriconzole or Posaconazole was used for IFD prophylaxis. Results: Eight patients were identified as having an IFD, including 2 with proven IFD, 1 with probable IFD, and 5 with possible IFD. The most common prophylaxis regimen is micafungin (68.2%, 43/63). The incidence rate of IFD in the primary antifungal prophylaxis (PAP) and secondary antifungal prophylaxis (SAP) groups were 11.3% and 25% (P=0.488). The OS of day at +100, six months and +200 in the IFD and No IFD groups were 62.5% vs. 86.9% (P=0.1), 50 % vs.78.2% (P=0.07), and 50% vs.75.9% (P=0.094), respectively. The 3-year probabilities of overall survival (OS) in the IFD and No IFD groups were 50% and 60.5%, respectively (p=0.316). The incidence rate of Non-relapse Mortality in the IFD and No IFD groups were 42.9% and 30%, respectively (p=0.319). Conclusion: Antifungal prophylaxis could help to reduce the incidence of IFD after CBT for patients with hematological malignancies. Figure Disclosures No relevant conflicts of interest to declare.

Objective: To evaluate the efficacy and safety of the combination of haploidentical hematopoietic with low-dose ATG, PTCy and cord blood competitive transplantation for hematologic malignancies. Methods: This study was conducted as a retrospective review of medical records of 31 patients with hematologic malignancies who received a combination of haploidentical hematopoietic with low-dose ATG, PTCy and cord blood competitive transplantation at Fujian medical university union hospital, from November 2016 to April 2019. Results: Among the 31 patients with hematologic malignancies, the median age was 22 (6-52) years, of which 17 were CR patients and 14 were No CR patients. The conditioning was modified FABuCy+ low-dose ATG (total dose 5mg/kg) based regimen. Cord blood units were selected based on the results of HLA typing and cell doses evaluated before freezing. Units with at least 5/10 matched HLA loci became the candidates. Prophylaxis for graft-versus host disease(GVHD) was by CsA(cyclosporine), MMF (mycophenolate mofetil) plus PTCy(post-transplant cyclophosphamide). The median values of absolute total nucleated cell counts were 123.0 (49.0-258.8) × 107 / kg in The haploidentical grafts and 2.5 (1.1-13.0 × 107 / kg in the cord blood units,respectively. The median Doses of CD34+ cells infused were 70.0 (11.8-297.6) × 105 / kg in the haploidentical grafts and 1.4 (0.1-13.0) × 105 / kg in the cord blood units, respectively. All patients attained complete engraftment , of which 19 were haploidentical engraftment and 12 were cord blood engraftment. The median durations of myeloid engraftment were 14 (12-37) days and 14.5 (10-48) days for Platelets. With a cumulative platelet engraftment incidence of 90.3%. During a median follow-up of 8.5 (1.3-30.7) months, the incidence of grade II-IV acute GVHD at 100-day was 47.5%, grade III-IV acute GVHD was 13.8%, respectally. The incidence of limited chronic GVHD at 1-year was 35.1%, severe chronic GVHD was 14.6 %, respectively. The estimated 1-year OS was 76.1%,DFS was 68.5% ,GRFS was 60.6%, NRM was 18.5% , RM was 6.7% , respectively. The estimated 1-year OS of CR patients and No CR patients were 92.9% and 55.0%(p=0.114), DFS were 83.6% and 51.9%(p=0.053),NRM were 7.1% and 31.2%(p=0.132),relapse were 10.0% and 30.4%(p=0.131), respectively. Conclusion:The results suggested that the combination of haploidentical hematopoietic with low-dose ATG, PTCy and cord blood competitive transplantation may potentially improve the outcome of HSCT. It offers a transplant alternative for patients with hematologic malignancies who lack matching donors. Disclosures No relevant conflicts of interest to declare.

Bloodstream infection (BSI) caused by multidrug‐resistant bacteria (MDRB) or extensively drug-resistant bacteria (XDRB) in immunocompromised and neutropenic patients after hematopoietic stem cell transplantation (HSCT) is a global threaten. However, effective treatment regimen is still controversial and inadequate due to the rapid deterioration, the horrific evolution of bacteria and high mortality that the mortality related to BSI caused by carbapenem-resistant Enterobacteriaceae (CRE) is 51% in adult neutropenic patients[1]. Here, we presented four cases that CRE was detected in blood of severe agranulocytosis patients undergoing HSCT. All patients ranged from 2 to 50 years old were diagnosed as hematologic disease. CREs were detected within 1 month from the date of engraftment. In case 1, a 6-year-old boy with high-risk B-cell acute lymphocyte leukaemia received halpo-HSCT after chimeric antigen receptor T-cell therapy and the 20th chemotherapy, ultimately, he died from acute graft versus host disease and BSI that tienam and tigecycline showed little effect though the MIC value of tigecycline was less than 0.5ug/ml. The second patient is a 25-year-old female who was diagnosed as hemophagocytic syndrome with recurrent fever and a salvage haploidentical transplantation combining with a unit of umbilical cord blood stem cells was performed. Finally, she died from BSI caused by CRE although a combination therapy using polymyxin B, tigecycline and doubled-dose Tienam was given, which subsequently was changed to another therapeutic regimen using a higher dose tigecycline and fosfomycin and polymyxin B in light of the resistance of tienam. Patients in case 3 and case 4 received a combination therapy with tigecycline, polymyxin B, fosfomycin, and Tienam intravenously. For the 29-month-old boy diagnosed as acute monocytic leukemia with high expression of WT1 and abnormal karyotype and treated with cord blood stem cell transplantation in case 3, the tigecycline was given with a dose of 2.4mg/kg at the first time, followed by doses of 1.2mg per dose every 12 hours, and a dose of 2.0mg/kg at the first time, followed by doses of 1.25mg/kg per dose every 12 hours of polymyxin B, 0.15g/kg per every 12 hours of fosfomycin, as well as 30mg/kg per dose every 6 hours of imipenem were administrated. For the 50-year-old woman with multiple myeloma receiving an autologous hematopoietic stem cell transplantation (auto-HSCT) in case 4, the dose of tigecycline was 100mg at the first time, followed by doses of 1.2mg per dose every 12 hours. Fosfomycin with the dose of 8.0g was used every 12 hours, and the dose of imipenem was1.0g per dose every 6 hours, also, polymyxin B was administrated with a dose of 2.0mg/kg at the first time, followed by doses of 1.25mg/kg per dose every 12 hours. Consequently, patients in case 3 and case 4 were survival. Therefore, a strong combination therapy, as well as the emergency of new drugs might be considered in immunocompromised and neutropenic ill patients with BSI caused by MDRB or XDRB. Disclosures No relevant conflicts of interest to declare.

Background: Acute lymphoblastic leukemia (ALL) is a neoplastic cancer characterized by clonal expansion of leukemic cells in lymph organs and bone marrow. Lots of kinds of different chromosomal translocation can be found in those leukemic cells. However, the role of the abnormal chromosomals and genes in leukemogenesis is not yet fully understood. Identifying new chromosomal translocation can facilitate a better understanding of pathogenesis of this disease. Case presentation: We report a rare case of acute lymphocytic leukaemia with t(3;13)(q29,q21). The patient was diagnosed pre-B-ALL with no abnormal chromosomal or gene fusion and achieved complete remission (CR) after induction chemotherapy. Ten months later, she relapsed in the consolidation with cytogenetics test showing 46,XX,t(3;13)(q29,q21). Given no CR after 2 chemotherapy regimens, she received salvage cord blood transplantation. Regular intrathecal methotrexate was applied to prevent central nervous system leukemia. Good graft-versus-leukemia (GVL) effect was induced by daily injection of low dose of IL-2 two months post transplantation. Minimal residual disease (MRD) negativity was maintained until central nervous system leukemia was found 8 months after transplantation. A whole exome sequencing was performed. Nine driver mutation genes and seven tumor genes were found. Conclusions: We highly suspect that the relapse in central nervous system after transplantation is associated with the rare chromosomal translocation. Disclosures No relevant conflicts of interest to declare.

Intravenous busulfan (IV-Bu) or total body irradiation (TBI) based regimens are currently the most widely used myeloablative conditioning regimens for patients with hematologic malignancies undergoing allogeneic stem-cell transplantation(allo-SCT). Numerous trials have been undertaken on the clinical outcomes between IV Bu and TBI, but there are no comparative data for cord blood transplantation(CBT). We conducted a prospective registry-based study to analysis the outcomes of IV Bu and TBI in CBT patients with hematologic malignancies. From May 1, 2008 to Mar 31, 2016, a total of 331 consecutive patients with hematologic malignancies recieved singe unrelated CBT were involved in the study. Eligibility criteria for this analysis included:(1)Weigh ≧35 kilograms and age ≦ 60 years; (2)All patients recieved a single unit CBT but not a double units CBT; (3)Consensus criteria preparative regimens were based on full dose IV Bu(total 12.8 mg/kg, 0.8mg/kg every 6 h for 4 days) or TBI(total 12 Gy, 4 fractions) combined with Cy(60mg/kg × 2d); (4)GVHD prophylaxis regimens include cyclosporine(CSA) and mycophenolate mofetil(MMF) without Antithymocyte Globulin(ATG). Patients who has recieved a previous autologous or allogeneic transplantation was excluded in the study. The cumulative incidence of neutrophil engraftment were 91.6% in IV Bu/Cy cohort and 98.0% in Cy/TBI cohort(P < .001), respectively. The median follow-up time in IV Bu/Cy and Cy/TBI cohorts was 28.7(range, 12.2 to 91.3) months and 55.5(range, 13.1 to 117.1) months, respectively(P Our resluts demonstrates that, compared with TBI, IV Bu regimen was associated with a higher incidence of graft rejection in CBT. But there was no difference in survival with no increased risk for NRM or relapse between two regimens. For those centers lack of radiation facilities, IV Bu may be a valid and efficient alternative to TBI. With the restriction of a retrospective registry analysis and limited patient munbers, rigorously designed prospective randomized controlled trials are needed to further investigated the availability of IV Bu and TBI for CBT. Disclosures No relevant conflicts of interest to declare.

Host dendritic cells (DCs) play a critical role in initiating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL), and separation of GVL from GVHD remains a major challenge in the treatment of hematologic malignancies by allogeneic hematopoietic cell transplantation (HCT). Here, we show that preconditioning with anti-CD3 monoclonal antibody before conditioning with total body irradiation (TBI) prevents GVHD but retains GVL in a HCT model of major histocompatibility complex (MHC)–mismatched C57BL/6 donor to BALB/c host. Prevention of GVHD is associated with inhibition of donor T-cell expression of homing and chemokine receptors, and inhibition of GVHD target tissue expression of chemokines. Furthermore, inhibition of donor T-cell expression of gut homing α4β7 and chemokine receptor (CCR)9 by anti-CD3 preconditioning results from a reduction of CD103+ DCs in draining mesenteric lymph nodes (LNs), which is associated with down-regulation of DC expression of CCR7, a receptor required for tissue DC migration to draining LNs. These results indicate that anti-CD3 preconditioning reduces not only tissue release of chemokines but also prevents tissue DC migration to draining LNs and subsequently reduces the capacity of DCs of draining LNs to imprint donor T-cell tissue tropism. Therefore, modulation of host DCs by anti-CD3 preconditioning before HCT represents a new approach for separating GVL from GVHD.

Natural killer T (NKT) cells recognize CD1d, and include invariant Jα18+ iNKT and non-invariant subpopulations. NKT prevent graft versus host disease (GVHD) via IL-4, but the mechanisms remain unclear. We have reported that donor CD8+ T cells are confined to host lympho-hematological tissues in anti-CD3-conditioned recipients, and the confinement is associated with presence of high percentage of residual host NKT. But it is unknown whether NKT can regulate T cell migration. In the current studies, using NKT deficient CD1d-/-, Rag-2-/-, and iNKT deficient Jα18-/- host-type mice, and by add-back of iNKT cells from wild-type or IL-4-/- host-type mice to in vivo transplantation and/or in vitro co-culture experiments, we observed that 1)host NKT down-regulate donor CD8+ T expression of α4β7, CCR5, CCR6, CXCR3 but upregulate expression of CCR3, as well as down-regulate GVHD target tissue expression of ccl3-5 and Cxcl9-11 chemokines in an IL-4 dependent manner. 2) iNKT and non-invariant NKT have redundant role in regulating donor T expression of chemokine receptors; but iNKT are required for down-regulating tissue release of chemokines. 3) iNKT down-regulate donor CD8+ T expression of α4β7 without reduction of IFN-g production. These indicate that NKT preventing GVHD can be via reducing donor T tissue migration capacity. (This work was supported by Nesvig Lymphoma foundation) Disclosures No relevant conflicts of interest to declare.

Abstract 3562 Poster Board III-499 A radiation-free conditioning regimen with anti-T cell globulin (ATG) + Busulfan (B) + Fludarabin (F) (ATG + BF) could replace sublethal total body irradiation (TBI) in clinic, but this regimen did not prevent induction of severe acute graft versus host disease (GVHD). We recently reported that anti-CD3 preconditioning markedly ameliorated acute GVHD in recipients conditioned with sublethal TBI in a mouse model of MHC-mismatched C57BL/6 (H-2b) donor to BALB/c (H-2d) host. In the current studies, we test whether replacing ATG with anti-CD3 prevented acute GVHD. We found that, when spleen and BM cells (50×106, each) from donors were transplanted, the recipients conditioned with ATG + BF developed severe acute GVHD and all (12/12) of them died by 30 days after transplantation, in contrast, the recipients conditioned with anti-CD3 + BF developed only moderate acute GVHD and 50% (6/12) of the recipients survived for more than 100 days. When the donor cells were titrated down to 25, 12.5, or 6.25 ×106, we found that none (0/8) of the recipients conditioned with ATG + BF developed chimerism, in contrast, all (8/8) of the recipients conditioned with anti-CD3 + BF developed complete chimerism, although the recipients showed mild to moderate clinical GVHD in a dose-dependent manner. Next, we tested whether depletion of donor CD4+ T cells could completely prevented GVHD in recipients conditioned with anti-CD3 + BF, and we found that, although all (8/8) of the recipients given 12.5 or 6.25×106 CD4+ T-depleted spleen (CD4−-SPL) cells developed complete chimerism and all survived for more than 100 days, the recipients still showed mild to moderate clinical GVHD (i.e. hair loss). Because vorinostat (V), a histone deacetylase inhibitor was previously reported to inhibit tissue release of proinflammatory cytokines, we tested whether replacing F with V could further ameliorated GVHD. We found when donor CD4−-SPL cells and BM cells (12.5 ×106 each) were transplanted, the recipients conditioned with anti-CD3 + BV showed no clinical GVHD, although the recipients conditioned with anti-CD3 + BF showed mild to moderate clinical GVHD. Furthermore, we found that donor CD4−-SPL and BM cells eliminated luciferase transfected BCL1 leukemia/lymphoma cells much more efficiently in the recipients conditioned with anti-CD3 + BV than in the recipients conditioned with anti-CD3 + BF, as revealed by in vivo bioluminescent imaging. In addition, we observed that the separation of GVL from GVHD was associated with prevention of donor T cell migration into GVHD target tissues, which resulted from reduction of CCR7+ DCs that induce donor T cell expression of tissue homing and chemokine receptors (i.e. a4b7, CCR9, E-Ligand, P-Ligand, CCR4, CCR10) in the recipients conditioned with anti-CD3 + BV. Taken together, anti-CD3 and vorinostat synergistically promote prevention of GVHD and retention of GVL in a clinically applicable radiation-free conditioning regimen. This work was supported by Marcus foundation. Disclosures: No relevant conflicts of interest to declare.

We reported that donor CD8+ T cells mediated graft versus leukemia (GVL) activity without causing graft versus host disease (GVHD) in anti-CD3-conditioned recipients, although the same dose of donor CD8+ T cells caused lethal GVHD in recipients conditioned with sublethal total body irradiation (TBI) (J. Immunol. 2007). We recently observed that donor CD8+ T cells from the liver of anti-CD3-conditioned recipients showed a marked reduction in proliferation in response to anti-CD3/CD28 stimulation, as compared to those from TBI-conditioned recipients, indicating that donor CD8+ T cells are tolerized in the tissues of anti-CD3-conditioned recipients. B7H1, a co-inhibitory molecule, is constitutively expressed by hematopoietic cells and expressed by parenchymal cells after IFN-γ induction. B7H1 tolerizes T cells by interaction with its ligand PD-1 on activated T cells. To explore the role of B7H1 in GVHD prevention in anti-CD3-conditioned recipients, donor C57BL/6 CD8+ T cells (20×106) and bone marrow cells (100×106) were transplanted into anti-CD3-conditioned wild-type or B7H1−/− BALB/c recipients. While the wild-type recipients all survived for more than 100 days without signs of GVHD, the B7H1−/− recipients developed severe GVHD with diarrhea, weight-loss, and hair-loss, and 70% of them died 60 days after transplantation. Similarly, while donor CD8+ T and BM cells induced little GVHD in unconditioned Rag-2−/− BALB/c recipients, they induced severe lethal GVHD in B7H1−/− Rag-2−/− BALB/c recipients. Furthermore, donor CD8+ T and BM cells still induced lethal GVHD in unconditioned chimeric B7H1−/−Rag-2−/− recipients reconstituted with B7H1+/+Rag-2−/− BM. In addition, we observed upregulation of B7H1 expression by hepatocytes and intestine epithelial cells of anti-CD3-conditioned BALB/c or unconditioned Rag-2−/− recipients after donor cells infusion, as judged by RT-PCR, flow cytometry analysis, and histoimmunostaining of B7H1. In vivo bioluminescent imagine showed much more severe tissue infiltration of donor T cells in B7H1−/− recipients as compared to B7H1+/+ recipients, and the in vitro proliferation of donor CD8+ T cells from the liver of the former recipients was much more vigorous than that of the latter recipients. These results demonstrate that B7H1 expression by tissue parenchymal cells rather than hematopoietic cells tolerizes infiltrating donor T cells in GVHD target tissues and prevents GVHD; and that the radiation-free anti-CD3-conditioning regimen can maintain this tissue protection mechanism. (This study is surpported by Marcus Foundation and NIH R01 AI 066008).

Host dendritic cells (DCs) play a critical role in initiating graft versus host disease (GVHD) and graft versus leukemia (GVL), and separation of GVL from GVHD remains a major challenge in the treatment of hematological malignancies by allogeneic hematopoietic cell transplantation (HCT). Here, we show that preconditioning with anti- CD3 mAb before conditioning with total body irradiation prevents GVHD but retains GVL in a HCT model of MHC-mismatched C57BL/6 donor to BALB/c host. Prevention of GVHD is associated with inhibition of donor T expression of homing and chemokine receptors and inhibition of GVHD target tissue expression of chemokines. Furthermore, inhibition of donor T expression of gut homing a4b7 and chemokine receptor CCR9 by anti-CD3 preconditioning results from reduction of CD103+ DCs in draining mesenteric lymph nodes, which is associated with downregulation of DC expression of CCR7, a receptor required for tissue DC migration to draining LN. These results indicate that anti-CD3 preconditioning reduces not only tissue release of chemokines but also prevents tissue DC migration to draining LN and subsequently reduces draining LN DC’s capacity in imprinting donor T tissue tropism. Therefore, modulation of host DCs by anti-CD3 preconditioning before HCT represents a new approach for separating GVL from GVHD. (Li and Chen contribute equally.

Donor T cell-mediated graft versus leukemia (GVL) and graft versus autoimmunity (GVA) plays a critical role in the treatment of hematological malignancies and refractory autoimmune diseases using allogeneic hematopoietic cell transplantation (HCT). However, graft versus host disease (GVHD) remains a major obstacle in classical HCT, where recipients are usually conditioned with total body irradiation or high dose chemotherapy. We have recently reported a radiation-free and GVHD preventive anti-CD3-conditioning regimen, which allows donor CD8+ T cells to facilitate engraftment and mediate GVL without causing GVHD (PNAS, 2007 and J. I. 2007). In order to promote the clinical application of the anti-CD3-conditioning regimen, we need to overcome the cytokine storm that causes flu-like syndrome in patients after anti-CD3 mAb injection and reduce the required donor bone marrow cell dose. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was reported by others to induce apoptosis of human T cell lymphoma cell lines and to reduce serum levels of pro-inflammatory cytokines in HCT recipients. Therefore, we tested whether conditioning with a combination of anti-CD3 and SAHA can reduce the cytokine storm caused by anti-CD3 and reduce the resistance of engraftment mediated by residual host T cells. Accordingly, titrated dose of SAHA (0.125–8 μM) were added to cultures of BALB/c spleen cells stimulated with plate-bound anti-CD3. We found that SAHA augmented apoptosis of anti-CD3 activated T cells in a dose-dependent manner. Although low-dose SAHA (0.125–0.25 μM) did not augment apoptosis, it rendered the residual live T cells partially unresponsive. At the same time, SAHA significantly reduced the pro-inflammatory cytokines (IL-2, IFN-γ, TNF-α, and IL-6) in the culture supernatants. Similarly, in vivo treatment with anti-CD3 and low-dose SAHA (40 μg/g) led to significant reduction of serum levels of those cytokines and partial unresponsiveness of the residual host T cells. Finally, conditioning with combined anti-CD3 and SAHA (40 μg/g) induced complete chimerism without GVHD in 12/12 of old BALB/c as well as in 8/8 of old autoimmune lupus NZB/NZW F1 recipients after two injections of BM cells (2 ×106/g) and CD4+ T-depleted spleen cells (4 ×106/g), although conditioning with anti-CD3 alone did not induce any chimerism (0/8). The chimeric NZB/NZW F1 recipients showed complete reversal of autoimmune glomerulonephritis and proteinuria. These results indicate that SAHA can not only reduce cytokine storm but also facilitate engraftment when combined with anti-CD3 for conditioning of recipients. This radiation free and GVHD preventive conditioning regimen may provide a novel approach for clinical HCT.

Hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies as well as refractory autoimmune diseases. However, graft versus host disease (GVHD) remains a major obstacle in classical HCT, where recipients are usually conditioned with total body irradiation or high dose chemotherapy. We recently reported that donor CD8+ T cells facilitated engraftment and mediated graft versus leukemia (GVL) without causing graft versus host disease (GVHD) in young (6–8 weeks old) MHC-mismatched mouse recipients conditioned with anti-CD3 mAb (Blood 2005). Thereafter, we observed that anti-CD3 conditioning alone was not sufficient for induction of chimerism in old (>12 weeks) recipients, due to the higher percentage of residual host CD8+ T cells in the old recipients. Romidepsin (Desipeptide), a histone deacetylase inhibitor, was reported to induce apoptosis of human T cell lymphoma lines. We hypothesize that depsipeptide will induce the apoptosis of anti-CD3 activated proliferating T cells, and that conditioning with a combination of anti-CD3 and depsipeptide will markedly reduce the residual host T cells and allow donor stem cell engraftment. To test our hypothesis, we first added Romidepsin (1.25–10 ng/ml) to cultures of T cells with or without anti-CD3 stimulation. We found that, although Romidepsin showed no effect on un-stimulated T cells, it augmented apoptosis of anti-CD3 activated T cells in a dose dependent manner, and the maximum augmentation was 5 fold. In addition, when Romidepsin (1.25–10 ng/ml) was added to a culture of mixed lymphocyte reaction (MLR), we found that it suppressed MLR in a dose dependent manner also, and the maximum suppression was greater than 98%. Second, old (> 12 weeks) BALB/c recipients were conditioned with one I.V. injection of anti-CD3 (20μg/g) and three I.P. injections (every other day) of Romidepsin at a dose of 0.4 μg/g. 7 days after anti-CD3 injection, recipients were injected with donor bone marrow cells (100×106) and CD4+- T depleted spleen (CD4−-SPL) cells (100×106). CD4−-SPL cells were injected again 7 days later. We found that, 4 weeks after HCT, 7/8 of the recipients conditioned with a combination of anti-CD3 and Romidepsin but only 1/8 of the recipients conditioned with anti-CD3 alone became chimeric. The recipients showed healthy appearance without signs of GVHD. The results are combined from two replicate experiments. This radiation free and GVHD preventive conditioning regimen may provide a novel approach for clinical HCT.