CD8 Tregs Promote Gvhd Prevention and Restore Impaired GVL Effect Mediated By CD4 Tregs in Mice

Abstract Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses. While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C)while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL). To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses. This work was supported by NIH grants: R01 CA118116 and R01 CA169116 Disclosures No relevant conflicts of interest to declare.

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Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽

10.1182/blood.v120.21.4670.4670 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4670-4670

Author(s):

Chang-Qing Xia ◽

Anna Chernatynskaya ◽

Clive Wasserfall ◽

Benjamin Looney ◽

Suigui Wan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Memory T Cells ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

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Role of STAT3 in Immunoregulatory Function of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v114.22.3637.3637 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3637-3637

Author(s):

Jinsun Yoon ◽

Seoju Kim ◽

Eun Shil Kim ◽

Byoung Kook Kim ◽

Young Lee

Keyword(s):

T Cells ◽

Conflicts Of Interest ◽

Hematologic Malignancies ◽

Treg Cells ◽

Human Mscs ◽

Hematopoietic Stem ◽

The One

Abstract Abstract 3637 Poster Board III-573 The one of the best curative treatment modality in hematologic malignancies is an allogeneic hematopoietic stem cell transplantation (HSCT). However, graft-versus-host disease (GVHD) is a major obstacle of allogeneic HSCT. BM derived human MSCs are known to have immunoregulatory effect in vitro and in vivo via inhibiting alloreactive T lymphocytes, leading to their clinical use for the prevention of GVHD in HSCT. However, the molecular mechanism of immunoregulatory effect of human MSCs is not fully understood. In this study, the signaling of immunoregulatory effect was investigated by co-culture of human MSCs with lymphocytes. The proliferation of allogeneic T cells was inhibited by MSCs. Among the STATs, STAT3 was a key molecule in MLR co-cultured with MSCs. STAT3 siRNA treated MSCs did not inhibit the lymphocyte proliferation. After MSCs were trasnsfected with STAT3 plasmid, the fraction of CD4+CD25+FOXP3+ cells (Treg cells) were increased, while the fraction of CD4+, CD8+, CD25+ was decreased. In addition, Th1-related cytokines (IL-2, IL-12 and INF-γ) and Th17-related cytokines (IL-6, IL-17 and IL-21) were down-regulated, and Th2-related cytokines (GATA-3, IL-4 and IL-10) were up-regulated in MLR co-cultured with STAT3-ablated MSCs, while vice versa in MLR co-cultured with STAT3-transfected MSCs. Furthermore, ELISA showed that concentration of Th1-related cytokine (IL-2) in the supernatant of MLR co-cultured with STAT3-ablated MSCs was higher than that of control; while concentration of Th2-related cytokine (IL-4) was lower than that of control. These results suggested that induction of Th1 to Th2 shift by MSCs might be mediated via STAT3 molecule. In summary, STAT3 may be an indispensable molecule in the immunoregulatory effect in human MSCs via modulation of regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

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Interruption of the IFN γR/CXCR3 Axis Results in Altered T Cell Trafficking In Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽

10.1182/blood.v118.21.2971.2971 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2971-2971

Author(s):

Jaebok Choi ◽

Edward Dela Ziga ◽

Julie Ritchey ◽

Julie Prior ◽

Lynne Collins ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Target Organs ◽

Donor T Cells

Abstract Abstract 2971 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. Using a MHC-mismatched GvHD model, B6 (H-2b) → Balb/c (H-2d), we demonstrated that infusion of IFN γR deficient allogeneic donor T cells induce significantly less GvHD, compared to WT T cells, determined by survival (74% vs. 0 % in overall survival; p =0.0004), weight and percentages of B220+ B cells (12.4% vs. 3.8%; p =0.0205), CD3+ T cells (14.3% vs. 4.3%; p =0.0025) in blood. Of note was that the IFN γR deficient donor T cells maintained a beneficial GvL effect, which was examined in both a systemic leukemia and a solid tumor model using luciferase-expressing A20 cells derived from Balb/c. We found that IFN γR deficient donor T cells responded normally to allogeneic antigens as measured by in vitro mixed lymphocyte reaction analyses, and express similar levels of granzyme B, compared to WT T cells. However, IFN γR deficient T cells trafficked predominantly to the spleen while WT T cells trafficked to gastrointestinal tract and peripheral lymph nodes, which are major GvHD target organs, based on in vivo bioluminescence imaging. All of these findings suggest that the reduced GvHD was not due to reduced function, altered subsets or relative deficiency of allogeneic donor T cells but from modification of in vivo trafficking of IFN γR deficient donor T cells compared to WT T cells. We further demonstrated that the IFN γR-mediated signaling in alloreactive donor T cells was required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. CXCR3−/− T cells demonstrated a reduction in GvHD while maintenance of the same robust GvL effect using the same MHC mismatched transplant model. Thus, the IFN γR-CXCR3 axis represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Current studies are focused on 1) whether forced expression of CXCR3 rescues the GvHD-inducing potential of IFN γR deficient donor T cells and 2) if inhibition of IFN γR signaling (IFN γR, JAK1 and/or JAK2, CXCR3 and STAT1) using both neutralizing antibodies and small molecule inhibitors can recapitulate the anti-GvHD and pro-GvL effects seen in IFN γR−/− and CXCR3−/− T cells. Disclosures: No relevant conflicts of interest to declare.

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Leukemia Cell-Derived Exosomes Targeted Dendritic Cells Can Induce More Strong Anti-Leukemia Response Than Exosomes Alone

Blood ◽

10.1182/blood.v118.21.4278.4278 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4278-4278

Author(s):

Chang Sheng ◽

Jing Sun ◽

Chun Wang ◽

Siguo Hao

Keyword(s):

Cell Line ◽

K562 Cell ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Disease Free Survival ◽

Membrane Vesicles ◽

Biological Properties ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Hematopoietic Stem

Abstract Abstract 4278 Exosomes are the newest family number of ‘bioactive vesicles’ that play important roles in antigen presentation. Recent clinical trials about exosomes have shown minor clinical benefit. However, exosomes derived from leukemia cell lines were less studied, In this study, we investigated the biological properties and anti-leukemia immunity of leukemia cell-derived exosomes (LEX). The results showed that like other tumor cells, similarly, leukemia cells can release exosomes too. Transmission electron microscopy showed that exosomes derived from NB4 cell, a promyloblastic leukemia cell line and K562 cell, a chronic myeloid leukemia cell line were membrane vesicles with diameter about 50–100μ m, and loading their specific moleculars such as RAR-α and ABL and adherent moleculars such as ICAM-1. Similarly, our data also demonstrated the presence of Hsp70 in these two leukemia cell-derived exosomes. The data from two-dimensional electrophoresis of K562 cell-derived exosomes showed that most of molecules expressing on leukemia cells were detected on their exosomes, indicating that LEX load most of proteins expressed on their original leukemia cells. Out data also demonstrated that LEX can induce anti-leukemia CTL response and LEX-targeted DC can induce more efficient anti-leukemia immunity compared with LEX alone. In conclusion, exosome-based therapies could be considered as a promising strategy to prolong disease-free survival in patients with leukemia after consolidation or hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of EZH2 Depletes MLL Fusion Leukemia Stem Cells Through Restoration of p16 Expression

Blood ◽

10.1182/blood.v120.21.3510.3510 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3510-3510

Author(s):

Koki Ueda ◽

Akihide Yoshimi ◽

Masahiro Nakagawa ◽

Satoshi Nishikawa ◽

Victor E Marquez ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

High Specificity ◽

Prolonged Survival ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Colony Forming Capacity

Abstract Abstract 3510 Leukemia stem cells (LSCs) are resistant to conventional chemotherapy and persistent LSCs after chemotherapy are supposed to be a major cause of disease relapse or refractoriness. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified or used in clinical settings so far. Epigenetic regulators are associated with many cellular processes such as cell cycle, proliferation, and apoptosis. Of note are polycomb group proteins, because they potentially control stemness including activity of cancer stem cells, and can be pharmacologically targeted by a selective inhibitor of H3K27, 3-deazaneplanocin A (DZNep). We first administrated DZNep to MLL-related leukemia mouse model in order to test whether DZNep has potential to eradicate LSCs of the leukemic mice. Remarkably, the leukemic granulocyte-macrophage progenitors (LGMPs) in MLL/AF9 positive cells were significantly decreased in number by administration of DZNep while AraC did not affect the number of LGMPs, which implied that LSCs were targeted by DZNep. These data were reproduced by transplantation assays using short hairpin RNA (shRNA)-mediated knockdown of EZH2, a major component of polycomb repressive complex 2 (PRC2) which is responsible for H3K27 tri-methylation. Significantly, DZNep administration to wild-type mice led to only mild suppression of hematopoiesis, suggesting that this agent spares normal hematopoietic stem cells while eliminating LSCs, which is consistent with a previous report that genetic depletion of EZH2 did not compromise adult hematopoiesis in mice. Serial replating assay of MLL/AF9-induced leukemia cells showed that DZNep treatment in vivo diminished their colony forming capacity. Limiting dilution transplantation assays revealed that frequency of LSCs was markedly reduced by DZNep administration. DZNep treatment or EZH2 knockdown significantly prolonged survival of MLL/AF9 and MLL/ENL leukemic mice. To elucidate a molecular mechanism underlying the effects of DZNep on LSCs, we investigated transcriptional or epigenetic changes during DZNep treatment and EZH2 knockdown. Gene expression profiling revealed that p16 was significantly upregulated by EZH2 knockdown or DZNep administration. Knockdown of p16 completely canceled the survival advantage of the leukemia mice which received DZNep in vivo and restored the colony forming capacity of leukemia cells transduced with shRNA for EZH2 in vitro. These results supported the idea that p16 upregulation derived from EZH2 attenuation is central to the LSC reduction. Next, we investigated epigenetic status around p16 promoter and transcription start site (TSS) by chromatin immunoprecipitation (ChIP) assays. In MLL/ENL leukemia cells, both H3K4 and H3K27 methylation marks were highly enriched around the TSS of p16, together with EZH2 and Bmi1, a component of PRC1. Therefore removal of EZH2 is supposed to convert the promoter of p16 from a bivalent to an active state. The results of ChIP assays also indicated that MLL/ENL fusion protein binds to p16 coding region. In order to clarify whether dependency on EZH2 is specific for MLL fusion leukemia or can be applied for other types of leukemia, we evaluated the consequence of EZH2 inhibition in several types of leukemia. DZNep or shRNA for EZH2 strongly suppressed the proliferation of leukemia cell lines and immortalized cells harboring MLL fusion genes with high specificity. Administration of DZNep or transduction of shRNA targeting EZH2 significantly prolonged survival of MLL/AF9 and MLL/ENL-induced leukemia mice while TEL/PDGFRA-AML1/ETO-induced leukemia was not sensitive to DZNep, although bone marrow (BM) cells from either mice became globally hypo-methylated on H3K27 by exposure to this drug. Serial replating assay with DZNep or EZH2-shRNA demonstrated high sensitivity to EZH2 inhibition of MLL/AF9-transduced BM cells but not of AML1/ETO-transduced BM cells, E2A/HLF-transduced BM cells, or normal c-kit+ BM cells. Thus, the anti-leukemia effect of EZH2 inhibition is thought to be specific for MLL fusion leukemia. Collectively, our findings indicate that EZH2 is a potential therapeutic target of LSCs of MLL fusion leukemia to overcome the poor prognosis, encouraging the development of inhibitors against EZH2 with high specificity. Disclosures: No relevant conflicts of interest to declare.

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Enhanced Expression of PD-1 Modulates CD4+Foxp3+ Regulatory T Cell Homeostasis during Low-Dose IL-2 Therapy in Patients with Chronic Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v124.21.662.662 ◽

2014 ◽

Vol 124 (21) ◽

pp. 662-662

Author(s):

Takeru Asano ◽

Haesook T. Kim ◽

John Koreth ◽

Robert J Soiffer ◽

Yusuke Meguri ◽

...

Keyword(s):

T Cells ◽

Low Dose ◽

Conflicts Of Interest ◽

Self Regulation ◽

In Vitro Proliferation ◽

Ki 67 ◽

Hematopoietic Stem ◽

Treg Population ◽

Significant Difference

Abstract CD4+Foxp3+ regulatory T cells (Treg) play a central role in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that low-dose IL-2 administration preferentially increased Treg in patients with active cGVHD and resulted in clinical improvement with only minor toxicities (NEJM 2011). In the clinical trial, IL-2 induced selective and rapid proliferation of Treg in the first week of therapy but proliferation subsequently returned to baseline levels despite continued daily administration of IL-2 (Matsuoka et al. Sci Trans Med 2013). Mechanisms that limit Treg proliferation may play an important role in Treg homeostasis as continuous high-level proliferation may increase Treg susceptibility to apoptosis resulting in overall reduction of the Treg population (Matsuoka et al. JCI 2010). To examine mechanisms for negative regulation of Treg proliferation we examined expression of Programed Death-1 (PD-1) on Treg during IL-2 therapy. Serial blood samples from 14 patients who received daily subcutaneous IL-2 (3x105-3x106IU/m2/day) for 8 weeks were studied before and at 1, 2, 4, 6, 8, 10 and 12 weeks after starting IL-2. Treg were compared to conventional CD4+Foxp3- T cells (Tcon) within individual patient samples and examined for expression of PD-1. Treg and Tcon were further divided into subpopulations by the expression of CD45RA. Before IL-2 therapy, both CD45RA+ naïve Treg and Tcon showed little expression of PD-1 (%PD-1+ cells; median 2.1%, 1.8%, respectively). However, both CD45RA- activated/memory Treg and Tcon showed significantly higher expression of PD-1 than their naïve counterparts (%PD-1+ cells: median 22%, p<0.0001; median 19%, p<0.0001, respectively) and there was no significant difference in PD-1 expression between CD45RA- Treg and Tcon. After starting IL-2, expression of PD-1 rapidly increased, mainly on the CD45RA- activated/memory Treg subpopulation and this was sustained during 8 weeks of IL-2 therapy (%PD-1+ Treg; median 38.7% at 4 weeks, p<0.002 vs baseline). In contrast, Tcon PD-1 expression did not change during IL-2 therapy. To confirm the inhibitory function of PD-1 on Treg in patients receiving IL-2, we purified Treg and Tcon by cell-sorting, labeled cells with CFSE and cultured each subset separately with or without blocking anti-PD-L1 antibody, in the presence of anti-CD3 and anti-CD28 stimulation for 4 days. Treg obtained during IL-2 therapy showed resistance to in vitro proliferation, but vigorous proliferation was regained in the presence of anti-PD-L1 mAb. In contrast the effect of PD-L1 blockade on Tcon was relatively small. These data suggest that the PD-1 selectively limits Treg proliferation during IL-2 therapy. Based on these findings, we explored the possibility of a novel immune strategy to enhance Treg expansion during low-dose IL-2 by combination with PD-1 blockade using a murine BMT model. Lethally irradiated B6D2F1 mice were transplanted with 1x106 spleen cells from the control B6 mice together with 5x106 TCD-BM from either PD-1 KO B6 or control B6 donors. Recipients were treated with 5x103 IU IL-2 or control vehicle from day 35 to 49. As expected, recipients transplanted with cells from control donors showed significantly higher expression of PD-1 on Treg than Tcon when treated with IL-2 (%PD-1+ cells; median 47.2%, 8.9%, respectively, p<0.0001) and the increase of Treg after IL-2 treatment was more evident in recipients transplanted from PD-1 KO donors (p<0.05). To examine this clinically, we tested whether anti-PD-1 mAb can boost Treg proliferation during IL-2 administration and we confirmed that IL-2 induced Treg proliferation was significantly enhanced with the combined use of anti-PD-1 antibody (%Ki-67 + cells; median 22.9% vs 33.2%, p<0.05). In conclusion, these findings indicate that the self-regulation of Treg homeostasis through the PD-1/PD-L1 pathway can limit the expansion of Treg mediated by exogenously administered IL-2. Blockade of PD-1/PD-L1 pathway may provide an opportunity for in vivo manipulation of Treg homeostasis. Our data provide important information for developing therapeutic strategies to modulate Treg homeostasis in vivo to promote immune tolerance. Disclosures No relevant conflicts of interest to declare.

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Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

PLoS ONE ◽

10.1371/journal.pone.0076607 ◽

2013 ◽

Vol 8 (10) ◽

pp. e76607 ◽

Cited By ~ 26

Author(s):

Erin Hertlein ◽

Kyle A. Beckwith ◽

Gerard Lozanski ◽

Timothy L. Chen ◽

William H. Towns ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

In Vivo Studies ◽

Leukemia Cell Line ◽

Chronic Lymphocytic Leukemia Cell ◽

Lymphocytic Leukemia Cell

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A Selective TNFR2 Agonist Expands Host Treg Cells in Vivo to Protect from Acute Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v124.21.1099.1099 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1099-1099 ◽

Cited By ~ 1

Author(s):

Andreas Beilhack ◽

Martin Chopra ◽

Marlene Biehl ◽

Martin Vaeth ◽

Andreas Brandl ◽

...

Keyword(s):

T Cells ◽

Graft Versus Host Disease ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Peripheral Tissues ◽

Host Disease ◽

Graft Versus Host ◽

Pre Treatment

Abstract Donor CD4+Foxp3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) while maintaining the anti-tumoral effect of transplanted conventional T cells in preclinical mouse models. Current clinical study protocols with donor Tregs for treatment or prophylaxis of GVHD rely on their ex vivo expansion and infusion in high numbers. Here we present a fundamentally novel strategy for inhibiting GVHD that is based on the in vivo expansion of recipient Tregs prior to allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in Treg biology. To this end we constructed a recombinant nonameric TNFR2-specific agonist, mimicking the activity of murine membrane-bound TNF on TNFR2 without TNFR1 stimulation, thereby avoiding the inflammatory side effects observed with conventional TNF. In vitro, this TNFR2-agonist expanded natural Tregs from wild type but not from TNFR2 KO mice. Accordingly, a human variant of this TNFR2-specific agonist expanded human Tregsin vitro. In vivo treatment of healthy mice with the murine TNFR2-agonist significantly increased Treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute GVHD. Next, we pre-treated recipient mice with this novel TNFR2-agonist to expand host-type radiation resistant Tregs prior to of allo-HCT in two models across MHC barriers (C57BL/6, H-2b->Balb/c, H-2d and FVB/N, H-2q->C57BL/6, H-2b). TNFR2-agonist pre-treatment resulted in significantly prolonged survival and reduced GVHD severity when compared to TNFR2-deficient recipients or untreated allo-HCT recipients. This was accompanied by reduced donor T cell proliferation and infiltration into GVHD target organs as assessed by in vivo and ex vivo bioluminescence imaging, flow cytometry and immunofluorescence microscopy. While in vivo TNFR2-agonist pre-treatment protected allo-HCT recipients from GVHD, anti-tumor effects of transplanted T cells remained unaffected in two different murine B cell leukemia models. In vivo depletion of host derived Tregs completely abrogated the protective effect of TNFR2-agonist pre-treatment. Our study shows that the expansion of host Tregs by selective in vivo TNFR2-activation significantly improves the outcome after allo-HCT and results in prolonged tumor-free survival. Disclosures No relevant conflicts of interest to declare.

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Busulphan Metabolism Via Flavin-Containing Monooxygenase 3 (FMO3) Can Explain Several Interactions with Other Drugs

Blood ◽

10.1182/blood.v124.21.1150.1150 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1150-1150

Author(s):

Ibrahim El Serafi ◽

Manuchehr Abedi-Valugerdi ◽

Seán Naughton ◽

Maryam Saghafian ◽

Jonas Mattsson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Plasma Concentrations ◽

Liver Microsomes ◽

In Vivo Studies ◽

High Dose ◽

Disappearance Rate ◽

Hematopoietic Stem ◽

Kinetics Of

Abstract Hematopoietic stem cell transplantation (HSCT) is a curative treatment for several malignant and non-malignant diseases. Busulphan (Bu) is an alkylating agent that is used in high doses as a part of the conditioning regime prior to HSCT. Busulphan is metabolized mainly in the liver by conjugation with glutathione (GSH) by glutathione transferases (GST), in particular, GSTA1. Busulphan interactions with drugs metabolized by hepatic enzymes other than GSH/GST have been reported. The aim of the present study was to investigate the role of other enzymes than GSH/GST in the busulphan metabolism including FMO3 and cytochrome P450 enzymes (CYPs). In our in vitro studies, human liver microsomes were incubated with busulphan first core metabolite, tetrahydrothiophene (THT). The incubation results showed a rapid disappearance of THT and gradual formation of the other metabolites. THT incubation with recombinant enzymes showed FMO3 to give the highest Initial THT disappearance rate (v0, 6.87 µmol/min/mL) and the highest intrinsic clearance(CLint, 0.26 µL/min/mg protein) followed by other CYPs. Comparing CYPs based on the initial THT disappearance rate (v0) per POR/CYP ratio, CYP2C8 had the highest rate for THT metabolism (5.03 nmol/min/(POR/CYP ratio) followed by CYPs 2C9, 2C19, 2E1 and 3A4. These results showed that THT is metabolized by microsomal enzymes and FMO3 is the main enzyme. For in vivo studies, we determined the kinetics of Bu in mice in the presence or absence of phenylthiourea (PTU), an inhibitor of FMO3. Moreover, the effect of THT accumulation on Bu metabolism was also determined in the same species. Treatment with PTU resulted in increase of Bu plasma concentrations as expressed as AUC. Furthermore, synchronized treatment of mice with THT and Bu also enhanced the plasma levels of Bu as compared to animals receiving Bu alone. After THT dosing, THT concentrations and AUC significantly (P < 0.05) increased after concurrent PTU injection. THT concentrations and AUC for mice treated with Bu and PTU also significantly (P < 0.05) increased compared to mice injected with Bu alone. To evaluate the role of FMO3 in the kinetics of Bu in the clinical settings, we investigated twelve patients undergoing HSCT and conditioned with busulphan / cyclophosphamide. A significant up-regulation (P < 0.05) of mRNA was found for FMO3 after Bu conditioning as confirmed using qRT-PCR. The up-regulation observed for FMO3 was similar to what was observed for GSTA1 (P < 0.05). To further confirm the involvement of FMO3 on in vivo Bu kinetics in humans, blood samples were drawn routinely from a patient treated with high dose busulphan and concomitant administration of voriconazole. Voriconazole is an antimycotic drug is wieldy used in hematology and HSCT and is known to be metabolized via FMO3. High busulphan levels that were not relevant to the administered dose were detected in this patient. Measuring THT in the samples showed that THT levels detectable with high accumulation and slow oxidation rate. In conclusion, our results showed that FMO3 and, to a lesser extent, CYPs are involved in busulphan metabolism through metabolism of its first core metabolite, THT. FMO3 is up regulated during Bu conditioning. FMO3 inhibition affects Bu kinetics. Our findings may offer valuable explanations into several drug interactions involving busulphan during HSCT conditioning therapy and thus, lower treatment related toxicity reported in HSCT. Disclosures No relevant conflicts of interest to declare.

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Use of FoxP3 Diptheria Toxin Receptor (Foxp3DTR) Donor T Cells to Define the Mechanism Involved in the Inhibition of GvHD by Azacytidine in Vivo

Blood ◽

10.1182/blood.v120.21.4113.4113 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4113-4113

Author(s):

Matthew L Cooper ◽

Jaebok Choi ◽

Julie Ritchey ◽

John F. DiPersio

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Conflicts Of Interest ◽

Similar Rate ◽

Primary Treatment ◽

Hematopoietic Stem ◽

Donor T Cells ◽

Toxin Receptor ◽

Apoptotic Effects

Abstract Abstract 4113 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired graft versus leukemia (GvL) effect is frequently accompanied by Graft versus Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression or,1 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells.2 To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT).3 To induce GvHD, 1×107 panT cells obtained from Foxp3 DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16 +18 +20 (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery. Mice injected with PBS (n=5) or DT (n=5) (without azaC) developed severe GvHD and died by day 26 post HSCT. In sharp contrast, 100% of mice (n=5) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC +DT (n=5) resulted in significantly prolonged survival compared to the PBS treated mice (p=0.003) but in contrast to the azaC group, failed to survive beyond day 60 (p=0.003). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=0.001), CD3+ T cells (p=0.002), and B220+ B cells (p=0.02) than the azaC +DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. These results suggest that BOTH pharmacologic conversion of alloreactive FoxP3- donor T cells to FoxP3-expressing Tregs and direct toxicity to allogeneic T cells by azaC represent important mechanisms involved in the amelioration of GvHD by azaC in vivo. Disclosures: No relevant conflicts of interest to declare.

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