Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

Abstract The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

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Correction of Murine Hemophilia A by Hematopoietic Stem Cell Gene Therapy.

Blood ◽

10.1182/blood.v106.11.1291.1291 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1291-1291

Author(s):

Robert G. Hawley ◽

Morvarid Moayeri ◽

Teresa S. Hawley

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Hemophilia A ◽

Bone Marrow Cells ◽

Sublethal Dose ◽

Peripheral Blood Cells ◽

Hematopoietic Stem ◽

Fviii Inhibitor ◽

Marrow Cells

Abstract A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally-delivered FVIII transgene offers the possibility to permanently correct the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based oncoretroviral vector encoding a secretion-enhanced B domain-deleted human FVIII transgene linked to a downstream EGFP reporter gene into immunocompetent hemophilia A mice (FVIII exon 16 knockout mice on a C57BL/6 background) that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy) or a nonmyelablative preparative regimen involving busulfan (two intraperitoneal doses of 10 mg/kg). Both groups of irradiated mice were transplanted with 2 × 106 sorted EGFP+ bone marrow cells. At 26 weeks, 48 ± 24% (n = 10) and 18 ± 11% (n = 12) EGFP+ nucleated peripheral blood cells were detected in mice conditioned with 800 and 550 cGy irradiation, respectively. The busulfan-conditioned mice (n = 4) were transplanted with 15–20 × 106 transduced unsorted bone marrow cells. One mouse died at 4 weeks posttransplant due to an unknown cause. The reconstitution kinetics of the remaining three mice was very similar to the group of mice conditioned with 550 cGy irradiation (18 ± 7% of their nucleated peripheral blood cells were EGFP+ at 26 weeks posttransplant). Broad transcriptional activity of the vector was observed in cells belonging to both the myeloid and lymphoid lineages in peripheral blood, and in donor-derived cells residing within the bone marrow, spleen and thymus. Importantly, therapeutic levels of FVIII (42%, 18% and 11% of normal, respectively, by COATEST assay) were detected in the plasma of all recipients 22–26 weeks posttransplant. When the mice were subsequently challenged with high doses of recombinant human FVIII (up to eight intravenous injections of 5–10 IU of recombinant full-length human FVIII at weekly intervals) to investigate the durability of tolerance induction, only minimal levels of inhibitor antibodies were detected in a subset of the corrected animals (0.8 ± 0.6, 0.7 ± 0.5 and 3 ± 4 Bethesda units per ml) in contrast to the robust anti-FVIII inhibitor response seen following immunization of naive hemophilia A mice (98 ± 48 Bethesda units per ml; n = 11). Suppression of the immune response to human FVIII was specific, as gene-treated mice mounted a normal humoral immune reaction to an unrelated antigen, tetanus toxoid. While we make no claims as to the nature of the hyporesponsive states elicited in each case, the results obtained in the small cohort of busulfan-conditioned animals are particularly exciting since the experimental protocol more closely approximates a clinically-acceptable situation, both in terms of a mild conditioning regimen as well as the lack of a preselection step for transduced bone marrow cells. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

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Bone Marrow Memory CD8+ T Cells Play a Supporting Role in Hematopoiesis

Blood ◽

10.1182/blood.v124.21.4370.4370 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4370-4370

Author(s):

Sulima Geerman ◽

Fernanda M Pascutti ◽

Sudeep Bhushal ◽

Martijn A. Nolte

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Conflicts Of Interest ◽

Lymphoid Organ ◽

Strong Impact ◽

Activated T Cells ◽

Hematopoietic Stem ◽

Memory Cd8 T Cells

Abstract The bone marrow (BM) has an important function as primary lymphoid organ through the process of hematopoiesis. This process is sustained by hematopoietic stem cells (HSCs) and their life-long production of new blood cells, which is made possible by their unique ability to self-renew. Next to this, BM also functions as a secondary lymphoid organ, as it can mediate primary T cell responses against invading pathogens. We and others have shown that activated T cells can influence the hematopoietic process through the production of pro-inflammatory cytokines. This indicates that adaptive immune responses and hematopoiesis are intertwined in the BM, though most of the cellular and molecular interactions that occur during this crosstalk are yet unknown. Based on previous observations that T cell deficient mice have altered hematopoiesis and that depletion of T cells from allogeneic BM grafts compromises HSC engraftment, we questioned to what extent BM T cells can directly affect the function of HSCs. To test this, we sorted and co-cultured murine BM T cells with HSCs (Lin-Sca-1+c-Kit+CD48-CD150+). We found that particularly BM CD8+ central memory (CD44+CD62L+) T cells (Tcm) enhance the capacity of HSCs to self-renew. Furthermore, we found that TCR-transgenic mice, which do not have memory T cells, have lower numbers of HSCs, which could subsequently be increased by transferring BM CD8+ Tcm. Remarkably, an increase in HSC numbers was also observed when HSCs were cultured with only supernatant derived from BM CD8+ Tcm. Moreover, HSCs cultured with supernatant from BM CD8+ Tcm and later transplanted in myeloablated hosts displayed a strongly enhanced ability to restore hematopoiesis. Importantly, the strong impact of BM T cells on HSCs was not only apparent in the steady state situation, but also following a viral infection with either acute or chronic lymphocytic choriomeningitis virus (LCMV). We could establish that both acute and chronic LCMV-specific CD8+ T cells or supernatant from these cells could increase the HSC self-renewal capacity. In conclusion, our findings demonstrate that BM memory CD8+ T cells can positively influence the function of HSC through soluble mediators. We postulate that this process is particularly relevant after immune activation, in order to protect and/or restore the HSC pool and the subsequent hematopoietic recovery. We are currently using a proteomics approach to identify these soluble mediator produced by CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

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Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis

Blood ◽

10.1182/blood.v116.21.4314.4314 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4314-4314

Author(s):

Akm Khyrul Wara ◽

Kevin Croce ◽

ShiYin Foo ◽

Xinghui Sun ◽

Basak Icli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Hindlimb Ischemia ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Deficient Mice ◽

Tgf Β1 ◽

Impaired Angiogenesis

Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P<0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P<0.0001; GMPs: WT 32.8% vs. KO 8.97%, P<0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P< 0.0004; GMPs: WT 275.25 vs. KO 108.75, P <0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P<0.00004; GMPs: WT 635.66 vs. KO 263.66, P<0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P <0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P<0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Targeting FVIII Expression to Platelets Induces Immune Tolerance in Hemophilia A Mice with or without Pre-Existing Anti-FVIII Immunity,

Blood ◽

10.1182/blood.v118.21.4170.4170 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4170-4170

Author(s):

Yingyu Chen ◽

Erin L. Kuether ◽

Jocelyn A. Schroeder ◽

Robert R. Montgomery ◽

David W. Scott ◽

...

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Immune Tolerance ◽

Hemophilia A ◽

Conflicts Of Interest ◽

High Titer ◽

Control Group ◽

Hematopoietic Stem ◽

Therapy Strategy ◽

Inhibitory Antibodies

Abstract Abstract 4170 Our previous studies have shown that targeting FVIII expression to platelets (2bF8) can correct murine hemophilia A phenotype even in the presence of inhibitory antibodies. In the present study, we wanted to explore 1) whether platelets containing FVIII can act as an immunogen; and 2) whether platelet-derived FVIII can induce immune tolerance in a hemophilia A mouse model. To investigate whether platelets containing FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets to naïve FVIIInull mice weekly for 8 weeks. These platelets were between 30 to 50% of total platelets upon infusion and the levels of platelet-FVIII in the infused animals were 0.11 ± 0.01 mU/108 platelets (n = 6) one week after infusion. No anti-FVIII inhibitory antibodies were detected in the infused mice during the study course. All animals developed inhibitors following further challenged with recombinant human FVIII (rhFVIII) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger immune response in hemophilia A mice. To explore whether platelet-derived FVIII will act as an immunogen in the presence of primed spleen cells (from mice already producing inhibitory antibody), we co-transplanted splenocytes from highly immunized FVIIInull mice and bone marrow (BM) cells from 2bF8 transgenic mice into 400 cGy sub-lethal irradiated FVIIInull recipients. We monitored the levels of inhibitory antibodies in recipients for up to 8 weeks and found that inhibitor titers declined with time after transplantation. We then challenged co-transplantation recipients with rhFVIII and found that inhibitor titers in the control group co-transplantat of FVIIInull BM cells increased 103.55 ± 64.83 fold (n = 4), which was significantly more than the group receiving 2bF8 transgenic BM cells (14.34 ± 18.48, n = 5) (P <.05). The inhibitor titers decreased to undetectable in 40% of 2bF8 transgenic BM cells co-transplantation recipients even after rhFVIII challenge, indicating immune tolerance was induced in these recipients. To further explore the immune response in the lentivirus-mediated platelet-derived FVIII gene therapy of hemophilia A mice, we transduced hematopoietic stem cells from pre-immunized FVIIInull mice with 2bF8 lentivirus (LV) followed by syngeneic transplantation into pre-immunized lethally irradiated FVIIInull recipients and monitored the levels of inhibitor titers in recipients. After full BM reconstitution, platelet-FVIII expression was sustained (1.56 ± 0.56 mU/108platelets, n = 10), but inhibitor titers declined with time, indicating that platelet-derived FVIII does not provoke a memory response in FVIIInullmice that had previously mounted an immune response to rhFVIII. The t1/2 of inhibitor disappearance in 2bF8 LV-transduced recipients (33.65 ± 11.12 days, n = 10) was significantly shorter than in untransduced controls (66.43 ± 22.24 days, n = 4) (P <.01). We also transplanted 2bF8 LV-transduced pre-immunized HSCs into 660 cGy sub-lethal irradiated naïve FVIIInull mice. After BM reconstituted, recipients were assessed by platelet lysate FVIII:C assay and tail clip survival test to confirm the success of genetic therapy. Animals were then challenged with rhFVIII. Only 2 of 7 2bF8 LV-transduced recipients developed inhibitory antibodies (55 and 87 BU/ml), while all untransduced control developed high titer of inhibitors (735.50 ± 94.65 BU/ml, n = 4). In conclusion, our results demonstrate that 1) platelets containing FVIII are not immunogenic in hemophilia A mice; and 2) platelet-derived FVIII may induce immune tolerance in hemophilia A mice with or without pre-existing inhibitory antibodies. This tolerance induction would add an additional significant benefit to patients with platelet-derived FVIII gene therapy strategy because protein infusion could be administered in some special situations (e.g. surgery in which a greater levels of FVIII may be required) with minimized risk of inhibitor development. Disclosures: No relevant conflicts of interest to declare.

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The Role of Bone Marrow T Cells in Regulating Hematopoietic Stem Cell Function.

Blood ◽

10.1182/blood.v120.21.2349.2349 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2349-2349

Author(s):

Claudia Brandao ◽

Alexander M. de Bruin ◽

Martijn A. Nolte

Keyword(s):

Bone Marrow ◽

T Cells ◽

Immune System ◽

T Cell ◽

Cd8 T Cells ◽

Feedback Mechanism ◽

T Cell Subsets ◽

Effector Memory ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2349 After immune activation, effector/memory T cells, including virus-specific CD8 T cells, are known to migrate to the bone marrow (BM), where they can be maintained by the production of IL-15 by the stroma; however, it is not yet known whether these T cells also have a function at this site. Since depletion of T cells from allogenic BM grafts compromises HSC engraftment, we hypothesize that T cells can directly influence the balance between differentiation and self-renewal of hematopoietic stem cells (HSCs). To test the ability of T cells to affect hematopoiesis, we performed co-cultures of HSCs and T cells isolated from murine BM. We found that T cells localized in the BM are able to enhance HSC differentiation as well as their self-renewal capacity. This feature is specific for BM central memory (CM) CD8 T cells, since other T cell subsets are not able to affect HSCs to the same extent. Moreover, depletion of CM CD8 T cells from the total BM T cell pool abrogates the impact on HSC differentiation and self-renewal, indicating that this particular T cell population is both sufficient and required for the observed effects. BM CM CD8 T cells do not affect quiescence of HSCs, but do enhance their proliferative capacity, and we found that supernatant from CM CD8 T cells is sufficient for this effect. Interestingly, competitive transplantation assays showed that HSCs cultured with CM CD8 T cells-derived supernatant contribute much better to leukocyte formation than medium-treated HSCs. This effect is seen in both the myeloid and lymphoid compartment, indicating that CM CD8 T cells are able to release soluble factors that support and enhance the multilineage reconstitution capacity of HSCs. Functional studies with blocking antibodies or knock-out mice showed that the supernatant-mediated effect is not caused by the hematopoietic cytokines IL3, IL6, IL21, GM-CSF, RANTES, TNFα or IFNγ. Preliminary data indicate that this feedback mechanism of the immune system on the hematopoietic process in the bone marrow is also present in the human situation, since autologous BM T cells increase the numbers of human HSCs, as well as their differentiation capacity. Overall, these findings demonstrate that T cells have an important function in the BM and that especially CD8 TCM cells can directly influence HSC homeostasis. We postulate that this feedback mechanism of the immune system on the hematopoietic process in the BM is particularly relevant during viral infection, as the efficient migration of virus-specific CD8 T cells to the BM could well benefit the replenishment of the HSC/progenitor cell compartment and restoration of blood cell numbers that got lost upon infection. Disclosures: No relevant conflicts of interest to declare.

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Prolonged Thrombocytopenia After Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Its Association with an Increase in CD8+ CX3CR1+ Cells in Bone Marrow.

Blood ◽

10.1182/blood.v120.21.3077.3077 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3077-3077

Author(s):

Xiao-hui Zhang ◽

Guo-xiang Wang ◽

Yan-rong Liu ◽

Lan-Ping Xu ◽

Kai-Yan Liu ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Surface Expression ◽

T Cell Subsets ◽

Control Group ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Abstract 3077 Background: Since prolonged thrombocytopenia (PT) is an independent risk factor for poor clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the underlying mechanisms need to be understood in order to develop selective treatments. Previous studies1–4 have suggested that abnormalities in B cells may play a role in the pathogenesis of PT. However, abnormalities in B cells alone do not fully explain the complete pathogenic mechanisms of PT. Our previous studies5 showed that the frequency of megakaryocytes with a ploidy value ≤ 8N was significantly increased in patients who developed PT after allo-HSCT compared to the control group. Mechanisms concerning the megakaryocyte hypoplasia in PT after allo-HSCT are not well understood. Design and Methods: PT was defined as a platelet count ≤80 × 109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia, such as aGVHD, disease recurrence, CMV infection, or antiviral drug treatment at three months post-HSCT when all other blood cell counts had return to normal.5 We analyzed T cell subsets in bone marrow (BM) and peripheral blood (PB) from allo-HSCT recipients with and without PT (n = 23 and 17, respectively) and investigated the expression characteristics of homing receptors CX3CR1, CXCR4 and VLA-4 by flow cytometry. Futhermore, Mononuclear cells (MNCs) from PT patients and controls were cultured with and without autologous CD8+ T cells in vitro, and clarify the effect of activated CD8+ T cells on the ploidy and apoptosis of megakaryocytes in the bone marrow. Results: The results demonstrated that the percentage of CD3+ T cells in the BM was significantly higher in PT patients than the experimental controls (76.00 ± 13.04% and 57.49 ± 9.11%, respectively, P < 0.001), whereas this difference was not significant for the PB (71.01 ± 11.49% and 70.49 ± 12.89%, respectively, P = 0.911). While, some T cell subsets in the BM and PB from allo-HSCT recipients with PT were not significantly different from that of the experimental control group, such as CD8+ T cells, CD4+ T cells, CD4+ CD25bright T cells (regulatory T cells), CD44hi CD62Llo CD8+ T cells and naive T cells (CD11a+ CD45RA+). Furthermore, the surface expression of homing receptor CX3CR1 on BM T cells (64.16 ± 14.07% and 37.45 ± 19.66%, respectively, P < 0.001) and CD8+ T cells (56.25 ± 14.54% and 35.16 ± 20.81%, respectively, P = 0.036), but not in blood, were significantly increased in PT patients compared to controls. For these two groups of patients, the surface expression of CXCR4 and VLA-4 on T cells and CD8+ T cells from both BM and PB did not show significant differences. Through the study in vitro, we found that the activated CD8+ T cells in bone marrow of patients with PT might suppress apoptosis (MNC group and Co-culture group: 18.02 ± 3.60% and 13.39 ± 4.22%, P < 0.05, respectively) and Fas expression (MNC group and Co-culture group: 21.10 ± 3.93 and 15.10 ± 2.33, P <0.05, respectively) of megakaryocyte. In addition, megakaryocyte with a ploidy value ≤ 8N (MNC group: 40.03 ± 6.42% and 24.54 ± 4.31%, respectively, P < 0.05) was significantly increased in patients with PT compared to the control group. Conclusions: In conclusion, an increased surface expression of CX3CR1 on T cells may mediate the recruitment of CD8+ T cells into the bone marrow in patients with PT who received an allo-HSCT. Moreover, CD8+CX3CR1+ T cells, which can have significantly increased numbers in bone marrow of patients with PT, likely caused a reduction in the megakaryocyte ploidy, and suppressed megakaryocyte apoptosis via CD8+ T cell-mediated cytotoxic effect, possibly leading to impaired platelet production. Therefore, treatment targeting CX3CR1 should be considered as a reasonable therapeutic strategy for PT following allo-HSCT. 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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Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v120.21.513.513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 513-513

Author(s):

Pekka Jaako ◽

Shubhranshu Debnath ◽

Karin Olsson ◽

Axel Schambach ◽

Christopher Baum ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.363.363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 363-363

Author(s):

Jing Qi ◽

Qi Cai ◽

Sandeep Singh ◽

Ling Li ◽

Hongjun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Cell Activity ◽

Disease Free Survival ◽

Hematopoietic Stem ◽

The Impact ◽

Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

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