Host Hematopoietic Stem Cells Research Articles

Tetramer Formation of BCL11A Is Required for Both Protein Stability and Fetal Hemoglobin Silencing

The zinc-finger (ZnF) repressor protein BCL11A regulates the switch from fetal (HbF, α 2γ 2) to adult hemoglobin (HbA, α 2β 2) through direct binding at the γ-globin promoter. Clinical trials of gene-based reduction of BCL11A with ex vivo modified hematopoietic stem cells (HSCs) have yielded transformative outcomes in patients with sickle cell disease and β-thalassemia, establishing BCL11A as a therapeutic target. Unless in vivo methods for delivery of gene-based therapies become efficient enough to modify a substantial fraction of host HSCs, small molecule drugs will be required to reduce overall disease burden. With the goal of targeting BCL11A with small molecules, we have characterized its native state. The protein is largely unstructured, except for 7 ZnFs, numbered 0-6. Previously we showed that BCL11A protein appears stable in cells with a T 1/2 of ~ 24 hrs. Here we report the unexpected finding that the protein assembles and functions as a tetramer. Recombinant BCL11A behaves as a tetramer in biophysical assays. Gene editing of the endogenous BCL11A locus in CD34-derived, immortal HUDEP2 cells has been employed to correlate protein structure and expression with consequences of a given modification. We found that BCL11A lacking ZnF0 is unstable, leading to protein loss and derepression of γ-globin. Recombinant ZnF0 self-assembles into a stable tetramer, whose X-ray structure reveals a hydrophobic interface between subunits. Mutation of residues in the hydrophobic core disrupts tetramerization and markedly reduces steady-state protein in cells. Reciprocal immunoprecipitation in cells reveals that ZnF0 is both necessary and sufficient to mediate BCL11A mutlimer assembly. Collectively, these results indicate that BCL11A tetramerization is required for protein stability. In cells expressing BCL11A lacking ZnF0, the steady-state protein level is partially restored by addition of proteasome inhibitors, suggesting that mutant protein is actively degraded by the ubiquitin-proteasome system. In contrast to findings with ZnF0, discrete removal of ZnF1 within the context of full-length BCL11A results in a marked increase in steady-state protein in cells. In vitro purified ZnF1 forms a dimer and only dimeric ZnF1 can be stably expressed in cells. These findings re consistent with a model in which assembly of the BCL11A tetramer is required to mask a degron in ZnF1 that becomes obscured on its dimerization. Masking of a degron in ZnF1 predicts that discrete removal of both ZnF0 and ZnF1 within full-length BCL11A will lead to production of a monomer. Indeed, HUDEP2 cells expressing BCL11A lacking both ZnFs exhibit an elevated level of steady-state, monomeric protein, consistent with the degron masking model. However, despite an elevated level, cells expressing monomeric BCL11A are defective in HbF silencing, indicating that the tetramer structure is critical for γ-globin repression. Our findings provide novel insights into BCL11A action and suggest new strategies for therapeutic targeting. Combining structural, biochemical, and cellular approaches, we have shown that ZnF-mediated tetramer formation shields BCL11A from proteolytic degradation, and that the tetramer state itself is required for effective γ-globin repression. Of relevance to BCL11A as a therapeutic target, the tetramer of ZnF0 constitutes a structured, stable domain in an otherwise largely unstructured protein, and thereby offers possibilities for small molecule targeting. The multimer state of BCL11A is a vulnerability for HbF repression.

Selective Bcl-2 inhibition promotes hematopoietic chimerism and allograft tolerance without myelosuppression in nonhuman primates.

Hematopoietic stem cell transplantation (HSCT) has many potential applications beyond current standard indications, including treatment of autoimmune disease, gene therapy, and transplant tolerance induction. However, severe myelosuppression and other toxicities after myeloablative conditioning regimens have hampered wider clinical use. To achieve donor hematopoietic stem cell (HSC) engraftment, it appears essential to establish niches for the donor HSCs by depleting the host HSCs. To date, this has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. An approach that is capable of more selectively depleting host HSCs is needed to widen the clinical application of HSCT. Here, we show in a clinically relevant nonhuman primate model that selective inhibition of B cell lymphoma 2 (Bcl-2) promoted hematopoietic chimerism and renal allograft tolerance after partial deletion of HSCs and effective peripheral lymphocyte deletion while preserving myeloid cells and regulatory T cells. Although Bcl-2 inhibition alone was insufficient to induce hematopoietic chimerism, the addition of a Bcl-2 inhibitor resulted in promotion of hematopoietic chimerism and renal allograft tolerance despite using only half of the dose of total body irradiation previously required. Selective inhibition of Bcl-2 is therefore a promising approach to induce hematopoietic chimerism without myelosuppression and has the potential to render HSCT more feasible for a variety of clinical indications.

Streptavidin-Drug Conjugates Streamline Identification of Optimal Toxic Payloads for Antibody-Based Hematopoietic Stem Cell Transplantation Conditioning

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides the best and possibly only chance for cure for acute myeloid leukemia (AML). In preparation for allo-HSCT, patients undergo conditioning with chemotherapy and/or irradiation to deplete host hematopoietic stem cells (HSCs), suppress the host immune system, and destroy residual leukemia cells. However, conditioning associated toxicities impose a formidable barrier to patients undergoing this potentially curative therapy. Antibody-drug conjugates (ADCs) have emerged as a promising and potentially safer alternative for allo-HSCT conditioning by allowing targeted depletion of the host HSC niche. Previously, we used saporin-conjugated CD45- or cKit-ADCs as the backbone of a novel conditioning regimen which enabled engraftment between fully mismatched mice. However, saporin ADCs were nonmyeloablative and showed modest antitumor activity. We hypothesized that fully myeloablative ADCs capable of enhanced antileukemia activity could be produced using alternative toxic payloads to saporin. However, direct conjugation of multiple candidate payloads and/or antibodies is costly, time-consuming, and laborious. Here, we describe the production of a panel of streptavidin (SAv)-drug conjugates which allows efficient ADC generation from any biotinylated protein. This flexible platform enabled the rapid production of CD45-ADCs conjugated to multiple toxic payloads, facilitating in vitro screening of their ability to target HSCs and leukemia cells. METHODS: Azide-alkyne cycloaddition ("Click" chemistry) was used to covalently link SAv-azide (Protein Mods, Inc) with dibenzocyloocytne (DBCO)-coupled drug payloads (Levena Biopharma) (Fig. 1). We conjugated three toxic payloads to SAv for these studies: monomethyl auristatin E (MMAE), Duocarmycin SA (Duo), and PNU-159682 (PNU). All of these payloads contain cleavable valine-citrulline linkers for payload release following ADC internalization. For the conjugation reactions, SAv-azide was combined with DBCO-coupled payload and incubated 2 hours at 20oC, with 20% DMSO added as cosolvent. Free payload and DMSO were removed using size exclusion chromatography and dialysis. ADCs were freshly prepared before use by mixing SAv-drug conjugates in a 1:1 molar ratio for 15 minutes with biotinylated antibodies. For our initial studies, we evaluated murine anti-CD45.2 (Clone 104) and human anti-CD45 (Clones BC8 and HI30). Non-biotinylated antibodies combined with SAv-drug conjugates were used as negative controls. All ADCs were tested for cytotoxicity in vitro against 1) murine YAC-1 (mCD45.2+) and human Jurkat (mCD45.2-) cell lines using XTT viability assays, 2) primary HSCs from B6 mice using methylcellulose assays, and 3) AML1 primary murine leukemia cells (Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Tim Ley) using methylcellulose assays. IC50 values for each assay were derived with nonlinear regression of dose response data. RESULTS: Mouse CD45-Duo and CD45-PNU were highly effective at killing AML1 cells (IC50 values: 39.2pM and 61.1pM, respectively; Fig. 2), as well as B6 HSCs (IC50 values: 0.85nM and 0.13nM, respectively) in vitro. Notably, mouse CD45-MMAE was ineffective against YAC-1 cells, B6 HSCs, and AML1 cells, suggesting that MMAE is not an effective payload for ADC-based conditioning in the mouse. Importantly, nonbiotinylated antibodies mixed with SAv-drug conjugates were largely ineffective, indicating the need for the antibody to bind the SAv-drug conjugate to achieve specific toxicity. Preliminary studies using human CD45-MMAE and CD45-Duo demonstrated that while both payloads could yield ADCs able to kill Jurkat cells, ADCs made using human antibody clone BC8 were considerably more effective than ADCs made with clone HI30. CONCLUSIONS: Our novel SAv-drug conjugate successfully enabled rapid generation of ADCs with a number of potential toxic payloads. This system identified multiple effective candidate payloads for use in mouse and human ADCs for targeting HSCs and leukemia cells in vitro and in vivo. Our ongoing and future studies with this system are focused in three areas: 1) production and testing of mouse and human cKit ADCs, 2) using mouse ADCs to condition for murine HSCT and target AML1 cells in vivo, and 3) evaluating killing of human cord blood-derived HSCs and patient-derived AML cells with human ADCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fully Myeloablative Antibody-Drug Conjugates Condition for Hematopoietic Stem Cell Transplantation and Provide Durable Antileukemia Benefit

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) holds the potential for cure of numerous blood diseases and provides the best chance for durable control of acute myeloid leukemia (AML). Successful allo-HSCT requires that host hematopoietic stem cells (HSCs) are depleted to make bone marrow (BM) space for the incoming donor HSCs, and that host T and NK cell mediated immunity is suppressed to prevent donor HSC rejection. To meet these requirements, conditioning regimens comprised of chemotherapy and/or irradiation are used, which carry risks of severe toxicities that may prevent patients from undergoing this potentially curative therapy. Thus, there is an unmet clinical need for novel conditioning regimens that minimize toxicity without sacrificing therapeutic efficacy. Previously, we showed in mouse models that saporin-based antibody-drug conjugates (ADCs) targeting CD45 or cKit combined with Janus kinase 1/2 (JAK1/2) inhibitors enabled stable, high-level engraftment in fully mismatched HSCT with ≥99% donor myeloid lineage chimerism. However, saporin ADCs are not fully myeloablative, which limited their antitumor efficacy in prior studies and would likely limit the ability of these conditioning agents to provide protection against AML relapse. We hypothesized that alternative toxic payloads to saporin would enable generation of fully myeloablative ADCs capable of enhanced antileukemia benefit. METHODS: We conjugated anti-mouse CD45.2 (clone 104), anti-mouse cKit (clone 2B8), and their respective isotype control antibodies with pyrrolobenzodiazepine (PBD) dimers via maleimide-thiol chemistry. PBD-conjugated ADCs were tested for cytotoxicity in vitro against the murine YAC-1 (CD45.2+) or P815 (cKit+) cell lines, as well as the human Jurkat (CD45.2-cKit-) cell line using XTT viability assays. Cytotoxicity against BM HSCs from B6 mice and AML1 primary leukemia cells (B6-derived, Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Timothy Ley) was tested using methylcellulose assays. HSC depletion in vivo was assessed 7 days after treatment with a single dose of CD45- or cKit-PBD. Syngeneic and fully mismatched HSCT studies with CD45-PBD were done by conditioning B6 mice with 50 µg ADC 7 days before transplant (d-7), then infusing 10x106 B6-GFP (syngeneic) or 20x106 BALB/c (allogeneic) BM cells on d0. For in vivo leukemia killing studies, B6 mice were infused with 5x105 AML1 cells on d-6, treated with 50 mg CD45-PBD or 75 mg CD45-saporin on d-2, then transplanted on d0 with 10x106 T cell depleted BM cells from 129/Sv mice. In allo-HSCT studies, mice received oral ruxolitinib (2 mg/g chow) starting on d-3. RESULTS: CD45-PBD targeted murine YAC-1 cells and cKit-PBD targeted P815 cells in vitro with picomolar-range potencies; neither ADC was effective against human Jurkat cells. Target-specific killing was confirmed with blocking studies using excess unconjugated anti-CD45.2 or anti-cKit. While CD45-PBD and cKit-PBD showed similarly high efficacy and picomolar-range potencies at targeting AML1 leukemia cells in vitro, cKit-PBD was approximately 40-fold more potent at depleting B6 HSCs in vitro than CD45-PBD (IC50 values: 243 fM for cKit-PBD versus 10 pM for CD45-PBD). In vivo, a single 50 µg dose of CD45-PBD or 25 µg dose of cKit-PBD completely ablated the HSC niche in B6 mice. Initial transplantation studies done with CD45-PBD confirm that this ADC enables both syngeneic and allogeneic HSCT with near complete donor chimerism in all recipient mice (Fig. 1). Finally, a single CD45-PBD dose provided durable protection against AML1 leukemia, whereas the nonmyeloablative CD45-saporin was only minimally protective (Fig. 2). CONCLUSIONS: We successfully generated fully myeloablative CD45- and cKit-ADCs using PBD as the toxic payload. Notably, CD45-PBD enabled HSCT with near total conversion to donor hematopoiesis and was highly protective against the aggressive AML1 primary leukemia. Studies thus far with cKit-PBD have shown superior killing of murine HSCs in vitro and in vivo compared to CD45-PBD; testing of cKit-PBD in HSCT models and in vivo AML1 leukemia killing studies are in progress. Given the high potency of the PBD payload, we are conducting extensive histopathologic studies and serum chemistry testing to evaluate the acute and chronic organ toxicities of conditioning with PBD-based conjugates. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of hematopoietic stem cells residing in the meninges of adult mice at steady state.

Steady-state extramedullary hematopoiesis during adulthood is an emerging field of great interest. The meninges contain both innate and adaptive immune cells, which provide immunosurveillance of the central nervous system (CNS). Hematopoietic progenitors that give rise to meningeal immune cells remain elusive. Here, we report that steady-state meninges of adult mice host hematopoietic stem cells (HSCs), as defined by long-term, efficient, multi-lineage reconstitution and self-renewal capacity in the meninges, blood, spleen, and bone marrow of sublethally irradiated adult recipients. HSCs lodge in the meninges after birth with local expression of pro-hematopoietic niche factors. Meningeal HSCs are locally maintained in homeostasis and get replenished from the blood only when the resident pool is reduced. With a tissue-specific expression profile, meningeal HSCs can provide the CNS with a constant supply of leukocytes more adapted to local microenvironment.

231.2: Selective Bcl-2 Inhibition Promotes Hematopoietic Chimerism and Allograft Tolerance Without Myelosuppressive Complications

Introduction: Hematopoietic stem cell transplantation (HSCT) has been an only approach that reproducibly achieved renal allograft tolerance in humans. However, severe myelosuppression and toxicities after myeloablative conditioning have hampered wider clinical application. To achieve hematopoietic stem cell (HSC) engraftment, it is essential to make space in the bone marrow (BM) niches by depleting host HSCs. This has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. Therefore, a novel approach more selectively capable of depleting HSCs is needed to widen the clinical application of HSCT. Based on murine studies that showed successful induction of mixed chimerism and allograft tolerance without irradiation by Bcl-2 inhibition, we evaluated an FDA approved Bcl-2 inhibitor (Venetoclax, Vntx) in our mixed chimerism inducing conditioning regimen. Methods: Cynomolgus monkeys received kidney and bone marrow transplantation after conditioned with various nonmyeloablative regimens which included total body irradiation (TBI), thymic irradiation (TI) and anti-thymocyte globulin (ATG), followed by a short post-transplant course of costimulatory blockade (CoB) and Cyclosporine. The study groups were consisted of 3Gy TBI, no Vntx (Group A), 1.5Gy TBI, no Vntx (Group B), TBI 1.5Gy with Vntx(10 mg/kg x 11 on days -4 to 6) (Group C), TBI 1.5Gy with Vntx but Belatacept as CoB (Group D), no TBI with Vntx (Group E), no TI with Vntx (Group F), no CoB with Vntx (Group G). Results: Most recipients treated with 3Gy TBI (Group A) developed mixed chimerism and long-term immunosuppression (I.S.)-free renal allograft survival (Table 1), but suffered from transient but severe pancytopenia. With reduced TBI (1.5Gy), all recipients failed to develop chimerism, and 4/5 developed acute rejection. By adding Vntx, despite the dose of TBI being reduced to half (1.5Gy), all six recipients (Group C) developed significantly superior chimerism (Figure 1) than regimens without Bcl-2 inhibition (Groups A and B), and 5/6 achieved long-term I.S.-free allograft without pancytopenia (Figure 2). With Belatacept in place of anti-CD154 mAb, 2/3 achieved chimerism and long-term I.S. free allograft survival. The current studies also revealed that a minimal non-toxic dose of TBI, TI and CoB remain to be essential for long-term I.S.-free renal allograft survival. Without TBI (Group E) or CoB (Group G), the recipients failed to develop chimerism and rejected allografts despite administration of Vntx. Without TI (Group F), all recipients developed mixed chimerism but failed to achieve tolerance. Conclusion: Bcl-2 inhibition with Vntx significantly promoted mixed chimerism and renal allograft tolerance without myelosuppressive complications. A minimal non-toxic dose of TBI, TI, and CoB were still required for chimerism and allograft tolerance induction even with Bcl-2 inhibition.

Non-genotoxic Restoration of the Hematolymphoid System in Fanconi Anemia

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant Using a Human Antibody Drug Conjugate Targeting C-KIT

For patients undergoing hematopoietic stem cell transplantation (HSCT), conditioning remains a primary source of transplant related morbidity and mortality and limits the broader application of this life-saving therapy. Current conditioning regimens, including total body irradiation and high dose chemotherapy, cause acute toxicity, cytokine storm, increased graft-versus-host disease, and organ damage. In addition, these regimens are also associated with the development of secondary malignancies, autoimmunity, and infertility.To address this significant unmet medical need, we have developed a conditioning regimen using an antibody drug conjugate (ADC) targeting C-KIT (CD117) to specifically deplete host hematopoietic stem cells. CD117 is highly expressed on hematopoietic stem and progenitor cells (HSPCs) and binding to its ligand, stem cell factor, promotes hematopoiesis, self-renewal, and rapid internalization of the receptor. The rapid internalization and selective expression of CD117 on HSPCs make it an ideal target for an ADC-based approach to depleting HSPCs, while limiting systemic toxicity. In addition, CD117 is frequently overexpressed on malignant cells in patients with acute myelogenous leukemia. Thus, an ADC targeting CD117 may provide an opportunity for disease control prior to or concomitant with an HSCT. Previous studies have demonstrated that an antagonistic antibody against CD117 lead to high levels of HSPC depletion and donor engraftment in immunocompromised mice, but failed to facilitate this effect in immunocompetent hosts. To overcome the engraftment barrier, additional studies were conducted with an anti-CD117 antibody conjugated to the ribosome inactivating toxin, saporin, which allowed for robust HSPC depletion and high levels of congenic donor chimerism in immunocompetent mice (Czechowicz et.al., Blood 2016, 128:493).To advance this technology toward the clinic, we have developed a fully human anti-CD117 antibody conjugated to a small molecule toxin capable of depleting proliferating and quiescent cells (CD117-ADC), that demonstrated greater than 90% killing of the leukemia cell line Kasumi-1, and was equally effective at killing primary human CD34+ bone marrow cells during in vitro culture. Humanized NSG mice treated with CD117-ADC had greater than 90% depletion of human HSPCs in the bone marrow, 21 days following a single administration of the ADC. The specificity of CD117-ADC for HSPCs was confirmed by the presence of stable peripheral human lymphocyte populations, and absence of human myeloid cells; indicating a lack of stem and progenitor cells capable of replenishing these short-lived cells. In addition to the ability of CD117-ADC to deplete HSPCs in humanized mice, preliminary data suggests that CD117-ADC is also effective at reducing tumor burden in mice challenged with CD117 expressing tumor cells.Eliminating HSPCs from the stem cell niche with a targeted ADC has the promise to enable the engraftment of donor HSCs, while preventing the systemic toxicity associated with irradiation and chemotherapy. We have demonstrated that an ADC targeting CD117 is capable of depleting human HSPCs in vitro and in vivo and has the potential to reduce tumor burden in mice. Ultimately, this novel approach for safer conditioning prior to HSCT could greatly increase the number of patients eligible for transplant and significantly reduce the damaging side effects associated with current protocols and may provide effective non-genotoxic conditioning for all patients suffering from malignant or non-malignant diseases who require a HSCT. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

5-Azacytidine Depletes Hematopoietic Stem Cells and Synergizes with an Anti-CD117 Antibody to Augment Engraftment of Donor Stem Cells in Immunocompetent Mice

Monoclonal antibody (mAb)-targeting of CD117 (c-Kit) present on hematopoietic stem cells (HSC) is an emerging conditioning strategy for hematopoietic cell transplantation (HCT) to replace standard-of-care alkylator and radiation therapy used to deplete recipient HSC and permit donor HSC engraftment. In preclinical and clinical studies anti-CD117 mAbs, which inhibit stem cell factor (SCF) from binding to CD117, have shown single agent efficacy in permitting donor HSC engraftment in immunocompromised recipients. However, preclinical data suggest that anti-CD117 mAb, ACK2, used as a single agent is insufficient to enable HSC engraftment in immunocompetent mice. Hence, there is a desire to potentiate the effect of naked anti-CD117 mAbs to permit targeted elimination of host HSC for a range of immune competent bone marrow (BM) states, from monogenic disorders, e.g., hemoglobinopathies, to BM clonal malignant diseases. Here, we show that ACK2 synergizes with the widely used hypomethylating agent, 5-Azacytidine (AZA), to deplete HSC and enable robust long-term HSC engraftment in immunocompetent mice. The cell depleting effect of AZA or ACK2+AZA was first tested on mature and primitive hematopoietic cells of C57BL/6 (B6) mice. Mice received AZA at 5mg/kg i.p. for 5 days either alone or combined with ACK2 at 500 µg i.v. 5 days prior to AZA. BM and spleen were harvested days 6, 10 and 20 after the first AZA dose and analyzed by extended immunophenotype. For HCT studies both congenic [B6 (H-2b, Thy1.1, CD45.1) into B6 recipients (H-2b, Thy1.1, CD45.1/CD45.2)] and allogeneic [B6 (H-2b, Thy1.1, CD45.1) into BALB.B recipients (H-2b, Thy1.2, CD45.2)] transplantation mouse models were tested. Single agent AZA induced rapid depletion of CD117+ cells in the BM, including LT-HSC (Lin-Sca+Kit+[LSK]CD150+CD48-) and ST-HSC (LSKCD150-CD48-). In vivo HSC depletion by AZA has not been previously reported, and was surprising given that most HSC are quiescent, and non-proliferating cells are known to be insensitive to the toxicities of AZA. Examination of HSC populations in the spleen did not suggest that mobilization of LT- or ST-HSC to secondary organs caused the decrease in BM. Rather, AZA-induced myelosuppression was followed by rapid expansion of myeloid-biased MPP and increased HSC proliferation as assessed by Ki67 intracellular protein expression. We hypothesized, that the in vivo HSC-depletive effects of AZA might result from increased sensitivity of proliferating HSC to AZA. Hence, the in vitro effects of different AZA concentrations were tested on proliferating HSC. In both mouse and human HSC cultures, supplemented with SCF and TPO, we observed that AZA reduced the number of cells in a dose dependent manner and decreased cell viability at concentrations of ≥ 0.5 µg/ml. In vivo, combined ACK2+AZA led to more robust HSC depletion and prolonged time to HSC recovery compared to AZA alone. We then evaluated if blockade of CD117 with its ligand SCF by ACK2 was the cause of this prolonged HSC-depletion. 2B8 mAb, which also binds mouse CD117, but only partially inhibits CD117/SCF signaling, did not result in augmented in vivo HSC depletion (Figure 1A). We further tested if immunocompetent mice treated with ACK2+AZA could be "rescued" with exogenous recombinant SCF. SCF-treatment resulted in faster recovery of LT-HSC. Together these studies suggest that the synergy of ACK2+AZA on the depth and duration of HSC-depletion depends of the disruption of CD117/SCF binding. Finally, we tested ACK2+AZA conditioning on donor stem cell engraftment in immunocompetent mice. Transplantation of 15-20x106 WBM cells following ACK2+AZA conditioning led to significantly increased stable mean multilineage donor chimerism of >60% in the congenic (Figure 1B) and >20% in the allogeneic setting. Single agent AZA enabled sustained donor engraftment with levels of myeloid donor chimerism of 10-15%, serving as proof of principle that AZA treatment can indeed clear HSC niches and permit engraftment of true HSC. Similar to WBM grafts, 5x104 purified HSC (LSK) engrafted successfully in the ACK2+AZA group, reaching long-term (26 weeks) mean donor myeloid chimerism >25% in both congenic and allogeneic settings. Our studies reveal a previously unknown effect of AZA on BM HSC and provide a platform for the clinical use of a novel less toxic non-myeloablative conditioning strategy for HCT, which is aimed at the rapid translation into clinical practice. Disclosures Pang: Jasper Therapeutics, Inc: Current Employment, Current equity holder in private company. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Non-Genotoxic Anti-CD117 Antibody Conditioning Results in Successful Hematopoietic Stem Cell Engraftment in Patients with Severe Combined Immunodeficiency

Non-Genotoxic Anti-CD117 Antibody Conditioning Results in Successful Hematopoietic Stem Cell Engraftment in Patients with Severe Combined Immunodeficiency

INCOMPLETE REJUVENATION OF AGED HSCS IN YOUNG BONE MARROW NICHE

Hematopoietic stem cell (HSCs) give rise to all kinds of blood cells throughout life. As they age, HSCs exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation. HSC niche, which is essential for the maintenance of HSCs, also undergo considerable changes with aging. However, detailed molecular mechanisms that regulate HSC aging have not been fully understood. Although it is technically difficult to directly evaluate the relationship between aging of HSCs and BM niche, we assumed that engrafting aged HSCs into young niche would rejuvenate them. In this study, we transplanted 20-month-old aged HSCs into 10-week-old young mice without irradiation following the report by Shimoto et al. that the number of empty HSC niches available for engraftment is larger than the number of niches occupied by host HSCs (Blood, 2017). Two months after transplantation, we collected aged HSCs engrafted in young mice (Aged/Y), then performed secondary transplantation and RNA sequence analysis. Secondary transplantation revealed no rejuvenation of aged HSCs in terms of repopulating capacity and non-biased differentiation. However, the gene expression profiles of Aged/Y HSCs were reprogrammed to a large extent similar to those of young HSCs. We detected many genes, the aberrant expression of which in aged HSCs was restored close to the levels of young HSCs. These genes were related to KEGG pathways such as phosphorylation, chemokine signaling pathway, and proteolysis. We are currently engaged in the epigenome profiling of Aged/Y HSCs engrafted in the young niche. These results indicate that a significant portion of changes in transcriptional profiles of HSCs with aging is dependent on aging of niche. Rejuvenation of niche can largely restore the age-related transcriptional profiles of HSCs but cannot restore the functional defects of aged HSCs.

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

Toxicity-Free Hematopoietic Stem Cell Engraftment Achieved with Anti-CD117 Monoclonal Antibody Conditioning

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit donor HSC engraftment to provide life-long hematopoietic and immune function. Currently HCT relies on DNA damaging radiation or chemotherapy to achieve HSC niche clearance. We have pursued a non-toxic approach to target and deplete HSC using humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit). We opened a Phase 1 dose escalation trial using AMG 191 as the sole conditioning agent to achieve donor HSC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID). The primary endpoint is to assess the safety of administering AMG 191. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor granulocyte chimerism at 24 weeks. We have completed the first dose cohort of patients receiving 0.1 mg/kg AMG 191 and treated the first two patients in the second cohort (0.3 mg/kg). All five patients tolerated the AMG 191 infusion and the subsequent infusion of their CD34-selectd donor cells without clinical problems. Here we report efficacy in the first two patients, who have reached the 24-week timepoint. Both patients had T-B-NK+ SCID with mutations in the DCLRE1C (Artemis) gene. Both previously received unconditioned HCT as infants, failed to develop donor B cells and remained dependent on exogenous immunoglobulin. CD34-selected mobilized peripheral blood cells from the original donors were infused when the AMG 191 serum level was Conclusion: These data are proof of concept that a humanized monoclonal antibody targeting CD117 can safely clear human HSC niches and facilitate donor HSC engraftment. This study is ongoing and open for enrollment.

CD117-Amanitin Antibody Drug Conjugates Effectively Deplete Human and Non-Human Primate HSCs: Proof of Concept As a Targeted Strategy for Conditioning Patients for Bone Marrow Transplant

Background: Genotoxic conditioning prior to allogeneic and autologous bone marrow transplantation (BMT) limits the use of these potentially curative treatments due to risks of regimen-related morbidities and mortality, including risks of organ toxicity, infertility, and secondary malignancies. CD117, which is specifically expressed on hematopoietic stem cells (HSCs) and progenitors is rapidly internalized and is an ideal target for an antibody drug conjugate (ADC) based approach to conditioning. We have previously shown that a single dose of an anti-CD117 ADC depleted >95% of bone marrow HSCs in a humanized mouse model and reduced disease burden while extending survival in an AML tumor model (Hartigan et al., Blood 2017 130:1894). The aim of this translational study was to develop a potent anti-CD117 ADC highly effective in eliminating host HSCs with a short half-life and minimal adverse side effects in a non-human primate (NHP) model.Methods: Three different DNA-damaging cytotoxic payloads and amanitin (AM) were site specifically conjugated to an anti-CD117 antibody. The ADCs were titrated and evaluated for in vitro cytotoxicity using human bone marrow CD34+ cells. The ADCs were administered in ascending doses to humanized NSG mice. HSC depletion and immunophenotype of the human cells in the peripheral blood was determined by flow cytometry. For amanitin conjugates, NHP HSC depletion was evaluated in male cynomolgus monkeys in single ascending doses (3/group). HSC content in the bone marrow was monitored by flow cytometry and colony-forming unit (CFU) analysis on day 7 or 14 and 56 post dosing. Hematology and clinical chemistries were evaluated throughout the two-month study.Results: Of the toxins evaluated, only anti-CD117 conjugated with the RNA polymerase II inhibitor amanitin resulted in >90% depletion of human HSCs in humanized NSG mice at 0.3 mg/kg. The AM-conjugates also demonstrated a broad therapeutic window in this model (therapeutic index of >120). As a proof-of-concept for the depletion of HSCs in large animals, a single i.v. dose escalation study was performed with the cross-reactive anti-CD117-AM in NHP. On-target, dose-dependent decreases in phenotypic HSCs and CFUs were observed in the bone marrow at day 7 post anti-CD117-AM dosing with >95% HSC depletion observed with a single dose of 0.3 mg/kg (Fig. 1). In the periphery, a dose-dependent transient decrease in reticulocytes was observed at day 4 with a neutrophil and monocyte nadir at day 18. The depth and duration of the depletion was also dose-dependent. The anti-CD117-AM induced depletion was on target and amanitin dependent as the unconjugated antibody and isotype-AM had no effect. Notably, white blood cell and lymphocyte counts were stable through day 56, demonstrating that this strategy will spare the adaptive immune system. Thrombocytopenia occurred 4-8 days post infusion and was dose-dependent, transient and reversible. This also occurred with the isotype-AM, suggesting the effect was off-target.Because the half-life of the anti-CD117-AM was 5 days, a second dose escalation study with anti-CD117-AM engineered to have a short half-life (~18 h) was performed in NHPs. The short half-life anti-CD117-AM demonstrated similar potency on all cell parameters at 0.3 mg/kg and was well tolerated at the effective dose. As expected, the short half-life anti-CD117-AM was rapidly cleared with a half-life of 15-18 h. In both studies, a transient dose dependent elevation of liver enzymes was observed in groups treated with the highest doses of isotype-AM, anti-CD117-AM, and the short half-life anti-CD117-AM.Conclusions: Anti-CD117-AM exhibited potent elimination of NHP HSCs and progenitors in vivo. The potency of short half-life anti-CD117-AM was comparable, providing a model for target cell depletion and rapid clearance prior to BMT. Both ADCs were well tolerated at the efficacious doses. This strategy preserves the adaptive immune system with delayed onset of neutrophil nadir (18 days), potentially shortening the period of neutropenia. Targeted depletion of hematopoietic cell subtypes with limited off-target effects could provide a significant improvement in standard-of-care approaches to patient preparation prior to HSC transplant. [Display omitted] DisclosuresPearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Non-Genotoxic Conditioning Efficiently Depletes Host Hematopoietic Stem Cells and Facilitates Robust Multi-Lineage Engraftment in a Mouse Model of Fanconi Anemia

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality.Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A).Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis.In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.

The effect of temperature on white spot disease progression in a crustacean, Pacifastacus leniusculus

The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6 °C was zero, meanwhile the animals kept at 22 °C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6 °C to 22 °C the mortality reached 100% indicating that the virus is not cleared at 6 °C. Moreover, the VP28 expression at 6 °C was significantly lower compared to animals kept at 22 °C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6 °C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6 °C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully.

Novel Conditioning Regimens for Blood Stem Cell Transplantation

Hematopoietic stem cell (HSC) therapies rely on chemotherapy and/or radiation to overcome host barriers to permit cell engraftment and provide life-long donor derived hematopoiesis. In conventional allogeneic transplantation these modalities function to both suppress immune rejection and create sufficient niche space to allow HSC to engraft. For gene-corrected or gene-modified cells that are derived from autologous patient sources, access to the HSC niche constitutes the primary barrier. Non-genotoxic approaches are being developed with the goal to target molecules expressed on hematopoietic stem and progenitors. We have studied the use of monoclonal antibodies (mAbs) that target the molecule CD117, also known as cKit, to deplete endogenous stem cells and safely allow engraftment of purified HSC. CD117 is a cell surface molecule expressed at high levels on HSC and early myeloid progenitors as well as other non-hematopoietic cell types. It is a receptor tyrosine kinase that upon binding to its ligand, stem cell factor (SCF), propagates intracellular signaling pathways that regulate activities including cell survival, proliferation and differentiation. Studies in immune deficient mice showed that a single dose of anti-CD117 mAb transiently depletes HSC, creating a therapeutic window for donor HSC to engraft1. We have developed for use in clinical transplantation an anti-human CD117 (anti-hCD117) mAb. This antibody significantly inhibits in vitro proliferation of human HSC and effectively depletes human HSC in mice xenografted with human cord blood cells2, and is effective in depleting HSC and progenitors from the bone marrow of non-human primates (NHP). Although CD117 is expressed on non-hematopoietic cells including mast cells, melanocytes, and cells in the gastrointestinal tract, antibody administration in animals and in healthy human volunteers, show little to no off-target toxicity. We are currently testing in a Phase I clinical trial the anti-hCD117 mAb as the sole conditioning agent for the transplantation of children with severe combined immunodeficiency (SCID). We have observed that the effect of the mouse anti-CD117 mAb is more potent in immune deficient mice compared to immune sufficient wildtype mice as evidenced by markedly inferior HSC engraftment results in wildtype recipients. Thus, we are exploring next generation approaches to achieve deeper and more extended depletion of HSC and progenitors in immune competent animals. In vivo HSC clearance by the mouse anti-CD117 mAb is dependent on Fc-mediated antibody effector functions. We therefore tested combination therapy with anti-CD117 mAb plus biologic agents that inhibit CD47, a myeloid-specific immune checkpoint, with the goal to promote phagocytosis of target cells that bind the anti-CD117 mAb. The combined treatment led to elimination of >99% of host HSCs and robust multilineage blood reconstitution following HSC transplantation3. These studies show that a conditioning approach using anti-CD117 antibodies is feasible, and has the potential to replace genotoxic agents with targeted and safer biologic agents. 1. Czechowicz A, Kraft D, Weissman IL, et al, Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches. Science. 2007; 318: 1296. 2. Logan AC, Czechowicz A, Kelley BV, et al. Anti-CD117 (c-Kit) monoclonal antibodies deplete human hematopoietic stem cells and facilitate their replacement in humanized NOD/SCID/IL2Rg-/- mice: A non-toxic conditioning regimen for allotransplantation. Blood . 2012; 120: 4099. 3. Chhabra A, Ring AM, Weiskopf K, et al.Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci Transl Med . 2016; 8: 351RA105. Disclosures No relevant conflicts of interest to declare.

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015

Background aimsWith the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis. MethodsIn 2015 [respectively 2014], 205 [276] teams from 32 countries responded to the cellular and tissue-engineered therapy survey; 178 [126] teams reported treating 3686 [2665] patients. ResultsIndications were musculoskeletal/rheumatological disorders (32% [33%]), cardiovascular disorders (12% [21%]), hematology/oncology (predominantly prevention or treatment of graft versus host disease and HSC graft enhancement; 20% [20%]), neurological disorders (4% [6%]), gastrointestinal disorders (<1% [1%]) and other indications (31% [20%]). The majority of autologous cells (60% [73%]) were used to treat musculoskeletal/rheumatological (44% [36%]) disorders, whereas allogeneic cells were used mainly for hematology/oncology (61% [68%]). The reported cell types were mesenchymal stromal cells (40% [49%]), chondrocytes (13% [6%]), hematopoietic stem cells (12% [23%]), dermal fibroblasts (8% [3%]), dendritic cells (2% [2%]), keratinocytes (1% [2%]) and others (24% [15%]). Cells were expanded in vitro in 63% [40%] of the treatments, sorted in 16% [6%] of the cases and rarely transduced (<1%). Cells were delivered predominantly as suspension 43% [51%], intravenously or intra-arterially (30% [30%]), or using a membrane/scaffold (25% [19%]). DiscussionThe data are compared with those from previous years to identify trends in a still unpredictably evolving field. Perspectives of representatives from plastic surgery practitioners, Iran and ISCT are presented (contributing authors D.A. Barbara, B. Hossein and W.L. Mark, respectively).

Blood Stem Cell Activity Is Arrested by Th1-Mediated Injury Preventing Engraftment following Nonmyeloablative Conditioning.

T cells are widely used to promote engraftment of hematopoietic stem cells (HSCs) during an allogeneic hematopoietic cell transplantation. Their role in overcoming barriers to HSC engraftment is thought to be particularly critical when patients receive reduced doses of preparative chemotherapy and/or radiation compared with standard transplantations. In this study, we sought to delineate the effects CD4+ cells on engraftment and blood formation in a model that simulates clinical hematopoietic cell transplantation by transplanting MHC-matched, minor histocompatibility-mismatched grafts composed of purified HSCs, HSCs plus bulk T cells, or HSCs plus T cell subsets into mice conditioned with low-dose irradiation. Grafts containing conventional CD4+ T cells caused marrow inflammation and inhibited HSC engraftment and blood formation. Posttransplantation, the marrows of HSCs plus CD4+ cell recipients contained IL-12-secreting CD11c+ cells and IFN-γ-expressing donor Th1 cells. In this setting, host HSCs arrested at the short-term stem cell stage and remained in the marrow in a quiescent cell cycling state (G0). As a consequence, donor HSCs failed to engraft and hematopoiesis was suppressed. Our data show that Th1 cells included in a hematopoietic allograft can negatively impact HSC activity, blood reconstitution, and engraftment of donor HSCs. This potential negative effect of donor T cells is not considered in clinical transplantation in which bulk T cells are transplanted. Our findings shed new light on the effects of CD4+ T cells on HSC biology and are applicable to other pathogenic states in which immune activation in the bone marrow occurs such as aplastic anemia and certain infectious conditions.