Hematopoietic Progenitor Cell Transplantation Research Articles

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Ongoing production of tissue-resident macrophages from hematopoietic stem cells in healthy adult macaques

AbstractMacrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac–derived cells that populate the tissue before birth, and bone marrow–derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.

Efficacy, Safety, and Health-Related Quality of Life (HRQOL) in Patients with Sickle Cell Disease (SCD) Who Have Received Lovotibeglogene Autotemcel (Lovo-cel) Gene Therapy: Up to 60 Months of Follow-up

Introduction: Lovo-cel gene therapy uses autologous transplantation of hematopoietic stem and progenitor cells (HSPC) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbA T87Q. The phase 1/2 HGB-206 (NCT02140554) and phase 3 HGB-210 (NCT04293185) studies of lovo-cel are the largest clinical trials of gene therapy in SCD to date. We report efficacy and safety from these studies, including the first data from adult and pediatric patients (age 12 to <18 years) in HGB-210, and HRQOL data from HGB-206. Methods: This analysis includes patients from HGB-206 Group C and HGB-210 who received lovo-cel using the current HSPC mobilization and manufacturing process. Patients with SCD and recurrent severe vaso-occlusive events (VOEs) or history of overt stroke underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. After 24 months of follow-up post lovo-cel infusion, patients enrolled in the long-term study LTF-307 (NCT04628585). Lab evaluations, SCD-related outcomes (eg, resolution of VOEs), globin response (a composite endpoint evaluating HbA T87Q percentage and total Hb), and safety are reported up to 60 months. An independent Event Adjudication Committee confirmed VOEs met protocol criteria. HRQOL data from Patient-Reported Outcomes Measurement Information System (PROMIS)-57 domains of pain interference, fatigue, and pain intensity are reported up to 48 months. Results:This analysis includes47 patients (HGB-206 Group C, n=36; HGB-210, n=11; male, 59.6%; median [range] age, 23 y [12-38]) who received a lovo-cel infusion as of Feb 13, 2023. Median (range) follow-up was 35.5 months (0.3-61.0). Median (range) time to neutrophil and platelet engraftment was 20 days (12-35) and 35 days (19-136). Peripheral blood vector copy number remained stable (median >1 c/dg through follow-up). Median HbA T87Q levels were ≥4.5 g/dL from 6 months post infusion to last study visit. Median (range) total Hb level increased from 8.7 g/dL (6.1-12.5) at relative baseline to 11.8 g/dL (8.4-15.0) at last visit; median percent HbA T87Q of nontransfused total Hb was ≈40% or more ( Figure). Of 33 evaluable patients (ie, ≥18 months follow-up and ≥4 VOEs in the 2 years before enrollment), 30 (90.9%) and 32 (97.0%) had complete resolution of VOEs and severe VOEs during the 6-18 months post infusion, vs a median (range) of 3.5 (1.5-16.5) and 3.0 (0.5-13.0) events/year in the 2 years before enrollment. Additional efficacy analyses in subgroups (eg, stroke history, α-globin genotype) will be presented. Among patients who had globin response or who had ≥18 months follow-up (n=38), 33 (86.8%) achieved globin response. Hemolysis markers (eg, reticulocytes, total bilirubin, lactate dehydrogenase) approached normal levels. Among all 47 patients, 44 (93.6%) had ≥1 AE of grade ≥3 after lovo-cel infusion; the most common of which were stomatitis (33 [70.2%] patients) and thrombocytopenia (28 [59.6%]). Serious AEs were reported for 26 (55.3%) patients, the most common of which was chronic pain/acute exacerbation of chronic pain (3 [6.4%]; 2 chronic neuropathic pain and 1 chronic pain associated with anxiety). One patient with -α3.7/-α3.7 genotype was diagnosed with myelodysplastic syndrome with stable complete blood counts 30 months post infusion; updated data to be presented. No veno-occlusive liver disease, graft failure, replication-competent lentivirus, or vector-mediated insertional oncogenesis was observed. Lovo-cel treatment regimen largely reflected known side effects of HSPC collection and busulfan conditioning regimen. A total of 25 patients (all from HGB-206 Group C; age ≥18 y) had evaluable HRQOL data. Mean scores for PROMIS-57 domains of pain intensity, pain interference, and fatigue improved (ie, decreased) over time up to 48 months ( Table). Conclusion: One-time treatment with lovo-cel resulted in sustained HbA T87Q production and near-complete resolution of VOEs and severe VOEs up to 18 months post treatment; the safety profile was consistent with underlying SCD and myeloablative conditioning. Patients reported sustained improvements in pain intensity, pain interference, and fatigue. Ongoing long-term follow-up will continue to provide important information on efficacy, safety, and patient experience post lovo-cel treatment.

CD33-Deleted Hematopoietic Cells (Trem-Cel) Are Protected from CD33xCD3 Bispecific Antibody Treatment and Produce Significantly Reduced Levels of Inflammatory Cytokines in Preclinical Studies

INTRODUCTION: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia. Currently, allogeneic hematopoietic stem cell transplant (HCT) is the standard of care for high-risk patients yet ~40% patients will relapse, emphasizing the need for new therapeutic approaches. Effective targeted therapy in AML has been hampered by a lack of cancer specific targets. One described AML target is CD33, a glycoprotein expressed predominantly on normal myeloid cells and the majority of AML cells. However, CD33-directed therapies have been challenged by cytopenias due to on-target, off-tumor toxicity and, in the case of bispecific antibodies, cytokine release syndrome (CRS). Tremtelectogene empogeditemcel (trem-cel) is a gene-edited hematopoietic stem and progenitor cell (HSPC) transplant product currently in clinical trial (NCT04849910) whereby CD33, shown to be dispensable for normal hematopoiesis and function, is deleted to allow for post-HCT treatment with CD33-directed immunotherapies with reduced myelotoxicity as the reconstituted hematopoietic compartment lacks CD33. JNJ-67571244, a bispecific antibody that binds to both CD3 and CD33, is capable of inducing T cell recruitment for CD33-directedtumor cytotoxicity. This study examines whether CD33 neg hematopoietic cells are protected from JNJ-67571244 and if CD33 neg human HSPC (hHSPC) xenotransplanted mice have reduced levels of inflammatory cytokines associated with CRS when treated with JNJ-67571244. METHODS: In vitro proof-of-concept (POC) studies were carried out using CD3 + T cells co-cultured with JNJ-67571244 and AML target cell lines. Further studies were conducted with CD34 + hHSPCs that were either CRISPR/Cas9 gene edited to delete CD33 or treated with a non-targeting gRNA control (gCtrl) followed by differentiation towards the monocytic lineage. These in vitro differentiated monocytes (MIVD) were assayed for editing frequency, CD33protein expression and myeloid phenotype by flow cytometry prior to co-culture with donor matched T cells and JNJ-67571244 ranging from 0.5 pM to 500 nM. Cytotoxicity was analyzed using flow cytometry at 48 and 72 hr and cytokine concentrations in the supernatants were measured using Luminex. For in vivo POC studies, CD33 or gCtrl-edited CD34 + hHSPCs were xenotransplanted into NSG-SGM3 mice followed by treatment with either control bispecific (CD3xNull) or JNJ-67571244 at 0.5 mg/kg. Plasma cytokine concentration were measured via Luminex and hematopoietic tissues were analyzed by flow cytometry to measure protection from JNJ-67571244 cytotoxic killing. RESULTS: Annexin V and Live/Dead staining showed that JNJ-67571244 was able to induce AML target cell killing by human T cells. Importantly CD33 neg MIVD cells co-cultured with T cells and JNJ-67571244 were significantly more viable at all doses compared to gCtrl cells. The EC 50 of gCtrl MIVD was 0.0208 nM whereas the CD33 neg MIVD EC 50 was at least 20,000-fold higher (>430.8 nM). T cell activation, measured by CD25 and CD69 positivity, and release of cytokines that are associated with CRS such as IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in the CD33 neg MIVD cells treated with JNJ-67571244 compared to the gCtrl group at all doses. Analysis of blood and bone marrow from xenotransplant studies revealed that JNJ-67571244 was effective at killing CD33 + cells as well as CD14 + monocytes in the gCtrl group treated with JNJ-67571244. Conversely, CD14 + monocytes and CD15 + neutrophils derived from CD33-edited hHSPCs, were not significantly reduced in cell number when treated with JNJ-67571244 suggesting that they were protected from JNJ-67571244 specific killing. IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in mice transplanted with CD33-edited hHSPCs compared to gCtrl group in response to JNJ-67571244. CONCLUSIONS: Taken together, these studies demonstrate that CD33 deleted hematopoietic compartment is protected from the CD33 directed immuno-therapy JNJ-67571244 both in in vitro cytotoxicity assays and preclinical xenotransplantation studies, with decreased concentrations of inflammatory cytokines associated with CRS. These findings enable the development of a next-generation AML treatment strategy by pairing trem-cel transplant with a subsequent CD33-directed bispecific compound to potentially improve safety and efficacy while minimizing myelotoxicity.

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor. Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function. Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting. Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.

Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases

Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

CD45.1/CD45.2 Congenic Markers Induce a Selective Bias for CD8+ T Cells during Adoptive Lymphocyte Reconstitution in Lymphocytopenia Mice.

CD45.1/CD45.2 congenic markers have been used to track hematopoietic lineage differentiation following hematopoietic stem and progenitor cell (HSPC) transplantation. However, several studies suggest that a bias exists in CD45.1 versus CD45.2 hematopoietic cell reconstitution from HSPCs. Meanwhile, no definitive comparison has been reported for mature immune cells as to whether the CD45.1/CD45.2 disparity can skew the immune cell response. In this study, using lymphocytopenia Rag1-/- CD45.2 mice as hosts, we assessed the reconstitution potential of CD45.1 versus CD45.2 lymphocytes following adoptive transfer of mature T and B cells. We have found a selective bias for CD8+ T cells in that CD45.1 cells showed significantly higher reconstitution compared with CD45.2 cells, whereas CD4+ T cells and CD19+ B cells showed equivalent reconstitution. These results suggest that CD45.1/CD45.2 markers may induce an alloreactive response or a survival bias specific to CD8+ T cells, and they therefore call for caution for using them as congenic markers in immunologic models.

A retrospective analysis to evaluate if KIR B haplotype donors associate with a reduced risk of relapse in patients with haematological malignancies following haploidentical transplantation at the Blood and Bone Marrow Transplant Unit at Hammersmith Hospital ICHNHST.

Relapse is a major cause of treatment failure in haploidentical haematopoietic progenitor cell transplant (HPCT) with PTCy. Natural killer cells suppress graft versus host disease and mediate the graft versus leukaemia effect, driven by killer cell immunoglobulin-like receptors (KIRs). Emerging research suggests that donor KIR genotype may influence graft outcome in haploidentical transplants with varying impacts between patient cohorts. This study investigates whether donors with greater KIR B motifs associate with outcomes such as greater relapse-free survival (RFS), overall survival (OS), nonrelapse mortality (NRM), acute graft versus host disease (GvHD) and infection. The study cohort included 98 haploidentical donor-recipient (D/R) pairs (myeloablative n = 37, RIC n = 61) with various haematological malignancies, receiving primary T-cell replete haploidentical HSCT with PTCγ. Following KIR SSO genotyping, donors are categorised into neutral (n = 63) or better and best (n = 35), based on KIR B motif content. Kaplan-Meier and Cox regression survival functions are performed to investigate associations with outcomes. Our results show that the better and best category has significantly poorer RFS (p = 0.013; hazard ratio [HR] 3.16, 95% CI 1.21-8.24: p = 0.018). The greater risk of relapse associated with poorer OS (p = 0.011; HR 2.24, 95% CI 1.18-4.24: p = 0.01) in the better and best category. The competing KIR receptor-ligand and missing licensing proof models failed to predict transplant outcomes. Here, we show neutral donors associate with favourable outcomes in T-cell replete haplo-HPCT with PTCγ after categorisation using the KIR B content model, due to the increased risk of relapse associated with the use of better and best donors.

Developing an Allogeneic Hematopoietic Progenitor Cell Transplant Service in a Resource-Limited Country: Challenges and Outcomes.

Introduction Allogeneic stem cell transplant has curative potential for many hematological disorders. Building an allogeneic hematopoietic progenitor cell transplant (HPCT) unit requires huge investment, infrastructure, equipment, medical supplies, and training of health care professionals. The key objective of this study is to share our experience of developing an allogeneic HPCT service at our tertiary care cancer hospital in a low-middle-income country. In addition, this study presents the outcomes of the first 30 allogeneic HPCTs done at our center. Methods This retrospective observational study included adult patients 18 years old or older with hematological malignancies who underwent allogeneic HPCT between July 2019 and April 2023 at Shaukat Khanum Memorial Cancer Hospital and Research Centre. Result Of the 30 patients, 24 underwent matched sibling donor (MSD) transplants in which a myeloablative-conditioning regimen (MAC) was used in 19, and a reduced conditioning regimen (RIC) was used in one. Of the six haploidentical-related donor transplants, four received MAC, and two received RIC. The median recipient age at HPCT was 23 and 21 years for MSD and Haplo-related donor transplants, respectively. The median follow-up duration was 12 months (Range: 10 days - 33 months). The overall survival rate at one yearwas 71.3% among all allogeneic stem cell transplant patients, whereas the disease-free survival rate at one yearwas 63.7%. In the acute lymphoblastic leukemia group, the disease-free survival rate at one year post allograft was 51.5%, while in the acute myeloid leukemia group, it was 78.7%. Conclusion This study demonstrates the successful development of an allogeneic bone marrow transplant unit at our hospital despite significant financial constraints. This has allowed us to provide a potentially curative and life-saving treatment to a substantial number of cancer patients. The bone marrow transplant outcomes of this study are comparable to those reported by international bone marrow transplant registries.

An NFIX-mediated regulatory network governs the balance of hematopoietic stem and progenitor cells during hematopoiesis.

The transcription factor (TF) nuclear factor I-X (NFIX) is a positive regulator of hematopoietic stem and progenitor cell (HSPC) transplantation. Nfix-deficient HSPCs exhibit a severe loss of repopulating activity, increased apoptosis, and a loss of colony-forming potential. However, the underlying mechanism remains elusive. Here, we performed cellular indexing of transcriptomes and epitopes by high-throughput sequencing (CITE-seq) on Nfix-deficient HSPCs and observed a loss of long-term hematopoietic stem cells and an accumulation of megakaryocyte and myelo-erythroid progenitors. The genome-wide binding profile of NFIX in primitive murine hematopoietic cells revealed its colocalization with other hematopoietic TFs, such as PU.1. We confirmed the physical interaction between NFIX and PU.1 and demonstrated that the 2 TFs co-occupy super-enhancers and regulate genes implicated in cellular respiration and hematopoietic differentiation. In addition, we provide evidence suggesting that the absence of NFIX negatively affects PU.1 binding at some genomic loci. Our data support a model in which NFIX collaborates with PU.1 at super-enhancers to promote the differentiation and homeostatic balance of hematopoietic progenitors.

Rescue of Alzheimer’s disease phenotype in a mouse model by transplantation of wild-type hematopoietic stem and progenitor cells

Alzheimer's disease (AD) is the most prevalent cause of dementia; microglia have been implicated in AD pathogenesis, but their role is still matter of debate. Our study showed that single systemic wild-type (WT) hematopoietic stem and progenitor cell (HSPC) transplantation rescued the AD phenotype in 5xFAD mice and that transplantation may prevent microglia activation. Indeed, complete prevention of memory loss and neurocognitive impairment and decrease of β-amyloid plaques in the hippocampus and cortex were observed in the WT HSPC-transplanted 5xFAD mice compared with untreated 5xFAD mice and with mice transplanted with 5xFAD HSPCs. Neuroinflammation was also significantly reduced. Transcriptomic analysis revealed a significant decrease in gene expression related to "disease-associated microglia" in the cortex and "neurodegeneration-associated endothelial cells" in the hippocampus of the WT HSPC-transplanted 5xFAD mice compared with diseased controls. This work shows that HSPC transplant has the potential to prevent AD-associated complications and represents a promising therapeutic avenue for this disease.

CD45.1/CD45.2 congenic markers induce a selective bias for CD8+ T cells during adoptive lymphocyte proliferation in lymphocytopenia mice

Abstract CD45.1/CD45.2 congenic markers have been frequently used to track hematopoietic lineage differentiation following hematopoietic stem and progenitor cell (HPSC) transplantation. However, several studies suggest that a bias exists in CD45.1 versus CD45.2 hematopoietic cell reconstitution from the transplanted HPSCs. Meanwhile, no definitive comparison has been reported for mature immune cells as to whether the CD45.1/CD45.2 disparity can skew immune cell response. In this study using lymphocytopenia Rag1−/− CD45.2 mice as hosts, we assessed the proliferative potential of CD45.1 versus CD45.2 lymphocytes following adoptive transfer of mature T and B cells harvested from the spleens of CD45.1 and CD45.2 mice. We have found that a selective bias for CD8+ T cells in that CD45.1 CD8+ T cells showed significantly higher proliferation and higher expression of PD-1 compared CD45.2 CD8+ T cells in the Rag1−/− CD45.2 hosts. This bias is likely due to MHC-I restricted allogeneic stimulation as CD45.1 versus CD45.2 CD4+ T cells and CD19+ B cells contained the same graft as CD8+ T cells showed equivalent proliferation. These results suggest that CD45.1/CD45.2 markers induce an alloreactive response specific to CD8+ T cells, and therefore call for caution for using them as congenic markers in immunologic models. This study was supported by funds through the National Cancer Institute (R01CA184728), the Maryland Department of Health's Cigarette Restitution Fund (CRF) Program and used shared core facilities supported by University of Maryland Greenebaum Comprehensive Cancer Center (UMGCC) Support Grant (P30CA134274).

The Humanized Mouse Model: What Added Value Does It Offer for HIV Research?

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

The efficiency of a continuous versus an intermittent apheresis method for collection of hematopoietic progenitor stem cells: A systematic review.

Peripheral blood hematopoietic progenitor stem cells (HPSCs) are the most common source of stem cells for autologous and allogenic transplantation. Currently, systematic reviews comparing the collection efficiency of a continuous to an intermittent method are lacking despite the existence of primary studies. Therefore, the objective of this review was to synthesize the best available evidence to compare the efficiency of the continuous vs the intermittent method for the collection of hematopoietic progenitor stem cells required for HPC transplantation. A search using MEDLINE, CINAHL, EMBASE, Google scholar, and MedNar for both published and unpublished studies was conducted in December 2021. The systematic review was conducted in accordance with JBI methodology. A critical appraisal of the studies was undertaken by two independent reviewers using the JBI quasi-experimental critical appraisal checklist. A total of six studies were included in the review. The findings of this review demonstrated that there was no statistically significant difference in the collection efficiency, length of procedure time, and total blood volume processed between the continuous and intermittent programs. The evidence suggests that the continuous method is as safe and effective as the intermittent method to collect HPSCs. Until further evidence becomes available clinicians should be guided by the policies of their individual hospitals.

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS 2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of thesickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome

AbstractWHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele–inactivated cells were enriched over wild-type allele–inactivated cells. The WHIM allele–inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.

595 - Reflections on Equity in Brazilian Health Care:- Racial and Social Issues Related to the Population Undergoing Hematopoietic Progenitor Cell Transplantation in a Private Cell Therapy Service in São Paulo/ Brazil

595 - Reflections on Equity in Brazilian Health Care:- Racial and Social Issues Related to the Population Undergoing Hematopoietic Progenitor Cell Transplantation in a Private Cell Therapy Service in São Paulo/ Brazil