Umbilical Cord Blood CD34 Research Articles

Impact of fetal umbilical cord blood CD34+ cells on breast cancer cell lines: a mechanism of fetal microchimerism?

Introduction Fetal microchimerism could be involved in the regulation of breast cancer oncogenesis. CD34+ cells could be of a particular interest as up to 12% of the CD34+ population in maternal blood are of fetal origin. The aim of this research was to analyze the impact of umbilical cord blood (UCB) CD34+ on MCF-7 and MDA-MB-231 breast cancer cell lines, in order to uncover novel biological mechanisms and suggest novel treatment options for breast cancer. Methods UCB CD34+ cells were obtained from healthy women at full-term delivery. Direct cultures were grown with MCF-7 and MDA-MB-231 cells. Proliferation, migration, invasion, and transcriptomic analysis of breast cancer cells were compared between cultures exposed and non-exposed to UCB CD34+ cells. Interactions between UCB CD34+ and breast cancer cells were analyzed under fluorescent microscopy. Functional analyses were generated with QIAGEN's Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results Direct contact between UCB CD34+ and breast cancer cell lines induced a reduction in the proliferative capacities of MCF-7 and MDA-MB-231 and diminished the migration abilities of MDA-MB-231 cells. In 3D co-culture, UCB CD34+ cells were attracted by tumor spheroids and incorporated into tumor cells. These cell-to-cell interactions were responsible for transcriptome modifications coherent with observed functional modifications. Among the cytokines secreted by UCB CD34+, IFN was identified as a potential upstream regulator responsible for the molecular modifications observed in transcriptomic analysis of MCF-7 breast cancer cells exposed to UCB CD34+ cells, as was IL-17A in MDA-MB-231 cells. Conclusion Direct cell-to-cell contact induced functional modifications in breast cancer cells. Interactions between UCB CD34+ and breast cancer cells could induce cell fusion and signal transmission via cytokines. Further analysis of direct cell-to-cell interactions should be performed at a molecular level to further understand the potential role of fetal CD34+ cells in breast cancer.

Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the ex vivo expansion and maintenance of hematopoietic stem/progenitor cells

BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell–cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

RasGRP3 Role in Umbilical Cord Blood CD34+ Cell Homing and Engraftment

RasGRP3 Role in Umbilical Cord Blood CD34+ Cell Homing and Engraftment

Human Pro-T-Cell Manufacturing in Vitro Is a Safe Procedure for Hematopoietic Stem Cell Transplantation with Delayed T-Cell Reconstitution: Interim Results from 2 Different Clinical Trials

Introduction: Alternative donor Hematopoietic stem cell transplantation (HSCT) strategies are followed by a prolonged period of immune deficiency due to delayed T-cell reconstitution. Either Umbilical Cord (CB) blood transplant or CD34+ selected haplo-transplant provided similar T-cell lymphocytes (CD3+ and CD4+) recoveries at any time point, and recipients had significantly fewer CD4+ T-cells during the first six months compared to HLA-matched donors. The GMP generation of Human T lymphoid Progenitors (HTLP) that are competent to rapidly become in vivo differentiated functional and thymic-educated T-cells may overcome the post-transplant delayed immune reconstitution. Method: HTLP safety and efficacy to accelerate immune reconstitution is currently evaluated in 2 multicentric and single-arm clinical trials: after umbilical cord blood in adult hematologic malignancy (Eudract 2019-004883-23) and after partially HLA compatible CD34+ immune-selected allogeneic hematopoietic stem cell transplantation in Severe Combined Immune Deficiency (SCID) patients (Eudract 2018-001029-14). Results: We enrolled and treated six patients, 4 SCID and 2 adults with malignancy, with an average follow-up of 8.00 (range, 1.60 to 37.7) months. HTLP cell product was infused between D7 to D14 post-HSCT for SCID patients after ensuring the decrease of the ATG level while meaning for the adult patient; the expanded pro-T cells were injected at D0. The Pro-T manufacturing process allows to robustly expand CD34+ cells to HTLP with a median of 79.7% (range 54.6; 98.9) of differentiation (CD7+ CD3-) and a median fold of proliferation of 14.7 (range 8.80; 18.5). No chromosome instability was induced during the expansion. Patients received increasing doses ranging from 0.1 to 0.5 10 6 HTLP/kg. The amount of CD3+/kg was less than 11.2 (range 0; 588)/ kg. No early or mid-term toxicity due to the administration of HTLP has been reported for evaluable patients (6/6). One SCID patient died on D53 because of severe VOD. Primary engraftment was confirmed in 4 out of 5 patients. Interestingly, despite the gradual increase of doses, there was no aGVHD over grade >2. Preliminary efficacy results suggest that circulating CD3+ CD4+ TCRαβ+ T cells > 50/μl before M4 depended on the increased infused dose of HTLP and an intrathymic paracrine effect. Notably, there was no viral infection disease or relapse during the outcome. Discussion conclusion: First infusions of HTLP in both clinical trials confirmed the safety and reproducible GMP manufacturing. HLTP offers an exciting perspective for improving immune reconstitution in alternative hematopoietic transplants.

Phase 1b/2 Trial of Enasidenib in Lower-Risk MDS and Nonproliferative CMML without Isocitrate Dehydrogenase Type 2 Mutations

Introduction: Enasidenib is an FDA approved selective-inhibitor of mutant IDH2 ( mIDH2) enzyme commonly found in acute myeloid leukemia (AML). Previous studies found enasidenib unexpectedly improved red blood cell and platelet transfusion needs in mIDH2 AML patients without a reduction in AML disease burden (Pollyea DA, et al. Leukemia 2019; 33(11):2575-2584; DiNardo CD, et al. Lancet Oncol 2021; 22(11):1597-1608). We subsequently demonstrated enasidenib robustly enhanced human erythropoiesis from umbilical cord-blood and bone marrow-derived CD34+ hematopoietic progenitor cells independent of IDH2 status and the oncometabolite R-2-hydroxyglutarate (R-2-HG), thus prompting us to investigate enasidenib as an erythropoiesis-stimulating agent (Dutta R, et al. J Clin Invest 2020; 130(4):1843-1849). Aim: To evaluate the safety, tolerability, and efficacy of daily enasidenib in patients with lower-risk MDS or nonproliferative CMML without IDH2 mutations. Methods: In this ongoing two-part phase 1b/2 single-center, investigator-initiated, open-label trial, patients with a diagnosis of IDH2 wildtype lower risk MDS and nonproliferative CMML self-administered continuous daily dosing of enasidenib. Two dose levels were planned at 100mg and 200mg daily for Cohort A and B, respectively based on a standard 3 x 3 dose escalation schema. All patients were evaluated for adverse events (AEs) to determine dose-limiting toxicities (DLTs) and establish the recommended phase 2 dose (RP2D). Patients were screened in a consecutive manner to reduce selection bias by the investigators and all consents were translated into participant's native language to ensure full understanding and voluntary participation. Eligible patients are 18 years or older, ECOG ≤3, negative for IDH2 mutation, have adequate organ function, and symptomatic anemia (hemoglobin <10.5g/dL). Other causes such as iron/nutritional deficiencies and bleeding were ruled out during screening. Prior treatment with ESA, G-CSF, and luspatercept was allowed with a 30-day washout period. Each cycle is 28 days. Patients were evaluated by the PI on Day 1 (±3 days), and laboratory evaluation was conducted every 15 ±3 days. Results: Seven patients were enrolled between July 6, 2022 and December 21, 2022 with three patients in Cohort A receiving 100mg and 4 patients in Cohort B receiving 200mg (Table 1A). No DLTs were seen during the first 6 cycles of treatment. One treatment was discontinued due to the need for treatment of vasculitides related to a new diagnosis of VEXAS made during cycle 2 of the trial (unrelated to study drug). Another patient discontinued treatment after 1 cycle to pursue allogeneic stem cell transplant. All patients experienced at least one treatment-related adverse event (TRAE) which were limited to grade 1 or 2. TRAEs noted were consistent with those previously reported with enasidenib use (Stein EM, et al. Lancet Haematol 2020; 7(4):e309-e319), most commonly hyperbilirubinemia observed in 6 patients (85.7%), nausea in 4 patients (57.1%), and rash (inflammatory dermatoses, previously well-described) in 5 patients (71.4%) (Table 1B) (Parisi R, et al. Leuk Res 2022; 123:106970). No grade 3 or higher TRAE were observed. Two patients (28.7%) experienced a Grade 3 adverse event of pneumonia. Four patients developed an infection (57.1%) with only one requiring a hospitalization. One grade 4 event occurred (duodenal hemorrhage) in a patient with history of duodenal ulcers and concurrent aspirin use. As above, a patient in Cohort A was diagnosed with VEXAS syndrome after enrollment and experienced a complicated course of vasculitis and opportunistic infections in the setting of high dose steroid use. Interestingly, one newly diagnosed patient in the 200mg Cohort B without prior growth factor or ESA is enjoying a sustained trilineage improvement with increase of absolute neutrophil count, hemoglobin, and platelets from baseline (10 cycles at the time of data cutoff of June, 16, 2023). Conclusions: Enasidenib at 200mg dose was safe and well tolerated in patients with lower-risk MDS. Thus far, one patient has seen trilineage improvement in blood counts and continues on study. The Phase 2 portion of the study is ongoing to further characterize safety and evaluate for efficacy to improve anemia and decrease transfusion needs.

Human ILC1s Target Leukemia Stem Cells and Control Development of AML

Acute myeloid leukemia (AML) is a devastating disease with a median 5-year survival of only 40-45% for patients younger than age 65 who are treated with standard chemotherapy. Although in some cases allogeneic stem cell transplantation has proven to be curative, the treatment-related mortality and the risk for disease relapse due to persistence of leukemia stem cells (LSCs) remains relatively high. Therefore, safer and more effective novel therapeutic approaches are needed to improve the clinical outcomes of patients with AML. Innate lymphoid cell (ILC) is critical in mediating immune responses and regulating tissue homeostasis and inflammation. We recently reported that mouse ILC1s contribute to the control of AML by eliminating LSCs and inhibiting their differentiation into myeloid blasts, and functional impairment of mouse ILC1s in AML leads to the outgrowth of LSCs and disease relapse (Li et al., Nature Immunology. 2022). However, the full role and mechanistic characterization of human ILC1s in anti-tumor responses to AML remains to be fully explored. Upon analysis of ILC1s in the blood of patients with AML at the onset of disease, we observed a highly significant reduction in the total ILC1s count among lineage-negative cells (Lin −, defined asdepletion of CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FceRI, and CD56 positive cells) relative to healthy donors ( p = 0.031, n = 6 healthy donors; n = 4 patients with AML). Further, functional ILC1s positive for IFNγ and DNAM-1 were significantly reduced in the patients with AML compared to healthy donors. Analysis of 106 AML cases from the Cancer Genome Atlas (TCGA) showed that AML patients with a high ILC1 gene signature had a significantly prolonged overall survival compared to AML patients with a low ILC1 gene signature. By directly interacting with LSCs, human ILC1s can eliminate LSCs via the production of IFNγ. Through Wright-Giemsa staining, we observed that ILC1s blocked the differentiation of CD34 +CD38 − cells into macrophage-like leukemia-supporting cells, which were previously reported to support the growth of leukemic cells rather than inhibit them. Flow cytometry of these differentiated cells showed that some exhibited the tumor-promoting phenotype with expression of CD11b and CD206. These macrophage-like leukemia-supporting cells significantly decreased when co-cultured with ILC1s. This differentiation is at least partially dependent on TNF secreted by ILC1s. We also performed an in vivo transplantation experiment, in which human CD34 +CD38 − cells and human ILC1s were co-injected intravenously (i.v.) into NOD.Cg- Prkdc scid Il2rg tm1WjlTg (NSG-SGM3) mice that express human IL3, GM-CSF, and SCF to support the stable engraftment of myeloid lineages. Injection of human ILC1s from healthy individuals reduced the LSC engraftment into these mice and suppressed the progression of AML. This was evidenced by a significant decrease in the number of CD45 +CD33 + blast cells, CD34 +CD38 − LSCs, and significantly prolonged survival of the mice ( p = 0.0118, n = 5/group). These results were all in comparison to mice that did not receive an injection of ILC1s. Although ILC1s target LSCs, they have no toxicity on normal HSCs. For a therapeutic purpose, we were able to derive ILC1s from umbilical cord blood (UCB) CD34 + hematopoietic stem cells (HSCs) with over 700-fold expansion and the expanded ILC1s demonstrate authentic ILC1 phenotypes and functions. Moreover, arming the expanded ILC1s with a chimeric antigen receptor (CAR) targeting FLT3, which we previously constructed (Chen et al., Leukemia. 2017), enhanced their effector function. In conclusion, ILC1s in patients with AML are impaired, while a high expression of the ILC1 gene signature is associated with better overall survival. Human ILC1s can both eliminate LSCs via production of IFNγ and block LSC differentiation into tumor-promoting macrophage-like cells through TNF. These effects converge to limit leukemogenesis in vivo. The use of UCB CD34 + HSCs to generate ILC1s, especially after being engineered with a CAR, could allow for a readily available supply of ILC1s to be produced for human adoptive transfer studies. Our findings provide evidence that targeting human ILC1s may be a promising therapeutic approach for extending disease-free survival in patients with AML.

Abstract 32 Expanded Umbilical Cord Blood CD34+ Cells Are Immunomodulatory in a Rat Model of Neonatal Brain Injury

Abstract Introduction Hypoxic ischemic (HI) insults during pregnancy and birth can result in neurodevelopmental disorders, including cerebral palsy. We have previously shown that multiple doses of umbilical cord blood (UCB) cells are needed to effectively reduce long-term neuropathology and behavioural outcomes. Unfortunately, for multiple doses it is likely that UCB expansion will be required to increase the number of cells available for treatment. Objectives In this study, we aimed to investigate the efficacy of expanded UCB derived CD34+ cells in modulating perinatal brain injury. Methods UCB derived CD34+ cells were expanded over a 14-day period using a previously optimised expansion protocol. HI injury was induced in postnatal day (P) 10 rats by left carotid artery ligation, followed by 90min hypoxia (8% oxygen). Pups were administered 1 million expanded UCB cells (eUCB) on P11, 13 and 20. Rats underwent extensive behavioural testing and post-mortem was performed on either P17 or P50 where tissue was collected for flow cytometry or immunohistochemistry analysis. Results After the 14-day expansion there was a ~800-fold increase in cell number with 70.7±2.18% CD34+ cells. Flow cytometry at P17 showed a significant increase in peripheral regulatory T-cells following eUCB administration (P=0.047). At P50, HI brain injury resulted in significant behavioural deficits and adverse neuropathologies. Multiple doses of eUCB significantly reduced microglial activation compared to HI in the cortex (P=0.02) and the hippocampus (P=0.018). Discussion Results from this study show for the first time that expanded UCB-derived CD34+ cells are a promising treatment option for perinatal brain injury as they have significant anti-inflammatory properties.

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

A Microwell Device for the Efficient Generation of Arrays of Microtissues and Humanized Bone Marrow Micro-Ossicles

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.

Evaluation of Expansion and Maintenance of Umbilical Cord Blood CD34+ Cells in The Co-Culture with Umbilical Cord Blood-Derived Mesenchymal Stem Cells in The Presence of Microcarrier Beads.

Umbilical cord blood (UCB) is an accessible and effective alternative source for hematopoietic stem cell (HSC) transplantation. Although the clinical application of UCB transplantation has been increased recently, quantitative limitation of HSCs within a single cord blood unit still remains a major hurdle for UCB transplantation. In this study we used microcarrier beads to evaluate the ex vivo expansion of UCB-derived HSCs in co-cultured with UCB-derived mesenchymal stem cells (MSC). In this experimental study, we used microcarrier beads to expand UCB-derived MSCs. We investigated the simultaneous co-culture of UCB-derived CD34+ cells and MSCs with microcarrier beads to expand CD34+ cells. The colony forming capacity and stemness-related gene expression on the expanded CD34+ cells were assessed to determine the multipotency and self-renewal of expanded cells. Our results indicated that the microcarrier-based culture significantly increased the total number and viability of UCB-derived MSCs in comparison with the monolayer cultures during seven days. There was a significant increase in the UCB-derived CD34+ cells expanded in the presence of microcarrier beads in this co-culture system. The expanded UCB-derived CD34+ cells had improved clonogenic capacity, as evidenced by higher numbers of total colony counts, granulocyte, erythrocyte, monocyte, megakaryocyte colony forming units (CFU-GEMM), and granulocyte-monocyte colony forming units (CFU-GM). There were significantly increased expression levels of key regulatory genes (CXCR4, HOXB4, BMI1) during CD34+ cells self-renewal and quiescence in the microcarrier-based co-culture. Our results showed that the increase in the expansion and multipotency of CD34+ cells in the microcarrierbased co-culture can be attributed to the enhanced hematopoietic support of UCB-derived MSCs and improved cell-cell interactions. It seems that this co-culture system could have the potential to expand primitive CD34+ cells.

13P Universal prospects of cryopreserved umbilical cord blood CD34+ progenitor cell-derived NK cells: Clinical and preclinical evaluation of non-engineered and genetically engineered candidates

13P Universal prospects of cryopreserved umbilical cord blood CD34+ progenitor cell-derived NK cells: Clinical and preclinical evaluation of non-engineered and genetically engineered candidates

Human serum albumin promotes self-renewal and expansion of umbilical cord blood CD34+ hematopoietic stem/progenitor cells.

We investigated the effect of human serum albumin (HSA) on human umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells (HSPCs) cultured in vitro and transplanted in vivo. Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation. CD34+ cells were then sorted by CD34 conjugated magnetic microbeads. The sorted cells were cultured with or without HSA for 8 days in vitro. After 8 days, all cells were harvested for flow phenotyping and colony formation cell (CFC) experiments. The cells were injected into immunodeficient mice (NOD/Shi-scid/IL2Rγnull, NOG) via intravenous injections. From 4 weeks post-transplantation, flow cytometry was used to calculate human cell chimerism in the peripheral blood (PB) every 2 weeks. Flow phenotyping of human cell chimerism in bone marrow and spleen was calculated 16 weeks post-transplantation. Compared to the control group, CD34+ cells cultured with HSA increased significantly in vitro. The long-term engraftment of HSPCs and the hematopoietic multilineage reconstruction capacity were preserved by HSA. Normal engraftment of human cells could be maintained via HSA treatment could maintain normal engraftment of human cells in recipient PB. Here, we found that HSA was beneficial to maintaining CD34+ cell expansion and short-term colony formation in vitro and optimizing multilineage reconstitution in immunodeficient mice in vivo.

The Investigation of Antitumor Effect of Humanized Bispecific BCMA/CD38 CAR-T Cells on Multiple Myeloma

Background Multiple myeloma (MM) is an incurable disease that will eventually deteriorate. Chimeric antigen receptor (CAR) T cell therapy has been widely used in the treatment of hematological malignancy and showed a promising effect. BCMA (CD269) is exclusively expressed on the MM cells, making it an ideal target for immunotherapy. CD38 is highly expressed on the surface of MM cells, but is also expressed in immune cells and a minority of hematopoietic stem/ progenitor cells. Humanization reshaped antibody may reduce the potential risk of immunogenicity associated with the animal-derived antibodies. In this study, we constructed humanized BCMA CAR-T, CD38 CAR-T and bispecific BCMA/CD38 CAR-T, and explored their killing efficiency towards MM cells, providing the basis for clinical application. Methods Mouse anti-human BCMA and CD38 single chain fragment variable (scFv) sequences were obtained from previously established monoclonal antibody hybridoma cells (69G8 and HIT2) in our institution, respectively. The sequences were then humanized. Single-target CAR (BCMA CAR, CD38 CAR) and dual-target BCMA/CD38 CAR (LoopBC38 CAR) plasmids were constructed. Phenotype of CAR-T cells was detected. CAR-T cells and tumor cell lines were co-cultured in vitro, followed by the detection of the percentage of residual tumor cells, the degranulation and cytokine release level of T cells. Bone marrow mononuclear cells (BMMNC) from MM patients were co-cultured with CAR-T cells. MM cells were intravenously injected into immunodeficient mice, followed by CAR-T cell therapy. After co-culturing with peripheral blood mononuclear cells (PBMC) from healthy donors and CD34+ umbilical cord blood (UCB) stem cells, the proportion, cell subsets of residual cells and colony formation ability were assessed to verify the safety of CAR-T cells. Results The proliferation ability of CD38 CAR-T and LoopBC38 CAR-T was weaker than that of VEC-T and BCMA CAR-T. The residual H929 cells (BCMA+CD38+) after 48h cocultured with VEC-T and LoopBC38 CAR-T at E:T ratio of 1:1 was (20.82±9.773) % vs. (0±0) % (p<0.0001). After 48h at E:T ratio of 1:1, residual K562-BCMA cells (BCMA+CD38-) cocultured with CD38 CAR-T were (49.53±9.773) %, while that of LoopBC38 CAR-T were (8.49±4,766) % (p<0.0001). After 48h at E:T ratio of 1:4, residual Raji cells (BCMA-CD38+) were (62.69±22.2) % in BCMA CAR-T and (19.81±12.22) % in LoopBC38 group (p<0.01). The secretion level of cytokines such as IFN-γ, TNF-α, IL-2 was significantly higher in CAR-T cells than that of VEC-T cells. After co-cultured with H929, the IL-6 secretion level of LoopBC38 CAR-T cells was (7.28±0.4558) %, which was lower than that of CD38 CAR-T cells [(259.4±3.039) %, p<0.0001]. When cocultured with BMMNC derived from MM patients, BCMA CAR-T could kill BCMA+ cells, CD38 CAR T could kill CD38+ cells, while LoopBC38 CAR-T has both effects. In H929 xenograft NSG mice, the median survival time of MM mice was 35 days for VEC-T, 82 days for CD38 CAR-T. Until now, more than 50% mice are alive in BCMA CAR-T group and LoopBC38 CAR-T group, so they didn't reach the median survival time yet. LoopBC38 CAR-T did not significantly kill PBMC form healthy donors, with the residual PBMC cells after 24h co-culturing was (48.26±1.708) % in VEC-T and (49.0±2.517%) in LoopBC38 CAR-T (p>0.05), but had killing efficiency towards mononuclear-macrophages and NK cell. A slight killing effect on UCB CD34+ cells was observed in LoopBC38 CAR-T. The residual CD34+ cells in LoopBC38 CAR-T were (29.47±4.077) %, while that of VEC-T was (48.5±11.88) % (p>0.05). The number of CFU-GEMM in LoopBC38 CAR-T was (3.0±1.512) %, which was lower than that of VEC-T [(4.667±3.386) %, (p<0.001)]. Conclusion All CAR-T cells showed strong killing efficiency toward MM cells both in vitro and in vivo. Humanized BCMA CAR-T and CD38 CAR-T can specifically kill multiple myeloma cell lines and primary cells of patients. Bispecific CAR-T was able to target both antigens. CD38 CAR-T and bispecific CAR-T cells showed self-killing ability and altered their phenotypes. They can kill NK cells, mononuclear-macrophages in peripheral blood of healthy donor and have certain effects on the formation of CFU-GEMM, which may be a safety problem that requires further improvement for the application of CD38 CAR-T and bispecific BCMA/CD38 CAR-T cells.

Primary HSPC-Derived Monocytes with DNMT3A p.R882H Exhibit Activated Myeloid Cell Gene Signatures

Clonal hematopoiesis (CH) is a common age-associated phenomenon of specific somatic clonal changes in blood cells. CH has been found to increase the risk of hematopoietic malignancies and cardiovascular disease, as well as an assortment of other chronic medical conditions. DNMT3A, which encodes a de novo DNA methyltransferase, is the most commonly mutated gene in CH and has a mutational hotspot at p.R882. Here, we used CRISPR knock-in to engineer primary hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) with DNMT3A p.R882H in order to evaluate the impact of this mutation on cellular phenotypes. To model the R882H mutation, we employed electroporation assisted transduction using Cas9-gRNA RNP along with single-stranded AAV6 as a homology donor. In addition to the desired amino acid substitution, we inserted a self-cleaving fluorescent marker under control of the endogenous DNMT3A promoter. With this construct, we achieved high transduction efficiency (range 39-51%) of UCB CD34+ HSPCs. We then used FACS to isolate mCherry+/BFP+ homozygous-edited CD34+ cells for culture and downstream experiments. Following a week of culture in HSPC expansion media (SFEM II with Stemspan supplement, SR-1, & UM-729) and a week of culture in monocyte differentiation media (IMDM with 20% FBS, SCF, M-CSF, IL-3, & FLT3L), we performed negative selection to enrich for CD14+ monocytes without CD16+ depletion. Given that the R882H mutation has been found to associate with atherosclerosis, we then asked if the transcriptome of the R882H population showed any differences in immune signaling pathways at baseline. We therefore performed bulk RNAseq on unstimulated cells (N = 3 biological replicates) and analyzed results with DESeq2. Comparing R882H samples to isogenic wild-type controls revealed 304 differentially expressed genes (FDR < 0.05), including higher expression of CCL8, a monocyte chemoattractant, and MAOB, a mediator of myeloid IL-1β expression and mitochondrial ROS production in response to NLRP3-activating stimuli (Fig 1). Pathway enrichment analysis was also suggestive of a highly primed immunologic posture in the R882H cells. Examination of the MSigDB Immunological Signatures (V7.5.1) pathways with the highest and lowest normalized enrichment scores showed a pattern consistent with myeloid cell activation (Fig 2). Among the most highly enriched was a set of genes with lower expression in myeloid cells of control subjects compared to those from patients with lupus (GSE10325, NES = 2.32, padj = 6.1e-08, red diamonds in Fig 1). Also enriched were genes with lower expression in pre-vaccine PBMCs vs. PBMCs 7 days after subject vaccination with the attenuated yellow fever 17D vaccine, which is known to generate a strong innate immune response (GSE13485, NES = 2.61, padj = 6.1e-08). Other enriched pathways included genes upregulated when macrophages were stimulated with LPS compared to LPS plus IL-10 (GSE9509, NES = 2.34, padj = 6.1e-08), genes upregulated in WT compared to Pparg knockout macrophages after rosiglitazone treatment (GSE25123, NES = 2.27, padj = 6.1e-8), and genes downregulated in healthy dendritic cells compared to those infected with Newcastle virus (GSE18791, NES = 2.24, padj = 9.9e-08). In line with these, there was also de-enrichment of genes that are upregulated in PBMCs from healthy donors compared to PBMCs from infants with flu (GSE34205, NES = -2.03, padj = 6.1e-6). The preponderance of significantly enriched gene sets describing myeloid activation across numerous contexts suggest that monocytes with R882H are at baseline immunologically primed, even in the absence of a specific insult. Prior research has demonstrated severe myeloid inflammatory dysfunction in DNMT3A-mutant cells, but these studies often rely on provocative stimuli such as LPS or have used samples taken from patients with numerous comorbid conditions. In this study, we used high-efficiency CRISPR knock-in to introduce DNMT3A p.R882H into primary UCB HSPCs that we subsequently differentiated into monocytes. In doing this, we showed that this mutation may cause a dysregulated immune response in myeloid cells prior to any inflammatory insult. Furthermore, the techniques used in this study can be extended to examine other CH mutations, various hematopoietic lineages, and responses to exogenous stimuli. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 1640: Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L

Abstract Humanized mice are being applied widely to study human immune system homeostasis, function, and as a testing platform for cancer immunotherapies. A major limitation for many humanized mouse models is the lack of functional and mature human innate immune cells, which are critical for effective human immune system-tumor interactions. Previous studies have demonstrated that delivery of human FLT3L into immunodeficient mice that lack mouse FLT3, promotes the development of human innate immune cell subsets following engraftment with human hematopoietic stem cells (HSC). Here we describe a NOD-scid IL2rgnull (NSG) mouse that lacks the expression of mouse FLT3 and expresses human FLT3L transgenically (NSG-mFLT3nullTg (HuFLT3L) or NSG-FLT3L. In these studies, NSG-FLT3L and NSG mice were engrafted with human umbilical cord blood (UCB) CD34+ HSC and compared for human immune system development and function. HSC-engrafted NSG-FLT3L and NSG mice show similar levels of total human CD45+ cells in blood over the course of 18 weeks post-engraftment. However, HSC-engrafted NSG-FLT3L mice show significantly higher levels of human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes, CD56+ NK cells and CD3+ T cells in the blood as compared to NSG mice. CD34-FLT3L mice also show increased levels of human immune cell infiltration into the gut mucosa. CD56+ NK cells and CD3+ T cells from HSC-engrafted NSG-FLT3L mice express granzyme A and granzyme B, indicating cytotoxic activity. Following treatment of HSC-engrafted NSG-FLT3L mice with LPS, heightened levels of human cytokines were detected in serum samples, confirming innate immune system function. The growth kinetics of tumor cells from a triple negative breast cancer cell line MDA-MB-231 and a lung PDX model LG1306 in HSC-engrafted NSG-FLT3L mice are work in progress and will be compared with HSG-engrafted NSG mice. Overall these results demonstrate that the NSG-FLT3L model supports enhanced development of functional, human innate immune cells and is a novel tool to study human immuno-oncology. Citation Format: Li-Chin Yao, Shantashri Vaidya, Pali Kaur, James G. Keck, Leonard D. Shultz, Dale L. Greiner, MIchael A. Brehm. Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1640.

Effect of expansion of human umbilical cord blood CD34\u2009+\u2009cells on neurotrophic and angiogenic factor expression and function

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90.

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood–brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays >2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate >350% relative cell killing is achieved in SCI-101–treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.

Ex Vivo Production of Large Numbers of Genetically Modified NK Cells from Cord Blood or Mobilized Peripheral Blood CD34 + Cells Using Notch Ligand Delta-like 4 Culture System

Natural killer (NK) cells are an essential component of human innate immune, with a remarkable ability to provide protection against cancer and viral infections. NK-based immune therapy has several advantages over T cell immunotherapy: NK cells do not cause graft-versus-host disease and do not induce T-cell driven inflammatory cytokine storm. Moreover, unlike T cell, NK cells do not need prior sensitization, specific antigen recognition and clonal expansion for their cytotoxic effector functions and they can rapidly trigger cytotoxicity against their targets. NK cells can be expanded from multiple sources including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), or mobilized peripheral blood (mPB) CD34 + cells. Yet the lack of efficient methods for in-vitro NK cell expansion, purification and genetic modification limits the clinical use of NK cell therapy.We aimed at developing a simple, scalable and clinically feasible technique for therapeutic NK cell production based on our recently published culture system that can produce large numbers of unmodified or genetically modified CD7 + T lymphoid progenitors from UCB and mPB CD34 + cells in 7 days on immobilized Notch-ligand delta-like 4 recombinant protein DLL4. Since T cells and NK cells share a common T/NK cell progenitor, we are interested to investigate the NK cell potential of DLL4 culture-generated T lymphoid progenitors.Firstly, we tested the NK cell potential of the transduced or non- transduced UCB or mPB CD34 +-derived progenitors from DLL4 culture by subjecting them to a feeder free NK cell differentiation for 3 weeks. HSPC-derived progenitors can be efficiently transduced with lentiviral vectors (average transduction efficacy: 50%). The transduced/ non-transduced progenitors were able to efficiently differentiate into NK (CD3 -CD56 +) cells beginning from one week of differentiation, reaching a frequencies of NK cells of >90% without any detectable T cell contamination at week 2. The phenotypic characterization of the NK cells demonstrates the presence of activation receptors such as NKG2D, DNAM-1, NKp30, NKp44 and NKp46 while lacking the inhibitory receptors like KLRG1, KIR2DL2/DL3 and KIR3DL1/DL2. Interestingly, these cells express the transcription factors known to be essential for NK cell differentiation and functions such as Eomes, T-bet and ID2. Additionally, the CB or mPB HSPC-derived NK cells (both transduced/ non-transduced) express perforin and granzyme B reflecting their ability to show cytotoxic potential.Further, the stimulation of UCB or mPB derived NK cells with myelogenous leukemia cell line K562 cells showed high level of degranulation, which is the key step for Interferon gamma (IFNg) induction. An analysis cytotoxic activity of the CB or mPB derived NK cells against K562 cells and monocytic leukemia cell line THP1 cells showed their ability to efficiently kill K562 cells compared to THP1 cells, however the UCB derived NK cells were highly cytotoxic compared to mPB derived NK cells.These data suggests that our DLL4 culture system, along with feeder-free NK cell differentiation is a unique combination that is able to give rise to high number of pure NK cell population with high cytotoxic potential. These results lay a foundation towards an easier approach to NK cell therapy for effective treatment of cancers and viral infections, which will be developed in collaboration with Smart Immune Inc. DisclosuresCavazzana: Smart Immune: Other: co-founder.

Loss of Lamin B1 in Myeloid Neoplasms with 5q Deletion Causes Myeloid-Biased Hematopoiesis and Pelger-Huet Nuclear Anomaly

Hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations and cytogenetic abnormalities leading to myeloid neoplasms (MDS/AML). A subgroup of 10-20% MDS and AML cases undergo complex chromosomal rearrangements associated with TP53 mutations and poor prognosis. One of the most common chromosomal events in high-risk myeloid neoplasms is deletion of chromosome 5q (del(5q)). In contrast to 5q- syndrome, characterized by a distal commonly deleted region (CDR) and good prognosis, the critical CDR in high-risk MDS/AML centers on 5q31. However, the key genes in the high-risk 5q-deleted region that contribute to dysregulation of hematopoiesis, chromosomal instability, and disease progression remain poorly characterized.Progression to high-risk MDS/AML is often accompanied by morphologic dysplasia in the form of Pelger-Huët cells, abnormal neutrophils with bilobed or unilobed nuclei and coarse clumping of the nuclear chromatin. The inherited form of Pelger-Huët anomaly (PHA) is caused by mutations in LBR, which encodes lamin B receptor. Acquired PHA or pseudo-PHA is commonly seen in myeloid malignancies, often in high risk MDS and AML with TP53 mutations. While LBR is not mutated in MDS/AML, its main interaction partner lamin B1/LMNB1 is encoded in the high-risk 5q-deleted region. Lamins organize chromatin into perinuclear compartments and regulate diverse biological processes, including gene expression and DNA repair. The frequent deletion of LMNB1 in 5q-deleted MDS and AML led us to hypothesize that LMNB1 loss drives both nuclear dysmorphology and functional HSC defects in malignant transformation.LMNB1 expression was broadly decreased in MDS and AML, particularly in del(5q) cases, 83% of which had deletions of the LMNB1 locus. We show that in a clinical cohort of MDS patients, the Pelger-Huët nuclear phenotype is strongly associated with del5q. To understand the role of LMNB1, we performed lentiviral shRNA knockdown in normal umbilical cord blood (CB) CD34 + HSPCs followed by transplantation into immunodeficient NSG mice. To study the role of lamin B1 in 5q-deleted MDS, we established an in vitro patient-derived induced pluripotent stem cell (iPSC) model by reprograming an MDS patient with TP53 mutation and complex karyotype, and deriving MDS HSPCs with TP53+/R209fs alone or TP53+/R209fs with an isolated del5q spanning 5q22-5q31.1 encoding LMNB1 (TP53;del5q).Using these model systems we show that LMNB1 loss is both necessary and sufficient to cause Pelger-Huët nuclear morphology in normal and MDS neutrophils. Additionally, we find that LMNB1-deficient human HSCs display increased self-renewal and profound myeloid lineage bias in vitro and in vivo. Single cell RNA expression data from LMNB1-deficient cells in vivo shows mis-expression of lineage-specifying transcription factors in single multipotent progenitors. LMNB1 deficiency also results in poor marking and propagation of unrepaired DNA breaks, leading to genome instability. The role of LMNB1 in maintaining chromatin organization prompted us to use chromosome conformation capture (Hi-C) to visualize the 3D chromatin organization of LMNB1-deficient HSPCs. We show that lamin B1 regulates enhancer-promoter loops suggesting that lamin B1 loss dysregulates HSC function by altering enhancer-promoter interactions at lineage-specifying transcription factor loci.Together, we identify lamin B1 as a novel 5q gene that contributes to malignant transformation by driving enhanced self-renewal, myeloid-bias, and genome instability. Furthermore, lamin B1 deletion is the genetic cause of acquired PHA in myeloid malignancies, providing a potential biomarker for high risk neoplasms. DisclosuresBecker: Glycomimetics: Research Funding; CVS Caremark: Consultancy; Abbie: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Cardiff Oncology: Research Funding; SecuraBio: Research Funding.