Subset Of Hematopoietic Stem Cells Research Articles

An epigenetically distinct HSC subset supports thymic reconstitution.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). The Kit lo subset of HSCs is enriched for multipotential precursors, 1, 2 but their T-cell lineage potential has not been well-characterized. We therefore studied the thymic reconstituting and T-cell potential of Kit lo HSCs. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs support better thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. We find the frequency of the Kit lo subset declines with age, providing one explanation for the reduced frequency of T-competent HSCs and reduced T-lymphopoietic potential in BM precursors of aged mice. 3, 4, 5 Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs with enhanced lymphoid potential have a distinct underlying epigenetic program.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p<0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p<0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p<0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p<0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p<0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Relationship between Sarcopenia and the Heterogeneity and Microenvironment of Acute Myeloid Leukemia Based on Single-Cell Transcriptome Sequencing

Background: High intratumoral heterogeneity heterogeneity (ITH) and changes in bone marrow microenvironment (BMM) components are important for drug resistance and recurrence in patients with acute myeloid leukemia (AML), and may also influence the phenotype of sarcopenia. This study aims to determine the relationship between sarcopenia and the heterogeneity and microenvironment of AML. Method: Body composition was assessed by bioelectrical impedance analysis before treatment. Sarcopenia was diagnosed by low muscle quantity according to European Working Group on Sarcopenia in Older People 2 (EWGSOP2). Bone marrow samples from 9 patients with relapsed or refractory AML were used to perform single-cell transcriptome sequencing (scRNA-seq) to analyze the differences in tumor cell subsets and microenvironmental components between AML patients with or without sarcopenia. The samples of 19 newly diagnosed AML patients were further collected for bulk RNA-seq, and deconvolution was done. Bioinformatics methods were used to verify the differences in cell subsets of microenvironmental components in patients with and without sarcopenia, as well as the genes and signaling pathways of tumor cells with differential expression. Results: Nine leukemia-like cell subsets were identified in AML patients by scRNA-seq. In AML patients with sarcopenia, hematopoietic stem cell (HSC) subsets accounted for a significantly higher proportion of malignant cells, while progenitor cells and monocytes showed a higher proportion in patients without sarcopenia. In sarcopenic group, there were significantly fewer neutrophils, megakaryocytes, and CD8 + Tcm cells in the microenvironment component, while significantly more immunosuppressed Treg cells. After deconvolution of bulk RNA-seq data from 19 newly diagnosed AML patients, it was confirmed that Treg cells were more distributed in sarcopenic patients. Among them, 167 genes were significantly up-regulated, and the most significantly up-regulated 5 genes were ME3, UBE2E3, PCSK5, LAMC1 and PTPRF. A total of 224 significantly down-regulated genes were identified, including RNF18B, TNFAIP3, GALNT2, ORM2 and IL1R2. Pathway enrichment of differentially expressed genes was found, including signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, protein digestion and absorption pathway, etc. Conclusion: Sarcopenia may be related to immunosuppressive microenvironment and affect the prognosis of AML patients.

Cytotoxic Stress Transiently Increases the Contribution of Platelet-Biased Hematopoietic Stem Cells to Platelet Production

While hematopoietic stem cells (HSCs) and megakaryocyte (Mk)/platelet lineages exhibit substantial phenotypic and transcriptional overlap, direct Mk production from HSCs is still under investigation. In particular, the hypothesis that specific HSC clones might facilitate rapid megakaryopoiesis and their physiological roles are yet to be elucidated. Previous clonal studies using either single cell transplantations (Yamamoto, Cell 2013; Carrelha, Nature 2018) or barcoding assays (Pei, Cell Stem Cell 2020; Rodriguez-Fraticelli, Nature 2020) have identified subsets of HSCs with Mk-biased output, designated as Mk-biased HSCs or platelet-biased HSCs (P-HSCs), in contrast to multi-lineage HSCs (MUL-HSCs) with balanced output. Moreover, P-HSCs exhibit up-regulated platelet-lineage gene expression and chromatin accessibility, and they generate platelets at a faster rate than MUL-HSCs, pointing towards a direct platelet formation pathway (Rodriguez-Fraticelli, Nature 2020; Meng, 2023 Nat Cell Biol). Notably, under inflammatory stress, direct Mk progenitor (MkP) production and Mk formation from HSCs have been shown to accelerate (Haas, Cell Stem Cell, 2015; Morcos, Nat Commun 2022), suggesting that P-HSCs could drive rapid platelet production during hematopoietic emergencies. To probe this premise, we re-analysed previously published RNA-seq data on HSC subtypes and discovered that the gene Vldlr is markedly expressed in MUL-HSCs relative to P-HSCs. We subsequently created a Vldlr-Cre transgenic mouse model for lineage tracing of platelet production from MUL-HSCs and P-HSCs. Flow cytometry analysis across the HSC-MPP2-preMegE-MkP-platelet developmental sequence revealed a notable increase in the percentages of Tomato- HSCs and platelets, in comparison to MPP2-preMegE-MkP, suggesting that Tomato- HSCs significantly bypass the conventional MPP2-preMegE-MkP pathway in platelet formation. Transplantation of labelled and non-labelled HSCs confirmed that Vldlr-Cre predominantly labelled MUL-HSCs, whilst Tomato- HSCs were rich in P-HSCs and largely generated Tomato- platelets, implying that platelets derived from the direct P-HSC pathway can be identified as Tomato- through Vldlr-Cre lineage tracing. We exploited this model to examine the distinct roles of classical and direct pathways during emergency thrombopoiesis, induced by either antibody-mediated platelet depletion mimicking immune thrombocytopenia or a cytotoxic drug 5-fluorouracil (5-FU) that causes severe hematopoietic disruption. Both interventions precipitated a significant decrease in platelet counts, as previously demonstrated. However, the proportion of Tomato- platelets did not change during platelet recovery following antibody intervention, while the percentage of Tomato- platelets rose up to four-fold in early recovery stage following 5-FU treatment, suggesting an acceleration of the direct pathway under this stressor. The percentage of Tomato- platelets returned to baseline following platelet count normalization. Further analysis of bone marrow progenitors post-intervention revealed that the proportion of Tomato- MkP was maintained during recovery from antibody treatment but significantly expanded after 5-FU treatment. Nevertheless, the fraction of Tomato- HSCs remained unaltered under both interventions, indicating that neither reagent selectively activates P-HSCs or MUL-HSCs. We hypothesize that the 5-FU treatment, by significantly eliminating bone marrow progenitors, permits P-HSCs to rapidly replenish the MkP pool and platelets due to their faster platelet formation kinetics, whereas progenitors are not affected by the antibody, leaving both pathways to contribute to platelet recovery. In summary, our findings suggest that P-HSCs provide a fast-track for platelet formation, enabling them to contribute to platelet recovery more rapidly following progenitor depletion induced by cytotoxic stress.

Low CD117 Expression Identifies Hematopoietic Stem Cell Subsets with Enhanced Lymphoid Potential in Mice and Humans

Background: Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective de novo T-cell generation and thymic recovery, restoring the adaptive immune system after Hematopoietic Cell Transplantation (HCT). Recent studies have brought new insights into functional heterogeneity within HSCs, revealing an organized and predictable framework governing the adoption of lineage-restricted fates. However, the gene-regulatory networks underlying lymphoid determination and their conservation with aging in HSCs need to be elucidated. This study aims to comprehensively investigate the molecular profile of young and old HSCs and identify HSC subsets with multilineage transcriptional programs. Results: We conducted single-cell multiomic RNA and ATAC sequencing of young and old HSCs defined by Lineage-CD34-CD48-CD150+Sca-1+Kit+. We identified two CD117 (Kit) HSC subsets, Kit lo and Kit hi, with unique transcriptional profiles. Kit hi HSCs were characterized by quiescence, platelet bias, and low-output gene signatures, while Kit lo HSCs enriched for multi-lineage and high-output gene signatures. We observed a decreased frequency of Kit lo HSCs in aged mice. Although Kit lo HSCs have been previously described to exhibit increased self-renewal (Shin et al. JEM, 2014), their T-cell potential and the molecular underpinnings governing lymphoid differentiation programs are unknown. We performed in vitro studies, S17 stromal assay, and artificial thymic organoids (ATOs), which revealed an enhanced output of lymphoid progenitors and T-cells from Kit lo HSCs, consistent across all age cohorts. We next compared Kit lo vs. Kit hi HSCs in a competitive allogeneic HCT model. Kit lo HSCs demonstrated improved thymic recovery and post-HCT T-cell reconstitution, independent of age. To orthogonally evaluate post-HCT thymic function, we assessed Recent Thymic Emigrants (RTEs) output, which further substantiated superior recovery in mice receiving Kit lo HSCs. We further observed that Kit lo HSC-derived T-cells exhibited better proliferation in response to an acute Listeria monocytogenes infection. Next, we transplanted middle-aged mice to evaluate the rejuvenation potential of different Kit HSC subsets. We observed that Kit lo HSCs partly mitigated age-related changes in the thymic microenvironment, specifically by enhancing the regeneration of thymic epithelial cells (TECs) in middle-aged mice, thereby reversing the decline in T-cell production. We found differential expression and activity of key transcription factors (TFs), including Runx3, Zbtb7a, and Ezh1, associated with lymphoid differentiation through integrative transcriptional and chromatin accessibility analyses of Kit lo HSCs. These TFs notably showed distinct patterns in Kit lo HSCs independent of age, indicating their potential role in driving their enhanced T-cell potential. To establish the existence of a comparable human subset, we interrogated a human BM CITE-Seq dataset (Sommarin et al. Biorxiv 2021) for the mouse Kit lo gene signature. In concordance with our findings in mice, we observed an enrichment of the Kit lo gene program in young HSCs and decreased frequency of Kit lo HSCs in old BM. Next, we used ATOs to validate differential lymphoid potential. We found increased T-cell output originating from Kit lo HSCs, thus underscoring their enhanced lymphoid capacity and potential clinical relevance. Conclusion: Collectively, we demonstrate a distinct HSC subset, Kit lo HSC, with an age-conserved lymphoid gene program that enhances T-cell production and facilitates thymic regeneration. Importantly, we demonstrate the presence of an analogous HSC subset in humans, revealing the relevance of these insights to human health and disease. Thus, Kit lo HSCs have therapeutic potential to counteract age-associated immune senescence and treatment-related immune suppression.

Investigation of CD83 As a Novel Human Hematopoietic Stem Cell Inflammatory Activation Marker

Hematopoietic stem cells (HSC) are responsible for life-long blood production and must balance homeostasis with sudden demand situations such as infections. However, the heterogeneity of molecular programs engaged by acute inflammatory stimuli to activate emergency hematopoiesis at the level of HSC are poorly understood. In a recent study linking human HSC heterogeneity to aging, recovery from severe COVID-19 infection, and clonal hematopoiesis (CH), we discovered two HSC subsets through multiomic analysis, denoted HSC-I and -II, in a xenograft model of inflammation-recovery. While HSC-I had few inflammation-driven molecular changes, HSC-II showed sustained epigenetic and transcriptional alterations in response to dual tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) challenges despite a 2.5-month recovery period. Importantly, a signature of long-term memory T cells (Akondy et al 2017) was enriched in HSC-II (Zeng, Nagree, et al, in submission). We interrogated the human HSC multiomic signatures uncovered from this human xenograft model to identify potential regulators associated with HSC response to and recovery from acute inflammation. We identified CD83 as a candidate that is differentially expressed between our HSC subsets. Similarly, we found CD83 expression to be enriched in a subset of adult-derived HSCs from patients where CH status was established and enrichment of an HSC-II signature observed (Jakobsen et al, submitted). CD83, first identified as a dendritic cell marker, is expressed on many immune cells, with linkage to controlling murine T regulatory (T-Reg) vs memory cell fate (Doebbeler et al, 2018), but has not previously been shown to be relevant for human HSC biology. Flow cytometry analysis of cord blood (CB) hematopoietic stem and progenitor cells (HSPC) showed CD83 to be low or absent in HSC (Figure A) and their downstream progeny at steady state. However, robust CD83 expression was observed in multiple HSPC subpopulations including long-term (LT-) and short-term (ST-) HSC within 16 hours of ex vivo culture, with highest expression in LT-HSC (Figure A). Importantly, CD83 surface expression on LT-HSC was enhanced following culture with TNFα (Figure A). Bystander effects were excluded by demonstrating similar findings after culture of purified HSC populations. Acute in vivo LPS treatment of mice xenografted with CB-derived HSC was sufficient to upregulate CD83 surface expression after 16 hours on LT-HSC/HSPC. To elucidate the potential function of CD83-expressing HSC/multipotent progenitors (MPP), CD19-CD34+CD38-CD45RA- cells were sorted from xenografts following acute inflammatory challenge for low and high CD83 expression and subjected to bulk RNA sequencing. We found enrichment of a gene expression signature specific to activated human HSCs (Garcia-Prat et al, 2021) within CD83-positive HSC/MPP (Figure B). Strikingly, enrichment of a signature of stimulated vs resting T cells (Galetti et al, 2020) and of effector T cells (Akondy et al 2017) was found in CD83-positive compared to CD83-negative HSC/MPP (Figure B). T cell effector function is in part dictated by metabolic reprogramming. We thus examined reactive oxygen species (ROS) levels in LT-HSC following ex vivo culture with TNFα and found lower ROS levels with CD83 expression. We therefore hypothesize that CD83 may be an ‘effector’ marker defining a distinct HSC subset during homeostatic or inflammatory activation that is subsequently fated to become memory-like HSC-II. We are undertaking studies to determine the mechanistic role of CD83 in HSC function and HSC-II formation via Cas9-sgRNA-mediated knockout or lentivirus-mediated overexpression of CD83. In summary, CD83 represents a long-sought marker of inflammatory activation at the HSC level. CD83 may have significant clinical potential as a biomarker of aberrant activation in gene therapy manufacturing protocols or for HSC transplantation. Future studies will shed light on whether CD83 can functionally regulate the molecular programs underlying human HSC heterogeneity.

CD11a regulates hematopoietic stem and progenitor cells.

Integrin αLβ2 (CD11a/CD18, CD11a) is a critical leukocyte adhesion molecule in leukocyte arrest and immunological synapse formation. However, its role in the bone marrow has not been investigated in depth. Here we showed that CD11a was expressed on all subsets of hematopoietic stem and progenitor cells (HPSCs). CD11a deficiency enhanced HSPCs activity under lipopolysaccharide (LPS) stimulation as demonstrated by a higher HSPC cell count along with an increase in cell proliferation. However, our mixed chimera experiment did not support that this phenotype was driven in a cell-intrinsic manner. Rather we found that the production of IL-27, a major cytokine that drives HSPC proliferation, was significantly upregulated both in vivo and in vitro. This adds a novel role of CD11a biology.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Endometrial hyperplasia with loss of APC in a novel population of Lyz2-expressing mouse endometrial epithelial cells.

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of β-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Functional and Molecular Heterogeneity of Aging Hematopoietic Stem Cells

Background: Aging hematopoiesis is characterized by increased numbers of hematopoietic stem cells (HSCs) that exhibit impaired self-renewal and long-term reconstitution potential. We previously demonstrated that differences in surface expression of c-Kit in young mouse HSCs could identify distinct functional HSC subsets, with c-Kithi HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation. We therefore sought to determine if similar functional and molecular differences may be observed in old HSCs. Methods: We evaluated c-Kithi and c-Kitlo subsets of HSCs (CD34-CD150+LSK) from young (8-12 wk), and old (18-20 mo) C57BL/6J mice. For competitive transplantation assays, 250 double sorted c-Kithi or c-Kitlo HSCs from aged CD45.2 mice were mixed with 5x105 unfractionated bone marrow mononuclear cells (BMMCs) from young CD45.1 mice. In parallel, we generated the chromatin accessibility and transcriptional profiles of c-Kithi and c-Kitlo HSCs from young and old mice. Results: We first performed competitive transplants of old c-Kithi and c-Kitlo HSCs into young recipients. These studies demonstrated age-related myeloid bias exhibited by both HSC subsets. In addition, similar to young HSCs, old c-Kithi HSCs exhibit significantly reduced long-term reconstitution capacity compared to old c-Kitlo HSCs. Next, to evaluate age-related functional differences, we performed equal competitive transplants in which equal numbers of c-Kithi or c-Kitlo old and young HSCs were transplanted into the same young recipient. These studies demonstrated that c-Kithi HSCs exhibit similar reconstituting capacity independent of age. In contrast, old c-Kitlo HSCs exhibited significantly reduced long-term reconstitution compared to their young counterparts, though with better-preserved self-renewal capacity, as reflected in their self-renewal quotients (SRQ). However, when we quantified the capacity of transplanted HSCs to give rise to downstream committed progenitors and mature hematopoietic cells, calculated as their differentiation quotient (DQ), old c-Kitlo HSCs exhibited markedly reduced DQ compared to their young counterparts. In contrast, c-kithi recipients showed that old c-Kithi HSCs gave rise to mature cell lineages more efficiently. To investigate molecular mechanisms driving heterogeneity in HSC aging, we evaluated the transcriptional profiles of young and old HSCs. Old c-Kitlo HSCs showed significant enrichment of genes associated with inflammation, interferon responses, and the quiescent state in comparison to old c-Kithi HSCs. On the other hand, old c-Kithi HSCs were enriched for cell cycle related and MYC target genes compared to old c-Kitlo HSCs and young HSCs. Old c-Kithi HSCs also exhibited increased expression of genes associated with high cell output state and high mitochondrial membrane potential, which is associated with a more active HSC cell state. To investigate the potential role of chromatin accessibility in determining the observed transcriptional changes, we performed ATAC-seq of young and old HSC subsets. Old HSCs exhibited more open chromatin peaks than young HSCs, with the number of peaks decreasing in the order of old c-Kitlo > old c-Kithi > young c-Kithi > young c-Kitlo HSCs. Evaluation of predicted transcription factor binding sites showed enrichment for CTCF and PU.1 sites in young c-Kitlo and c-Kithi HSCs, respectively, with PU.1 associated with high cell output states. In contrast, old c-Kitlo and c-Kithi HSCs were enriched for ERG and CTCF sites, respectively, with predicted binding sites enriched in genes associated with low and high cell output, respectively. These data suggest that differential ERG binding of quiescence and cellular output-related gene signatures regulate functional differences between old c-Kitlo and old c-Kithi HSCs. Conclusion: Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. Identifying and prospectively fractionating old HSCs offers a novel approach for investigating the molecular mechanisms underlying HSC aging.

Systematic Evaluation of AML-Associated Antigens Identifies Novel Anti-U5 snRNP200 Therapeutic Antibodies for the Treatment of AML

Numerous antibody-based immunotherapies are in development for AML including antibodies targeting CD47, CD70, CD123, CD33, and TIM3. However, systematic evaluation of the distribution of AML-associated antigens in combination with Fc receptors and the immune microenvironment required for antibody-mediated therapeutic responses have been limited. Additionally, efforts to maximize antibody Fc receptor engagement on immune effector cells in AML patients via Fc region engineering have not been explored. Here we applied a custom 36-color flow cytometry panel tailored for AML patient bone marrow along with proteogenomic assessment via CITE-seq to simultaneously assess known and novel antibody target expression on AML cells, immune microenvironment, and Fc receptor expression across immune subsets. Application to samples from 7 age-matched healthy donors and 51 newly diagnosed AML patients allowed for unbiased identification of the malignant blast population via UMAP projection. Quantification of differential antigen expression on blasts and leukemic stem cells versus normal hematopoietic stem and progenitor cell (HSPC) subsets, identified that out of 9 AML putative antibody targets (CD123, TIM3, CD33, CD47, CD90, CD38, CD25, CD70, U5 snRNP200), only U5 snRNP200 is expressed on blasts but not normal HSPCs (Fig. A). Characterization of the distribution of Fc receptors revealed that AML patients exhibit reduced cell-surface activating Fc receptors (CD16, CD32A) on NK and non-classical monocytes with concomitant increases in inhibitory (CD32B) Fc receptors on monocytes and B-cells. Moreover, AML patients had fewer classical monocytes and type 2 conventional dendritic (cDC2) cells than normal age-match control marrow. Given the unique cell surface expression of U5 snRNP200 on AML blasts compared with normal HSPCs, we deeply interrogated the distribution of this marker throughout normal and malignant hematopoiesis. While U5 snRNP200 expression was not seen on normal HSPCs, U5 snRNP200 was present on mature B-cells and a subset of NK cells in normal adult human and mouse hematopoietic cells. U5 snRNP200 is an RNA helicase which is an essential conserved component of the spliceosome. Its known function is limited to the nucleus and it is therefore unexpected that it would be expressed on the cell surface. To rigorously confirm expression of U5 snRNP200 on the cell membrane, we knocked in a HaloTag at the N-terminus of the locus encoding U5 snRNP200 within K562 cells and traced expression of tagged endogenous U5 snRNP200 protein across cellular compartments. This clearly revealed cell surface localization of the full-length protein on the surface of AML cells. We next conducted genome-wide CRISPR screens in two human AML cell lines to identify regulators of surface U5 snRNP200 expression. In both cell lines, the top gene required for cell surface U5 snRNP200 expression was CD32A. CRISPR knockout of CD32A in U937 cells reduced U5 snRNP200 surface expression, while restoration of CD32A rescued U5 snRNP200 surface expression. AML-associated expression of U5 snRNP200 was present on a number of syngeneic immunocompetent mouse models of AML including MLL-AF9, RN2 (MLL-AF9 + NRAS G12D), transgenic EVI1 rearranged/SF3B1K700E mutant, and Tet2/Zrsr2 double knockout mice. To test the therapeutic potential of targeting cell surface U5 snRNP200, we engineered several anti-murine U5 snRNP200 antibody variants featuring modified Fc regions to assess the possible roles of engagement of Fc receptors on immune cell subsets to enhance AML cell killing. In both the RN2 and EVI1/SF3B1 mutant models, anti-U5 snRNP200 antibodies featuring the Fc region modified to engage activating Fc receptors and induce antibody-dependent cellular cytotoxicity (IgG2a) provided superior survival benefit compared to IgG1 or IgG2b U5 snRNP200 antibodies or a mutant IgG1 antibody (IgG1 D265A) which does not bind Fc receptors (Fig. B). These data provide a high-density roadmap of the distribution of known and novel AML-associated antigens together with the Fc receptor distribution and immune microenvironment in AML. The results motivated discovery of a novel antibody-therapeutic targeting aberrant cell-surface U5 snRNP200 and research into mechanisms for cell surface trafficking of U5 snRNP200. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification and Separation of Distinct Subpopulations of Hematopoietic Stem Cells and Multipotent Progenitors By Deep Learning

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are crucial for maintaining lifelong hematopoiesis. Several decades of successful clinical HSC transplantation have demonstrated the therapeutic importance of HSCs and MPPs. For basic research, HSCs and MPPs are routinely analyzed and separated by fluorescence-activated cell sorting (FACS) based on cell surface marker staining, a procedure that requires incubation with various antibodies and expensive laser-equipped flow cytometers. Both antibody staining and laser impair cell viability and stem cell activity. To develop a new staining-free, laser-free method for predicting and separating subpopulations of HSCs and MPPs, we used a deep learning approach that can extract minutiae from large-scale datasets of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) to distinguish subpopulations of hematopoietic precursor solely based on their morphology. Remarkably, our deep learning model can achieve predictable identification of subsets of HSCs with at least 85% accuracy; this demonstrates for the first time that HSC subpopulations can be differentiated solely based on their morphology exhibited in light microscopic images through deep learning. We anticipate that our robust and accurate deep-learning-based platform for hematopoietic precursors will provide a basis for the future development of a next-generation cell sorting system.

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Zeb1 sustains hematopoietic stem cell functions by suppressing mitofusin-2-mediated mitochondrial fusion

Metabolic status is essential in maintaining normal functions of hematopoietic stem cells (HSCs). However, how the dynamic of the mitochondrion, as a central organelle in metabolism, is molecularly regulated to orchestrate metabolism and HSC stemness remains to be elucidated. Here, we focus on the role of Zeb1, a well-characterized epithelial-to-mesenchymal transition (EMT) inducer which has been demonstrated to confer stem-cell-like characteristics in multiple cancer types in stemness regulation of HSCs. Using a Zeb1-tdTomato reporter mouse model, we find that Zeb1+Lin−Sca-1+c-Kit+ cells (Zeb1+-LSKs) represent a subset of functional long-term HSCs. Zeb1+LSKs exhibit a reduced reactive oxygen species (ROS) level, low mitochondrial mass, low mitochondrial membrane potential (MMP), and particularly small, round fragmented mitochondria. Of note, ectopic expression of Zeb1 leads to a fragmented mitochondrial morphology with a low mitochondrial metabolic status in EML cells. In addition, Zeb1-knockout (Zeb1-KO) LSKs from fetal liver display an exhausted stem-cell activity. Zeb1 deficiency results in elongated and tubulated mitochondria with increased mitochondrial mass, elevated MMP, and higher ROS production. Mechanistically, Zeb1 acts as a transcriptional suppressor on the key mitochondrial-fusion protein Mitofusin-2 (encoded by Mfn2). We highlight an important role of Zeb1 in the regulation of mitochondrial morphology in HSC and the metabolic control of HSC stemness by repressing Mfn2-mediated mitochondrial fusion.

Single-cell analyses reveal early thymic progenitors and pre-B cells in zebrafish.

The zebrafish has proven to be a valuable model organism for studying hematopoiesis, but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single-cell RNA sequencing on ∼70,000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus, elucidated B- and T-cell developmental trajectories, and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic-like cell populations and provided evidence in support of the existence of a pre-B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies.

Melanoma-derived small extracellular vesicles remodel the systemic onco-immunity via disrupting hematopoietic stem cell proliferation and differentiation

Hematopoiesis and the immune system beyond the tumor microenvironment are typically dysregulated in cancer. Tumor-derived small extracellular vesicles (sEVs) containing exosomes are emerging contributors to tumor progression and immunomodulation. However, a comprehensive definition of how tumor-derived sEVs impacts systemic immunity is lacking. In this study, we used mass cytometry with extensive antibody panels to determine the expression of 24 immune cell markers, eight intracellular proteins, and seven immune checkpoint proteins in systemic immune cell lineages. The systemic immune landscape in response to tumor-derived sEVs across three immune organs in a melanoma mouse model was then characterized. Melanoma-derived sEVs significantly and extensively influenced the composition and intracellular pathways of immune lineage and T cells. An immunosuppressive immune system with decreased natural killer and CD8 T cells in the spleen and bone marrow (BM), increased regulatory T cells in lymph nodes, and increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in the BM, was induced by melanoma-derived sEVs. Additionally, melanoma-derived sEVs significantly enhanced the PD-1/PD-L1 axis in CD4 T cells and myeloid cell subsets. These sEVs largely promoted the proliferation of multiple hematopoietic stem and progenitor cell subsets and accelerated their differentiation towards MDSCs in naive mice and mice undergoing hematopoietic reconstruction. Moreover, melanoma-derived sEVs directly promoted the survival and activation of MDSCs in vitro. Collectively, our work examines the effects of tumor-derived sEVs on the systemic onco-immune macroenvironments and highlights the contribution of these sEVs to the dysregulation of hematopoiesis and systemic immune landscape in cancer.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion

AbstractAdult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil–induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

FGD5 marks a subpopulation of hematopoietic stem and progenitor cells that resist interferon-γ-mediated differentiation

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of all immune and blood cells in both steady state and emergency settings. The rates at which HSPCs divide and differentiate vary widely in accordance with both cell intrinsic and cell extrinsic factors. However, the kinetics of these events remain poorly understood. In prior work, we determined that the inflammatory cytokine interferon-γ (IFN-γ) induces HSPC division and differentiation. Here, we report that a subset of hematopoietic stem cells (HSCs) that express Fgd5 do not divide or differentiate in response to IFN-γ. This suggests that FGD5 marks a subset of HSCs that remains unperturbed during emergency hematopoiesis and is potentially a mechanism of preservation of the HSC compartment.

CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells.

SummaryHematopoiesis is maintained by functionally diverse lineage-biased hematopoietic stem cells (HSCs). The functional significance of HSC heterogeneity and the regulatory mechanisms underlying lineage bias are not well understood. However, absolute purification of HSC subtypes with a pre-determined behavior remains challenging, highlighting the importance of continued efforts toward prospective isolation of homogeneous HSC subsets. In this study, we demonstrate that CD49b subdivides the most primitive HSC compartment into functionally distinct subtypes: CD49b− HSCs are highly enriched for myeloid-biased and the most durable cells, while CD49b+ HSCs are enriched for multipotent cells with lymphoid bias and reduced self-renewal ability. We further demonstrate considerable transcriptional similarities between CD49b− and CD49b+ HSCs but distinct differences in chromatin accessibility. Our studies highlight the diversity of HSC functional behaviors and provide insights into the molecular regulation of HSC heterogeneity through transcriptional and epigenetic mechanisms.