cMPL-Based Purification and Depletion of Human Hematopoietic Stem Cells: Implications for Pre-Transplant Conditioning.

The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies.

Social Determinants modulate NK cell activity via obesity, LDL, and DUSP1 signaling.

Adverse social determinants of health (aSDoH) are associated with obesity and related comorbidities like diabetes, cardiovascular disease, and cancer. Obesity is also associated with natural killer cell (NK) dysregulation, suggesting a potential mechanistic link. Therefore, we measured NK phenotypes and function in a cohort of African-American (AA) women from resource-limited neighborhoods. Obesity was associated with reduced NK cytotoxicity and a shift towards a regulatory phenotype. In vitro, LDL promoted NK dysfunction, implicating hyperlipidemia as a mediator of obesity-related immune dysregulation. Dual specific phosphatase 1 (DUSP1) was induced by LDL and was upregulated in NK cells from subjects with obesity, implicating DUSP1 in obesity-mediated NK dysfunction. In vitro, DUSP1 repressed LAMP1/CD107a, depleting NK cells of functional lysosomes to prevent degranulation and cytokine secretion. Together, these data provide novel mechanistic links between aSDoH, obesity, and immune dysregulation that could be leveraged to improve outcomes in marginalized populations.

Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response.

Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials.

Mono-ADP-ribosyltransferase 1 ( Artc1 )-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival.

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.

Tissue- and cell-specific properties of enterochromaffin cells affect the fate of tumorigenesis toward nonendocrine adenocarcinoma of the small intestine.

Small intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. However, EC cell-derived tumorigenesis remains poorly understood. Here, we examined whether the gain of Myc and the loss of RB1 and Trp53 function in EC cells result in SI-NET using tryptophan hydroxylase 1 (TPH1) Cre-ERT2-driven RB1fl Trp53fl MycLSL (RPM) mice. TPH1-Cre-induced gain of Myc and loss of RB1 and Trp53 function resulted in endocrine or neuronal tumors in pancreas, lung, enteric neurons, and brain. Lineage tracing indicated that the cellular origin for these tumors was TPH1-expressing neuroendocrine, neuronal, or their precursor cells in these organs. However, despite that TPH1 is most highly expressed in EC cells of the small intestine, we observed no incidence of EC cell tumors. Instead, the tumor of epithelial cell origin in the intestine was exclusively nonendocrine adenocarcinoma, suggesting dedifferentiation of EC cells into intestinal stem cells (ISCs) as a cellular mechanism. Furthermore, ex vivo organoid studies indicated that loss of functions of Rb1 and Trp53 accelerated dedifferentiation of EC cells that were susceptible to apoptosis with expression of activated MycT58A, suggesting that the rare dedifferentiating cells escaping cell death went on to develop adenocarcinomas. Lineage tracing demonstrated that EC cells in the small intestine were short-lived compared with neuroendocrine or neuronal cells in other organs. In contrast, EC cell-derived ISCs were long-lasting and actively cycling and thus susceptible to transformation. These results suggest that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation, affect the fate and rate of tumorigenesis induced by genetic alterations and provide important insights into EC cell-derived tumorigenesis.NEW & NOTEWORTHY Small intestinal neuroendocrine tumors are of putative enterochromaffin (EC) cell origin and are the most common malignancy in the small intestine, followed by adenocarcinoma. However, the tumorigenesis of these tumor types remains poorly understood. The present lineage tracing studies showed that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation affect the fate and rate of tumorigenesis induced by genetic alterations toward a rare occurrence of adenocarcinoma.

Kruppel-like factor 1-GATA1 fusion protein improves the sickle cell disease phenotype in mice both in vitro and in vivo.

Sickle cell disease (SCD) and ß-thalassemia are among the most common genetic disorders worldwide, affecting global health and mortality. Hemoglobin A2 (HbA2, α2δ2) is expressed at a low level in adult blood due to the lack of the Kruppel-like factor 1 (KLF1) binding motif in the δ-globin promoter region. However, HbA2 is fully functional as an oxygen transporter, and could be a valid antisickling agent in SCD, as well as a substitute for hemoglobin A in ß-thalassemia. We have previously demonstrated that KLF1-GATA1 fusion protein could interact with the δ-globin promoter and increase δ-globin expression in human primary CD34+ cells. We report the effects of 2 KLF1-GATA1 fusion proteins on hemoglobin expression, as well as SCD phenotypic correction in vitro and in vivo. Forced expression of KLF1-GATA1 fusion protein enhanced δ-globin gene and HbA2 expression, as well as reduced hypoxia-related sickling, in erythroid cells cultured from both human sickle CD34+ cells and SCD mouse hematopoietic stem cells (HSCs). The fusion proteins had no impact on erythroid cell differentiation, proliferation, and enucleation. Transplantation of highly purified SCD mouse HSCs expressing KLF1-GATA1 fusion protein into SCD mice lessened the severity of the anemia, reduced the sickling of red blood cells, improved SCD-related pathological alterations in spleen, kidney, and liver, and restored urine-concentrating ability in recipient mice. Taken together, these results indicate that the use of KLF1-GATA1 fusion constructs may represent a new gene therapy approach for hemoglobinopathies.

Pembrolizumab and decitabine for refractory or relapsed acute myeloid leukemia.

BACKGROUND: The powerful 'graft versus leukemia' effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic cell transplantation in acute myeloid leukemia (AML) provides rationale for investigation of immune-based therapies in this high-risk blood cancer. There is considerable preclinical evidence for potential synergy between PD-1 immune checkpoint blockade and the hypomethylating agents already commonly used for this disease. METHODS: We report here the results of 17 H-0026 (PD-AML, NCT02996474), an investigator sponsored, single-institution, single-arm open-label 10-subject pilot study to test the feasibility of the first-in-human combination of pembrolizumab and decitabine in adult patients with refractory or relapsed AML (R-AML). RESULTS: In this cohort of previously treated patients, this novel combination of anti-PD-1 and hypomethylating therapy was feasible and associated with a best response of stable disease or better in 6 of 10 patients. Considerable immunological changes were identified using T cell receptor ß sequencing as well as single-cell immunophenotypic and RNA expression analyses on sorted CD3+ T cells in patients who developed immune-related adverse events (irAEs) during treatment. Clonal T cell expansions occurred at irAE onset; single-cell sequencing demonstrated that these expanded clones were predominately CD8+ effector memory T cells with high cell surface PD-1 expression and transcriptional profiles indicative of activation and cytotoxicity. In contrast, no such distinctive immune changes were detectable in those experiencing a measurable antileukemic response during treatment. CONCLUSION: Addition of pembrolizumab to 10-day decitabine therapy was clinically feasible in patients with R-AML, with immunological changes from PD-1 blockade observed in patients experiencing irAEs.

Early Myeloid Derived Suppressor Cells (eMDSCs) Are Associated With High Donor Myeloid Chimerism Following Haploidentical HSCT for Sickle Cell Disease.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

Personalized Single-Cell Proteogenomics to Distinguish Acute Myeloid Leukemia from Non-Malignant Clonal Hematopoiesis.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.

Comparative engraftment and clonality of macaque HSPCs expanded on human umbilical vein endothelial cells versus non-expanded cells.

Ex vivo hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and in vitro models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias. We investigated the impact of ex vivo expansion of macaque HSPCs on the engineered endothelial cell line (E-HUVECs) platform regarding safety, engraftment of transduced and E-HUVEC-expanded HSPC over time compared to non-expanded HSPC for up to 51 months post-transplantation, and both clonal diversity and lineage distribution of output from each engrafted cell source. Short and long-term engraftment were comparable for E-HUVEC expanded and the non-expanded HSPCs in both animals, despite extensive proliferation of CD34+ cells during 8 days of ex vivo culture for the E-HUVEC HSPCs, and optimization of harvesting and infusion of HSPCs co-cultured on E-HUVEC in the second animal. Long-term hematopoietic output from both E-HUVEC expanded and unexpanded HSPCs was highly polyclonal and multilineage. Overall, the comparable HSPC kinetics of macaques to humans, the ability to study post-transplant clonal patterns, and simultaneous multi-arm comparisons of grafts without the complication of interpreting allogeneic effects makes our model ideal to test ex vivo HSPC expansion platforms, particularly for gene therapy applications.

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis.

BACKGROUND: Inflammation plays an important role in the development of cardiovascular disease (CVD). Patients with chronic inflammation diseases have high levels of inflammation and early fatal myocardial infarction due to early, unstable coronary plaques. Cholesterol crystals (CC) play a key role in atherogenesis. However, the underlying mechanisms of endothelial cell (EC)-derived CC formation are not well understood in chronic inflammation. METHODS: We utilized a combination of a mouse psoriasis model (K14-Rac1V12 mouse model) and human psoriasis patients to study the effect of inflammatory cytokines on CC formation in ECs. Lysosomal pH, alterations in lipid load and inflammatory proteins were evaluated as potential mechanisms linking inflammatory cytokines to CC formation. Coronary CT angiography was performed (n = 224) to characterize potential IFNγ and TNFα synergism on vascular diseases in vivo. FINDINGS: We detected CC presence in the aorta of K14-Rac1V12 mice on chow diet. IFNγ and TNFα were found to synergistically increase LDL-induced CC formation by almost 2-fold. There was an increase in lysosomal pH accompanied by a 28% loss in pH-dependent lysosomal signal and altered vATPaseV1E1 expression patterns. In parallel, we found that LDL+IFNγ/TNFα treatments increased free cholesterol content within EC and led to a decrease in SOAT-1 expression, an enzyme critically involved cholesterol homeostasis. Finally, the product of IFNγ and TNFα positively associated with early non-calcified coronary burden in patients with psoriasis (n = 224; ß = 0.28, p < 0.001). INTERPRETATION: Our results provide evidence that IFNγ and TNFα accelerate CC formation in endothelial cells in part by altering lysosomal pH and free cholesterol load. These changes promote early atherogenesis and contribute to understanding the burden of CVD in psoriasis. FUNDING: Funding was provided by the Intramural Research Program at NIH (NNM) and the National Psoriasis Foundation (NNM and YB).

GL261 luciferase-expressing cells elicit an anti-tumor immune response: an evaluation of murine glioma models.

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 104 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 105 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 104 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.

Dendritic Cell-Restricted Progenitors Contribute to Obesity-Associated Airway Inflammation via Adam17-p38 MAPK-Dependent Pathway.

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely, how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK), which is blocked with a selective p38α/ß MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically, we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether, these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation.

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics.

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.

Circulating Lymphangioleiomyomatosis Tumor Cells With Loss of Heterozygosity in the TSC2 Gene Show Increased Aldehyde Dehydrogenase Activity.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a destructive metastasizing neoplasm of the lung characterized by proliferation of LAM cells in specialized lung nodules. LAM cells are characterized by expression of the prometastatic and cancer-initiating hyaluronan receptor CD44v6, and loss of heterozygosity (LOH) of TSC1 and TSC2. The circulating neoplastic LAM cells are thought to be involved in metastasis. Because LAM cells display properties of neoplastic, metastatic, and stem cell-like cancer cells, we hypothesized that elevated aldehyde dehydrogenase (ALDH) activity, characteristic of cancer and stem cells, is a property of LAM cells. METHODS: We performed an in silico search of ALDH genes in microdissected LAM lung nodules. To identify circulating LAM cells, we osmotically removed red blood cells from whole blood to obtain peripheral blood mononuclear cells, which were then sorted by fluorescence-activated cell sorting based on their level of ALDH activity. RESULTS: Microdissected LAM lung nodules possess a distinctive ALDH gene profile. The cell subpopulation with high ALDH activity, isolated from circulating cells, possessed TSC2 LOH in 8 of 14 patients with LAM. Approximately 60% of the circulating cells with high ALDH activity expressed CD44v6. Cells with TSC2 LOH from patients with LAM and LAM/TSC exhibited different properties in different body locations, but all cell types showed high ALDH activity. CONCLUSIONS: This new procedure allows for isolation of circulating LAM cells from cultured cells, blood, and chylous effusions and shows that circulating LAM cells are heterogeneous with neoplastic, metastatic, and cancer-stem cell-like properties.

Human bone marrow assessment by single-cell RNA sequencing, mass cytometry, and flow cytometry.

New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from 20 healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated a strong correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference data set and highlights opportunities for further refinement.

Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation.

In this study we demonstrate the feasibility of direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors following a brief ex vivo stimulation. Cytokine-secreting cells were identified using cell surface "catch" reagents and single cell data were obtained by sorting of individual cytokine-secreting cells into 96 well plates containing lysis buffer followed by analysis using ultrasensitive immunoassays for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). CD8 cells negative for cytokine production, as determined by the cell surface catch reagents were used as negative controls. Furthermore, studies were undertaken to compare the mean fluorescence intensity (MFI) values of cytokine staining by flow cytometry with the quantification of cytokines using the current method. This study demonstrates that it is feasible to quantify cytokines from individual primary cells. A shift from qualitative to quantitative determinations of cytokine protein levels in single cells will permit more precise and reproducible studies of heterogeneity in the immune system and can be accomplished with readily available instrumentation.