High-speed Cell Sorting Research Articles

Image-Enabled Cell Sorting Using the BD CellView Technology.

This chapter is an extension of the original publication by Schraivogel et al. (Science 375:315-320, 2022) which described, for the first time, image-enabled and high-speed cell sorting based on the BD CellView technology. It summarizes the technical aspects of the instrument in an easy-to-digest form and provides example-based guidance toward implementation of the CellView-based image cell sorting technology. As an example, it explains how to use the image-enabled cell sorter to analyze the chemically induced fragmentation of the Golgi apparatus in HeLa cells-an experiment that was alluded to in the original publication but was not included in the manuscript due to space constraints. The chemically induced Golgi fragmentation sort illustrates an elegant example of the utility of image-enabled cell sorting as a significant expansion of the single-cell toolbox. It is such a striking phenotype when analyzed with image cytometry but undetectable when using conventional flow cytometry. Described in a straightforward and concise manner, this experiment serves as a standard system assurance for image-based cell sorters.

Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening.

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.

High-speed fluorescence image-enabled cell sorting.

Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.

Isolation and characterization of eosinophils in bovine blood and small intestine

An effective method to isolate functional eosinophils from blood and tissues is required to analyze the multiple roles eosinophils play in innate immunity and tissue homeostasis. Highspeed cell sorting was used to isolate bovine eosinophils from blood polymorphonuclear (PMN) cells and from small intestine intraepithelial leukocytes. Eosinophils and neutrophils were purified from bovine blood with highspeed cell sorting after gating on autofluorescence (FL1) high and low PMN subpopulations. Highspeed sorting of intestinal eosinophils was accomplished by using a combination of positive (CD45+, CD11cLow, side scatterHigh) and negative (CD3−) selection parameters. Eosinophils sorted from blood PMNs were 88.6 ± 5.8 % (mean + 1 SD; n = 4) pure and yielded significantly (p < 0.05) more RNA than purified neutrophils. Analysis of Toll-like receptor (TLR) gene expression and TLR ligand-induced pro-inflammatory cytokine (IL-1, IL-6, IL-8, and TNFα) gene expression demonstrated significant (p < 0.01) functional differences between blood eosinophils and neutrophils. Eosinophils varied between 14.7 % to 29.3 % of CD45+ IELs and purity of sorted intestinal eosinophils was 95 + 3.5 % (mean + 1SD; n = 5). A comparison of mucosal and blood eosinophils revealed significant (p < 0.01) differences in TLR gene expression, supporting the hypothesis that functionally distinct eosinophil populations are present in blood and tissues. In conclusion, highspeed cell sorting provides an effective method to isolate viable eosinophils from blood and tissues that can then be used for transcriptome analyses and in vitro function assays.

Adrenergic receptor gene expression in bovine leukocytes

The α- and β-adrenergic receptors (ARs) bind the stress hormones epinephrine (E), norepinephrine (NE), and dopamine and activate diverse physiological responses. A lack of information on AR gene expression in leukocytes limits our understanding of how stress alters immune function. Quantitative analyses of AR gene expression was completed for bovine leukocytes. Individual leukocyte lineages and subpopulations within lineages were isolated with high-speed cell sorting to facilitate a targeted analysis of AR gene expression. These analyses confirmed all 9 AR genes were expressed in bovine leukocytes with marked differences in AR gene expression when comparing among leukocyte lineages. Furthermore, separation of polymorphonuclear cells into neutrophils and eosinophils revealed these key innate immune cells also differ significantly in AR gene expression. This study provides the first comprehensive survey of AR gene expression in immune cells of any mammalian species and provides insight into conflicting reports that stress can either activate or suppress immune function.

Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS).

Flow cytometrists have long observed a spectrum of cell‐type‐specific changes ranging from minor functional defects to outright cell destruction after purification of cells using conventional droplet cell sorters. We have described this spectrum of cell perturbations as sorter induced cellular stress, or SICS (Lopez and Hulspas, Cytometry, 2020, 97, 105–106). Despite the potential impact of this issue and ubiquitous anecdotes, little has been reported about this phenomenon in the literature, and the underlying mechanism has been elusive. Inspired by others' observations (Llufrio et al., Redox Biology, 2018, 16, 381–387 and Binek et al., Journal of Proteome Research, 2019, 18, 169–181), we set out to examine SICS at the metabolic level and use this information to propose a working model. Using representative suspension (Jurkat) and adherent (NIH/3T3) cell lines we observed broad and consistent metabolic perturbations after sorting using a high‐speed droplet cell sorter. Our results suggest that the SICS metabolic phenotype is a common cell‐type‐independent manifestation and may be the harbinger of a wide‐range of functional defects either directly related to metabolism, or cell stress response pathways. We further demonstrate a proof of concept that a modification to the fluidic environment (complete media used as sheath fluid) in a droplet cell sorter can largely rescue the intracellular markers of SICS, and that this rescue is not due to a contribution of metabolites found in media. Future studies will focus on characterizing the potential electro‐physical mechanisms inherent to the droplet cell sorting process to determine the major contributors to the SICS mechanism.

Positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo.

The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis-based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min-1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min-1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid-rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman-independent phenotypes greatly expands RACS' application.

Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10–100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) – microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

Single Cell Transcriptomic Analysis of the Multiple Myeloma Bone Marrow Identifies a Unique Inflammatory Stromal Cell Population Associated with TNF Signaling

Background: In multiple myeloma, tumor cell survival, disease progression and therapy response are influenced by signals derived from the non-malignant bone marrow niche. This notwithstanding, a detailed in-vivo definition of the cells that define the multiple myeloma niche is lacking. Mesenchymal stromal cells are important niche constituents. Recent progress made with single cell transciptomics suggests that mesenchymal stromal cells are a dynamic population of cells that can exist as several subsets with functionally distinct activation and differentiation profiles. Aim: To identify mesenchymal stromal cell subsets specific for the multiple myeloma bone marrow niche, by comparing stromal cells from myeloma patients to non-cancer controls. Methods: The non-hematopoietic bone marrow niche was isolated from viably frozen bone marrow aspirates from 10 newly diagnosed multiple myeloma patients (6 hyperdiploid, 3 t(11;14) and 1 with deletion of 17p) and 2 non-cancer controls using high speed cell sorting. The purified cells were analyzed by 10X Genomics single cell sequencing directly post-thawing, without prior cell culture. From 10 multiple myeloma patients we generated single cell transcriptomes with an average read-depth of 20,000 reads per cell of in total 12,000 niche cells and from the 2 non-cancer controls a total of 3,500 niche cells. Transcriptomes were pooled and subjected to clustering analyses using the Seurat package for R to identify genetically distinct clusters of niche cells and changes in these clusters associated specifically with multiple myeloma. Results: The bioinformatical analyses generated 10 distinct clusters of niche cells, all of which were present in both non-cancer and multiple myeloma bone marrow. One of these clusters contained CDH5+ endothelial cells while the remaining 9 clusters were subsets of CXCL12+LEPR+ mesenchymal stromal cells. Because samples were taken from the central marrow by aspiration, peripheral endosteal or neuronal lineage cells were not represented in these clusters. Gene Set Enrichment Analysis (GSEA) of the stromal cell clusters from myeloma versus non-cancer controls revealed two significantly altered pathways: TNF signaling via NF-kB and Inflammatory response. Detailed analyses of the individual stromal cell clusters identified two clusters that were responsible for the inflammatory changes identified by GSEA. Both clusters were present in all myeloma patients, constituted on average 20% of total stromal cells and were defined by transcription of the inflammatory chemokines CXCL2, CXCL3 and CXCL8 the cytokine IL6. All these transcripts were absent from the equivalent clusters in control bone marrow. The presence of inflammatory stroma in the multiple myeloma niche indicates either the appearance of a novel stromal cell subset, or activation of pre-existing stromal cells. GSEA analyses suggested inflammatory signaling, and to functionally confirm this, we tested whether activation of stromal cells would induce the inflammatory stromal phenotype. Stimulation of primary human stromal cells in vitro with recombinant TNF was sufficient to induce transcription of CXCL2, CXCL3 and CXCL8, recapitulating the inflammatory transcriptome. Moreover, manual removal of these TNF target genes from the in-silico clustering analyses led to a merging of the inflammatory clusters with non-inflammatory clusters. This indicates that the major distinguishing feature of the myeloma-specific stromal cells are genes induced upon stromal cell activation. Conclusion: Through single cell transcriptomic analyses we have identified the presence of activated inflammatory stromal cells associated with TNF signaling in the multiple myeloma stromal niche. These inflammatory stromal cells are reminiscent of pathogenic cancer-associated fibroblasts found in solid tumors, where these cells create a pro-tumorigenic niche that favors tumor survival and proliferation while simultaneously inhibiting anti-cancer immunity. These findings represent the first description of myeloma-specific stromal cell subsets, and provide novel cellular targets for interventions aimed at disrupting the pro-tumorigenic microenvironment in multiple myeloma. Disclosures Broyl: Celgene, amgen, Janssen,Takeda: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; SkylineDx: Research Funding; Takeda: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding.

Mutational Profile of Leukemic Stem Cells in FLT3-ITD Mutated AML

Introduction Persistence of leukemia stem cells (LSCs) in patients with Acute Myeloid Leukemia (AML) achieving complete remission (CR) after chemotherapy leads to disease recurrence and poor outcome. Therefore, the identification of LSCs driving resistance to therapy represents an important challenge. LSCs reside within the CD34+/CD38- cells and it has been recently demonstrated that co-expression of CD99 allows for separation of LSCs from functionally normal hematopoietic stem cells in AML. We showed that the presence of a CD123/CD99/CD25+ population within CD34+ cells strongly correlates with FLT3-ITD-positivity. The aim of this study was to deeply characterize the molecular profile of CD34+/CD123+/CD99+/CD25+ LSCs to shed light on the subclonal architecture of FLT3-ITD+ AML and to track the expansion of mutated clones. Methods Molecular status of FLT3-ITD and NPM1 were investigated at the DNA level in a cohort of 150 de novo AML patients at diagnosis and at relapse, using standard procedures. Clonal evolution of FLT3-ITD mutations was studied at diagnosis in 14 FLT3-ITD mutated AML on sorted bone marrow populations, purified by the Cytoflex High-Speed Cell Sorter using a sequential gating strategy, to separate CD34/CD123/CD99/CD25+ LSCs-, CD34+ precursors (CD123/CD99/CD25-) and T-lymphocytes. Moreover, to characterize the genetic profile of FLT3-ITD-mutated clones, DNA targeted sequencing was performed on 22 cell populations isolated from 7 AML patients, using the Oncomine™ Myeloid Research Assay panel on the Ion Torrent™ S5 sequencer. Results 39 of 150 AML cases were FLT3-ITD+, at a median allelic ratio (AR) of 0.36 (range 0.05-7.9). The FLT3-ITD AR was significantly higher within the rare LSCs compartment as compared to MNC (median AR: 0.78, range 0.42-19.3, vs 0.09, range 0.05-0.6m p&amp;lt;0.0001, n=14), and to CD34+/CD123+/-/CD99low/-/CD25- precursors (Ctrl) (median AR=0.02, range 0-0.59) (p=&amp;lt;0.0001) (Fig 1A). In particular, a FLT3-ITD mutated minor subclone was present (median AR=0.05, range 0.05-0.08) in the MNC of 6 patients, and was highly enriched in the corresponding CD34+/CD123+/CD99+/CD25+ sorted LSCs (median AR=0.5, range 0.4-0.8). FLT3-ITD positive clones may also persist in sorted LSCs at the time of complete remission, being undetectable using standard techniques and driving later relapse (UPN1, Fig 1B). Furthermore, FLT3-ITD LOH may occur during disease progression, leading to AR &amp;gt;1, while the number of LSCs do not seem to expand (Fig 1B). These data confirm that LSCs may persist at rare frequency during progression, still representing the treatment-resistant FLT3-ITD reservoir, driving disease relapse with expansion of more mature, FLT3-ITD positive populations. Aiming at shedding light on the molecular heterogeneity and subclonal structure of AMLs, using targeted NGS we compared the mutational profiles of MNCs to that of highly purified LSCs and/or to the CD34+/CD123-/CD25-/CD99- counterpart (n=7 patients). In 4 cases the same mutation pattern was present in both MNCs and LSCs. In 3 cases we found additional mutations in NRAS, BCOR and KRAS in MNCs, indicating acquisition of other transforming events during maturation (Fig 2A). Furthermore, the variant allele frequency (VAF) of mutations was similar in MNCs and LSCs (p=0.12), while FLT3-ITD and TKD burden was enriched in LSCs (p=0.03) (Fig 2B). Conversely, the NPM1 mutation burden remained almost stable in the different cell subsets (Fig 2C). In conclusion, our study shows that FLT3 mutations occur in early LSCs characterized by the CD34+/CD123+/CD99+/CD25+ immunophenotype, which represent the leukemic reservoir. These data may be the rational for CD99-targeted treatments in FLT3-ITD mutated AML to achieve durable disease eradication. Disclosures Venditti: Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Buccisano:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees.

High-speed On-chip Cell Sorting

High-speed On-chip Cell Sorting

K-RAS Mutant Gene Found in Pancreatic Juice Activated Chromatin From Peri-ampullary Adenocarcinomas.

External pancreatic duct stents inserted after resection of pancreatic head tumors provide unique access to pancreatic juice analysis of genetic and metabolic components that may be associated with peri-ampullary tumor progression. For this pilot study, portal venous blood and pancreatic juice samples were collected from 17 patients who underwent pancreaticoduodenectomy for peri-ampullary tumors. Portal vein circulating tumor cells (CTC) were isolated by high-speed fluorescence-activated cell sorting (FACS) and analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) for K-RAS exon 12 mutant gene expression (K-RASmut). DNA, chromatin, and histone acetylated active chromatin were isolated from pancreatic juice samples by chromatin immunoprecipitation (ChIP) and the presence of K-RASmut and other cancer-related gene sequences detected by quantitative polymerase chain reaction (PCR) and ChIP-Seq. Mutated K-RAS gene was detectable in activated chromatin in pancreatic juice secreted after surgical resection of pancreatic, ampullary and bile duct carcinomas and directly correlated with the number of CTC found in the portal venous blood (P = .0453). ChIP and ChIP-Seq detected acetylated chromatin in peri-ampullary cancer patient juice containing candidate chromatin loci, including RET proto-oncogene, not found in similar analysis of pancreatic juice from non-malignant ampullary adenoma. The presence of active tumor cell chromatin in pancreatic juice after surgical removal of the primary tumor suggests that viable cancer cells either remain or re-emerge from the remnant pancreatic duct, providing a potential source for tumor recurrence and cancer relapse. Therefore, epigenetic analysis for active chromatin in pancreatic juice and portal venous blood CTC may be useful for prognostic risk stratification and potential identification of molecular targets in peri-ampullary cancers.

Spark-generated microbubble cell sorter for microfluidic flow cytometry.

High-speed and accurate cell sorting is of great significance for cell analysis regarding both bioresearch and clinical application. Different from the jet-in-air sorting of commercial flow cytometers, sorting in fully enclosed and disposal microfluidic chips can avoid aerosols and crosscontamination, thus contributing to the improvement of biosafety and test accuracy. However, current microfluidic sorters usually require complicated structures, or otherwise cannot attain high throughput. In this article, a sorting mechanism for microfluidics is proposed for the first time based on the jet flow induced by the spark-generated cavitation microbubble that can be easily realized by a pair of electrodes. The sorter was integrated into a microfluidic chip based on three-dimensional (3D) hydrodynamic focusing and a binary optical element (BOE) for laser illumination. Besides, several aspects of the sorting mechanism were studied to optimize the device. It achieved a switching time of 250 μs at the sample flow velocity of 5 m/s and performed the continuous operation at 200 Hz. Both the stability of fluorescence signals and the viability of cells were basically maintained. To conclude, this work explores a new on-chip sorting mechanism which possesses the merits of simple structure, easy control, and fast switching. © 2018 International Society for Advancement of Cytometry.

A Phase I Study of Donor Regulatory T Cells As Treatment for Steroid Dependent/Refractory Chronic Graft Versus Host Disease

Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant morbidity and mortality, limiting quality of life and survival in the allogeneic transplant survivor. CD4+CD25+CD127dimFoxP3+ regulatory T cells (Treg) are a naturally occurring cell population isolatable in the peripheral blood (PB) with key immune regulatory function. Treg have been shown to protect against GVHD in a number of murine models and in patients. Further, clinical correlations have been observed between the presence of CGVHD and a decreased frequency of Treg post allogeneic hematopoietic cell transplant (HCT) as well as the use of low dose IL-2 resulting in clinical responses with an associated increase in Treg. Based on these observations we embarked on a phase I safety and tolerability trial of the treatment of steroid-refractory or dependent CGVHD with infusion of donor Treg in matched related donor recipients.Methods: Eligible patients were ³ 18 years of age with steroid refractory or dependent CGVHD with a matched related HCT donor. All patients were on systemic immunosuppression at the time of enrollment. Donor derived Treg were enriched from a non-mobilized PB apheresis product over 2 days of apheresis. The fresh apheresis product underwent enrichment by CD25+ immunomagnetic selection on the CliniMACS¨ Cell Selection System (Miltenyi Biotec) and Treg were purified by high-speed flow cytometry and gated on the CD4+CD127dim population. The functional activity of the Treg product was assessed via a mixed lymphocyte reaction (MLR). The trial dosing was through a 3+3 dose escalation design with three Treg dose cohorts: 1 x 10E5 Treg/kg, 5 x 10E5 Treg cells/kg and 1.5 x 10E6 Treg cells/kg.Results: Ten patients were enrolled and received donor Treg cell infusions. Isolation of highly purified Treg was successful from all donors. A median of 1.9x10E8 (range 0.9-2.78x10E8) Treg were collected with a purity that ranged from 87.1-99.3% FoxP3+ (Table 1). Function of the Treg enriched apheresis product confirmed Treg suppressive activity with a median suppression of 69.1% (48.7-77.2%) at a ratio of responder cells:CD4+/CD8+:Treg cells of 1:2:1, with sustained suppression to a ratio of 1:2:0.1. All 10 pateints were treated with three patients treated on all three dose cohorts and an additional patient was treated on the third cohort due to the planned cell dose being not reached with the third patient-donor pair. There were no adverse toxicities with the Treg infusion and no incidence of acute GVHD or acute exacerbation of CGVHD. The ratio of Treg/CD4+ cells in the patients’ PB ranged from 2-4.4% at the time of the infusion and remained stable <3 months post infusion. Six of 10 patients have had stable to improved CGVHD with one of these patients having had a flare of CGVHD in the setting of a lapse in immunosuppressive medication and response with reinstitution of immunosuppression. Four patients have had unresponsive or progressive disease requiring the addition of new immunosuppressive agents <2 months post Treg cell infusion. All enrolled patients remain alive at a follow up of >150 days post Treg cell infusion.Summary: This study is the first clinical experience utilizing highly purified donor derived Treg isolated through clinical scale high speed cell sorting. Donor Treg cell therapy for the treatment of steroid refractory or dependent CGVHD at doses of 1.5x10E5-1.5x10E6 Treg/kg were administered without toxicity including absence of AGVHD or exacerbation of CGVHD. The Treg dose of 1.5 x 10E6/kg of recipient weight may be the maximum feasible dose attainable without ex vivo Treg cell expansion. Treg mediated suppression in the MLR documented the in vitro suppressive capability of the Treg cells with investigations ongoing to characterize the functionality of the Treg cells infused. Although this trial was designed to describe the feasibility, safety and tolerability of Treg cell infusions in patients with active CGVHD, the preliminary outcomes of clinical responses and survival are encouraging and comparable to other therapies for steroid refractory/dependent CGVHD with only a single infusion of cells. Further strategies such as multiple infusions or the addition of low dose IL-2 could be considered to attempt to augment the clinical efficacy of Treg based cellular immunotherapy. [Display omitted] DisclosuresMeyer:Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties.

The functional relationship between hematopoietic stem cells and developing T lymphocytes

In contrast to all other blood and immune cells, T lymphocytes do not develop in the bone marrow (BM), but in the specialized microenvironment provided by the thymus. Similar to the other lineages, however, all T cells arise from multipotent hematopoietic stem cells (HSCs) that reside in the BM. Not all HSCs give rise to T cells; but how many and what kind of developmental checkpoints are located along this intricate differentiation path is the subject of intense research. Traditionally, this process has been studied almost exclusively using mouse cells, but recent advances in immunodeficient mouse models, high-speed cell sorting, lentiviral transduction protocols, and deep sequencing techniques have allowed these questions to be addressed using human cells. Here we review the process of thymic seeding by BM-derived cells and T cell commitment in humans, discussing recent insights into the clonal composition of the thymus and the definition of developmental checkpoints, on the basis of insights from human severe combined immunodeficiency patients.

Identification of a Novel Gene Expression Signature in Mantle Cell Lymphoma from the Fondazione Italiana Linfomi (FIL)-MCL-0208 Trial: A Focus on the B Cell Receptor Pathway

Background. The aggressive clinical behavior of mantle cell lymphoma (MCL) is attributed to specific genetic and molecular mechanisms involved in its pathogenesis, mainly the t(11;14)(q13;q32) traslocation and cyclin D1 (CCND1) overexpression. Nevertheless, evidence of a certain degree of clinical/biological heterogeneity has been disclosed by gene expression profile (GEP) and (immuno)genetic/immunohistochemistry studies.Aim. To use a GEP approach to identify MCL subsets with peculiar clinical/biological features in the context of MCL patients treated homogeneously with an autologous transplantation-based program.Methods. The study was based on a cohort of 42 MCL cases enrolled in the Fondazione Italiana Linfomi (FIL)-MCL-0208 randomized Italian clinical trial. Purified clonal CD19+ MCL cells were obtained by high-speed cell sorting of peripheral blood MCL samples. GEP experiments were performed in 30 cases, with Agilent platform. Bioinformatics analyses were performed by Gene Springs and Gene Set Enrichment Analysis (GSEA) software. Gene signature validations were performed by quantitative real time PCR (QRT-PCR).Results. i)Unsupervised and supervised analyses. Unsupervised analysis by principal component analysis (PCA) was able to divide the cohort in two main subgroups named PCA1 (12 cases) and PCA2 (18 cases). Supervised analysis by segregating cases according to the PCA1 and PCA2 classification defined a gene expression signature of 710 gene (234 up-regulated) that highlighted a constitutive overexpression of genes of the BCR signaling pathway. Consistently,GSEA showed a significant enrichment of genes belonging to 3 gene sets related to BCR signaling. ii) Identification of a "PCA2-type" gene signature. By merging the list of differentially expressed genes according to supervised analysis of GEP data and the gene list related to BCR signaling according to GSEA, a group of 9 genes, all overexpressed in PCA2 cases, i.e. AKT3, BLNK, BTK, CD79B, PIK3CD, SYK, BCL2, CD72, FCGR2B, was obtained. Among these genes, a subgroup of 6 genes, i.e. AKT3, BLNK, BTK, CD79B, PIK3CD, SYK, was selected for the direct involvement in the BCR pathway, and utilized for further validations. iii) Generation of a 6-gene prediction model. The selected 6 genes were then utilized to generate a prediction model by using 20 cases as training sub-cohort and the remaining 10 cases as validation cohort. By this approach, 9/10 cases of the validation cohort were correctly assigned according to the PCA2/PCA1 classification. The model was re-tested by QRT-PCR in 24 cases used in the GEP (16 for training and 8 for validation), and again, 7/8 cases of the validation sub-cohort were correctly classified. QRT-PCR was then utilized to classify further 12 cases (7 cases defined as PCA2) not employed for GEP analysis. Overall, in the 42 cases, 23 cases were considered as PCA2 with the GEP/QRT-PCR approach. iv) Clinical/biological correlations. No association was found between the 6-gene signature and IGHV status (22/30 unmutated IGHV cases) or between the signature and the overexpression of SOX11 (17/30 cases over the median value). In addition, no association was found with the presence of the main recurrent mutations of the ATM, BIRC3, CCND1, KMTD2, NOTCH1, TP53, TRAF2, WHSC1 genes. Finally, an "ad-interim" analysis of progression free survivals (PFS) (Cortelazzo et al EHA, 2015) suggested a trend for a shorter PFS (2-years PFS 45% vs 72%, p=0.08) for cases classified as PCA2 by the GEP/QRT-PCR approach. v) 6-gene signature and sensitivity to the BCR inhibitor ibrutinib. The finding that PCA2 cases overexpressed BCR-related genes and had a more aggressive clinical course prompted us to investigate the 6-gene signature in the context of ibrutinib sensitive/resistant MCL cell lines. To do this, the proliferation rate of the MCL cell lines REC1, JEKO1, UPN1, GRANTA, JVM2, Z138 was investigated either in presence or in absence of ibrutinib 10 nanoM for 7 days. REC1, JEKO1 were selected as responsive by showing ≥80% inhibition upon ibrutinib. Of note, responsive cell lines showed higher expression levels of the 6-gene signature then the resistant counterpart, as evaluated by QRT-PCR.Conclusions. A novel 6-gene expression signature related to the BCR pathway has been found to characterize MCL cells with peculiar clinical/biological features and sensitivity to BCR inhibitors. DisclosuresLuminari:Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

Monocyte-Derived Dendritic Cells from Feline Immunodeficiency Virus Positive Cats are Productively Infected and Maintain CD8+ T Cell Stimulatory Capacity

Dendritic cells (DCs) have been utilized to enhance CD8+ T cell responses to pathogen-associated peptides for enhancement of vaccine efficacy. CD8+ T cells play a central role in the elimination of viruses during acute viral infection and control of viremia during chronic viral infection. For lentiviral infections such as HIV and FIV, dendritic cell vaccines may be useful for augmenting CD8+ T cell function. Therefore, we asked if monocyte-derived dendritic cells (moDCs) from FIV+ chronically infected cats maintained the ability to stimulate CD8+ T cells in the absence of exogenous antigen, when compared to uninfected controls. Using high speed cell sorting, monocytes were isolated from PBMCs based on forward versus side scatter gating. Cells were then cultured with IL-4 and GM-CSF over the course of 6 days, and LPS was added to stimulate maturation after the first 72 hours. We confirmed the identity and maturation status of cells by cytologic examination and FITC-dextran uptake. We then assessed differences in CD8+ T cell proliferation in the presence of monocytes / macrophages (M/M), immature dendritic cells (iDCs), and mature dendritic cells (mDCs). We demonstrated that CD8+ T cell proliferation is maintained in the presence of DCs from FIV+ cats and that production of inflammatory cytokines and chemokines by DCs from FIV+ cats is retained in vitro. Further, we confirmed the presence of virus in co-culture. These results suggest that moDCs from FIV-infected animals serve as viral reservoirs. More importantly, these results support our hypothesis that moDCs maintain the ability to stimulate CD8+ T cells during chronic lentiviral infection.

A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells.

Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creER(T2) transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5(+) stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.

Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm

Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80μM, 120μM, 160μM, 200μM, 240μM, or 320μM) for 45min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160μM Hoechst33342 for various durations (0min, 15min, 30min, 45min, 60min, 75min, or 90min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160μM. Moreover, when the staining duration was equal to or longer than 45min, the frozen-thawed sperm can be successfully sorted in the presence of 160μM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro fertilizing capability. Accordingly, the minimum values of Hoechst33342 concentration and staining duration can be set at 160μM and 45min respectively. However, the maximum values of Hoechst33342 concentration and staining duration were not determined based on the current study. Further research on how to reduce injuries caused by freezing, thawing, and Hoechst33342 staining on frozen-thawed ram sperm is needed.