Intercellular nanotube-mediated mitochondrial transfer enhances T cell metabolic fitness and antitumor efficacy.

Mitochondrial loss and dysfunction drive T cell exhaustion, representing major barriers to successful T cell-based immunotherapies. Here, we describe an innovative platform to supply exogenous mitochondria to T cells, overcoming these limitations. We found that bone marrow stromal cells establish nanotubular connections with T cells and leverage these intercellular highways to transplant stromal cell mitochondria into CD8 T cells.

LIM-domain-only 4 (LMO4) enhances CD8 T-cell stemness and tumor rejection by boosting IL-21-STAT3 signaling.

High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8 T cells. Using IFN-γ production as a proxy for CD8 T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells.

Multiparametric Analysis of Apoptosis by Flow Cytometry.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process.

Laser Sources for Traditional and Spectral Flow Cytometry.

Lasers for light scattering measurement and fluorescence excitation are essential components of all flow cytometers. Flow cytometers now typically rely on multiple laser wavelengths allowing excitation of a constantly increasing variety of fluorescent probes. The expanding use of spectral flow cytometry to increase the magnitude of multiparametric analysis is also changing the significance of laser choice in cytometry.

Basic Principles of Flow Cytometer Operation: The Make your Own Flow Cytometer.

Flow cytometry is a critical technology for biomedical analysis and is an essential component of almost any study of the immune system. Widespread usage and increasing instrument complexity have, however, led to increasing neglect of education in their basic operating principles, a common situation with many technologies. This chapter describes the basics of flow cytometer operation using the Make Your Own Flow Cytometer ( https://www.

Deep ultraviolet 266 nm laser excitation for flow cytometry.

High dimensional flow cytometry relies on multiple laser sources to excite the wide variety of fluorochromes now available for immunophenotyping. Ultraviolet lasers (usually solid state 355 nm) are a critical part of this as they excite the BD Horizon™ Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have increased the number of simultaneous fluorochromes available for practical high-dimensional analysis to greater than 40 for spectral cytometry.

Flow cytometry and cell sorting.

While flow cytometry is a critical single cell analytical technique in biomedical science, the technology of flow cytometry associated cell sorting is equally important. Physical separation of cells analyzed by flow cytometry was recognized as an important goal even in the field's beginning, and many of the earliest cytometers were also cell sorters. Isolation of cells based on flow cytometric analysis has formed the foundation of immune cell differentiation and development and continues to grow importance as techniques for genomic and proteomic analysis expand.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries.

Novel CDK12/13 Inhibitors AU-15506 and AU-16770 Are Potent Anti-Cancer Agents in EGFR Mutant Lung Adenocarcinoma with and without Osimertinib Resistance.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients.

Increased Activity of a NK-Specific CAR-NK Framework Targeting CD3 and CD5 for T-Cell Leukemias.

NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model.

Surface NKG2C Identifies Differentiated αβT-Cell Clones Expanded in Peripheral Blood.

T cells that express CD56 in peripheral blood of healthy humans represent a heterogeneous and poorly studied subset. In this work, we analyzed this subset for NKG2C expression. In both CD56 and CD56 subsets most of the NKG2C T cells had a phenotype of highly differentiated CD8 TEMRA cells.

Intrabone transplantation of CD34+ cells with optimized delivery does not enhance engraftment in a rhesus macaque model.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow.

CD56 CD57 NKG2C NK cells retaining proliferative potential are possible precursors of CD57 NKG2C memory-like NK cells.

Formation of the adaptive-like NK cell subset in response to HCMV infection is associated with epigenetic rearrangements, accompanied by multiple changes in the protein expression. This includes a decrease in the expression level of the adapter chain FcεRIγ, NKp30, and NKG2A receptors and an increase in the expression of NKG2C receptor, some KIR family receptors, and co-stimulating molecule CD2. Besides, adaptive-like NK cells are characterized by surface expression of CD57, a marker of highly differentiated cells.

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis.

High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers.

The Role of O-Antigen in LPS-Induced Activation of Human NK Cells.

NK cells can be stimulated by bacterial lipopolysaccharides (LPS). Unlike macrophages, human NK cells do not express or express very low level of surface TLR4 receptor normally required for the LPS stimulation. This has led to the assumption that the mechanisms of stimulating action of LPS on macrophages and NK cells differs.

The transcription factor c-Myb regulates CD8 T cell stemness and antitumor immunity.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8 T cell memory compartment. Following viral infection, CD8 T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells.

Recurrent Stimulation of Natural Killer Cell Clones with K562 Expressing Membrane-Bound Interleukin-21 Affects Their Phenotype, Interferon-γ Production, and Lifespan.

A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones.

Analysis of NK cell clones obtained using interleukin-2 and gene-modified K562 cells revealed the ability of "senescent" NK cells to lose CD57 expression and start expressing NKG2A.

In this work, we analyzed the phenotype and growth of human NK cell clones obtained by the stimulation of individual NK cells with IL-2 and gene-modified K562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). We generated clones from NK cells at distinct differentiation and activation stages, determined by CD56, CD57 and HLA-DR expression levels. Less differentiated CD56bright NK cell subsets showed higher cloning efficiency compared with more differentiated CD56dim subsets, especially with the CD57bright subset.

Deep ultraviolet lasers for flow cytometry.

Modern flow cytometers require multiple laser wavelengths to excite the wide variety of fluorescent probes now available for high-dimensional analysis. Ultraviolet (UV) lasers (typically solid state 355 nm) have become a critical excitation source for the Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have pushed the number of fluorescent probes available for simultaneous analysis to nearly 30, allowing an unprecedented level of precision for immune cell analysis.