Flow Cytometric Characterization of Antigen-Specific T Cells Based on RNA and Its Advantages in Detecting Infections and Immunological Disorders

Abstract

Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.

Analysis of Tumor-Infiltrating T-Cell Transcriptomes Reveal a Unique Genetic Signature across Different Types of Cancer

“…During the last decades, the former T-cell subsets have been identified with the analytical method of flow cytometry by using fluorescent-labeled antibodies against proteins such as CD3, CD8, CD4, CD25, CD127 and FOXP3 [ 15 ]. This technique is widely used in clinical samples for the monitoring of these cells in cancer immunotherapies [ 16 , 17 ]. However, nowadays, novel genetic sequencing techniques have allowed the identification of T-cells based on their genetic signature, including the same markers classically used in flow cytometry.…”

Impedimetric Microfluidic Sensor‐in‐a‐Tube for Label‐Free Immune Cell Analysis

“…[ 13,14 ] However, the need of bulky and expensive optics in these techniques hampers their broad application potential into personal care. Previous works related to in‐flow detection of single cells, [ 15 ] and in particular of immune cells have been already reported with some of those techniques, [ 16 ] as well as the detection of expressed cell proteins (cytokines). [ 17 ] Among these proteins, cytokines TNF‐α, [ 18 ] IL‐1β, IL‐2, [ 19 ] IL‐6, IL‐8, Il‐10, and IL‐12, [ 20 ] expressed by, that is, lipopolysaccharide (LPS)‐activated monocytes CD14+, [ 21–23 ] are the most prominent markers which provide insights about different stages of immune cell activation, being of crucial importance in immune diseases diagnosis.…”

“…[13,14] However, the need of bulky and expensive optics in these techniques hampers their broad application potential into personal care. Previous works related to in-flow detection of single cells, [15] and in particular of immune cells have been already reported with some of those techniques, [16] Analytical platforms based on impedance spectroscopy are promising for non-invasive and label-free analysis of single cells as well as of their extracellular matrix, being essential to understand cell function in the presence of certain diseases. Here, an innovative rolled-up impedimetric microfulidic sensor, called sensor-in-a-tube, is introduced for the simultaneous analysis of single human monocytes CD14+ and their extracellular medium upon liposaccharides (LPS)-mediated activation.…”