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T-Cell Assay Types

Key Points

  • T-cell viability and activity can be assessed using assays that are specific for different functions and states (eg. infiltration, exhaustion, cytotoxicity, and proliferation).
  • T-cells are an important lymphocyte for anti-tumour activity but are also implicated in certain autoimmune diseases. This makes an accurate understanding of their viability and effector function critical across multiple physiological contexts.

Contents

Background

  What is a T-cell?

  What do T-cells do?

  What is an assay?

Usage

Types of T-cell Assays

  Infiltration

  Exhaustion

  Cytotoxicity

  Proliferation

Glossary

T-Cell Assay Background

What is a T-cell?

T-cells, also called T-lymphocytes, are a type of white blood cell that constitutes part of the adaptive immune response. The name for T-cells comes from the fact that these cells mature in the thymus, where they undergo two separate types of selection. This is to ensure each cell can recognise MHC molecules, which present antigens to T-cells, but is not self-reactive (sensitive to autologous compounds). T-cells bear special receptors called T-cell receptors, often abbreviated to simply ‘TCR’. TCRs interact with MHC molecules on most nucleated cells. As a member of the lymphoid lineage, they are related to NK cells and B cells, and together the three strains are commonly called lymphocytes.

What do T-cells do?

T-cells are generally divided into two groups by their primary function: CD8+ “cytotoxic” T-cells, and CD4+ “helper” T-cells. To put it concisely, cytotoxic T-cells directly kill other cells they encounter if those cells are presenting an antigen which binds to their antigen specific TCR. Helper T-cells have wider purposes ranging from recruiting other immune cells to activating B-cells to produce antibodies (Source).

“CD” stands for cluster of differentiation, an immunophenotyping term used to classify cells by key surface marker molecules. For T-cells, these markers allow distinction between the helper and cytotoxic variants of the cells, and for further subdivision by function.

In order to effectively proliferate and produce cytokines, T-cells require two co-stimulatory signals in addition to TCR binding. To become fully activated, the T-cell surface ligand CD28 must bind with CD80/86 on the antigen-presenting cell (APC), and inflammatory cytokines from the APC must bind with their cognate receptor on the T-cell.

Active cytotoxic T-cells are then able to kill cells directly via CD8+/MHC I recognition with co-stimulatory signals and subsequent effector function. In the case of helper T-cells, once activated by the CD4+/MHC II interaction, these lymphocytes can go on to help B-cells generate antibodies against a compound or to recruit other immune cells to the area the antigen was found.

T-cells secrete different cytokines depending on their subtype. CD8+ cytotoxic T-cells secrete interferon-gamma, tumour necrosis factor alpha, and interleukin 2 (IFNγ, TNFα, and IL-2) in high levels, whereas the secretion of cytokines by CD4+ helper T-cells is more dependent on the subset of the helper cell. The image below shows the variants of helper T-cells, along with the cytokines they secrete upon activation.

A picture showing how a CD4+ Helper T-cell can differentiate into a variety of more specific helper cells, such as Th1, Th2, Th17, and others. The picture also shows the cytokines each helper cell produces, and the transcription factors and cytokines which cause it to differentiate from the naive to specialised helper T cell.

Fig 1. T-cell differentiation, The Ansel Lab, UCSF, accessed Feb 2020

What is an assay?

An assay is a procedure used to assess the relative or absolute quantity of a substance of interest or to measure an aspect of the effector function of a cell. They can provide a valuable source of reproducible data when performed correctly. Key features of a high performing assay are detailed below.

Specificity is the property of an assay being able to measure only the targeted compound or function. When choosing an assay, it is important to consider whether the analyte being measured is specific to a single process or cell, or whether there could be other factors which would produce the same compound outside of the experimental design.  

Sensitivity refers to the ability of an assay to detect the substance of interest over a range of concentrations. Different assays confer different levels of sensitivity, and the choice to use a fluorescence rather than an absorbance assay can be influenced by the sensitivity one desires (Source). The dynamic range of an assay is linked to its sensitivity, and is the upper and lower concentration limits that a test can accurately measure ​(Source). Accuracy (sometimes called precision) is how true an assay’s measurement is to the actual value of the sample.

Robustness is the extent to which an assay is resilient to slight changes in sample preparation or handling. This includes human user variations. Similarly, it is important that an assay be consistently reproducible across time and location (Source).

Usage

T-cell assays can be used to determine whether T-cells are present, the extent to which a cell is performing its effector function, the state of a T-cell, and its sensitivity to growth or proliferation factors. Assays present a reliable and reproducible method of assessing these functions and states, and ultimately allow a robust way to measure aspects of the strength or success of an immune response in a biological system or in vitro. Direct or indirect indicators of presence and function can be determined with specific activation markers, detailed below.

Types of T-cell Assays

1) Infiltration

2) Exhaustion

3) Cytotoxicity

4) Proliferation

T-Cell Infiltration Assays

    T-cell infiltration is the ability for a cell to travel inside of solid tissue to enact effector functions. Especially in the tumour microenvironment, it is crucial that anti-tumour cells be able to effectively penetrate the solid mass of malignant cells. Infiltration is part of the reason chimeric antigen receptor T-cell and autologous T-cell therapy (CAR-T/ACT) has been so successful in blood cancers but less advantageous in solid tumours. In order for a cell to enact its effector function—to kill— it must be in close physical proximity so that it can engage with the dysfunctional cell via direct contact. T-cell infiltration assays measure the level of CD3, a TCR co-receptor, to determine the presence of T-cells in a sample. To learn more about this useful marker for measuring infiltration, check out the protocol for our CD3e human ELISA kit.

T-cell Exhaustion Assays

T-cell exhaustion is a dysfunction caused by the constant stimulation of T-cells, often due to a chronically unresolved inflammatory response. T-cell exhaustion assays measure the level of inhibitory receptors present on the cell surface of CD4+ and CD8+ cells, which often are upregulated during the exhaustion phase of the T-cell life cycle. Common markers include PD-1 (human assay here) and CTLA-4 (human assay here). The causes, course, and reversal of T-cell exhaustion is an area of great interest in the scientific community today (Source). This is, in part, because of the role that CAR-T and ACT have come to play in the treatment of certain cancers, and the growing interest in the complex role of the immune system in cancer therapy.

T-cell Cytotoxicity Assays

T-cell cytotoxicity is the ability of a T-cell to directly cause the death of another cell. T-cell cytotoxicity assays measure the level of cells bearing CD8a, the glycoprotein which interacts with the MHC Class 1 present on most nucleated cells. CD8+ cytotoxic T-cells are thought to mediate cytotoxicity. Our human CD8 alpha/ CD8A ELISA kit allows for the measurement of this type of effector cell in humans. Similarly, ADCC (antibody-dependent cell-mediated cytotoxicity) is a way to determine if a helper T-cell response has allowed for antibody production via B-cell to plasma B cell differentiation. Our ADCC assay kit can help to identify effector function by labelling late apoptotic and necrotic target cells.

T-cell Proliferation Assays

T-cell proliferation refers to the ability of a T-cell to divide in vivo. Because of the restrictive tumour microenvironment, nutrient and oxygen availability is often low. Such conditions can culminate in the failure of lymphocytes to survive once they reach their target tissues. T-cell proliferation assays measure the division response of T-cells when proliferation-stimulating factors are used (Source). CD7 is an early marker on T-cells, present from the pre-thymic stage all the way through to maturity. Check out this kit to learn more (Source: Atlas of Hematopathology (Second Edition), 2018, Pages 29-56)

Glossary

CD: “Cluster of differentiation” refers to an immunophenotyping marker used to differentiate the origin and function of a cell.

MHC: Major histocompatibility complex, also called human leukocyte antigen (HLA) complex, are a set of genes that code for the proteins which interact with TCRs via CD4 or CD8.  

Antigen: A substance which can generate an immune response in vivo, and later be recognised by the adaptive immune system (ex vivo or in vitro).

27th Mar 2020 Paige Dougherty, MSc. Immunotherapeutics

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