T cells can be problematic to characterise because they have a wide variety of subtypes and because of the technical difficulties of studying the membrane-bound T cell receptor, but there are situations where you want to be able to do this such as analysing the degree to which immunological memory has been induced to measuring how well an individual mounts a response to a particular antigen.

If you do want to study T cell responses, don’t fear! There are various ways to achieve this, and these assays fall into two main categories: those that detect the activation of T cells in response to a stimulus by measuring a characteristic response, such as cytokine secretion and those that analyze cells by the specificity of their T cell receptor.

## 1. Limiting dilutions culture

This technique involves measuring the frequency of T cells that mount a response to a specific antigen by using various dilutions of cells and then measuring the number of wells with no response.

Not to go into too much of the maths but in short the distribution of active T cells in each well should follow a Poisson distribution (as these cell should be randomly distributed). We can use this information to then calculate the number of active T cells we have.

In short, we know from Poisson distribution that if the proportion of wells that are negative for a response is 37% then there is an average of one antigen-specific T cell per well.

So read of the number of cells that produced 37% negative wells, and then we can calculate the frequency of the responding cells. E.g. If seeding cells at 5000 cells per well resulted in 37% of cells being negative we know that we have a frequency of 1/5000 active cells.

After priming or immunizing the mice, the frequency should increase showing that the antigen is driving proliferation.

## 2. ELISPOT

If you want to perform more detailed studies, such as phenotyping cells, limited dilution culture is pretty laborious. In these cases you can use ELISPOT (enzyme-linked immunospot) assay. This is a variation on ELIZA. Using this technique, you measure T cell responses by their cytokine production.

The steps:

In short you coat a PVDF (polyvinylidene fluoride)-covered microplate with monoclonal or polyclonal antibodies before blocking using a non-reactive serum and culturing the T cells on the plate along with a substance or cells to activate cytokine secretion. The cytokineis then captured by the coated antibodies. Next, you wash the plate to remove debris, unbound antibodies and media before adding a secondary antibody, which is coupled to a chromogenic (color-making) substrate (often streptavidin with horseradish peroxidase or alkaline phosphatase), before washing again. You can then see your cytokines of interest as visible spots and count them manually with a microscope or automatically using an automatic reader respectively.

This method does have its drawbacks as while it is more convenient and useful in quantitating the number of T cell producing an antibody it is still limited when describing the cytokine producing profile of a single cell. But don’t worry, for those wanting to characterise at the single cell level there is intracellular staining!

## 3. Intracellular Staining

There is a great article on intracelluar staining which provides more information on this technique, but in short you inhibit cytokine secretion by treating with a protein transport inhibitor (such as Monensin or Brefeldin) before fixing and permeabilising your cells. You can then use fluorescently labelled antibodies specific to your cytokine and measure by flow cytometry. There is of course a drawback to this method – that is your cells are killed in the process.

## 4. Cytokine Capture

An alternative to intracellular staining which also overcomes the issues with the ELISPOT is cytokine capture, which uses hybrid antibodies. Here, the two heavy and light chain pairs from different antibodies are combined to give a hybrid antibody with two antigen-binding sites for 2 different ligands. These bispecific antibodies are used to detect cytokines. One ligand is a marker specific to T cells and the other is specific for the cytokine of interest. Activated T cells are treated with a protein transport inhibitor and so cytokines accumulate within the cell.

You then use the hybrid antibody to capture and detect the labelled cytokines on the surface of the T cells. A hybrid antibody can be used to do this. Hybrid antibodies are made from antibodies specific for both the cytokine of interest and a common cell surface protein, like MHC class I. The hybrid antibodies bind activated T cells. If the T cells secrete cytokines, the hybrid antibody that is bound to the surface of the cell captures them. Cytokine secreting T cells are then detected using a labelled second antibody against the cytokine of interest.

## 5. Tetramer Staining

T cell responses can also be measured by the specificity of their receptors, using fluorochrome-tagged tetramers or specific MHC:peptide complexes. MHC:peptide tetramers are made from recombinant MHC molecules with specific peptides bound to biotin-streptavidin. T cells that express the corresponding specificity bind these tetramers. These are then stained for detection and can be analysed by flow cytometry!

## 6. Spectratyping and Biosensor Assays

The diversity of the T cell population can be defined by spectratyping. Here, CDR3 regions are visualised by gel electrophoresis allowing you to compare receptors expressing the same V segment. Biosensor assays can then be used to measure the rate of association and dissociation of ligands to their complementary antigen receptors.

The ligand to be tested is fixed to a gold-plated surface. Soluble T cell receptors are then brought into contact with the bound ligands and allowed to bind.  After a period, they dissociate. The rate at which this occurs can be measured in real time using biosensors. The rate of attachment and detachment is monitored by biosensors capable of measuring the binding of molecules on the surface of the gold-plated glass chips by the effects that binding has on the total internal reflection of a polarised light source on the surface of the glass chip. It detects any fluctuations in the angle and intensity of the beam of light reflected and this is plotted against time to create a ‘sensorgram.’

The receptor can also be bound rather than the ligand. After the maximum binding has been achieved when the system is either saturated or has reached a state of equilibrium between binding and dissociating the graph line will plateau to show that no more binding is happening. The unbound molecules can then be washed away and after continued washing they all begin to come off the protein they have bound. The curve measuring the interaction will then begin to decline showing the rate at which dissociation occurs.

What methods do you use for analysing T-cells?

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1. Rohit says:

Lovely summarisation. In fact, a different version of the limiting dilution culture is used for estimating viral load- the plaque assay. But the link for the math for limiting dilution culture is not opening. Can you please look into it? Thank you.

2. Geo says:

What about proliferation assays? That`s a nice method. And there are a lot of dye that can be used.

3. Olwen Reina says:

That’s a great idea, Max! Thank you for your input! I’ll make this suggestion to our lovely editor :).

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