Dramatic Footage of Immune System at Work Caught on Tape

By Jason Socrates Bardi

Using a new technique that allows scientists to see the internal machinery of a living cell, a team of researchers at The Scripps Research Institute (TSRI) addressed one of the most fundamental issues in immune research: the early events in the immune system's recognition of foreign invaders, such as bacteria and viruses, in the body.

In the latest issue of the journal Immunity, a team led by TSRI Associate Professor of Immunology Nicholas R.J. Gascoigne and Senior Research Associate Tomasz Zal used fluorescence resonance energy transfer (FRET) to look at the close interaction of immune molecules that recognize foreign antigens, which are small molecule markers that are components of the pathogens. Specifically, the researchers focused on the main receptor on the surface of mature T cells, called the T cell Receptor, and one important T cell surface "coreceptor" molecule, CD4.

In particular, Zal and Gascoigne were interested in demonstrating vividly through FRET how other "antagonist" molecules in the bloodstream that can bind to the T cell receptor can block the interaction of the CD4 with the antigen, inhibit the signaling cascade that leads to T cell activation, and reduce the effectiveness of an immune response.

"We can look at positions of [CD4 and T cell receptor] proteins and whether or not they are interacting," says Gascoigne.

"That allows us to see whether or not you are getting T cell activation by a particular ligand—the very earliest events in T cell recognition," he adds.

Recognition Key to Immune Response

The immune system long ago evolved ways to recognize pathogenic invaders through their antigens. For instance, these antigens, or fragments of the pathogens, may come from pathogenic proteins that have been taken up and processed into small peptides a few amino acids long, which are then taken up by specialized antigen-presenting cells (APC). The APCs "present" the antigens on their surfaces by displaying them in molecular complexes with the so-called major histocompatibility complex (MHC) proteins.

When a pathogen invades the immune system, APCs alert T cells by displaying the pathogenic antigens. When specific T cells see the antigen in the MHC, they generate a systemic immune response designed to lead to the destruction of the pathogen, starting with a cascade of internal activation events.

The first event in this cascade is the positive recognition of the MHC and antigen peptide by the T cell receptor and coreceptors. The coreceptor is crucial for this recognition because it stabilizes the binding of the T cell receptor to the MHC.

Once that positive recognition occurs, the T cells become activated as killer and helper T cells, aggressively destroying infected cells, stimulating an inflammatory response in infected tissue, and producing chemicals that induce other cells to make and release soluble antibodies that target the pathogen in the bloodstream. Such immune reaction regularly keeps us alive as we go through life in constant contact with the bacteria, viruses, and infectious microbes of the world.

Significantly, the immune system has also evolved caution about activating its T cells. Excessive or inappropriate immune responses can be lethal to an organism, and so the cells of the immune system are highly discriminating in their ability to recognize foreign antigen and only foreign antigen. T cells can tell the difference between foreign peptide antigen and a "self" peptide that only differ by a single amino acid. That one amino acid makes all the difference.

"The immune system can tell the difference," says Gascoigne, "and it makes a totally different response."

However, the immune system can also be tricked into missing the foreign peptide when other molecules—antagonists—block the binding of the coreceptor to the MHC. Without this crucial step, the T cell will not become activated even if the T cell receptor sees the foreign antigen in the MHC.

In Gascoigne and Zal's study, they use FRET to look at the recognition of MHC by the T cell receptor and the coreceptor CD4. They are able to see the interaction of MHC/CD4/T cell receptor live on the screen, and find that they can block this critical early event in immune recognition by adding antagonists.

Fluorescence Resonance Energy Transfer

Using FRET, scientists can now look at protein–protein interactions anywhere in a living cell in real time. FRET works on the same basis of traditional fluorescence microscopy, in which fluorophores—small molecules like green fluorescent protein (GFP) that absorb and reemit photons of a particular wavelength—are attached to proteins in the cell. One can then illuminate the cells with a monochromatic light source and train a microscope camera to capture the reemitted photons.

In FRET, two different fluorescent molecules are used. Under the microscope, these two will have different emission wavelengths and therefore different colors, cyan and yellow, for instance. However, the emission wavelength of the cyan overlaps with the excitation of the yellow, and so when the two molecules are very close together, within 10 nanometers or so (a millionth of a centimeter), the cyan molecule will donate its energy to the yellow molecule, and yellow instead of cyan fluorescence will result.

The new color indicates that the molecules to which the cyan and yellow fluorophors are attached are interacting. In the case of the Gascoigne lab's work, the CD4 molecules had yellow fluorescent protein attached, and part of the T cell receptor complex had a cyan fluorescent protein attached.

When the CD4 and the T cell receptor are working properly and both recognizing the MHC, their two fluorescent proteins are close enough to interact, which is visible as reduced cyan fluorescence and increased yellow fluorescence upon exciting the cyan fluorophore under the microscope. And when T cell receptor antagonists are mixed in, there is no yellow fluorescence from the activation of the cyan protein, which would indicate that the fluorescent proteins—and therefore the CD4 molecules and the T cell receptors—are not interacting.

The research article "Inhibition of T-cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse" is authored by Tomasz Zal, M. Anna Zal, and Nicholas R.J. Gascoigne and appears in the April 17, 2002 issue of Immunity.

The research was funded by the National Institutes of Health and the Human Frontier Science Program Organization.


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