Furnishing A Foundation For Vaccines

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An effective vaccine is premised on the actions of the body's protective immune response, which implies that we understand the nature of that response.

Nevertheless, our understanding often remains sorely limited, especially in the case of difficult diseases like cancer or HIV. We therefore need an experimental approach that would identify the characteristics of a protective immune response.

If such an approach is to succeed, it must take into account the wide range of cell types and mediators that confer immunity. Instead, most available immune monitoring assays measure just one or two parameters associated with the function of antigen-specific T cells, or the titers of antibodies or immune complexes. The results end up providing only a limited, incomplete picture of the immune response.

Going with the flow

Flow cytometry-based assays can address this shortcoming. Recent advances in these techniques offer new prospects for identifying immune protection correlates. For example, many flow cytometers can discriminate more than 11 fluorochromes, making it possible to study multiple parameters and determine the capacity of a particular T cell subset to produce single, double, or multiple cytokines.

This kind of analysis can be conducted with respect to vaccines for yellow fever or Hep A, examining the capacity of different memory T cell subsets obtained from immunized subjects to secrete cytokines such as IL-2, IFN-g, TNF-a, IL-10, and IFN-a. Peripheral Blood Mononuclear Cells (PBMC), which are critical components of the immune system, can be extracted and stimulated with cognate peptides, then stained for surface markers to reveal these memory T cell subsets along with antibodies.

In the same way, using cytokine production, the success and the efficacy of experimental vaccines and immunotherapies can be studied.

Drawing a map

Another major breakthrough in the development of these assays has been PhosFlow technology. In this case, phosphorylated molecules are detected using specific anti-phospho antibodies simultaneously with cell surface markers. The identity of the cell subset harbouring this phosphorylated molecule is then revealed.

Traditionally, such events have been examined through kinase assays, phosphoamino acid labeling, and immunoprecipitation/Western blotting. However, PhosFlow technology has several advantages because of the ability to perform single-cell analysis, to measure several fluorescent parameters simultaneously, and to analyze rare cell subsets and small samples. Even more importantly, two or three phosphoproteins can be detected simultaneously at the single cell level.

Tweaking T cells

In addition to simply characterizing the behaviour of immune system cells as they cope with disease, such information could make it possible to breathe new life into those cells. That is the conclusion of two Oxford-based researchers, Sarah Rowland-Jones and Tao Dong, writing in Nature this fall2.

They suggest that the way in which HIV-specific killer T cells appear to lose their function could be reversed, a conclusion that follows from a careful review of findings surrounding a programmed death molecule (PD-1) found on the surface of these cells.

This molecule appears to regulate T cell response to acute infection, but a chronic infection like HIV likewise appears to dysregulate that signal into a winding-down of the immune response. In fact, when antibodies block the action of PD-1, these apparently exhausted T cells revert to their full range of immune duties, including killing cells and proliferating.
Although the authors caution against unwarranted optimism about the therapeutic prospects of this work, the way ahead for the use of flow cytometry in this context appears to be all too clear.

"The work will undoubtedly prompt therapeutic strategies to restore responding T cells to full health and function, not only in HIV-1 infection, but in other settings where T-cell exhaustion is believed to play a part in disease, such as chronic infection with hepatitis B and C viruses. Similar approaches are being explored in cancer immunotherapy, because upregulation of the PD-1 ligands is a feature of many tumours and is associated with a poor prognosis."

With files from Elias Haddad, PhD Université de Montreal.

References
1. Jonathan M. Irish, Nikesh Kotecha & Garry P. Nolan, “Mapping normal and cancer cell signalling networks: towards single-cell proteomics”, Nature Reviews, v. 6, February 2006, pp. 146-155

2. Sarah Rowland-Jones & Tao Dong, “Tired T cells turn around”, Nature, v. 443, 21 September 2006, pp. 282-3