The Science Behind the Test

T Cell Immunology

Recent discoveries in immunology have enabled the classification of T cells into two groups: effector T cells and memory T cells.

Memory Cells

Memory T cells carry the body’s long-term immunity or ‘memory’ of an infection-causing pathogen and are present after any infection.

Effector Cells

Effector T cells fight the pathogen directly and come in various forms, designed to kill different pathogens in different ways. Most are short-lived and die off when the pathogen is cleared from the body.

Due to the short life of effector T cells, their continuing presence indicates the cellular immune response is currently encountering and fighting a pathogen somewhere in the body. Measuring the presence of effector T cells in a sample therefore diagnoses an ongoing infection. (In contrast, the presence of memory T cells, for example, would only indicate that someone had been infected with the pathogen at some point in the past).


Interferon-gamma is released by T cells, along with a number of other cytokines, to fight the infecting organism which has been recognized as foreign. Specific epitopes on the infecting organism have to be recognized by the T cells to initiate this release. Therefore the T-SPOT.TB test uses very specific antigens (ESAT-6 and CFP 10) to stimulate the effector T cells.

ESAT-6 and CFP 10

One of the major drawbacks of the Tuberculin Skin Test is its cross reaction with BCG vaccination. The Purified Protein Derivative that is injected in the skin test is a crude mixture of around 200 peptides extracted from dead Mycobacterium tuberculosis cells. Many of these proteins have common epitopes to BCG (and environmental mycobacteria) so the skin test will cross react with these.

An analysis of the Mycobacterium tuberculosis genome identified an area that was lost from M. bovis when it was being passaged by Calmette and Guerin to produce the BCG vaccine. This lost area of the Mycobacterium tuberculosis genome is know as the Region of Difference 1 or RD1. Nine proteins are made by this genomic area including ESAT-6 and CFP 10. Therefore if the T cells of someone with a Mycobacterium tuberculosis infection encounter these proteins they will recognise them as foreign and will respond by secreting interferon-gamma and other agents.

Since these RD1 proteins are not found in BCG or most environmental mycobacteria, T cells from someone who has been given the BCG vaccination or is infected with environmental mycobacteria will not respond to the ESAT-6 or CFP10 antigens as the T cells will not recognise them.