Antigen titration for T cell stimulation

T lymphocytes don't 'naturally' respond to an antigen; for an antigen to elicit a T cell response

1. A T cell clone with that particular receptor must be present

2. There must be antigen presenting cells (dendritic cells/ macrophages/ B cells)

In vitro, in isolated mononuclear cell preparations from whole blood, the second point is not usually a problem. However, the first condition, presence of antigen specific T cell clone, varies. 

For example, for a disease like tuberculosis which is very prevalent in India, one might expect that everyone has at least a few T cell clones for tubercular antigens. However, if the person has never got actual tuberculosis disease, this clone will not be very large. For rare diseases (like Scrub typhus), one would expect an even lower proportion of antigen specific T cells.

Empirically, it is prudent to do a titration of a specific antigen - if only to see which dose does the T cell respond the most. It is not a very tightly controlled experiment, because

1. The total number of lymphocytes vary between people

2. The size of antigen specific T cell clones vary between person to person

3. The T cell response is not dose dependent

However, like all biological phenomenon, T cell stimulation is not an 'all or none' phenomenon, and some correspondence with dose of antigen is always observed. In this case, a whole cell lysate of Mycobacterium tuberculosis (the antigen) was used in increasing doses to stimulate T cells from a healthy person. Production of interferon gamma (IFNG) was used as a marker for T cell response.

1. CD4+ T cells were selected (first panel): the anti-CD4 antibody is tagged with APC

2. The IFNG producing CD4+ T cells are shown in sequence, with increasing doses of the antigen producing a higher proportion of IFNG+ T cells

The unstimulated population is used as a control to create the box ('gate') for IFNG+ T cells

The proportion of IFNG+ T cells plateaus at 7.5 microL of antigen dose, and thus this dose was selected.

Thanks Uddeep & Dr Abhinav 

Visualising cytokines

Cytokine production by cytotoxic T cells

T cells stimulated with tubercular antigen show a visible increase in the proportion of interferon-gamma producing CD8+ T cells; (flow cytometric data, PE Cy5 = CD8, FITC = IFN gamma)

Before stimulation; note the very small fraction of cells above the horizontal line (i.e. those producing interferon)

After stimulation; 2-4 % of cells have now been activated and moved above the line

The CD8 negative cell population (i.e. left of the vertical line) are a mix of B cells and helper (CD4+) T cells; note that CD4+ T cells will also produce interferons, as seen in the top left quadrant.