Fig. A.24 The frequency of specific lymphocytes can be determined using limiting-dilution assay. Various numbers of lymphoid cells from normal or immunized mice are added to individual culture wells and stimulated with antigen and antigen-presenting cells (APCs) or polyclonal mitogen and added growth factors. After several days, the wells are tested for a specific response to antigen, such as cytotoxic killing of target cells. Each well that initially contained a specific T cell will make a response to its target, and from the Poisson distribution one can determine that when 37% of the wells are negative, each well contained, on average, one specific T cell at the beginning of the culture. In the example shown, for the unimmunized mouse 37% of the wells are negative when 160,000 T cells have been added to each well; thus the frequency of antigen-specific T cells is 1 in 160,000. When the mouse is immunized, 37% of the wells are negative when only 1100 T cells have been added; hence the frequency of specific T cells after immunization is 1 in 1100, an increase in responsive cells of 150-fold.
number of methods for doing this, either by detecting the cells directly by the specificity of their receptor, or by detecting activation of the cells to provide some particular function, such as cytokine secretion or cytotoxicity.
The response of a lymphocyte population is a measure of the overall response, but the frequency of lymphocytes able to respond to a given antigen can be determined by limitingdilution culture. This assay makes use of the Poisson distribution, a statistical function that describes how objects are distributed at random. For instance, when a sample of heterogeneous T cells is distributed equally into a series of culture wells, some wells will receive no T cells specific for a given antigen, some will receive one specific T cell, some two, and so on. The T cells in the wells are activated with specific antigen, antigen-presenting cells, and growth factors. After allowing several days for their growth and differentiation, the cells in each well are tested for a response to antigen, such as cytokine release or the ability to kill specific target cells (Fig. A.24). The assay is replicated with different numbers of T cells in the samples. The logarithm of the proportion of wells in which there is no response is plotted against the number of cells initially added to each well. If cells of one type, typically antigen-specific T cells because of their rarity, are the only limiting factor for obtaining a response, then a straight line is obtained. From the Poisson distribution, it is known that there is, on average, one antigen-specific cell per well when the proportion of negative wells is 37%. Thus, the frequency of antigen-specific cells in the population equals the reciprocal of the number of cells added to each well when 37% of the wells are negative. After priming, the frequency of specific cells goes up substantially, reflecting the antigen-driven proliferation of antigen-specific cells. The limiting-dilution assay can also be used to measure the frequency of B cells that can make antibody against a given antigen.