Lymphocytes: Precursor Frequencies


The frequency of antigen‐specific cells will vary during development and in response to the first and subsequent encounters with an antigen, either through infection or immunisation, and is an important indicator of adaptive immune function. Antigen‐specific lymphocyte prevalence can be estimated indirectly and directly using several different techniques. Functional assays have been used for several decades to indirectly calculate the frequency of antigen‐specific lymphocytes by measuring a proliferative response, cytokine production or cytolytic activity in response to antigenic stimulation. The functional activity of individual lymphocytes is also able to be measured directly, which allows phenotypic analyses of antigen‐specific cells. More recently, labelled MHC (major histocompatibility complex)/peptide multimers have allowed researchers to directly enumerate and comprehensively phenotype lymphocytes using multiparameter flow cytometry.

Key Concepts

  • Antigen‐specific precursors may be undetectable in naive individuals but are increased in frequency after immunisation.
  • Antigen‐specific lymphocyte prevalence is dependent on immune status, the tissues from which cells are isolated and the immunogenicity of the antigen.
  • Indirect functional assays may detect less abundant precursors, but direct methods will detect cells rendered nonfunctional by anergy or exhaustion.
  • Indirect methods for determining lymphocyte precursor prevalence rely on the single hit Poisson model.
  • ELISPOT and flow cytometry are the principal techniques for direct characterisation of lymphocyte precursors.

Keywords: precursor frequency; lymphocyte; antigen specific; adaptive immunity; flow cytometry

Figure 1. The single‐hit Poisson model. (a) The probability mass function for the Poisson distribution. (b) Graphical representation of the Poisson distribution where λ = 1. (c) Derivation of the zero term of the Poisson probability mass function indicates that the proportion of empty wells and average number of events per interval (i.e. cells per well) have a logarithmic relationship. (d) Solving the equation, the average cells per positive well will be one (λ = 1) when the natural log (ln) of the % empty wells is −1 or the proportion of empty wells is approximately 0.368 (36.8%).
Figure 2. Functional assays of precursor prevalence. In a functional assay to determine precursor prevalence, potential responder cells are plated at a range of cell concentrations (a) 6.25 × 105 to 1 × 106 cells well−1. The percentage of wells that are negative for precursor activity (b) are counted. To determine at which cell concentration the proportion of positive wells will have an average of 1 cell well−1, the natural log (ln) % of negative wells (c) is calculated and correlated with total potential responder cells (Figure).
Figure 3. Determination of precursor prevalence. (A) Plotting the ln % of negative wells (c, y‐axis) and corresponding total cells per well (a, x‐axis) produces a straight line described by the equation indicated. Calculating (a) where (c) is equal to −1 indicates the prevalence of antigen‐specific cells. (B) Alternatively, the logarithmic relationship between % of negative cells (b, y‐axis) and total cells per well (a, x‐axis) can be calculated and solved to determine the value for (a) where (c) is equal to e−1 or ∼36.8%. Using either method results in a prevalence of 1 precursor cell per 340 700 cells.


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Further Reading

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Clanchy, Felix IL, Williams, Richard O, and Mageed, Rizgar A(Jul 2017) Lymphocytes: Precursor Frequencies. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000949.pub2]