Lymphocytes: Precursor Frequencies

Abstract

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.
close

References

Barnden MJ, Allison J, Heath WR and Carbone FR (1998) Defective TCR expression in transgenic mice constructed using cDNA‐based alpha‐ and beta‐chain genes under the control of heterologous regulatory elements. Immunology & Cell Biology 76 (1): 34–40.

Grailer A, Sollinger HW and Burlingham WJ (1988) A rapid assay for measuring both colony size and cytolytic activity of limiting dilution microcultures. Journal of Immunological Methods 107 (1): 111–117.

Guillaume P, Dojcinovic D and Luescher IF (2009) Soluble MHC‐peptide complexes: tools for the monitoring of T cell responses in clinical trials and basic research. Cancer Immunity 9: 7.

Heeg K, Steeg C, Schmitt J and Wagner H (1987) Frequency analysis of class I MHC‐reactive Lyt‐2+ and class II MHC‐reactive L3T4+ IL 2‐secreting T lymphocytes. Journal of Immunology 138 (12): 4121–4127.

Karimi MA, Lee E, Bachmann MH, et al. (2014) Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium‐51 release assay. PLoS One 9 (2): e89357.

Kay HD, Fagnani R and Bonnard GD (1979) Cytotoxicity against the K562 erythroleukemia cell line by human natural killer (NK) cells which do not bear free Fc receptors for IgG. International Journal of Cancer 24 (2): 141–150.

Kodituwakku AP, Jessup C, Zola H and Roberton DM (2003) Isolation of antigen‐specific B cells. Immunology & Cell Biology 81 (3): 163–170.

Lefkowits I and Waldmann H (1979) Limiting Dilution Analysis of Cells in the Immune System. London and New York: Cambridge University Press.

Mannering SI, Morris JS, Jensen KP, et al. (2003) A sensitive method for detecting proliferation of rare autoantigen‐specific human T cells. Journal of Immunological Methods 283 (1–2): 173–183.

Marcus A and Raulet DH (2013) Evidence for natural killer cell memory. Current Biology 23 (17): R817–R820.

Massilamany C, Upadhyaya B, Gangaplara A, Kuszynski C and Reddy J (2011) Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers. BMC Immunology 12: 40.

Matza D, Lantner F, Bogoch Y, et al. (2002) Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity. Proceedings of the National Academy of Sciences of the United States of America 99 (5): 3018–3023.

Newell EW, Sigal N, Nair N, et al. (2013) Combinatorial tetramer staining and mass cytometry analysis facilitate T‐cell epitope mapping and characterization. Nature Biotechnology 31 (7): 623–629.

Pinna D, Corti D, Jarrossay D, Sallusto F and Lanzavecchia A (2009) Clonal dissection of the human memory B‐cell repertoire following infection and vaccination. European Journal of Immunology 39 (5): 1260–1270.

Ryser JE and MacDonald HR (1979) Limiting dilution analysis of alloantigen‐reactive T lymphocytes. I. Comparison of precursor frequencies for proliferative and cytolytic responses. Journal of Immunology 122 (5): 1691–1696.

Sabatino JJ Jr, Huang J, Zhu C and Evavold BD (2011) High prevalence of low affinity peptide‐MHC II tetramer‐negative effectors during polyclonal CD4+ T cell responses. Journal of Experimental Medicine 208 (1): 81–90.

Spitzer MH and Nolan GP (2016) Mass cytometry: single cells, many features. Cell 165 (4): 780–791.

Whitmire JK, Eam B and Whitton JL (2008) Tentative T cells: memory cells are quick to respond, but slow to divide. PLoS Pathogens 4 (4): e1000041.

Wong P and Rudensky AY (1996) Phenotype and function of CD4+ T cells in mice lacking invariant chain. Journal of Immunology 156 (6): 2133–2142.

Wu P and Ward RE (1993) Ig repertoire expression of BALB/c primary and secondary B cell precursors specific for phosphorylcholine. Journal of Immunology 150 (9): 3862–3872.

Wylie DE and Klinman NR (1981) The murine B cell repertoire responsive to an influenza‐infected syngeneic cell line. Journal of Immunology 127 (1): 194–198.

Further Reading

Cerwenka A and Lanier LL (2016) Natural killer cell memory in infection, inflammation and cancer. Nature Reviews. Immunology 16 (2): 112–123.

Phetsouphanh C, Zaunders JJ and Kelleher AD (2015) Detecting antigen‐specific T cell responses: from bulk populations to single cells. International Journal of Molecular Sciences 16 (8): 18878–18893.

Taswell C (1981) Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. Journal of Immunology 126 (4): 1614–1619.

Trouw LA, Huizinga TW and Toes RE (2013) Autoimmunity in rheumatoid arthritis: different antigens–common principles. Annals of the Rheumatic Diseases 72 (Suppl 2): ii132–ii136.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

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