A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens.
Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.
Givan
A L
AL
Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. givan@dartmouth.edu
Fisher
J L
JL
Waugh
M
M
Ernstoff
M S
MS
Wallace
P K
PK
D000941:Antigens / Q000008:administration & dosage*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002454:Cell Differentiation; D002455:Cell Division; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007158:Immunologic Techniques; D016130:Immunophenotyping; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D009930:Organic Chemicals; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D013745:Tetanus Toxoid / Q000008:administration & dosage
D000941:Antigens; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; D013745:Tetanus Toxoid