title: Estimating point and interval frequency of antigen-specific CD4+ T cells based on short in vitro expansion and improved poisson distribution analysis.
abstract: BACKGROUND
  Knowledge of antigen-specific CD4(+) T cells frequencies is pivotal to the choice of the antigen to be used in anti-viral and anti-tumor vaccination procedures and for monitoring of immune responses. Methods that employ small cell numbers from patient samples, are easy to perform and do not require complex techniques/instrumentations and therefore standardization are desirable.
  
  
  RESULTS
  Purified blood CD4(+) T cells from healthy donors were cultured with autologous antigen presenting cells in several replicate wells in equal numbers in the absence (un-stimulated wells) or in the presence of synthetic peptides corresponding to viral antigens promiscuous HLA-DR epitopes (antigen-stimulated wells). At day 7 of culture low dose IL-2 was added and at day 14 IFN-γ and IL-5 release in the supernatant was measured. A statistical analysis approach, based on Poisson distribution, was then implemented to calculate the frequency of viral-specific CD4(+) T cells. We first determined a patient-specific exceptionality threshold of cytokine release in the un-stimulated wells and then, based on this threshold, we counted the inactive/active wells within the antigen-stimulated wells. This number, along with the number of cells per well, allowed the point and interval estimates of frequencies. A ready-to-use Excel worksheet template with automatic calculations for frequencies estimate was developed and is provided as a supplemental file (Table S9).
  
  
  CONCLUSIONS
  We report a simple experimental procedure combining short term in vitro cell culture with statistical analysis to calculate the frequency of antigen-specific CD4(+) T cells. The detailed experimental procedure along with the Excel applicative are a valuable tool for monitoring immune responses in the clinical practice.
authors.affiliation: Tumor Immunology Unit, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Di Lullo
authors.forename: Francesca
authors.initials: F
authors.lastname: Ieva
authors.forename: Renato
authors.initials: R
authors.lastname: Longhi
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Paganoni
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Protti
mesh_terms: D018791:CD4 Lymphocyte Count; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D007371:Interferon-gamma / Q000378:metabolism; D015848:Interleukin-5 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology; D016012:Poisson Distribution; D015203:Reproducibility of Results
chemical_list: D000939:Epitopes; D019309:Epstein-Barr Virus Nuclear Antigens; D015848:Interleukin-5; D007371:Interferon-gamma