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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.    Methodology/Principal Findings  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/Significance  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.

Estimating Point and Interval Frequency of Antigen-Specific CD4+ T Cells Based on Short In Vitro Expansion and Improved Poisson Distribution Analysis