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

Giulia Di Lullo, Francesca Ieva, Renato Longhi, Anna Maria Paganoni and Maria Pia Protti

PLOS ONE, 2012, vol. 7, issue 8, 1-9

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. 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.

Date: 2012
References: View complete reference list from CitEc
Citations:

Downloads: (external link)
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042340 (text/html)
https://journals.plos.org/plosone/article/file?id= ... 42340&type=printable (application/pdf)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:plo:pone00:0042340

DOI: 10.1371/journal.pone.0042340

Access Statistics for this article

More articles in PLOS ONE from Public Library of Science
Bibliographic data for series maintained by plosone ().