Abstract
During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.
Highlights
The immune system provides protection from various infections and tumors, but can mediate allergies, autoimmunity and transplant rejection
peripheral blood mononuclear cells (PBMC) from healthy human donors were selected from the ePBMC library (CTL, Shaker Heights, OH, USA)
The cells were tested within 2 h of thawing because we found that “overnight resting” does not improve the functionality of properly cryopreserved PBMC [26] and it was reported that overnight resting changes the cytokine signatures of antigen-specific T cells [27]
Summary
The immune system provides protection from various infections and tumors, but can mediate allergies, autoimmunity and transplant rejection. There have been occasional observations of delayed IL-4, IL-5 and IL-17 secretion kinetics by memory T cells [18], but the detection of these effector T cell classes in PBMC has mostly been unsuccessful, likely because of the low frequency of such T cells, and possibly because too short antigen-stimulation cultures have been used. The data showed that, invariably, IL-4, IL-5 and IL-17 secretion by CD4 cells is delayed by several days compared to the production of IFN-γ This notion has profound implications for reliable detection of Th2 and Th17 cells as these T cell subsets would go undetected in assays that do not extend an 8 h antigen stimulation period. Accounting for the delayed secretion kinetics of Th2 and Th17 cells, should be part of any immune monitoring approach
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