Fluorescent Cell Barcoding as a Tool to Assess the Age-Related Development of Intracellular Cytokine Production in Small Amounts of Blood from Infants

Abstract

Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 µl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4+ T-cells, and for PMA, PHA, SEB, and TT in CD8+ T-cells. The intracellular cytokine production by CD4+ and CD8+ T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants.

T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca2+ Influx

“…To analyze the activation of the MAPK-ERK pathway, which leads to AP-1 translocation into the nucleus, we used CB as an example for the “youngest” T cells, which produced only low amounts of IL-2 [2,3], in comparison to adult CD31 + naive T cells. Strikingly, the MAPK-ERK axis of naive CD4 + T cells responded robustly by phosphorylation of ERK.…”

“…Many yet unknown antigens from dietary compounds, commensal bacteria, and environmental sources flood the immune system in the first month of life, so the prevention of immune memory formation in this period would appear to be a prudent strategy. Our work thus may help us understand the paradoxical observation that T cell responses in infants are strong but are difficult to induce [3,6,8,9,22–25,75–78]. …”

“…In this challenging time period for the immune system, epidemiological studies have shown that neonates and infants are especially susceptible to infections; this period of life is also decisive for directing immune responses and pathologies later in life [1]. T cell functions, such as cytokine production, are downregulated in response to antigens of acute infections during the neonatal and early infancy periods [2,3]. This has been attributed to reduced numbers of neonatal lymphocytes [4,5] and to those cells’ attenuated ability to be fully activated because of limited expression of activation-associated molecules such as CD40L and NFAT [6–9].…”

Loss of ADAM17 is associated with severe multiorgan dysfunction

“…The cytokine pattern of T-cell subsets was determined using fluorescent cell barcoding as recently described by our group [4]. Heparinized blood was stimulated and incubated within 3 hours after collection.…”

Rapid comparative immunophenotyping of human mesenchymal stromal cells by a modified fluorescent cell barcoding flow cytometric assay

“…To date, cell barcoding has been achieved using antibody‐mediated, amine reactive cytosolic dyes, streptavidin‐based, multicolor bead based, and genetic fluorescent barcoding approaches with respective advantages and disadvantages . The assay has been successfully utilized for immortal cell lines, freshly isolated (primary) cell samples, and blood cells . The technique tags several cell samples with unique fluorescent signatures enabling multiplexed simultaneous analyses.…”