Sanne Hoeks

Chapter 4 50 Journal of Allergy and Clinical Immunology 2014 133891-894.e5DOI: (10.1016/j.jaci.2013.09.022) FIGURE E2. T cell subsets ex vivo. A, CD4 + FOXP3 + cell percentages ex vivo. B, PBMC were stimulated with PMA and Ionomycin. Supernatants were collected and analyzed for IFNg. *P < .05. To investigate the capacity of T cells to become activated and differentiate into different T-cell subsets, we cultured PBMCs for 6 days with anti-CD3. We focused on Treg cells and T H 17 cells because it has been shown that CB cells differ from adult cells in their capacity to differentiate into these cells. 2, 6 In addition, the gut microbiota has an important role in the development of these cells and colonization occurs soon after birth. 7-9 We therefore hypothesized that the infant samples would reflect an adult phenotype with regard to T H 17 and Treg-cell differentiation. First, we assessed the proliferation of PBMCs on anti-CD3 activation by 3H-labeled thymidine incorporation. Cells derived from adults showed the highest proliferation, although only significantly different from the samples obtained at 3 months (Fig E3, A ). The inability of CB cells to produce IL-17 was confirmed in these assays (Fig 2, A and B ). The number of T cells producing IL-17 was already increased at age 3 months, and the amount of IL-17 in the supernatant of these cell cultures gradually increased from CB to adulthood. On activation, CB cells easily upregulated FOXP3 when compared with adult cells (Fig 2, C ). In contrast to the differences in T H 17 development, this phenomenon was still observed in samples derived from children aged 3 to 12 months. So, the propensity of CB T cells to become Treg cells remained until at least age 1 year, while the inability to become T H 17 is overcome within the first months of life. Furthermore, we investigated the production of other T-cell–derived cytokines in these cultures. No difference in IL-13 production was found. The assumed T H 2 bias in infants was mainly present in the form of a significantly reduced IFN-γ production, corresponding with a lower IFNγ/IL-13 ratio compared with that in adults (see Fig E3, B-D ).