Sanne Hoeks

Chapter 3 40 As the development of T H 17 cells inversely correlates to the development of Treg cells, 5 we considered that the induction of T H 17 cells in the APB could be responsible for the diminished induction of Treg cells in APB cells as compared with CB cells. Pharmacologic inhibition of proinflammatory cytokine signaling, through the addition of IL-1RA– or IL-6R– blocking antibody (anakinra and tocilizumab, respectively), decreased the number of IL-17–producing T cells and lowered concentrations of supernatant IL-17 (Fig E2, C - E ). Nevertheless, the number of APB cells expressing FOXP3 after 6 days of culture remained stable and was not affected by these treatments. Because CB cell cultures did not produce IL-1β and IL-6, blockade of the signaling pathways did not affect the percentages of IL-17 + or FOXP3 + T cells. Finally, as CB precursor cells did not form T H 17 cells, while we did see a signaling effect of inflammatory cytokines, we hypothesized that the blockade in T H 17 differentiation should be upstream of the transcription of IL-17 but downstream of cytokine signaling. We therefore analyzed the T H 17 defining transcription factor RORγT, in humans encoded by transcript variant 2 of the RORC gene. 9, 10 Indeed, quantitative PCR of RORC2 mRNA after 3 and 4 days of activation shows significantly lower RORC2 mRNA content in CB as compared with APB T cells (Fig 2, D ), resulting in a reduced IL-17 mRNA (Fig E2, F ) and prevention of IL-17 production. We propose that neonatal T cells have an intrinsic mechanism that prevents T H 17 differentiation through the regulation of RORγT expression. This may be of functional relevance to prevent adverse immune reactions against self or nonpathogenic microbes during the establishment of T-cell repertoire and bacterial colonization. Exact mechanisms preventing RORC transcription in CB cells are not known yet but might be found in upstream signaling or DNA methylation and histone acetylation. Appropriate activation, for example, via innate signaling, can overcome this deficiency. 1 We thank Dr Wilco de Jager for technical assistance in the cytokine measurements. FIGURE 2. CB T cells cannot be forced to Th17 development and do not upregulate RORC. APB and CB naïve T cells were activated by plate bound anti-CD3 in the presence of viable APC and recombinant cytokines as indicated for 6 days. A, Representative cytometry plots. B, IL-17 in the culture supernatants (n = 6 left plot and n = 4 APB, n = 5 CB right plot). C, Percentage of FOXP3 + CD4 + cells (n = 6 APB, n = 5 CB left plot, n = 4 APB, n = 5 CB right plot). D, Relative RORC2 mRNA expression (n=4). *p<0.05, *P < .05, Kruskal-Wallis with Dunn post hoc (Fig 2, B, C) and Student t test (Fig 2, D). ▶