Sanne Hoeks

Defective neonatal TH17 development and reduced RORC2 mRNA 39 3 ◀ FIGURE E2. A, CB cell cultures produce low amounts of proinflammatory cytokines. APB and CB-naive T cells were activated by anti-CD3 in the presence of viable APCs. Culture supernatants were harvested from separate wells daily and analyzed for IL-1β, IL-6, and IL-17 concentration. n = 4. B, Addition of recombinant human cytokines does not influence APB cell culture supernatant concentration of IL- 10, IFN-γ, and IL-13, but does so in CB cell cultures. n = 4 for APB and 5 for CB. C, Blockade of proinflammatory cytokine signaling in CB cells does not affect FOXP3 upregulation. APB and CB-naive T cells were activated by anti-CD3 in the presence of viable APC and blocking antibodies toward IL-1β (1 μg/mL anakinra) and IL-6 (1 μg/mL tocilizumab) signaling. After 6 days, IL-17–producing and FOXP3-expressing cells were assessed by flow cytometry. Representative cytometry plots for n = 4. D, IL-17 concentration in the culture supernatants after 6 days of culture, n = 4. E, Percentage of FOXP3- expressing CD4 T cells. n = 4. F, IL-17 mRNA content is lower in CB cells than in APB cells after 6 days of culture. n = 4. *P < .05, **P < .01, ***P < .001, Student t test of the area under the curve (Fig E2, A), Kruskal-Wallis with Dunn post hoc (Fig E2, B and D, comparing the effect of blocking antibodies to medium control) and Student t test (Fig E2, F). When IL-6 (mouse) or IL-1β, IL-6, and IL-23 (human) are present during T-cell activation, FOXP3 is downregulated and RORγT enforces T H 17 development. Neonatal innate cells differ in their activation-induced cytokine production, compared with APB cells. 2 Does this biased cytokine production profile account for the difference in T H 17 development capacity? Indeed, culture supernatants of CB cells show reduced concentrations of both IL-1β and IL-6 during the culture period of 6 days as compared with APB cultures where these cytokines were readily produced. APB cultures yield an increased concentration of IL-17 at the end of the culture (see Fig E2, A ). To further investigate whether the absence of these cytokines in CB cell cultures had a role in the disability of T H 17 development, we added recombinant human cytokines to the cultures of naive APB and CB CD4 + T cells and monitored again both IL-17 production and FOXP3 expression. Addition of IL-1β, IL-6, and IL-23 resulted in an increased number of IL- 17–producing APB T cells without affecting the number of cells that upregulated FOXP3 (Fig 2, A - C ). Only the combination of IL-1β, IL-6, and IL-23 induced IL-17 production in CB cells but never to the extent observed in APB cells. The number of FOXP3 + cells was reduced when these cytokines were included alone or in combination in the cultures. Addition of GM-CSF or TGFβ did not clearly affect IL-17 production alone, nor in combination with the inflammatory cytokines (Fig 2, B ). The combination of inflammatory cytokines and these cytokines did show a reduced FOXP3 expression in APB cells. Thus, CB T cells respond to the cytokines as shown by reduced FOXP3 expression and the increase in IL-10 and IFNγ production (Fig E2, B ), suggesting that the lack of T H 17 differentiation in these experiments is not due to the absence of the specific receptors for these cytokines.