Sanne Hoeks

Chapter 2 28 CB showed a peak of FOXP3 + cells around 4 days of culture, after which the percentage of FOXP3 + cells decreased to stable expression at day 6 (Fig 1, D ). Both APB and CB cells had a similar stable FOXP3-expressing population compared with the initial peak around day 4; 65% of the FOXP3 + T cells at day 4 remain stable FOXP3-expressing cells during the 8-day culture. The induction of Treg cells in the periphery on activation can be mediated by several factors. Cytokines like TGFβ and IL-2 are known to induce FOXP3, 5 whereas inflammatory cytokines prevent this. To test whether possible differences in secreted mediators, such as cytokines and chemokines, can explain the difference in Treg cell induction, we exchanged culture supernatants between CB and APB every day during the 6-day culture period of naive T cells with APCs and anti-CD3. This approach did not result in altered differences between CB and APB Treg cell induction, making a role for secreted mediators unlikely (not shown). Fig 1 Journal of Allergy and Clinical Immunology 2011 1281369-1371DOI: (10.1016/j.jaci.2011.08.006) FIGURE 1. CB APCs stimulate Treg induction. A, FOXP3 + Treg cell percentages ex vivo or after 6-day naive (CD4 + CD25 - CD45RO - ) T-cell stimulation, as indicated in the figure (left to right, n= 3, 6, 4, and 10). ns, Not significant. B, Treg cell percentage after 6-day alloreaction between naive T cells and APCs (n= 4). C, Treg cell percentage of anti-CD3–activated naive T cells after 6-day culture in the presence of sorted indicated APCs (left to right, n= 5, 3, and 3). cDC, Conventional dendritic cells; pDC, plasmacytoid dendritic cells. D, Time course of Treg cell expansion of anti-CD3–activated naive T cells cultured with viable APCs (n= 4). *P ≤.05. Another factor influencing FOXP3 induction is the strength of the T-cell receptor (TCR) signal and costimulation. 6, 7 In our settings the TCR signal was standardized by plate-bound anti- CD3, but co-stimulatio is provided by the APCs. High co-stimulation through CD28 signaling prevents the upregulation of FOXP3. We therefore investigated the role of the CD28 ligands CD80 and CD86 on APCs. We observed no difference in the expression of these molecules. When blocking antibodies toward CD80, CD86, or both were added to the cultures, this did not result in a difference in FOXP3 expression (Fig 2, A ). This finding was confirmed by adding increasing concentrations of cytotoxic T lymphocyte–associated antigen 4-immunoglobulin (CTLA4- Ig) to the cultures, blocking both CD80 and CD86, which had no obvious effect (data not shown).