Foxp3 induction requires soluble factors from monocyte-CD4 T cell interaction, but not necessarily direct contact of monocytes with converting T cells.

<p>To determine contact-dependency for converting T cells, T cells were separated from iRBC-co-cultured whole PBMC or T cells+monocytes through a 20 nm pore size transwell and analysed by flow cytometry on day 6. Bar graphs show data of 5 donors (Mean+/−SEM), analysed as fold change in the induction of CD25^hiFoxp3^hi or CD25^hiFoxp3^int CD4 T cells (A). Representative FACS plots for each condition are shown (B). Foxp3^hi∶Foxp3^int ratios were calculated for T cells in direct co-cultures as well as inside and outside the transwell (C). Foxp3^hi and Foxp3^int levels, as well as IL-2 and IL-10 levels in culture supernatants were monitored on day 2, 4, 5 and 6 and are shown for 1 out of 2 donors (D). To determine which T cell subpopulation has to interact with monocytes to induce Foxp3 expression in transwell-separated T cells, T cells that were added back to monocytes were depleted of CD4⁺, CD8⁺, or CD25⁺ cells prior to culture. Proportions of CD25^hiFoxp3^hi and CD25^hiFoxp3^int cells of CD4 T cells induced in the transwell-separated T cell compartment (on day 6) as well as IL-2 and IL-10 levels in the culture supernatant (on day 2 and 6, respectively) were measured and are shown as fold changes compared to whole T cells (E). Fold changes (black bars) were calculated by normalizing for each donor the percentage of each of the two cell types of CD4⁺ T cells or cytokine levels on the respective values obtained for monocytes+T cells directly cultured with iRBCs (A) or for cultures with whole T cells added back to monocytes within the transwell (E) (white bars). The T cell compartment analysed and depicted in each individual graph is highlighted in bold red. Experiments were set up with a 2∶1 iRBC∶PBMC ratio.