Input function for Th1/Th2 cell differentiation under mixed input conditions reveals a tunable mixed phenotype.

<p>(A) Histograms of T-bet levels measured in populations of cells cultured with decreasing levels of IL-4 and increasing levels of IL-12 (yellow to blue color). Dashed curve, cells from a T-bet knockout mouse. (B) Histograms of GATA3 levels measured in populations of cells cultured with increasing levels of IL-4 and a constant level of IL-12 (yellow to blue color). Dashed curve, GATA3 isotype control staining. (C, D) Measured median fluorescence intensities (MFI) for T-bet (C) and GATA3 (D), in response to a matrix of orthogonal gradients of the two input signals IL-12 and IL-4. Regions 1 and 2 represent standard polarizing conditions used to generate a Th1 or Th2 response, respectively. Region m represents a state with mixed inputs, resulting in expression of both T-bet and GATA3. (E–G) Scatter plots showing normalized measured expression patterns of T-bet and GATA3, under the conditions marked by 1,m,2 in panels (C, D) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001616#pbio.1001616.s020" target="_blank">Text S1</a> for details). A single, unimodal population is observed, which shifts in the T-bet-GATA3 plane in response to input signals. Colored dots in each panel show the population median. (H) Distributions of the parameter α, representing the ratio between expression levels of T-bet and GATA3 (see F and definition in the main text), for cell populations cultured under Th1, Th2, and mixed input conditions. The distributions all show a single peak, and continuously shift from a Th1 (α≈90°) to a Th2 state (α≈0°). (I) Representative images of cells cultured under various input conditions as indicated, fixed and stained for T-bet (blue, pseudo-color) and GATA3 (red, pseudo-color). Images were acquired using fluorescent flow microscopy (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001616#s4" target="_blank">Materials and Methods</a>). Three cells for each condition are shown in the bright-field (BF), T-bet, and GATA3 channels. (J) The input function of T-bet is well described by separation of variables, with each input influencing the output in an independent manner. Shown are the calculated dependencies of T-bet on the two inputs, F₁(IL-12) and F₂(IL-4), and the calculated input function given by F₁(IL-12)×F₂(IL-4), which shows a high similarity with the measured data (C). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001616#pbio.1001616.s003" target="_blank">Figure S3</a> for similar results for GATA3, IFN-γ, and IL-4.