Gene Regulatory Network of Human GM-CSF-Secreting T Helper Cells

Figure 1

Experimental setup and quality control for human CD4 T cell transcriptome and chromatin accessibility analysis. (a) Human PBMCs for each donor were split in three fractions, and memory CD4 T cells, naïve CD4 T cells, and bulk (total) CD4 T cells were isolated in parallel by negative (untouched) magnetic isolation (cell purities; see Supplementary Figure S1). From bulk CD4 T cells, GM-CSF-secreting cells (GM-CSF positive) were captured and the negative fraction was used as the corresponding GM-CSF-negative population. The five indicated cell populations were used for molecular profiling by ATAC-seq and RNA-seq. Differentially accessible DNA regions (DAR) and differentially expressed genes (DEG) were determined for the comparison of memory versus naïve CD4 T cells and for GM-CSF-positive versus GM-CSF-negative CD4 T cells, respectively. (b, c) High purity of isolated (captured) GM-CSF+ CD4 T cells was confirmed by flow cytometry. The histograms show the signal of the GM-CSF capture construct for the indicated cell populations, pregated on live singlet lymphocytes based on forward scatter, side scatter, and pulse width. Here, bulk CD4 T cells represent an aliquot taken after labeling with the GM-CSF-PE capture construct, but without further capture assay procedure. (b) shows a representative donor and (c) shows summarized data for donors from 4 independent experiments (mean and SEM are indicated in grey). Each donor is represented by a symbol; same symbol shape (but filled or unfilled) indicates donors processed within the same experiment.