Journal: Nature Communications

Article Title: Gene correction for SCID-X1 in long-term hematopoietic stem cells

doi: 10.1038/s41467-019-09614-y

Figure Lengend Snippet: Evaluation of IL-2 receptor function in IL2RG cDNA targeted T cells. a Schematic of signaling (pSTAT5—bottom) and proliferation (CFSE—top) in vitro assays. b pSTAT5 assay derived FACS plots. Top: healthy male-derived T cells genome targeted with IL2RG cDNA tNGFR (KI) or with tNGFR+ only cassette integrated at the IL2RG endogenous locus (KO). In red are the percent of double positive IL2RG -tNGFR + pSTAT5 + [4.42%/(4/42% + 3.18%)]×100. We compare 58.2% cells (IL2RG targeted T cells) with 58.7% ( IL2RG from WT T cells), ( n = 3 biological replicates). c Quantification of IL-2R signaling through phosphoSTAT5 pathway. d pSTAT5 MFI for WT, KI, and KO experiments from ( b ) p = 0.02, Welch’s t -test. WT T cells (gray circles, n = 6), IL2RG KI (blue circles, n = 3) and IL2RG KO (orange circles, n = 3). e Proliferation profile of CFSE labeled, TCR stimulated IL2RG cDNA tNGFR+ sorted or mock-targeted T cells. Mock-targeted T cells are WT T cells cultured for the same amount of time as the tNGFR+ targeted cells and have been nucleofected in the absence of RNP or absence of transduction with AAV6. Shown FACS analysis at days 2, 4, 6, and 8. pSTAT5 phosphorylated STAT5, CFSE carboxyfluorescein succinimidyl ester, KI knocked in, KO knocked out, tNGFR truncated nerve growth factor receptor, IL-2 interleukin 2. Source data are available in the Source Data file

Article Snippet: Intracellular antigens were stained with pSTAT5 AF-488 (pY694, BD Bioscience) or isotype control (BD Biosciences).

Techniques: In Vitro, Derivative Assay, Labeling, Cell Culture, Transduction