Lymphocyte transcriptional profile and memory markers expressed on CAR⁺ T-cell surface.

<p>After 29 days of expansion on clone#1 AaPC/IL-2/IL-21, ROR1RCD28 and ROR1RCD137 cells were (i) lysed for mRNA expression analysis using bar-codes or (ii) phenotyped for T-cell surface markers by flow cytometry. (a) RNA lysates were profiled for expression of a selected group of lymphocyte genes with non-enzymatic digital multiplex array of mRNA transcripts (NanoString) where transcription factors are shown on the left, genes associated with survival, co-stimulation, and trafficking are shown on the middle, and genes associated with effector function are shown on the right. Dashed line represents the limit-of-detection calculated by mean + 2xSD of negative controls. (b) Flow cytometry of ROR1RCD28 and ROR1RCD137 T cells showing co-staining for CD3 and CD56, CD4 and CD8, CD28 and CD27, CD62L and CCR7, CD45RO and CD62L, or CD95 and CD57 in cells gated for CAR expression based staining with Fc-specific antibody. ROR1RCD28⁺ T cells are shown in the top panels and ROR1RCD137 T cells are displayed in the bottom panels. One of 3 representative donors is displayed and quadrant frequencies are shown in the upper right corners. (c) Cumulative frequencies of cells staining positive for each memory marker within an extended memory phenotype panel. (d) Multi-parameter flow cytometry was used to determine frequencies of cells staining positive for combinations of CD45RA, CD27, CD28, and CCR7. For (c) and (d) ROR1RCD28 are in open shapes/bars and ROR1RCD137 are in closed shapes/bars and lines displayed in (c) are means (n = 3) and in (d) are mean ± SD (n = 3). Student’s two-tailed t-tests were used for statistical analysis between the two groups. *p<0.05 Expression and phenotype data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128151#pone.0128151.s001" target="_blank">S1 Dataset</a>, Fig 3 tab.