Fig. 1: Fox19CAR-Treg in vitro generation and validation. | Nature Communications

Fig. 1: Fox19CAR-Treg in vitro generation and validation.

From: Regulatory T cells expressing CD19-targeted chimeric antigen receptor restore homeostasis in Systemic Lupus Erythematosus

Fig. 1

A Fox19CAR lentiviral vector (LV) and FoxP3 LV schematic representations. The FoxP3 gene and an anti-CD19 second-generation CAR are inserted in an unidirectional LV and under the control of a Phosphoglycerate Kinase (PGK) promoter. The vector encoding for the FoxP3 gene alone is a bi-directional LV with the FoxP3 gene in sense (PGK promoter) and the enhanced Green Fluorescent Protein (eGFP) in antisense (minimal CMV or mCMV promoter). LTR long terminal repeat, SD splice donor, SA splice acceptor, GA gag-pol element, RRE REV responsive element, cPPT central polypurine tract, pA polyadenilation signal, CTE constitutive transport element, WPRE woodchuck hepatitis virus post-transcriptional regulatory element. B Transduction efficiency of CAR19.28z LV, Fox19CAR LV and FoxP3 LV measured as percentage of either CAR+ or GFP+ cells among CD4+CD25+CD127-FoxP3+ lymphocytes at day +14. Recombinant CD19 for UT-, 19CAR- and Fox19CAR-Tregs and GFP for FoxP3 LV respectively, were employed to assess the transduction percentage. N = 5 for UT- and 19CAR-Tregs. N = 10 for Fox19CAR-Tregs. N = 3 for FoxP3-Tregs. One-way ANOVA with Tukey correction for multiple comparison. ***p-value 0.0008, ****p-value < 0.0001. C Representative flow cytometry plot for co-localization of the CAR construct and FoxP3 in Fox19CAR-Tregs compared to untransduced (UT) cells. CAR expression was assessed using the human rCD19. D Expansion rate of UT-, 19CAR-, Fox19CAR- and FoxP3-Tregs, assessed at day +14 since the initial stimulation. N = 6 for UT-, Fox19CAR- and 19CAR-Tregs. N = 3 for FoxP3-Tregs. One-way ANOVA with Tukey correction for multiple comparison. E Polyclonal suppressive capacities of UT-, 19CAR- and Fox19CAR-Tregs. Results are expressed as Suppression Index, calculated as Suppression index = [1-(PBMCs’ proliferation with Tregs)/(PBMCs’ proliferation alone)] * 100. N = 3 for UT-, Fox19CAR- and 19CAR-Tregs. N = 3 for UT conventional T cells. Two-way ANOVA with Tukey correction for multiple comparison. F Antigen-specific suppressive capacities of UT-, 19CAR-, Fox19CAR- and FoxP3-Tregs. Results are expressed as Suppression index = [1-(B cell proliferation with Tregs)/(B cell proliferation alone)] * 100. N = 6 for UT- and Fox19CAR-Tregs. N = 3 for 19CAR- and FoxP3-Tregs. Two-way ANOVA with Tukey correction for multiple comparison. The exact p-values are reported in the graph. All the results are expressed as mean ± standard deviation.

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