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. 2018 Mar 21;9:507. doi: 10.3389/fimmu.2018.00507

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Copyright © 2018 Casucci, Falcone, Camisa, Norelli, Porcellini, Stornaiuolo, Ciceri, Traversari, Bordignon, Bonini and Bondanza.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Generation of nerve-growth-factor receptor (NGFR)-spaced CD44v6 chimeric antigen receptor (CAR)-T cells. (A) Structure of the low-affinity NGFR extracellular region and of the four NGFR isoform-spaced CD44v6 CAR constructs (NWL, NGFR wild-type long; NWS, NGFR wild-type short; NML, NGFR mutated long; NMS, NGFR mutated short). Gray boxes: single-chain variable fragment (scFv); white box: CD28 costimulatory/transmembrane endodomain; black box: CD3 zeta chain. Primary T cells from healthy donors were stimulated with CD3/CD28-beads, transduced with RVs encoding for CD44v6 CARs spaced with NWL, NWS, NML, or NMS, enriched with immuno-magnetic beads (see Materials and Methods) and cultured with IL-7/IL-15. (B) Left: FACS plots of CAR-T cells from a representative donor (of n = 8) before (pre) and after (post) enrichment. Right: NGFR expression on CAR-T cells showed as relative fluorescence intensities (RFIs) over isotype-matched controls (means ± SEM from n = 8 donors). Results from a paired t-test are indicated when statistically significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). (C) Left: FACS plots of resting CAR-T cells from a representative donor stained with NGFR (x axes) and Protein L (y axes). Right: NGFR and Protein L expression on CAR-T cells showed as RFIs over isotype-matched controls (means ± SEM from n = 3 donors). (D) Expansion of NGFR isoform- or CH2CH3-spaced CD44v6 CAR-T cells expressed as fold increase at day 16–20 after bead stimulation (see Materials and Methods, means ± SEM from n = 5 donors). (E) Memory phenotypes of NGFR isoform-enriched CD44v6 CAR-T cells analyzed at day 16–20 after bead stimulation. Left: FACS plots from a representative donor. Right: data obtained from n = 7 donors (means ± SEM). Results from a paired t-test on TSCM are indicated when statistically significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). TSCM, CD45RA+/CD95+/CD62L+ stem memory T cells; TCM, CD45RA−/CD95+/CD62L+ central memory T cells; TEM, CD45RA−/CD95+/CD62L− effector memory T cells; TEMRA, CD45RA+/CD95+/CD62L− effector memory RA T cells. (F) Exhaustion/activation phenotypes of NGFR isoform-enriched CD44v6 CAR-T cells analyzed at day 16–20 after bead stimulation. Left: FACS plots from a representative donor. Right: data obtained from n = 3 donors (means ± SEM). Results from a paired t-test on HLA-DR−/PD1− cells are indicated when statistically significant (**P ≤ 0.01). (G) NGFR isoform-enriched CD44v6 CAR-T cells were labeled with CFSE and analyzed by FACS after 6-day culture in the absence of cognate antigen stimulation. Proliferation was expressed as percentages of CFSE-diluting cells (means ± SEM from n = 3 donors). Results from a paired t-test are indicated when statistically significant (*P ≤ 0.05; **P ≤ 0.01). (H) Mortality rate of NGFR isoform-enriched CD44v6 CAR-T cells expressed as percentages of dead DAPI + cells at day 16–20 after bead stimulation. Left: FACS plots from a representative donor. Right: data obtained from n = 7 donors (means ± SEM). Results from a paired t-test are indicated when statistically significant (*P ≤ 0.05; ***P ≤ 0.001).