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. 2021 Jul 19;32(13-14):744–760. doi: 10.1089/hum.2020.216

Figure 2.

Figure 2.

Immune-selection of CAR T cells. (A) CAR T cells endowed with NWL and NWN2 spacers were immune-selected by the use of either a single-step protocol with the mAb ME20.4 coupled to magnetic MicroBeads (CliniMACS CD271-LS MicroBeads; Miltenyi), or by a two-step method using mAb ME20.4 followed by selection with beads conjugated to anti-mouse antibodies (Dynabeads; Dynal). Both immunoselection processes allowed the isolation of highly enriched (>90%) populations of CAR T cells endowed with NWL spacer, whereas CD44v6-NWN2.CAR T were purified only with the two-step method. (B) Pure populations of CAR T cells expressing the NWL or the NWN2 CAR constructs were stained with (1) the anti-LNGFR mAbs C40-PE (left panels); (2) the ME20.4 mAb followed by an anti-mouse-PE (middle panels); and (3) the ME20.4 mAb coupled to MicroBeads followed by an anti-MicroBead-PE (right panels). NWL expressing CAR T cells are recognized by all the combinations tested (upper panels). Concerning the NWN2 construct (lower panels), ME20.4 mAb is able to bind the transduced cells and can be in turn recognized by an anti-mouse mAb, thus justifying the successful selection obtained with the two-step procedure. On the contrary, when directly coupled to the MicroBeads, the ME20.4 mAb is no more able to bind the NWN2.CAR. mAb, monoclonal antibody. Color images are available online.