Two patients presented with cellular immune reactivity toward retrovirally transduced T cells independent of CAR transgene. (A) Patient 5 developed both anti-CAR and antivector immune reactivity. Patient 5 stimulator T cells were generated either by transduction or nucleofection with the transgenes CAIX-CAR or huCD24 (see “Methods”). Transduced T cells were enriched for transgene positivity by MACS-selection, whereas nucleofected T cells were used within 48 hours after nucleofection, without enrichment. Of note, the percentage CAR T cells was lower after nucleofection (23%) versus transduction (97%), whereas the percentages huCD24⁺ T cells were similar after nucleofection (78%) and transduction (74%). Responder cells were day 50 PBMCs cocultivated for 5 weekly cycles with CAR T cells. Propagated PBMCs were stimulated with transduced or nucleofected stimulator T cells, and at 2 hours and 24 hours assayed for CD107 and CD137 expression, respectively. The dot plots show CD107/CD137 versus CD8 expression within the CD3 T-cell population and values indicate the percentage positivity. Controls were stimulation by medium alone or NTD T cells. Of note, discrepancy with Figure 3C in the levels of immune recognition of CAIX CAR and huCD24 T cells might be attributed to biologic variation in the batches of responder cells used for both tests and assay sensitivity (cytotoxicity and flow cytometry). (B) Patient 10 developed antivector immune reactivity only. Patient 10 stimulator T cells were prepared and assayed as described for patient 5. Graphs show individual and mean results of 3 independent experiments obtained with PBMCs collected at day 86. The proportion of transgene expression on stimulator T cells was for CAR generated by transduction 94%-98% versus 36%-40% by nucleofection, and for huCD24 generated by transduction 88%-94% versus 56%-68% by nucleofection. TD, transduced stimulator T cells; NF, nucleofected stimulator T cells.