Persistence of NY-ESO-1 SPEAR T cells in the peripheral blood and bone marrow. (A) Persistence of NY-ESO-1 SPEAR T cells in PBMC through 1 year after T-cell infusion measured by quantitative polymerase chain reaction methodology is shown for responders (left) and nonresponders (right). Note: Responder/nonresponder status based on BOR up to year 1. (Assumptions: average of 1 vector copy per cell based on transduction efficiency; 1 μg DNA = ∼158 000 cells. Lower limit of quantification of the assay was established at 50 copies/μg DNA at the University of Pennsylvania, and 100 copies/μg DNA at Cambridge Biomedical.) (B) Persistence of NY-ESO-1 SPEAR T-cells in PBMC through 100 days after T-cell infusion was confirmed via flow cytometry measurements. Box plot shows the mean, upper, and lower quartiles. (C) Persistence of NY-ESO-1 SPEAR T cells was compared in PBMC and bone marrow (BM) from patients with paired samples, and is shown as a percentage of the total CD4⁺ (or CD8⁺) T cells. Box plot shows the mean, upper, and lower quartiles. (D) NY-ESO-1 SPEAR T-cell proliferation in the BM and PBMC was demonstrated by Ki-67 staining. Box plot shows the mean, upper, and lower quartiles. Day 21 was selected because of patient numbers (evaluable BM samples, n = 8) and robust data at this time. Note: the pentamer is less efficient for CD4⁺ T-cell binding and, therefore, may underestimate this compartment.