Safety profile of CD44v6-targeted T cells toward hematopoietic cells. (A) CD44v6 messenger RNA (mRNA) expression in primary leukemic blasts, peripheral blood (PB)–derived CD14⁺ monocytes (mono), and CB-derived CD34⁺ cells was analyzed by qPCR. (B) CD44v6 expression on CB HSCs and progenitors; BM HSCs, progenitors, and monocytes; and PB monocytes, T cells, and B cells was analyzed by FACS and expressed as RFI. HSCs and progenitors were identified as follows: HSCs/multipotent progenitors (MPPs), CD34⁺/CD38^–/CD45RA^–; common lymphoid progenitors (CLPs), CD34⁺/CD10⁺; common myeloid progenitors (CMPs), CD34⁺/CD38⁺/CD123⁺/CD45RA^–; granulocyte/monocyte progenitors (GMPs), CD34⁺/CD38⁺/CD123⁺/CD45RA⁺; and myeloid erythroid progenitors (MEPs), CD34⁺/CD38⁺/CD123^–/CD45RA^–. CD44v6 expression on tumor cells from AML and MM patients is shown for comparison. The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2). (C) Human lymph node, brain, liver, and skin sections stained with hematoxylin and eosin were analyzed by immunohistochemistry for expression of CD44v6 and the CD68R macrophage marker. (D) CD44v6.CAR28z⁺ and CTR.CAR28z⁺ T cells were tested in chromium-release assays against circulating monocytes, T cells, and B cells. Specific lysis is shown at different E:T ratios (mean ± SD from n = 4 donors). (E) CB CD34-selected cells were incubated for 4 hours with autologous CD44v6.CAR28z⁺ or CTR.CAR28z⁺ T cells, or medium alone, before being assayed in a standard colony-forming assay (see “Material and methods”). Absolute numbers (a.n.) of erythroid- (E-CFU) and granulocyte/monocyte-colony forming units (GM-CFU) at 14 days (mean ± SD from n = 3 CB units). (F) CD44v6.CAR28z⁺ or CTR.CAR28z⁺ T cells from healthy donors (n = 2) were cultured with whole BM mononuclear fractions from MM patients (n = 6). After 4 days, residual malignant plasma cells (CD38⁺/CD45^–), HSCs (CD34⁺/CD38^–), and hematopoietic progenitor cells (HPCs, CD34⁺/CD38⁺) were counted and analyzed by FACS. Left: results from a representative experiment. Right: antimyeloma effects by CD44v6.CAR28z⁺ T cells measured as the elimination index for each combination (see “Material and methods”). Results from a 1-way ANOVA are shown when statistically significant (***P < .001). (G) After irradiation, NSG-3GS mice were infused with CB CD34-selected cells and, after 4 weeks, treated with autologous CD44v6.CAR28z⁺ T cells or CD19.CAR28z⁺ T cells (n = 4 mice/group) or saline (n = 3). Absolute numbers of CD19⁺ cells (left panel) and CD14⁺ cells (right panel) in the peripheral blood of mice at different time points after T-cell infusion. Results from a 1-way ANOVA are shown when statistically significant (*P < .05; **P < .01).