Siglec-6 is expressed by AML cell lines and Siglec-6 CAR T cells recognize and eliminate AML cells in vitro. (A) Flow cytometric analysis of Siglec-6 expression on AML cell lines (U937, MV4;11, MOLM13, and Kasumi-1). Histograms show staining with anti-Siglec-6 mAb (red) and isotype control antibody (blue). Inset numbers indicate the NMFI calculated by dividing MFI obtained after staining with anti-Siglec-6 mAb by MFI of isotype control. (B) Detection of Siglec-6 expression on AML cell lines using dSTORM super-resolution microscopy. Each representative image depicts Siglec-6 molecules on the basal membrane of a cell. Scale bars represent 5 µm. (C) Real-time qPCR was performed to assess Siglec-6 messenger RNA (mRNA) transcript levels in AML cell lines. Data are normalized to MOLM-13 Siglec-6 mRNA transcript values. (D) Quantification of Siglec-6 expression on AML cell lines by dSTORM super-resolution microscopy. Each point represents a cell. (C-D) Data are representative of 3 independent experiments. (E) Specific cytolytic activity of CD8⁺ Siglec-6_28z CAR, Siglec-6_BBz CAR, CD19_BBz CAR, and UTD T cells against AML cell lines in a luminescence-based assay (4 hours of coculture). The assay was performed in triplicate wells with 5000 target cells per well. Data are presented as mean ± standard deviation (SD). (F) Enzyme-linked immunosorbent assay (ELISA) was performed to detect interferon-γ (IFN-γ) and interleukin-2 (IL-2) in supernatant obtained after 24-hour coculture of CD8⁺ Siglec-6_28z CAR, Siglec-6_BBz CAR, or UTD T cells with target cells. T cells and target cells were seeded at a 2:1 E:T ratio in triplicate wells. Data are represented as mean concentration ± SD. (G) Proliferation of CD8⁺ Siglec-6_28z CAR and Siglec-6_BBz CAR T cells examined by carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution after 72 hours of coculture with target cells. The assay was performed in triplicate wells at a (2:1) E:T ratio. Histograms show proliferation of live (7-amino-actinomycin D [7-AAD^–]) T cells. No exogenous cytokines were added. Data shown in panels E-G are representative of results obtained with CAR and control T-cell lines prepared from at least 5 healthy donors. (H) Correlation between specific lysis by CD8⁺ Siglec-6_BBz CAR T cells (after 4-hour coculture; 2.5:1 E:T ratio) and Siglec-6 normalized expression on AML cell lines. Simple linear correlation was calculated (R² = 0.91; P = .0009). test **P < .01; ***P < .001, Student t test. ns, not significant.