AML-directed T-cell immunotherapies upregulate MHC-II expression on human AML cell lines and primary human AML cells in vitro and in vivo. (A) Human THP-1 and primary AML cells from patient 250167, 329614, 1220117, or 1592619 were treated with vehicle (PBS), 10 ng/mL FLZ, 50 ng/mL IFN-γ, human HLA-mismatched CD3⁺ T cells (E:T of 1:1), or FLZ and human T cells (FLZ + T) for 48 hours. Twenty-five thousand AML cells were plated per well. MHC-II relative median fluorescence intensity (rMFI; A) on the THP-1 and AML cells was measured via multiparametric flow cytometry. (B) THP-1, AML-250167, AML-329614, 1220117, or 1592619 cells were treated with vehicle (PBS), 50 ng/mL IFN-γ, human UTD T cells (UTD; E:T of 0.1:1), or human T cells expressing a CAR targeting CD123 (CART-123; E:T of 0.1:1) for 48 hours. MHC-II rMFI was measured by flow cytometry. (C-J) NSG-S mice were sublethally irradiated with 250 rads and injected with 10⁶ human AML-250167 cells per mouse. After 5.5 weeks, mice were treated with vehicle (PBS), 2 mg/kg FLZ, 1 × 10⁷ human HLA-mismatched CD3⁺ T cells, or FLZ (1 hour after T-cell injection) and human T cells (FLZ + T) for 48 hours. (C) A Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plot depicts scRNA-seq data from 34 793 high-quality human AML and T cells harvested from the bone marrow. (D) Proportions of cells (size of circle) for each single cell cluster were broken down by treatment group. (E) Differentially expressed genes (DEG) in AML_3 cluster relative to all other AML clusters. (F) MSigDB hallmark gene sets enriched from AML_3 DEGs. (G) Plasma levels of IFN-γ. (H-J) MHC-I (H) and MHC-II (I) MFI and the percentage of MHC-II positive (J) human AML cells in the bone marrows of NSG-S mice were determined by flow cytometry. Bars represents means and error bars represent standard errors above and below (when applicable) the mean. P values were calculated using an unpaired, 2-sided Student t test. ∗P < .05; ∗∗P < .001; ∗∗∗P < .0001; ∗∗∗∗P < .00001.