Effects of in vivo treatment with miRNA mimics on CLL cells engrafted in NSG mice. Mice were injected with 50 × 10⁶ CLL cells prestimulated with autologous activated T cells. After 4 to 6 weeks, blood samples were evaluated for the presence of circulating leukemic cells by flow cytometry, and after CLL cell engraftment was achieved, the mice were treated with 3 doses of miR-146b-5p or miR CTR mimic. Mice were sacrificed after 3 days from the last inoculum, and cell suspensions from the spleen, bone marrow (BM), and peripheral blood (PBL) were analyzed by flow cytometry for the percentage of CD19⁺CD5⁺ CLL cells or CD19⁻CD5⁺ T cells over the total of human CD45⁺ cells. (A) Flow cytometry analysis of 2 representative mice injected with CLL GE1-PM129 and treated with miR CTR mimic (NSG 13) or miR-146b-5p (NSG 5). (B) Summary of the flow cytometry analyses of 14 mice injected with CLL cells from 2 different cases (GE1-PM129 and GE1-RO148) and treated as in (A). Percentages of CD19⁺CD5⁺ CLL cells or (C) CD19⁻CD5⁺ T cells are shown. (D) Apoptotic neoplastic cells and T cells in the spleen from a miR-146b-5p (NSG 16) and a miR CTR (NSG 13) mimic-treated mouse. Apoptotic cells were detected by flow cytometry as annexin-V–positive cells by gating CD45⁺CD19⁺CD5⁺ CLL cells (solid red histogram profiles) or CD45⁺CD19⁻CD5⁺ T cells (solid blue histogram profiles). (E) Summary of the flow cytometry tests carried out in 14 mice treated as in (D). Percentages of apoptotic CLL and T cells are expressed as mean ± SD. (F) High-power magnification (200×) images of longitudinal sections of paraffin-embedded mouse spleen stained with αCD20-Ab or αCD3-Ab showing the infiltrating foci present in mice treated with miR CTR (NSG 13) or miR-146b-5p mimics (NSG 16).