(A) Experimental schema. NSG mice engrafted with control or CD33 KO HSPC were treated with autologous anti-CD33 CAR T cells (CART33), followed by serial retro-orbital bleeding. Mice were euthanized and bone marrow (BM) and spleen were analyzed 4 weeks after CART33 infusion. A subset of mice in each group did not receive CART33. (Control-no CART33: n=4, CD33 KO-no CART33: n=4, Control+CART33: n=15, CD33 KO+CART33: n=15, 2 independent experiments, 2 donors). (B) Numbers of CD14+ monocytes in the peripheral blood of CD33 KO HSPC-engrafted mice after CART33 treatment are similar to control HSPC mice without CART33 treatment (one-way ANOVA) (left). Representative flow cytometry plot (right) shows complete eradication of CD33+ cells in both groups after CART33 treatment, which leads to loss of CD14+ monocytes in the control HSPC-engrafted mice, while CD14+CD33− cells are still detected in CD33 KO HSPC-engrafted mice. Gated on live singlet human CD45+CD19−CD3− cells. (C) CD14+ cells are present in significantly higher numbers in the spleen and bone marrow of CD33 KO HSPC-engrafted mice after CART33 treatment compared to controls (unpaired t-test). (D) Bone marrow human stem cells (CD34+38−) and progenitors (CD34+38+) are significantly higher after CART33 treatment in CD33 KO HSPC mice compared to controls (unpaired t-test). Representative flow cytometry plot showed on right, gated on live singlet human CD45+ lineage-negative cells. (E) Experimental schema. Control or CD33 KO HSPC-engrafted mice were injected with Molm14-GFP/luciferase prior to treatment with CART33 (n=26 mice, 2 independent experiments, 3 donors). (F) Mice show the expected reduction in AML burden as measured by bioluminescent imaging (BLI) after CART33 treatment (unpaired t-test). (G) Co-engraftment of AML does not impair the survival of CD33− negative myeloid cells in vivo after CART33 treatment in the peripheral blood (top; one-way ANOVA), spleen and bone marrow (bottom; unpaired t-test). (H) Peripheral blood CD33 levels decline after CART33 treatment in both cohorts of mice, resulting in undetectable levels of CD14+ cells in the control mice, while CD14+ cells persist in CD33 KO HSPC-engrafted mice (shown is one representative cohort of mice, single donor, n=4/group). (I) Bone marrow human stem and progenitor cells continue to survive CART33-mediated attack even in the setting of coexisting AML (unpaired t-test). All data are represented as means ± SD. ns: not significant (p>0.05); **p<0.01; ***p<0.001; ****p<0.0001.