Fig 2: Axl-deficient macrophages trigger a robust NK-cell and T-cell immune response that suppresses leukemia. A and B,Axlf/f and Csf1r-Cre+ Axlf/f mice were challenged with 103 B-ALL cells and treated with either an anti-NK1.1 antibody or a mouse IgG2a isotype control (50 µg/mouse) every 5 days as indicated. Leukemic burden (% GFP+) in the bone marrow and spleen on day 14 is depicted (B). ns, not significant. ***, P < 0.001, unpaired two-tailed Student t test. C, Kaplan–Meier survival analysis of mice of the indicated genotypes challenged with 103 B-ALL cells and treated as in A. Treatments stopped once all anti-NK1.1–treated mice were dead. ns, not significant. **, P < 0.01, log-rank (Mantel–Cox) test. D and E, Same as in A and B using 105MLL–ENL AML cells. Leukemic burden (% Tomato+) on day 25 is depicted. ns, not significant. **, P < 0.01; ***, P < 0.001, unpaired two-tailed Student t test. F, Kaplan–Meier survival analysis of mice of the indicated genotypes challenged with 105MLL–ENL AML cells and treated as in D. Treatments stopped once all anti-NK1.1–treated mice were dead. ns, not significant. **, P < 0.01; ***, P < 0.001, log-rank (Mantel–Cox) test. G, Kaplan-Meier survival analysis of mice of the indicated genotypes challenged with 103 B-ALL cells. Data are pooled from two independent experiments as indicated in the scheme. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, log-rank (Mantel–Cox) test. H, Leukemic burden (% GFP+) in all terminally ill animals that could be analyzed from G. Note that burden from animals found dead cannot be depicted. ns, not significant. *, P < 0.05; **, P < 0.01, unpaired two-tailed Student t test. I, Kaplan–Meier survival curve of mice of the indicated genotypes challenged with 105 MLL–ENL cells. Survival of the reference groups (Axlf/f and Csf1r-Cre+ Axlf/f) is also depicted in Fig. 2I. ns, not significant. **, P < 0.01; ***, P < 0.001, log-rank (Mantel–Cox) test. J, Leukemic burden (% tomato+) in all terminally ill animals that could be analyzed from I. Note that burden from animals found dead cannot be depicted. ns, not significant, unpaired two-tailed Student t test.

Fig 3: Axl-deficient macrophages prevent the establishment of an immune suppressive environment in response to leukemia. A, Nonleukemic (GFP-) spleen leukocytes were FACS purified from control Axlf/f mice and Csf1r-Cre+ Axlf/f mice that were either naïve (Axlf/f n = 2; Csf1r-Cre+ Axlf/f n = 2) or challenged with 103 B-ALL cells (n = 2 Axlf/f + B-ALL; n = 2 Csf1r-Cre+ Axlf/f+ B-ALL) for 8 days and subjected to 10X Genomics scRNA-seq. Data clustering, UMAP visualization of 36,000 individual cells (pooled from all conditions) followed by marker-based cell type annotation identified 10 broad immune subsets across all profiled single cells. B, Dot plot of selected cluster-specific marker genes. C, Relative abundance of identified cell types across conditions. D, Volcano plots showing the DEG (Padj < 0.01 and fold change >1.5) in macrophages comparing Axlf/f and Csf1r-Cre+ Axlf/f under steady-state conditions (left) and upon leukemia challenge (right), with the significant genes (max 10) annotated. E, Real-time PCR expression data in F4/80+ spleen macrophages purified using magnetic beads from naïve WT mice (n = 4) and mice transplanted with 103 B-ALL (WT n = 4; Csf1r-Cre+ Axlf/f n = 4). Data are normalized to a reference gene, Ubc, and are mean ± SEM. *, P <0.05, unpaired two-tailed Student t test. F, Real-time PCR expression data in dendritic cells (DC) isolated by flow cytometry as CD45+GFP-MHC-II+CD11c+ from the spleen of B-ALL challenged Axlf/f (n = 4) and Csf1r-Cre+ Axlf/f mice (n = 3). Data are normalized to a reference gene, Sdha, and are mean ± SEM. *, P < 0.05; ****, P < 0.0001, unpaired two-tailed Student t test. G, Representative gating and flow cytometry based quantification of total classical dendritic cells (DCs: CD45+GFP-MHC-II+CD11c+) as well as subsets: cDC1 (CD8+DCs: MHC-II+CD11c+CD8+CD11b-) and cDC2 (CD11b+DCs: MHC-II+CD11c+CD8-CD11b+) within nonleukemic splenocytes (GFP-CD45+) from B-ALL challenged Axlf/f (n = 6) and Csf1r-Cre+ Axlf/f (n = 4) mice. ns, not significant; ***, P < 0.001, unpaired two-tailed Student t test.

Fig 4: Axl deficient macrophages trigger antileukemic immunity and elicit PD-1 checkpoint blockade. A, Kaplan–Meier survival analysis of WT mice challenged with 103 B-ALL cells and treated with either anti–PD-1 (n = 8) or isotype control (n = 7). B, GFP+ blasts (left) and PD-1+ cells (right) by IHC in the spleen of Csf1r-Cre+ Axlf/f mice that succumbed to B-ALL with a delayed latency of >40 days (Mice depicted in Fig. 2C). C and D,Csf1r-Cre+ Axlf/f from three independent experiments were followed by weekly bleeding to identify mice with late disease recurrence (n = 7). Flow cytometry data depicting PD-1 expression in peripheral blood lymphocytes (CD4 and CD8 T cells, NK cells) and corresponding PD-1 mean fluorescence intensity (MFI) from Csf1r-Cre+ Axlf/f mice showing signs of relapse (detectable GFP+ cells, representative data in G). ****, P < 0.0001, unpaired two-tailed Student t test. E, PD-1 ligand (PD-L1) expression by IHC in bone marrow cells with both stromal and hematopoietic morphology (left), as well as on cytospined B-ALL cells (right). F and G,Csf1r-Cre+ Axlf/f mice with late disease recurrence (n = 7, depicted in C and D) were either left untreated (n = 3) or subjected to 7 cycles of anti–PD-1 treatment (n = 4; 200 µg/mouse every 4 days). Representative FACS plot depicting leukemic burden (GFP+ B220dim) in the peripheral blood of the same mouse before and after one shot of anti–PD-1 treatment. H, Kaplan–Meier survival analysis of mice from F. *, P < 0.05, log-rank (Mantel–Cox) test.

Fig 5: Bemcentinib, a clinical-grade AXL inhibitor, triggers effective antileukemic immunity in B-ALL that depends on IL12, TNFa, and engagement of CD8 T cells. A, Representative phospho-AXL (white) expression and IBA1+ leukemia-associated macrophages (red) by immune fluorescence in frozen bone sections from vehicle and bemcentinib-treated leukemia-bearing mice depicted in B, at final analysis. B, Leukemic burden (% GFP+ B220dim in bone marrow, spleen, and peripheral blood) and spleen pictures of WT mice challenged with 103 B-ALL cells and treated twice daily with either vehicle or bemcentinib at 50 mg/kg. Treatment was initiated on day 4 post–leukemia injection and mice were analyzed on day 11. *, P < 0.05; **, P < 0.01, two-tailed Student t test. C, Same as B, using CD8-deficient mice. ns, not significant, two-tailed Student t test. D, Day 10 leukemic burden (% GFP+ B220dim in peripheral blood) in WT mice challenged with 103 B-ALL cells treated as in B, in the presence or absence of blocking antibodies against IL12 (300 µg/mouse) and TNFa (400 µg/mouse). Blocking antibodies for IL12 and TNFa were administered daily starting from day 4 post–leukemia challenge. Each dot represents an individual mouse and mean value is depicted. ns, not significant, *, P < 0.05, unpaired two-tailed Student t test.