Figure 7. Tumoricidal activities of microglia are dependent on TAM receptor (AXL/MER).

(A) Median survival of IgG (29 days; n = 7) was compared with αCTLA-4 (45 days; n = 5, p = 0.009). Median survival of Ciita sgRNA (27.5 days, n=5) was compared with Ciita sgRNA treated with αCTLA-4 (39 days; n = 9, p = 0.0085). Similarly, comparison of αCTLA-4 treatment of WT 005 with that of Ciita sgRNA 005 (p = 0.876) were done by log-rank analysis. (B) Median survival of IgG (27 days; n = 4) was compared with αCTLA-4 (49.5 days; n = 4, p = 0.00673). Median survival of Ifngr1 sgRNA (21 days, n=9) was compared with Ifngr1 sgRNA treated with αCTLA-4 (21 days; n = 9, p = 0.4679). Similarly, comparison of αCTLA-4 treatment of WT 005 with that of Ifngr1 sgRNA 005 (p = 0.00213) were done by log-rank analysis. (C) tdTomato 005 GSCs cells were treated with IFNγ (10 ng/ml) or Rotenone (100 nM) overnight. Microglia isolated from Cx3cr1^GFP/+ mice were co-cultured with tdTomato 005 GSCs from different conditions in-vitro for 4 hours. tdTomato signal from microglia was measured by flow cytometry. (D) Heatmap showing the level of apoptotic cell clearance associated genes in microglia from bulk RNA-seq in different treatment among IgG vs. αCTLA-4 vs. αCTLA-4+ αCD4. (E) The localization of Iba1⁺, AXL⁺ cells in the brain were analyzed by immunofluorescence confocal imaging. The colocalization of Iba1⁺ and AXL⁺ was indicated as white. The average MFI of AXL was quantified. (F) Rag1^−/− recipient mice were transferred with or without CD4⁺ T cells (1×10⁶) cells on day 0. All Rag1^−/− mice were implanted with 3 × 10⁵ 005 GSCs on day 1. AXL expression on microglia within tumor tissue were analyzed and quantified on day 35. (G) Axl^F/FMertk^F/FCx3cr1^CreERT mice were treated with 150 mg/kg tamoxifen for two doses 48 hours apart. The efficiency of AXL and MER depletion was checked. (H) tdTomato 005 GSCs cells were treated with IFNγ (10 ng/ml) overnight and then incubated with microglia isolated from Axl^−/−Mertk^−/−Cx3cr1^GFP/+or Cx3cr1^GFP/+ mice for 4 hours and the tdTomato signal from microglia was measured. (I) Axl^F/FMertk^F/FCx3cr1^CreERT mice were treated with 150 mg/kg tamoxifen for two doses 48 hours apart 1 week before, and then implanted with 3 × 10⁵ 005 GSCs on day 0 with indicated treatment. The efficacy of depletion of AXL and MER on microglia was checked by flow cytometry (left). Median survival of IgG (27 days; n = 5) was compared with αCTLA-4 (45 days; n = 5, p = 0.019). Median survival of Axl^F/FMertk^F/FCx3cr1^CreERT (29 days, n=8) was compared with Axl^F/FMertk^F/FCx3cr1^CreERT treated with αCTLA-4 (25.5 days; n = 9, p = 0.8502). Similarly, wild-type B6 mice treated with αCTLA-4 was compared with Axl^F/FMertk^F/FCx3cr1^CreERT treated with αCTLA-4 (p = 0.016) by log-rank analysis (right). All results were pooled from or representative of 2–4 experiments with n = 3 mice / group (D), n = 3 mice / group (E), n = 4–6 mice / group (F). Data are expressed as mean ± SEM.