Figure S1.
Engineering an inhibitor-resistant human CSF1R variant. (A) Example of rendered dot plot derivation from a WT mouse 14 d after receiving a GFP+ microglia transplant. Left image, brain tile scan with magnified inset showing few donor-derived microglia engrafted (GFP/green). Right image, rendered dot plot of engrafted cells overlaid onto DAPI channel. Scale bars, 1,000 µm (brain tile), 50 µm (insets). (B) Quantification of microglia depletion expressed as IBA1+ cell density after 7 d of PLX3397 treatment (25 mg/kg/d, subcutaneous, red bars) relative to saline-treated littermates (black bars). Data represented as mean values ± SEM calculated from three matched sagittal sections from n = 2 independent biological replicates per group. (C) Baseline engraftment of GFP+ microglia, BMDMs, and CIMs intracerebrally transplanted into WT animals on P0, P1, P2, or P3 with or without 2 d of 25 mg/kg PLX3397 pretreatment. CIMs are further described starting in G. All animals were harvested 14 d after transplantation. Data represented as mean values ± SEM calculated from three matched sagittal sections from n = 2–3 independent biological replicates per group. Percent engraftment measured as shown in J. (D) Relative abundance of Ba/F3s transduced with WT-CSF1R (black), G795A-CSF1R (green), or D802V-CSF1R (constitutively active, gray) with or without 50 ng/ml CSF1h, 48 h after transduction. Representative data plotted as mean values ± SEM from two to three technical replicates per group from n = 2 independent experiments. (E) Relative abundance of WT or G7595-transduced Ba/F3s 0–72 h after provision with CSF1h and PLX3397 (247 nM). Data represented as mean values ± SEM from three technical replicates from n = 2 independent biological replicates per group. (F) Data table showing PLX3397 IC50, log IC50, standard error of LogIC50, and R2 of IC50 of Ba/F3s transduced with CSF1R variants. + denotes ligand independent mutant, ++ denotes constitutively active control, # denotes variants unable to sustain growth, and ## denotes catalytically dead control. Values calculated from the average of n = 3 independent replicates. (G) Representative image of CIM engraftment in the Csf1r−/− murine brain. P2 cell injection, P16 harvest. IBA1 (red), DAPI (blue). Scale bars, 1,000 and 50 µm (inset). (H) Brightfield images of WT- and G795A-expressing macrophages derived from Csf1r−/− CIMs, 7 d after estrogen removal and CSF1h treatment. (I) IC50 curve of WT- and G795A-transduced BMDMs from FVB WT mice. Data represented as mean values ± SEM from three technical replicates from n = 3 independent biological replicates per group. (J) Method used to calculate percent engraftment depicting total brain area (pink outline) and engrafted area (yellow outline). Scale bar, 1,000 µm. (K) Top: Experimental paradigm depicting timing of CIM injection, PLX3397 treatment, and endpoint. Bottom: Representative rendering of G795A-CIM distribution (green) 14 d after injection into PLX3397 treated Rag2+/+;Il2r𝛾+/+;CSF1h+/+ host. Scale bar, 1,000 µm. (L) Top: Experimental paradigm depicting timing of BMDM injection, PLX3397 treatment, and endpoint. Bottom: Rendering showing G795A-BMDM distribution, 28 d after injection into PLX3397 treated Rag2+/+;Il2r𝛾+/+;CSF1h+/+ host. Image is from most well engrafted animal. Scale bar, 1,000 µm. (M) Quantification of percent area engrafted by GFP+ donor macrophages. Dots represent biological replicates calculated as mean engraftment ± SEM across three matched sagittal sections. G795A-CIMs on PLX3397, n = 18; WT-CIMs on PLX3397, n = 6; G795A-CIMs untreated (no PLX3397), n = 4; G795A-BMDMs on PLX3397, n = 3; WT-BMDMs on PLX3397, n = 4. For CIMs, one-way ANOVA (P = 0.0009). For BMDMs, two-tailed unpaired t test (P = 0.1152). (N and O) Percent engraftment of whole brain (N) and cortical cell density (O) in PLX3397-treated adult Rag2−/−;Il2r𝛾−/−;CSF1h+/+ mice transplanted with G795A- or WT-CIMs. Dots represent biological replicates calculated as mean engraftment ± SEM across three matched sagittal sections. G795A-CIMs on PLX3397, n = 7; off PLX3397, n = 3; WT-CIMs on PLX3397, n = 5; and G795A untreated (no PLX3397), n = 2. One-way ANOVA (% engraftment: P = 0.0010; cell density: P = 0.0014). (P) Top: Experimental paradigm depicting timing of stereotactic BMDM injection, PLX3397 treatment, and endpoint using 9–12-wk-old Rag2−/−;Il2r𝛾−/−;CSF1h+/+ mice. Bottom: Representative rendering of G795A-BMDM distribution. (Q) Quantification of percent engraftment of G795A- and WT-BMDMs. Dots represent biological replicates calculated as mean engraftment ± SEM across two matched sagittal sections. G795A-BMDMs, n = 4; WT-BMDMs, n = 2. Unpaired two-tailed t test (P = 0.0324). Unless otherwise noted, P values calculated as Tukey’s HSD: #P < 0.12, *P < 0.05, **P < 0.01.

Engineering an inhibitor-resistant human CSF1R variant. (A) Example of rendered dot plot derivation from a WT mouse 14 d after receiving a GFP+ microglia transplant. Left image, brain tile scan with magnified inset showing few donor-derived microglia engrafted (GFP/green). Right image, rendered dot plot of engrafted cells overlaid onto DAPI channel. Scale bars, 1,000 µm (brain tile), 50 µm (insets). (B) Quantification of microglia depletion expressed as IBA1+ cell density after 7 d of PLX3397 treatment (25 mg/kg/d, subcutaneous, red bars) relative to saline-treated littermates (black bars). Data represented as mean values ± SEM calculated from three matched sagittal sections from n = 2 independent biological replicates per group. (C) Baseline engraftment of GFP+ microglia, BMDMs, and CIMs intracerebrally transplanted into WT animals on P0, P1, P2, or P3 with or without 2 d of 25 mg/kg PLX3397 pretreatment. CIMs are further described starting in G. All animals were harvested 14 d after transplantation. Data represented as mean values ± SEM calculated from three matched sagittal sections from n = 2–3 independent biological replicates per group. Percent engraftment measured as shown in J. (D) Relative abundance of Ba/F3s transduced with WT-CSF1R (black), G795A-CSF1R (green), or D802V-CSF1R (constitutively active, gray) with or without 50 ng/ml CSF1h, 48 h after transduction. Representative data plotted as mean values ± SEM from two to three technical replicates per group from n = 2 independent experiments. (E) Relative abundance of WT or G7595-transduced Ba/F3s 0–72 h after provision with CSF1h and PLX3397 (247 nM). Data represented as mean values ± SEM from three technical replicates from n = 2 independent biological replicates per group. (F) Data table showing PLX3397 IC50, log IC50, standard error of LogIC50, and R2 of IC50 of Ba/F3s transduced with CSF1R variants. + denotes ligand independent mutant, ++ denotes constitutively active control, # denotes variants unable to sustain growth, and ## denotes catalytically dead control. Values calculated from the average of n = 3 independent replicates. (G) Representative image of CIM engraftment in the Csf1r−/− murine brain. P2 cell injection, P16 harvest. IBA1 (red), DAPI (blue). Scale bars, 1,000 and 50 µm (inset). (H) Brightfield images of WT- and G795A-expressing macrophages derived from Csf1r−/− CIMs, 7 d after estrogen removal and CSF1h treatment. (I) IC50 curve of WT- and G795A-transduced BMDMs from FVB WT mice. Data represented as mean values ± SEM from three technical replicates from n = 3 independent biological replicates per group. (J) Method used to calculate percent engraftment depicting total brain area (pink outline) and engrafted area (yellow outline). Scale bar, 1,000 µm. (K) Top: Experimental paradigm depicting timing of CIM injection, PLX3397 treatment, and endpoint. Bottom: Representative rendering of G795A-CIM distribution (green) 14 d after injection into PLX3397 treated Rag2+/+;Il2r𝛾+/+;CSF1h+/+ host. Scale bar, 1,000 µm. (L) Top: Experimental paradigm depicting timing of BMDM injection, PLX3397 treatment, and endpoint. Bottom: Rendering showing G795A-BMDM distribution, 28 d after injection into PLX3397 treated Rag2+/+;Il2r𝛾+/+;CSF1h+/+ host. Image is from most well engrafted animal. Scale bar, 1,000 µm. (M) Quantification of percent area engrafted by GFP+ donor macrophages. Dots represent biological replicates calculated as mean engraftment ± SEM across three matched sagittal sections. G795A-CIMs on PLX3397, n = 18; WT-CIMs on PLX3397, n = 6; G795A-CIMs untreated (no PLX3397), n = 4; G795A-BMDMs on PLX3397, n = 3; WT-BMDMs on PLX3397, n = 4. For CIMs, one-way ANOVA (P = 0.0009). For BMDMs, two-tailed unpaired t test (P = 0.1152). (N and O) Percent engraftment of whole brain (N) and cortical cell density (O) in PLX3397-treated adult Rag2−/−;Il2r𝛾−/−;CSF1h+/+ mice transplanted with G795A- or WT-CIMs. Dots represent biological replicates calculated as mean engraftment ± SEM across three matched sagittal sections. G795A-CIMs on PLX3397, n = 7; off PLX3397, n = 3; WT-CIMs on PLX3397, n = 5; and G795A untreated (no PLX3397), n = 2. One-way ANOVA (% engraftment: P = 0.0010; cell density: P = 0.0014). (P) Top: Experimental paradigm depicting timing of stereotactic BMDM injection, PLX3397 treatment, and endpoint using 9–12-wk-old Rag2−/−;Il2r𝛾−/−;CSF1h+/+ mice. Bottom: Representative rendering of G795A-BMDM distribution. (Q) Quantification of percent engraftment of G795A- and WT-BMDMs. Dots represent biological replicates calculated as mean engraftment ± SEM across two matched sagittal sections. G795A-BMDMs, n = 4; WT-BMDMs, n = 2. Unpaired two-tailed t test (P = 0.0324). Unless otherwise noted, P values calculated as Tukey’s HSD: #P < 0.12, *P < 0.05, **P < 0.01.

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