Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 1. Aberrant localization of FLT3 ITD in intracellular membranes. (A) The human leukemia cell lines RS4-11 and MV4-11 harboring endogenous wild-type FLT3 or FLT3 ITD, respectively, were subjected to immunostaining with anti-FLT3 antibodies (red). Plasma membranes were decorated with Alexa-Fluor-488-labeled wheat germ agglutinin (WGA, green). Nuclei were stained with HOECHST 33347 (blue). Scale bars: 5 mm. (B) Quantitative analysis of FLT3 distribution, by flow cytometry. FLT3 was detected using antibodies (anti-CD135) recognizing a surface epitope. Cells were stained either intact, or after permeabilization to reveal the total FLT3 amounts, as indicated. The relative amount of surface FLT3 in the two cell lines is also given.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Immunostaining, Clinical Proteomics, Labeling, Staining, Flow Cytometry

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 2. FL stimulation of wild-type FLT3-expressing myeloid cells causes K-Ras and N-Ras activation. (A,B) Wild-type murine FLT3 (mFLT3)- expressing 32D cells (A) and human RS4-11 cells (B) endogenously expressing wild-type FLT3 were treated with FL (20 ng/ml) or were left untreated. At the indicated time points, cells were lysed and subjected to Ras– GTP pulldown (PD). The activation status of Ras, FLT3 and ERK was sequentially determined in the same lysates, as described in Materials and Methods. The same membrane sections were reprobed to detect the different Ras isoforms and to detect total proteins with pan-specific antibodies. Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of panels) are indicated. Four (A) or two (B) independent experiments, respectively, yielded consistent results. pErk1/2, anti-phospho-p44/42 MAPK; pTyr591, anti-phospho-FLT3Y591. Note that FLT3 gives rise to two species, a 130 kDa immature form, and a 150 kDa complex glycosylated form (Schmidt-Arras et al., 2005). Only the latter is available at the cell surface for the ligand and autophosphorylates in response to ligand stimulation. Owing to high expression levels of mFLT3 in the 32D cell line employed for the biochemical experiments (Grundler et al., 2005), relatively large amounts of immature 130 kDa FLT3 are detectable in these cells, also showing some basal autophosphorylation.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Activation Assay, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 3. FL stimulation of wild-type FLT3-expressing myeloid cells promotes K-Ras and N-Ras activation at the plasma membrane. RS4-11 cells expressing endogenous wild-type FLT3 were co-transfected with constructs encoding mCherry–K-Ras (A) or mCherry–N-Ras (B) (both shown in red), along with E3-R3(A/D) (green, RBD), a trivalent fluorescent reporter probe for Ras–GTP. Cells were deprived of serum for 2 hours, challenged with FL (100 ng/ml), and analyzed by confocal laser-scanning microscopy. Of at least 20 monitored cells each, ,40% (A) and ,70% (B) showed FL- dependent redistribution of E3-R3(A/D) to the plasma membrane, indicating Ras activation at that site, as shown in these examples. Scale bars: 10 mm. Original magnification 636. In A, a larger area is shown to depict several cells with similar reactions. Note that some of the probe accumulates in the nucleus. The reason for this is not known, but it is unrelated to Ras activation (Augsten et al., 2006).

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Activation Assay, Clinical Proteomics, Membrane, Transfection, Construct, Confocal Laser Scanning Microscopy

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 4. Constitutive K-Ras activation in FLT3-ITD-expressing myeloid cells depends on FLT3 kinase activity. Murine FLT3-ITD-expressing 32D cells (A) or human MV4-11 cells endogenously expressing FLT3 ITD (B) were serum starved for 2 hours, then treated with the selective FLT3 inhibitor cpd.102 (1 mM) for 1 hour or left untreated (cpd.102 2, washout 2). Then cells were washed twice with medium again containing cpd.102 (cpd. 102 +, washout 2) or vehicle. At the indicated time points after washing and further incubation, cells were lysed and subjected to a Ras–GTP pulldown. The activation status of Ras, and ERK was sequentially determined in the same lysates, as described in the Materials and Methods. FLT3 activation was assessed in separate experiments under identical conditions. The same membrane sections were reprobed to detect the different Ras isoforms and to detect total proteins with pan-specific antibodies. Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of panels) are indicated. pERK1/2 denotes anti-phospho-p44/42 MAPK; pTyr591, anti-phospho-FLT3 tyrosine 591. Six (A) or three (B) independent experiments, yielded consistent results. (C) Quantification of Ras-GTP levels in 32D mFLT3 ITD cells. Five blots for K-Ras and four blots for N-Ras were quantified by densitometry, and intensities of Ras-GTP after inhibitor cpd.102 treatment were normalized to levels without treatment.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Activation Assay, Expressing, Activity Assay, Incubation, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 5. FLT3 ITD causes constitutive K-Ras activation at the plasma membrane. 32D cells expressing human FLT3 ITD, or human MV4-11 cells endogenously expressing FLT3 ITD (as indicated) were co-transfected with plasmids encoding either mCherry– K-Ras or –N-Ras (red) together with the E3-R3(A/D) reporter for Ras–GTP (green, RBD). Cells were deprived of serum for 2 hours, treated with the selective FLT3 inhibitor cpd.102 or left untreated, and subjected to image analysis by confocal laser scanning microscopy. (A) Representative images of FLT3-ITD- expressing 32D cells co-transfected with the mCherry– K-Ras-encoding plasmid and E3-R3(A/D), and treated with the selective FLT3 inhibitor cpd.102 or left untreated, as indicated. (B) Corresponding quantitative comparisons of cpd.102-treated and non-treated cells. (C) A 32D cell line harboring an ITD version that is anchored to the endoplasmic reticulum by a C-terminal tag (FLT3 ITD R3) was also analyzed. (D,E) Representative images of (D) MV4-11 cells co- transfected with mCherry–K-Ras-encoding plasmid and E3-R3 and (E) corresponding quantitative comparisons of cpd.102-treated and non-treated cells. (F,H) Representative images of FLT3-ITD-expressing 32D cells (F) or MV4-11 cells (H) co-transfected with the mCherry–N-Ras-encoding plasmid and E3-R3 (A/D), and treated with the selective FLT3 inhibitor cpd.102 or left untreated, as indicated. (G,I) Corresponding quantitative analysis of the effect of inhibitor treatment on N-Ras activation in intracellular compact structures (‘spots’). (J,K) Quantitative analysis of Ras isoform activation in different compartments in 32D cells expressing FLT3 ITD (J) or MV4-11 cells (K) transfected with either mCherry–K-Ras- or mCherry–N-Ras-encoding plasmids and E3-R3(A/D). PM, plasma membrane. For all quantifications and statistic analyses, 60 cells from three independent experiments were analyzed for each condition. Scale bars: 10 mm. Original magnification 636. Note that some of the probe accumulates in the nucleus. The reason for this is not known, but it is unrelated to Ras activation (Augsten et al., 2006). *P,0.05; **P,0.01; n.s., not significant.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Expressing, Transfection, Confocal Laser Scanning Microscopy, Plasmid Preparation

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 6. Inhibition of FLT3-ITD-driven cell proliferation and induction of apoptosis by interference with Ras activation at the plasma membrane. (A) Dn K-RasS17N inhibits FLT3-ITD-dependent cell proliferation. MV4-11 cells were transduced with lentiviral particles driving expression of EYFP-tagged wild-type (WT) K-Ras, dn K- RasS17N, or the yellow fluorescent control protein Venus, as indicated. The fraction of cells in S phase was determined by DNA labeling using a Click-iT DNA synthesis kit after 72 hours. Alexa Fluor 647 labeling was scored for the EYFP/Venus positive cells (n54, means 6 s.e.m.; *P,0.05, **P,0.01, t-test). (B,C) Apoptosis induction by dn K- RasS17N. MV4-11 cells transduced with the indicated expression constructs were subjected to flow cytometric apoptosis assays using the annexin-V PE method. (B) Example of analysis. (C) Quantification of multiple experiments. The numbers represent the fraction of yellow fluorescent cells that were positive for PE-labeled annexin-V (n54, means 6 s.e.m.; *P,0.05, **P,0.01, t-test).

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Inhibition, Activation Assay, Clinical Proteomics, Membrane, Transduction, Expressing, Control, DNA Labeling, DNA Synthesis, Labeling, Construct

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 1. Aberrant localization of FLT3 ITD in intracellular membranes. (A) The human leukemia cell lines RS4-11 and MV4-11 harboring endogenous wild-type FLT3 or FLT3 ITD, respectively, were subjected to immunostaining with anti-FLT3 antibodies (red). Plasma membranes were decorated with Alexa-Fluor-488-labeled wheat germ agglutinin (WGA, green). Nuclei were stained with HOECHST 33347 (blue). Scale bars: 5 mm. (B) Quantitative analysis of FLT3 distribution, by flow cytometry. FLT3 was detected using antibodies (anti-CD135) recognizing a surface epitope. Cells were stained either intact, or after permeabilization to reveal the total FLT3 amounts, as indicated. The relative amount of surface FLT3 in the two cell lines is also given.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Immunostaining, Clinical Proteomics, Labeling, Staining, Flow Cytometry

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 2. FL stimulation of wild-type FLT3-expressing myeloid cells causes K-Ras and N-Ras activation. (A,B) Wild-type murine FLT3 (mFLT3)- expressing 32D cells (A) and human RS4-11 cells (B) endogenously expressing wild-type FLT3 were treated with FL (20 ng/ml) or were left untreated. At the indicated time points, cells were lysed and subjected to Ras– GTP pulldown (PD). The activation status of Ras, FLT3 and ERK was sequentially determined in the same lysates, as described in Materials and Methods. The same membrane sections were reprobed to detect the different Ras isoforms and to detect total proteins with pan-specific antibodies. Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of panels) are indicated. Four (A) or two (B) independent experiments, respectively, yielded consistent results. pErk1/2, anti-phospho-p44/42 MAPK; pTyr591, anti-phospho-FLT3Y591. Note that FLT3 gives rise to two species, a 130 kDa immature form, and a 150 kDa complex glycosylated form (Schmidt-Arras et al., 2005). Only the latter is available at the cell surface for the ligand and autophosphorylates in response to ligand stimulation. Owing to high expression levels of mFLT3 in the 32D cell line employed for the biochemical experiments (Grundler et al., 2005), relatively large amounts of immature 130 kDa FLT3 are detectable in these cells, also showing some basal autophosphorylation.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Activation Assay, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 3. FL stimulation of wild-type FLT3-expressing myeloid cells promotes K-Ras and N-Ras activation at the plasma membrane. RS4-11 cells expressing endogenous wild-type FLT3 were co-transfected with constructs encoding mCherry–K-Ras (A) or mCherry–N-Ras (B) (both shown in red), along with E3-R3(A/D) (green, RBD), a trivalent fluorescent reporter probe for Ras–GTP. Cells were deprived of serum for 2 hours, challenged with FL (100 ng/ml), and analyzed by confocal laser-scanning microscopy. Of at least 20 monitored cells each, ,40% (A) and ,70% (B) showed FL- dependent redistribution of E3-R3(A/D) to the plasma membrane, indicating Ras activation at that site, as shown in these examples. Scale bars: 10 mm. Original magnification 636. In A, a larger area is shown to depict several cells with similar reactions. Note that some of the probe accumulates in the nucleus. The reason for this is not known, but it is unrelated to Ras activation (Augsten et al., 2006).

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Activation Assay, Clinical Proteomics, Membrane, Transfection, Construct, Confocal Laser Scanning Microscopy

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 4. Constitutive K-Ras activation in FLT3-ITD-expressing myeloid cells depends on FLT3 kinase activity. Murine FLT3-ITD-expressing 32D cells (A) or human MV4-11 cells endogenously expressing FLT3 ITD (B) were serum starved for 2 hours, then treated with the selective FLT3 inhibitor cpd.102 (1 mM) for 1 hour or left untreated (cpd.102 2, washout 2). Then cells were washed twice with medium again containing cpd.102 (cpd. 102 +, washout 2) or vehicle. At the indicated time points after washing and further incubation, cells were lysed and subjected to a Ras–GTP pulldown. The activation status of Ras, and ERK was sequentially determined in the same lysates, as described in the Materials and Methods. FLT3 activation was assessed in separate experiments under identical conditions. The same membrane sections were reprobed to detect the different Ras isoforms and to detect total proteins with pan-specific antibodies. Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of panels) are indicated. pERK1/2 denotes anti-phospho-p44/42 MAPK; pTyr591, anti-phospho-FLT3 tyrosine 591. Six (A) or three (B) independent experiments, yielded consistent results. (C) Quantification of Ras-GTP levels in 32D mFLT3 ITD cells. Five blots for K-Ras and four blots for N-Ras were quantified by densitometry, and intensities of Ras-GTP after inhibitor cpd.102 treatment were normalized to levels without treatment.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Activation Assay, Expressing, Activity Assay, Incubation, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 5. FLT3 ITD causes constitutive K-Ras activation at the plasma membrane. 32D cells expressing human FLT3 ITD, or human MV4-11 cells endogenously expressing FLT3 ITD (as indicated) were co-transfected with plasmids encoding either mCherry– K-Ras or –N-Ras (red) together with the E3-R3(A/D) reporter for Ras–GTP (green, RBD). Cells were deprived of serum for 2 hours, treated with the selective FLT3 inhibitor cpd.102 or left untreated, and subjected to image analysis by confocal laser scanning microscopy. (A) Representative images of FLT3-ITD- expressing 32D cells co-transfected with the mCherry– K-Ras-encoding plasmid and E3-R3(A/D), and treated with the selective FLT3 inhibitor cpd.102 or left untreated, as indicated. (B) Corresponding quantitative comparisons of cpd.102-treated and non-treated cells. (C) A 32D cell line harboring an ITD version that is anchored to the endoplasmic reticulum by a C-terminal tag (FLT3 ITD R3) was also analyzed. (D,E) Representative images of (D) MV4-11 cells co- transfected with mCherry–K-Ras-encoding plasmid and E3-R3 and (E) corresponding quantitative comparisons of cpd.102-treated and non-treated cells. (F,H) Representative images of FLT3-ITD-expressing 32D cells (F) or MV4-11 cells (H) co-transfected with the mCherry–N-Ras-encoding plasmid and E3-R3 (A/D), and treated with the selective FLT3 inhibitor cpd.102 or left untreated, as indicated. (G,I) Corresponding quantitative analysis of the effect of inhibitor treatment on N-Ras activation in intracellular compact structures (‘spots’). (J,K) Quantitative analysis of Ras isoform activation in different compartments in 32D cells expressing FLT3 ITD (J) or MV4-11 cells (K) transfected with either mCherry–K-Ras- or mCherry–N-Ras-encoding plasmids and E3-R3(A/D). PM, plasma membrane. For all quantifications and statistic analyses, 60 cells from three independent experiments were analyzed for each condition. Scale bars: 10 mm. Original magnification 636. Note that some of the probe accumulates in the nucleus. The reason for this is not known, but it is unrelated to Ras activation (Augsten et al., 2006). *P,0.05; **P,0.01; n.s., not significant.

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Expressing, Transfection, Confocal Laser Scanning Microscopy, Plasmid Preparation

Journal: Journal of cell science

Article Title: Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

doi: 10.1242/jcs.131789

Figure Lengend Snippet: Fig. 6. Inhibition of FLT3-ITD-driven cell proliferation and induction of apoptosis by interference with Ras activation at the plasma membrane. (A) Dn K-RasS17N inhibits FLT3-ITD-dependent cell proliferation. MV4-11 cells were transduced with lentiviral particles driving expression of EYFP-tagged wild-type (WT) K-Ras, dn K- RasS17N, or the yellow fluorescent control protein Venus, as indicated. The fraction of cells in S phase was determined by DNA labeling using a Click-iT DNA synthesis kit after 72 hours. Alexa Fluor 647 labeling was scored for the EYFP/Venus positive cells (n54, means 6 s.e.m.; *P,0.05, **P,0.01, t-test). (B,C) Apoptosis induction by dn K- RasS17N. MV4-11 cells transduced with the indicated expression constructs were subjected to flow cytometric apoptosis assays using the annexin-V PE method. (B) Example of analysis. (C) Quantification of multiple experiments. The numbers represent the fraction of yellow fluorescent cells that were positive for PE-labeled annexin-V (n54, means 6 s.e.m.; *P,0.05, **P,0.01, t-test).

Article Snippet: Polyclonal goat anti-FLT3 antibody used for immunoprecipitation and immunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3 (MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Inhibition, Activation Assay, Clinical Proteomics, Membrane, Transduction, Expressing, Control, DNA Labeling, DNA Synthesis, Labeling, Construct

Journal: Cell reports

Article Title: Precision off-the-shelf natural killer cell therapies for oncology with logic-gated gene circuits.

doi: 10.1016/j.celrep.2024.114145

Figure Lengend Snippet: Figure 5. Combining OR and NOT gates to protect primary healthy human hematopoietic stem cells (A) Applying NOT gate architecture to the OR-gated CAR yields an OR-NOT gate circuit that targets multiple AML subpopulations while protecting HSCs. Three- dimensional antigen space reveals how 3-input logic gating is required to achieve this clinical goal by targeting cells defined by specific combinations of the three antigens. (B) Construct design incorporating OR gate aCAR, aEMCN iCAR, and a modified form of IL-15 (to enhance NK cell persistence) into a single tricistronic payload. (C) OR gate aCAR and iCAR from construct design in (B) were co-expressed at high levels. Gate was set based on an untransduced NK cell control. (D) Killing of blasts and LSCs from multiple AML patient samples by OR-NOT gate NK cells (green) is enhanced compared to untransduced NK cells (black). (E) OR-NOT gate CAR-NK cells (medium green) reduce killing of EMCN+ HSCs from freshly thawed primary human CD34-enriched bone marrow cells, compared to OR gate CAR-NK cells without the NOT gate (light green). No statistically significant reduction of killing occurs in response to SEM leukemia cells. Values represent the mean of three technical replicates, and error bars represent ±SE of mean. Welch’s test was used to discern significant differences (**p < 0.01).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data Affymetrix Human Genome U133 Plus 2.0 Array datasets (various) Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) GSE13159, GSE15434, GSE17054, GSE24006, GSE28490, GSE28491, GSE42519, GSE49910, GSE63270, GSE6891, GSE93777 Human (NCBITaxon:9606) ‘‘cell surface’’ proteins Gene Ontology Resource (https://geneontology.org) GO:0009986 Human (NCBITaxon:9606) ‘‘membrane’’ proteins Gene Ontology Resource (https://geneontology.org) GO:0016020 Antibody-based annotations of protein localization Human Protein Atlas (https:// www.proteinatlas.org/) N/A AML bulk RNAseq data https://www.cancer.gov/ccg/research/ genome-sequencing/tcga N/A The gene and gene product information of KLRG1 Uniprot (https://www.uniprot.org/) Q96E93 The gene and gene product information of BTLA Uniprot (https://www.uniprot.org/) Q7Z6A9 The gene and gene product information of KIR3DL1 Uniprot (https://www.uniprot.org/) P43629 The gene and gene product information of NKG2A Uniprot (https://www.uniprot.org/) P26715 The gene and gene product information of SIGLEC-2 Uniprot (https://www.uniprot.org/) P20273 The gene and gene product information of SIGLEC-10 Uniprot (https://www.uniprot.org/) Q96LC7 The gene and gene product information of LIR-2 Uniprot (https://www.uniprot.org/) Q8N423 The gene and gene product information of LIR-3 Uniprot (https://www.uniprot.org/) O75022 The gene and gene product information of LAIR1 Uniprot (https://www.uniprot.org/) Q6GTX8 The gene and gene product information of KIR2DL1 Uniprot (https://www.uniprot.org/) P43626 The gene and gene product information of LIR1 Uniprot (https://www.uniprot.org/) Q8NHL6 The gene and gene product information of CD33 Uniprot (https://www.uniprot.org/) P20138 The gene and gene product information of FLT3 Uniprot (https://www.uniprot.org/) P36888 The gene and gene product information of EMCN Uniprot (https://www.uniprot.org/) Q9ULC0 Experimental models: Cell lines Human: SEM (ALL, female) DSMZ ACC 546 Human: MOLM13 (AML, male) AddexBio C0003003 Human: MV4-11 (AML/ALL, male) ATCC CRL-9591 Human: Lenti-X 293T (transformed, female) Takara 632180 Human: GP2-293 (transformed, female) Takara 631458 Experimental models: Organisms/strains NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ Jackson Laboratories 030890 Recombinant DNA Gene circuit constructs This paper N/A Antigen expression constructs This paper N/A Software and algorithms Prism GraphPad https://www.graphpad.com/ scientific-software/prism/

Techniques: Construct, Control