congenic recipient mice  (Charles River Laboratories)

Journal: Blood

Article Title: Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation

doi: 10.1182/blood.2021012519

Figure Lengend Snippet: Asxl1 −/− co-operates with Nras G12D/+ to promote CMML progression and AML transformation. (A) The absolute numbers of monocytes in peripheral blood of control ( Vav-Cre ), Asxl1 −/− , Nras, and NA mice were monitored regularly. Monocytosis (red dash line) is defined as threefold over the average number of control monocytes. (B) Kaplan-Meier survival curves were plotted against days after birth. P values were determined using the log-rank test. (C-I) Moribund Nras and NA mice and age-matched control mice were analyzed. (C-F) Quantification of (C) spleen weight, (D) numbers of WBCs, (E) numbers of red blood cells (RBCs), and platelets (PLT), and (F) frequencies and numbers of monocytes in peripheral blood. Green triangles: NA mice with CMML; purple triangles, NA mice with AML. Red dashed line (F) indicates monocytosis as described above. (G) Quantification of disease incidence (χ 2 analysis). (H) Representative spleen hematoxylin and eosin–stained sections from Control, Nras, and NA mice. Black scale bar, 500 μm; red scale bar, 20 μm. (I) Mac1 and c-Kit analysis of bone marrow and spleen cells from control and NA mice with CMML-like or AML-like disease. (J) Malignant cells from moribund NA mice with CMML-like or AML-like disease were transplanted into sublethally irradiated recipient mice (CD45.1 + ). Kaplan-Meier survival curves were plotted against days after transplantation. P values were determined using the log-rank test. Data are presented as mean + SD. * P < .05; ** P < .01; *** P < .001.

Article Snippet: 10,18,19 CD45.1 + congenic C57BL/6 recipient mice were purchased from The Jackson Laboratory (stock #002014).

Techniques: Transformation Assay, Control, Staining, Irradiation, Transplantation Assay

Journal: Blood

Article Title: Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation

doi: 10.1182/blood.2021012519

Figure Lengend Snippet: AP-1 TF Jun regulates PD-L1 and CD86 expression and contributes to the suppressive immune microenvironment in NA-AML mice. (A) Quantification of bone marrow Mac1 + c-Kit + cells expressing immune checkpoint ligands PD-L1/PD-L2, CD155, and CD80/CD86 in moribund NA mice with CMML (NA-CMML) or AML (NA-AML) and age-matched control ( Vav-Cre ), Asxl1 −/− , and Nras mice. (B) Enrichment of Jun binding at PD-L1 and CD86 enhancer/promoter regions was analyzed using chromatin immunoprecipitation (ChIP)-qPCR in bone marrow cells of moribund NA-AML and age-matched controls, Asxl1 −/− , and Nras mice. Immunoglobulin G (IgG) was used as a negative control. The enrichment was normalized to 5% input (n = 3). (C-D) NA-AML cells were cultured in vitro and infected with pGIPZ lentiviral vectors encoding short hairpin Control (shControl) or shJun. At 72 hours after infection, (C) mRNA levels of Jun, PD-L1, and CD86 were analyzed in sorted c-Kit + GFP + cells using qRT-PCR, and (D) surface expression of PD-L1 and CD86 were analyzed in Lin – c-Kit + GFP + cells using flow cytometry. (E) NA-AML cells were cultured in vitro and treated with dimethyl sulfoxide (DMSO) or 4 μΜ SR11302 (AP-1 inhibitor) for 5 days. Surface expression of PD-L1 and CD86 were analyzed in Lin – c-Kit + cells using flow cytometry. (F-I) Sublethally irradiated mice were transplanted with 2.5 × 10 5 bone marrow cells from moribund NA-AML mice or age-matched control mice. (F) In NA-AML recipients, donor-derived (CD45.2 + ) cells were exclusively Mac1 + leukemia cells, whereas T cells (Thy1.2 + ) were derived from the host (CD45.1 + ). (G-H) Quantification of (G) bone marrow and (H) spleen CD4 and CD8 T cells expressing immune checkpoint receptors PD-1, TIGIT, and CTLA4. (I) Quantification of exhausted T cells (PD-1 + TIGIT + LAG3 + ) in spleen CD4 and CD8 T cells in NA-AML and control recipients. Data are presented as mean + SD. * P < .05; ** P < .01; *** P < .001. SSC, side scatter.

Article Snippet: 10,18,19 CD45.1 + congenic C57BL/6 recipient mice were purchased from The Jackson Laboratory (stock #002014).

Techniques: Expressing, Control, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Negative Control, Cell Culture, In Vitro, Infection, Quantitative RT-PCR, Flow Cytometry, Irradiation, Derivative Assay

Journal: Blood

Article Title: Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation

doi: 10.1182/blood.2021012519

Figure Lengend Snippet: Combined inhibition of MEK and BET downregulates inhibitory immune checkpoint pathways and AML transformation gene signature and prolongs the survival of Nras G12D/+ ; Asxl1 −/− AML mice. Sublethally irradiated mice (CD45.1 + ) were transplanted with 2.5 × 10 5 bone marrow cells from moribund primary NA-AML recipients. Two weeks after transplantation, mice were randomly separated into (A-G) 2 groups and treated with vehicle (Veh) or a combination (Combo) of trametinib and GSK525762 once per day or (H) 4 groups treated with vehicle, trametinib (Tra), and/or GSK525762 (GSK) once per day. (A-F) Recipients were euthanized at ∼5 weeks posttransplant. Quantification of (A) spleen and liver weights; (B) bone marrow leukemia cells (CD45.2 + ) expressing PD-L1, CD86, CD80, PD-L2, or CD155 using flow cytometry; (C) bone marrow CD4 and CD8 T cells expressing PD-1, TIGIT, or CTLA-4; and (D) exhausted T cells (PD-1 + TIGIT + LAG3 + ) in spleen CD4 and CD8 T cells. (E) T-cell cytotoxicity assay. CD45.2 + leukemia cells (Target) were sorted from moribund NA-AML recipients treated with vehicle. The same number of Target cells were cocultured with splenic CD8 T cells (Effector), which were sorted from recipients treated with vehicle (Veh) or combination (Com) of drugs at different ratios. The live leukemia cells were quantified 48 hours after coculture. (F) Quantification of AP-1 complex genes ( Fos , Fosb , Jun , Junb , Jund , and Atf3 ) and Flt3 mRNA levels using qRT-PCR in sorted leukemia cells (CD45.2 + ). (G-H) Kaplan-Meier survival curves were plotted against days after transplantation using NA-AML cells from different donors. (I) 1.78 million bone marrow mononuclear cells from a RAS and ASXL1 double-mutant CMML patient were transplanted into irradiated NRGS mice (n = 4 mice per group). Five days after transplantation, mice were randomly separated into 2 groups and treated with vehicle or trametinib plus GSK525762 until moribund stage as described in “Materials and methods.” Kaplan-Meier survival curves were plotted against days after transplantation. (G-I) Data are presented as mean + SD. P values were determined using the log-rank test. * P < .05; ** P < .01; *** P < .001.

Article Snippet: 10,18,19 CD45.1 + congenic C57BL/6 recipient mice were purchased from The Jackson Laboratory (stock #002014).

Techniques: Inhibition, Transformation Assay, Irradiation, Transplantation Assay, Expressing, Flow Cytometry, Cytotoxicity Assay, Quantitative RT-PCR, Mutagenesis