Figure 5.

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 × 10⁵ 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.