Fig 2: Myeloid cells express SBSN in bone marrow of MDS patients. Immunofluorescence detection of CD11b (green) using anti-CD11b-BV421, SBSN (red) using anti-SBSN and Alexa Fluor 568 goat anti-rabbit in bone marrow smears of (A) MDS (EB-2) and (B) MDS 5q-syndrome patients. Nuclear DNA is stained by SiR-DNA (blue), with n = 3 per group. Scale bar, 10 µm. (C) Gating strategy used for isolation of eMDSCs, MDSCs, and monocyte populations from the bone marrow of MDS (n = 6) and ‘hematological malignancy’ patients (HM; multiple myelomas, T-cell lymphoproliferation, chronic anemia; n = 4). (D) SBSN mRNA levels of BM myeloid subpopulations eMDSCs [Lin- (CD3, 14, 16, 19, 20, 56) HLA-DR-, CD33+], PMN-/MN-MDSCs (MDSCs; Lin+, HLA-DR-, CD33+), monocytes (Lin+, HLA-DR+, CD33++), adipocytes, and granulocyte-containing high-density fraction in MDS (n = 4) and HM (multiple myelomas and T-cell lymphoproliferation; n = 3) estimated after cell sorting by RT–qPCR. (E) Comparison of the bone marrow subpopulation percentage in MDS (n = 6) and HM (multiple myelomas and chronic anemia; n = 3) patients. For patients' samples, one-way ANOVA was used for multigroup comparisons followed by the Tukey's post hoc test. Results are the means ± SD; *P < 0.05, **P < 0.01.

Fig 3: Microglial surface expression of CX3XR1, CD11b, MHC-II, and CD206 molecules. A representative gating strategy for CX3CR1-(A) and CD11b-(D) positive microglia population. MFI of CX3CR1 in cerebellum (B) and hippocampus (C). MFI of CD11b in cerebellum (E) and hippocampus (F). Percentages of (G) MHC-II and (H) CD206 and (I) ratios of MHC-II versus CD206 microglia in cerebellum. Percentages of (J) MHC-II and (K) CD206 and (L) ratios of MHC-II versus CD206 microglia in the hippocampus: * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001.

Fig 4: Antitumor immunity induced by the a-PDL1/PEIGel in a 4T1 allograft murine model. a Infiltration of T lymphocytes into tumors from different treatment groups. Nuclei were stained with DAPI (blue fluorescence), CD3+ cells were identified by green fluorescence, and CD8+ cells were identified by red fluorescence; scale bar ?= ?200 ?µm. 4T1 tumor-bearing mice were intratumorally injected with 100 ?µL PBS, PEIGel, a-PDL1 or a-PDL1/PEIGel (dose equivalent to 100 ?µg a-PDL1) on day 0, and the subcutaneously transplanted tumors were removed on day 14 for immunofluorescence and flow cytometric analysis. a Infiltration of T lymphocytes into tumors from different treatment groups. Nuclei were stained with DAPI (blue fluorescence), CD3+ cells were identified by green fluorescence, and CD8+ cells were identified by red fluorescence; scale bar ?= ?200 ?µm and 50 ?µm. a-e Flow cytometric analysis and f-i quantitative analysis of CD8+ T cells after gating on CD45+CD3+ cells, IFN-?+ T cells after gating on CD45+CD3+CD8+ cells, Treg cells after gating on CD4+FOXP3+ cells, and M2-like macrophages (CD206+) after gating on CD45+CD11b+F4/80+ cells; n ?= ?5 biologically independent mice per group. Data are presented as the mean ?± ?SD. Statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test. *P ?< ?0.05, **P ?< ?0.01, ***P ?< ?0.001, ****P ?< ?0.0001.

Fig 5: LPS-induced hippocampal microglial changes in WT and miR-146b deficient mice. Flow cytometry analysis with indicated markers was performed using brain cells from mice were injected with LPS in saline or equal volume of saline (control). (A) Representative gating strategy for quantification of microglial percentage. (B) Percentage of microglial cells among total brain cells. (C) Representative gating strategy for quantification of macrophages and MHCII+ microglia. (D) Percentage of macrophages (E) Percentage of MHCII+ microglia among microglial cells. (F, G) Mean fluorescent intensity (MFI) of CD11b and Csf1r (CD115) among microglial cells. Number of animals = 6-7. Data are represented as mean ± SEM; * p < 0.05, ** p < 0.01, **** p < 0.0001 (Two-way ANOVA with Tukey’s multiple comparison test).