Fig 1: Fc?RIIB promotes gMDSCs generation from HPCs via Stat3 signaling. (A) Volcano plots of differentially expressed genes in HPCs from WT or Fc?RIIB KO mice at 14 days post-grafting of MC38 cells (adjusted P = 0.01 and fold change (FC) = 2), n = 4. (B) Signaling pathway enrichment analysis of differentially expressed genes. (C) Heatmap of differentially expressed genes that function as transcription factors involved in differentiation of HPCs into GMPs, n = 4. (D) Heatmap of differentially expressed genes in Jak-Stat signaling from WT or Fc?RIIB KO HPCs, n = 4. (E, F) WT or KO Mice were sacrificed at day 14 post-grafting, and tumor-infiltrating MDSCs were assessed for Stat3 (E) and S100A8 (F) expression; n =5. (G) WT mice BMs were treated with Veh (PBS), GM-CSF (20ng/mL) for 48 hrs, in the presence or absence of STAT3-IN-1 (GM-Stat3i, 1µM), and MDSCs proportion were analyzed. (H, I) Isolated Gr-1+ cells from BM were treated with GM-CSF (20ng/mL) for 48 hrs, in the presence or absence of STAT3-IN-1 (GM-Stat3i, 1µM), DCF-DA (H) and PD-L1 (I) expression were assessed using FCM; n = 4. (J, K) BM derived MDSCs were treated with STAT3-IN-1 (GM-Stat3i, 1µM) for 48 hrs, and then cocultured with CFSE-labeled CD8+ T lymphocytes isolated from WT mice (1:2) for 3 days, the production of IFN-? in CD8+ T cells (J, n = 4), anti-CD3 and anti-CD28 induced proliferation (K, n = 4) were measured by FCM; n = 4. Data are expressed as means ± SD. **P <0.01, by Mann-Whitney test.
Fig 2: GM-CSF induces Fc?RIIB expression on MDSCs via Sp1 signaling. (A) Fc?RIIB expression on HPCs and tumor-infiltrating MDSCs were analyzed, representative histogram plots were shown. (B, C) WT and KO BM cells were treated with 10% tumor supernatants (from MC38 cells) for 48 hrs, the percentage of CD11b+Gr-1+ cells in BM cells (B), Fc?RIIB expression on MDSCs (C) was determined. (D) WT mice BM cells were treated with PBS (Control) or GM-CSF (20 nM) for 48 hrs, the expression of Fc?RIIB were determined with flow cytometry. (E) GM-CSF protein levels in supernatants of MC38 tumor were measured using ELISA, n =5. (F) GM-CSF protein levels in serum of tumor-free or MC38 tumor-bearing WT and Fc?RIIb-/- mice were measured using ELISA, n = 4 each group. (G) In silico analysis predicted two Sp1 binding site in the promoter of Fcgr2b, TSS; transcription start site. (H) ChIP assay analyzed recruitment of Sp1 to Fcgr2b gene locus in WT MDSCs. The prepared chromatin from MDSCs was immunoprecipitated with an anti-Sp1 antibody or control IgG, and pulled-down DNA was subjected to qPCR using the specific primers designed for Sp1 binding region. (I) Luciferase report assay of Fcgr2b promoter containing WT or mutant Sp1 binding site in Sp1 overexpression HPCs. (J) Sp1 expressions in CD11b+Gr-1+ cells from BM and paired tumor were measured by FCM, n = 5. (K) Sp1 expression in WT and KO MDSCs were measured by FCM, n = 4. (L, M) CD11b+Gr-1+ cells from BM were treated with 10% tumor supernatants or GM-CSF (20 nM) for 48 h, the expression of Sp1 was determined by western blot (L) and FCM (M), n = 5. (N) CD11b+Gr-1+ cells from BM were transfected with control (sh-NC) or virus expressing shRNA against Sp1 (sh-Sp1), in the presence or absence of 20 nM GM-CSF for 48 hrs, Fc?RIIB expression on MDSCs were analyzed. (O, P) BM cells were treated with GM-CSF for 48 hrs, in the presence or absence of 1 µL PBS (Con) or Mithramycin A (Mith, 20 nM), Fc?RIIB expression on MDSCs (O) and the percentages of MDSCs (P) in BM cells were analyzed by FCM. Data are expressed as means ± SD. **P <0.01, by Mann-Whitney test (A to F, I, K to P) or Wilcoxon matched-pairs signed rank test (J).
Fig 3: Fc?RIIB deficiency impairs the differentiation of gMDSCs from HPCs in the tumor-bearing mice. (A) The percentage of proliferating (Ki67+) MDSCs in WT and KO tumor tissues were analyzed with flow cytometry after Ki67 staining, n = 5. (B) Representative staining and frequencies of AnnexinV+7AAD+ cells in MDSCs from WT and KO tumor tissues were assessed by FCM. (C) The gene expression of MDSC-related chemokines within whole tumor tissues from WT and KO mice were detected by qPCR; n = 4. Data are expressed as means ± SD. (D) WT and KO BMs were treated with GM-CSF/IL-6 (20ng/mL) to induce MDSCs differentiation, MDSCs ratio and numbers were analyzed after 72 hrs. (E) Gating strategy for granulocyte/macrophage progenitors (GMP; Lin-Sca-1-C-kit+CD16/32+CD34+), common myeloid progenitors (CMP; Lin-Sca-1-C-kit+CD16/32intCD34+), megakaryocyte/erythrocyte progenitors (MEP; Lin-Sca-1-C-kit-CD16/32-CD34-), and percentages of these HPCs subpopulations rates in BMs from WT and KO tumor bearing mice were detected, n = 5. (F) Representative photograph of femurs dissected from WT and KO tumor-free or MC38 tumor-bearing mice on day 21 after tumor cells implantation. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference, by Mann-Whitney test.
Fig 4: NKR-P1B signaling correlates with the rate of surfactant lipid uptake by AM.a RT-qPCR of select metabolic genes from WT and Nkrp1b-/- AM from 2-week-old mice. Data normalized to WT levels. b RT-qPCR analysis of WT AM crosslinked with 2D12 or isotype antibody. Data normalized to isotype control. c Fluorescence of WT and Nkrp1b-/- AM after 40 min NBD-PC incubation. d MFI of WT and Nkrp1b-/- AM after 0, 20, 40, and 60 min NBD-PC incubation. e Fluorescence of WT and Nkrp1b-/- AM after 20 and 60 min incubation with NBD-PG. f MFI of WT and Nkrp1b-/- AM after 0, 20, 40, and 60 min NBD-PG incubation. g NBD-PC uptake in WT and Nkrp1b-/- AM crosslinked with 2D12 or isotype control. h MFI of NBD-PC uptake in (g). i NBD-PG uptake in WT and Nkrp1b-/- AM crosslinked with 2D12 or isotype control. j MFI of NBD-PG uptake in (i). k NBD-PC uptake in WT AM crosslinked with 2D12 or isotype control in the presence of GM-CSF or vehicle control. l MFI of NBD-PC uptake in (k). Data in a–d, g–j (n = 3 independent experiments). Data in e, f, k, l (n = 5 independent experiments). m Images of WT and Nkrp1b-/- AM after 0 or 6 days of in vitro culture post-ORO staining. 100 µm scale bars shown. ORO-staining indicated (arrows). n Quantification of ORO-positive AM/700 µm2 shown in (m). Data in m and n represent (n = 2; day 0) or (n = 3; day 6) independent experiments. Data in a and b show mean ± SD and d, f, h, j, l, n show mean ± SEM. Statistics for d and f represent an unpaired, two-tailed Student’s t-test of data prior to normalization where *p < 0.05 and **p < 0.01. Statistics for h, j, l, and n represent a two-way ANOVA with Tukey’s correction where **p < 0.01, ***p < 0.001, and ****p < 0.0001. All graphs and histograms indicate WT in blue and Nkrp1b-/- in orange. Source data are provided as a Source Data file.
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