Fig 1: Epigenetically imprinted lung microenvironment by viral infection promotes lung tumor growth(A) Schematic of experimental setup for bone marrow transplantation and tumor implantation.(B) Representative image of tumor-bearing lung lobes (left) and quantification of tumor burden (right) at week 3 in PBS- or IAV-bone marrow transferred KPi.t. mice.(C) Tumor growth curves of PBS- or IAV-infected mice s.c. implanted with KP tumor cells.(D) Schematic of the tumor parabiosis experiment. Wild-type female CD45.1+ and CD45.2+ congenic mice were treated with PBS or infected with IAV, respectively, and maintained for 4 weeks prior to surgical parabiosis (forming PBS-IAV pairs). PBS-PBS pairs served as controls. After 3 weeks of parabiosis recovery, both mice in each pair were i.t. inoculated with KP tumor cells. Tumor burden and neutrophil populations were analyzed 3 weeks later.(E and F) Frequency and cell number of SiglecFhi neutrophils in the lung parenchyma (E) and blood (F) among parabiosis pairs.(G) Frequency and cell number of SiglecFhi neutrophils in the lung parenchyma of parabiosis pairs, stratified by CD45.1+ and CD45.2+ origin to distinguish host- vs partner-derived cells.(H and I) Representative image of tumor-bearing lung lobes (H) and quantification of tumor burden (I) at week 3 among parabiosis pairs.(J) Heatmap showing upregulated cytokines and chemokines in BAL from IAV_Cre, IAV_KPi.t. and SCV2_KPi.t. mice compared to their respective PBS-treated controls.(K) Representative scATAC-Seq genomic browser tracks showing chromatin accessibility at the Csf3 locus in AM, IM, epithelial cells, fibroblasts and endothelial cells from PBS- and SCV2-infected lungs at 28 d.p.i.(L) Representative bulk ATAC-Seq genomic browser tracks displaying chromatin accessibility at the Csf3 locus in AM isolated from BAL of PBS- and IAV-infected mice at 35 d.p.i.(M) G-CSF production by SCV2- or IAV-infected lung cells or AMs at 35 d.p.i. following overnight stimulation with LPS or UV-irradiated KP cells. Culture supernatants were collected and analyzed by ELISA.(N) Flow cytometry dot plots showing the induction of SiglecF+ neutrophils following recombinant G-CSF or TGF-β treatment, as well as G-CSF/TGF-β and GM-CSF/TGF-β co-treatment (left), and the proportion of SiglecF+ neutrophils (right).(O) Blockade of G-CSF receptor (G-CSFR) reduced both the frequency and number of SiglecFhi neutrophils in tumor-bearing lungs of mice previously infected with IAV.(P) Representative image of tumor-bearing lung lobes and quantification of tumor burden from (O).(Q) Schematic model illustrating how prior severe respiratory viral infection epigenetically reprograms lung microenvironment via G-CSF production, promoting the local accumulation of SiglecFhi neutrophils and contributing to accelerated tumor growth.Data represent two independent experiments or pooled from two experiments. Graphs display mean ± SEM. Statistical significance was assessed by Mann-Whitney test (B and J), two-way ANOVA (E, F, G, and I), and one-way ANOVA (N). (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)
Fig 2: Methodology for generating glioma-educated tumor-associated macrophages (gTAMs) in vitro. (A) Protocol for generating gTAMs by culturing healthy murine bone marrow in sterility with tumor-conditioned media (TCM) from different glioma cell lines (GL261, U87, CT2A, and U251) with cytokines M-CSF, IL-4, and IL-10 for 7 d. For example, bone marrow cells educated into gTAMs using GL261 TCM are referred to as GL261 gTAMs. M1 and M2 macrophage controls were generated for comparison by culturing murine bone marrow cells in supplemented Hibernate media with GM-CSF or M-CSF, respectively. On day 6, M1 and M2 macrophages were polarized with LPS and IFNy or IL-4, respectively. (B) The development of bone marrow cells into gTAMs or M1/M2 control macrophages was captured on days 3, 5, and 7 by a 20X light microscope. (C) Mass cytometry (CyTOF) of CD45+ CD11b+ singlets and downstream unsupervised clustering of the gTAMs or M1/M2 macrophages via tSNE analysis revealed heterogeneity in the different gTAMs compared to M1/M2 macrophage controls. The color of the plots indicates density.
Fig 3: Effects of transfected plasmid DNAs or expressed IFNB1 on H-2Kb and H-2Kb-bound SIINFEKL expression: (A) Transfection of plasmid DNAs (right panel), leading to increased H-2Kb expression in MOC2 cells compared to control MOC2 cells (left panel). (B) Quantification of H-2Kb positive cells in control and plasmid DNA transfected MOC2 cells (n = 4). (C) Transfected plasmid DNA (right panel) increases H-2Kb-bound SIINFEKL in MOC2 cells compared to control cells (left panel) after pulsing with the SIINFEKL peptide. (D) Quantification of the increase in H-2Kb-bound SIINFEKL-positive cells in the plasmid DNA transfected cells compared to control cells (n = 4). (G) Viral-mediated transduction of IFNB1 (illustrated in orange, bottom panel) but not GM-CSF (blue, middle panel) increases H-2Kb expression compared to the control (Pink, top panel) MOC2 cells. (H) Mean fluorescence intensity (MFI) of H-2Kb expression in control, GM-CSF-expressing MOC2 cells, and IFNB1-expressing MOC2 cells (n = 3). (E) Expression of IFNB1 (Ed) in mK2-SIIN-MOC2 cells, which express the fluorescent protein mKate2 and SIINFEKL, significantly increases H-2Kb-bound SIINFEKL presentation compared to either the vector control (Eb) or the GM-CSF-expressing (Ec) cells. mK2-MOC2 cells (Ea) were used to indicate mKate2 expression as a control. mK2: mKate2; SIIN: SIINFEKL. (F) Quantification of H-2Kb-bound SIINFEKL positive cells in mk2-MOC2, vector control-expressing, GM-CSF-expressing, IFNB1-expressing-mK2-SIIN-MOC2 cells (n = 3). p-values are indicated.
Fig 4: B cells and DCs both generated effector memory CD8+ T cells upon stimulation and resulted in CD8+ T cell proliferation. B cells and DCs isolated from C57Bl/6 spleen were either LPS treated or not and then loaded with SIINFEKL peptide. CD8+ T cells were labeled with CFSE and co-cultured with APCs in the presence or absence of GM-CSF and IL-4 for 48 h. Flow cytometry was performed to identify (A) central memory CD8+ T cells (CD44+ CD62L+) and (B) effector memory CD8+ T cells (CD44+ CD62L−). Each data point represents a biological replicate, the graph plotted shows the mean and standard deviation for each treatment group. (C) Representative proliferation histogram plots of CD8+ T cells as measured by loss in CFSE. Asterisks **** indicate p < 0.0001. Results presented are from one experiment and are representative of at least two similar, independent experiments.
Fig 5: Intrinsic TCF4 is required for mature AM development and maturationA. Representative western blot for TCF isoforms in primary mouse AMs, CD4 T cells, and peritoneal macrophages (PM).B. Representative western blot for TCF4 in primary mouse AMs without or with GM-CSF, TGF-β or both stimulation after 48h.C. Representative western blot for TCF4 in primary mouse monocytes without or with GM-CSF, TGF-β or both stimulation after 24h and 48h.D. Representative flow cytometry plots showing AM population in BAL cells isolated from Tcf7l2fl/fl or Tcf7l2cKO mice.E. Total AM numbers, Siglec Fhigh, Siglec Flow AM, and total BAL protein were determined in naïve Tcf7l2fl/fl (n=5) and Tcf7l2cKO(n=6) mice.F. Bulk RNA-seq analysis of AM isolated from Tcf7l2fl/fl or Tcf7l2cKO mice. A heatmap of k-means clustering of differentially expressed AM-associated transcription factors is shown.G. Schematic for lung immune cells isolation from PND3 mice. Down bar graph showing lung immune cells (AM, pre-AM, Fetal macrophages, and Fetal monocytes) from naïve PND3 Tcf7l2fl/fl (n=5) or Tcf7l2cKO(n=5) mice.H. Schematic diagram for bone marrow (BM) chimera experiment (I-K). WT mice were lethally irradiated and reconstituted with CD45.1+ WT (50%) mixed with CD45.2+ Tcf7l2fl/fl (50%) or CD45.2+ Tcf7l2cKO (50%) BM cells. 8 weeks later, immune cell reconstitution in BAL fluid and blood was examined.I. Percentage of total frequency of CD45.1 and CD45.2 population from WT:Tcf7l2fl/fl (n=3) and WT:Tcf7l2cKO (n=5) mice in blood.J. Flow cytometry plot showing CD45.1 and CD45.2 population from WT:Tcf7l2fl/fl and WT:Tcf7l2cKO mice pre-gated on Siglec F+ and CD11c+ AM in BAL fluid.K. Percentage of CD45.1 and CD45.2 population from WT:Tcf7l2fl/fl and WT:Tcf7l2cKO mice pre-gated on Siglec F+ and CD11c+ AM in BAL fluid.L. Schematic for IAV infection model in Tcf7l2fl/fl and Tcf7l2cKO mice.M. Relative original body weight in percentages of Tcf7l2fl/fl (n=5) or Tcf7l2cKO(n=9) mice post infection.N. Survival plot in Tcf7l2fl/fl (n=5) or Tcf7l2cKO(n=9) mice after IAV infection.Data are representative of at least two independent experiments with similar results except F. p-values are represented as *p< 0.05, **p< 0.01, ***p< 0.005, ****p< 0.001.
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