Fig 2: FGL2 suppresses NF-?Bp65 and STAT1/5 signaling for CD103 induction. a, Expression levels of key proteins in the TAK1-NF-?B/p38 cascades and JAK2/STAT1/5 signaling were examined by western blotting in bone marrow cells cultured with conditioned medium (CM) from control tumor cells (Ctrl), FGL2KO tumor cells, Ctrl-CM + IgG, Ctrl-CM + anti-FGL2, FGL2KO-CM + IgG, or FGL2KO-CM + anti-GM-CSF for 3 days. Data were summarized as ratio changes from at least three independent experiments. The t-test was used to calculate the two-sided P values. Significant results were presented as *P < 0.05, **P < 0.01, ***P < 0.001. b Representative FACS analysis of CD103 expression on CD11c+B220- dendritic cells (DCs), which were pretreated for 1 h with I?Ba inhibitor Bay 11-7085 (Bay), NF-?B inhibitor 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ), JAK2 inhibitor JSI-124 (JSI), or p38 inhibitor PH797804 (PH) in bone marrow cells cultured with CM for 5 days. Plots are representative of three independent experiments. c Schematic illustration of cellular and molecular events underlying FGL2-regulated GBM progression. Tumor cells secreted GM-CSF and FGL2 simultaneously. GM-CSF-induced CD103+ DCs development was blocked in the presence of FGL2, so that CD8+ T cells were not primed and activated because of small CD103+ DC populations. Less CD103+ DC differentiation and subsequent lack of CD8+ T cell priming and activation resulted in GBM progression. For molecular signaling, TRAF6/TAK1/NF-?B/p38 signal and JAK2/STAT1/5 were activated in response to GM-CSF, contributing to CD103 induction. These signaling cascades were blocked by FGL2, thereby suppressing CD103 induction on DCs

Fig 3: FGL2 suppresses GM-CSF-dependent CD103 induction on cDCs in vitro. a Expression of CD103 on CD11c+B220- DCs cultured with conditioned medium (CM) for 5 or 15 days. Bone marrow cells were isolated from C57BL/6 mice and then cultured with Ctrl-CM (CM from Ctrl tumor cells) or FGL2KO-CM (CM from FGL2KO tumor cells) for 5 days or 15 days. Non-adherent cells were recovered, stained, and analyzed by FACS to determine the expression levels of CD103, Clec9A, and MHCII on the CD11c+B220- DCs. b OT-I T cell activation by OVA-pulsed CM-cultured bone marrow DCs. Non-adherent bone marrow DCs were recovered from CM-cultured bone marrow DCs at 15 days. The bone marrow DCs (2 × 104) were incubated with soluble OVA protein (100 µg/mL) for 2 h and then washed twice before co-culturing with the CFSE-labeled OT-I cells (5 × 104). OT-I activation was measured by CD69 expression level, which was determined by flow cytometry. c Protein levels of GM-CSF and FLT3L in medium conditioned by tumor cells were detected by ELISA. Data were summarized from five independent experiments and were analyzed by paired t-test. d Representative FACS assay of CD103 expression on CD11c+B220- DCs cultured with FGL2KO-CM in the presence of neutralization antibody anti-GM-CSF (5 µg/mL) or anti-FLT3L (5 µg/mL) for 5 days. Data were summarized from four independent experiments and were analyzed by paired t-test. **P < 0.01 comparing with IgG. e Representative FACS assay of CD103 expression on wild-type (WT) and FGL2KO bone marrow (BM) cells cultured with rmGM-CSF (10 ng/mL) for 7 days with or without rFGL2 (200 ng/mL). All plots are representative of three independent experiments

Fig 4: Receiver operating characteristic (ROC) curves of predictors of lung cancer lymph node metastasis. ROC curves of (A) cytology, (B) methionyl-tRNA synthetase (MRS)/CD45 immunofluorescence (IF), and (C) the combination of cytology, positron emission tomography (PET), and MRS/CD45 IF were created using core biopsy results as a reference value. lr.eta, linear predictor eta; PV(+), positive predictive value; PV(-), negative predictive value; s.e., standard error; est., estimates.

Fig 5: Intracellular trafficking of RA1-CB[6] @AuNRs as evaluated by immunocytochemistry. a Schematic representation of the experiment. b Confocal images of leukemic cells incubated with RA1-CB[6] @AuNR-PEG-TRITC. Cells were incubated for 4 h with AuNR (50 µg/mL), washed to remove the non-internalised AuNRs, added new cell culture media, irradiated for 2 min with a 780 nm laser (power: 2 W/cm2) and finally fixed with PFA (4%). The cell membrane was immunostained for CD45, while endosomes were immunostained for EEA1. White scale bar corresponds to 30 µm. c Intracellular localization of RA1-CB[6] @AuNRs before NIR light irradiation was confirmed by a Z-stack scan. Scale bar corresponds to 10 µm. d The co-localization between RA1-CB[6] @AuNR-PEG-TRITC and early endosomes before and after irradiation was quantified by image analyses. Results are average ± SEM (n = 3). Statistical analysis was performed using a t-test. ***Denotes statistical significance (P < 0.001)