Fig 1: XAF1 secretion stimulates T cell-mediated tumor regression. A Regression of 4T1-Xaf1−/− tumors (n = 6 per group) in response to rXAF1 injection. The tumors were exposed to recombinant mouse Xaf1 (2.5 mg/kg) or saline at day 10, 11, and 12. B Representative photographs of syngeneic tumors at day 18 after inoculation. C, D IHC assay showing significantly higher tumor infiltration of CD3+ T cells (dark brown) in rXaf-injected versus saline-injected tumors. Number of CD3+ T cells was counted in high-power field (HPF). E, F Flow cytometry analysis of tumor-infiltrating CD4+ and CD8+ cells and their CD69 and IFN-γ expression. G, H Regression of Xaf1+/+ tumor by IFN−γ and its impediment by co-injection of α-Xaf1 (n = 4 per group). The tumors exposed to saline, IFN-γ (0.1 mg/kg), and α-Xaf1 (1 mg/kg) at day 14, and 16. I, J IHC assay showing IFN-γ effect on tumor infiltration of CD3+ T cells and its impediment by α-Xaf1. K, L IFN-γ effect on tumor infiltration of CD4+ and CD8+ T cells and their CD69 expression and its blockade by α−Xaf1. Data represent the mean ± SD. Student's t test was performed to determine the statistical significance. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 2: Secretion of stress-induced XAF1. A, B Detection of XAF1 in the condition media (CM) derived from etoposide-exposed HT1376 cells. Cells were exposed to an increasing dose or time of etoposide as indicated. Immunoblot (IB) assay was performed to detect XAF1 and BAX in 10 % of total cell lysate and CM. Eto, etoposide. C, D XAF1 secretion from IFN-γ-treated HCT116 cells. Cells were exposed to an increasing dose or time of IFN-γ. E XAF1 secretion from XAF1-overexpressed HCT116 cells. Cells were transfected with an increasing dose of XAF1 and treated with or without TNFα. F XAF1 secretion from XAF1-overexpressed HCT116 cells. Cells were transfected with an increasing dose of XAF1 or BAX. IB assay was performed at 12 h after transfection. G Cytosolic LDH release assay for CM from HT1376 cells exposed to IFN-γ. Total LDH content was determined following triton-based permeabilization of cells. Data represent means ± SD of triplicate assays. H No induction of necroptosis (P-MLKL) and pyroptosis (IL-1β) marker proteins in HT1376 cells exposed to IFN-γ (200 ng/ml) and etoposide (50 μM). I Comparison of secreted XAF1 level in four discontinuous CM batches of 4 h interval after IFN-γ addition.
Fig 3: XAF1 association with overall survival of T cell-enriched patients. A, B TCGA data analysis of cancer patient database showing an association of XAF1 expression with overall survival in CD8+ and CD4+ T cell-enriched cancer patients. Stomach cancer patients (n = 375), liver cancer patients (n = 371), bladder cancer patients (n = 405), and breast cancer patients (n = 1090). C An association of XAF1 expression with overall survival of patients following anti-PD-1 (n = 851), anti-PD-L1 (n = 486), and anti-CTLA-4 (n = 131) therapy. D Schematic representation of XAF1 secretion from stressed tumor cells and its roles in the tumor microenvironment. XAF1 is secreted through the endo-lysosomal trafficking pathway. Secreted XAF1 enhances T cell-mediated tumor surveillance.
Fig 4: T cell activation by secreted XAF1. A, B A transwell assay showing stimulation of Jurkat cell migration by CM derived from HT1376 and HCT116 cells incubated with IFN-γ (0.2 μg/ml, 36 h). The CM was added to Jurkat cells through lower chamber for 20 h. C rXAF1 stimulation of Jurkat cell migration. Cells were incubated with rXAF1 for 20 h. D Immunodepletion of secreted XAF1 from CM of HT1376-XAF1+/+ cells using α-XAF1. E, F Loss of migration-promoting activity of XAF1-immunodepleted CM of HT1376-XAF1+/+ cells. G rXAF1 stimulation of Jurkat cell proliferation and IL-2 production. H RT-PCR assay showing rXAF1 upregulation of CD69 and CD25 mRNA expression in Jurkat cells. I, J Flow cytometry analysis showing rXAF1 induction of CD69 expression in mouse PBMCs. Data represent means ± SD of triplicate assays. Student's t test was performed to determine the statistical significance. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 5: XAF1 secretion through the endo-lysosomal pathway. A Effect of conventional secretion inhibitors (Tunicamycin and Brefeldin A) and an endocytic pathway inhibitor (Dynasore) on the secretion of XAF1 proteins induced by tetracycline addition in DU145 (Tet-XAF1) cells. B Inhibition of XAF1 secretion by lysosome inhibitors (NH4Cl, Chloroquine, and Bafilomycin A1). C A dose-associated inhibition of XAF1 secretion by Dynasore. Cells were exposed to IFN-γ and then incubated with an increasing dose of Dynasore as indicated. D Inhibition of XAF1 secretion by VAMP-7 depletion. E Microscopic analysis showing XAF1 colocalization with RAB7 and LAMP1 and its disruption by Dynasore. F XAF1 constructs and its secretion status. ZF zinc finger. G, H IB assays showing no secretion of XAF1 mutants lacking the ZF4 domain. I A proteinase K assay showing secretion of XAF1 in a soluble form. CM derived from IFN-γ-exposed cells were incubated with proteinase K for 30 min with or without Triton X-100.
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