Fig 2: ZQT attenuated the immunosuppressive effect of MDSCs on T cells in the tumor microenvironment. (a, b) CD3+ T cells isolated from the tumor tissue of tumor-bearing C57BL/6 mice treated with normal saline or ZQT were cocultured with LLC cells for 4 h before incubating with CD3, CD8, and CD107a antibodies. Events shown were finally gated on CD3 and CD8. (c) ELISA analysis for the expression of IFN-? in cells from the tumor tissue. (d, e) RT-qPCR quantitative analysis of Granzyme B, Perforin 1, Arg-1, iNOS, and S100A9 gene expression. (f) ROS assay for ROS production in tumor tissue from ZQT-treated mice or normal saline-treated mice. (g, h) T-lymphocyte in the spleen of wild-type C57BL/6 mice and MDSCs in tumor tissue of tumor-bearing mice treated with normal saline or ZQT were isolated using the MojoSort Mouse Isolation Kit. T-lymphocyte proliferation assay was used to show dose-dependent suppression of T cell proliferation by MDSCs isolated from tumor tissue of tumor-bearing mice treated with normal saline or with ZQT. CD3+ T cells isolated from tumor tissue stained with 5 µM CFSE were incubated with MDSCs for 72 h. Meanwhile, CD3 and CD28 antibodies were added for CD3+ T cell stimulation. Flow cytometry analysis was performed to quantify 72 h CFSE dilution. n = 7. Data are expressed as the mean ± SD. n/s: nonstatistical significance. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: IL-3-deficient mice have increased asthmatic trait(A) Experimental design for the induction of allergic asthma.(B) ELISA analysis of the IL-3 level in the supernatant obtained from total lung cells (n = 13/12 (unst., OVA), n = 14/13 (aCD3/CD28)).(C) Il3/Hprt mRNA expression in total lung cells of BALB/c WT mice with and without asthma (n = 4/4 (unst.), n = 4/5 (aCD3/CD28)).(D) Airway resistance to increasing doses of methacholine measured by non-invasive plethysmography. Airway resistance was analyzed as Penh (n = 16/16/17/18).(E) IgE serum level of mice with and without asthma measured by ELISA (n = 13/13/13/12).(F) Pathological score of the lung inflammation of mice with and without asthma (n = 12/13/11/12). A representative light microscopic picture of the H&E staining of lung sections is shown for each group.(G and H) Total cell count in the BAL of WT and IL3-/- mice with and without asthma (n = 11/11/11/13), and (h) cell count of mononuclear cells in the BALF of mice with and without asthma (n = 14/14/12/13).(I, and J) Flow cytometry analysis of CCR3+ Gr-1+ eosinophils (i) and CCR3- Gr-1+ neutrophils (j) in total lung cells (n = 9/9/9/10). A representative dot plot is shown for each group.(K) ELISA analysis of the IL-5 level in the BALF (n = 7/10/9/11). Data are presented as means ± SEMs. Two-tailed Student’s t test or two-way ANOVA was used to calculate statistical significance. *p = 0.05; **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: Tumor-derived extracellular vesicles (TEVs) promote tumor-infiltrating Treg cell accumulation.(A) The number of induced Foxp3-eGFP+ cells (iTreg) generated from naive T cells by using anti-CD3, anti-CD28, Interleukin-2 (IL-2), and Transforming growth factor-ß (TGF-ß). These iTreg cells were cocultured with TEV-stimulated bone marrow-derived dendritic cells (BMDCs) in the presence of IL-2 and TGF-ß for 5 days with a control mAb (n = 7) or an anti-interferon-ß (IFN-ß) mAb (n = 5). (B) Tumor growth curves of Cd300afl/fl and Cd300afl/fl;ItgaxCre mice treated with an anti-IFN-ß mAb or a control mAb (control, n = 7; anti-IFN-ß, n = 7 for each genotype mouse) three times (days 7, 11, and 14) and after inoculation of B16 melanoma cells. (C) Representative fluorescence micrographs of tumor sections from Foxp3-eGFP wild-type (WT) (phosphate-buffered saline (PBS), n = 4; GW4869, n = 6) and Foxp3-eGFP Cd300a-/-- mice (PBS, n = 5; GW4869, n = 6) in the absence or presence of GW4869, and stained with an anti-GFP mAb (green) and the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI, left). The number of Foxp3+ cells was quantified from four high-power fields (LPF) (right). White arrow shows Foxp3-positive cells. Scale bar, 200 µm. Tumor growth curves of WT (PBS, n = 6; GW4869, n = 9) (D) or Cd300afl/fl (n = 4 each) (E) and Cd300a-/- mice (PBS, n = 7; GW4869, n = 9) (D) or Cd300afl/fl;Itgax-Cre (n = 6 each) (E) that were treated with GW4869 or PBS three times (days 14, 18, and 21). Data are given as means ± standard error of the means (SEMs). RQ: relative quantification; N.S.: not significant. **p < 0.01 and ***p < 0.001. p values were obtained by using a one-way analysis of variance (ANOVA) (A and C) and a two-way ANOVA followed by Bonferroni’s post-test (B, D, and E). Data were pooled from two (A and C) or three (B, D, and E) independent experiments. Figure 4—source data 1.Source data for Figure 4A-E.

Fig 5: Homotypic recruitment of human polyclonal and CAR T cells.(A) Transmigration of human T cells towards supernatant from T cells conjugated with IgG-coated beads (control beads), recombinant human CCL3 and CCL4, or supernatant from T cells conjugated with aCD3/CD28-coated beads in the absence or presence of CCR2/CCR5 dual antagonist (Cenicriviroc), relative to transmigration towards basal medium. Bars: mean from four independent experiments (data points). Error bars: SEM. p-values from ANOVA and Tukey’s multiple comparison test. (B) Cell trajectories of primary polyclonal human T cells adjacent to a tumouroid containing human T cells stimulated with control or aCD3/CD28 beads. Random representative tracks from dataset of three independent experiments, quantified in (C). (C) FMI of polyclonal human T cells as per (B), also assessed in the presence of Cenicriviroc. Box-whiskers indicate medians and the interquartile range (IQR). Red bars: mean of pooled data from three independent experiments. Data points: mean of each individual experiment. n: number of tracks for each experimental condition. ns: p>0.05, p-value from two-tailed Wilcoxon signed rank test compared to hypothetical median of 0. (D) Human polyclonal T cells (green) responding to a tumouroid containing T cells (magenta) stimulated with control (top) or aCD3/CD28 beads (bottom). Insets highlight CTLs infiltrating the tumouroid. Scale bars: 500 µm. (E, F) Swarming index and density kymographs quantify human T cell movements with respect to tumouroids in (D). r: distance from tumouroid edge. (G) Human CAR T cells (green) responding to tumouroids containing additional CAR T cells (magenta) and control fibroblast cells (top) or target glioblastoma cells (bottom). Insets highlight CTLs infiltrating the tumouroid. Scale bars: 500 µm. (H, I) Swarming index and density kymographs quantify CAR T cell movements with respect to tumouroids in (G). r: distance from tumouroid edge.Figure 6—source data 1.Source data file for Figure 6.