Fig 2: The diverse features of GC classified by signature score. The immunotherapy‐related features (MSI/EBV status) and tissue HER2 positivity were classified by EV‐score in (a) training and (b) validating cohorts. The mutual correlations among tissue‐based transcriptomic ARG1, CD3, PD‐L1, PD‐L2 expressions and tissue‐score were assessed in (c) TCGA and (d) GSE62254 datasets. The immunotherapy‐related features (MSI/EBV status) and ERBB2 amplification were classified by tissue‐score in (e) TCGA and (f) GSE62254 datasets. The tissue‐based TH1/TH2 ratio, activated T cell and NK cell were classified by tissue‐score in (g) TCGA and (h) GSE62254 datasets. The tissue‐based transcriptomic IFN‐γ, perforin and granzyme A/B/H/K/M expressions were classified by tissue‐score in (i) TCGA and (j) GSE62254 datasets

Fig 3: Smad7 Deficiency in DCs Protects Mice against DSS-Induced Colitis(A) Percentage body weight changes (left) and linear regression analysis (right) of Smad7fl/fl and DC-Smad7−/− mice treated with 3% DSS in drinking water for 7 days (n = 8).(B) Disease activity index, a composite of body weight loss, blood in stool, and consistency of stool (higher score corresponds to colitis severity) (left) and linear regression analysis (right) of Smad7fl/fl and DC-Smad7−/− mice treated with 3% DSS in drinking water for 7 days (n = 9).(C) Representative histological sections with H&E (left) and scores (n = 10) (right) of colitic mice based on degree of ulceration at day 7, scored blinded by a pathologist at HRHCF. Scale bars represent ~1 mm (top) and ~100 μm (bottom).(D) Colon lengths of colitic mice at day 7 (n = 13).(E) Representative NanoString (immunology panel) heatmap of inflammatory and regulatory genes in distal colon from colitic Smad7fl/fl and DC-Smad7−/− mice. Important upregulated and downregulated genes of interest in DC-Smad7−/− are highlighted in red and green, respectively.(F) qRT-PCR validation of selected genes, including IL-1β, IL-6, TNF-α, and IL-18 expression in these samples (n = 3).(G) qRT-PCR of TGF-β, CD103, IL-12, IL-1β, IL-6, IL-23, and TNF-α expression in MLN CD11c+ DCs from colitic mice (n = 5).(H) Representative FACS histograms (left) and MFIs (right) of PDL2, PDL1, and CD103 in MLN CD11c+ DCs from colitic mice (n = 3). MFI data reflective of CD11c+ population and expressed as FC from Smad7fl/fl condition.(I) qRT-PCR of Foxp3, IFN-γ, and IL-17a in MLN CD4+ T cells (n = 5).(J) Representative FACS plots (left) and frequencies (right) of Foxp3+, IFN-γ+, and IL-17a+ populations in MLN (top) and lamina propria (LP) (bottom) CD4+ T cells (n = 4–10).(K) Percentage of body weight scores (left) and linear regression analyses (right) of Smad7fl/fl and DC-Smad7−/− mice treated during DSS administration, as indicated. Mice were treated with either immunoglobulin (Ig) control, (top) anti-CD25 (500 μg) (center), or anti-PD1 (250 μg) (bottom), given i.p. every other day 4 times, starting with DSS administration at day 0 (n = 3–4). Only control colitis body weight loss data for anti-CD25 are shown. Control data for anti-PD1 are identical (data not shown).Data representative of ≥3 independent experiments. qRT-PCR data expressed as FC from Smad7fl/fl condition. Means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t test. See also Figure S3.

Fig 4: A four protein‐based plasma EV‐score at treatment baseline robustly predicted immunotherapeutic outcomes. The prognostic correlations of EV‐derived (a) ARG1, (b) CD3, (c) PD‐L1 and (d) PD‐L2 at BL were assessed by Kaplan‐Meier survival analysis. These four genes were further combined to generated an EV‐score. (e) ROC curve was used to measure the specificity and sensitivity of EV‐derived ARG1, CD3, PD‐L1, PD‐L2 and EV‐score in characterizing patients reached an irPFS (progression) or irOS (survival) longer than 6 months. (f) Prognostic indications of scattered EV‐scores (left panel), or for the cut‐off value of EV‐score≥1 (right panel). (g) Timeline‐scaled changes of tumour volume and (h) the best changes of tumour volume for all patients were stratified by EV‐score. CT images and changes of tumour volume were displayed for representative cases defined as (i) EV‐score≥1 or (j) EV‐score < 1. (k) Correlation between EV‐score and IFN‐γ content in paired peripheral blood samples at BL

Fig 5: EV‐derived PD‐L1 and PD‐L2 exerted opposite impacts on the tumour growth inhibition of anti‐PD‐1 agent. (a) The expression of PD‐L1 or PD‐L2 in mouse cancer cell lines MC38/CT26. (b) Western Blot assay for EV markers of isolated cellular EV samples. (c) ELISA quantification for the concentration of PD‐L1 or PD‐L2 expression in 5 μg extracted EVs. The time‐scaled changes of (d) mice body weight, (e) tumour volume and (f) images of xenograft tumours on the last day of treatment were recorded for MC38/CT26 xenografts. (g) The staining of HE, ki‐67 and CD8 in MC38/CT26 xenograft tumours were displayed. The (h) TH1/TH2 ratio and the expression of (i) IFN‐γ, (j) TNF‐α, (k) granzyme B in CT26 xenograft tumours were quantified. *, P < 0.05. ns, P value not significant