Fig 2: PD-1+ lymphocytes are increased in PBMCs from GEP-NET patients with progressive disease. PBMCs from 32 patients and 32 controls were isolated and incubated with conjugated antibodies directed against CD3, CD4, CD8, CD25, FOXP3 and PD-1. Measurements were made by flow cytometry as stated in ‘Materials and Methods’ and values represent percentage of positive cells for each marker depicted as boxplots. (a–d) Percentage of PD-1+ cells in healthy controls and patients. (e–h) Percentage of PD-1+ cells in controls and patients classified according to disease status in non-residual disease (ND), stable disease (SD) or progressive disease (PD). Asterisks indicate significant differences (p-values for Tukey’s test: *p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: Expression of PD-1 is associated to increased malignancy in patients with GEP-NETs. (a,e) CD3, (b,f) FOXP3, (c,g) PD-1 and (d,h) PD-L1 were measured by IHC in a set of TMAs (n = 164) of GEP-NETs. Values represent boxplot of CD3 IHC score and bar graphs of the percentage of samples with positivity for PD-1, FOXP3 and PD-L1 expression. (a–d) Samples were classified based on metastases status at diagnosis: patients with metastasis (MET) or without metastasis (No MET). (e–h) Samples were classified based on the disease status at follow-up evaluation: (1) non-residual disease, if a complete resection after surgery had been achieved and no tumor relapse/recurrence was evidenced; (2) stable disease, in cases of residual but non-progressive tumor burden; and (3) progressive disease, if tumor growth or new lesions were detected. The median of follow-up was 4.9 years (p25: 2.6–p75: 8.6 years). Asterisks indicate significant differences (p-value for Fisher’s Exact Test: *p < 0.05).

Fig 4: Heatmap analysis of γδ T cells. The cytokine and cytotoxic and immune marker frequencies were normalized across all LTB groups, and the expression data are shown. Each column indicates a single person. We show the heatmap expression of (A) GMCSF and Th1 cytokines, (B) PD-1 and Th17 cytokines, and (C) PD-1 and cytotoxic markers in LTB patients coinfected with DM (n = 20), PDM (n = 20), and NDM (n = 20) individuals upon UNS, Mtb (PPD, WCL), and positive antigen (P/I) control stimulation with continuous color shading.

Fig 5: CD226 expression and association with clinical response to PD-L1 checkpoint blockade treatment in NSCLC(A) Relative gene expression of CD226, CD28, PDCD1, or TIGIT in three different atezolizumab clinical trials (BIRCH, OAK, or POPLAR) in NSCLC.(B) Co-expression of CD226 (left) or CD28 (right) with PDCD1 or TIGIT are positively correlated. Shown is the Spearman correlation coefficient.(C–F) Survival based on CD226 (C), CD28 (D), PDCD1 (E), or TIGIT (F) gene expression. Kaplan-Meier plots of overall survival (OS) are shown for the atezolizumab arm in the indicated clinical trial (BIRCH, OAK, or POPLAR), with patients separated on the basis of gene expression. Patients were dichotimized into top 50% (high, green line) or bottom 50% (low, red line) relative to the median expression over all patients in the corresponding clinical trial. Two-sided p values and hazard ratios from a Cox proportional hazards model are indicated for each plot.