Fig 1: P3‐(CD80+PDL1−)pDCs accumulate within the tumor and were linked to a bad clinical outcome. PBMC and tumor‐infiltrating cells from melanoma patients together with PBMC from HD were labelled with specific antibodies allowing depicting the pDC subsets and submitted to flow cytometry analysis. (a) Concatenated tSNE plots showing CD11c, BDCA2, CD80 and PDL1 expression in CD45+Lin−HLA‐DR+BDCA3− cells from HD blood (n = 20), Pt blood (n = 17) and tumor infiltrate (n = 27). Presented tSNE plots were based on proportional sampling and constructed after clustering with CD11c, BDCA2, BDCA1, CD80 and PDL1 markers by the viSNE algorithm (Cytobank). Black polygons encase P3‐(CD80+PDL1−)pDCs seen only in melanoma patients. (b) Gating strategy for depicting P0‐, P1‐, P2‐ and P3‐pDCs. After cell debris exclusion, single‐cell gating and dead cell exclusion, CD45+ cells were selected. Human plasmacytoid dendritic cells (pDCs or P0‐pDCs) were then depicted as CD45+Lin−HLA‐DR+CD11c−BDCA2+ cells. CD80 and PDL1 allowed to further differentiate subpopulations amongst pDCs. P1‐pDCs were defined as CD80−PDL1+, P2‐pDCs as CD80+PDL1+ and P3‐pDCs as CD80+PDL1−. Representative flow cytometry dot plots displaying the frequencies of pDC subpopulations found on HD or patient blood and tumor infiltrate from melanoma patients. (c) Comparative frequencies of P0‐, P1‐, P2‐ and P3‐pDCs within alive pDCs in the blood of healthy donors (HD, open circles, n = 20) or patients (Pt, filled circles, n = 25), and tumor infiltrate of melanoma patients (filled triangles, n = 28). Results are expressed as percentages of positive cells. Only significant statistics are shown on the graphs. Bars indicate median. P‐values were calculated using the Mann‐Whitney (dashed lines) and Kruskal–Wallis (straight lines) non‐parametric tests. *P‐value ≤ 0.05, **P‐value ≤ 0.01, ***P‐value ≤ 0.001. (d) Comparative OS and PFS (from sampling time) of patients with low or high circulating P1‐(left panel) or tumor‐infiltrating P3‐(right panel)pDCs. Groups were separated according to the median percentage of circulating P1‐pDCs (2.1%, n = 12 or 13 patients/group) and tumor‐infiltrating P3‐pDCs (1.7%, n = 8 to 10 patients/group). Comparisons between groups were made using the Log‐rank test. (e) Hazard ratios from comparative PFS (from sampling time) of gender, Breslow depth and frequencies of P1, P2‐ and P3‐pDCs in the tumor infiltrate of melanoma patients (n = 27). For pDC frequencies, groups were separated using the median percentage of tumor‐infiltrating P1‐, P2‐ and P3‐pDCs (0.50%, 0.04% and 1.7%, respectively). HD, healthy donor; Pt, patient.