Fig 2: Tregs populate perivascular CCR7+ DC niches in human and mouse tumors(A) Heatmaps depict the enrichment of immune and non-immune cell types in the immediate neighborhood of CCR7+ DCs in NSCLC spatial transcriptomic data (n = 4).47(B) (Left) Representative FOV displaying CCR7+ DCs (HLA-DR+LAMP3+; yellow) located near BVs (CD31+PDPN−; magenta) and Tregs (CD4+FOXP3+; white) in one HNSCC sample using high-plex whole-tissue imaging. Scale bar represents 20 μm. (Right) Box plots display the frequencies of BV-associated, LV-associated, and non-vessel-associated CCR7+ DCs close (<5 μm) to Tregs among all tumor CCR7+ DCs with nearby Tregs. Wilcoxon test, whiskers represent min to max; *p < 0.05.(C) Correlations between CCR7+ DCs and Tregs within CD45+ cells, as determined by scRNA-seq in multiple human cancer types. Spearman rank correlation; significant correlations are shown with a fitted red line.(D) (Left) Scheme outlining the analyses of CCR7+ DCs and Tregs in NSCLC samples. Patients with numerous (>5) CCR7+ DC clusters (n = 12) were selected for downstream analyses. (Right) Frequency of CCR7+ DCs (CD11c+LAMP3+) with at least one nearby (<50 μm) Treg (CD4+FOXP3+) in each individual patient. Numbers of FOVs analyzed per sample are as follows: NR01, n = 126; NR06, n = 455; NR09, n = 180; NR12, n = 79; NR26, n = 293; R11, n = 122; R15, n = 205; R35, n = 175; R37, n = 459; R45, n = 276.(E) (Left) Scheme outlining the analysis of tumor biopsies from HNSCC patients before immunotherapy (pre-IO). Patients were divided into non-responders (NR, n = 5) and responders (R, n = 5) based on the assessment of clinical response at 6 months. (Right) CCR7+ DC shortest distance to Tregs, TCONV, and CD8+ T cells in NR versus R tumors. Data are shown for all CCR7+ DCs compiled (NR tumors, n = 1,457 cells; R tumors, n = 1,324 cells). Unpaired t test, whiskers represent min to max; ****p < 0.0001. Numbers of FOVs analyzed per sample as in (D).(F) (Left) Scheme outlining the analyses of CCR7+ DC-CD8+ T cell niches. (Right) Frequencies of CCR7+ DC-CD8+ T cell niches with or without Tregs in their proximity (<100 μm). Two-way ANOVA with multiple comparisons, whiskers represent min to max; *p < 0.05. Numbers of FOVs analyzed per sample as in (D).(G) Representative FOV displaying CCR7+ DCs (FSCN1+ cells; FSCN1 in yellow) located near BVs (CD31+LYVE-1− cells; CD31 in magenta) and Tregs (FOXP3+ cells; FOXP3 in white) in untreated MC38 tumors. Scale bar represents 50 μm.(H) Correlations between the numbers of CCR7+ DCs and Tregs per mg of tumor tissue, as determined by fluorescence-activated cell sorting (FACS) analyses of MC38 and D4M3. A tumors. Spearman rank correlation; significant correlations are shown with a fitted red line.(I) Box plots show the frequencies of tumor CCR7+ DCs close (<5 μm) to Tregs that are associated to BVs or LVs in MC38 tumors (n = 7). Whole-tumor sections were analyzed. Paired t test, whiskers represent min to max; ****p < 0.0001.See also Figures S5 and S6.