Figure 5.

TAN/T‐cell interactions and TANs polarisation in TDLNs. Graphs illustrate the frequency of different immune cell types (a) and the percentage of TANs interacting with T cells (b) in the metastatic nest (M‐TDLN^M, black bars/circles) and nodal parenchyma (M‐TDLN^P, white bars/circles) M‐TDLN compartments quantified by using a digital microscopy algorithm by ImageJ software. Section of M‐TDLN from gastric carcinoma illustrating a triple interaction between T cells, CD66b⁺ TANs and S100⁺ interdigitating dendritic cells (c, d) in M‐TDLN^M (c) and M‐TDLN^P (d) nodal compartments. The graphs illustrate the percentage of ki‐67⁺CD3⁺ proliferating T cells (e) and of foxp3⁺CD3⁺ regulatory T cells (h) based on counts performed on triple stains (f, g, j, k). The analysis has been performed in the metastatic nest (M‐TDLN^M, black circles, f, j) and nodal parenchyma (M‐TDLN^P, white circles, g, k) compartments of M‐TDLNs CD66b⁺ TAN^Hi with worse prognosis (n = 10) compared with CD66b⁺TAN^Lo with good prognosis (n = 10). As control group, dermatopathic lymphadenitis has been included (grey triangles, d–l). Boxplots reporting median and interquartile ranges of proliferating ki‐67⁺ T cells (%, h) and of foxp3⁺ regulatory T cells (%) (l) in M‐TDLN^M, M‐TDLN^P or D‐LD; P‐values were estimated by the Kruskal–Wallis test and pairwise comparisons using the Dunn’s procedure with a Bonferroni correction for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). M‐TDLNs shown are from OSCC cases (n = 4) (f, g, j, k, m) and stained as labelled. In m, images are taken as snapshot (from Aperio Scanscope) of digitalised slides and resized (bottom panels). Green arrow heads indicate ki‐67⁺CD3⁺foxp3⁺ T cells, and yellow arrow heads indicate ki‐67⁺CD3⁺foxp3⁻ T cells. Sections are counterstained with haematoxylin. Original magnification: 400× (c, d, scale bar 50 μm), 600× (f, g, j, k, scale bar 33 μm).