Figure 3.

Analysis of the functional phenotype of intratumoral CD8⁺ and CD4⁺ T cells. The co-expression of GZMB and CD8 was evaluated by sequential IHC. AEC color signals were extracted from each digitized single-marker image by color deconvolution, followed by pseudo-coloring. A representative image is shown (Pt#16). Nuclei (blue), GZMB (red), CD8 (green). Scale bars, 100 μm (A). The GZMB expression between matched pre- and post-therapy samples, either in the total patient cohort or in any of the defined patient groups is illustrated in graphs (all patients n = 14 p = 0.5830, naïve/CT/RT n = 9 p = 0.2500, immuno_treated n = 5 p = 0.4375) (B). Correlation between the GZMB ÷ CD8 ratio and the percentage of PDL1 over total cells in the naïve/CT/RT patient cohort (Spearman r = −0.8667, **p = 0.0045) (C). FFPE pre-therapy sections were analyzed by multiplex IHC. Results from a representative patient (Pt#3) are shown. Nuclei (blue), GZMB (yellow), CD8 (gray), CD4 (green), and Foxp3 (red). Scale bars, 100 μm (D). Changes in the expression of Foxp3 marker in matched pre- and post-therapy lesions are shown in graphs for all patients (n = 12, p = 0.2334) as well as for the two defined patient sub-groups (naïve/CT/RT n = 9, p = 0.7334, immuno_treated n = 3, p = 0.2500) (E). CR and PR Patients are displayed in green, SD in black, and PD in red. Open circles denote on-treatment samples, open triangles post-treatment ones. Statistical comparisons are based on the non-parametric two-tailed Wilcoxon signed-rank test. Only values statistically significant are reported.