Fig. 4: Spatial relationship between CD4⁺ T cells, Tregs and tumor cells predicts pembrolizumab response in CTCL.

a SpatialScore schematic. The SpatialScore is calculated by taking the ratio of the physical distance between each CD4⁺ T cell and its nearest tumor cell (distance “right”) relative to its nearest Treg (distance “left”). [1] A lower SpatialScore (i.e., CD4⁺ T cells closer to tumor cells than Tregs) suggests increased T cell effector activity. [2] A higher spatial score (i.e., CD4⁺ T cells closer to Tregs than tumor cells) suggests increased T cell suppression. b–c SpatialScore calculated per cell for all CD4⁺ T cells (b) and PD-1⁺ CD4⁺ T cells (c) across patient groups (mean ± s.e.m.). P values calculated with a linear mixed-effect model taking a patient identifier as a random effect. d GZMB protein expression on PD-1⁺ CD4⁺ T cells by CODEX per tissue microarray spot (mean fluorescence intensity (arbitrary units, a.u.), red bar). P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. e CODEX images showing contact between a tumor cell (cross) and GZMB-expressing PD-1⁺ CD4⁺ T cell (arrow) in responder patient 13 post-treatment. Scale bars, 10 µm. f Cytotoxicity gene scores, computed on bulk RNA-seq data, per tissue microarray spot. Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. g–h Pre- to post-treatment changes in tumor therapy resistance gene scores, computed on bulk RNA-seq data, per patient in responders (g) and nonresponders (h). Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values were calculated by two-sided Wilcoxon’s signed-rank tests. i Ki-67⁺ tumor cell frequencies per tissue microarray spot (mean, red bar). P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. j SpatialScore calculated from Vectra mIHC data per cell for all PD-1⁺CD4⁺ T cells (mean ± s.e.m.). P values calculated with a linear mixed-effect model taking a patient identifier as a random effect. k Vectra mIHC images (left panels) and corresponding spatial plots (right panels)from responder patient 13 (R) and nonresponder patient 14 (NR) pretreatment. Scale bars, 20 µm. l SpatialScore calculated from Vectra mIHC data per patient in responders and nonresponders pretreatment(mean, red bar). P value calculated by a two-sided Wilcoxon’s rank-sum test, with no adjustments for multiple hypotheses. Source data are provided as a Source Data file.