Quantification of CD8 Immunostaining

Digital images of stained slides were acquired using Leica’s Aperio VERSA 8 automated microscope. TMA spots were automatically identified and analyzed using the Image Scope 12.3.3 software package (Leica Microsystems; Wetzlar, Germany) according to the following procedure: every TMA slide was scanned at 40 × magnification. Digital images were segmented using the Image Scope brightfield TMA-Tool, and the segmentation was corrected manually. Two Aperio ePathology Image Analysis macros (Leica Microsystems) were adjusted to determine the number of CD8⁺ cells in each tissue spot and to measure the corresponding area of each tissue spot. The number of stained cells and the area in square millimeters of each individual spot was used to calculate the density of CD8⁺ stained cells/mm² (number of cells per square mm). Schematic representation of the workflow to detect CD8 positive cell density is shown in supplementary Fig. 1.