Halo software

B-cell clusters that were identified as TLS were further subdivided from the surrounding tissue using HALO Software (Indica Labs, Albuquerque, New Mexico, USA) for analyses of intra-TLS lymphocytes in this study. Specifically, an individual box was drawn around the TLS in HALO for both PDM and NDMM specimens. B- and T-cell areas were bounded, using PNAd⁺ vasculature to define the border periphery. This enabled analyses of intra-TLS lymphocytes from other intra-tumoral lymphocytes in PDM, and enabled a uniform analysis of TLS T-cell regions in both PDM and NDMM. Immune cells were quantified using HALO Software (Indica Labs, Albuquerque, New Mexico, USA). Lymphocyte density was quantified as density of cells expressing a given marker (cells/mm²) within the ROI. Fractions of lymphocytes expressing a given activation marker were calculated by normalizing the density of cells expressing the lymphocyte marker and the activation marker by the density of cells expressing the lymphocyte marker (Example: Fraction of Ki67⁺CD8⁺ T-cells = CD8⁺Ki67⁺/CD8⁺). Fractions where the denominator was zero were treated as zeros. TLS areas reported in Supplemental Table S1 were quantified using HALO Software. Measurements of the total specimen areas reported in Supplemental Table S1 were obtained on H&E stained specimens and analyzed using ImageJ software.^{14 (link)}