Figure 4.

CD19-CD28 increases proinflammatory T-cell signatures in tumors and facilitates transendothelial migration. (A) Experimental design of in vivo efficacy study. OCI-Ly18 tumor-bearing humanized NSG mice were treated with vehicle (histidine buffer, 10 mice per group), glofitamab (0.15 mg/kg or 1 mg/kg, 25 mice per group), and CD19-CD28 (1 mg/kg, 25 mice per group). On day 19 and 21, five scouts per group were euthanized for analysis. Gpt: Gazyva (obinutuzumab) pretreatment, 30 mg/kg. (B) Gene signature analysis via RNA sequencing from frozen tumor tissue at different time points. Color code shows change between glofitamab monotherapy vs combination with CD19-CD28 (red denotes higher and blue lower expression in the combination). Statistical significance was calculated using ∗P < .05, ∗∗P < .01 (adjusted for multiple testing using FDR correction). Individual genes are shown in supplemental Figure 5E (C) Immunofluorescence microscopy of tumors on study day 29, showing ICAM1 (red), CD31 (green), and CD8 (cyan). Images were captured on a Leica SP8 inverted confocal microscope using a 40× lens, with a resolution of 512 × 512 pixels and a z-spacing of 1.5 μm. (D) Image quantification was performed with Imaris 9.6. Dot plots show spots created on ICAM1 signal per μm³ (left) and spots created on ICAM1-expressing endothelial (CD31⁺) cells of total ICAM1-expressing cells in tumors (right) on study day 29, quantified by fluorescence microscopy. Dots represent intratumoral regions from 1 or more mice. One-way ANOVA with Tukey multiple comparison test: ∗P < .05; ∗∗P < .01. FDR, false discovery rate; ICAM1, intercellular adhesion molecule 1.