Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

Abstract

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T cells following in vitro activation and post immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM.

Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.

FIGURE 1

ICIs influence T-cell distribution in tumor and spleen. Mice (n = 28) were inoculated with GL261 cells, treated with ICI or vehicle control, and analyzed at the designated time points relative to treatment. A, Schematic showing tumor inoculation day, treatment days, and time points relative to treatment in which mice were analyzed. B, Percentage of CD8⁺ TILs (red dots) and CD4⁺ TILs (blue dots) in each time point relative to treatment, for vehicle-control group (left panel) and ICI-treated group (right panel) C, Absolute number of TILs per mm³ of tumor. CD8⁺ cells × 10² (upper panels; red dots) and CD4⁺ cells × 10² (bottom panels; blue dots) in each time point relative to treatment, for ICI-treated group and vehicle-control group. D, Frequencies of CD8⁺ (red dots) and CD4⁺ T-cells (blue dots) in spleen in each time point relative to treatment, for vehicle-control group and ICI-treated group. B–D, Shown are means ± SEM. Scattered dots represent individual mice. Shown are representative data of two independent experiments. n = 3–4 per group. CD8⁺ and CD4⁺ were gated from CD3⁺CD45⁺ cells. One-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001).

FIGURE 2

Kinetics of activation markers on T cells following ICI treatment. Mice with intracranial GL261 gliomas were treated with ICI, and analyzed at the designated time points after treatment, as in Fig. 1. A, Percentages of CD69⁺ of CD8⁺ (left) and CD4⁺ TILs (right). B, Percentages of CD69⁺ of CD8⁺ (left) and CD4⁺ T cells (right) in spleen. C, Trajectories of CD69⁺/CD8⁺ in TILs (green line) and spleen (purple line) ratios at the designated time point compared with the pretreatment group. D, Percentages of PD-1⁺ of CD8⁺ (left) and CD4⁺ TILs (right). E, Percentages of PD-1⁺ of CD8⁺ (left) and CD4⁺ T cells (right) in spleen. F, Trajectories of PD-1⁺/CD8⁺ in TILs (blue line) and spleen (red line) ratios at the designated time point compared with the pretreatment group. G, Percentages of TIM3⁺ of CD69⁺/CD8⁺TILs. For A, B, D, E, and G, mean ± SEM values are shown. Scattered dots represent individual mice. Shown are representative data of two independent experiments. n = 4 per group. One-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001). C and F, Two-way ANOVA with multiple comparison test is used to compare the ratios between TILs and spleen T cells at each time point (*, P < 0.05; **, P <0.01; ***, P <0.001).

FIGURE 3

CD69 expression increases on TILs following ICI treatment in patients with GBM. A–C, scRNA-seq analysis of sorted CD45⁺ immune cells (n  =  156,766 cells) from 40 patients with GBM: 14 GBM.new, 12 GBM.rec, and 14 GBM.pembro. A, UMAP cell clustering analysis of tumor cell cluster (SOX4; top right), myeloid cell cluster (CD14; middle right), and lymphocyte cluster (CD3D; bottom right). B, UMAP data color-coded by the expression of CD3 (red), CD69 (purple; top), and coexpression (merge) in each group of patients (bottom). C, Dot plot analysis of cells within the lymphocyte cluster; the size of the dots corresponds to the percentage of cells expressing the feature (gene). The color represents the average expression level.

FIGURE 4

Immuno-PET of ⁸⁹Zr-DFO-CD69 Ab visualizes the TME after ICI treatment in a GBM mouse model. Mice were inoculated with GL261 cells, and evaluated by immuno-PET of ⁸⁹Zr-DFO-CD69 Ab, comparing the treatment (ICI) group to the control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, tail vein injection of ⁸⁹Zr-DFO-CD69 Ab, immuno-PET, and BioD. B, Representative coronal head images from immuno-PET of ⁸⁹Zr-DFO-CD69 Ab of ICI-treated and control mice at designated time points. Scales show SUVs of PET (SUV; colored) and CT (HU; gray). C and D, Comparison between ICI-treated mice and control at designated time points of tumor-specific regions’ SUV_max (C) and SUV_max TBR (D). E, Representative coronal-ventral and sagittal 3D whole-body MIP PET images of ⁸⁹Zr-DFO-CD69 Ab of control and ICI-treated mice acquired 6 days after tracer administration. The scale shows SUVs of PET. F, Day 6 BioD results of blood- and tumor site–associated radioactivity, assessed as %ID/g. In C, D, and F, bars show means ± SEM. Scattered dots represent individual mice. Shown are representative data of three independent experiments, n = 5 per group. C and D, Multiple unpaired t test with Welch correction. F, Two-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001). IP, intraperitoneal.

FIGURE 5

Immuno-PET of ⁸⁹Zr-DFO-CD69 Ab as a prognostic predictor after ICI treatment in a GBM mouse model. Survival follow-up of mice inoculated with GL261 cells, and evaluated by immuno-PET of ⁸⁹Zr-DFO-CD69 Ab, comparing treatment (ICI) group to control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, MRI, tail vein injection of ⁸⁹Zr-DFO-CD69 Ab, immuno-PET, and survival follow-up. B, The overall survival (OS) rates of the ICI-treated group and control group are plotted using Kaplan–Meier survival curves, with log-rank (Mantel–Cox) curve comparison test. C, Right, heat map display of Pearson correlation coefficient analysis between immuno-PET signals and survival in the two groups. Results are shown for each SUV measurement method (SUV_max, SUV_max TBR, SUV_mean, and SUV_mean TBR) in each immuno-PET designated time point (days 1, 2, 3, 4, and 6). Shown are representative data of two independent experiments, n = 4–5 mice per group (*, P < 0.05; **, P <0.01). Left, examples of scatter diagrams of Pearson correlation coefficient taken from immuno-PETs on day 2 of SUV_max and SUV_max TBR. Scattered dots represent individual mice, n = 4–5 mice per group.