ICOS immunoPET enables visualization of activated T cells and early diagnosis of murine acute gastrointestinal GvHD

Abstract

Allogeneic hematopoietic cell transplantation (HCT) is a well-established and potentially curative treatment for a broad range of hematological diseases, bone marrow failure states, and genetic disorders. Acute graft-versus-host disease (GvHD), mediated by donor T cells attacking host tissues, still represents a major cause of morbidity and mortality following allogeneic HCT. Current approaches to diagnosis of gastrointestinal acute GvHD rely on clinical and pathological criteria that manifest at late stages of disease. New strategies allowing for GvHD prediction and diagnosis, prior to symptom onset, are urgently needed. Noninvasive antibody-based positron emission tomography (PET) (immunoPET) imaging of T-cell activation post-allogeneic HCT is a promising strategy toward this goal. In this work, we identified inducible T-cell costimulator (ICOS) as a potential immunoPET target for imaging activated T cells during GvHD. We demonstrate that the use of the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer allows in vivo visualization of donor T-cell activation in target tissues, namely the intestinal tract, in a murine model of acute GvHD. Importantly, we demonstrate that the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer does not affect GvHD pathogenesis or the graft-versus-tumor (GvT) effect of the transplant procedure. Our data identify ICOS immunoPET as a promising strategy for early GvHD diagnosis prior to the appearance of clinical symptoms.

Graphical abstract

Figure 1.

Identification of ICOS as a biomarker of acute GvHD. (A-B) Volcano plots summarizing the differential gene expression analysis performed on publicly available RNA-seq data comparing murine (A) and human (B) T cells recovered from murine models of acute GvHD with cells prior to adoptive transfer. Vertical dashed lines on volcano plots indicate a log₂ fold change of 1.5; horizontal dashed lines indicate an adjusted P value of .05. Genes encoding for selected activation markers previously employed as immunoPET targets are highlighted in red. (C) Representative FACS histograms (left panels) and summary of percentages (right panels) of ICOS expression on CD90.1⁺CD45.1⁺CD4⁺ (top panels) and CD8⁺ (bottom panels) T cells recovered from the spleen 4 and 7 days post–adoptive transfer of C57BL/6 T cells into allogeneic BALB/c recipients. Values are summarized as box plots, representing the range, first quartile, median, third quartile, and eventual outliers. Results are pooled from 2 independent experiments (n = 9-11 mice per group). Day 4 and 7 values were compared with day 0 values using a nonparametric Mann-Whitney U test. P values are indicated. (D) Representative FACS histograms (left panels) and summary of percentages (right panels) of ICOS expression on human-CD45⁺ murine-CD45⁻ CD3⁺ T cells recovered from spleen 14 days post–adoptive transfer of human PBMCs from healthy donors into sublethally irradiated NSG mice. Results are pooled for a total of 9 mice (n = 2-4 mice per group). Day 14 and 0 values were compared using a nonparametric Mann-Whitney U test. P values are indicated. (E-F) Uniform Manifold Approximation and Projection plots of FACS data obtained from the analysis of spleens recovered at day 7 after MHC-mismatch murine allogeneic HCT (E) or at day 14 upon induction of xenogeneic GvHD by adoptive transfer of PBMCs into NSG mice (F). ICOS-expressing cells are depicted in red.

Figure 2.

⁸⁹Zr-DFO-ICOS mAb immunoPET imaging of acute GvHD. (A) Representative BLI of a mouse with acute GvHD at day 4 post-HCT. (B) Reference atlas indicating the location of key clearance and lymphoid organs as well as GvHD target tissues. (C) Representative ⁸⁹Zr-DFO-ICOS mAb PET/CT images acquired 48 hours after tracer administration at day 4 after HCT in TBI control (n = 5), bone marrow control (n = 7), or GvHD (n = 11) mice. Key organs, namely spleen (red arrow), cervical lymph node (cLN) (black arrow), mLN (green arrow), and intestine (yellow arrow), are highlighted. Images are representative of 2 independent experiments. %ID/g, percent injected dose per gram.

Figure 3.

Quantitative PET ROI and BioD analysis of ⁸⁹Zr-DFO-ICOS mAb in acute GvHD. (A) Quantitative ROI analysis of PET images (upper panels) and BioD (lower panels) of spleen, mLN, intestine, liver, heart, kidney, bone, and muscle 48 hours after tracer injection in TBI (blue; n = 5), BM (green; n = 7), or GvHD mice (red; n = 11). Values are summarized as box plots, representing the range, first quartile, median, third quartile, and eventual outliers. Tracer uptake in GvHD and TBI or BM mice was compared using the Mann-Whitney U test. (B) Correlation between tracer uptake measured by PET ROI image analysis with that determined by BioD analysis, evaluated using a Spearman rank correlation coefficient test. Results are pooled from 2 independent experiments. %ID/g, percent injected dose per gram.

Figure 4.

Assessment of ⁸⁹Zr-DFO-ICOS mAb PET imaging as a potential tool for gastrointestinal GvHD early diagnosis. (A) Heatmap visualization and hierarchical clustering of normalized ICOS PET ROI values (rows) from single transplant recipients (columns). Data shown are pooled from 2 independent experiments. (B) PCA of normalized ICOS PET ROI values. Each arrow represents the relative contribution of each ROI. (C) ROC analysis showing sensitivity against 1 − specificity for distinguishing GvHD mice from other cohorts based on PET ROI values for organs identified by the PCA analysis (spleen, mLN, and intestine) and for muscle as a negative control.

Figure 5.

Administration of ICOS mAb at tracer doses does not impact murine acute GvHD or the GvT effect. GvHD score (A) and animal survival (B) after allogeneic HCT. Two-way analysis of variance was used to compare GvHD score and log-rank test to compare survival curve between GvHD mice receiving 10 μg of cold anti-ICOS mAb (continuous red line) and isotype mAb (dashed red line) IV at day 4 post–allogenic transplantation. (C) Representative in vivo BLI images of A20^luc+ cell burden 7 days post-HCT in GvHD mice and BM controls having received cold anti-ICOS mAb or isotope control at day 4 post-HCT. Results are pooled from 2 independent experiments (n = 10 mice/group) and compared using a nonparametric Mann-Whitney U test.