In vivo Imaging Technologies to Monitor the Immune System

Abstract

The past two decades have brought impressive advancements in immune modulation, particularly with the advent of both cancer immunotherapy and biologic therapeutics for inflammatory conditions. However, the dynamic nature of the immune response often complicates the assessment of therapeutic outcomes. Innovative imaging technologies are designed to bridge this gap and allow non-invasive visualization of immune cell presence and/or function in real time. A variety of anatomical and molecular imaging modalities have been applied for this purpose, with each option providing specific advantages and drawbacks. Anatomical methods including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound provide sharp tissue resolution, which can be further enhanced with contrast agents, including super paramagnetic ions (for MRI) or nanobubbles (for ultrasound). Conjugation of the contrast material to an antibody allows for specific targeting of a cell population or protein of interest. Protein platforms including antibodies, cytokines, and receptor ligands are also popular choices as molecular imaging agents for positron emission tomography (PET), single-photon emission computerized tomography (SPECT), scintigraphy, and optical imaging. These tracers are tagged with either a radioisotope or fluorescent molecule for detection of the target. During the design process for immune-monitoring imaging tracers, it is important to consider any potential downstream physiologic impact. Antibodies may deplete the target cell population, trigger or inhibit receptor signaling, or neutralize the normal function(s) of soluble proteins. Alternatively, the use of cytokines or other ligands as tracers may stimulate their respective signaling pathways, even in low concentrations. As in vivo immune imaging is still in its infancy, this review aims to describe the modalities and immunologic targets that have thus far been explored, with the goal of promoting and guiding the future development and application of novel imaging technologies.

Keywords: computed tomography (CT); imaging; magnetic resonance imaging (MRI); optical imaging (OI); positron emission tomography (PET); scintigraphy; single-photon emission computed tomography (SPECT); ultrasound.

Figure 1

Currently utilized whole-body immune imaging technologies. Depending on the imaging modality, radioactive symbols may be interchanged with the appropriate tag (i.e., fluorescence, superparamagnetic iron oxide nanoparticles, nanobubbles, etc.). (Left to right) Cells can be directly labeled to track their location and migration. Antibody-based tracers can target molecules or receptors on the cell surface, as well as soluble protein such as cytokines. Proteins including cytokines, ligands, and peptides can also be directly labeled to detect cell populations expressing their receptors. Finally, reporter gene technology can be utilized to achieve specific labeling of adoptive cell populations (e.g. expression of the sodium/iodide symporter as a means of ^99mTc pertechnetate uptake).

Figure 2

Direct cell labeling was utilized to examine acute rejection in rats with renal allografts (aTx) compared to control kidneys (CTR), syngeneic xenografts (sTx), and models of ischemia-reperfusion injury (IRI), and acute Cyclosporin A toxicity (CSA) by examining ¹⁸F-FDG-labeled T cells uptake. They identified significantly higher CD3 accumulation in the acute rejection model compared to the aforementioned models. (A) Maximum intensity projection (MIP) whole-body PET images of rats imaged with ¹⁸F-FDG-labeled T cells to examine renal allograft rejection. (B) The accumulation of the T lymphocytes present in the kidneys is expressed as percent injected dose ± standard error of the mean (%ID ± SEM). This research was originally published in Grabner et al. (48). Permission to reproduce this image has been obtained from the Journal of Nuclear Medicine.

Figure 3

Reporter gene technology. Tregs were transduced with a reporter gene for the Sodium Iodide Symporter (NIS) for specific ^99m${\text{TcO}}_4^{-}$ uptake. C57BL/6 mice were injected with NIS Tregs and injected 1 day later with ^99m${\text{TcO}}_4^{-}$ to examine Treg uptake in the spleen, demonstrating in vivo radiolabeling of Tregs. Mice were imaged by NanoSPECT/CT with a focus on the spleen (white arrow). This research was originally published in Sharif-Paghaleh et al. (74). Permission to reproduce this image has been obtained from the PLOS One.

Figure 4

Antibody-based tracers. Antibody based tracers have been developed to target immune cell populations. However, one hurdle to overcome for some antibodies is Fc-mediated depletion. Tavaré et al. developed two ⁶⁴Cu-NOTA anti-murine CD8 minibodies. (A) ⁶⁴Cu-NOTA-2.43Mb exhibited targeted spleen uptake in B/6 mice. (B,C) Both the blocking cohort and CD8 depleted cohort displayed decreased spleen uptake (Upper images—Coronal MIPs, Lower images—Transverse). This research was originally published in Tavaré et al. (89). Permission to reproduce this image has been obtained from the Proceedings of the National Academy of Sciences of the United States of America.

Figure 5

Antibody based tracers in non-cancerous diseases. ImmunoPET tracer development has also extended to immunogenic diseases such as Colitis. Freise et. al. imaged CD4⁺ T cells with ⁸⁹Zr-malDFO-Gk1.5 cys-Diabody (cDb), to non-invasively monitor inflammation of the intestines caused by specific cell subsets. Ex vivo representative images of the colons, ceca and mesenteric lymph nodes (MLNs) are shown comparing tracer uptake in a dextran sulfate sodium (DSS) induced colitis model compared to control. The DSS mice had 3.1-, 3.9-, and 3.0-fold increased uptake in the colons, ceca, and MLNs, respectively. This research was originally published in Freise et al. (91). Permission to reproduce this image has been obtained from the Journal of Nuclear Medicine.

Figure 6

Directly labeled cytokines, ligands and peptides. A high affinity consensus (HAC) of PD-1, ⁶⁴Cu–DOTA–HAC, is an example of a directly labeled ligand that was used to target PD-L1. (A) The PET-CT images were acquired 1 h post injection in NSG bearing mice. The specificity of the tracer was evaluated by imaging CT26 tumors that were PD-L1⁺ (red dashed line), PD-L1⁻ (white dashed line), PD-L1⁺ blocked or dual tumors (PD-L1⁺ left, PD-L1⁻ right). (B) The uptake was quantified in %ID/g. Error bars represent SD. The decreased uptake in the blocked hPD-L1(+) tumors and the hPD-L1- tumors indicated specificity of the tracer. **P < 0.01. This research was originally published in Maute et al. (115). Permission to reproduce this image has been obtained from the Proceedings of the National Academy of Sciences of the United States of America.