Summary

CD8 + T cells can be classified into distinct subsets based on their functional properties, homing preferences and differentiation status, typically defined by the expression of surface markers such as CD45RA, CD45RO, CCR7, CD28, and CD62L. Naïve CD8 + T cells (N, CD45RA + CCR7 + CD28 + CD62L+) not yet encountering antigen, primarily circulate in lymphoid tissues. In contrast, central memory CD8 + T cells (CM, CD45RO + CCR7 + CD28 + CD62L+) while also residing in lymphoid tissues provide long-term immune protection by retaining the ability to proliferate rapidly upon antigen re-exposure. Effector memory CD8 + T cells (EM, CD45RO + CCR7 − CD28 − CD62L−) are primed for rapid effector responses and circulate in peripheral tissues, albeit with reduced longevity compared to their central memory counterparts. Finally, the terminally differentiated T cells that re-express CD45RA (EMRA, CD45RA + CCR7 − CD28 − CD62L−) comprise an important fraction of CD8 + T cells in the blood, exhibit features of replicative senescence and are significantly expanded in older individuals. CD8 + EMRA cells tend to increase in blood and blood-rich sites such as the bone marrow, spleen and lungs as individuals age. Thus, the accumulation of EMRA cells with age reflects a shift in the CD8 + T cell compartment contributing to the functional alterations observed in adaptive immunity during ageing.
Given these systemic changes that occur with age, it is increasingly important to employ advanced imaging techniques that can capture the dynamic interplay of immune cells throughout the body. Total body positron emission tomography (TB-PET) imaging allows for clinical non-invasive total body imaging with a 10- to 40-fold increase in sensitivity compared to conventional PET systems. TB-PET therefore provides the means to analyse immune cell localisation in the whole body in detail, using small numbers of radionuclide-labelled cells. Using radionuclides for PET imaging makes the detection and quantification of discrete cell populations, including radiosensitive T cells, within defined tissue locations possible.
PET radionuclides with a long half-life, such as ⁸⁹Zr (t_1/2 = 78.4 h), can be used for (pre)clinical imaging and enable the detection of cells longitudinally from repeat PET scans. Indirect radiolabelling through ⁸⁹Zr-labelled antibodies has been used to demonstrate the role of CD8 + T cells in tumour immunity and their response to therapy. However, direct labelling of immune cells with ⁸⁹Zr provides increased sensitivity and specificity and can be achieved through the chelation of ⁸⁹Zr with four oxine (8-hydroxyquinoline) molecules to generate a metastable complex that can cross cell membranes. This complex dissociates inside cells, depositing ⁸⁹Zr bound to intracellular proteins. A usual caveat with direct labelling is that lymphocytes are prone to radiation damage, senescence, and cell death. The advent of TB-PET has meant that T cells can now be labelled with ⁸⁹Zr at lower activity levels without compromising detectability; this approach showed no reduction in cytokine production or migration when subjected to 70 kBq/10⁶ cells. However, it remains unexplored whether CD8 + EMRA cells responds differently to cell labelling and whether homing abilities are altered. This is important as most chronic diseases requiring analysis by PET generally occur at an older age. Therefore, it is of interest to determine whether the build-up of CD8 + EMRA T cells with age influences the ability of ⁸⁹Zr labelling for imaging.
The authors show here the radiolabelling efficiencies and retention of human CD8 + T cells isolated from young and older individuals with [⁸⁹Zr]Zr(oxinate)₄ and assess the distribution of these radiolabelled CD8 + T cells in vivo. They found that cryopreservation does not influence the ability of CD8 + T cells to be labelled with ⁸⁹Zr and that cryopreserved ⁸⁹Zr-CD8 + T cells were readily detected using PET imaging for at least up to three days in mice. Longitudinal assessment by in vivo PET scanning indicated retention of ⁸⁹Zr within CD8 + T cells albeit it with different migratory patterns observed when using young and old T cells. This study suggests that the age of CD8 + T cells should be considered in future longitudinal PET imaging studies using in vivo cell labelling as it may influence the migratory capacity of the studied cells.

Results from nanoScan® PET/CT

For the imaging studies, ⁸⁹Zr-labelled CD8 + T cells were injected intravenously into NOD scid gamma (NSG) mice (3 × 10⁶ cells/animal in 100 µL PBS, 63 ± 44 kBq, single CD8 + T donor per experiment) under anaesthesia (1.5–2.5% isoflurane in oxygen). Three hours post-injection, mice were re-anaesthetised and placed in a preclinical nanoScan® PET/CT scanner (Mediso) where anaesthesia was maintained, and the bed was heated to maintain a normal body temperature. Two hours of PET acquisition (1:5 coincidence mode; 5 nanoseconds coincidence time window) were followed by CT. PET-CT was repeated at t = 24 and 72 h (2 and 3 h, respectively). Kinetic information from the PET/CT images were reconstructed using a Monte Carlo-based full-3D iterative algorithm (Tera-Tomo, 400–600 keV energy window, 1–3 coincidence mode, 4 iterations and subsets) at a voxel size of (0.4 × 0.4 × 0.4) mm³ at 2 frames per minute and corrected for attenuation, scatter, and decay. Images were co-registered and analysed using VivoQuant v.3.0 (InVicro LLC) capturing maximum intensity projection (MIP) and transverse plane images. Image analysis was conducted on the summed images for each time point with background, not associated with anatomy, being removed manually. Spherical volumes of interest (VOIs) were placed on organs based on the CT image when tissue, e.g. spleen, was not visible. Percentage injected activity (%IA) was calculated as follows %IA = (Activity remaining at scan time / Initial injected activity) * 100 to assess overall injected activity in each animal and %IA/mL to determine injected activity distribution and concentration in selected organs.
Owing to the loss of ⁸⁹Zr from aged CD8 + T cells (Fig. 1C), significantly less activity was injected when using these cells (Young CD8 + T cells: 105 ± 32 kBq; Old CD8 + T cells: 22 ± 17 kBq). Despite this difference, PET imaging successfully detected CD8 + T cells from both age groups, with CD8 + T cells initially localising to the lung and liver (3 h, Fig. 3A-D). While young and old CD8 + T cells gradually accumulated in the spleen, a higher proportion of old CD8 + T cells remained in the liver and lung (Fig. 3C, D). The organ weight and injected radioactive activity were normalised for all imaging timepoints. Biodistribution showed the highest concentration of ⁸⁹Zr labelled CD8 + T cells were found in the spleen (Fig. 3E). However, CD8 + T cells from older individuals accumulated in the spleen at a significantly lower rate compared to cells isolated from young individuals (Fig. 3E). Uptake in other organs showed no major differences between the two groups, suggesting that the radiolabel remains stably associated with T cells in vivo (Supplementary Fig. 1B). Additionally, the absence of significant bone uptake, where free ⁸⁹Zr typically accumulates further indicates cell-bound activity rather than free ⁸⁹Zr. It is possible that free ⁸⁹Zr may be distributed diffusely throughout the body, resulting in low-level, sub-threshold activity that is not readily detectable in individual organs. However, the authors believe that the loss of ⁸⁹Zr occurred prior to injection, suggesting an age-related decline in the migratory capacity of CD8 + T cells from older individuals.

• In summary, work presented here highlights the importance of considering cellular senescence when applying specific diagnostic modalities.
• In particular, these findings have implications for human CD8 + T cell tracking and detailed localisation preference using TB-PET imaging.
• This study established a method for labelling and tracking cryopreserved CD8 + T cells, though further research is needed to understand the differences in migratory behaviour between cells from young and older individuals.

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