Comparing a Novel PET Agent, Yttrium-86 Oxine, with Biopsy and FACS Analysis for Tracking in Vivo Migration Patterns of Different Preparations of DC from a Single Donor

Aim: The objective of this study was compare the in vivo distribution of different preparations of dendritic cells (DC) from a single donor after intradermal (ID) administration and correlate FACS analysis of excised tissue and imaging findings.

Method: DC were generated from a single healthy donor apheresis product. The DC types were GM-CSF + IL-13 (IL-13 imDC), then further matured with FMKp+IFNg (IL-13mDC), GM-CSF + IL-4 (IL-4 imDC) and matured (IL-4 mDC) and GM-CSF+IFN-α (IFN-α DC). Antigen naive DC preparations were labelled with Yttrium-86 oxine (prepared on site) by incubation at room temperature for 30 minutes followed by centrifugation and resuspension. 5 × 105 radio-labelled cells in 50_L was injected ID into the footpad and the volar aspect of the foreleg of Balb/c nude mice (n=5 per group). A cohort (n=5) of control mice were similarly injected with Yttrium-86 oxine only. Images were acquired on a small animal PET (SAPET) (Mosaic, Philips Medical Systems) at 2, 24 and 45 hours post administration. Mice were then euthanized and popliteal, axillary and inguinal lymph nodes (LN) harvested. LN were processed into single cell suspension, stained with anti-human HLA-DR and CD11c and analysed by flow cytometry. SAPET images were graded: 0 – no signal, 1 – marginal, 2 – definite nodal signal, 3 – strong signal. DC detection by FACS was graded: 0 – no detection, 1 – <0.5% DC, 2 – 0.5.⋄1.5% DC, 3 – >1.5% DC of all viable cellular events. Results: Pre-injection analysis of DC showed expression of HLA-DR, HLA-ABC, CD11c, CD80 and CD86, low levels of CD83, CCR5 and CCR7 along with down regulation of CD14. Matured DC populations showed increased levels of CD83 and CD86. Similar phenotypic expression on the DC populations were observed prior to cryopreservation and upon thawing. Mean DC radiolabelling efficiency of 72% (54% – 88%). There was a minor decrease in cell numbers and viability after labelling most likely due to oxine related toxicity. There were no substantial differences in tracking between cell types. In 28 of 43 nodes assessed, FACS and SAPET results were congruent (27 FACS and SAPET positive and 1 FACS and SAPET negative). Of the 15 discrepant samples, 13 were SAPET positive and FACS negative, while 2 were FACS positive and PET negative. Average FACS and SAPET grades at 45 hours were higher for popliteal nodes than axillary nodes. No false positive nodal accumulation was visualised at 45 hours in control mice. Conclusion: This novel PET technique provides a robust, reproducible method for quantification of in vivo functional DC tracking to LN. Detection of DC migration using this novel PET agent is a more reliable technique than biopsy and FACS analysis, which has an inherent sampling error.