In Vivo Bioluminescence Imaging (BLI) and Sequential 18F]FHBG microPET Imaging Studies of Human T Cell (huT) Trafficking, Expansion and Xenogeneic Graft-Versus-Host-Disease (XGVHD) Following Different Routes of T Cell Administration.
Authors: Nervi Bruno , Rettig Michael P. , Ritchey Julie K. , Wang Hanlin L. , Bauer Gerhard , Walker Jon , Prior Julie L. , Piwnica-Worms David , Nolta Jan A. , DiPersio John F. 
Published: October 2019

Abstract
GVHD is the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. We developed a consistent and sensitive model of human GVHD by retroorbitally (ro) injecting huT cells into sublethally irradiated NOD/SCID-β2mnull mice (β2). Using this model, 59% (20/34) mice conditioned with 250cGy and injected ro with 107 huT cells developed lethal XGVHD and exhibited huT cell engraftment, weight loss, and infiltration in multiple target tissues. β2 mice injected with huT through the lateral tail vein (iv) had only small and transient huT cell engraftment and failed to develop lethal XGVHD. To evaluate why the ro versus iv route affected huT cell engraftment and the development of lethal XGVHD, we performed in vivo BLI and sequential [18F]FHBG microPET imaging studies. We used huT cells transduced with a click beetle red luciferase/enhanced green fluorescent protein (CBRluc/egfp) BLI reporter gene (huT*) and/or a chimeric CD34-thymidine kinase fusion suicide/PET gene. We injected 107 huT* cells either iv or ro into 250cGy irradiated β2 mice. Mice injected iv (n=5) failed to develop lethal XGVHD, whereas 60% of the mice injected ro (n=5) developed lethal XGVHD. During the first week, mice injected ro presented early expansion of the huT cells in the retroorbital cavity and local regional draining lymph nodes followed by widespread dissemination throughout the whole body, with increased percentage of huT in the blood (p<0.04), and lost significant body weight (p<0.02) in comparison to mice injected iv. Serial BLI revealed very different trafficking and expansion profiles between the two routes of administration. HuT*cells injected iv immediately trafficked to the lungs and failed to expand during the first 3 weeks. In contrast, the ro-injected cells remained in the ro cavity and trafficked to secondary lymphoid organs during the first week. This altered trafficking of the ro-injected cells was associated with >10-fold increase in the BLI signal during the first 2 weeks. This homing pattern and strong BLI signal of the ro-injected huT* cells remained until death from GVHD. The BLI signal of the iv-injected huT* cells increased between days 21 and 45. However, this late expansion was not associated with the development of XGVHD. We also generated huT cells that co-expressed the suicide gene CD34-TK75 and CBRluc/egfp (huT**). We observed the same results after we injected 0.25×107 huT** cells into 250cGy irradiated and anti-CD122 mAb-treated NOD/SCID mice followed with BLI and PET. These studies demonstrated the ability to image trafficking of the transduced T cells in vivo with BLI and PET. In summary, all mice that received huT iv showed initial clearing of the huT cells in the lungs and a significant delay in their in vivo expansion abrogating their potential to cause XGVHD. In contrast, the development of lethal XGVHD in immunodeficient mice was dependent upon the initial retention and early expansion of huT in the retroorbital cavity.