258. Multi-Modality Non-Invasive Imaging of NK-92 Cells Trafficking and Tumor Targeting in Mice
Authors: Bruno Nervi, Michael Rettig, Julie Ritchey, John Walker, Gerhard Bauer, Philip Herrbrich, Jan A. Nolta, John F. DiPersio
Journal: Molecular Therapy (2004)

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality following allogeneic bone marrow transplantation. We recently developed a novel chimeric suicide gene in which the extracellular and transmembrane domains of human CD34 were fused in frame to the entire coding region of HSV-tk (CD34-tk). In this study, we developed a new model for xenogenic GVHD and evaluated the ability of GCV to prevent GVHD after infusion of CD34-tk-modified human T cells (Hu T). To induce GVHD, sublethally irradiated immunodeficient mice were injected intravenously (i.v.) (tail vein) or retroorbitally (r.o.) with human PBMCs or purified T cells. Engraftment in the peripheral blood was evaluated weekly by FACS and mice were sacrificed if they lost > 20% body weight. NOD-SCID-β2M null mice conditioned with 250 cGy and injected i.v. with human PBMCs (107 T cells; n = 4) or T cells (0.5–2 × 107; n = 28) failed to engraft and did not develop GVHD. A similar pattern was observed in RAG2-/-γc-/- mice conditioned with 350 cGy and injected i.v. or r.o. with human PBMCs (n = 9) or T cells (n = 11). In contrast, NOD-SCID-β2M null mice conditioned with 250 cGy and injected r.o. with human PBMCs (107 T cells; n = 11) or T cells (1-2 × 107; n = 16) exhibited 17% Hu T engrafment (range 1.5–62%) 2 weeks post-infusion and developed lethal GVHD, with an overall survival of 75% and 25%, respectively, at 5 weeks. Consistent with GVHD, these mice lost weight (>20%) and exhibited decreased activity, hunched posture and ruffled fur. Histological, immunohistochemical, and FACS analyses demonstrated Hu T infiltration in the spleen (46%), liver (60%), kidney (40%), lung (49%) and bone marrow (11%). Furthermore, we observed a 10–15 fold increase in the expression of T cell activation markers CD25, CD30, and CD69 in both the peripheral blood and tissues. We then evaluated whether CD34-tk/GCV suicide gene therapy could be used to mitigate this xenogenic GVHD. High efficiency transfer (56%) of CD34-tk to primary Hu T was accomplished after a single exposure to VSV-G pseudotyped, MLV-based retrovirus 48 h post-activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Transduced T cells (Td T) were purified 2 days post-infection by CD34 immunomagnetic selection (98% CD34+) and 107 cells were injected r.o. into 250 cGy conditioned NOD-SCID-β2M null mice. Although we observed >15 % engraftment (range 2–37%) and expression of CD25, CD30, and CD69 in the peripheral blood of mice 4 weeks post-infusion of the Td T, we observed no GVHD. However, we were able to demonstrate that the Td T cells could be efficiently eliminated in vivo by treatment with GCV. Animals treated with GCV from days 1–7 had less than 1% engraftment at 4 weeks post-infusion. This selective elimination of the Td T cells was not sustained however, as evidenced by the 5% and 18% Hu T engraftment in the peripheral blood and spleen, respectively, of mice at week 10 post-infusion. In summary, we have developed a new model of human T cell induced GVHD in the NOD/SCID/B2M null mouse. This model provides a unique opportunity for preclinical testing of the CD34-tk/GCV suicide gene system as well as other methods of GVHD control.