1. ¹Oncology, Washington University School of Medicine, Saint Louis, MO
2. ²Xcyte Therapies, Inc., Seattle, WA

^|[ast]|MB is an employee of Xcyte Therapies, Inc. and has equity interest in the company.

Abstract

Our group has previously reported the development of novel CD34-TK chimeric suicide genes for optimal T cell engraftment, graft versus leukemia effect and minimal graft versus host disease (GvHD) in murine BMT models (Rettig et al., Mol. Ther. 2003; Rettig et al., J. Immunol. 2004). These studies have demonstrated critical functional impairment of murine T cells after selected methods of activation, transduction and selection. Unfortunately no in-vivo models exist to consistently examine the impact of ex-vivo manipulation of human T cells (HuT) on T cell function in-vivo. NOD SCID|[beta]|2M null mice (|[beta]|2 mice) were conditioned with 250cGy TBI on day -1 (n=31), or 300cGy on day 0 (n=22). 10⁷ naive HuT or CD3/28 bead activated (Xcyte|[trade]|Dynabeads|[reg]|) with 50 U/ml IL-2 for 4 days (Act 4d) or 8 days (Act 8d) HuT were injected retroorbitaly (ro), intravenous tail vein injection (iv) or lower HuT doses resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood (PB) of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. (See Table 1)

All mice receiving 300cGy and succumbing to lethal GvHD had well preserved CD4/CD8 ratios (1-1.2). Infiltration of murine tissues was greatest in those mice receiving 300cGy and Act d4 HuT (spleen, liver, lung, kidney: 50-70%). In mice receiving na|[iuml]|ve T cells, expression of activation markers (CD25,CD30,CD69) increased from 1% on day 0 to 28-39% on day 15. In contrast, ex-vivo Act 4d and Act 8d T cells expressed high levels of these same markers on day 0 (62-99%) followed by rapid down regulation on day 3 (2.2-7.8%) and subsequent increased expression similar to na|[iuml]|ve T cells. Of interest, serum human IFN|[gamma]| levels dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=0.27ng/ml and day 15=36ng/ml) compared to mice who did not develop lethal GvHD (day 10=0.04ng/ml and day 17=1.02ng/ml). Finally, we evaluated the HuT engraftment and GvHD potential of naive and Act 4d in RAG2|[gamma]| null mice conditioned with chlodronate liposomes on day -1 and 350cGy on day 0, as previously described by others. We injected 10⁷ and 1.5|[times]|10⁷ naive or Act 4d HuT iv. All mice exhibited low HuT engraftment and no lethal GvHD. (See Table 2)

In conclusion, we have developed a consistent and informative xenograft model of human T cell induced GvHD in NOD SCID|[beta]|2M null mice. This will allow us to study the effects of specific ex-vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on GvHD.