Comparison of the Proliferative Kinetics, GVHD Potential and GCV Sensitivity of Naive and Transduced and Selected Murine T Cells after Allogeneic BMT.
Authors: Rettig Michael , Ritchey Julie , Prior Julie , Piwnica-Worms David , Bonyhadi Mark , DiPersio John 
Published: April 2020

Abstract
We and others have previously demonstrated that early administration of the prodrug ganciclovir (GCV) protects mice from lethal GVHD after BMT with allogeneic naive (uncultured) thymidine kinase (TK)-expressing transgenic (Tg) T cells. However, the efficacy of GCV control of GVHD following BMT with ex-vivo activated, TK transduced and selected (Td) donor T cells has not been thoroughly characterized because of the significantly reduced GVHD-inducing potential of cultured cells compared to naive T cells. In this study we used the intracellular fluorescent dye CFSE and in vivo bioluminescence imaging (BLI) to monitor the proliferative kinetics, GVHD-inducing potential and GCV sensitivity of Td and naive T cells after allogeneic and syngeneic BMT. Murine T cells were transduced with either a CD34/TK chimeric fusion suicide gene or a click beetle red luciferase-EGFP (CBR/EGFP) dual function reporter gene. High efficiency (&gt;50%) Td of T cells was accomplished 24 h after activation with anti-CD3/anti-CD28 coated beads and gene-modified cells were purified to &gt; 98% using a MoFlo cell sorter or CD34 immunomagnetic selection 48h post-infection. Naive B6 CD34/TK+ Tg T cells were isolated the day of BMT. Purified T cells were then injected (4e6 total T cells), along with T cell depleted B6 (CD45.1+) BM, into lethally irradiated allogeneic (BALB/c, CD45.2+) or syngeneic (B6, CD45.2+) recipients. The CBR/EGFP BLI signal increased nearly 3 orders of magnitude between days 1 and 8 post-BMT, remained steady for a week, and then only gradually declined over the next month (only a 3-fold decrease between days 14 to 42 post-BMT). Consistent with this BLI data, we found, using CFSE, that the naive and Td CD34/TK+ T cell groups exhibited similar division kinetics during the first week after BMT. At 3 days post-BMT in the allogeneic recipients, CD4 and CD8 cells from both the naive and CD34-TK+ donor T cells were detected in the 8 divisions discernible by CFSE, with approximately equal percentages (5%–15%) of cells in each division cycle. By the next day, however, virtually all of the CD4 and CD8 cells from both donor T cell groups had divided more than 7 times. In contrast, less than 10% of the CD4 or CD8 cells of either donor T cell group had undergone more than 7 divisions in the syngeneic recipients. Most importantly, untreated animals (No GCV) that received either naive or Td T cells died of GVHD, with a median survival of 25 days and 26 days, respectively. To evaluate the ability of GCV to prevent this GVHD, we administered a 7 d course of GCV beginning 1, 4, or 10 days after BMT. Treatment with GCV from days 1–7 after BMT significantly decreased the BLI signal intensity and protected the allogeneic recipients from lethal GVHD, irrespective of the donor T cell source. We observed similar protection from lethal GVHD with significantly reduced weight gain in animals that received Td T cells and treatment with GCV from days 4–10. In contrast, lethal GVHD was evident in day 4–10 GCV treated mice that received naive T cells and in all recipients (irrespective of donor T cell source) that were treated with GCV from days 10–16 after BMT. These observations may have important implications in future clinical trials using TK-expressing T cells to control GVHD after BMT.