Monitoring of developing graft-versus-host disease mediated by herpes simplex virus thymidine kinase gene-transduced T cells.

Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.

Biodistribution and retention time of retrovirally labeled T lymphocytes in mice is strongly influenced by the culture period before infusion.

T lymphocytes used for adoptive immunotherapy are often cultured before transfer to generate sufficient amounts of effector cells with desired specificity. Modification of lymphocytes induced by in vitro activation and expansion may influence their potential effector capacity by altering the survival and trafficking patterns after transfer. In this report, the authors show that the culture period of T cells after ConA/IL-2 stimulation strongly influences the retention and tissue distribution of these cells after infusion into syngeneic C57BL/6 mice. Infused labeled cells that have been cultured for 3 days remained in the peripheral blood and organs in at least a ten-fold higher number than cells cultured for 8 days. In addition, cells cultured for 3 days preferentially migrate to lungs and liver shortly after infusion and subsequently to lymph nodes and spleen. Cells cultured for 8 days preferentially migrate to liver and can be hardly detected in lymph nodes. In contrast, labeled cells cultured for 3 days are predominantly present in lymph nodes starting from day 8 until day 28. We showed that accurate monitoring of transferred cells is feasible, which may contribute to understanding response to adoptive immunotherapy.