Alla Nikitine

Mesenchymal stromal cells fail to prevent acute graft-versus-host disease and graft rejection after dog leukocyte antigen-haploidentical bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have been shown to have immunosuppressive effects in vitro. To test the hypothesis that these effects can be harnessed to prevent graft-versus-host disease (GVHD) and graft rejection after hematopoietic cell transplantation (HCT), we administered a combination of 3 different immortalized marrow-derived MSC lines (15-30 × 10⁶ MSCs/kg/day, 2-5 times/week) or third-party primary MSC (1.0 × 10⁶ MSCs/kg/day, 3 times/week) to canine recipients (n = 15) of dog leukocyte antigen-haploidentical marrow grafts prepared with 9.2 Gy of total body irradiation. Additional pharmacological immunosuppression was not given after HCT. Before their in vivo use, the MSC products were shown to suppress alloantigen-induced T cell proliferation in a dose-dependent, major histocompatibility complex-unrestricted, and cell contact-independent fashion in vitro. Among 14 evaluable dogs, 7 (50%) rejected their grafts and 7 engrafted, with ensuing rapidly fatal acute GVHD (50%). These observations were not statistically different from outcomes obtained with historical controls (n = 11) not given MSC infusions (P = .69). Thus, survival curves for MSC-treated dogs and controls were virtually superimposable (median survival, 18 vs 15 days, respectively). Finally, outcomes of dogs given primary MSCs (n = 3) did not appear to be different from those given clonal MSCs (n = 12). In conclusion, our data fail to demonstrate MSC-mediated protection against GVHD and allograft rejection in this model.

Lentiviral vector conferring resistance to mycophenolate mofetil and sensitivity to ganciclovir for in vivo T-cell selection

Several clinical studies of gene-modified T cells have shown limited in vivo function of the cells, immunogenicity of the transgene, and lack of a selective advantage for gene-modified T cells. To address these problems, we developed a lentiviral vector (LV) that provides a selectable, proliferative advantage and potentially decreases immunogenicity for transduced T cells. The bicistronic vector expressed two genes linked with an internal ribosomal entry site. One gene is a variant of the inosine monophosphate dehydrogenase 2, inosine monophosphate dehydrogenase (IMPDHIY), conferring resistance to the immunosuppressive drug mycophenolate mofetil (MMF). The other is a suicide gene, herpes simplex virus thymidine kinase (HSV-TK), rendering proliferating cells sensitive to ablation with ganciclovir, fused to the selectable transmembrane marker ΔCD34 (ΔCD34/TK). Cells transduced with LV-ΔCD34/TK.IMPDHIY were efficiently enriched by immunomagnetic selection for CD34, proliferated in 0.5–5 μM MMF, and were killed by 0.5–25 μg ml−1 ganciclovir. We demonstrate efficient selection and killing of gene-modified cells and suggest LV-ΔCD34/TK.IMPDHIY-transduced T cells could be used to facilitate allogeneic hematopoietic cell engraftment. The expression of IMPDHIY would allow in vivo selection with MMF, and ΔCD34/TK expression would allow rapid and safe elimination of transduced T cells if graft-versus-host disease developed.