Selective depletion of alloreactive donor T cells leads to elimination of graft-versus-host reactivity and stimulates graft-versus-leukaemia/myeloma effect.

Graft-versus-host disease is a severe complication of allogeneic stem cell transplantation. The major role is played by alloreactive donor T-cell clones leading to host tissue damage. Selective depletion is a strategy to eliminate host-reactive donor T cells from haematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. We have used irradiated peripheral blood mononuclear cells from cancer patients and healthy donor cells as responder cells in primary mixed leukocyte reaction. To prepare graft-versus leukaemia/myeloma-specific T cells, alloreactive T cells in primary mixed leukocyte reaction were depleted with anti-CD25 immunotoxin. The remaining T cells had insignificant alloreactivity in secondary mixed leukocyte reaction. Then, allodepleted donor T cells were repeatedly stimulated using purified leukaemia/tumour cells from the same cancer patient. Leukaemia/tumour-reactive donor T cells were purified using cell sorter on the basis of CD4 and CD8 activation. Their specificity was tested in nonradioactive cytotoxicity test. We performed 22 reactions (15 samples with leukemic and 7 samples with multiple myeloma cells). Selective depletion of alloreactive donor T cells with anti-CD25 immunotoxin led to significant depletion (99.2-100 %, median 99.7%). The effect of donor T cells was well preserved, while the graft-versus-host reactivation of donor cells was negligible, even after repeated stimulation with patient's non-tumour cells. Thus, it is possible to selectively deplete donor alloreactive T cells with anti-CD25 immunotoxin. In the cases of leukaemia patients, a strong graft-versus-leukaemia reactivity was noticed in allodepleted donor T cells; in myeloma patients, graft-versus-myeloma reactivity was less significant.

Tumor-specific histone signature and DNA methylation in multiple myeloma and leukemia cells.

Understanding the epigenetics of tumor cells is of clinical significance for the treatment of cancer, and thus, chemists have focused their efforts on the synthesis of new generation of inhibitors of histone deacetylases (HDACs) or methylation-specific enzymes as novel important anti-cancer drugs. Here, we tested whether the histone signature and DNA methylation in multiple myeloma (MM) and leukemia cells is tumor-specific as compared with that in non-malignant lymphoblastoid cells. We observed a distinct histone signature in c-myc, Mcl-1, and ribosomal gene loci in MOLP8 MM and K562 leukemia cells, when compared with lymphoblastoid cells. Histone and DNA methylation patterns in MOLP8 cells were partially modified by the clinically promising HDAC inhibitor, vorinostat. In comparison with lymphoblastoid WIL2NS cells, MOLP8 cells and K562 cells were characterized by an absence of the gene silencing marker H3K9me2 in the c-myc and ribosomal genes. However, high levels of H3K27me3 were detected in the promoters and coding regions of selected genomic regions in these cells. Treatment by vorinostat increased the level of DNA methylation at the c-myc promoter, and this alteration was accompanied by a decrease in c-MYC protein. In MOLP8 cells, vorinostat significantly increased the H3K9 acetylation in the Mcl-1 coding regions and promoter. Both MOLP8 and K562 leukemia cells were characterized by decreased levels of H3K9me2 in the Mcl-1 gene as compared with lymphoblastoid WIL2NS cells. Lower levels of H3K9me1 in the Mcl-1 promoter, however, were specific for MM cells as compared with the other cell types studied. In other MM and leukemia cell lines, COLO677, OPM2, and U937, the ribosomal genes were less prone to epigenetic heterogeneity as compared to the c-myc and Mcl-1 proto-oncogenes. Taken together, these data describe both tumor-specific and loci-specific histone signature and DNA methylation profiles.