Definition of the variables affecting efficacy of immunodepletion ex vivo of peripheral blood progenitor cell grafts by alemtuzumab (Campath in the bag).

The immunodepleting effects of alemtuzumab on peripheral blood progenitor cell (PBPC) grafts for stem cell transplantation need to be better defined. The optimal graft cell concentration, antibody dose, need for complement, and whether alemtuzumab is infused with the graft during transplantation remain unclear. PBPC from 6 normal allogeneic stem cell donors harvested by apheresis were first quantitated and the cellular content defined by flow cytometry. Mononuclear cells were then incubated with incremental concentrations of alemtuzumab (.00001, .0001, .001, and .01 mg/mL) for 30 minutes at 20°C or in cell dose responses with 1, 5, and 10 × 10(6) mononuclear cells/mL added to a fixed dose of .001 mg/mL of alemtuzumab with or without a source of complement. Cells were enumerated and analyzed by flow cytometry before and after exposure to alemtuzumab. To determine the presence of unbound anti-CD52, the supernatant of the cell dose responses were tested using the ELISA assay. Selected CD34+ lineage-negative cells were incubated with antibody at the same working concentrations and conditions and cultured in granulocyte-macrophage colony-forming unit assay. The colony numbers were compared with control cultures devoid of the antibody. Incremental concentrations of alemtuzumab led to a significant (2 log) reduction in CD3, CD4, and CD8 populations, which plateaued at .001 mg/mL. Addition of complement led to a further significant reduction in the CD4 and CD8 cells. The maximum CD4 (3 log) and CD8 (2 log) cell death was obtained at 10 × 10(6) cells/mL. Analysis of supernatants for soluble alemtuzumab by ELISA showed a significant reduction in the free antibody concentration when the cell number was increased from 1 to 10 × 10(6) cells/mL implying utilization/binding of the antibody by target cells. Incremental concentrations of alemtuzumab did not affect the number of granulocyte-macrophage colony-forming units. Alemtuzumab depletes all cells expressing the CD52 antigen and has higher activity on CD3, CD8, and particularly on CD4 cells, which are depleted in excess of 2 logs. From this study, we were able to derive that the optimal cell kill in the graft without detectable free alemtuzumab in the supernatant can be achieved with 1 mg of antibody per 100 mL containing 10 × 10(9) cells and active complement (AB serum).

Monocyte derived dendritic cells have reduced expression of co-stimulatory molecules but are able to stimulate autologous T-cells in patients with MDS.

INTRODUCTION: Research has implied that the immune system plays a role in the pathogenesis of MDS and that T-cells are reacting to tumour antigen present on the surface of the malignant cells. This could imply that the immune system could be utilized to generate immune based therapy. The aim of this pilot study was to examine the feasibility of studying this further by analysing the interaction of dendritic cells with T-cells in a small cohort of MDS patients. METHODS: Dendritic cells were generated in 6 MDS patients and 9 controls by culturing monocytes with GM-CSF and IL-4. After activation with LPS and TNFα, the dendritic cells were analyzed for expression of co-stimulatory and activation antigens. Thereafter, they were co-cultured with T-cells and the T-cell response was examined by measuring the % change in expression of the activation antigen CD69. RESULTS: MDS MoDC had reduced expression of HLA-DR (p=0.006), CD11c (p=0.0004), CD80 (p=0.03) and CD86 (p=0.003), while resting T-cells from MDS patients had higher expression of the activation antigen CD69 on all subsets. The % change in CD69 expression increased significantly for both the control and MDS T-cells after co-culture with allogeneic dendritic cells, however this change was lower in the MDS group. Despite the increased CD69 expression prior to culture, MDS MoDC significantly up-regulated CD69 expression on autologous T-cells to values that were statistically higher than control cells. CONCLUSION: This initial study suggests that the T-cells in MDS are able to respond to dendritic cells and are therefore probably not part of the malignant clone. It further implies that the dendritic cell population could be capable of presenting antigen and initiating an immune response and therefore further study is both feasible and warranted.

The clonogenic potential of selected CD34+ cells from patients with MDS appear preserved when tested ex vivo.

Our aim was to examine in 17 patients with MDS the effects of PMA activated and non-activated autologous lymphocytes on selected bone marrow CD34+ progenitors, in dose response studies. We used a double layer culture technique. Compared with controls, there was no difference in the colony growth promoting capacity of autologous PMA stimulated or unstimulated blood lymphocytes from MDS patients. In addition, similar to control studies, increasing numbers of lymphocytes, (0, 1×10(5), 1×10(6)) led to a corresponding increase in the number of CFU-GM (p=0.04). We conclude that MDS blood mononuclear cells have the ability to stimulate colony growth of autologous CD34+ cells while these selected progenitors show a proliferative capacity that is similar to normal when they are isolated from the bone marrow accessory cells.