A Randomized Trial To Evaluate the Impact of Cytokines (G-CSF or GM + G-CSF) on Dendritic Cell and T-Cell Content and Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant.

Introduction: Optimal cellular immunity following allogeneic HPC transplant is a balance between effective anti-tumor immunity and the avoidance of life threatening GvHD. Our group previously demonstrated that DC content following allogeneic BMT can impact EFS (Waller et al, Blood 2001), and that mobilization cytokines can impact DC content of the autologous PBSC graft (Lonial et al, BBMT 2004). The current trial was designed with the hypothesis that the use of G + GM-CSF will result in fewer tolerogenic DC2 cells within the graft, and increased Th1 phenotype among the mobilized T-cell when compared with normal donors mobilized with G-CSF alone.

Methods: 43 normal donors were randomized to receive G-CSF alone (7.5 mcg/kg BID) or G-CSF (7.5mcg/kg qd) and GM-CSF (7.5 mcg/kg qd) which was administered for 5 days or longer if additional days of apheresis were necessary. Side effects between the 2 cytokine regimens were documented using a questionnaire that was administered within 2 weeks of successful collection. Graft content was evaluated using multi-color flow cytometry, and pre-mobilization blood was collected to test for T-cell and DC content as well as T-cell function. DC1 (myeloid DC) were defined as Lin−/HLA-DR+/CD11C+/CD123−, while DC2 (lymphoid DC) were defined as Lin−/HLA-DR+/CD11C−/CD123+. T-cell proliferation was assessed using thymidine incorporation assays following mitogen exposure, and T-cell activation profile was assessed using ELISA assays of secreted cytokines and intracellular cytokine measurements following mitogen stimulation.

Results: 19 patients received G+GM-CSF and 24 patients received G-CSF alone. All 43 donors were successfully mobilized, though more patients in the G-CSF arm required multiple days of collection (mean number of collections 1.4 G-CSF vs 1.1 G+GM-CSF, p=0.06). There was no difference in %CD34+ or CD34+ content of the grafts between groups. There was a marked reduction in DC2 content as measured by percent (0.33% vs 0.45%, p=.03) absolute DC2 content (1.98e6 vs 3.66e6, p=0.0003), and delivered DC2 dose e6/kg (2.8 vs 5.2, p=.0002) for the donors randomized to received the combination of GM/G-CSF vs G-CSF alone. There was no statistical difference in DC1 content measured in an analogous fashion. Additionally, grafts collected with G-CSF alone had significantly greater numbers of total T-cells than compared with G+GM-CSF (379e6/kg vs 230e6/kg, p=0.0007). This percent reduction in total T-cell content was identical to the reduction in the CD4 and CD8 subsets between the 2 randomized groups. Interestingly, cells collected from the recipients of G+GM-CSF on the day of mobilization secreted higher amounts of IL-2 at rest, though when stimulated with PMA, G-CSF mobilized cells secreted significantly higher amounts of IL-2. Other secreted cytokines were measured, and to date do not show a clear trend towards a difference. Side effects between the 2 randomized arms were similar with 1 patient in the G-GM-CSF arm having to stop growth factors secondary to bone pain and fever.

Conclusion: The combination of G+GM-CSF mobilizes significantly fewer DC2 cells as well as 40–50% fewer T-cells, and is more likely to result in a successful single PBSC collection than the use of G-CSF alone. Further laboratory evaluation of the graft, survival, and GvHD data following transplant will also be presented.