Structural and Functional Characterization of the NHR2 and Runt Domains of AML1/ETO.

Introduction:Optimal cellular immunity following allogeneic HPC transplant represents a balance between the induction of sufficient anti-tumor immunity to eradicate residual cancer cells without the induction of life-threatening GvHD. Dendritic cells are potent APCs with the ability to regulate immune responses. Our group has previously reported that increased numbers of donor DC2 result in inferior EFS following allo BMT (Waller et al, Blood 2001), and that myeloid cytokines used for mobilization modulate the DC content of the auto graft (Lonial et al, BBMT in press). The current trial was designed to evaluate the impact of different cytokine combinations on DC content and T-cell function in normal donors mobilized with either G-CSF or the combination of G-CSF + GM-CSF. Methods: 32 normal donors were randomized to mobilization with G-CSF (7.5 mcg/kg BID) or the combination of GM-CSF (7.5 mcg/kg qAM) + G-CSF (7.5 mcg/kg qPM) until completion of the stem cell collection. Side effects between the 2 regimens were documented using a questionnaire filled out by the donors within 2 weeks of stem cell collection. DC, T-cell, and other cell subsets were measured from the graft using flow cytometry. T-cell function was evaluated by measuring T-cell proliferation in response to PMA, Con A, PHA, and PWM. Cytokines (IL2, IL4, IL10,IL12, TNF, and INF) secreted in response to antigens were measured by ELISA. DC1 (myeloid DC) were defined as Lin-/HLA-DR+/CD11c+/CD123- while DC2 (lymphoid DC) were defined as Lin-/HLA-DR+/CD11c-/CD123+. Results: 28 patients have been successfully collected to date (G-CSF n=15, GM+G-CSF n=13). No donor has failed to mobilize in either group. Among the 15 donors mobilized with G-CSF alone, 5 required multiple days of apheresis as compared with 1 of 13 donors who received GM+G-CSF who required multiple days of apheresis (p=0.06). There was no difference in baseline values of T-cells or DC subsets in the peripheral blood prior to cytokine administration. Grafts collected with GM-CSF+ G-CSF contained significantly fewer DC2 cells and T-cells (median DC2 dose of 2.1 x 10E6/kg and CD3 dose of 197x 10E6/kg) compared with grafts from donors who received G-CSF alone (median DC2 dose of 3.8 x 10E6/kg (p=.01) and CD3 dose of 320 x 10E6/kg (p=0.001)). There was no difference in the content of CD34+ or DC1 in the grafts, nor in the ratio of CD4:CD8 T-cells between grafts collected with the 2 cytokine combinations. T-cell proliferation and cytokine secretion in response to mitogens was not different between grafts collected from the two groups. To date, there is no difference in the frequency of GvHD or relapse between the patients transplanted with the grafts collected from the 2 cytokine cohorts. Conclusions: The addition of GM-CSF to the mobilization regimen results in significantly fewer DC2 cells and T-cells in the blood HPC graft which could impact immune function and GvL following allogeneic HPC transplant. Clinical outcomes and further analysis of TH1/TH2 polarization of T-cells in grafts collected with either G-CSF or G-CSF+GM-CSF are in progress..