Detection And Culture In Vitro Of Dendritic Cells From The Patients With B Lineage Of Childhood Acute Lymphoblastic Leukemia

Object 1) By detecting the percentages of CD11c+ myeloid DC(mDC) and CD123+ plasmacytoid DC(pDC)from the children's patients with B lineage of acute lymphoblastic leukemia (B-ALL), the study is to understand the relationship between DC and MRD and seek the appropriate time point so as to purge MRD by using DC vaccine in the future. 2) This study also will lay the foundation of DC's vaccine by cultivating in vitro mononuclear cells (MNCs) of bone marrow(BM) from childhood B-ALL.Methods 1) The percentages of mDC and pDC from peripheral blood (PB) and BM in childhood B-ALL (35 cases at diagnosis, 20 cases with treatment on the thirty- third day ) were tested by flow cytometry. At the same time, MRD of B-ALL on the thirty-third day was evaluated by flow cytometry. 2) MNCs were separated from BM of 12 cases with B-ALL (7 cases at diagnosis, 5 cases during complete remission) and were cultivated for 7-10 days by using different cytokines in vitro. Cells was observed by inverted microscope. Immunophenotypes, such as CD80, CD86, CD1a, CD83, CCR7 and HLA-DR, were tested by flow cytometry. The ability to stimulate mixed lymphocyte reaction (MLR) were evaluated by the MTT assay.Results 1) The percentages of CD11c+DC and CD123+DC in childhood B-ALL at diagnosis were significantly reduced than normal controls (P<0.001) and cases with treatment on the 33rd day (P<0.01).After treatment of 33 days,the percentage of CD123+DC in PB of childhood B-ALL decreased than normal controls(t=-2.539,P=0.02). The levels of DC(including CD11c+DC and CD123+DC) in PB have significant correlation with the levels of DC in BM at diagnosis, no significant correlation with BM after treatment of 33 days. On the 33rd day of treatment, the level of PB123+DC in PB is lower than in BM(t=-2.539,P=0.02). The levels of CD123 + DC and CD11C + DC have no significant correlation with MRD after treatment of 33 days. 2) Cells owned typical DC-like shape by culture in vitro from MNCs of B-ALL. The expressions of CD1a,CD80,CD83,CD86,HLA-DR and CCR7 were significiantly increased. The abability of stimulation MLR gradually went up.Conclusions 1)The levels of mDC and pDC significantly decreased in children with B-ALL at diagnosis,but increased after treatment.The level of DC have no significant correlation with MRD in childhood B-ALL after treatment of 33 days. The combination with testing two kinds of DC in PB and BM are better than only in PB or BM. 2) Cells from B-ALL could be cultivated to DCs in vitro, they owned DC's immunophenotypes and functions. It will lay the foundation of purging MRD by using DC vaccine in the future.