AB44. Comparison research of the morphology, phenotypes and functions of the dendritic cells derived from hematopoietic stem cells and peripheral monocytes

Background: As a kind of promising anti-tumor immunotherapy, autologous dendritic cell (DC) vaccine derived from peripheral blood monocytes faced limitations including deficiency of function and shortage of cell number. CD34+ progenitors are another ideal source of DC generation and different methods were explored and resulted in different cell number, various function of DC.

Objective: Present study aimed to explore the differences between DCs derived from CD34+ hematopoietic stem cells (CD34-DC) of the cord blood and those derived from monocytes (Mo-DC) of the peripheral blood, in their cell morphologies, cell phenotypes, and the function of inducing antigen-specific cytotoxic T lymphocytes (CTLs), which could specifically induce tumor lyses, and explore different sources DC ultrastructural changes related to its antigen processing and antigen-presenting function of differences, in order to explore more powerful therapeutic DC vaccines.

Methods: Monocytes were obtained from peripheral blood of healthy volunteers by using density gradient centrifugation, and the dendritic cells derived from monocytes (Mo-DC) were harvested by adherent assay. CD34+ hematopoietic stem cells were harvested from the cord blood of healthy full-term pregnant women, and purified by using magnetic bead separation, and expanded in GM-CSF/SCF medium for 8-10 days. DC progenitors were differentiated continuously in the GM-CSF/IL-4 medium, and CD34+ stem cell derived DCs (CD34-DC) were harvested every 3 days. Take d3, d5, d7 days immature DC (CD34-imDC, Mo-imDC) and d7 harvested CD34-mDC, Mo-mDC (DC vaccine) in the TEM compare their cell processes, nuclear size and endosomal vesicles quantity. Taken after different incubation time (d1, d3, d5, d7) CD34-DC and Mo-DC surface molecules detected by flow cytometry expression, some cells pulsed with FITC-OVA^257-264 and continued to culture 24 hours, respectively, detecting the antigen-presenting ability by flow cytometry. Take culture 5^th day DCs load CEA protein and tumor necrosis factor (TNF-α) was added. Preparation of the mature CD34-DC, Mo-DC (DC vaccine), in vitro induced CTL generation, both vaccines induced the CTL were of high CEA-expressing tumor cell lines Ls174-T do killing assay, using MTS cytotoxicity assay.

Results: After stimulated by cytokine and tumor antigen, they displayed the relative consistency in cell appearance. Antigen loaded DCs were floated with no dendrite, round-like shape, and the membrane was clear and smooth. Number of endosomal vesicles was determined and results showed CD34-DC had more endosomal vesicles than Mo-DC at each time points (P<0.05). CD34-mDC and Mo-mDC generated from CD34-DC and Mo-DC stimulated by antigen and cytokine can become mature (P<0.05). CD34-DC has more antigen presenting ability than Mo-DC. CD34-DC induced higher amount of additional lymphocytes over Mo-DC, but there are no significant differences (P>0.05). CD34-DC and Mo-DC displayed an increased oncolytic efficacy and CC-CTLs showed stronger cytotoxicity than CM-CTLs (P<0.05).

Conclusions: With GM-CSF/SCF medium can induce cord blood CD34+ hematopoietic stem cell expansion and generate DC precursor cells and cultured adherent growth d8 beginning, a group can be harvested every 3 days DC precursor cells with GM-CSF/IL-4-induced medium can further generate CD34-DC, no phenotypic differences between CD34-DC and Mo-DC. Umbilical cord blood -derived CD34-DC has a stronger antigen-presenting, promoting lymphocyte proliferation and the ability to induce activation of CTLs. By electron microscope, changes of the number of dendritic protrusions, nuclear size and the number of endosomal vesicles are to assess in DCs’ micro-mechanism. Umbilical cord blood-derived CD34-DC is expected to become the new ideal raw cells for DC vaccine.