Prolonging the Incubation of Monocytes with GM-CSF and IL-4 for More Than 3 Days during the Generation of Dendritic Cells (DCS) In Vitro Markedly Diminishes IL-12 and Increases IL-10 Production When DCS Are Subsequently Matured.
Authors: Tawab Abdul , Fan Yong , Mai Thao , Read Elizabeth J. , Kurlander Roger J. 
Published: October 2019

Abstract
Dendritic cells (DCs) are potent antigen presenting cells with therapeutic potential in stimulating host T cell responses against cancer and microbial pathogens. For this purpose, monocytes can be transformed into DCs by incubation with GM-CSF and IL-4. The resulting immature DCs are usually “matured” before use to enhance the expression of MHC products (for antigen presentation), CD83 (for optimal T response costimulation), chemokine receptor 7 (CCR7) (for migration to T cell rich areas within lymph nodes), and IL-12 (for induction of type 1 immune T cell responses). There is still no consensus on the best protocol for generating clinical grade DCs. In practice, monocytes are differentiated using GM-CSF/IL-4 for up to a week and then matured using a wide variety of agents for another 8 to 48 h. The current studies address how the duration of the differentiation and maturation steps affect DC expression of CD83, CCR7, and IL12. DCs were grown in RPMI 1640 with 10% human serum containing 2000 units/ml each of IL-4 and GM-CSF for 2 to 7 days with periodic refeeding to promote differentiation. Cells were then matured using 100 ng/ml of LPS plus 1000 units/ml of γ-IFN. DCs exposed to LPS/γ-IFN rapidly upregulated CD83, CCR7, and IL-12 expression. CD83 levels were near maximal within 8 h, while CCR7 expression and IL-12 expression reached peak levels within 18 h. Variations in the duration of DC incubation with GM-CSF/IL-4 had little impact on CD83 and CCR7 expression, but markedly affected cytokine secretion. IL-12 production by DCs during maturation and after restimulation with CD40L dropped progressively as the length of GM-CSF/IL-4 incubation was prolonged. In matched studies using 4 separate donors, DCs incubated with CSF/IL-4 for 3 days produced a log mean of 23,400 pg/ml of IL-12 during maturation and 17,900 pg/ml after CD40L restimulation. After incubation with GM-CSF/IL-4 for 7 days, DCs produced 1,500 pg/ml of IL-12 on direct stimulation and only 100 pg/ml on restimulation. IL-10 production showed the opposite pattern. DCs incubated for 7 days with GM-CSF/IL-4 produced a log mean of 370 pg/ml of IL-10 on restimulation compared to 20 pg/ml by matched cells incubated for only 3 days. These differences in IL-12 and IL-10 production were all significant in a paired t-test at p<0.01. This pattern was not unique to cells matured using LPS/γ-IFN. Similarly designed studies comparing IL-12 and IL-10 production by DCs matured using two other common maturation mixtures, poly-(I:C) (20 μg/ml) plus γ-IFN and CD40L (1 μg/ml) plus γ-IFN demonstrated the same pattern of reduced IL-12 production and increased IL-10 production when cells were cultured for extended periods. The results suggest it may be advantageous to avoid incubation of monocytes in GM-CSF/IL-4 beyond 3 days when generating DCs intended to promote type 1 T cell responses. Indeed, if endogenous DCs and DCs generated ex vivo regulate IL-12 production similarly, DC age could be a significant factor determining how DCs shape type 1 and 2 T cell polarization in vivo.