TGF-β1, in Synergy with M-CSF Induce Generation of a Novel Type of Human Cord Blood Dendritic Cell That Is Both Anti-Inflammatory and Capable of Ex-Vivo Expansion of Myeloid Progenitor Cells.
Authors: Li Geling , Campbell Timothy B. , Graham-Evans Barbara , Cooper Scott , Broxmeyer Hal E. 
Published: April 2020

Abstract
Tolerogenic dendritic cells (DCs) could be a promising cellular therapeutic tool in human transplantation due to their potential for silencing unwanted immune reactions. However, it is not known whether tolerogenic DCs are capable of regulating human hematopoiesis. We recently reported that M-CSF, in combination with IL-4, induced cord blood monocyte differentiation into a population of dendritic cells (DC) with tolerogenic potential (M-DC) (Li et.al. J. Immol.174: 4706, 2005.). TGF-β1 has potent immune regulatory functions on DCs. In this context, we investigated effects of TGF-β1 on cord blood M-DC differentiation by characterizing phenotype, cytokine production and allogeneic mixed lymphocyte reaction (MLR) of the resulting DC population (TGF/M-DC). We also evaluated effects of the resultant cells on expansion and growth factor withdrawal-induced apoptosis of human CD34+ progenitors isolated from cord blood. The effects were compared to those of TGF-β1, GM-CSF and IL-4-induced DCs (TGF/DC). Addition of TGF-β1 to highly purified cord blood monocytes cultured with M-CSF and IL-4 changed M-DC morphology to that of a homogeneous DC morphology (TGF/M-DC). Similar to M-DC, TGF/M-DC negatively stained for monocyte/macrophage markers CD14 and CD16, and positively stained for CD86 and HLA-DR, molecules involved in antigen presentation. It appeared that TGF/M-DC were bona fide DC with typical DC morphology and phenotype. In comparison with TGF-DC, TGF/M-DC produced high amounts of IL-10, but displayed reduced capacity in induction of allogeneic MLR, features categorizing them more appropriately into an anti-inflammatory DC subset. We generated conditioned medium (CM) from both types of DC by stimulation of these DC with LPS and studied effects of their CM on ex-vivo expansion of CD34+ progenitors isolated from cord blood. In the absence of DC CM, the combination of SCF, Flt3 ligand and TPO (SFT, a cytokine combination widely used for cell expansion) induced a 10.5 ± 1.8 fold expansion of myeloid progenitors (CFU-GM) in 1 week culture. TGF/M-DC CM (25% v/v), but not TGF DC CM, in synergy with SFT further expanded myeloid progenitors (CFU-GM) 19.4 ± 2.7 fold beyond that seen with SFT alone. The effects of TGF/M-DC CM was accompanied with a significant increase of CD34+ cell numbers after 1 week (TGF/M-DC CM in combination with SFT, TGF-DC CM in combination with SFT, and SFT alone expanded CD34+ cells by 8.4 ± 2.8 fold, 3.8 ± 2.8 fold and 5.9 ± 1.1 fold respectively). TGF/M-DC CM and TGF/DC CM prevented growth factor withdrawal-induced apoptosis of CD34+ cells, as analyzed by annexin-V staining. Therefore, TGF/M-DC might be capable of in vivo silencing of unwanted immune responses while promoting myeloid recovery. These features of TGF/M-DC make them an interesting cellular therapeutic candidate to potentially facilitate hematopoietic recovery as well as down-modulate unwanted immune responses after hematopoietic stem cell transplantation.