ALBERT

Description: M-CSF Drives Human Cord Blood Monocyte Differentiation along an Alternative Pathway into Suppressive CXCR4highIL-10highIL-12absentTNF-αdefective Dendritic cells Geling Li and Hal E. Broxmeyer. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA; Walther Cancer Institute, Indianapolis, IN, USA Macrophage colony-stimulating factor (M-CSF) in cooperation with TGF-β1 induces CD34+ hematopoietic progenitor cells to differentiate into Langerhans cells, a subset of immature dendritic cells (DCs) specifically localized in the epidermis. M-CSF levels are dramatically elevated in immunosuppressive conditions and M-CSF deficient mice are osteopetrotic. Therefore, we hypothesized that M-CSF might be involved in the generation of a novel subtype of suppressive DCs which may be located primary in bone marrow where large amounts of SDF-1 are constitutively produced. We also speculated that M-CSF-induced DCs might express the only known ligand of SDF-1, CXCR4 and migrate towards SDF-1. Highly purified umbilical cord blood monocytes (〉 98% purity) cultured in the presence of M-CSF, IL-4 and TGF-β1 differentiated into a highly homologous population of cells with typical dendrites and acquired a langerhan cell (LC) phenotype by up-regulated expression of CD1a and E-cadherin. We termed these cells M-LC to distinguish them from regular LCs cultured in the presence of GM-CSF, IL-4 and TGF-β1. In comparison with LC, M-LCs produced a much higher level of IL-10, no IL-12 (detection limit, 4 pg/ml) and a minimal level of TNF-α (21.6 ± 37.5 pg/ml) upon LPS stimulation. LPS-activated M-LCs induced lower allogeneic mixed lymphocyte reaction (MLR) than LPS-activated LCs. Furthermore, exposure to LPS-activated M-LCs rendered a state of low-responsiveness in cord blood naïve CD4+ T cells to further re-stimulation with alloantigens in secondary MLR. M-LCs expressed much higher surface levels of CXCR4 than LCs regardless of their maturation status. M-LCs started to migrate towards SDF-1 at a lower concentration (≥10 fold lower than that required for LCs) and displayed enhanced responsiveness to SDF-1-induced chemotaxis. Our results demonstrate that M-CSF drives monocyte differentiation along an alternative pathway into suppressive CXCR4highIL-10highIL-12absentTNF-αdefective DCs. These novel DCs may be of use to suppress unwanted immune responses in vivo.

Description: Cord blood has been successfully used as a transplantable source of hemopoietic stem cells with a relatively low incidence of graft-versus-host disease (GVHD). Mature dendritic cells (DC), as the most efficient antigen presenting cells, play a critical role in the pathogenesis of GVHD and their function is intimately linked to their capacity to migrate. Chemokines and their G-protein linked receptors are of particular importance in DC migration. CCR7 and CXCR4 are two chemokine receptors expressed on mature DC. Binding of CCR7 ligand, MIP-3β/CCL19 and CXCR4 ligand, SDF-1/CXCL12 induce chemotaxis of mature DC and are pivotal for DC to initiate their functions in vivo. We hypothesized that the relatively lowered level of GVHD elicited by cord blood, compared to adult cells, might be due, at least in part, to a reduced capability of cord blood DC to migrate to lymphoid organs to initiate immune reactions and to organs such as liver and skin where GVHD pathogenesis usually occurs. To begin testing this hypothesis, we randomly picked 16 cord blood donors and 14 adult blood donors and did paired comparison study on chemotactic responses of cord blood and adult blood DC to CXCL12 and CCL19. Monocytes were isolated from both cord blood and adult blood and cultured with GM-CSF and IL-4 for 5 days to generate immature DC. LPS was added into cell culture for an additional 1 day to induce the maturation of these DC. Mature DC were harvested on day 6. We found that cord blood DC expressed significantly reduced surface levels of CCR7 compared with that of adult blood DC (p = .00067) and migrated at significantly lower efficiency towards the CCR7 ligand, CCL19 than did adult blood DC (p=0.029). Cord blood DC expressed significantly higher surface expression of CXCR4 than adult blood DC (p = .0018), but migrated at significantly lower efficiency towards CXCR4 ligand, CXCL12 than adult blood DC (p=0.00012). The lowered responsiveness of mature cord blood DC to chemotaxis by CXCL12 may reflect the enhanced truncation of CXCR4 we found on these cells. Also, cord blood DC migrated less well than adult blood DC toward the combination of CXCL12 and CCL19 than adult blood DC (p = .0014). These results suggest that low incidence and pathogenesis of GVHD observed in cord blood might be due, at least in part, to reduced capability of cord blood mature DC to migrate to critical organs where immune reaction and inflammation occur, effects reflecting lowered expression of CCR7 and enhanced truncation of CXCR4 on mature cord blood DC.

Description: 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.

Description: Tolerogenic dendritic cells (DCs) may be valuable in transplantation for silencing immune reaction. Macrophage colony-stimulating factor (M-CSF)/IL-4 induces differentiation of cord blood (CB) monocytes into DCs (M-DCs) with tolerogenic phenotype/function. We assessed whether factors produced by tolerogenic DCs could modulate hematopoiesis. TGF-β1 added to CB M-DC cultures induced bona fide DC morphology (TGF-M-DCs), similar to that of DCs generated with TGF-β and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-4 (TGF-GM-DCs). Of conditioned media (CM) produced from TGF-M-DCs, TGF-GM-DCs, M-DCs, and GM-DCs, TGF-M-DC CM was the only one that enhanced SCF, Flt3 ligand, and TPO expansion of myeloid progenitor cells ex vivo. This effect was blocked by neutralizing anti–M-CSF Ab, but protein analysis of CM suggested that M-CSF alone was not manifesting enhanced expansion of myeloid progenitors. LPS-stimulated TGF-M-DCs induced T-cell tolerance/anergy as effectively as M-DCs. TGF-M-DCs secreted significantly lower concentrations of progenitor cell inhibitory cytokines and were less potent in activating T cells than TGF-GM-DCs. Functional differences between TGF-M-DCs and TGF-GM-DCs included enhanced responses to LPS-induced ERK, JNK, and P38 activation in TGF-M-DCs and their immune suppressive–skewed cytokine release profiles. TGF-M-DCs appear unique among culture-generated DCs in their capability for silencing immunity while promoting expansion of myeloid progenitors, events that may be of therapeutic value.

Description: Dendritic cells (DCs) are important regulators in graft-versus-host disease (GVHD). To gain insight into cord blood (CB) DC immunology, we compared chemotactic responses of mature monocyte-derived DCs and maturation agent lipopolysaccharide (LPS)–induced signaling between CB and adult blood (AB). Mature CB DCs expressed reduced CCR7, but increased CXCR4. This was associated with reduced migratory efficiency toward both CCR7 ligand CCL19 and CXCR4 ligand CXCL12. LPS induced higher extracellular signal-regulated kinase (ERK) phosphorylation in CB than in AB DCs. Specific inhibition of ERK during CB DC maturation enhanced LPS-induced up-regulation of CCR7 and CXCR4 on CB DCs and their chemotaxis toward CCL19 and CXCL12, to a level similar to that of mature AB DCs. Overall, monocyte-derived CB DCs responded to LPS with stronger and sustained ERK activation, which negatively correlated with LPS-induced up-regulation of CCR7 and CXCR4 on CB DCs and their migratory responses. These findings may have potential relevance to better understanding DC function in CB transplantation.