Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

Normal differentiation and functions of mouse dendritic cells derived from RAG-deficient bone marrow progenitors

Dendritic cells (DC) mature upon infectious agent detection to elicit immune responses. It has been suggested that T cells influence peripheral DC function. However, it is not known if lymphocytes influence DC progenitors. Therefore, we determined the ability of bone marrow progenitors from T and B cell-deficient mice to generate functional DC. We report that bone marrow-derived DC from RAG-2(-/-) mice differentiate and proliferate normally. Moreover, such generated DC efficiently internalize particles, mature in response to various Toll-like receptor engagement, and activate allogenic T cells. This work strongly supports that early signals delivered during DC ontogeny by mature lymphocytes do not influence the functional differentiation of DC progenitors.     

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Background

Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.

Methods

Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2^d) prior to transplanting into C57BL/6 mice (H-2^b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).

Results

Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.

Conclusion

Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.     

Modulation of dendritic cell differentiation in the bone marrow mediates sustained immunosuppression after polymicrobial sepsis

Murine polymicrobial sepsis is associated with a sustained reduction of dendritic cell (DC) numbers in lymphoid organs and with a dysfunction of DC that is considered to mediate the chronic susceptibility of post-septic mice to secondary infections. We investigated whether polymicrobial sepsis triggered an altered de novo formation and/or differentiation of DC in the bone marrow. BrdU labeling experiments indicated that polymicrobial sepsis did not affect the formation of splenic DC. DC that differentiated from bone marrow (bone marrow-derived DC [BMDC]) of post-septic mice released enhanced levels of IL-10 but did not show an altered phenotype in comparison with BMDC from sham mice. Adoptive transfer experiments of BMDC into naive mice revealed that BMDC from post-septic mice impaired Th1 priming but not Th cell expansion and suppressed the innate immune defense mechanisms against Pseudomonas bacteria in the lung. Accordingly, BMDC from post-septic mice inhibited the release of IFN-γ from NK cells that are critical for the protection against Pseudomonas. Additionally, sepsis was associated with a loss of resident DC in the bone marrow. Depletion of resident DC from bone marrow of sham mice led to the differentiation of BMDC that were impaired in Th1 priming similar to BMDC from post-septic mice. Thus, in response to polymicrobial sepsis, DC precursor cells in the bone marrow developed into regulatory DC that impaired Th1 priming and NK cell activity and mediated immunosuppression. The absence of resident DC in the bone marrow after sepsis might have contributed to the modulation of DC differentiation.     

Induction on differentiation and modulation of bone marrow progenitor of dendritic cell by methionine enkephalin (MENK)

Methionine enkephalin (MENK), the endogenous neuropeptide, is known to exert direct effects on the neuroendocrine and the immune systems and participates in regulation of various functions of cells related to both the innate and adaptive immune systems. Dendritic cells (DCs) play important role in initiating and regulating T cell responses. The aim of this work is to investigate the effects of MENK on differentiation, maturation, and function of DCs derived from murine bone marrow progenitors (BM-derived DCs). Our result showed that MENK could induce BM-derived DCs to polarize predominantly to mDC subtype, rather than pDC both in vivo and in vitro, and this was in favor of Th1 response. BM-derived DCs, after treatment with MENK, up-regulated the expressions of MHC class II and key costimulatory molecules. Result by RT-PCR showed MENK could increase expressions of delta and kappa receptors on BM-derived DCs. Also MENK promoted BM-derived DCs to secret higher levels of proinflammatory cytokines of IL-12p70, TNF-α. Furthermore, differentiated BM-derived DCs treated with MENK displayed higher activity to induce allogeneic T cell proliferation and MENK also inhibited tumor growth in vivo and induced apoptosis of tumor cells in vitro. Thus, it is concluded that MENK could be an effective inducer of BM-derived DCs and might be a new therapeutic agent for cancer, as well as other immune handicapped disease. Also we may consider MENK as a potential adjuvant in vaccine preparation.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multi-dimensional flow cytometry.

A detailed analysis of normal myeloid differentiation was performed using mutlidimensional flow cytometry. Based on two light scattering and three color immunofluorescence signals, the normal maturation pathways of both the monocyte and neutrophil lineages could be elucidated. Gradual changes of light scattering properties and cell surface antigen expression defined the pathways of each of the lineages. The consistency of the location of these lineage specific pathways found in normal individuals provided the basis for the discrimination between normal and leukemic cells in acute myeloid leukemia (ANLL). The position of leukemic cells in patients with ANLL in a five-dimensional space was compared with the position of the maturation tracks in normal individuals. The expression of normal antigens on leukemic cells provided the tools to: (1) distinguish normal from clonal populations of leukemic cells in all 15 patients; (2) detect a lineage predominance, either monocytic or neutrophilic, in all 15 patients; (3) detect maturation heterogeneity in all 15 patients. Although maturation pathways of the monocytic and the neutrophilic lineages were analogous to the normal patterns they were distinct in several ways. The expression of normal antigens on leukemic cells may provide the tools to: (1) obtain a new frame-work for classification of leukemia based on the ability to quantify the aberrant antigen expression and to define a 'distance from normal' based on the characteristics studied (the maturation heterogeneity of the leukemic cells also can be correlated with the clinical outcome of the patients); (2) detect minimal residual disease using the difference in locations of the leukemic cells in the multidimensional space from the normal maturation pathways (3) monitor relapse and changes in phenotypes which may accompany chemotherapy, suggesting the appearance of variant or new clones.     

Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver

It has been unclear whether the progenitors colonizing the thymus are multipotent or T cell lineage restricted. We investigated the developmental potential of hematopoietic progenitors in various populations of liver and blood cells from day 12 fetuses using the recently established in vitro experimental system effective in determining the capability of individual progenitors to generate T, B, and myeloid cells. Multipotent progenitors (p-Multi) were exclusively found in the Sca-1 high-positive (Sca-1high) subpopulation of lineage marker (Lin)-c-kit+CD45+ fetal liver cells. Restriction of developmental capacity begins at the Sca-1high stage, and a large majority of progenitors in the Sca-1low or Sca-1- population are restricted to generate T, B, or myeloid cells. Such a lineage commitment or restriction taking place in the fetal liver is independent of the thymus, because no difference in the proportion of different types of progenitors were seen between nu/nu and nu/+ fetuses. T cell lineage-restricted progenitors (p-T) were abundant in the blood of day 12 fetuses, whereas p-Multi were undetectable. It was further shown that the p-Multi generated a large number of B and myeloid cells in the thymic lobe. These results strongly suggest that it is p-T but not p-Multi that migrate into the thymus.     

IL-3 induces differentiation of bone marrow precursor cells to osteoclast-like cells

IL-3, a cytokine with hematopoietic differentiating capability, induced murine bone marrow cells to differentiate into cells resembling osteoclasts. The cells resulting from treatment with IL-3 were multi-nucleated and demonstrated tartrate-resistant acid-phosphatase activity, as do resident osteoclasts found in bone. IL-3-induced osteoclast-like cell development in the absence of serum-derived vitamin D metabolites, and a mAb that inhibited IL-3-induced proliferation of an addicted cell line also inhibited the development of osteoclasts in the presence of IL-3. The same Ab had no effect on 1 alpha, 25-dihydroxyvitamin D3-induced differentiation of osteoclasts. This newly described function of IL-3 may indicate a role for activated T cells in the bone resorption seen with rheumatoid arthritis.     

Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205(+)CD11b(+)Gr-1(+) bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205(+)CD11b(+)Gr-1(low) cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-alpha, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205(+)CD11b(+)Gr-1(high) cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205(+)CD11(+)Gr-1(high) cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.     

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Osteogenic growth peptide C-terminal pentapeptide [OGP(10-14)] acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. Whether OGP(10-14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. In the present study, we investigated the roles of OGP(10-14) in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that OGP(10-14) promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. OGP(10-14) increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of core-binding factor 1 (cbfa1). In contrast, OGP(10-14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2). These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is regulated by OGP(10-14).     

Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8alpha expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8alpha+) and "myeloid" (CD8alpha-). DCs from mice genetically deficient in CD8alpha expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8alpha- DCs from CD8alpha-sufficient mice are better APCs compared with CD8alpha+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.     

Glycogen synthase kinase-3 acts upstream of ADP-ribosylation factor 6 and Rac1 to regulate epithelial cell migration

Cell sheet movement during epithelial wound closure is a complex process involving collective cell migration. We have found that glycogen synthase kinase-3 (GSK-3) activity is required for membrane protrusion and crawling of cells at the wound edge and those behind it in wounded Madin-Darby canine kidney (MDCK) epithelial cell monolayers. RNA interference-based silencing of GSK-3alpha and GSK-3beta expression also results in slowed cell sheet migration, with the effect being more pronounced with knockdown of GSK-3beta. Both GSK-3alpha and GSK-3beta are in activated states during the most active phase of cell migration. In addition to having a positive control or permissive, rather than negative, function in MDCK cell migration, GSK-3 appears to act upstream of the small GTPases ADP-ribosylation factor 6 (ARF6) and Rac1. Expression of constitutively active ARF6 restores a protrusive, migratory phenotype in cells treated with GSK-3 inhibitors. It does not, however, restore to normal levels the directional polarization of cells behind the wound edge toward the wound area, implying the existence of a separate ARF6-independent branch of the GSK-3 pathway that regulates proper wound-directed polarization of these cells. Finally, inhibition of GSK-3 also strongly reduces activation of Rac1 and cell scatter in response to hepatocyte growth factor/scatter factor, which triggers dispersal and migration of cells in monolayer culture as fibroblast-like individual cells, a mode of epithelial cell motility distinct from the collective migration of wound closure.