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PubMed2004

The expression of P-glycoprotein in AML cells with FLT3 internal tandem duplications is associated with reduced apoptosis in response to FLT3 inhibitors.

P-glycoprotein (pgp), a membrane efflux pump, is recognized to have an anti-apoptotic function. Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) receptor are the most common mutations in acute myeloid leukaemia (AML). Both ITDs and pgp positivity confer an adverse clinical prognosis. FLT3 inhibitors induce variable apoptosis in cell lines transfected with FLT3 ITDs. We studied the effect of herbimycin A, AG1296 and PKC412 on primary AML blasts. All compounds showed significantly higher cell kill after 48-h incubation in samples with an ITD compared with wild type (Herbimicin P < 0.001; AG1296 P = 0.001, PKC412, P = 0.002). Pgp-positive samples were significantly less sensitive to herbimycin and AG1296 than pgp-negative samples, although neither molecule inhibited the efflux function of pgp. The concurrent incubation with the pgp inhibitor PSC833 resulted in an enhanced cell kill in 4/5 ITD pgp-positive samples versus two of nine ITD pgp-negative samples. PKC412 inhibited pgp function and induced cell death in FLT3 ITD/pgp-positive samples. We conclude that AML samples with a FLT3 ITD are more susceptible to these inhibitors than wild-type samples. However, the expression of pgp in cells with FLT3 ITDs can reduce their sensitivity to FLT3 inhibitors and therefore pgp expression should be assessed in clinical trials of FLT3 inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells.

The cytokine tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. Through its ability to promote ex vivo expansion and oncoretroviral transduction of primitive human hematopoietic progenitors, the flt3 ligand (FL) has emerged as a key stimulator of candidate human hematopoietic stem cells (HSCs). However, recent studies in the mouse suggest that though it is present on short-term repopulating cells, flt3 is not expressed on bone marrow long-term reconstituting HSCs, the ultimate target for the development of cell replacement and gene therapy. Herein we demonstrate that though only a fraction of human adult bone marrow and cord blood CD34+long-term culture-initiating cells (LTC-ICs) express flt3, most cord blood lymphomyeloid HSCs capable of in vivo reconstituting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice are flt3+. The striking difference in flt3 and c-kit expression on mouse and candidate human HSCs translated into a corresponding difference in flt3 and c-kit function because FL was more efficient than SCF at supporting the survival of candidate human HSCs. In contrast, SCF is far superior to FL as a viability factor for mouse HSCs. Thus, the present data provide compelling evidence for a contrasting expression and response pattern of flt3 and c-kit on mouse and human HSCs.

Animals↗

PubMed2004

FLT3-ITD-TKD dual mutants associated with AML confer resistance to FLT3 PTK inhibitors and cytotoxic agents by overexpression of Bcl-x(L).

FLT3 (fms-like tyrosine kinase 3) is constitutively activated in about 30% of patients with acute myeloid leukemia (AML) and represents a disease-specific molecular marker. Although FLT3-LM (length mutation) and TKD (tyrosine kinase domain) mutations have been considered to be mutually exclusive, 1% to 2% of patients carry both mutations. However, the functional and clinical significance of this observation is unclear. We demonstrate that FLT3-ITD-TKD dual mutants induce drug resistance toward PTK inhibitors and cytotoxic agents in in vitro model systems. As molecular mechanisms of resistance, we found that FLT3-ITD-TKD mutants cause hyperactivation of STAT5 (signal transducer and activator of transcription-5), leading to upregulation of Bcl-x(L) and RAD51 and arrest in the G(2)M phase of the cell cycle. Overexpression of Bcl-x(L) was identified as the critical mediator of drug resistance and recapitulates the PTK inhibitor and daunorubicin-resistant phenotype in FLT3-ITD cells. The combination of rapamycin, a selective mTOR inhibitor, and FLT3 PTK inhibitors restored the drug sensitivity in FLT3 dual mutant-expressing cells. Our data provide the molecular basis for understanding clinical FLT3 PTK inhibitor resistance and point to therapeutical strategies to overcome drug resistance in patients with AML.

Acute Disease↗

PubMed2007

A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells.

The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I-restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results demonstrate that AML leukemic blasts can in principle process and present immunogenic FLT3-ITD neoepitopes. Therefore, FLT3-ITD represents a potential candidate target antigen for the immunotherapy of AML.

Amino Acid Sequence↗

PubMed1999

p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells.

Flt3/Flk2 belongs to class III receptor tyrosine kinases. Like other members of type III receptor tyrosine kinases, murine Flt3 induces tyrosine phosphorylation of p85 and subsequently activation of PI3 kinase upon FL binding. While p85 binds murine Flt3 at Y958 in the carboxyl terminus of the receptor, human Flt3 does not have a potential p85-binding site in the carboxyl terminus. In this study, we examined whether p85 binds to human Flt3 in Baf3/Flt3 and THP-1 cells. In contrast to murine Flt3, p85 is not tyrosine phosphorylated after FL stimulation, nor does it bind Flt3 in both cell lines. Instead p85 associates inducibly with tyrosine phosphorylated SHP-2 and constitutively with SHIP and two tyrosine phosphorylated proteins with molecular weights about 100-kDa (p100) and 120-kDa (p120) in Baf3/Flt3 cells. The p100 associates with both p85 and SHP-2. In THP-1 cells, p85 associates inducibly with tyrosine phosphorylated SHIP, p100 and p120. These results indicate that p85 does not bind human Flt3, but forms a complex with SHP-2, SHIP, p100 and p120 in hematopoietic cells.

Amino Acid Sequence↗

PubMed2001

Effects of dexamethasone on Flt3 receptor expression and proliferation induced by recombinant human Flt3 ligand in malignant hematopoietic cells.

AIM: To investigate the effect of dexamethasone (DXM) on the expression of Flt3 receptor and the proliferation mediated by recombinant human Flt3 ligand (rhFL) in leukemia cells. METHODS: Eighteen malignant hematopoietic cell lines and 10 leukemia blasts from leukemia patients were examined by flow cytometry for the expression of Flt3 receptor before and after incubation with DXM 0.1 micromol/L for 24 h. The effect of DXM on the proliferation of malignant hematopoietic cells was measured by MTT assay. RESULTS: (1) Expression of the Flt3 receptor in malignant hematopoietic cell lines and leukemia blasts was widespread and extremely heterogeneous; (2) The presence of receptor on the surface of malignant hematopoietic cell didn't necessarily imply a significant ligand-induced response, at least in terms of proliferation. Conversely, some Flt3 receptor-negative malignant hematopoietic cells responded to rhFL; (3) DXM down-regulated the expression of Flt3 receptor and inhibited the proliferation induced by rhFL in some malignant hematopoietic cell lines and fresh leukemia cells. CONCLUSION: DXM may down-regulate the expression of Flt3 receptor on the surface of malignant hematopoietic cells and inhibit the proliferation induced by rhFL. A combination of rhFL and DXM may serve to control hematopoietic defects in malignant hematopoietic diseases.

Antineoplastic Agents, Hormonal↗

PubMed2005

In vitro activity of the flt3-inhibitor su5614 and standard cytotoxic agents in tumour cells from patients with wild type and mutated flt3 acute myeloid leukaemia.

The correlation between drug sensitivity in vitro and the mutation status of the FLT3 receptor gene was evaluated in tumour cells from 17 previously untreated AML patients. Tumour cells with internal tandem duplication (ITD) in the FLT3 receptor gene were significantly more sensitive to the FLT3 inhibitor SU5614 than tumour cells with wild type FLT3. Combinations of SU5614 with etoposide and amsacrine showed better effect (p<0.05) compared with the respective single drugs. Our results suggest that the FLT3 inhibitor SU5614 may have a therapeutic potential, especially in combination with other cytotoxic agents, in patients with FLT3-ITD positive AML.

Acute Disease↗

PubMed1999

Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3-L.

The biological effects of flt3-L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG-CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low-level expression, flt3-L exerted synergistic effects with a combination of c-kit ligand, IL-3, IL-6, GM-CSF and G-CSF, mainly to induce proliferation of CD34+/CD38- cells. In addition, flt3-L increased the detection of HPP-CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3-L is active on circulating early mobilized haemopoietic progenitors, despite the low- level expression of its receptor.

ADP-ribosyl Cyclase↗

PubMed2004

Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412.

Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.

Adult↗

PubMed2004

Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1alpha(CXCL12)/CXCR4 axis.

Flt3 ligand (FL) enhances hematopoietic cell proliferation and facilitates hematopoietic stem cell mobilization in vivo, while the stromal-derived factor 1alpha (SDF-1alpha, CXC ligand 12 [CXCL12])/CXC receptor 4 (CXCR4) axis is critical for their homing and trafficking. We investigated if FL and its receptor, Flt3, functionally interact with CXCL12/CXCR4 to regulate hematopoietic cell migration. FL stimulated chemokinetic activity when used alone, but synergistically enhanced short-term migration of CD34+ cells, Ba/F3 cells expressing human Flt3 (Ba/F3-Flt3), and human RS4;11 acute leukemia cells, induced by CXCL12. Moreover, overexpression of constitutively activated internal tandem duplication (ITD)-Flt3 mutants in Ba/F3 cells dramatically enhanced migration toward CXCL12. In Ba/F3-Flt3 cells, synergistic cell migration to FL plus CXCL12 was associated with enhanced phosphorylation of mitogen-activated protein kinase p42/p44 (MAPK(p42/p44)), cyclic adenosine monophosphate response element binding protein (CREB), and Akt, and was partially inhibited by pretreatment of cells with selective inhibitors for MAPK(p42/p44), protein kinase A (PKA), or phosphatidylinositol 3-kinase (PI3-kinase), implicating these pathways in migration to FL plus CXCL12. In contrast, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulated CXCR4 expression, inhibited CXCL12-mediated phosphorylation of MAPK(p42/p44), CREB, and Akt, and impaired migration toward CXCL12. These findings suggest that FL/Flt3 may facilitate hematopoietic cell migration/homing and mobilization by enhancing or inhibiting CXCL12/CXCR4 signaling pathways and that the FL/Flt3 axis participates in trafficking of normal and transformed hematopoietic cells.

Acute Disease↗

PubMed2006

Antineoplastic effect of a single oral dose of the novel Flt3 inhibitor KRN383 on xenografted human leukemic cells harboring Flt3-activating mutations.

Activating mutations of Fms-like tyrosine kinase 3 (Flt3) are the most common genetic abnormalities found in acute myeloid leukemia (AML) and represent potential therapeutic targets. The novel Flt3 inhibitor KRN383 inhibited the autophosphorylation of Flt3 bearing internal tandem duplications (ITDs) and the Asp835Tyr (D835Y) point mutation with half-maximal inhibitory concentration (IC(50)) values of < or =5.9 and 43 nM, respectively. KRN383 also inhibited the proliferation of the ITD-positive cell lines with IC(50) values of < or =2.9 nM. A single oral administration of 80 mg/kg of KRN383 eradicated ITD-positive xenograft tumors in nude mice and prolonged the survival of SCID mice carrying ITD-positive AML cells. The effectiveness of a single oral dose of KRN383 suggests that it has the potential to be used in a wide variety of clinical regimens, including multicycle and combination therapies.

Administration, Oral↗

PubMed2003

FLT3-activating mutations in acute promyelocytic leukaemia: a rationale for risk-adapted therapy with FLT3 inhibitors.

Our understanding of the genetic basis of acute myeloid leukaemias has been enhanced through cloning of recurring chromosomal translocation breakpoints. However, the remarkable observation, more than a decade ago, that all-trans retinoic acid (ATRA) induced remission in patients with t(15;17) acute promyelocytic leukaemia (APL) was a driving force in the subsequent cloning and characterization of the PML-RARalpha fusion that is causally implicated in the pathogenesis of this disease. Major improvements in treatment and outcome of APL patients have been made since that time by incorporating ATRA in conventional chemotherapy but 30% of APL patients still succumb to complications of their disease or their therapy. Recent information that the haematopoietic receptor tyrosine kinase FLT3 is mutated in about 30% of APL patients suggests strategies for further improving treatment and outcome in this subset of APL patients using small-molecule inhibitors of FLT3. The role of FLT3 mutations in APL and other AML will be discussed.

Animals↗

PubMed1998

Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: comparison with stem cell factor.

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)-derived CD34+ cells expressing different levels of flt3 or c-kit tyrosine kinase (TK) receptor in clonal cell culture. The c-kit receptor was expressed by 58.5+/-16.7% of CB CD34+ cells (n=19), in which c-kit(high), c-kit(low) and c-kit cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6+/-8.3% (n=9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU-E) formation by any subpopulation of CD34+ cells. However, SCF + Epo supported BFU-E and erythrocyte-containing mixed (CFU-mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU-mix colony formation supported by interleukin (IL)-3 + Epo when CD34+c-kit(low) or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU-mix colony formation supported by IL-3 + Epo when CD34+c-kit(high) or low and CD34+FR+ cells were used. The replating potential of CFU-mix supported by IL-3 + Epo+ FL was greater when CD34+c-kit(low) or CD34+FR+ cells were used. When the CD34+c-kit(low) cells were used, the number of lineages expressed in secondary cultures of CFU-mix colonies derived from primary cultures containing IL-3 + Epo + FL or SCF was significantly larger than when the primary cultures contained IL-3 + Epo. Furthermore, the number of long-term culture-initiating cells found in CD34+FR+ cells was larger than that in FR cells. CB-derived CD34+c-kit(low) cells represent a less mature population than c-kit(high) cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB-derived CD34+FR+ cells are less mature than CD34+FR- cells.

Antigens, CD34↗

PubMed2001

A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations.

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 have been found in 20% to 30% of patients with acute myeloid leukemia (AML). These mutations constitutively activate the receptor and appear to be associated with a poor prognosis. Recent evidence that this constitutive activation is leukemogenic renders this receptor a potential target for specific therapy. In this study, dose-response cytotoxic assays were performed with AG1295, a tyrosine kinase inhibitor active against FLT3, on primary blasts from patients with AML. For each patient sample, the degree of cytotoxicity induced by AG1295 was compared to the response to cytosine arabinoside (Ara C) and correlated with the presence or absence of a FLT3/ITD mutation. AG1295 was specifically cytotoxic to AML blasts harboring FLT3/ITD mutations. The results suggest that these mutations contribute to the leukemic process and that the FLT3 receptor represents a therapeutic target in AML. (Blood. 2001;98:885-887)

Acute Disease↗

PubMed1998

Structure-function analysis of FLT3 ligand-FLT3 receptor interactions using a rapid functional screen.

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.

Amino Acid Sequence↗

PubMed1996

Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells.

The novel hematopoietic growth factor FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell factor), and FMS. the receptor for M-CSF (macrophage colony- stimulating factor). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced growth of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell growth underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean 20%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic growth factor- dependent cell lines increased their proliferation upon incubation with FL, whereas all growth factor-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic fibroblast growth factor), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming growth factor-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.

Adult↗

PubMed2002

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF.

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime naïve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.

Adjuvants, Immunologic↗

PubMed2006

Flt3-ligand-mobilized peripheral blood, but not Flt3-ligand-expanded bone marrow, facilitating cells promote establishment of chimerism and tolerance.

Facilitating cells (CD8+/TCR-) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor-plasmacytoid dendritic cells (p-preDCs), a tolerogenic cell expanded by Flt3-ligand (FL). In this study, we evaluated the function and composition of FL-expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL-expanded FCs were transplanted with c-Kit+/Sca-1+/Lin- (KSL) cells into allogeneic recipients, BM-FCs exhibited significantly impaired function whereas PB-FCs were potently functional. A significant upregulation of P-selectin expression and downregulation of VCAM-1 (vascular cell adhesion molecule 1) were present on FL-expanded PB-FCs compared with FL BM-FCs. Stromal cell-derived factor-1 (SDF-1), and CXCR4 transcripts were significantly increased in FL PB-FCs and decreased in FL BM-FCs. Supernatant from FL PB-FCs primed HSC migration to SDF-1, confirming production of the protein product. The FL PB-FCs contained a predominance of p-preDCs and natural killer (NK)-FCs, and NK-FCs were lacking in FL BM-FCs. The impaired function for BM-FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF-1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

Animals↗