Mobilization of human lymphoid progenitors after treatment with granulocyte colony-stimulating factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+ CD10+ CD19- Lin- and CD34+ CD10+ CD19+ Lin- cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.     

Differentiation of CD34+ cells from human cord blood and murine bone marrow is suppressed by C6 beta-chemokines

Several recently identified chemokines, Lkn-1, CKbeta8-1, MRP-2, and Mu C10 (MRP-1), are classified as C6 beta-chemokines. All of these chemokines have been found to suppress colony formation by bone marrow (BM) myeloid progenitors. Since cord blood (CB), like BM, contains CD34-positive cells, we examined the effects of these chemokines on CD34+ cells isolated from human CB. Lkn-1 and CKbeta8-1 suppressed colony formation by multi-potential granulocyte erythroid mega-karyocyte macrophages (CFU-GEMM), granulocyte-macrophages (CFU-GM), and erythroid (BFU-E) cells among the CD34+ cells from CB. CC chemokine receptor 1 (CCR1) that is known to be a receptor for Lkn-1 and CKbeta8-1 in neutrophils, monocytes, and lymphocytes, was also present on the surface of CD34+ cells from CB. Taken together these results suggest that Lkn-1 and CKbeta8-1 are active in inhibiting myeloid progenitor cells from both BM and CB. Macrophage inflammatory protein related protein-2 (mMRP-2) and Mu C10 (mMRP-1), which are murine C6 beta-chemokines, also inhibited colony formation by CB CD34+ cells. The inhibitory activity of these chemokines suggests that they may protect hematopoietic progenitors from the cytotoxic effects of the antiblastic drugs used in cancer therapy.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.     

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.     

Expression of CD24 on CD19- CD79a+ early B-cell progenitors in human bone marrow

CD24 is a surface marker expressed in immature and mature B cells and involved in cellular adhesion and apoptosis. There are no data, which delineate the stage in early development of human B cells, which marks the expression of CD24. We studied lymphopoiesis in normal pediatric bone marrow (BM) and found that 1.5+/-0.2% of WBC were CD24(+) lymphocytes which did not express CD19. A significant fraction of these cells expressed low levels of CD45 (CD19- CD24+ CD45low cells). Small numbers of CD19- CD24+ CD45low cells were found in the regenerating BM of children with acute lymphoblastic leukemia after the completion of chemotherapy and in normal adult BM. Flow cytometric analyses have shown that CD19- CD24+ CD45low lymphocytes express CD10, CD34, CD79a, CD179a (VpreB), and TdT markers, i.e., displayed antigenic properties of early B-cell progenitors. Our data indicate that CD19- early B-cell progenitors in human BM express CD24, and that the expression of CD24 in human B-cell development precedes the expression of CD19.     

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33

BACKGROUND: AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS: In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS: The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION: These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.     

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.     

FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI(2), endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.     

Follicular dendritic cell regulation of CXCR4-mediated germinal center CD4 T cell migration

Follicular dendritic cells (FDCs) up-regulate the chemokine receptor CXCR4 on CD4 T cells, and a major subpopulation of germinal center (GC) T cells (CD4(+)CD57(+)), which are adjacent to FDCs in vivo, expresses high levels of CXCR4. We therefore reasoned that GC T cells would actively migrate to stromal cell-derived factor-1 (CXCL12), the CXCR4 ligand, and tested this using Transwell migration assays with GC T cells and other CD4 T cells (CD57(-)) that expressed much lower levels of CXCR4. Unexpectedly, GC T cells were virtually nonresponsive to CXCL12, whereas CD57(-)CD4 T cells migrated efficiently despite reduced CXCR4 expression. In contrast, GC T cells efficiently migrated to B cell chemoattractant-1/CXCL13 and FDC supernatant, which contained CXCL13 produced by FDCs. Importantly, GC T cell nonresponsiveness to CXCL12 correlated with high ex vivo expression of regulator of G protein signaling (RGS), RGS13 and RGS16, mRNA and expression of protein in vivo. Furthermore, FDCs up-regulated both RGS13 and RGS16 mRNA expression in non-GC T cells, resulting in their impaired migration to CXCL12. Finally, GC T cells down-regulated RGS13 and RGS16 expression in the absence of FDCs and regained migratory competence to CXCL12. Although GC T cells express high levels of CXCR4, signaling through this receptor appears to be specifically inhibited by FDC-mediated expression of RGS13 and RGS16. Thus, FDCs appear to directly affect GC T cell migration within lymphoid follicles.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells

CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Valpha24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with alpha-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX(3)CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.     

Tumor necrosis factor-alpha regulates the expression of inducible costimulator receptor ligand on CD34(+) progenitor cells during differentiation into antigen presenting cells

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.     

CXCR7 Is Highly Expressed in Acute Lymphoblastic Leukemia and Potentiates CXCR4 Response to CXCL12

Recently, a novel CXCL12-binding receptor, has been identified. This CXCL12-binding receptor commonly known as CXCR7 (CXC chemokine receptor 7), has lately, based on a novel nomenclature, has received the name ACKR3 (atypical chemokine receptor 3). In this study, we aimed to investigate the expression of CXCR7 in leukemic cells, as well as its participation in CXCL12 response. Interesting, we clearly demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic cells compared with myeloid or normal hematopoietic cells and that CXCR7 contributed to T-acute lymphoid leukemic cell migration induced by CXCL12. Moreover, we showed that the cellular location of CXCR7 varied among T-lymphoid cells and this finding may be related to their migration capacity. Finally, we hypothesized that CXCR7 potentiates CXCR4 response and may contribute to the maintenance of leukemia by initiating cell recruitment to bone marrow niches that were once occupied by normal hematopoietic stem cells.