ALBERT

Description: Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient. Figure Figure

Description: Multiple hematopoietic defects have been defined in NOD mice and in humans with type I diabetes, including defects in myeloid cells and antigen presenting cells that correlate with diabetes progression. Since the replacement of HSC in NOD mice can eliminate the progression of autoimmunity and control on-going autoimmune responses, we characterized the function of HSC from NOD mice. We found that purified HSC from NOD mice have an autonomous behavior when transplanted in allogeneic recipient strains as reflected by significantly enhanced engraftment in allogeneic recipients. NOD HSC were able to compete for engraftment with syngeneic HSC even when the NOD and syngeneic HSC were given at a 1:1 ratio. NOD BMC produced a higher number of splenic colonies compared to B10.BR BMC in the allogeneic day 12 CFU-S assay. We also demonstrated that NOD HSC had a high resistance to irradiation, as reflected by the cell survival 20 hours after irradiation and in the in vitro CFC assay. These data suggest that NOD HSC escape alloreactivity and compete with normal HSC. The enhanced engraftment ability in allogeneic recipients of NOD HSC was not due to an increase in frequency of primitive HSC, enumerated by day 35 cobblestone area forming cells (CAFC). This finding was further confirmed by the fact that there was no difference in the long-term repopulating cell phenotype (CD49e+/CD49ddim) between HSC obtained from NOD, B10.BR or C57BL/10 mice. Notably, NOD bone marrow cells exhibit significantly enhanced chemotaxis to SDF-1 in vitro and significantly increased HSC adhesion to primary stroma. This was associated with an increase in the expression of VCAM-1, ICAM-1 and ICAM-2 on NOD HSC. Using NOD mice congenic at selected Idd loci with C57BL/10, we determined that the enhanced engraftment potential of NOD HSC mapped to the Idd9 (insulin-dependent diabetes) locus and, as such, the TNF receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSC. In conclusion, NOD HSC exhibit increased autonomy in vivo and in vitro compared to non-diabetic strains, and engraft better in allogeneic recipients, possibly due to enhanced migration and adherence to the microenvironment. This finding may be of interest for a better understanding of disease pathogenesis and in developing cell-based strategies to cure diabetes.

Description: Approaches to enhance engraftment of hematopoietic stem cells (HSC) are of primary interest in BM transplantation. CD8+TCR− facilitating cells (FC) improve HSC engraftment in allogeneic recipients without causing graft versus host disease. FC also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients, suggesting that FC act directly on HSC. We therefore analyzed the mechanism for FC-mediated effects on HSC. We found that FC increased the ability of purified HSC to generate colonies in both the CFC and CAFC/LTC-IC assays, confirming a direct effect of FC on different HSC compartments. Co-incubation of HSC with FC for 18 or 40 hours significantly increased the survival of HSC and their subsequent ability to generate CFC at these same time points. We determined that the anti-apoptotic effect of FC on HSC was associated with the up regulation of anti-apoptotic Bcl-3 transcripts. We postulated here that the effect of FC on HSC was due to cytokine secretion. As FC produce TNFα after CpG ODN stimulation and TNFα has various activities on HSC, we evaluated the role of TNFα on FC function. FC were sorted from TNFα deficient mice and the facilitative activity of FC on HSC engraftment was assessed. FC from TNFα deficient mice were impaired in facilitating HSC engraftment in both the syngeneic and allogeneic models, suggesting a role for TNFα in FC function. Notably, TNFα transcripts were present in FC by 16 hours of co-incubation of FC + HSC and FC produce TNFα (surface and intra-cellular) when in contact with HSC. Furthermore, when TNFα was blocked (using anti-TNFα mAb), FC from wild type mice lost the ability to increase HSC clonogenicity (from 38.2±13.6 CFC/1000 HSC in HSC alone to 65.7 ± 22 for HSC + FC and 38.2 ± 19.6 for FC pre-incubated 1 hour with anti-TNFα mAb before incubation with HSC). Moreover, anti-TNFα mAb also blocked the ability of FC to up-regulate Bcl-3 transcripts in HSC. In conclusion, FC act directly on HSC via several mechanisms to maintain the balance between proliferation/differentiation/survival of HSC. One central mechanism implicates TNFα production by FC, which may protect HSC from undergoing apoptosis by up-regulating anti-apoptotic transcripts (e.g. Bcl-3). These findings may have great impact for the use of accessory cells in HSC transplantation, especially when numbers of HSC are limiting.

Description: Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA–band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.

Description: Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.

Description: Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

Description: Recipient sensitization to MHC antigens from transfusion therapy and prior graft rejection is among the most critical of problems in clinical transplantation. Sensitized patients reject vascularized organ or bone marrow transplants within minutes to hours as a result of preformed anti-donor Abs. Preventing allosensitization at the time recipients are exposed to donor alloantigens would be of obvious clinical benefit. B cell activation and the generation of memory B cells depends upon T cell responses via signaling from the co-stimulatory molecule CD154 (on activated T cells) to CD40 (on B cells). We have demonstrated in an allogeneic mouse model [BALB/c (H2Kd) to B6 (H2Kb)] that blockade of T and B cell interactions with anti-CD154 induces B cell tolerance, as defined by complete abrogation of the generation of donor-specific Ab after skin grafting. Furthermore, anti-CD154 treatment promotes successful subsequent bone marrow transplantation in these recipients, confirming that sensitization was prevented. In this study, we evaluated the effect of anti-CD154 mAb on T- and B-cell populations, activation state, and cytokine expression by T cells. B6 recipients were treated with anti-CD154 (day 0 and +3) or isotype hamster IgG control around the time receiving BALB/c skin grafts (day 0), and the number of T-cells (CD4+ and CD8+), total B-cells (CD19+), immature B-cells (CD19+CD24highCD23low), and follicular B-cells (CD19+CD24lowCD23high) in the spleen was enumerated by 4 color flow cytometry at day 7, 15 and 25 after skin grafting. No significant difference in absolute number of T- and B-cell subpopulations was seen between anti-CD154 and control IgG treated groups at the time points tested. By measuring the percentage CD71+ cells in the CD8+ or CD4+ gate or CD69+ in the CD19+ gate, activated T and B cell populations were evaluated. In vivo blockade of CD154 resulted in a significantly reduced activation of alloreactive T- and B-cells: the percentage of CD8+/CD71+ T cells was significantly lower at day 7 and the percentage of CD4+/CD71+ T cells was significantly lower at all time points compared with control mice (P 〈 0.05). The percentage of CD19+/CD69+ B cells at day 7 and 25 was significantly lower compared with control IgG treated mice (P 〈 0.05). To determine the effect of anti-CD154 treatment on Th1 and Th2 cytokine production, intracellular IFN-γ and IL-10 expression was analyzed. The IFN-γ expression in both CD8 and CD4 T-cells was inhibited at day 7 and reached significance (P 〈 0.01) by day 15 compared with control IgG treated group. IL-10, a cytokine which promotes B-cell activation and differentiation expression, was similar at day 7 between the two groups, but significantly decreased in both CD8 and CD4 T-cells at day 15 in mice treated with anti-CD154. Therefore, these data suggest that blockade of CD154 during initial antigen exposure mechanistically interferes with activation of both allo antigen-specific T and B-cells and inhibits the generation of allogeneic Ab (allosensitization). These effects are associated with suppression of IFN-γ and IL-10 cytokine secretion. Figure Figure

Description: Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

Description: Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

Description: Human band 3 Walton is an AE1 mutation that results in the deletion of the 11 COOH-terminal amino acids of the protein and is associated with dominant distal renal tubular acidosis. The properties of band 3 Walton expressed with normal band 3 in the heterozygous mutant erythrocytes and the kidney isoform expressed in Xenopusoocytes and in the Madin-Darby canine kidney cell line were examined. The mutant erythrocytes have normal hematology but have reduced band 3 Walton content. Transport studies showed that erythrocyte band 3 Walton has normal sulfate transport activity, and kidney band 3 Walton has normal chloride transport activity when expressed inXenopus oocytes. The mutant protein is clearly able to reach the cell surface of erythrocytes and oocytes. In contrast, while normal kidney band 3 was expressed at the cell surface in the kidney cell line, the Walton mutant protein was retained intracellularly within the kidney cells. The results demonstrate that band 3 Walton is targeted differently in erythrocytes and kidney cells and indicate that the COOH-terminal tail of band 3 is required to allow movement to the cell surface in kidney cells. It is proposed here that the mutant band 3 gives rise to dominant distal renal tubular acidosis by inhibiting the movement of normal band 3 to the cell surface. It is suggested that this results from the association of the normal and mutant proteins in band 3 hetero-oligomers, which causes the intracellular retention of normal band 3 with the mutant protein.