Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells.

The proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture was examined with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation.HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry.Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL + TPO/ML did not significantly increase CD34(+) peripheral blood stem cell (PBSC) numbers.UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. Additional studies are needed to establish the functional activity of expanded UCB-HSC.

Altered development and cytokine responses of myeloid progenitors in the absence of transcription factor, interferon consensus sequence binding protein.

Mice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP(-/-) mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP(-/-) myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP(-/-) colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP(-/-) mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP(-/-) progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP(-/-) bone marrow to fibronectin. In summary, ICSBP(-/-) myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.

Protective effects of BB-10010 treatment on chemotherapy-induced neutropenia in mice.

Chemotherapy-induced neutropenia is a major dose-limiting factor in the management of cancer patients. Most chemotherapeutic agents are active against proliferating cells, interfering with DNA replication and/or mitosis. A number of chemokines, notably macrophage inflammatory protein-1 alpha [MIP-1alpha], have been reported to induce cell-cycle arrest in immature hematopoietic progenitors, raising the possibility that chemokines, such as MIP-1alpha, could be used to reduce or even eliminate the hematologic toxicity of cycle-active chemotherapy. We tested the effectiveness of BB-10010 [a genetically engineered analog of human MIP-1alpha] in vivo against three different cytotoxic drugs [cyclophosphamide (Cy), 5-fluorouracil (5-FU) and cytosine arabinoside (Ara-C)] commonly used in cancer therapy. BB-10010 treatment reduced the toxicity of all three agents, though the precise mode of protection varied with the cytotoxic drug used. BB-10010 reduced the neutropenic interval in Cy-treated mice without affecting the neutropenic nadir, whereas the absolute neutrophil counts [ANC] of both 5-FU and Ara-C treated mice were significantly higher throughout the neutropenic interval for mice receiving BB-10010 prior to chemotherapy. These findings indicate that the ability to manipulate the cell cycle of hematopoietic progenitors with chemokines, such as BB-10010/MIP-1alpha and other negative regulators, may be exploited to reduce chemotherapy-induced neutropenia; furthermore, the fact that BB-10010 is effective against several different cytotoxic agents is cause for guarded optimism that this approach may be generally applicable, and, once optimized for patient use, may prove to be of significant clinical benefit.

Development of macrophage anticryptococcal activity in vitro is dependent on endogenous M-CSF.

We previously showed that nonactivated resident murine peritoneal macrophages (PM) from five strains (e.g., BALB/c) have C'-dependent fungistasis for Cryptococcus neoformans in 24-h coculture, but not CD-1 PM unless culture time was extended or M-CSF treatment was used. We studied effect of a rat IgG1 monoclonal (m) antibody (Ab) to murine M-CSF receptor on this anticryptococal activity. Culture of BALB/c PM with mAb, diluted 1:10, prechallenge reduced fungistasis from 58 to 21% (P < 0.01), whereas further 10-fold dilutions did not. Moreover, M-CSF pretreatment (5000 U/ml) significantly enhanced fungistasis (to 85%), whereas adding mAb 1:10 or 1:100 reduced that (to 58 and 77%, respectively, P < 0.01). In 48-h culture CD-1 PM had 39% fungistasis, reduced to 0% by mAb, M-CSF treatment of CD-1 PM increased fungistasis to 72%, which was reduced to 13 or 58% (P < 0. 001) by 1:10 or 1:100 mAb, respectively. Complete blocking by mAb of CD-1 PM activity was consistent with lack of measurable early endogenous CD-1 M-CSF production. Increasing exogenous M-CSF could overcome the inhibition by mAb (64% fungistasis BALB/c PM reduced to 11% with inhibition by mAb or increased to 94% with 5000 U/ml M-CSF; 37% with both mAb and M-CSF, 51% with mAb and 10,000 U/ml; P < 0.05, 5000 U/ml + mAb vs 10,000 U/ml + mAb). Moreover, rabbit Ab to M-CSF significantly reduced anticryptococcal activity of untreated BALB/c macrophages. In summary, development of PM fugistatic activity is dependent on endogenous M-CSF, since it is blocked by anti-receptor mAb (as is exogenous M-CSF stimulation) or anti-M-CSF Ab, and macrophages of the mouse strain with delayed activity had no measurable early M-CSF production.

Mobilization and transplantation of peripheral blood stem cells.

Two hundred nineteen patients underwent peripheral blood stem cell (PBSC) transplantation from 1990 to 1997. Stem cells were mobilized with cyclophosphamide (CY), or with CY plus Taxol or etoposide, followed by cytokines, and collected when leukocyte counts > or = 1,000/microl, or when CD34+ counts > or = 20/microl. On average, four to five collections were needed to obtain sufficient PBSC for engraftment. When CD34+ counts were used, the average number of collections decreased from 5.4 to 4.2. A discrepancy was noted in the extraction ratios and number of collections that depended on the optical density (I/O) setting of the leukapheresis machine. Patients collected at a setting of 100 had higher extraction ratios and required fewer collections (mean = 2.7) than those collected at 150 (mean = 4.4). This result was unexpected, because the entire mononuclear cell layer is collected at the higher I/O setting. Further analysis revealed that a larger volume of red cells was collected at 150 than at 100. These procedures used a small-volume collection chamber, so the chamber was apparently overloaded by RBC at the higher setting. More rapid recovery of neutrophil counts and platelet counts was seen in PBSC transplants than in autologous marrow transplants; moreover, PBSC transplant patients required fewer RBC and platelet transfusions. Sixteen out of 21 normal donors for allogeneic PBSC transplants gave adequate collections (> 2.5 x 10(6) CD34+ cells/kg), but three donors failed to yield > or = 1.5 x 10(6) CD34 cells/kg. This suggests an inherent difference among certain normal donors that may make PBSC mobilization difficult.

Recovery from retrovirus-induced immune suppression in BDP/J mice: dominance of the "regressor' phenotype.

Murine acquired immune deficiency syndrome (MAIDS) is an immunosuppressive disease of mice induced by infection with the LP-BM5 murine leukemia virus (MuLV) retrovirus isolate. Certain inbred strains of mice are resistant to disease, but F1 crosses between sensitive and resistant strains are predominantly sensitive to MAIDS. One inbred strain, BDP, demonstrates a novel disease phenotype, recovery of immune function after a period of profound immune suppression. This trait is genetically dominant in crosses between BDP and either sensitive or resistant strains. The 'regressor' phenotype reveals the existence of a mechanism for recovery from immunosuppressive retrovirus infections, which may be of import in developing therapies for AIDS patients.

UVB radiation interrupts cytokine-mediated support of an epidermal-derived dendritic cell line (XS52) by a dual mechanism.

We have established long-term dendritic cell lines from the epidermis of newborn mice. These cell lines (XS series) proliferate maximally in response to granulocyte/macrophage-colony stimulating factor, as well as to CSF-1, which is produced by skin-derived NS fibroblast lines and by keratinocytes (albeit in smaller amounts). The purpose of this study was to examine the impact of UVB radiation on CSF-1-mediated interaction of dendritic cells with fibroblasts and keratinocytes. Exposure of NS cells to UVB radiation (unfiltered FS20 sunlamp) decreased CSF-1 production at mRNA and protein levels. Both changes occurred in a dose-dependent fashion, with 50 J/m2 causing a significant reduction. UVB radiation also downregulated CSF-1 mRNA expression by Pam 212 keratinocytes. UVB exposure of XS cells diminished the surface expression of CSF-1 receptors, with 50 J/m2 causing a significant reduction. Thus, UVB radiation interrupts CSF-1-mediated cell-cell interaction by a dual mechanism: downregulating CSF-1 production and abrogating CSF-1 receptor expression. Importantly, granulocyte/macrophage-colony stimulating factor receptor expression by XS cells was also inhibited by UVB radiation, once again, with 50 J/m2 producing significant inhibition. We propose that the resulting CSF-1 deficiency in epidermal microenvironment and unresponsiveness by dendritic cells to relevant growth factors may contribute to UVB-mediated loss of resident epidermal dendritic cells (i.e., Langerhans cells) in skin.

Adoptive transfer of resistance to murine retrovirus-induced immune suppression.

Infection of certain strains of mice, such as C57BL/6 and C57BL/10 [B10], with LP-BM5 murine leukemia virus (MuLV) rapidly causes a profound and lethal immune suppression. The H2d congenic strain of B10, B10.D2, is resistant to disease, but B10 x B10.D2 F1 mice are susceptible, indicating that disease sensitivity is dominant. To determine whether disease resistance could be adoptively transferred to a sensitive host, radiation chimeras (B10.D2 --> B10 x B10.D2 F1 and F1 --> F1) were challenged with LP-BM5 virus. Infected B10.D2 --> F1 chimeras showed no loss of immune function, whereas F1 --> F1 chimeras infected with LP-BM5 MuLV developed MAIDS and became completely immune suppressed. These results, coupled with previous studies, indicate resistance or sensitivity to disease is an inherent property of the hematopoietic system that can be transferred by bone marrow grafts.

Paradoxical stimulation of normal and leukemic rat hematopoiesis by monoclonal antibody to CSF-1 receptor.

We have recently developed a rat monoclonal antibody directed against the murine M-CSF receptor (c-fms). This reagent also inhibits in vitro colony formation by leukemic and normal rat splenocytes in response to M-CSF. At high antibody concentrations, the antibody augments, rather than inhibits, colony formation by rat cells in the presence of M-CSF, an effect that is not seen when murine cells are used as responders. The costimulatory and inhibitory activities of the monoclonal antibody preparations copurify in a number of purification methods, indicating that the costimulatory activity is intrinsic to the antireceptor antibody. Conversion of the antibody into monovalent Fab fragments by papain digestion destroys costimulatory activity. This finding raises a cautionary note for the in vivo use of intact monoclonal antibodies directed against growth factor receptors for the treatment of hematologic malignancies.

Enhancement of monocytopoiesis by granulocyte colony-stimulating factor: evidence for secondary cytokine effects in vivo.

Granulocyte colony-stimulating factor (G-CSF) is used with increasing frequency to reduce the neutropenic interval following dose-intensive chemotherapy. In mice, exogenous G-CSF reduces neutrophil recovery after 5-fluorouracil (5-FU) treatment from 14 to 8 days. G-CSF treatment also enhances recovery of blood monocytes; colony assays show increased numbers of macrophage and granulocyte-macrophage progenitors (CFU-M and CFU-GM) in the marrow. This unexpected effect of G-CSF treatment is dependent on endogenous M-CSF; antiserum to murine M-CSF inhibits both peripheral monocyte and monocytic progenitor recovery without affecting neutrophil or CFU-GM recovery. Conversely, the effect of M-CSF depletion is seen only in G-CSF-stimulated recovery; monocyte levels in mice treated with antiserum to M-CSF after 5-FU are indistinguishable from mice given 5-FU alone. No synergy between G-CSF and M-CSF can be demonstrated in vitro with either normal or 5-FU-treated marrow, indicating this G-CSF/M-CSF interaction must be indirect. These results reveal an unpredicted beneficial effect of G-CSF treatment on monocyte recovery and a role for endogenous M-CSF in rebound hematopoiesis.

Identification of a growth factor for primary murine stroma as macrophage colony-stimulating factor.

Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as the sole factor in the supernatant of the stromal cell line AC3.U that promotes stroma formation. Culture of marrow, for as little as 1 week, depleted M-CSF-dependent SIC while increasing the incidence of replatable, factor-independent SIC. This suggests that culture changes the properties of SICs, perhaps by inducing differentiation into mature stromal cells. Thus, our results show a novel function of M-CSF as an important modulator of stroma formation.

Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series) derived from murine epidermis.

We have established recently from mouse epidermis long-term dendritic cell lines (XS series) that resemble epidermal Langerhans cells (LC) by their surface phenotype, Ag-presenting profile and cytokine mRNA profile. The growth of XS lines was promoted maximally by granulocyte-macrophage-CSF or by a factor secreted by NS lines, which are fibroblastic cell lines established from dispase-separated specimens of mouse epidermis. The purpose of this study was to determine the identity of XS cell growth factor secreted by NS cells. We report the following: 1) NS cells express constitutively mRNA for CSF-1; 2) XS cells express the CSF-1R at mRNA and protein levels; 3) rCSF-1 mimics NS culture supernatant in its ability to promote XS cell growth; 4) NS supernatant-dependent XS cell growth is blocked completely by each of two Abs against the CSF-1R. We conclude that CSF-1 is responsible for the XS growth-promoting activity secreted by NS lines. We also report the following: 5) LC freshly isolated from skin express CSF-1R mRNA; and 6) fibroblasts derived from specimens of dermis also express mRNA and secrete large amounts (50-100 ng/ml) of CSF-1. These observations give rise to a new concept that dermal fibroblasts may support the survival and growth of LC (and their precursors) through the paracrine effect of elaborated CSF-1.

Inhibition of day-12 spleen colony-forming units by a monoclonal antibody to the murine macrophage/monocyte colony-stimulating factor receptor.

Primitive hematopoietic stem cells differentiate into committed progenitors that are thought to selectively express hematopoietic growth factor receptor(s), thereby acquiring hematopoietic growth factor responsiveness. To assess whether hematopoietic stem cells express hematopoietic growth factor receptors, the progenitor activity of bone marrow (BM) fractions, isolated by expression of receptors for macrophage/monocyte colony-stimulating factor (M-CSF), were examined. Recovery of day-12 spleen colony-forming units (CFU-S) is diminished in both M-CSF receptor-positive (M-CSFR+) and M-CSFR- fractions, indicating antibody inhibition of day-12 CFU-S. Incubation of BM cells with antibody without fractionation inhibits 50% to 60% of day-12 CFU-S. This inhibition is specific (control antibodies have no effect) and reversible by removal of bound antibody at low pH. Incubating BM cells with control or antireceptor antibody does not affect day-8 CFU-S, which are predominantly erythroid. Treating sublethally irradiated mice with antibody inhibits endogenous day-12 CFU-S. These results indicate that some early progenitors express M-CSFRs, and blocking M-CSFRs inhibits the ability of these progenitors to form colonies, possibly because of inactivation caused by prolonged receptor blockade.

LP-BM5 murine retrovirus-induced immunodeficiency disease in allogeneic SCID chimeric mice. Inability to recognize a putative viral superantigen does not prevent induction of disease.

T cell recognition of viral superantigens has been postulated to contribute to the pathogenesis of the immunodeficiency disease induced in mice by infection with the LP-BM5 murine leukemia virus complex. A candidate superantigen has been identified in the B cell lymphoma line B6-1710 derived from an LP-BM5-infected C57BL/6 (H-2b) mouse. We have asked whether the stimulatory activity expressed by B6-1710 behaves as a superantigen by assessing the ability of T cells from fully allogeneic H-2b-->H-2d SCID chimeric mice to respond to the line. T cells from allochimeric mice failed to respond to B6-1710, whereas they responded normally to Staphylococcus enterotoxin B, a well characterized superantigen. Despite this finding, allochimeric mice were fully susceptible to the immune deficiency disease induced by LP-BM5 virus infection. These findings show that the role of superantigen expression in retrovirus-induced immune deficiency disease remains to be defined.

Homozygous scid/scid;beige/beige mice have low levels of spontaneous or neonatal T cell-induced B cell generation.

The autosomal recessive scid mutation results in defective immunoglobulin and T cell receptor gene rearrangement. The scid mutation occurred in the allotype congenic C.B-17 line, and up to 25% of C.B-17 scid mice spontaneously produce both T cells and immunoglobulin, a phenotype known as "leaky." Moreover, introduction of neonatal T cells into C.B-17 scid mice leads to immunoglobulin production by 100% of animals. We have produced mice homozygous for both the scid and beige mutations. By contrast with C.B-17 scid mice, BALB/c scid.beige mice have a < 2% incidence of "leakiness." This percentage does not increase with age, and introduction of neonatal T cells fails to rescue immunoglobulin production. This suggests that a gene (or genes) closely linked to the beige locus regulates B and/or T cell development.

CD4 is expressed on murine pluripotent hematopoietic stem cells.

We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock-Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.

Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.