Megakaryocyte progenitors derived from bone marrow or G-CSF-mobilized peripheral blood CD34+ cells show a distinct phenotype and responsiveness to interleukin-3 (IL-3) and PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)

In the present study we investigated the proliferative response of megakaryocyte progenitor cells (CFU-MK) derived from peripheral blood stem cell (PBSC) collections of patients with haematological malignancies and normal donors. Highly purified CD34⁺ cells and mononuclear cell fractions were assayed in the presence of recombinant interleukin-3 (IL-3) and pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), alone or in combination, and megakaryocyte colony formation was evaluated in the plasma clot. In comparison, steady-state bone marrow samples from normal donors were highly enriched in CD34⁺ cells and tested with the cytokines studied. Our results showed that IL-3 was able to stimulate CFU-MK colony formation from bone marrow and peripheral blood CD34⁺ cells. Similarly, PEG-rHuMGDF stimulated, in a dose–response manner, CD34⁺ cells from the bone marrow. However, normal mobilized peripheral blood CD34⁺ cells were not induced to generate CFU-MK colonies by PEG-rHuMGDF. The same lack of response was observed when patients peripheral blood CD34⁺ cells primed with chemotherapy plus G-CSF or with G-CSF alone were assessed. In contrast, PEG-rHuMGDF stimulated CFU-MK growth when mononuclear cells, either from the bone marrow or from mobilized peripheral blood, were grown in plasma clot. Moreover, we analysed by flow cytometry the expression of Mpl receptor on the cell membrane of normal mobilized peripheral blood and normal steady-state bone marrow CD34⁺ cells. Our results showed a reduced expression of Mpl receptor on mobilized peripheral blood progenitor cells in comparison with bone marrow cells.     

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells

Summary. A large expansion of activated T cells (CD3⁺CD25⁺) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25⁺) CD4⁺ and CD8⁺ cells in nearly equal proportions. Kinetics studies showed that CD4⁺CD25⁺ cells proliferated more rapidly and peaked earlier than CD8⁺CD25⁺ cells. When experiments were performed with purified subpopulations by removing CD4⁺ cells (resulting in CD8⁺ BMMCs) or by removing CD8⁺ cells (resulting in CD4⁺ BMMCs), T-cell activation and autologous plasma cell decrease were observed in CD4⁺ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8⁺ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4⁺ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Thl-like profile of CD3-activated CD4⁺ cells.
These data indicate that CD4⁺ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4⁺ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

Second transplantation with CD34+ bone marrow cells selected from a two-loci HLA-mismatched sibling for a patient with chronic myeloid leukaemia

A 43-year-old man with chronic myeloid leukaemia underwent a second transplant with CD34⁺ bone marrow cells selected from his two-loci HLA-mismatched sibling after rejection of the first graft from an HLA-matched unrelated donor. By immunomagnetic positive selection, CD34⁺ marrow cells at 0.95×10⁶/kg with 97% purity and CD3⁺ T lymphocytes at 1.3×10⁴/kg were collected and transplanted. Engraftment was confirmed to be of CD34⁺ cell-donor origin. The patient developed only grade I acute graft-versus-host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34⁺ bone marrow cells are capable of reconstituting haemopoiesis and that CD34⁺ selection could be applicable to T-cell depletion.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia

Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo . We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61⁺ cells in 7 d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61⁺ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61⁺ cells were increased 8–10-fold by the same dose. Although increased CFU-Mk growth was only seen in 1/10 AML and 6/16 MDS marrows, CD61⁺ cell numbers in suspension culture were increased in 9/13 AML and 12/15 MDS samples, responses ranged from very limited to normal magnitude. There was no correlation between platelet count and CFU-Mk number, CD61⁺ cell number or response to PEG rHuMGDF. We did not find any increased CFU-GM colony or cluster growth in response to PEG rHuMGDF and the CD61⁺ cells produced in suspension culture had features of megakaryocytic differentiation. These data suggest that PEG rHuMGDF can enhance megakaryocyte proliferation in some patients with MDS and AML, and may have a role in the treatment of thrombocytopenia in these patients.     

Impaired survival of bone marrow GPIIb/IIIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia

Glycoproteic (GP) IIb/IIIa⁺ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV-1 seropositive thrombocytopenic patients, eight HIV-1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV-1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa⁺ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa⁺ cells from HIV-1 seropositive thrombocytopenic patients showed a significant (  P  < 0.001) increase of apoptosis with respect to both HIV-1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa⁺ cells purified from HIV-1 seropositive thrombocytopenic patients was inversely (  P  < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV-1 disease nor a direct infection with HIV-1 seems to play a primary role in the impaired survival of BM GPIIb/IIIa⁺ megakaryocytic cells. These findings strengthen the notion that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV-1 related thrombocytopenia.     

Haemopoietic progenitor cells are reduced in aplastic anaemia

Summary We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double-colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS), AA CD34⁺ cells were reduced by 68% compared to normal. In addition, AA CD33⁺ cells and the three progenitor subsets (CD34⁺/CD33^–, CD34⁺/CD33⁺ and CD34^–/CD33⁺) were reduced by 44–80%. Our data lend further support for an early stem cell deficiency in AA.     

Cytokine activity after allogeneic bone marrow transplantation, V. Analysis of IL-2 and IFN production by isolated CD4+ and CD8+ cells

Summary. Previous studies from this laboratory have shown that PBMC from recipients of an HLA-identical sibling bone marrow transplant produce levels of IL-2 which are 10–100-fold lower than those produced by the same number of PBMC from healthy controls, whereas production of IFN-γ is normal. The present study examined IL-2 and IFN production over a range of cell numbers for PBMC and for isolated CD4⁺ and CD8⁺ cells for controls and marrow transplant recipients. There was a 5-fold lower IL-2 production in marrow transplant recipient CD8⁺ cells compared with equivalent numbers of control cells, whereas no difference was found in IL-2 production by CD4⁺ cells. In contrast, IFN production by CD4⁺ cells from marrow transplant recipients was 4-fold higher than in controls, whereas CD8⁺ cells from both populations produced similar amounts of IFN. When the observed production of cytokine by PBMC was compared with the expected production based on the CD4⁺ and CD8⁺ content of the PBMC, control values were similar, but the expected values for both cytokines were approximately 2-fold higher than the observed values for marrow transplant recipients. The results suggest that the capacity of T cells from marrow transplant recipients to produce IL-2 and IFN is not impaired, but that the frequency of cytokine-producing cells may be reduced, and that a negative interaction present in recipient PBMC, eliminated by isolating T-cell subsets, is responsible for the observed low levels of cytokine production.     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.     

Alloreactive CD4+ T lymphocytes can exert cytotoxicity to chronic myeloid leukaemia cells processing and presenting exogenous antigen

Existing evidence supports that CD4⁺ T lymphocytes play a role in the graft-versus-leukaemia (GVL) reaction after allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML), not only as initiators of the immune response but also as effectors of GVL. In BMT between HLA-identical pairs this CD4-mediated GVL would require CML cells to process and present antigens through MHC class II molecules. To investigate whether CML cells are capable of processing and presenting antigens, and suitable targets for CD4⁺ T-cell-mediated cytotoxicity, we generated HLA-DR1-restricted CD4⁺ cytotoxic T-cell clones that specifically recognized tuberculous purified protein derivative (PPD). We have shown that CML cells and B lymphoblastoid cell line (B-LCL) cells but not PHA-blasts from patients with CML processed exogenous antigen, PPD, and induced proliferative and cytotoxic CD4⁺ T-cell responses. Antigen presentation was blocked by antibodies to HLA-DR but not to MHC class I and by treatment with chloroquine and brefeldin. This indicates that CML cells use a classic MHC class II antigen processing pathway to present PPD antigens to CD4⁺ T cells. Cytotoxicity to CML was shown by antibody blocking studies to be mediated mainly through fas antigen. These findings indicate that donor CD4⁺ T cells alone are sufficient to mediate GVL effects following allogeneic BMT for CML.     

Cell-mediated suppression of megakaryocytopoiesis in acquired amegakaryocytic thrombocytopenic purpura

Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a disorder of hematopoiesis characterized by severe thrombocytopenia due to a selective reduction or total absence of megakaryocytes in an otherwise normal-appearing bone marrow. Although the development of autoantibodies directed against cells in the megakaryocyte progenitor cell pool has been implicated in the pathogenesis of this disorder, cell-mediated suppression of megakaryocytopoiesis has not been described. Accordingly, we report two cases of AATP in which in vitro suppression of megakaryocyte colony formation by autologous ancillary marrow cells was demonstrable. Light-density bone marrow mononuclear cells (MNCs) obtained from both patients were either plated directly into plasma clot cultures, or after first being depleted by adherent monocytes (M phi) or T lymphocytes using standard methodologies. In some experiments, the depleted ancillary marrow cells were recovered for autologous co-culture studies with the MNCs from which they had been depleted. Megakaryocyte colony formation was detected in the cultures using an indirect immunofluorescence assay with a rabbit anti- human platelet glycoprotein antiserum. Removal of M phi (n = 6), or T lymphocytes (n = 4) from normal marrow MNCs had no apparent effect on colony formation. In contrast, depleting T lymphocytes from the MNCs of patient 1 significantly augmented megakaryocyte colony formation; a similar effect was observed after depleting M phi from the MNCs of patient 2. This observed augmentation in colony formation could be abrogated by autologous co-culture with the putative suppressor cell at effector cell/target cell ratios of 1:10 in the case of T lymphocytes or 1:5 in the case of M phi. Neither suppression nor stimulation of megakaryocyte colony formation was observed after culturing normal MNCs with autologous T cells (n = 4) or M phi (n = 3) at similar or greater ratios. We also observed inhibition of megakaryocyte colony formation after culturing normal MNCs in the presence of tissue culture medium conditioned by the M phi of patient 2. This effect was shown to be specific for megakaryocytes since this same conditioned medium had no significant effect on BFU-E and CFU-E-derived colony formation by autologous marrow mononuclear cells. These results suggest that: both T cells and M phi are capable of exerting a regulatory effect on the proliferation of human megakaryocyte progenitor cells (CFU-Meg); in the case of M phi, a soluble factor elaborated by these cells may be responsible for suppressing CFU-Meg growth; and aberrant ancillary cell- megakaryocyte progenitor cell interactions may lead to clinically significant disease.     

SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells

Summary. We investigated sialylated Lewis x (sLe^x) antigen expression on CD 34 positive (CD 34⁺) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34⁺ bone marrow progenitors strongly expressed the sLe^x antigen. This contradicts previous publications which reported sLe^x expression on malignant blast cells but not on normal CD 34⁺ progenitor cells.     

Haemopoietic progenitor cell differentiation: flow cytometric assessment in bone marrow and thymus

Summary. We have recently shown that expression of any of the lineage-associated molecules CD2, CD7, CD10, CD19 or CD33 does not ensure lineage-commitment of CD34⁺ progenitor cells. Further, normal progenitor cells and leukaemic blast cells have been shown to coexpress molecules associated with more than one haemopoietic lineage. Five-dimensional flow cytometric analysis of normal bone marrow cells was exploited to investigate the hypothesis of a developmental stage in haemopoiesis comprising CD34⁺ cells coexpressing CD2, CD5, CD7, CD10, CD19 and CD33 or any combination of these molecules. We report on a subpopulation of CD34⁺ bone marrow cells constituting < 5% of the CD34⁺ cells and characterized by extensive coexpression of several molecules associated with the B lymphoid, T lymphoid and myeloid lineages. There is every probability that some cells display the CD34+ CD2⁺ CD5⁺ CD7⁺ CD10⁺ CD19+ CD33+ phenotype. Studies on postnatal thymocytes suggest that this may be the phenotype or one of a few phenotypes of a candidate thymus-seeding progenitor cell population. Finally, our findings that CD34⁺ as well as CD34⁺ CD5⁺ thymocytes can be driven into non-T-lymphoid differentiation by cytokines, support the notion that the thymus is seeded by uncommitted progenitors.     

Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection

CD8⁺ T lymphocyte responses are important in the clearance of viral infections. In chronic infections they may contribute to pathogenesis. To investigate the role of CD8⁺ T lymphocyte responses in viral clearance and chronic hepatitis C we have compared hepatitis C virus (HCV) specific cytotoxicity and interferon-gamma (IFN-γ) production in patients with resolved-acute, and chronic HCV infection. CD8⁺ T cell responses to a panel of 13 HCV T cell peptide epitopes were studied using Elispot assays of IFN-γ production and chromium release cytotoxicity assays. Responses of seven patients with resolved acute HCV infection were compared with those of 14 chronically infected patients. HCV-specific cytotoxicity differentiated the two populations of patients. The majority (71%) of patients with resolved acute infection tested positive to 42% of relevant peptides compared with the minority (28%) of patients with chronic hepatitis C ( P =0.03) who responded to only 8% of relevant peptides ( P =0.0009). In contrast, HCV-specific IFN-γ production was detected in 86% of patients with either resolved or chronic infection in response to 42% and 35%, respectively, of relevant peptides tested (not significant). In patients with chronic infection the magnitude of the HCV-specific IFN-γ production was inversely correlated to viral load ( R ²=0.52; P =0.042). Failure to clear HCV infection may be attributable to the presence of noncytolytic IFN-γ producing CD8⁺ T lymphocytes in chronically infected patients. However these CD8⁺ T cells may play a beneficial role in contributing to the control of viral load in chronic hepatitis C.     

Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine–RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by ¹²⁵I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34⁺CD38⁻ cells (approximately 1600 sites/cell), and decreased in the following order: CD34⁺CD38⁻ cells > CD34⁺CD38⁺ cells > CD34⁻CD38⁺ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34⁺ fraction expressed more EpoR than their CD34⁻ fraction. However, the expression levels of EpoR of these fractions were lower than CD34⁺CD38⁻ cells. These results indicated that EpoR was highly expressed on CD34⁺ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

Influence of bone marrow graft B lymphocyte subsets on outcome after HLA-identical sibling transplants

The potential role of the infused B cell subset after Hematopoietic Stem Cell Transplantation has not been yet studied. The present study analyzed the impact of B cells on transplant outcome in 254 patients who received a bone marrow graft from a human leucocyte antigen-identical sibling donor. The influence of B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) and B cells (immature and mature B cells, CD34⁻ CD19⁺) was also analyzed. All included patients received a myeloablative regimen. The cumulative incidence function of acute graft-versus-host (GvHD) grade II to IV was 48% and was inversely associated with the number of CD34⁺ CD19⁺. There were no statistically significant associations between B cell subsets and chronic GvHD or survival. The CD34⁺ CD19⁺ B cell subset remained significantly associated with acute GvHD in multivariate analysis (Relative risk = 0·32, 95% confidence interval: 0·11–0·92, P  =   0·035). In conclusion, a higher B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) cell dose is associated with a significant decrease incidence of acute GvHD.     

Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA-5, VLA-4, LFA-1, ICAM-1, L-selectin and c-kit) on bone marrow CD34⁺⁺ cells from 16 CML patients by three-colour flow cytometry. The mean percentage of cells expressing L-selectin in the CD34⁺⁺CD38^{+  ∼  ++} fraction from untreated CML patients was significantly lower, and that in the CD34⁺⁺CD38⁻ fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon-α (IFN-α), the mean percentage of the cells expressing L-selectin in the CD34⁺⁺CD38⁻ fraction from three patients with a low percentage of Ph¹(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph¹(+) cells. In addition, L-selectin expression rate was inversely correlated to the percentage of Ph¹(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34⁺⁺ fraction except LFA-1. These data suggest that decreased L-selectin expression in CML CD34⁺⁺ cells reflects one of the features of malignant CML progenitors.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients ( P  = 0.039), which are absent in both CD34⁻ MDS cells ( P  = 0.53) and also CD34⁺ cells from normal subjects ( P  = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34⁻ cells ( P  = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34⁻ bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.