Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts.

The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia (B-lineage ALL) was compared with that on the myeloid and B-lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B-lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B-lineage ALL showed a lower expression of VLA-2 and VLA-3 and a higher expression of ICAM-1 and LFA-3 than their normal bone marrow counterparts. AML CD34+ cells had less L-selectin but more VLA-5 on their surface membrane than normal myeloid CD34+ cells. B-lineage ALL CD34+ cells showed an overexpression of LFA-3. In individual patients deficiencies or over-expression of the beta1 integrin chain, VLA-4, PECAM-1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.     

Different expression of adhesion molecules on myeloid and B-lymphoid CD34+ progenitors in normal bone marrow

Abstract: The expression of adhesion molecules was studied on B lymphoid and myeloid CD34⁺ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34⁺ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B-lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34⁺ cells in the 3 subpopulations expressed VLA-4, VLA-5, LFA-3 and H-CAM. B-lymphoid progenitors showed a higher density of VLA-4 and VLA-5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B-lymphoid progenitors. The majority of the CD34⁺ cells also expressed LFA-1 and L-selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L-selectin. We conclude that B lymphoid and early and late myeloid CD34⁺ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34⁺ precursors to circulate.     

Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony- stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady- state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = - .86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = - .70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.     

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.     

Distribution of surface-membrane molecules on bone marrow and cord blood CD34+ hematopoietic cells.

We have investigated the distribution of membrane molecules on CD34+ hematopoietic cells isolated from human bone marrow (BM) and cord blood (CB). A distinct CD10+ population was present in BM, but it was not detected in CB. Most CD34+ CD10+ cells in BM were B-cell precursors (BCP), because they expressed CD19. However, CD40 and CD37 were found on the majority of CD34+ cells from either BM or CB, demonstrating that these antigens are not restricted to B-lineage CD34+ cells. CD40 and CD37 were lost during culture of CD34+ cells in the presence of interleukin 3 (IL-3), indicating transient expression early in myeloid development. CD13 antigen was detected on virtually all CD34+ cells from BM and CB. Accordingly, CD13 was present on CD34+ CD10+ cells, demonstrating that this structure is not restricted to myeloid CD34+ cells. In contrast, myeloid CD33 antigen was not detected on CD34+ CD10+ cells. Expression levels of CD13 and of CD33 were heterogeneous in BM, reflecting diversity within the resident CD34+ population. CD25 and CD71 were found on a proportion of CD34+ cells from either BM or CB and maintained during culture in IL-3, consistent with a distribution on activated cells. Finally, a variety of adhesion receptors were present on CD34+ cells. These included the alpha 4 beta 1 (VLA-4), alpha 5 beta 1 (VLA-5), and alpha L beta 2 (LFA-1) integrins, as well as ICAM-1, LFA-3, H-CAM, and LAM-1. Expression of adhesion receptors was remarkably similar in BM and CB, and it followed an all-or-nothing pattern that failed to delineate CD34+ subsets. Taken together, our data show that although CD34+ cells from BM constitute a more heterogeneous population, resident and circulating CD34+ cells largely display the same cell-surface molecules.     

Adhesion molecule expression on CD34+ progenitor cells from normal and aplastic anaemia bone marrow

Summary. Aplastic anaemia (AA) is a disease of bone marrow failure. Evidence has been produced for both a stem cell and a stromal cell defect in this disease. The contribution of deficient or defective cell adhesion molecules (CAMs) has not been determined. CAMs have been shown to be important in stem cell-stromal cell interactions and maintenance of haemopoiesis.
In this study the expression of CAMs (LFA-1, LFA-3, ICAM-1, VLA-4, CD44, sLe^x and L-selectin) on CD34+ progenitor cells from 10 normal donors and eight patients with AA was investigated using double immunofluorescence. There was no significant difference in the percentage of CD34⁺ cells that were CAM⁺ between normal and AA bone marrow, suggesting that abnormal CAM expression on AA progenitor cells is not responsible for nor contributes to the pathogenesis of the disease. However, these findings do not exclude abnormal CAM function on progenitor cells, or abnormal expression or function of CAM ligands or counter-receptors on AA stromal cells.     

Adhesion molecules on CD 34+ hematopoietic cells in normal human bone marrow and leukemia

Summary Expression of selected adhesion molecules of the integrin and immunoglobulin family was investigated on CD 34+ leukemic cells in 19 AML and 11 ALL cases to evaluate phenotypic differences in adhesive properties of malignant hematopoietic precursor cells in comparison to normal bone marrow CD 34+ cells. Of the 2-integrin family, CD 11a was expressed on > 50% of CD 34+ cells in normal bone marrow and almost all leukemias, whereas CD 11 b and CD 11 c were not expressed on CD 34+ cells in normal bone marrow, but were found on CD 34+ blasts in some leukemias of a heterogeneous immunophenotype. Of the 1-family, CDw 49d (VLA-4) was strongly expressed on normal CD 34+ bone marrow cells and on the blasts of all 30 CD 34+ leukemic samples, whereas CDw 49 b (VLA-2) was absent on CD 34+ cells in normal bone marrow, but detected on CD 34+ cells in a few leukemias which did not constitute a clinical or phenotypic entity according to the FAB classification or immunocytological analysis. The lymphocyte-homing-associated adhesion molecule CD 44 (HCAM) and CD 58 (LFA-3) were expressed on CD 34+ cells in all investigated cases of normal and leukemic bone marrow. ICAM-1 (CD 54), the inducible receptor ligand for CD 11 a/CD 18, although present on CD 34+ cells in normal bone marrow, was lacking on blast cells of some ALL and AML cases. So far, the variable expression of 2-integrins as well as of VLA-2 and of ICAM-1 could indicate distinct differences in cell-cell or cell-matrix adhesion of leukemic cells in ALL and AML patients.     

Growth factor receptor profile of CD34 cells in normal bone marrow, cord blood and mobilized peripheral blood

Growth factors regulate the proliferation and differentiation of hemopoietic cells. Their effect on hemopoietic precursors differs according to the ontogenic source of the cells. Cord blood and mobilized blood CD34(+) cells have a higher sensitivity for growth factors than bone marrow CD34(+) cells. This could be due to a higher expression of growth factor receptors. Therefore, we examined the expression of receptors for stem cell factor (SCF), interleukin-6 (IL-6), IL-3, granulocyte colony-stimulating factor (G-CSF) and IL-7 on the CD34(+) cells of cord blood, mobilized peripheral blood and bone marrow. The receptors were detected with monoclonal antibodies and flow cytometry. The majority of the CD34(+) cells in bone marrow clearly expressed SCFR; they showed a moderate positivity for IL-3Ralpha and a weak staining for G-CSFR and IL-6 Ralpha. Less than 10% of the cells were IL-7R positive. Cord blood CD34(+) cells showed a higher expression of SCFR and a lower positivity for G-CSFR and IL-6Ralpha. Mobilized blood CD34(+) cells showed a lower expression of SCFR and G-CSFR, and a higher positivity for IL-3Ralpha. This was not solely due to the presence of more myeloid precursors in mobilized blood, as the growth factor receptor profile did not correspond to that of early or late myeloid CD34(+) precursors in normal bone marrow. Changes induced by the mobilization procedure occurred as well. In conclusion, the higher sensitivity for growth factors of hemopoietic precursors in cord blood and mobilized blood cannot be explained by a general increase of the growth factor receptor expression on the CD34(+) cells.     

Decrease of L-selectin expression on human CD34+ cells on freeze-thawing and rapid recovery with short-term incubation

In the homing process of hematopoietic progenitor cells (HPCs) to bone marrow, several adhesion molecules play important roles. However, HPCs are subjected to dramatic alteration of freeze-thawing that could affect these molecules. In this study, we investigated the effect of cryopreservation on the expression of adhesion molecules on HPCs.The expression of different adhesion molecules on CD34+ cells was examined by flow cytometry using an immunofluorescence technique. Changes in expression before and after cryopreservation, and that of L-selectin on addition of dimethylsulfoxide (DMSO) or with serum incubation, were investigated. The relationship between expression and function was also determined using a transmigration assay system. L-selectin expression on CD34+ cells derived from mobilized peripheral blood (MPB), bone marrow (BM), and umbilical cord blood (CB) was significantly decreased from 37% to 23%, 48% to 11%, and 67% to 19%, respectively, by the freeze-thawing process, while the expression of other adhesion molecules was not appreciably changed. Within 30 minutes of incubation with DMSO, L-selectin expression on CD34(+) cells significantly decreased from 65% to 22%. Furthermore, this was completely prevented by the matrix metalloproteinase (MMP) inhibitor, KB-R8301, indicating that the loss of L-selectin was due to shedding mediated by an MMP. To determine if L-selectin expression can be upregulated after cryopreservation, thawed samples were cultured overnight with serum. Values were observed to return to or rise higher than those of the fresh samples, this being particularly rapid and pronounced when the CD34+ cells were cocultured with the human BM stromal cell line, HS-27A. Therefore, this adhesion molecule could possibly be restored in vivo after transplantation in a way similar to the in vitro case. Despite considerable damage to HPCs during cryopreservation, changes in these cells are reversible, in line with successful restoration of hematopoiesis after transplantation.     

Expression of adhesion molecules on CD34 cells does not correlate with property to adhere to normal marrow-derived stroma

The differences of engraftment kinetics after umbilical cord blood (CB) and mobilized peripheral blood (MPB) transplantation are not yet fully understood. Since homing into bone marrow microenvironment would certainly play crucial role during engraftment, we have investigated adhesion capacities of CB and MPB CD34⁺ cells to bone marrow stromal cells (SC) and expression of several molecules known to be important during homing process. Cells, at day 0 and after 48 hour culture with SCF+ IL-3, are plated for 2 hours on BM confluent stromal layer. The non adherent (na) and adherent (a) fractions are then recovered. Cells are counted and evaluated for the coexpression of CD34 and VLA-4 (CD49d), VLA-5 (CD49e), L-selectin (CD62L) or CXCR4. The adhesion molecule expression is expressed in term of antigen density (Mean Equivalent Soluble Fluorescence (MESF)x 10³).Our results show that a) cell adhesion capacity is significantly increased after culture in CB (78.6±4.0% vs 58.2±7.4%) as well in MPB (72.5±5.9% vs 51.5±5.4%) and is not different between the two sources of progenitors; b) VLA-4, VLA-5, CD62L and CXCR4 expression increased significantly after culture, in CB and in MPB; c) no specific expression on adherent cells could be observed except for L-selectin after 48h culture; d) the only significant difference between CB and MPB concerns the CXCR4 expression,lower on PB cells but independent of cell adhesion capacities. In conclusions, it seems that neither adhesion capacities nor expression of evaluated adhesion molecules could explain differences between CB and MPB engraftment.     

Induction of LFA-1 on pluripotent CD34+ bone marrow cells does not affect lineage commitment

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis remains obscure. Since LFA-1 is predominantly expressed by leukocytes, it is considered as a marker of late stage stem cell maturation when expressed on CD34+ bone marrow cells, and represents more mature hematopoietic progenitor cells. We observed that freshly isolated CD34+ bone marrow cells express LFA-1, and that the level of expression is highly variable. Interestingly, the expression of LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrates the LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Culturing sorted CD34+ LFA-1+ cells in single cell per well assays in medium supplemented with SCF, Epo, IL-3, Il-6, GM-CSF, and G-CSF revealed that they gave rise to dispersed macrophage-like colonies, supporting the notion that CD34+LFA-1+ cells indeed consist of a mature committed cell population. In contrast, sorted CD34+LFA-1- cells had high proliferative potential and developed into large multilineage colonies within 14 days of culture. Unanticipated, in time course experiments we observed that these CD34+LFA-1- cells expressed LFA-1 within 24 hours upon culture. This induction was neither caused by the monoclonal antibody used to tag CD34 cells, nor dependent on growth factors present in the medium. These findings demonstrate that two populations of CD34+LFA-1+ cells can be discriminated: leukocyte lineage committed CD34+ cells in freshly isolated bone marrow cells, and multipotent CD34+ cells that acquired LFA-1 upon in vitro culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA- 1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, providing those cells with adhesion receptors enabling migration in the periphery.     

VLA-2 and VLA-5 cell adhesion molecules expression in CD34+ cells from umbilical cord blood and from bone marrow

BACKGROUND/AIMS: Umbilical cord blood contains a large number of early hematopoietic cells with high proliferating capacity, that has been used as an alternative to bone marrow transplantation. The aim of this study is to investigate the number of two cell adhesion molecules in cord blood and in bone marrow. METHODS: We investigated two integrins, named VLA-2 and VLA-5 (Very Late Appearing Antigen), expressed in the surface of CD34+ cells. The CD34+ cells, isolated with MACS CD34+ isolation kit, were labelled with the appropriate monoclonal antibodies. RESULTS: Cell adhesion molecules showed highly expressed in both cord blood and bone marrow CD34+ cells. CONCLUSION: There are no significant differences between the two sources of CD34+ populations.     

Granulocyte colony-stimulating factor and lineage-independent modulation of VLA-4 expression on circulating CD34+ cells

Although the use of allogeneic transplants of peripheral blood stem/progenitor cells (PBSCs) is increasing, the precise mechanism of PBSC mobilization has not yet been fully clarified. We examined the expression of some adhesion molecules on CD34+ cells from steady-state bone marrow (BM), granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs, and cytotoxic drugs plus G-CSF-mobilized PBSCs. Irrespective of mobilization method, very late antigen (VLA)-4 expression on circulating CD34+ cells was significantly lower than on steady-state BM CD34+ cells. To elucidate the influence of lineage commitment on VLA-4 expression of circulating CD34+ cells, we analyzed VLA-4 expression on different subsets of CD34+ cells with or without CD33, CD38, CD5, or CD10 antigens, or Glycophorin A in G-CSF-mobilized PBSCs and steady-state BM from related donors, using 3-color flow cytometry. VLA-4 on circulating CD34+ subsets was less expressed than on each corresponding subset of steady-state BM CD34+ cells. Furthermore, VLA-4 positive rates showed no significant difference among the CD34+ subsets. Finally, the data comparing CD34+ cells from steady-state and G-CSF-mobilized PBSCs revealed no differences in terms of VLA-4 expression. These data suggest that reduced expression of VLA-4 may be a result of peripheralization of CD34+ cells from bone marrow, which occurs in a G-CSF- and lineage-independent fashion.     

L-selectin expression is low on CD34+ cells from patients with chronic myeloid leukemia and interferon-a up-regulates this expression

BACKGROUND AND OBJECTIVE: Altered adhesive interaction between bone marrow (BM) stroma and progenitors in chronic myeloid leukemia (CML) may be in part caused by abnormal expression of cell adhesion molecules (CAMs) on malignant progenitor cells. Treatment of CML with interferon-a (IFN-a) re-establishes normal hemopoiesis in some patients in part by restoring normal adhesive interactions between CML progenitors and BM microenvironment, which may in turn be mediated by correcting CAM expression on progenitors. DESIGN AND METHODS: We investigated the expression of CAMs (L-selectin, b((2))-integrin, LFA-3, ICAM-1, ICAM-3, NCAM) on purified BM CD34(+) cells from CML patients (n= 34) and healthy adults (n= 15) by flow cytometry. Modulation of L-selectin expression on CD34(+) cells from CML after in vitro treatment with IFN-a was also investigated. RESULTS: The mean percentage of CD34(+ )cells expressing L-selectin was significantly lower in CML patients (25.4+/-12.8%) than in normal controls (68.7+/-8.3%, n=15). CD34(+)/HLA-DR(&endash;/low) and CD34(+)/ CD38(&endash;/low) co-expressing L-selectin were also significantly lower in untreated CML (27.4+/-21.5% and 39.8+/-26.7%, respectively, n=8) than in controls (61+/-17% and 83.7+/-10%, respectively, n=7). In vitro treatment with IFN-a of purified CD34(+) BM cells from untreated CML patients (n=8) induced a significant, dose and time-dependent increase in the L-selectin expression as indicated by FACS analysis. INTERPRETATION AND CONCLUSIONS: We hypothesize that this L-selectin deficiency reflects a cell surface adhesion defect of progenitors from CML that is partially restored by in vitro IFN-a treatment. These data may help to explain the adhesive abnormalities of CML progenitors to the BM microenvironment and the in vitro restoration of adhesion capacity after IFN-a treatment.     

Mobilization of CD34+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4

The beta1 integrin very late antigen 4 (VLA-4) plays a central role in mobilization and homing of CD34+ cells. In this study, we examined the activation state of VLA-4 on CD34+ cells from bone marrow (BM) and peripheral blood (PB) by flow cytometry using a vascular cell adhesion molecule I-immunoglobulin (VCAM-I/IgG) fusion protein as soluble ligand. In an intraindividual analysis, we found a significantly reduced affinity and avidity of the VLA-4 receptor on CD34+ cells from PB during granulocyte colony-stimulating factor (G-CSF)-enhanced marrow recovery in comparison with steady-state BM. Moreover, the amount of circulating CD34+ cells during marrow recovery was inversely related to the activation state but not to the expression level of VLA-4, suggesting that a modulation of the functional state of VLA-4 is involved in the mobilization of CD34+ cells. Moreover, VLA-4 function on CD34+ cells from BM was associated with the maturation state of CD34+ cells as high-affinity VLA-4 receptors were observed on the vast majority of more primitive CD34+ cells. In addition, we found that Mg2+ ions as well as co-incubation of CD34+ cells with endothelial cells resulted in an activation of the VLA-4 receptor. In conclusion, modulation of the functional state of VLA-4 appears to be of relevance for the mobilization and homing of CD34+ haematopoietic stem cells.     

Adhesion receptor expression by CD34+ cells from peripheral blood or bone marrow grafts: correlation with time to engraftment

OBJECTIVE: This study was undertaken to define the pattern of cell adhesion receptor expression by the CD34+ progenitor cells from mobilized peripheral blood and bone marrow from normal and autologous donors, and to correlate the adhesion receptor profile with time to blood cell recovery for patients undergoing autologous transplant. METHODS: Blood cell recovery was determined by absolute neutrophil count (> 500/microL), time to last red cell transfusion, and platelet count (> 50,000/microL and > 100,000/microL). The analysis for expression of adhesion receptors alphaL (CD11a), alpha2 (CD49b), alpha3 (CD49c), alpha4 (CD49d), alpha5 (CD49e), alpha6 (CD49f), beta1 (CD29), L-selectin (CD62L), ICAM-1 (CD54), PECAM-1 (CD31), HCAM (CD44), and P-selectin (CD62P) was performed by two-color flow cytometry. The Kruskal-Wallis test and Spearman's rank correlation were used for statistical analysis. RESULTS: Statistical analysis of adhesion expression by the CD34+ population for patients undergoing autologous transplant demonstrated that the higher percent expression of PECAM-1 correlated with longer time to platelet recovery (> 100,000/microL, p = 0.049). In contrast, the higher the percent expression of alpha6 (p = 0.013) and the increased density of expression of alpha2 (p = 0.035), alpha3 (p = 0.023), alpha4 (p = 0.044), beta1 (p = 0.027), and ICAM-1 (p = 0.010) correlated with shorter time to platelet recovery. Neutrophil recovery time decreased with increased density of expression of alphaL (p = 0.014) and P-selectin (p = 0.007) receptors. Increased density of expression of CD44 (HCAM) was associated with longer time to red blood cell recovery (p = 0.05). CONCLUSION: These data suggest that upregulation of specific adhesion receptors or selection of certain cell populations could result in earlier blood cell recovery after transplant.     

Human G-CSF-mobilized CD34-positive peripheral blood progenitor cells can stimulate allogeneic T-cell responses: implications for graft rejection in mismatched transplantation

To investigate mechanisms of stem cell graft rejection we studied the allo-stimulatory potential of G-CSF mobilized peripheral blood progenitor cells (PBPC). CD34+ cells were purified (>95%) in a two-step procedure using immunoaffinity columns for CD34 selection and T-depletion. The capacity of CD34+ cells to stimulate allogeneic T-cell responses was compared with other cells from the same individual. CD34+ cells induced potent proliferative responses at stimulator:responder ratios of 1:20, but were approximately 50-fold less efficient compared to dendritic cells. Furthermore, CD34+ cells primed responses from partially matched allogeneic T cells in bulk cultures. Dual-colour flow cytometry showed that the co-stimulatory molecules B7.1, CD40 and ICAM-1 were absent on resting CD34-positive progenitor cells, but were induced during incubation with allogeneic lymphocytes due to a cytokine-mediated effect. Up-regulation of accessory molecules on CD34+ cells was reproduced by incubation with interferon-gamma or GM-CSF which enhanced the allo-stimulatory activity of CD34+ cells. Blocking studies with inhibitory antibodies suggested co-stimulatory functions for B7.2, ICAM-3, CD40 and LFA-3. CD34+ cells were more efficient in inducing allogeneic T-cell responses when compared to the unprocessed leukapheresis products. The reduced allo-stimulatory ability of G-CSF mobilized PBPC could be explained by the presence of CD3+ 4+ and CD3+ 8+ lymphocytes with suppressor activity. We conclude that current methods of stem cell selection for transplantation do not avoid allosensitization of the recipient and that further graft manipulation with add-back of lymphocytes or selection of subsets of CD34+ cells with reduced allo-stimulatory ability may reduce graft rejection.     

Spontaneous and rapid reexpression of functional CXCR4 by human steady-state peripheral blood CD34+ cells

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37 degrees C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37 degrees C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin. This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide. When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37 degrees C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated. The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells. There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37 degrees C preincubation.     

分化抑制培养体系体外扩增脐血造血细胞的研究

目的:探讨分化抑制培养体系对脐血造血细胞体外扩增效应。方法:分化抑制培养体系与脐血单个核细胞(MNC)体外共同培养7 d,检测MNC细胞总数、CFC、CD34 细胞反应扩增效果,并检测CD34 细胞表面归巢相关黏附分子VLA-4(CD49d)、VLA-5(CD49e)、LFA-1(CD11a)、HCAM(CD44)、L-selectin(CD62L)的表达率。结果:分化抑制培养体系组明显扩增脐血MNC细胞总数、CFC、CD34 细胞(均P<0．05),对照组培养脐血MNC细胞总数明显下降,CFC和CD34 细胞完全死亡(均P<0．01)。CD34 细胞表面各黏附分子CD49d、CD44和C1362L表达与扩增前相当(均P>0．05),而CD49e和CD11a表达明显高于扩增前(均P<0．05)。结论:分化抑制培养体系体外显著扩增脐血造血细胞,并且扩增后的造血干(祖)细胞总体上保持其表面归巢相关黏附分子的表达,归巢功能不会减低,是一种安全有效的扩增体系。     

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.