Ontogeny-associated changes in the cytokine responses of primitive human haemopoietic cells

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34⁺CD38⁻ cord blood (CB) cells as was obtained in similar cultures of adult human CD34⁺CD38⁻ bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34⁺CD38⁻ CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.     

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.     

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.     

Amount of mpl on bone marrow haemopoietic precursor cells from healthy volunteers and patients with refractory anaemia

Using a non-isotopic ligand binding assay using multi-colour flow cytometry, we quantitatively examined the amount of mpl in megakaryocyte-platelet lineage cells. Firstly, we quantified the amount of mpl on cell lines. Mpl gene-transfected BaF3 cells expressed a large amount of mpl , whereas original BaF3, K562, HL-60 and NOMO-1 cells showed no mpl . In bone marrow cells from healthy volunteers, mpl was expressed on CD34⁺ cells from the very early stage of differentiation when they had no CD38 antigen. The amount of mpl increased with differentiation to CD34⁺CD41⁺ cells, but decreased with further differentiation to CD34⁻CD41⁺ cells. In CD34⁺CD41⁺ cells the amount of mpl varied according to cell size: abundant in large cells, moderate in medium-size cells and a little in small cells.
In bone marrow cells from patients with refractory anaemia (RA), the amount of mpl was decreased compared with that in bone marrow cells from healthy volunteers. When analysed by the same CD phenotype and same cell size, the amount of mpl was less in RA patients compared with that in healthy volunteers in all phenotypes and sizes tested. The proportion of large CD34⁺CD41⁺ cells was less in RA patients than in normal volunteers.     

High-resolution cell division tracking demonstrates the Flt3-ligand-dependence of human marrow CD34+CD38− cell production in vitro

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34⁺ (CD45RA/CD71)⁻, but not CD34⁺(CD45RA/CD71)⁺, human marrow cells initially recruited into division in vitro , shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34⁺CD38⁻ population through several (up to four) cell generations. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34⁺(CD45RA/CD71)⁻ cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

Cellular origin and extent of clonal involvement in multiple myeloma: genetic and phenotypic studies

The cellular origin and extent of clonal involvement in multiple myeloma (MM) are controversial. The third-complementarity-determining region (CDR3) of the immunoglobulin heavy chain gene is the target region of V_H replacements and somatic mutations. We analysed the CDR3 sequences of myeloma cells from eight newly diagnosed and three relapsed patients in order to elucidate the target cell of malignant transformation in MM. We also examined the extent of clonal involvement in MM using a CDR3 clone-specific nucleic acid probe. The peripheral lymphocytes from the five MM patients were separated into fractions such as CD34⁺, CD20⁺CD10⁺, CD20⁺CD21⁺, CD20⁺CD19⁻ and CD2⁺ cells. Amplified CDR3 DNAs from these subpopulations were hybridized with the probe specific to each patient's tumour cells. We found no evidence of ongoing V_H replacements or somatic mutations in CDR3 in MM. However, frequent nucleotide mutations in D and J_H segments were observed. Circulating malignant cells were detected in the CD34⁺ and all of the CD20⁺ subpopulations, but not in the CD2⁺ fraction. MM is a neoplasm originating from a B-lineage cell which has already undergone antigendependent selection. Nevertheless, the tumour cells are composed of heterogeneous subpopulations at various stages of differentiation, similar to normal B-lineage cells. Conversely, T cells were not involved in MM. These results imply that there is an analogous developmental pathway between the normal B-lineage cells and the tumour cells of MM.     

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.     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

Flow cytometric identification of candidate normal stem cell populations in CD45-negative B-cell precursor acute lymphoblastic leukaemia

CD45-negative B-cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34-positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 10⁶, storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34⁺CD19⁻ events. In eight of 12 patients (67%) with CD45-negative B-cell precursor ALL, a distinct CD34⁺CD19⁻CD45⁺ candidate normal stem cell population could be detected. In one patient analysed by four-colour staining, the CD34⁺CD19⁻CD45⁺ cells, unlike the CD45-negative leukaemic cells, expressed CD117 (c-kit), providing further evidence that these cells represent residual non-leukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34⁺CD19⁻CD45⁻cells, which were detected in 11 of the 12 patients within the CD34⁺CD19⁻ compartment.     

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.     

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.     

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.     

Expression of granulocyte colony-stimulating factor receptor increases with differentiation in myeloid cells by a newly-devised quantitative flow-cytometric assay

Summary. In order to develop a non-isotopic quantitative assay of granulocyte colony-stimulating factor (G-CSF) receptors on human or murine cells, we devised a flow-cytometric assay using cells stained with biotin-labelled G-CSF (b-G-CSF) and a streptavidin-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 fluorescence profiles according to the increase of fluorescence intensity due to the specific binding of b-G-CSF to G-CSF receptors. A good correlation was observed between the number of G-CSF receptors obtained by the radioisotopic binding assay and the number calculated from the D value by the flow-cytometric assay. Then, expression of G-CSF receptors on human bone marrow cells, peripheral blood granulocytes and blast cells from patients with acute myeloid leukaemia (AML) were studied. G-CSF receptors was expressed on CD34⁺CD33⁻, CD34+CD33+ and CD34⁻CD33+ cells in the following order: CD34 CD33⁺CD34⁺CD33⁺CD34⁺CD33 cells, indicating that the receptors increased with maturation. The receptor levels of CD34⁻CD33⁺ cells in bone marrow were apparently lower than those of CD34⁻CD33⁺ cells in peripheral blood granulocytes. On the other hand, an abnormal expression pattern of G-CSF receptors was noted in AML blast cells.     

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10⁺CD19⁺ cells and CD20⁻CD22⁺ cells in the CD34⁺ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34⁺CD10⁺CD19⁺ and CD34⁺CD20⁻CD22⁺ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.     

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis , on prolonged maintenance and expansion of cord blood CD34⁺ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34⁺ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34⁺, CD34⁺CD38⁻, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45⁺ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34⁺ cells for transplantation.     

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.