Low blood dendritic cells in chronic myeloid leukaemia patients correlates with loss of CD34+/CD38- primitive haematopoietic progenitors

Myeloid and plasmacytoid dendritic cells (MDC, PDC) play a key role in the initiation of immune responses. We found a reduction of DC subsets among 20 chronic myeloid leukaemia (CML) patients in chronic phase (MDC, mean, 0.10% +/- 0.10, P = 0.02; PDC, mean, 0.11% +/- 0.08, P = 0.006 versus controls). The maintenance of blood DC correlated with the presence of high percentages of circulating CD34+/CD38- progenitors that were able to give rise in vitro to CD1a+ DC. The reduced DC numbers may contribute to leukaemia escape from immune control and restoration of DC may be a goal for CML immunotherapy.     

Characterization of human blood dendritic cell subsets

Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin(-)) HLA-DR(+) cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14(+), CD16(+), and CD2(+) cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)-derived Lin(-) HLA-DR(+) preparations. Lin(-) cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin(-) HLA-DR(+) population into 5 nonoverlapping subsets (mean +/- 1 SD): CD123 (mean = 18.3% +/- 9.7%), CD1b/c (18.6% +/- 7.6%), CD16 (49.6% +/- 8.5%), BDCA-3 (2.7% +/- 1.4%), and CD34 (5.0% +/- 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin(-) HLA-DR(+) subsets, monocytes, and MoDCs. The poor viability of CD123(+) DCs in vitro was confirmed, but the CD16(+) CD11c(+) DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.     

Rapid induction of CD40 on a subset of granulocyte colony-stimulating factor-mobilized CD34(+) blood cells identifies myeloid committed progenitors and permits selection of nonimmunogenic CD40(-) progenitor cells.

CD40 antigen is a costimulatory molecule highly expressed on dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study, we evaluated CD40 expression on normal CD34(+) blood cells and functionally characterized CD34(+)CD40(+) and CD34(+)CD40(-) cell subsets. CD40, CD80, and CD86 antigens were constitutively expressed on 3.2% +/- 4.5%, 0%, and 1.8% +/- 1.2% CD34(+) blood cells, respectively. However, after 24 hours in liquid culture with medium alone, or with tumor-necrosis-factor-alpha (TNF-alpha), or with allogeneic mononuclear cells 10.8% +/- 3.8%, 75.3% +/- 15.0% and 53. 7% +/- 17.0% CD34(+) blood cells, respectively, became CD40(+). After incubation for 24 hours with TNF-alpha CD34(+)CD40(+) blood cells expressed only myeloid markers and contained less than 5% CD86(+) and CD80(+) cells. Also, a 24-hour priming with TNF-alpha or ligation of CD40 significantly increased the CD34(+) blood cells alloantigen presenting function. Finally, purified CD34(+)CD40(+) blood cells stimulated an alloreactive T-cell response in MLC, were enriched in granulocytic, monocytic, and dendritic precursors, and generated high numbers of DC in 11-14 d liquid cultures with GM-CSF, SCF, TNF-alpha and FLT-3L. In contrast, CD34(+)CD40(-) cells were poorly immunogenic, contained committed granulocytic and erythroid precursors and early progenitors, and differentiated poorly toward the DC lineage. In conclusion, a short incubation with TNF-alpha allows the selection of CD40(+) blood progenitors, which may be a useful source of DC precursors for antitumor vaccine studies, and also a CD34(+)CD40(-) blood cell fraction that could be exploited in innovative strategies of allogeneic transplantation across HLA barriers.     

Characterization of dendritic cell differentiation pathways from cord blood CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)     

Etoposide (VP-16) plus G-CSF mobilizes different dendritic cell subsets than does G-CSF alone

Dendritic cells (DCs) are antigen-presenting cells that are critical to the generation of immunologic tumor responses. Myeloid DCs (DC1) express myeloid antigen CD11c; lymphoid DCs (DC2) express CD123(+) and are CD11c(-). Analysis of DC subsets from peripheral blood progenitor cells (PBPC) collected from normal donors mobilized with G-CSF shows a predominance of DC2 cells. Whether PBPCs mobilization by chemotherapy yields different subsets of DCs has not been studied. We analyzed DC subsets in apheresis products from 44 patients undergoing autologous stem cell transplantation from 6/00 to 5/01. Patients received either G-CSF alone (10 microg/kg per day, n=11) or etoposide (2 g/m(2)) plus G-CSF (n=33) for progenitor cell mobilization. The patients were apheresed for 2-10 days (median 3) to reach a minimum of 2.0 x 10(6) CD34(+) cells/kg. Patients receiving G-CSF alone mobilized significantly more total DC2s than did those receiving etoposide plus G-CSF (median 6.2 x 10(6)/kg vs 2.9 x 10(6)/kg, P=0.001). The DC2/DC1 ratio was also significantly different in the two groups, with the G-CSF group having a higher ratio (median 1.2 vs 0.4, P<0.001). We conclude that the combination of chemotherapy plus G-CSF yields different mobilized dendritic cell subsets than does G-CSF alone.     

Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6

We studied concentration, phenotype, and function of peripheral blood (PB) dendritic cells (DCs) from patients with multiple myeloma (MM). The absolute number of circulating precursors of myeloid and plasmacytoid DCs was significantly lower in MM patients than in healthy subjects. After maturation, PBDCs from MM patients showed significantly lower expression of HLA-DR, CD40, and CD80 antigens and impaired induction of allogeneic T-cell proliferation compared with controls. Remarkably, they were not capable of presenting the patient-specific tumor idiotype to autologous T cells. Conversely, DCs generated in vitro from CD14(+) monocytes from the same patients, and PBDCs freshly isolated from healthy donors efficiently stimulated allogeneic and autologous T cells. To clarify the mechanism of PBDC deficiency in MM, we investigated the effects of the main plasma cell growth factor, interleukin-6 (IL-6), on the development of DCs from CD34(+) cells. IL-6 inhibited the colony growth of CD34(+) DC progenitors and switched the commitment of CD34(+) cells from DCs to CD14(+) CD1a(-) CD86(-)CD80(-) CD40(+/-)HLA-DR +/- monocytic cells exerting potent phagocytic activity but no antigen-presentation capacity. This effect was reversed by anti-IL-6 antibodies. Growing CD34(+) cells in the presence of autologous serum (without IL-6) also suppressed the development of functional DCs. This study demonstrates that PBDCs from MM patients are functionally defective, partially because of IL-6-mediated inhibition of development. This brings into question the advisability of using PBDCs as antigen carriers for immunotherapy trials in MM. The results also suggest a novel mechanism whereby myeloma cells escape immune recognition.     

Flow cytometric assessment of lymphocyte subsets, lymphoid progenitors, and hematopoietic stem cells in allogeneic stem cell grafts.

Currently, bone marrow (BM), cord blood (CB), and G-CSF-mobilized peripheral blood progenitor cells (PBPCs) are the most commonly used sources for allogeneic stem cell transplantation (SCT). The aim of this study was to assess the yields and distribution of lymphocyte subsets, lymphocyte progenitors and hematopoietic stem cells (HSC) in each type of allograft by three-color flow cytometry. The yields of CD34(+)CD38(-) HSCs did not differ significantly between BM grafts (2.80 +/- 0.74 x 10(6)) and leukapheresis products (LPs) (1.82 +/- 0.64 x 10(6)), and were lowest in CB grafts (0.21 +/- 0.05 x 10(6)). For most lymphocyte subsets yields were lowest in CB grafts and significantly higher in LPs than in BM grafts. BM grafts, however, contained the highest yields of CD34(+)CD19(+)CD20(-) B cell progenitors and CD19(+)CD20(-) B cells. The relative frequencies of the naive CD45RA(+)CD45RO(-) phenotype among CD4(+) and CD8(high) T cells were highest in CB grafts (P < or = 0.001), and higher in LPs than in BM grafts (P < or = 0.02). The latter finding was in accordance with a preferential G-CSF mobilization of naive T cells relative to the total lymphocyte population (P < or = 0.014). CD3(+)CD8(low) and CD3(+)CD8(low)CD4(-) subsets, which facilitate engraftment in murine transplantation models, demonstrated a tendency towards lower frequencies among T cells in CB grafts and LPs compared to BM grafts. This observation coincided with a significantly reduced mobilization of subsets potentially enriched for facilitating cells as compared to the total lymphocyte population (P < or = 0.036). The CD34(+) compartment of CB grafts contained a significantly higher percentage (12.1%) of CD34(+)CD7(+)CD3(-) T cell progenitors than those of BM grafts (5.1%) and LPs (3.6%). In addition, CB lymphocytes contained the highest fraction of CD3(-)CD16/56(+) NK cells (P < or = 0.013) and almost no CD3(+)CD16/56(+) NKT cells (P < 0.001) compared to adult cell sources. In summary, LPs, CB allografts and BM allografts differ widely with respect to the cellular composition of their lymphocyte compartments, which is partially affected by a varying mobilization efficiency of G-CSF for distinct lymphocyte subsets.     

Long-term culture of human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem cell factor induces extensive amplification of a CD34(-)CD14(-) and a CD34(-)CD14(+) dendritic cell precursor

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34(+) cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34(+) cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34(-)CD1a- CD14(+) (p14(+)) and CD34(-)CD1a-CD14(-) (p14(-)) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14(-), CD83(-)) macropinocytic DC. Mature (CD1a+, CD14(-), CD83(+)) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-alpha (TNF). In addition, p14(-) cells generated CD14(+) cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34(+) cells may improve future prospects for immunotherapy.     

Larger numbers of CD4(bright) dendritic cells in donor bone marrow are associated with increased relapse after allogeneic bone marrow transplantation

Relapse is the major cause of death after allogeneic bone marrow transplantation (BMT). This study tested the hypothesis that the numbers of donor mononuclear cells, lymphocytes, and CD34(+) cells influence relapse and event-free survival (EFS) after BMT. The study population consisted of 113 consecutive patients with hematologic malignancies who underwent non-T-cell-depleted BMT from HLA-matched siblings. Sixty-four patients had low-risk diagnoses (ALL/AML CR1, MDS RA/RARS, and CML CP1); 49 patients had high-risk diagnoses (all others). CD34(+) cells, T cells, B cells, natural killer cells, monocytes, and a rare population of CD3(-), CD4(bright) cells in the allografts were measured by flow cytometry. The CD3(-), CD4(bright) cells in bone marrow had the same frequency and phenotype as CD123(bright) type 2 dendritic cell (DC) progenitors, and they differentiated into typical DCs after short-term culture. Cox regression analyses evaluated risk strata, age, gender, and the numbers of nucleated cells, CD3(+) T cells, CD34(+) hematopoietic cells, and CD4(bright) cells as covariates for EFS, relapse, and nonrelapse mortality. Recipients of larger numbers of CD4(bright) cells had significantly lower EFS, a lower incidence of chronic graft-versus-host disease (cGVHD), and an increased incidence of relapse. Recipients of larger numbers of CD34(+) cells had improved EFS; recipients of fewer CD34(+) cells had delayed hematopoietic engraftment and increased death from infections. In conclusion, the content of donor CD4(bright) cells was associated with decreased cGVHD and graft-versus-leukemia effects in recipients of allogeneic bone marrow transplantation, consistent with a role for donor DCs in determining immune responses after allogeneic BMT.     

Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases

BACKGROUND/AIMS: Dendritic cells (DCs) are the most potent professional antigen-presenting cells. Although two subsets of circulating DCs, lineage(-)CD11c(+)CD4(low) (CD11c(+)DCs) and lineage (-)CD11c(-)CD4(+)CD123(+) (CD123(+)DCs) are identified in humans, the role of each DC subset in the immunopathogenesis of liver diseases is unknown.METHODS: We examined the numbers and activation status of each DC subset in the circulation and in the inflamed livers in patients with chronic liver diseases by flow cytometry and immunohistochemistry.RESULTS: The numbers of circulating CD11c(+)DCs were inversely correlated with serum alanine aminotransferase (ALT) levels in patients with chronic viral hepatitis, and that the expression of costimulatory molecules on circulating CD11c(+)DCs in patients with chronic viral hepatitis was significantly up-regulated in patients with high serum levels of ALT. Both DCs are also identified in the livers by flow cytometry, and the expression of costimulatory molecule CD40 on those DCs was significantly higher in liver DCs than that in circulating DCs. Moreover, the ratios of CD11c(+)DCs/CD123(+)DCs were higher in liver DCs (mean+/-SD, 7.2+/-6.0) than those of circulating DCs (4.0+/-4.6). Immunohistochemically, CD11c(+) or CD123(+) cells and CD83(+) activated DCs were observed mostly in portal areas with mononuclear cell infiltration in various liver diseases. These overall data suggest that DCs, especially CD11c(+)DCs, could be associated with the necroinflammatory response in the liver of chronic viral liver diseases.CONCLUSIONS: DCs, especially CD11c(+)DCs, may be involved in the immunopathogenesis of chronic liver diseases.     

来氟米特对狼疮患者树突状细胞作用机制的初探

目的　探讨来氟米特(LEF)处理前后系统性红斑狼疮(SLE)患者树突状细胞(DC)表面标志及功能的改变,揭示LEF治疗SLE的作用机制,为开展“抑制性DCs”治疗SLE奠定实验基础。方法　(1)分离SLE患者外周血单核细胞,用细胞因子诱导DC成熟, LEF组再加入A7717262(来氟米特的活性代谢产物)培养。第9天收集DC细胞,流式细胞仪检测CD80、CD83、CD86和HLA DR的表达。(2)分别将A771726处理或不处理的第9天DC和T细胞进行培养, 72h后用MTT法检测DC刺激淋巴细胞增殖的能力,FACS检测T细胞亚群和ELISA检测培养上清中IL 10和IFNγ水平。结果A771726处理后虽DC形态无改变,但DC表达CD83、CD86和HLA DR百分数较对照组均明显降低(72 70±1 77vs 79 36±4 80, 63 50±14 06vs. 83 91±9 81, 80 44±12 56vs. 90 51±8 63,P值均<0 01)。A771726处理后的DC,其刺激T细胞增殖相应的吸光度值明显降低,混合培养的上清液中IL 10水平较无A771726处理的DC与T细胞的混合培养上清液明显降低,而IFNγ两者间无显著差异;但见CD 4 CD 25CTLA 4 T细胞百分比增高。结论　LEF在体外可抑制SLE患者外周血DC的成熟;未成熟DC能抑制T细胞增殖及T细胞向Th2 细胞转化,诱导CD 4 CD 25CTLA 4 T细胞产生,从而纠正SLE患者的部分免疫紊乱。     

Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma

AIM: To investigate the influence of autologous cytokine-induced killer (CIK) cells on the phenotypes of CIK effector cells, peripheral T lymphocyte subsets and dendritic cell subsets in patients with primary hepatocellular carcinoma (HCC). METHODS: Peripheral blood mononuclear cells (PBMC) were collected by a blood cell separator from 13 patients with HCC, then expanded by priming them with interferon-gamma (IFN-gamma) followed by monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2) the next day. The phenotypic patterns of CIK cells were characterized by flow cytometry on d 0, 4, 7, 10, 13 and 15 of incubation, respectively. Then, 5 mL of venous blood was obtained from HCC patients before or 8-10 d after CIK cells were transfused into patients to assess the influence of CIK cells on the percentages of effector cells, and proportions of DC1 or DC2 in peripheral blood by flow cytometry. RESULTS: After two weeks of in vitro incubation, the percentages of CD3(+)CD8(+), CD3(+)CD56(+), and CD25(+) cells increased significantly from 33.5+/-10.1%, 7.7+/-2.8%, and 12.3+/-4.5% to 36.6+/-9.0% (P<0.05), 18.9+/-6.9% (P<0.01), and 16.4+/-5.9% (P<0.05), respectively. However, the percentages of CD3(+)CD4(+) and NK cells had no significant difference. The percentages of CD3(+) and CD3(+)CD8(+) cells were kept at high levels during the whole incubation period, but those of CD25(+), and CD3(+)CD56(+) cells began to decrease on d 7 and 13, respectively. The proportions of type I dendritic cell (DC1) and type II dendritic cell (DC2) subsets increased from 0.59+/-0.23% and 0.26+/-0.12% before CIK cell therapy to 0.85+/-0.27% and 0.43+/-0.19% (all P<0.01) after CIK cell transfusion, respectively. The symptoms and characteristics of HCC patients were relieved without major side effects. CONCLUSION: Our results indicated that autologous CIK cells can efficiently improve the immunological status in HCC patients, and may provide a potent approach for HCC patients as the adoptive immunotherapy.     

Blood dendritic cells in patients with acute lymphoblastic leukaemia

Myeloid and plasmacytoid dendritic cells (MDCs, PDCs) play a key role in the initiation of immune responses. In this study, we show a severe reduction of MDCs and PDCs in patients with B lineage acute lymphoblastic leukaemia (B-ALL; P = 0.01 vs. controls). DCs from patients with T lineage ALL (T-ALL) were quantitatively and functionally comparable to healthy donors, as demonstrated by secretion of interleukin (IL)-12p70 and interferon-alpha. In vitro, the circulating CD34(+) fraction of B-ALL cases did not generate either CD1a(+) MDCs or PDCs, suggesting that DC development is probably affected in B-ALL, but not in T-ALL.     

Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors

Distinct human dendritic cell (DC) subsets differentially control immunity. Thus, insights into their in vivo functions are important to understand the launching and modulation of immune responses. We show that nonobese diabetic/LtSz-scid/scid (NOD/SCID) mice engrafted with human CD34+ hematopoietic progenitors develop human myeloid and plasmacytoid DCs. The skin displays immature DCs expressing Langerin, while other tissues display interstitial DCs. Myeloid DCs from these mice induce proliferation of allogeneic CD4 T cells in vitro, and bone marrow human cells containing plasmacytoid DCs release interferon-alpha (IFN-alpha) upon influenza virus exposure. Injection of influenza virus into reconstituted mice triggers IFN-alpha release and maturation of mDCs. Thus, these mice may provide a model to study the pathophysiology of human DC subsets.     

不稳定性心绞痛患者循环内皮祖细胞与血管内皮功能的变化

目的探讨不稳定性心绞痛患者外周血循环内皮祖细胞(EPCS)与血管内皮功能的变化。方法采用高分辨率血管超声法检测30例不稳定性心绞痛患者与30例正常者作对照组肱动脉血流介导的内皮依赖性血管舒张功能(FMD)及硝酸甘油介导的非内皮依赖性血管舒张功能(NMD);流式细胞仪测定外周血中CD34+单个核细胞的水平;外周血分离单个核细胞一定条件下培养2周,免疫组织化学技术鉴定培养贴壁细胞表面标志CD34的表达;倒置荧光显微镜鉴定贴壁细胞FITC-UEA-I和DII-ACLDL双染色阳性细胞为正在分化的EPCS。结果不稳定性心绞痛组FMD明显低于对照组[(5·85±3·04)%比(8·81±4·48)%,P<0·05];NMD在两组中差异无统计学意义[(13·60±5·03)%比(14·18±4·50)%,P>0·05];CD34+细胞水平明显高于对照组[(0·13±0·05)%比(0·09±0·04)%,P<0·05];FMD与CD34+细胞水平呈负相关(R=-0·385,P<0·05)。培养的贴壁细胞免疫组化显示CD34阳性,倒置荧光显微镜显示这些贴壁细胞FITC-UEA-I和DII-ACLDL双染色阳性。结论不稳定性心绞痛患者CD34+细胞增加和血管内皮功能受损,提示循环EPCS增加可能是对急性冠状动脉缺血和内皮损伤的代偿反应。     

Flow cytometric evaluation of circulating CD34+ cell counts and apoptotic rate in children with acquired aplastic anemia and myelodysplasia

OBJECTIVE: Identification of a rapid and noninvasive test for the follow-up of aplastic anemia (AA) patients during immunosuppressive therapy (IST) to evaluate its functional effect on hematopoietic progenitors (HPC) and for early detection of progression to myelodysplasia or relapse. MATERIALS AND METHODS: Absolute count and apoptotic rate (AR) of peripheral blood (PB) CD34+ cells were evaluated by three-color flow cytometry for CD45, CD34, and annexin V in cord blood (CB), normal children, and adults, as well as in pediatric patients with AA at diagnosis and during IST, Fanconi anemia (FA), chronic immune cytopenia, and refractory anemia with excess blasts (RAEB). RESULTS: In normal subjects, the AR of PB CD34+ cells showed a progressive increase (p < 0.05), while their counts decreased (p < 0.05) from birth to adulthood. In very severe AA (vSAA) and severe AA (SAA) at diagnosis, the AR was 91.6% +/- 2.8%, higher than controls (p < 0.05), and PB CD34+ cell count was 2.6 +/- 2.4/microL. In FA patients, the PB CD34+ AR was again significantly increased (54.2% +/- 13.7%) with an absolute count of 3.7 +/- 1.2/microL. Conversely, in RAEB the AR was 11.7% +/- 3.5% and the absolute count 85.1 +/- 48.2/microL (p < 0.05). Chronic immune cytopenias did not significantly differ from controls. CONCLUSIONS: Flow cytometry evaluation of PB CD34+ AR and counts is a noninvasive and feasible first-step method for the differentiation of AA and myelodysplasia (MDS), and it might be useful for monitoring AA during IST to secure the early detection of relapse or transformation to MDS.     

Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment.

Dendritic cells (DCs) are responsible for the initiation of immune responses. Two distinct subsets of blood DCs have been characterized thus far. Myeloid DCs (MDCs) and plasmacytoid monocytes (PDCs) were shown to be able to promote polarization of naive T cells. This study shows a dramatic quantitative imbalance in both circulating blood DC subsets in 37 patients with acute myeloid leukemias. Eleven patients (30%) displayed a normal quantitative profile (MDC mean, 0.37% +/- 0.21%; range, 0.01% to 0.78%; PDC mean, 0.21% +/- 0.24%; range, 0.04% to 0.62%), whereas 22 (59%) showed a tremendous expansion of MDCs (9 patients: mean, 16.76% +/- 14.03%; range, 1.36% to 41%), PDCs (4 patients: mean, 7.28% +/- 6.84%; range, 1% to 14%), or both subsets (9 patients: MDC mean, 10.86% +/- 12.36%; range, 1.02% to 37.1%; PDC mean, 4.25% +/- 3.78%; range, 1.14% to 13.04%). Finally, in 4 patients (11%), no DC subsets were detectable. Both MDC and PDC subsets exhibited the original leukemic chromosomal abnormality. Ex vivo, leukemic PDCs, but not leukemic MDCs, had impaired capacity for maturation and decreased allostimulatory activity. Also, leukemic PDCs were altered in their ability to secrete interferon-alpha. These data provide evidence that DC subsets in vivo may be affected by leukemogenesis and may contribute to leukemia escape from immune control.     

Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab

CAMPATH antibodies recognize CD52, a phosphatidylinositol-linked membrane protein expressed by mature lymphocytes and monocytes. Since some antigen-presenting dendritic cells (DCs) differentiate from a monocytic progenitor, we investigated the expression of CD52 on dendritic cell subsets. Four-color staining for lineage markers (CD3, 14, 16, 19, 20, 34, and 56), HLA-DR, CD52, and CD123 or CD11c demonstrated that myeloid peripheral blood (PB) DCs, defined as lineage(-)HLA-DR(+)CD11c(+), express CD52, while expression by CD123(+) lymphoid DCs was variable. Depletion of CD52(+) cells from normal PB strongly inhibited their stimulatory activity in an allogeneic mixed lymphocyte reaction and also reduced the primary autologous response to the potent neoantigen keyhole limpet hemocyanin. CD52 is thus expressed by a myeloid subset of PBDCs that is strongly allostimulatory and capable of initiating a primary immune response to soluble antigen. Administration of alemtuzumab, a humanized monoclonal antibody against CD52, to patients with lymphoproliferative disorders or as conditioning for hematopoietic stem cell transplantation resulted in a marked reduction in circulating lineage(-)HLA-DR(+) DCs (mean 31-fold reduction, P =.043). Analysis of monocyte-derived DCs in vitro revealed a reduction in CD52 expression during culture in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, with complete loss following activation-induced maturation with lipopolysaccharide. In contrast to the findings in PB, epidermal and small-intestine DCs did not express CD52, suggesting either that transit from blood to epidermis and gut is associated with loss of CD52 or that DCs in these tissues originate from another population of cells.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.