Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective as with Dexa-BEAM plus G-CSF in lymphoma patients

We compared retrospectively the efficacy of granulocyte colony stimulating factor (G-CSF) alone with chemotherapy plus G-CSF in mobilizing CD34-positive cells in patients with malignant lymphoma. 35 patients underwent peripheral blood stem cell (PBSC) collection following mobilization either with 24 μg/kg G-CSF for 4 consecutive days (n = 18) or Dexa-BEAM chemotherapy plus 5 μg/kg G-CSF (n = 17). High-dose G-CSF was well tolerated with only slight bone pain and/or myalgia. The Dexa-BEAM therapy required hospitalization with a median duration of 21 d. The median number of apheresis procedures in both groups was two (range two to four), resulting in a median of 5.3 and 5.1 × 10⁶ CD34⁺ cells/kg. No patients in the G-CSF group, but one in the Dexa-BEAM group, failed to reach the target of collecting >2.0 × 10⁶ CD34⁺ cells/kg. The number of CFU-GM (10.4 v 6.0 × 10⁵/kg) and of BFU-E (10.6 v 4.5 × 10⁵/kg; P = 0.04) was higher in the G-CSF group than in the Dexa-BEAM group. A subset analysis of CD34⁺ cells was performed in 16 patients showing a higher mean of Thy-1 (CD90w) coexpression in the G-CSF than in the Dexa-BEAM group (4.8 v 1.8%, P = 0.12). Additionally the percentage of CD34⁺/CD38^? cells was higher in the G-CSF group (10.6% v 8.8%). However, these differences were not stastistically significant. The median time to leucocyte and platelet engraftment after high-dose chemotherapy was slightly shorter in the G-CSF than in the Dexa-BEAM group (9 v 10 and 12 v 13.5 d, respectively). These results demonstrate that high-dose G-CSF is as effective as Dexa-BEAM plus G-CSF in mobilizing peripheral blood stem cells and produces prompt engraftment. The major advantages of G-CSF mobilization were the safe outpatient self-application and the fixed-day apheresis.     

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets

Activated natural killer (A-NK) cells, a subset of CD56^dimCD3^– lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph⁺ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (±SD) of 83 ± 7% of CD56⁺CD3^– cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27 ± 33% CD56⁺CD3^– cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56⁺CD3^– cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl⁺ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl⁺ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P = 0.002 and P = 0.029, respectively) in the BCR/abl^– cultures than those in the six BCR/abl⁺ cultures. 5/6 BCR/abl^– cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56⁺CD3⁺ cells. Only 2/6 BCR/abl⁺ cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33⁺) were more frequently detected in the BCR/abl⁺ than BCR/abl^– A-NK cell cultures (P = 0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl⁺ cells from these cultures.     

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites

Increasing evidence indicates that hepatic CD8α⁺ dendritic cells (DCs) are important antigen cross‐presenting cells (APC) involved in the priming of protective CD8⁺ T‐cell responses induced by live‐attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α⁺ DCs in protective pre‐erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3^?/? mice, which lack functional CD8α⁺ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine‐induced immunity. We show that in wild‐type, but not in Batf3^?/?, mice CD8α⁺ DCs accumulate in the liver after immunization with live irradiation‐attenuated P. berghei sporozoites. IFN‐γ production by Plasmodium antigen‐specific CD8⁺ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC‐CD8+ T‐cell axis correlates with MHC class II upregulation on splenic CD8α^? DCs. Collectively, these findings underscore the essential role of CD8α⁺ DCs in robust protection induced by experimental live‐attenuated malaria vaccines.     

GM-CSF promotes differentiation of a precursor cell of monocytes and Langerhans-type dendritic cells from CD34+ haemopoietic progenitor cells

Epithelia-associated dendritic cells (DC) including Langerhans cells in the skin (LC) are precursors of lymph node located interdigitating DC (iDC). CD1a⁺ LC are known to be derived from CD34⁺ haemopoietic progenitor cells (HPC); however, cells of an intermediate differentiation state that are CD34^? and CD1a^? have not been identified. Monitoring the differentiation pathway of HPC in the presence of GM-CSF + IL-4, we observed the emergence of a distinct LC precursor population that was CD33⁺ CD13⁺ CD4⁺ CD38⁺ CD44⁺ CD34^? CD14^? CD1a^?. The cells could be separated by FACS due to a unique CD44/CD38 expression pattern or by CD44 expression in conjunction with the SSC profile. It was found that they were similarly generated in the presence of GM-CSF alone and were detectable in culture for at least a week. Irrespective of being generated in the presence of GM-CSF + IL-4 or GM-CSF alone, CD44/SSC-sorted precursor cells matured to MHC class II compartments (MIIC) and Birbeck granules (BG) expressing LC, when subsequently cultured in the presence of GM-CSF + IL-4. When IL-4 was omitted, however, the same cells matured to phagocytically active adherent macrophages (MΦ). These culture conditions were associated with a > 4-fold increase in the concentration of IL-6 when compared to those used for LC differentiation. The identification of a distinct oligopotent precursor cell population that can deliberately be induced to give rise to BG⁺ MIIC⁺ CD1a⁺ CD14^? LC or to adherent CD14⁺ MΦ further substantiates the close relationship of monocytes and DC and may help to identify its in vivo equivalent.     

Generation and functional characterization of human dendritic cells derived from CD34+ cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34⁺ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34⁺ cells, compared to that of BM CD34⁺ precursors in response to GM-CSF and TNF-α with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34⁺ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34⁺ cells enhanced both the percentage of total CD1a⁺ cells and CD1a⁺CD14^? cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34⁺ DC precursors into PB and circulating CD34⁺ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediate-late-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system

Introduction: One of the key factors critical for a successful human cord blood transplantation in treating patients with hematopoietic disorders is the number of hematopoietic stem/progenitor cells derived from human cord blood. Here, we report an alginate three‐dimensional (3D) culture system for the expansion of CD34⁺ cells in cord blood mononuclear cells (CBMCs). Methods: Cord blood mononuclear cells were isolated from human cord blood and encapsulated in 3D alginate beads. The cells were grown with different concentrations of cytokines. At day 0, 3, 6, 9, and 12, respectively, the percentage of CD34⁺ cells was quantified by flow cytometry. Colony‐forming cell assay was performed to determine the potential of hematopoietic reconstruction of the amplified cells under the 3D culture system. Results: After culturing for 12 days, the CBMCs encapsulated in the 3D alginate beads were amplified 5.89 ± 0.72 fold, CD34⁺ cells increased from 2.60 ± 0.52% to 13.27 ± 2.65%, and the colony‐forming assay showed that the colony‐forming unit‐granulocyte/granulocyte‐macrophage (CFU‐G/GM) increased from 363.34 ± 34.47/10⁵ cells to 3423.33 ± 645.14/10⁵ cells (P < 0.001). In comparison, the conventional two‐dimensional (2D) culture system showed that the CBMCs, CD34⁺ cells and the CFU‐G/GM were 0.68 ± 0.16 fold, 0.45 ± 0.17%, and 532.92 ± 82.97/10⁵ cells, respectively. Conclusion: This study demonstrates a new and efficient method to amplify the CD34⁺ human cord blood hematopoietic stem/progenitor cells in a 3D alginate culture system ex vivo for extended periods while retaining the hematopoietic reconstruction capacity.     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

Protection and immunological study on two tetraspanin‐derived vaccine candidates against schistosomiasis japonicum

Tetraspanins (TSPs) are proteins found on the surface of helminth parasites of the genus Schistosoma and are regarded as potentially protective antigens. The large extracellular loop of Schistosoma mansoni tetraspanin‐2, Sm‐TSP‐2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. It is well recognized that CD4⁺ T‐cell‐dependent immunity might play an important role against schistosomes; however, the contribution of CD8⁺ T cells against multicellular pathogen is still uncertain. The exogenous protein‐pulsed dendritic cells (DCs) can easily activate CD4⁺ T cells response, while CD8⁺ T cells response was relatively difficult to be induced. In this study, we evaluated the immunogenicity of TSP2HD antigen (hydrophilic domain of the S. japonicum tetraspanin‐2) and TAT (the protein transduction domain of HIV‐1)‐coupled TSP2HD protein. As TAT‐fused protein could promote major histocompatibility complex class I‐dependent antigen presentation in vitro, TAT‐TSP2HD‐pulsed DCs induced stronger proliferation of schistosome‐specific CD8⁺ T cells compared with DCs incubated with TSP2HD alone. Vaccination with TAT‐TSP2HD‐pulsed DCs in vivo could improve disease outcome in S. japonicum‐infected mice and was slightly superior to vaccination with DCs treated with TSP2HD. In summary, these data showed that TAT fusion proteins could help activate CD8⁺ cells and Th1 cells and provide part protection against schistosome.     

Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production

IL-22 is a cytokine that acts mainly on epithelial cells. In the skin, it mediates keratinocyte proliferation and epidermal hyperplasia and is thought to play a central role in inflammatory diseases with marked epidermal acanthosis, such as psoriasis. Although IL-22 was initially considered a Th17 cytokine, increasing evidence suggests that T helper cells can produce IL-22 even without IL-17 expression. In addition, we have shown the existence of this unique IL-22-producing T cell in normal skin and in the skin of psoriasis and atopic dermatitis patients. In the present study, we investigated the ability of cutaneous resident dendritic cells (DCs) to differentiate IL-22-producing cells. Using FACS, we isolated Langerhans cells (LCs; HLA-DR⁺CD207⁺ cells) and dermal DCs (HLA-DR^hiCD11c⁺BDCA-1⁺ cells) from normal human epidermis and dermis, respectively. Both LCs and dermal DCs significantly induced IL-22-producing CD4⁺ and CD8⁺ T cells from peripheral blood T cells and naive CD4⁺ T cells in mixed leukocyte reactions. LCs were more powerful in the induction of IL-22-producing cells than dermal DCs. Moreover, in vitro-generated LC-type DCs induced IL-22-producing cells more efficiently than monocyte-derived DCs. The induced IL-22 production was more correlated with IFN-γ than IL-17. Surprisingly, the majority of IL-22-producing cells induced by LCs and dermal DCs lacked the expression of IL-17, IFN-γ, and IL-4. Thus, LCs and dermal DCs preferentially induced helper T cells to produce only IL-22, possibly “Th22” cells. Our data indicate that cutaneous DCs, especially LCs, may control the generation of distinct IL-22 producing Th22 cells infiltrating into the skin.     

The immune-modulatory effects of a mixed herbal formula on dendritic cells and CD4+ T lymphocytes in the treatment of dust mite allergy asthma and perennial allergic rhinitis

Objective: Asthma and allergic rhinitis are chronic inflammatory diseases of the conducting nasal airway. Traditional Chinese medicine has long been used for supplemental therapy of allergic diseases, especially asthma and allergic rhinitis. We previously reported the effects of a mixed herbal formula in patients with allergic rhinitis. However, the immune-modulatory mechanism underlying the effects of herbal medicine for the treatment of allergic diseases remains unclear. Methods: We investigated the physiologic changes in dendritic cell (DC) and CD4⁺ T cell activity in patients with asthma and allergic rhinitis who were treated with a mixed herbal formula composed of Xin-yi-san?+?Xiao-qing-long-tang?+?Xiang-sha-liu- jun-zi-tang. Specifically, we set up in vitro autologous or heterologous co-culture experiments between DCs and CD4⁺ T cells, and used flow cytometry and ELISA to analyze the expression of surface molecules on DCs and the release of cytokines by CD4⁺ T cells. Results: Expression of HLA-DR on DCs was suppressed following treatment with the mixed herbal formula. Surface expression of CD40, CD54 and CD86 on DCs was also attenuated after treatment. In autologous co-cultures, CD4⁺ T cells increased their IL-10 production while decreasing TNF-α production. In heterologous co-cultures, IL-10 secretion by T cells was enhanced, while IL-12, IL-4, IL-5 and TNF-α secretion were reduced. Conclusion: Our mixed herbal formula attenuated the allergic reaction by modifying the physiologic function of the DC–CD4⁺ T cell interaction. Further investigations are necessary to understand the mechanism of immune modification mediated by the mixed herbal formula.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.     

Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205–mediated delivery of the self-peptide proteolipid protein (PLP)139–151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139–151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4⁺ Vβ6⁺ TCR T cells specific for PLP139–151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4⁺ T cell-mediated suppressor mechanism was obtained.     

CD70-silenced dendritic cells induce immune tolerance in immune thrombocytopenia patients

The hyper-response of dendritic cell (DC) is believed to participate in the pathogenesis of immune thrombocytopenia (ITP). The CD70 expression on the surface of DCs that takes part in the CD27-CD70 costimulation pathway is an important element of DC ‘licensing’, which may initiate a series of autoreactive immune responses. To elucidate the roles CD70 molecules play in the DCs of ITP patients, we first stimulated the CD70 molecules on the DCs of ITP patients and normal controls, and found that the stimulated DCs from ITP patients exhibited higher ability to induce CD4⁺CD25⁻ T lymphocytes proliferation, while lowering the ability of the induction of regulatory T cells (Tregs) from CD4⁺CD25⁻ T lymphocytes. Meanwhile, higher IFN-γ and lower IL-10 levels were found in the co-culture system of stimulated DCs and CD4⁺CD25⁻ T cells. We then silenced the CD70 genes on the induced DCs of ITP patients and normal controls by siRNA, and confirmed our suggestion that the silence of CD70 expression on the surface of DCs from ITP patients would decrease the CD4⁺CD25⁻ T lymphocytes proliferation and Tregs differentiation, simultaneously inducing higher IL-10 and lower IFN-γ levels. Thus, the interference with the CD27-CD70 costimulatory pathway might lead to the alleviation of the consequent immune reactions, polarisation of Th2, induction of immune tolerance as well as shed new light on treatment of autoimmune diseases.     

Protective role of interleukin-10-producing regulatory dendritic cells against murine autoimmune gastritis

Background Regulatory dendritic cells (Reg-DCs), which induce regulatory T cells and interleukin (IL)-10 in vitro, are capable of inducing immunogenic tolerance in vivo. In this study, we assessed whether Reg-DCs modulate the course of autoimmune processes in a murine model of autoimmune gastritis (AIG). Methods AIG mice were produced by neonatal thymectomy of 3-day old BALB/c mice followed by administration of polyinosinic:polycytidylic acid (poly I:C). Reg-DCs were produced by culturing bone marrow DCs with IL-10, lipopolysaccharide, and parietal cell (PC) antigen for 2 days. In the course of development of AIG, BALB/c mice were administered either Reg-DCs, mature DCs, or phosphate-buffered saline, intraperitoneally, four times. The levels of gastritis and autoantibody to PC antigen were assessed serially in these mice. Results The stages of gastritis and the titers of autoantibody to PC antigen were significantly lower in Reg-DC-treated mice than in mature DC-treated mice (P < 0.05). Spleen cells from Reg-DC-treated mice produced increased levels of IL-10 and decreased levels of IL-12p70 and interferon-γ (P < 0.05). Also, frequencies of IL-10-producing CD4⁺CD25⁺ T cells in the spleen and Foxp3⁺CD4⁺CD25⁺ T cells in the peripheral blood were significantly higher in Reg-DC-treated mice than in mature DC-treated mice (P < 0.05). Conclusions Taken together, these results suggest that increased induction of CD4⁺CD25⁺ regulatory T cells by Reg-DCs might contribute to downregulation of inflammatory processes and autoantibody production during AIG development in mice.     

Prolonged antigen survival and cytosolic export in cross-presenting human γδ T cells

Human blood Vγ9Vδ2 T cells respond to signals from microbes and tumors and subsequently differentiate into professional antigen-presenting cells (γδ T-APCs) for induction of CD4⁺ and CD8⁺ T cell responses. γδ T-APCs readily take up and degrade exogenous soluble protein for peptide loading on MHC I, in a process termed antigen cross-presentation. The mechanisms underlying antigen cross-presentation are ill-defined, most notably in human dendritic cells (DCs), and no study has addressed this process in γδ T-APCs. Here we show that intracellular protein degradation and endosomal acidification were significantly delayed in γδ T-APCs compared with human monocyte-derived DCs (moDCs). Such conditions are known to favor antigen cross-presentation. In both γδ T-APCs and moDCs, internalized antigen was transported across insulin-regulated aminopeptidase (IRAP)–positive early and late endosomes; however, and in contrast to various human DC subsets, γδ T-APCs efficiently translocated soluble antigen into the cytosol for processing via the cytosolic proteasome-dependent cross-presentation pathway. Of note, γδ T-APCs cross-presented influenza antigen derived from virus-infected cells and from free virus particles. The robust cross-presentation capability appears to be a hallmark of γδ T-APCs and underscores their potential application in cellular immunotherapy.     

Mesenchymal stem cells efficiently inhibit the proinflammatory properties of 6-sulfo LacNAc dendritic cells

Mesenchymal stem cells emerged as a promising treatment modality for steroid-refractory graft-versus-host disease, which represents a major complication of allogeneic hematopoietic stem cell transplantation. Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses and play a crucial role in the pathogenesis of graft-versus-host disease. Here, we investigated the impact of mesenchymal stem cells on the proinflammatory capacity of 6-sulfo LacNAc (slan) dendritic cells, representing a major subpopulation of human blood dendritic cells. Mesenchymal stem cells markedly impair maturation of slanDCs and their ability to secrete proinflammatory cytokines, which was dependent on prostaglandin E₂. In contrast, the release of anti-inflammatory IL-10 was improved by mesenchymal stem cells. Furthermore, mesenchymal stem cells efficiently inhibit slanDC-induced proliferation of CD4⁺ and CD8⁺ T cells and polarization of naïve CD4⁺ T lymphocytes into Th1 cells. These results indicate that mesenchymal stem cells significantly impair the high proinflammatory capacity of slanDCs and further substantiate their potential for the treatment of graft-versus-host disease.     

Alcohol Exposure Impairs Myeloid Dendritic Cell Function in Rhesus Macaques

Background: Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. Methods: Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL‐4 (10 ng/ml) and GM‐CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL‐1β (18 ng), IL‐6 (1800 U), TNF‐α (18 ng), and PGE₂ (1.8 μg) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. Results: EtOH‐treated animals had significantly lower numbers of myeloid DCs (lineage‐HLA‐DR+CD11c+CD123?) in both the PBMCs and BMCs compared to controls (5,654 ± 1,273/10⁶ vs. 2,353 ± 660/10⁶ PBMCs and 503 ± 34 vs. 195 ± 44/10⁶ BMCs). Under culture conditions, the number of lineage‐HLA‐DR+CD83+ cells was low in control wells (0.38 ± 0.08%). Alcohol inhibited the increase in the number of lineage‐HLA‐DR+CD83+ cells in iDC wells (2.30 ± 0.79% vs. 5.73 ± 1.40%). Alcohol also inhibited the increase in the number of lineage‐HLA‐DR+CD83+ cells in mature DC wells (1.23 ± 0.15% vs. 4.13 ± 0.62%). Conclusions: Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T‐cell expansion.     

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony-forming units (CFU-Mix), erythroid bursts (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), long-term culture-initiating cells (LTC-IC) and acute myelogenous leukaemia colony-forming units (CFU-AML). Continuous exposure of normal marrow and blood mononuclear non-adherent cells, blood CD34⁺CD45RA^? cells, and leukaemic blasts to increasing doses of genistein (1–100 μM ) resulted in a statistically significant (P ≤ 0.05) dose-dependent suppression of CFU-Mix, BFU-E, CFU-GM and CFU-AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID₅₀) of CFU-AML was significantly lower compared to CFU-GM ID₅₀ for marrow (19 v 32 μM P ≤0.017), unseparated blood (19 v 44 μM P ≤ 0.028) or CD34⁺CD45RA^? blood (19 v 36, P ≤ 0.04). Preincubation of leukaemic blasts with genistein (200 μM ) for 1–2 h confirmed that CFU-AML were significantly more sensitive than normal marrow and blood CFU-GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 ± 4%) and blood (40 ± 3%) LTC-IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose- and time-dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein-induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC-IC. The potent CFU-AML growth inhibition associated with the relative resistance of normal LTC-IC strongly supports the use of genistein for marrow purging.     

CCL17 and CCL22 chemokines within tumor microenvironment are related to infiltration of regulatory T cells in esophageal squamous cell carcinoma

It has been reported that an increased population of regulatory T cells (T‐regs) is one of the reasons for impaired anti‐tumor immunity. We investigated the frequency of Foxp3⁺ T‐regs in tumor‐infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) of patients with esophageal squamous cell carcinoma (ESCC). Furthermore, in order to elucidate the mechanisms behind T‐regs accumulation within tumors, we evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3⁺ T‐regs. CD4⁺CD25⁺Foxp3⁺ T‐regs as a percentage of CD4⁺ cells were counted by flow cytometry. The frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells in tumors was also evaluated by flow cytometry. Moreover, an in vitro migration assay using T‐regs derived from ESCC was performed in the presence of CCL17 or CCL22. The frequency of Foxp3⁺ T‐regs in TILs was significantly higher than that in the normal esophageal mucosa (24.6 ± 10.0 vs 7.1 ± 5.9%, P < 0.01). The frequency of Foxp3⁺ T‐regs in PBLs of ESCC patients was significantly higher than that in normal healthy donors (7.0 ± 4.2 vs 2.5 ± 1.0%, P < 0.01). Furthermore, the frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells within tumors was significantly higher than that of normal esophageal mucosa, and there was a significant correlation between the frequency of CCL17⁺ or CCL22⁺ cells and Foxp3⁺ T‐regs in TILs. In addition, the in vitro migration assay indicated that T‐regs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3⁺ T‐regs in ESCC.