Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

An increase in circulating mast cell colony-forming cells in asthma

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.     

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Background aimsPrevious studies have demonstrated that the combination of granulocyte–colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34⁺ hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets.MethodsWe characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor.ResultsThe number of aldehyde dehydrogenase (ALDH)^bright and CD34⁺ cells was significantly higher after plerixafor treatment (1.2–5.0 and 1.5–6.0 times; both P < 0.01) and an enrichment of the very primitive CD34⁺ CD38^? and ALDH^bright CD34⁺ CD38^? HSC subsets was detectable. Additionally, two distinct ALDH⁺ subsets could be clearly distinguished. The small ALDH^high subset showed a higher number of CD34⁺ CD38^? cells in contrast to the total ALDH^bright subpopulation and probably represented a very primitive subpopulation of HSC.ConclusionsA combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34⁺ cells but was also able to increase the proportion of more primitive stem cell subsets.     

Effects of granulocyte-colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor administration on T cell proliferation and phagocyte cell-surface molecules during hematopoietic reconstitution after autologous peripheral blood progenitor cell transplantation

Thirty-four ovarian and breast cancer patients received autologous peripheral blood progenitor cell transplantation after high-dose myeloablative chemotherapy and either granulocyte-colony-stimulating factor (G-CSF) or granulocyte/macrophage-colony-stimulating fictor (GM-CSF) in the immediate post-transplant period. The recovery of T cell functionality was monitored by a three-color flow-cytometric approach using carboxyfluorescein diacetate succinimidyl ester, a probe the fluorescence intensity of which halves at each round of cell replication, in conjunction with CD3 and CD25 monoclonal antibodies. There was no significant difference between the two treatments on days 12, 20, and 40, T cell proliferation always being considerably lower than that of control cultures from healthy donors. At day 80, a significantly higher proportion of mitogen-stimulated T cells from GM-CSF-treated patients expressed interleukin-2 receptor, and a higher proportion of these T cells were actively proliferating. This phenomenon did not reflect any difference in the relative proportion of various lymphocyte subsets (T cells, CD4 and CD8+ T cells, CD45RA+ and CD45RO- T cells, and natural killer cells). At the end of follow-up (1-1.5 years) T cell proliferation had returned to values typically observed in healthy individuals in both groups of patients. Soon after transplantation (day 12), neutrophils from G-CSF-treated patients had a more elevated Fcgamma receptor I density and monocytes from GM-CSF-treated patients had a more elevated Fcgamma receptor II and MHC class II molecules density. The up-modulation of Fcgamma receptor II was maintained until day 40. Thus, administering G-CSF and GM-CSF in the post-transplant period affects T lymphocyte proliferation and phagocyte membrane molecules differently.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

Stem cell transplantation for metastatic breast cancer: analysis of tumor contamination

The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination, and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination. Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m², thiotepa 500 mg/m², and carboplatin 800 mg/m² (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement, found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months. Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome in women with responding metastatic breast cancer.     

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime na?ve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.     

Combined administration of G-CSF and GM-CSF stimulates monocyte-derived pro-angiogenic cells in patients with acute myocardial infarction

Mobilization of endothelial progenitor cells has been suggested to contribute to neo-vascularization of ischemic organs. Aim of this study was to investigate whether the combination of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF may influence the expansion of circulating KDR+ cells in patients with acute myocardial infarction (AMI). KDR+ cells significantly increased in peripheral blood of AMI patients treated with G-CSF and GM-CSF compared to untreated patients. This KDR+ cells population was CD14+ but not CD34+ or CD133+. CD14+/KDR+ cells were also obtained in vitro by culturing mononuclear cells from healthy donors in a Rotary Cell Culture System in the presence of G-CSF + GM-CSF, but not of the individual growth factors. CD14+/KDR+ cells, obtained from patients or from in vitro culture, co-expressed hematopoietic (CD45, CD14) and endothelial markers (CD31, CD105, and VE-cadherin). CD14+/KDR+, but not CD14+/KDR- cells, stimulated the organization of human microvascular endothelial cells into capillary-like structures on Matrigel both in vitro and in vivo. The combination of G-CSF and GM-CSF induced a CD14+/KDR+ cell population with potential pro-angiogenic properties.     

Feasibility and cost analysis of day 4 granulocyte colony-stimulating factor mobilized peripheral blood progenitor cell collection from HLA-matched sibling donors

BackgroundGuidelines recommend treatment with 4–5 days of granulocyte colony-stimulating factor (G-CSF) for optimal donor peripheral blood progenitor cell (PBPC) mobilization followed by day 5 collection. Given that some autologous transplant recipients achieve adequate collection by day 4 and the possibility that some allogeneic donors may maximally mobilize PBPC before day 5, a feasibility study was performed evaluating day 4 allogeneic PBPC collection.MethodsHLA-matched sibling donors underwent collection on day 4 of G-CSF for peripheral blood (PB) CD34⁺ counts ≥0.04 × 10⁶/mL, otherwise they underwent collection on day 5. Those with inadequate collected CD34⁺ cells/kg recipient weight underwent repeat collection over 2 days. Transplant and PBPC characteristics and cost analysis were compared with a historical cohort collected on day 5 per our prior institutional algorithm.ResultsOf the 101 patient/donor pairs, 50 (49.5%) had adequate PBPC collection on day 4, with a median PB CD34⁺ cell count of 0.06 × 10⁶/mL. Day 4 donors were more likely to develop bone pain and require analgesics. Median collected CD34⁺ count was significantly greater, whereas total nucleated, mononuclear and CD3⁺ cell counts were significantly lower, at time of transplant infusion for day 4 versus other collection cohorts. There were no significant differences in engraftment or graft-versus-host disease. Cost analysis revealed 6.7% direct cost savings for day 4 versus historical day 5 collection.DiscussionDay 4 PB CD34⁺ threshold of ≥0.04 × 10⁶/mL identified donors with high likelihood of adequate PBPC collection. Day 4 may be the optimal day of collection for healthy donors, without adverse effect on recipient transplant outcomes and with expected cost savings.     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

Mobilization of dendritic cells from patients with breast cancer into peripheral blood stem cell leukapheresis samples using Flt-3-Ligand and G-CSF or GM-CSF

Treatment with myeloablative chemotherapy and autologous peripheral blood stem cell (PBSC) transplantation followed by vaccination with autologous dendritic cells (DCs) treated with tumor antigens is a promising therapeutic strategy for several types of cancer. Obtaining sufficient numbers of both PBSCs and DCs is central to this approach. Previously, it has been shown that administration of Flt-3-Ligand (FL) combined with either G-CSF or GM-CSF mobilizes large numbers of PBSCs in patients with cancer. In the current study, we sought to determine whether these same cytokines could simultaneously mobilize DCs into the PBSC leukapheresis collection. DCs were analysed in PBSC leukapheresis samples obtained from five patients with high-risk breast cancer who received G-CSF alone as priming prior to leukapheresis, four patients who received FL+G-CSF and five patients who received FL+GM-CSF. DCs were defined as cells with a lin(dim/-) HLA-DR+ CD11c+ phenotype. The proportions of DCs in the FL+G-CSF and FL+GM-CSF samples were significantly higher than in pre-mobilization peripheral blood and G-CSF leukapheresis samples. The mean yield of DCs/kg in the FL+GM-CSF samples was also significantly higher than the mean yield of DCs in the G-CSF samples. The FL+G-CSF and FL+GM-CSF mobilized DCs were immature by morphologic and phenotypic criteria but stimulated allogeneic T-cells at levels similar to DCs generated in culture from PBMCs. Overnight culture?of the immature DCs obtained from patients receiving either FL+G-CSF or FL+GM-CSF in TNF-alpha?resulted in the generation of mature DCs. In summary, administration of FL in combination with GM-CSF and G-CSF to patients with breast cancer can mobilize large numbers of immature DCs into PBSC leukapheresis collections.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

CD34+-derived CD11c+ + + BDCA-1+ + CD123+ + DC: expansion of a phenotypically undescribed myeloid DC1 population for use in adoptive immunotherapy

BACKGROUND: DC are commonly defined as HLA-DR+/Lin- cells that can be CD11c+ + + CD123+/ -, termed DC1/myeloid DC that induce a Th1 response, or CD11c- CD123+ + +, termed DC2/lymphoid DC that induce a Th2 response. However, significant heterogeneity within DC preparations is apparent and supports the existence of several distinct DC subpopulations. This study aimed to expand and characterize CD34+ DC for use in immunotherapy. METHODS: CD34+ cells were seeded at 1 x 10(5)/mL and expanded for 14 days in RPMI + 10% autologous plasma supplemented with GM-CSF, IL-4, Flt-3L and SCF. Maturation was induced with TNF-alpha and PGE2 for 2 days. DC were analyzed morphologically, phenotypically with a panel of MAb to lineage and DC markers, and functionally in MLR, T-cell assays and T-cell cytokine secretion by ELISA. RESULTS: Significant cellular expansion was observed: 60+/-5 x 10(6) DC from 1 x 10(6) CD34+ cells (n=28). Phenotypically DC were characterized as HLA-DR+ +, CD11c+ + +, CD80+ +, CD83+, CD86+ +, CD123+ +, CD15+ +, CD33+ +, BDCA-1+ +, CD4+ and Lin-. DC displayed potent allostimulatory capacity and efficient presentation of KLH and tetanus toxin. DC-primed T cells secreted IFN-gamma (Th1); however, no detectable IL-4 (Th2) was noted. DISCUSSION: We present features of CD34+ DC that have not been previously described. The CD34+ DC generated represent a population of myeloid DC functioning as DC1 but phenotypically expressing markers characteristic of both DC1 and DC2. This novel DC population is capable of inducing naive T-cell responses and can be expanded to clinically useful numbers. CD34+-derived DC represent attractive candidates for use in adoptive T-cell immunotherapy.     

Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

A randomized controlled clinical trial to determine the optimum duration of G-CSF priming prior to BM stem cell harvesting

BACKGROUND: Harvesting of hemopoietic stem cells (HSC) from G-CSF-primed BM for autologous transplantation is an alternative to collection of unprimed BM or G-CSF-primed peripheral blood (PB). However, the optimum number of days of G-CSF administration for this purpose is unknown. We set out to determine whether cell yields could be optimized by varying the number of days of G-CSF administration prior to BM stem cell harvesting. METHODS: We conducted a randomized controlled single-center trial of 6 days (the standard) vs. 4 days of G-CSF administration and compared yields of total nucleated cells (TNC), CD34(+) HSC and CFU-GM cells per kilogram patient body weight. Statistical analysis was by Student's t-test. RESULTS: Twenty-four patients were enrolled; 13 received 6 days and 11 received 4 days of G-CSF administration. Analysis of the first harvest aspirate showed higher proportions of CD34(+) HSC (P=0.02) and CFU-GM (P=0.03) in the 4-day group. For the 6-day and 4-day groups, respectively, the median yield of TNC/kg was 6.5 x 10(8) and 5.4 x 10(8) (P=0.28), of CD34(+) cells/kg 0.56 x 10(6) and 0.98 x 10(6) (P=0.04) and of CFU-GM cells/kg 1.66 x 10(5) and 1.55 x 10(5) (P=0.75). DISCUSSION: These results suggest that by 6 days the HSC-stimulating effect of G-CSF has passed its peak and that 4 days should be adopted as the standard for G-CSF priming prior to BM stem cell harvesting for autologous transplantation.     

Flow cytometric detection of circulating dendritic cells in healthy subjects

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.     

Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.     

Peripheral blood and BM CD34+ CD38- cells show better resistance to cryopreservation than CD34+ CD38+ cells in autologous stem cell transplantation

BACKGROUND: We and others have shown a critical role for CD34+ CD38- cells in hematopoietic recovery after autologous stem cell transplantation (ASCT), in particular for platelet reconstitution. Thus a routine assessment of CD34+ CD38- cells in freezing-thawing procedures for autografting could represent an important tool for predicting poor engraftment. METHODS: To compare the impact of cryopreservation on CD34+ CD38+ and CD34+ CD38- hematopoietic stem cell subsets, 193 autograft products collected in 84 patients with malignancies were assessed before controlled-rate cryopreservation in 10% DMSO and after thawing for autografting. RESULTS: Cell counts after thawing were significantly different from the pre-freezing counts for total CD34+ (P<0.0001) and CD34+ CD38+ (P<0.0001) cells, but not for CD34+ CD38- cells (P=0.252). Median losses for CD34+, CD34+ CD38+ and CD34+ CD38- cells were, respectively, 11.8%, 11.4% and 0.0%. The magnitude of fresh/post-thawing percentage cell variation was significantly different when comparing between the CD34+ CD38+ and CD34+ CD38- cell subsets (P<0.001). Moreover, CD34+ CD38- cells exhibited recovery values > or =100% in 85/160 graft products, compared with 51/193 in CD34+ CD38+ cells (P<0.0001). Also, recovery values > or =90% were significantly better in the CD34+ CD38- (98/160 grafts) than in the CD34+ CD38+ subsets (89/193 grafts) (P<0.01). DISCUSSION: In this work we have demonstrated that CD34+ cells that do not express the CD38 Ag show a significantly better resistance to cryopreservation. This could represent another example of the particular ability of less committed progenitor cells to overcome environmental injuries. Moreover, we consider routine assessment of CD34+ CD38- cells before freezing as clinically relevant, but post-thawing controls may be avoided because of their good resistance to freezing.