Circulating Blood Dendritic Cells Research Articles

Human Hepatitis B Virus Negatively Impacts the Protective Immune Crosstalk Between Natural Killer and Dendritic Cells.

Natural killer (NK) cells play a crucial role in the clearance of human viruses but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). Cooperation with dendritic cells (DCs) is pivotal for obtaining optimal NK cell antiviral function; thus, we investigated whether HBV might impact the ability of DCs to sustain NK cell functions. Human DCs were poor stimulators of interferon-gamma (IFN-γ) production by NK cells when exposed to HBV, while maintaining the capability to trigger NK cell cytotoxicity. HBV prevented DC maturation but did not affect their expression of human leukocyte antigen class I, thus allowing DCs to evade NK cell lysis. Tolerogenic features of DCs exposed to HBV were further supported by their increased expression of IL-10 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase, which contributed to the impairment of DC-mediated NK cell IFN-γ production and proliferation, respectively. HBV could also inhibit the expression of inducible immunoproteasome (iP) subunits on DCs. In fact, NK cells could induce iP subunit expression on DCs, but they failed in the presence of HBV. Remarkably, circulating blood DC antigen1 (BDCA1)+ DCs isolated from patients with CHB were functionally compromised, hence altering, in turn, NK cell responses. The abnormal NK-DC interplay caused by HBV may significantly impair the efficacy of antiviral immune response in patients with CHB.

Changes of post-operative peripheral blood dendritic cells in patients undergoing laparoscopy.

IntroductionSurgical intervention affects local and systemic immune responses, especially in obese individuals. Many studies have attempted to evaluate immunological response to surgical trauma. Surgery changes the quantity and phenotype of circulating blood dendritic cells (DCs), including a decrease of total DCs post-operatively. The study aimed to evaluate the percentage and changes of myeloid, lymphoid DCs, and myeloid to lymphoid DCs ratio in obese and normal weight patients undergoing laparoscopy.Material and methodsThe study enrolled asymptomatic patients with gallstones, who underwent laparoscopic cholecystectomy. Blood samples were obtained before the surgery as well as 24 and 48 hours after the surgery. Cells were collected using a FACSCalibur flow cytometry, and phenotypes were analyzed with CellQuest software.ResultsNo statistically significant differences were observed between obese and normal-weighted patients in all studied time periods, except for the myeloid to lymphoid DCs ratio assessed at 48-post-operative hour. The myeloid DCs percentage increased significantly in the post-operative period within both studied groups. The percentage of lymphoid DCs increased significantly in obese patients in all studied time periods.ConclusionsLaparoscopy induces immunomodulation, such as changes of myeloid and lymphoid dendritic cells, especially in obese patients. We describe new findings, in which minimally invasive surgical trauma promotes the increase of percentage of circulating DCs in the early post-operative period.

Glycan-binding profile of DC-like cells.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-"vector" it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1-3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Low Levels of Plasmacytoid Dendritic Cells at the Time of Engraftment Are a Valuable Severe aGVHD Indicator in Children Undergoing Allogeneic Stem Cell Transplantation

Graft-versus-host-disease (GVHD) remains a leading cause of transplant-related mortality (TRM) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients who do not respond to GVHD therapy are at high risk for death without relapse. Strategies that can predict the development of severe GVHD in transplant patients may improve risk stratification for steroids treatment refractory GVHD and TRM. Dendritic cells (DCs) play nonredundant roles in regulating both immunity and tolerance. Clinical and pre-clinical studies suggest that GVHD causes dysregulated development of functional DCs and impaired reconstitution of donor DCs is associated with increased risk of severe GVHD. We demonstrate that donor plasmacytoid DC (pDC) reconstitution around the engraftment time is a useful predictor for the development of severe GVHD in children after allo-HSCT. We examined reconstitution of donor DCs, including total DCs (lineage-negative (LIN-)HLADR+), pDCs (LIN-HLADR+CD123+CD1c-) and CD1c+DCs (LIN-HLADR+CD123-CD1c+), in peripheral blood of patients undergoing allo-HSCT, using age-matched healthy children as controls. The median time for engraftment was 14 days (range, 13.0~17 days), whereas clinical signs of GVHD occurred approximately 10 days later (median time, 24 days;range, 18~35 days). Thus, measuring the number and frequency of circulating blood DCs early at the engraftment time allowed us to evaluate the subsequent occurrence of GVHD. Transplant patients were followed up for 6 months after allo-HSCT. Of evaluable 45 transplant patients, 27 patients had no GVHD (grade 0) or mild GVHD (grade I), and 18 patients developed severe GVHD (grade II-IV GVHD). Patients who developed grade II-IV GVHD had no significant difference in frequency ( median, 0.2%; range, 0.04~0.35%) and number (median, 1.6 cell/ml; range, 1~3.8 cell/ml) of total DCs than patients with grade 0-I GVHD (median frequency, 0.41%; range, 0.15~0.63%, P<0.05; and median number, 2.6 cell/ml , range, 1~6.7 cell/ml,Figure1A,1B). Both pDCs and CD1c+DCs represent the major DC populations in human peripheral blood. Patients with grade II-IV GVHD showed significantly lower frequency and number of pDCs (median frequency, 3.4%; range, 0.64~14%, median number, 0.06/ml, range, 0.02~0.25 cell/ml) than patients with grade 0-I GVHD median frequency, 29.1%, range, 20.3%~39.2%, P<0.01; mediate number, 0.5 cell/ml, range, 0.2~1.9 cell /ml, P<0.01Figure1A,1B). In contrast, there was no significant difference in frequency and number of CD1c+DC between the two groups. These data suggest that the reconstitution of donor pDCs is linked to the occurrence of severe GVHD. Using Receiver operating characteristic (ROC) curve analysis, we explored thresholds of pDCs as a discriminator for grade II-IV GVHD patients versus grade 0-I GVHD patients. The median pDC level in all tested patients was 0.25 cell/ml (range, 0.1~0.9 cell/ml,Figure1C) at the engraftment time. We chose the threshold of pDC level at 0.3 cell/ml to evaluate the difference in patients with high (above the threshold) versus low (below the threshold) pDC levels. Of these 45 patients, 21 (46.7%) patients had high pDCs and 24 (53.3%) were low pDCs. Patients with low pDCs had a significantly higher probability of developing grade II-IV GVHD (p<0.01). The sensitivity of discriminating severe GVHD versus no/mild GVHD was 75% and the specificity was 94%. In addition, low engraftment pDC patients had higher TRM compared to high pDC patients (12.5% versus 0%) for median follow-up time 300 days (range, 210-465 days) after transplantation. However, there was no significant difference in overall survival rate between high pDC- and low pDC-group. Furthermore, the serum level of REG3α and ST2 at the engraftment time was not associated with the incidence of severe GVHD in these patients. Collectively, our findings identify that quantitation of pDCs at the engraftment time is a valuable prognostic predicator for severe GVHD in patients undergoing allo-HSCT. Given the importance of pDCs in modulating GVH reactions, it will be important to further determine whether impaired reconstitution of donor DCs may contribute to dysregulated immune tolerance against host tissues after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Identification of Lineage Relationships and Novel Markers of Blood and Skin Human Dendritic Cells

The lineage relationships and fate of human dendritic cells (DCs) have significance for a number of diseases including HIV where both blood and tissue DCs may be infected. We used gene expression profiling of human monocyte and DC subpopulations sorted directly from blood and skin to define the lineage relationships. We also compared these with monocyte-derived DCs (MDDCs) and MUTZ3 Langerhans cells (LCs) to investigate their relevance as model skin DCs. Hierarchical clustering analysis showed that myeloid DCs clustered according to anatomical origin rather than putative lineage. Plasmacytoid DCs formed the most discrete cluster, but ex vivo myeloid cells formed separate clusters of cells both in blood and in skin. Separate and specific DC populations could be determined within skin, and the proportion of CD14(+) dermal DCs (DDCs) was reduced and CD1a(+) DDCs increased during culture, suggesting conversion to CD1a(+)-expressing cells in situ. This is consistent with origin of the CD1a(+) DDCs from a local precursor rather than directly from circulating blood DCs or monocyte precursors. Consistent with their use as model skin DCs, the in vitro-derived MDDC and MUTZ3 LC populations grouped within the skin DC cluster. MDDCs clustered most closely to CD14(+) DDCs; furthermore, common unique patterns of C-type lectin receptor expression were identified between these two cell types. MUTZ3 LCs, however, did not cluster closely with ex vivo-derived LCs. We identified differential expression of novel genes in monocyte and DC subsets including genes related to DC surface receptors (including C-type lectin receptors, TLRs, and galectins).

Immunohistochemical characterisation of dendritic cells in human atherosclerotic lesions: possible pitfalls

Previously we demonstrated decreased blood myeloid (m) and plasmacytoid (p) dendritic cell (DC) counts in atherosclerotic patients. Therefore, we examined whether DCs, in particular DC precursors, accumulate in human plaques. Blood DC antigen (BDCA)-1, CD11c (mDCs), BDCA-2, CD123 (pDCs), langerin, fascin, S-100 (immature/mature DCs), and CD1a and CD83 (mature DCs) were investigated by immunohistochemistry of carotid arteries obtained by endarterectomy (EAS, frozen n = 11, fixed n = 11) or autopsy (fixed, n = 87). Fascin and S-100 required formaldehyde fixation, other markers needed cryo-preservation. BDCA-1, BDCA-2, langerin, and S-100 appeared specific for intimal DCs, unlike CD123 and fascin (staining endothelial cells), CD11c and CD1a (staining monocytes, foam cells) or CD83 (staining lymphocytes). BDCA-1 and BDCA-2 cells were detected in EAS, preferentially near microvessels. S-100 cells increased successively from intimal thickening, via pathological intimal thickening, fibrous cap atheroma and finally complicated plaques. Fascin cells followed the same pattern, but were more abundant. However, in lesions containing microvessels (complicated plaques, plaque shoulders and most EAS) this was partly explained by fascin positive endothelial cells. Even complicated plaques contained relatively few mature CD83 DCs. Accumulation of BDCA-1 and BDCA-2 around neovessels showed that mDCs and pDCs are recruited to advanced plaques, which is in line with the previously described decline of circulating blood DCs in patients with coronary artery disease. Unexpectedly, several DC markers yielded false positive signals. Hence, some accounts on numbers, trafficking and activation of DCs in atherosclerotic plaques may require re-evaluation.

Receiver operating characteristic curve analysis of circulating blood dendritic cell precursors and T cells predicts response to extracorporeal photopheresis in patients with chronic graft‐versus‐host disease

One proposed mechanism of extracorporeal photopheresis (ECP) in reducing chronic graft-versus-host disease (cGVHD) is alteration in numbers of circulating dendritic cells (DCs). This hypothesis was tested by correlating numbers of DC precursors and T cells in the blood before and during ECP therapy with response of cGVHD. Twenty-five patients with cGVHD were treated with ECP. Data were collected with emphasis on blood cellular markers, clinical response to ECP, and overall survival. Fourteen patients (56%) responded and had better 2-year survival than nonresponders (88% vs. 18%, p=0.003). Responders had higher baseline circulating myeloid DC (mDC) and plasmacytoid DC precursors and CD4+ and CD8+ T cells compared with nonresponders. Receiver operating characteristic curve analyses showed that the best baseline cutoff values to predict response to ECP were mDC counts of 3.7 cells/µL (79% sensitivity, 82% specificity) and CD4+ T-cell counts of 104 cells/µL (71% sensitivity, 82% specificity). CD4+ T cells declined in responders over time, but not in nonresponders, and no significant changes were seen in CD8 T-cell or DC numbers over a 12-month period in responder or nonresponder groups. Higher baseline numbers of circulating DCs and T cells may predict clinical response to ECP in patients with cGVHD.

CCR5 Expression On Circulating Blood DC Post-Allogeneic Hemopoietic Cell Transplant Is Highly Predictive for the Development of Clinically Significant Acute Graft Versus Host Disease.

Abstract 2251Poster Board II-228 Introduction:Dendritic cells (DC) are central to the development of acute graft-versus-host disease (GvHD) following allogeneic hematopoietic cell transplantation (alloHCT). We previously showed that the activation status, as assessed by CMRF-44 antigen expression, of blood CD11c+ myeloid DC is highly associated with the severity of acute GvHD, and that activated DC may be detected in the circulation prior to clinical presentation of GvHD (Transplantation 2007;83: 839–846). Because DC migration is tightly regulated by the interaction between chemokine receptors on DC and their ligands, we investigated the relationship between the severity of acute GvHD and the expression of the chemokine receptors CCR5 and CCR7 on CD11c+myeloid and CD11c- plasmacytoid from the peripheral blood of 32 patients post alloHCT. Methods:Peripheral blood was collected twice weekly up to day 100 post transplant from 22 patients who received transplants from matched siblings, 10 from unrelated donors. Nineteen transplants were performed with myeloablative conditioning, 13 with reduced intensity conditioning. The expression of each chemokine receptor on CD11c+ and CD11c- DC was calculated using multiparameter flow cytometry. The percentage of CD11c+ DC expressing a given receptor was added to the percentage of CD11c- DC expressing the same receptor to give a maximum score that could vary from 0 to 200%. Results:Eleven of 32 patients developed moderate to severe acute GvHD (grade II-IV), the remaining 21 patients developed either no GvHD or only grade I GvHD. The percentage of DC expressing either CCR5 or CCR7 was correlated with the development of acute GvHD. CCR7 expression was detected in 13 of 32 patients post-HCT with a median of 2.3% of DC positive (range 0 to 39%). CCR7 expression on DC showed no association with the severity of acute GvHD (p=1.0). In contrast, higher CCR5 expression was detected on DC in patients developing grade II-IV GvHD (median 98.0%, SEM 9.1%) than in those with grade 0-I GvHD (median 5.2%, SEM 5.1%) p<0.0001. All eleven patients with grade II-IV GvHD expressed CCR5 at a level of >35% of myeloid and plasmacytoid DC. Only two of 21 patients with grade I GvHD expressed CCR5 at the same level (66% and 94%). Most importantly, the expression of CCR5 preceded the development of moderate to severe GvHD in all patients by a median of 19 days (range 2 to 47 days, SEM 4.3 days). Using a receiver operator curve analysis, CCR5 expression above 35% demonstrated a sensitivity of 100% and a specificity of 90.5% for predicting grade II-IV GvHD. Conclusion:Expression of CCR5 on circulating DC post allo-HCT predicts for the development of moderate to severe GvHD, and detection could allow earlier therapeutic intervention prior to the development of clinical GVHD, if these findings are confirmed in a larger study Disclosures:No relevant conflicts of interest to declare.

Impaired IL-10–dependent Induction of Tolerogenic Dendritic Cells by CD4+CD25hiCD127lo/−Natural Regulatory T Cells in Human Allergic Asthma

Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However, the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. The study was performed in a cohort of 21 subjects with allergic asthma, 21 healthy control subjects, and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. Dendritic cells cultured with natural regulatory T cells up-regulated IL-10, down-regulated costimulatory molecules, and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore, this defective function of natural regulatory T cells was associated with their decreased IL-10 expression, disease severity, and could be reversed by oral corticosteroid therapy. These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma.

Comment on “The Anergic State in Sarcoidosis Is Associated with Diminished Dendritic Cell Function”

It was with great interest that we read the study by Mathew et al. ([1][1]). We thank the authors for their elegant and detailed functional analyses of circulating blood dendritic cells in sarcoidosis. From a clinical standpoint, one conclusion of the authors needs commenting and a word of caution

Alemtuzumab Depletes Dendritic Cells More Effectively in Blood Than in Skin: A Pilot Study in Patients With Chronic Lymphocytic Leukemia

The antibody alemtuzumab (anti-CD52) is effective in preventing acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). As well as depleting donor T cells, alemtuzumab may also work by targeting host dendritic cells (DC). To determine whether this second mechanism of action is significant, we investigated the effects of intravenous alemtuzumab by comparing skin and blood DC numbers of patients with chronic lymphocytic leukemia, before and after a 4-week course of alemtuzumab treatment. Although skin DC express CD52, the epitope is only weakly detectable and their numbers were not consistently reduced by alemtuzumab. In contrast, circulating blood DC, with stronger CD52 expression, were invariably diminished by alemtuzumab. Because DC depletion in the transplant recipient remains a promising approach for GvHD prophylaxis and therapy, more potent techniques, such as an antibody of different specificity, may be required for effective DC eradication in GvHD target organs.

Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease

Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented. Patients with angiographically proven coronary artery disease (n=18) and age and sex-matched controls (n=18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively. Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722+/-601/ml and 0.08+/-0.01%) than in controls (12,640+/-1289/ml and 0.21+/-0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11,857+/-1895/ml versus 15,226+/-928/ml and 0.17+/-0.03% versus 0.26+/-0.01% in controls. The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Dendritic Cell Immunotherapy for Melanoma

Dendritic cells (DC) are the most potent antigen-presenting cells that initiate T cell-mediated immune responses against cancer. It has been almost a decade since the first trial of DC-based cancer immunotherapy was published. Despite the many clinical trials conducted since, few solid conclusions have been reached, and no specific-immunotherapy has routinely demonstrated meaningful anti-tumour responses. Clinical-grade DC can be obtained from three distinct cell populations in the blood - monocytes, CD34(+) progenitors or direct isolation of circulating blood DC. This review discusses the science behind DC-based cancer immunotherapy, with a particular emphasis on the use of monocyte-derived DC in melanoma clinical trials, and the various potential avenues for improvement of patient clinical response rates.

Human Dendritic Cell Expression of HLA-DO Is Subset Specific and Regulated by Maturation

Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO. In this study, we report that, in fact, certain types of human primary DC express DO. These include Langerhans cells (LC) and some subtypes of circulating blood DC. Specifically, the majority of BDCA-3(+) DC, a small subset of uncertain function, are DO(+), while smaller proportions of CD11c(+), BDCA-1(+) (myeloid) DC, at most a minority of CD123(+)/BDCA-2(+) (plasmacytoid) DC, and no detectable CD16(+) (myeloid) DC, express DO. Immunohistochemistry of human tonsil sections demonstrates that tonsillar interdigitating DC are also DO(+). In a subset of immature LC with higher DO expression, an increased fraction of surface DR molecules carry CLIP peptides, indicating that DO functions as a DM inhibitor in these cells. LC expression of DO is down-regulated by maturation stimuli. DM levels also decrease under these conditions, but the DM:DO ratio generally increases. In the myeloid cell types tested, DO expression correlates with levels of DObeta, but not DOalpha, implying that modulation of DObeta regulates DO dimer abundance in these cells. The range of APC types shown to express DO suggests a broader role for DO in immune function than previously appreciated.

Human BDCA-1 Positive Blood Dendritic Cells in Immunosuppressed Patients: Phenotype, Function and Maturation.

Immunosuppressive drugs used in patients (pts) after stem cell / organ transplantation (Tx) as well as in pts with autoimmune disease are known to impair the cellular immune response. This results in an increased incidence of viral infections and viral associated malignancies which has been ascribed to the effect of immunosuppressive drugs on lymphocytes. However, in vitro data indicate that immunosuppressive drugs also target Dendritic Cells (DCs), the most potent antigen-presenting cells and initiators of lymphocyte responses. So far, most studies are based on in vitro data obtained with DC culture in the presence of different concentrations of single immunosuppressive drugs. To investigate the effect of immunosuppression on DC phenotype and function in vivo, we quantitatively and qualitatively analyzed freshly isolated human BDCA-1(CD1c) positive DCs from 15 solid organ transplant (SOT) recipients under immunosuppressive treatment. The percentage of BDCA-1 positive cells among total PBMCs was not statistically different in pts vs ctrls (0,52 vs 0,65, p<0,18). BDCA-1 positive DCs were analyzed for expression of HLA class I and II, CD14, costimmulatory molecules and chemokine expression. Interestingly, CD14 was found to be significantly higher expressed on pt-DCs vs ctrl-DCs suggesting a more immature DC-phenotype. We observed a trend toward a reduced expression of HLA-DR and CD86 on pts-DCs as compared to ctrls-DCs (p=0,059). Surface profile of BDCA-1 positive DCs was also analyzed after 48h of LPS and CD40L stimulation. Here we found a marked upregulation of HLA-DR and CD86 in pts- DCs as well as ctrl-DCs. Supernatant of stimulated DCs was analyzed with cytokine capture beads for secretion of inflammatory cytokines. High secretion of IL-6, IL-1 beta and partially of TNF-alpha by stimulated DCs was observed in both groups. Other Th2 type cytokines (IL-10, IL-4, IL-5) and Th1 type cytokines like IFN-gamma and Il-2 were not significantly secreted. We additionally addressed the question if mature and functionally competent DCs could be generated ex vivo from this pts cohort. After 9 days of culture with GM-CSF, IL-4, IL-1, IL-6, TNF-alpha and PGE2 fully mature DCs could be generated. Co-culture of EBV-peptide-pulsed DCs with autologous T-cells resulted in significant expansion of EBV-specific T cells in pts and ctrls. These T cells were fully functional as shown by IFN-γ secretion detected by ELISPOT. In summary, this is the first analysis of freshly isolated BDCA-1 positive DCs from immunosuppressed pts. Our data support the notion that immunosuppressive drugs target DCs and contribute to a maturation defect of circulating blood DCs which may help to understand the mechanism of impaired cellular immune responses in immunosuppressed pts. However, ex vivo generated DCs from immunosuppressed pts do not show an impairment in phenotype and function, suggesting that they could be efficiently be used in immunotherapeutic strategies.

Expression of Activation Marker, CMRF-44, on Blood CD11c+ Dendritic Cells Is Closely Associated with the Severity of Acute GVHD after Allogeneic Haematopoietic Stem Cell Transplant.

AIM: Dendritic cells (DC) are considered central to the development of graft versus host disease (GVHD) following allogeneic haematopoietic stem cell transplantation (alloHSCT). Both donor and host DC are thought to initiate allogeneic immune responses by presenting host antigens to donor T lymphocytes. We studied the potential of CMRF-44, a novel monoclonal antibody identifying activated circulating blood DC, as a predictive marker of acute GVHD.METHODS: In a prospective study, peripheral blood was taken from 40 patients twice weekly up to 100 days post-alloHSCT. Circulating myeloid (CD11chi) and plasmacytoid (CD123hi) DCs were enumerated and the expression of CMRF-44 was assessed on CD11chi DC by four colour flow cytometry. Multivariate analyses were performed using a non-parametric Mann-Whitney U-test and Receiver Operating Characteristic (ROC) curves.RESULTS: Following alloHSCT, the severity of acute GVHD correlated with the number of total DC in the blood (p=0.035). Furthermore, low myeloid and plasmacytoid DC numbers were significantly associated with grade 2-4 acute GVHD (p=0.046 and 0.017 respectively). In 40 alloHSCT patients, 27 developed acute GVHD. CMRF-44 was expressed on CD11c+ DC in all cases prior to the onset of acute GVHD. Of the 13 patients without GVHD, 8 had no circulating CMRF-44+ CD11c+ DC. CMRF-44 expression was independent of the reconstitution of myeloid DC (p=0.73). Patients who had CMRF-44+ CD11c+ DC in more than 20% of their post-transplant monitoring samples were more likely to develop acute GVHD (p=0.001, OR=37.1). In addition, patients with more severe grade 2–4 GVHD had significantly higher percentages of CMRF-44+ CD11c+ DCs (p=0.001). CMRF-44 expression at greater than or equal to 12% of CD11chi DCs had a sensitivity of 87.5 for prediction of grade 2–4 acute GVHD, and a specificity of 91.7.CONCLUSION: CMRF-44 expression on blood CD11c+ DC is highly associated with the onset of acute GVHD. These results suggest that CMFR-44 may be used as a predictive tool to identify patients at risk of severe GVHD and to direct therapy. Furthermore, it reinforces the potential application of suitably engineered CMRF-44 antibodies for the prevention or treatment of graft versus host disease.

Infection and dysfunction of circulating blood dendritic cells and their subsets in chronic hepatitis C virus infection.

To understand interactions between dendritic cells (DCs) and viruses, in vitro-cultured monocyte-derived DCs are usually used for functional analyses. However, several recent studies indicate that circulating blood DCs are different from monocyte-derived DCs, both phenotypically and functionally. Indeed, circulating DCs act as functional antigen-presenting cells in vivo. This study was conducted to evaluate the function of circulating blood DCs in patients with chronic hepatitis C and to examine whether circulating DCs from these patients were infected by hepatitis C virus (HCV). The phenotypes and biological functions of circulating DCs from patients with chronic hepatitis C ( n = 27), patients with non-HCV chronic liver disease ( n = 7), and normal volunteers ( n = 13) were analyzed. The presence of the HCV genome sequence in circulating blood DCs and in subsets of circulating DCs (myeloid DCs and plasmacytoid DCs) in patients with chronic hepatitis C was assessed. The stimulatory capacity of circulating DCs was significantly reduced in patients with chronic hepatitis C compared to patients with non-HCV chronic liver diseases and normal controls ( P < 0.01). HCV RNA was identified in the overall population of circulating DCs, and in myeloid DCs and plasmacytoid DCs. Nucleotide sequences of the 5' non-coding region of HCV RNA showed marked differences between paired samples of circulating DCs and sera from the same patients. Our results indicate the dysfunction and infection of circulatory blood DCs in chronic HCV infection. This may compromise the capacity of patients with hepatitis C to induce an effective antiviral immune response.

Growth factors, cytokines and dendritic cell development.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses. DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials. Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

Recovery of Lymphocyte and Dendritic Cell Subsets Following Reduced Intensity Allogeneic Bone Marrow Transplantation

Approaches using reduced conditioning regimens have been developed to obtain minimal procedure-related toxicity. Such novel therapeutic options are being explored with good preliminary results concerning feasibility and engraftment. However, many aspects remain under-evaluated and few data are available about immune and dendritic cell (DC) reconstitution after these highly immunosuppressive regimens.We present here our data in 20 patients receiving allogeneic bone marrow transplantation (allo-BMT) using a reduced preparative regimen. We evaluated in the first 3 months following allo-BMT, several immunological parameters including DC subsets, and compared these to historical results obtained in a group of myeloablative allo-BMT patients. We found an early recovery of leukocytes, CD8+ and NK lymphocytes. We also found a trend towards an improved B cell recovery. These results are somewhat in contrast to the altered immune recovery observed in the myeloablative setting. In addition, we found a significant early circulating DC recovery. Circulating blood DCs were also found to be of full donor origin as assessed by FISH in sex-mismatched pairs. Nevertheless, naive CD4+CD45RA+ T cells were found to be profoundly reduced following such regimens.Collectively, these data further enhance the overall benefits of reduced intensity regimens and the need for a stringent biological monitoring for assessment of the potential advantages of reduced intensity allo-BMT in comparison with conventional allo-BMT.

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

AbstractDendritic 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-α. These data provide evidence that DC subsets in vivo may be affected by leukemogenesis and may contribute to leukemia escape from immune control.