Resident human dendritic cells (DC) in vascularized lymphoid organs such as bone marrow (BM) and spleen (SP) may contribute significant T cell responses to the overall systemic immune responses. To address this possibility in vivo, we developed humanized (hu)NOD/SCID mice, which contain human resident DC in the BM and SP derived from engrafted cord blood CD34+ cells. When huNOD/SCID mice were injected with polyinosinic:polycytidylic acid [poly(I:C)], activation of the resident human myeloid (M)DC occurred concurrently, in the BM and SP, within 2–8 h of injection. Poly(I:C) activated human MDC in the BM and SP increased the proportions of CD86+MDC and their production of IL-6, IL-10 and IL-12. The presence of activated resident human MDC, prior to human allogeneic naïve T cell transplantation was associated with subsequent donor T cell activation, proliferation and effector cell generation in the BM and SP of recipient mice. However, the division kinetics of donor T cells appeared to be tissue-specific, with those in the BM undergoing six to eight divisions, whereas in the SP, the majority of donor T cells that entered a first division did not undergo subsequent division. Thus, human resident MDC in both BM and SP undergo rapid activation in response to systemic activator and contribute, according to their location, to distinctly different division kinetics of the locally recruited donor T cells. The findings in this humanized mouse model highlight the contribution of activated resident MDC in controlling local donor T cell responses and have direct application to investigations on the contribution of conditioning and DC activation to the pathogenesis of acute graft versus host disease following clinical allogeneic haematopoietic stem cell transplantation. Resident human dendritic cells (DC) in vascularized lymphoid organs such as bone marrow (BM) and spleen (SP) may contribute significant T cell responses to the overall systemic immune responses. To address this possibility in vivo, we developed humanized (hu)NOD/SCID mice, which contain human resident DC in the BM and SP derived from engrafted cord blood CD34+ cells. When huNOD/SCID mice were injected with polyinosinic:polycytidylic acid [poly(I:C)], activation of the resident human myeloid (M)DC occurred concurrently, in the BM and SP, within 2–8 h of injection. Poly(I:C) activated human MDC in the BM and SP increased the proportions of CD86+MDC and their production of IL-6, IL-10 and IL-12. The presence of activated resident human MDC, prior to human allogeneic naïve T cell transplantation was associated with subsequent donor T cell activation, proliferation and effector cell generation in the BM and SP of recipient mice. However, the division kinetics of donor T cells appeared to be tissue-specific, with those in the BM undergoing six to eight divisions, whereas in the SP, the majority of donor T cells that entered a first division did not undergo subsequent division. Thus, human resident MDC in both BM and SP undergo rapid activation in response to systemic activator and contribute, according to their location, to distinctly different division kinetics of the locally recruited donor T cells. The findings in this humanized mouse model highlight the contribution of activated resident MDC in controlling local donor T cell responses and have direct application to investigations on the contribution of conditioning and DC activation to the pathogenesis of acute graft versus host disease following clinical allogeneic haematopoietic stem cell transplantation.

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.

Dendritic cells (DCs) loaded with tumor-associated antigens are a promising treatment to prevent disease relapse in patients with multiple myeloma (MM). Early-phase clinical trials have shown safety, efficacy, and immunologic responses in MM, but a key issue now is the isolation of a functional, clinically relevant DC preparation. The authors have described a unique blood DC (BDC) isolation platform based on positive immunoselection with the CMRF-56 antibody. To validate this as a feasible source of BDCs for immunotherapy, the authors undertook a quantitative and functional analysis of BDCs in MM patients and healthy donors. These data show that MM patients have similar numbers of CD11c+CD16+ and CD11c+CD16- BDCs but about half the number of CD11c-CD123+ BDCs in whole blood compared with healthy donors. BDCs could be isolated by CMRF-56+ immunoselection from all MM patients tested, with similar yields and purity to healthy donors. These BDCs could be activated ex vivo with poly I:C or LPS. Furthermore, CMRF-56+ preparations could induce potent CD4+ and CD8+ T-lymphocyte responses in both MM patients and healthy donors. These data suggest that BDCs with in vitro functional integrity can be isolated from MM patients in sufficient numbers to justify a clinical trial.

Cytokines and antigen presenting cells (APC) maintain the mature T cell repertoire by homeostatic proliferation in the periphery. There are no conclusive data on the number and function of human memory CD4+T cells maintained in the periphery, in the absence of antigen, as a result of exposure to cytokines and access to blood APC. We showed that exposure to cytokines IL-7 and IL-15 and access to the MHC class II molecules on blood myeloid dendritic cells (MDC) allowed proliferation of memory CD4+T cells, which display a broad usage of the TCR V b repertoire. Memory CD4+T cells developed the capacity to suppress PWM-driven IgG and IgM antibody responses after exposure to IL-7 and IL-15 and independent of the access to blood MDC or Mo. The suppression of antibody responses by these memory CD4+T cells is dose-dependent: low numbers enhanced, whereas high numbers suppressed antibody responses. We conclude that IL-7, IL-15 and blood APC contribute to immunity by facilitating differentiation of regulatory memory CD4+T cells, which are capable of suppressing B cell differentiation and antibody production, as well as their role in controlling the number and effector function of memory CD4+T cells.

CD123(hi) CD11c(-) dendritic cells (CD123(hi) DC) are a distinct subset of human DC present in bone marrow, blood, lymphoid organs, and peripheral tissues. Pathogen stimulation, cytokine, or CD40 ligation induces CD123(hi) DC maturation, involving a shift from their innate immune to cognate antigen-presenting functions. In this study, we revealed that blood CD123(hi) DC in the presence of cytokine (granulocyte macrophage-colony stimulating factor and interleukin-3) undergo progressive, step-wise maturation through an "early" stage, delineated by expression of the antigen detected by the new monoclonal antibody CMRF58 (CD123(hi)CMRF58(+)CD40(-)CD86(-)CD83(-)) to the "late" stage with costimulatory antigen expression (CD123(hi)CMRF58(+)CD40(+)CD86(+)CD83(+/-)). In this early stage, cytokine-maintained CD123(hi) DC do not display changes in their morphology, no longer produce interferon-alpha (IFN-alpha) in response to bacteria, and develop the capacity to induce proliferation and polarization of allogeneic T cells. CD123(hi)CMRF58(+) DC, phenotypically similar to in vitro cytokine-maintained CD123(hi) DC, were not detected in tonsil but are present in allergen-challenged nasal mucosa of allergic individuals. Thus, CD123(hi) DC in certain tissue environments such as allergen-challenged nasal mucosa share a common CD123(hi)CMRF58(+) phenotype with in vitro cytokine-maintained blood CD123(hi) DC characterized by lack of IFN-alpha production.

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.

Changes in blood dendritic cell (BDC) counts (CD123(hi)BDC and CD11c(+)BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 microg/kg/d subcutaneously) and high-dose G-CSF in patients (30 microg/kg/d) increased CD123(hi)BDC (2- to 22-fold, mean 3.7 x 10(6)/L-17.7 x 10(6)/L and 1.9 x 10(6)/L-12.0 x 10(6)/L) in healthy donors and MM but decreased CD11c(+)BDC (2- to 10-fold, mean 5.7 x 10(6)/L-1.6 x 10(6)/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123(hi)BDC counts remained high, whereas low CD11c(+)BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123(hi)BDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.

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OBJECTIVE:Dendritic cells (DC) are the only antigen-presenting cells that can activate naïve T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease.METHODS:Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c+ and CD11c− DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin−= CD3−, CD19−, CD14−, CD16−) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86+ and CD40+lin− cells.RESULTS:Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c+ and CD11c− DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86+ and CD40+ DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44+ antigen was mainly associated with CD11c+ DC, and in UC was inversely related to the Clinical Activity Index (r = −0.69, p = 0.0002).CONCLUSIONS:There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86+ and CD40+ DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation.

ning microscopy.ANCA were identified with FITC and TRITC in nonapoptotic and apoptotic neutrophils respectively.Apoptotic and non-apoptotic DNA was labelled with mc and propidium iodide respectively.Cycloheximide was added to PMN culture to induce apoptosis.Results: Three patterns of scanning laser immufluorescence microscopy in non apoptotic neutrophils could be observed with respect to cellular UCassociated ANCA distribution: I) diffuse nuclear localisation (16.7%) 2) nuclear localisation in the nuclear periphery (50%) and 3) mixed nuclear and cytoplasmic localisation (33,4%).In all sera, ANCA fluorescence colocalises almost completely with apoptotic DNA, with persistence of a diffuse and intense fluorescence.No significant changes in ANCA titers were found when assessed with non-apoptotic neutrophils.Conclusions: The antigen(s) of ANCA associated to UC seem(s) to be localised in most cases in the neutrophil nucleus.The almost exact colocalisation between ANCA and apoptotic cleaved DNA, suggests that the intracellular DNA redistribution during neutrophil apoptosis might play a role for antigen exposure to immune system and ANCA production in Uc.