Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

High sensitivity of megakaryocytic progenitor cells contained in placental/umbilical cord blood to the stresses during cryopreservation.

In placental/umbilical cord blood (PCB) banking and PCB transplantation (PCBT), long-term cryopreservation of hematopoietic stem and progenitor cells is a unique requirement as compared to that for bone marrow transplantation and cytokine-mobilized peripheral blood transplantation. A long period of severe thrombocytopenia is a problem in many patients after PCBT. The object of this study was to define whether megakaryocytic progenitor cells (CFU-Meg), which produce platelets, are more sensitive to cryopreservation than the other myeloid progenitor cells in PCB. The leukocyte concentrates (LCs) obtained from clinical PCB banks were cryopreserved, and progenitor cell recoveries were determined by differential count of colony-forming cells (CFCs). The LCs were exposed to stresses which cells face during freezing, thawing, and washing out cryoprotectants. Most of the myeloid progenitor cells contained in the LCs showed good survival when cryopreserved at slow cooling rates, although cellular injury was observed at higher cooling rates and higher osmolalities. In contrast, the recovery rate of CFU-Meg was significantly lower than other progenitor cells, indicating a higher sensitivity to the various stresses they were exposed to during cryopreservation. Thrombocytopenia observed in patients receiving PCBT may be explained, at least in part, by the disappearance of CFU-Meg during cryopreservation.

Holoprosencephaly--topologic variations in a liveborn series: a general model based upon MRI analysis.

We present an MRI-based anatomic analysis of a series of 9 human brains, representing lobar, semilobar and alobar forms of holoprosencephaly. The analysis of these variable forms of the malformation is based upon a topologic systematics established in a prior analysis of a homogeneous set of semilobar malformations. This systematics has the dual advantage that it serves both as a uniform reference for qualitative description and as a quantitative descriptive base for mathematical correlations between parameters of topology and of growth and development. Within this systematics, the prosencephalic midline is divided from caudal to rostral into diencephalic (DD-right and left, subthalamus through suprachiasmatic junction with telencephalon), telencephalic (TT-right and left, suprachiasmatic border of telencephalon midline to hippocampal commissure) and diencephalic-telencephalic (DT-right and left-hippocampal commissure through temporal limb of choroid fissure) segments. The topologic abnormality of the initial semilobar series was expressed in an orderly rostral to caudal gradient along the TT segment. In each malformation, normal midline topology began with a small posterior corpus callosum. Although the topologic anomaly in the present series invariably also involved the TT segment, this involvement was not continuous and was variably associated with anomalies of the DD in 6 and unilaterally of the DT in 1 brain. In the present as well as with the earlier series of HPE malformations but not in "normative brains," total telencephalic growth is strongly correlated with the length of the midline telencephalic segment. We propose that this system of analysis will be sensitive to the developmental stage and locus of expression of genetic and non-genetic determinants of the formal origin of HPE. For all of the present series, karyotype anlyses were normal. Mutations in the Shh and Zic2 genes were excluded in 2 cases.

Bcl-2, Bcl-xL and c-FLIP(L) potentially regulate the susceptibility of human peripheral blood monocyte-derived dendritic cells to cell death at different developmental stages.

We examined the susceptibility of human monocyte-derived dendritic cells (DCs) to spontaneous and CD95-mediated cell death at different developmental stages. Time course experiments revealed that the susceptibility of mature dendritic cells (mDCs) to spontaneous cell death was significantly lower than that of immature dendritic cells (iDCs) in a long-term culture under cytokine-free conditions, and the treatment with GM-CSF rescued these cells from spontaneous cell death at the late culture period. iDCs and mDCs expressed similar levels of CD95 whereas both cell types were relatively resistant to CD95-mediated cell death. Antigen (Ag)-specific and nonspecific cognate interaction with T cells failed to cause cell death of iDCs and mDCs. iDCs constitutively expressed transcripts and intracellular products of Bcl-2 and Bcl-xL, but not cellular FLICE-inhibitory protein(long (c-FLIP(L)), while the increased expressions of Bcl-2, Bcl-xL and c-FLIP(L) were observed in mDCs. These results suggest that the selective expressions of Bcl-2, Bcl-xL and c-FLIP(L) may be involved in the difference in the susceptibility to cell death between iDCs and mDCs.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 x 10(7)/kg recipient body weight) were transplanted to an 8-10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte gamma-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 x 10(9)/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 x 10(9)/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.

Enhancement of migratory and aggregate activities of human peripheral blood monocyte-derived dendritic cells by stimulation with RANTES.

We examined the effects of various chemokines on the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (iDC). Stimulation of iDC with regulated on activation normal T cell expressed and secreted (RANTES) resulted in the promotion of their chemotactic migratory capacity in response to RANTES when compared with that of unstimulated cells. TNF-alpha induced a homotypic aggregated cluster formation of iDC in a dose-dependent manner, whereas the combination of TNF-alpha and RANTES exhibited more potent induction. IDC stimulated with RANTES were more efficient than unstimulated iDC in the production of endogenous RANTES. Treatment of iDC with the combination of TNF-alpha and RANTES was just little effective for the enhancement of allogeneic T-cell stimulatory capacity as compared with that of TNF-alpha treated iDC. These results suggest that endogenous secretions of RANTES from iDC stimulated with RANTES be potentially involved in RANTES-induced changes of properties with respect to morphology and function.

[Effects of glycosaminoglycans on the in vitro colony formation of CD34+ megakaryocytic progenitor cells in human placental/umbilical cord blood].

The in vitro effect of various glycosaminoglycans (GAGs) on the clonal growth of CD34+ megakaryocytic progenitor cells (CFU-Megs) isolated from human placental/umbilical cord blood (CB) was evaluated in human plasma containing semisolid culture stimulated by recombinant human thrombopoietin (TPO). The GAGs, including hyaluronic acid from human umbilical cords (HA-h), pig skins (HA-p) and rooster combs (HA-r), or keratan sulfate (KS), various chondroitin sulfates (CS-A, B, C, D, E), and heparan sulfate (HS), were tested. Each GAG alone did not affect the clonal growth of CFU-Meg. In the presence of TPO, adding of HA-p or HS (100 micrograms/ml) resulted in an approximately 1.3-fold increase, in the total number of colonies, due to an increase in large megakaryocyte colonies. In contrast, CS-E led to a marked decrease in CFU-Meg growth. At the end of the culture, the total number of cells increased 3.0-fold of the initial value of the control, but adding HA-p or HS showed an approximately 9.1-fold or 18.3-fold increase. Similarly, the total number of CFU-Meg detected in the harvested cells increased to 4.8-fold of the initial value, while, an approximately 18.3-fold or 38.8-fold increase was observed in the culture containing HA-p or HS, respectively. Flow cytometric analysis of the harvested cells showed no significant difference in the expression of surface antigens and DNA ploidy distribution of megakaryocytes between the control and GAG treatments. These results suggest that HA-p and HS promote the proliferation of immature CB CD34+ CFU-Meg in the presence of TPO.

Repopulating activities of human cord blood cells separated by a stem cell collection filter in NOD/SCID mice: a comparative study of filter method and HES method.

BACKGROUND: Volume reduction and removal of RBCs are essential for cost-efficient cord blood (CB) banking. It has previously been shown that a newly developed device, a stem cell-collection filter (SCCF), can reduce the CB volume and remove RBCs efficiently, giving high recovery rates for CD34+ cells, colony-forming cells, and long-term culture-initiating cells with short operation time. The aim of this study was to compare the quality of CB cells separated by SCCF and HES by analyzing repopulation in NOD/SCID mice. STUDY DESIGN AND METHODS: A total of 1 x 10(6) or 5 x 10(6) nucleated cells derived from SCCF- or HES-separated, cryopreserved, thawed, and washed CB were transplanted into NOD/SCID mice. Eight weeks after transplantation, bone marrow cells of the recipient mice were examined by flow cytometry and hematopoietic progenitor assay for the engraftment of human cells. RESULTS: Mice given human CB cells, separated by SCCF, showed degrees of engraftment similar to those in mice given HES-separated CB cells. There was no significant difference in the lymphohematopoietic reconstitution pattern in the two groups of mice. CONCLUSION: SCCF processing does not appear to reduce the number of repopulating cells in NOD/SCID mice or alter the number of HPCs. It is now shown that these cells can be captured by SCCF and removed, and that they will engraft.

Stem cell collection filter system for human placental/umbilical cord blood processing.

BACKGROUND AND OBJECTIVES: The hydroxyethyl starch method and the Top & Bottom method have been used worldwide for the volume reduction of human placental/umbilical cord blood (PCB) units. To simplify the preparation of nucleated cell (NC) concentrates, we developed a new filter device--the stem cell collection filter system (SCF SYSTEM)--which can collect mononuclear cells (MNC) at a high recovery rate. MATERIALS AND METHODS: The SCF SYSTEM consisted of a filter and two bags. Multilayered polyethylene terephthalate non-wovens, coated with a hydrophilic polymer, were used as filter media. PCB units were filtered by gravity (n = 12). Red blood cells, platelets and plasma were drained into the drain bag, and the NC trapped on the filter media was collected in the recovery bag by reverse washing. Recovered cell fractions were evaluated. RESULTS: The volume of cell concentrate recovered was 27.4 +/- 2.2 ml (mean +/- SD, n = 12). The whole time required for processing was less than 30 min, and handling was very simple. The viability of recovered NC was 97.8 +/- 3.2%. The recovery of lymphocytes, monocytes and granulocytes was 79.5 +/- 16.9%, 79.8 +/- 20.4% and 39.0 +/- 19.5%, respectively. The recovery rate of granulocytes was significantly lower than that of monocytes and lymphocytes (P < or = 0.0001). The recovery rates of CD3+ cells, CD19+ cells and CD56+ cells were almost the same as that of MNC. The recovery rates of CD34+ cells, total colony-forming cells and long-term culture-initiating cells were 81.7 +/- 27.0% (n = 11), 80.8 +/- 27.7% (n = 12) and 75.0 +/- 18.4% (n = 2), respectively. CONCLUSION: The new filter system was shown to be efficient for PCB processing, encompassing a very simple handling procedure with a good recovery of haematopoietic progenitor cells. Hence, the SCF SYSTEM is potentially useful for the volume reduction of PCB units for cord blood banking.

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide.

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2*- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2*- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.

Signaling events following chemokine receptor ligation in human dendritic cells at different developmental stages.

Responsiveness of dendritic cells (DC) to inflammatory CC chemokines is down-regulated during their maturation. We analyzed the mechanism underlying these events. Cell-surface expression of CC chemokine receptor (CCR)-1, -3 and -5 was increased during differentiation of immature DC (iDC) from monocytes. In contrast, these expressions were decreased during development of iDC into mature DC (mDC) to levels similar to those of monocytes. Transcriptional expression of CCR-1, -3 and -5 was increased during differentiation of iDC from monocytes, while the expression was decreased during development of iDC into mDC. Expression of CCR-7 transcript was detected in mDC, but not in monocytes or iDC. Both monocytes and iDC, but not mDC, migrated in response to inflammatory CC chemokines such as regulated on activation normal T cell expressed and secreted (RANTES)/CCL5, whereas mDC, but not monocytes or iDC, migrated to macrophage inflammatory protein (MIP)-3ss/CCL19. Receptor engagement of monocytes or iDC by RANTES (for CCR-1, -3 and -5) resulted in protein tyrosine phosphorylation events including activation of focal adhesion kinase as well as mitogen-activated protein kinase, whereas this stimulation induced little activation of these molecular events in mDC when compared with monocytes or iDC. On the other hand, stimulation with MIP-3ss (for CCR-7) induced tyrosine phosphorylation events in mDC, but not in monocytes or iDC. These results suggest that the down-regulation of cell-surface expression of CCR and of their downstream signaling events may be involved in the reduced chemotaxis of DC to inflammatory CC chemokines during their maturation.

Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture.

Long-term severe thrombocytopenia following human placental and umbilical cord blood (CB) transplantation is a significant clinical problem. We studied the ex vivo expansion of megakaryocytic progenitor cells (CFU-Meg) from cryopreserved/thawed leukocyte concentrates (LC) of CB prepared by the Tokyo Cord Blood Bank protocol. The LC cells were cultured in serum-free culture medium supplemented with a combination of early-acting cytokines including thrombopoietin (TPO), flt3-ligand (FL), and stem cell factor (SCF). Combination of TPO plus FL, TPO plus SCF, and all of these cytokines together resulted in 8.9-, 7.7-, and 8.4-fold increases in CFU-Meg, respectively, by Day 5 of culture. Our results showed that this simple expansion strategy has the potential for expanding CFU-Meg from cryopreserved/thawed LC cells from CB.

A putative chemoattractant receptor, C5L2, is expressed in granulocyte and immature dendritic cells, but not in mature dendritic cells.

Leukocyte chemoattractants are involved in the inflammatory response act via G protein-coupled receptors. We report the cloning of a novel human gene encoding the putative orphan receptor, C5L2, belonging to a subfamily of C3a, C5a and formyl Met-Leu-Ph receptors that are related to the chemokine receptor family. C5L2 transcript levels were abundant in granulocytes and immature dendritic cells but not in mature dendritic cells. We speculate that this receptor may regulate the activation of immature dendritic cells and play a role in the chemoattraction of leukocytes to inflammatory regions.

Effect of murine kidney extracts on the proliferation of hematopoietic progenitor cells in human umbilical cord blood.

We examined the effect of murine kidney extract (MKE) on the clonal growth of highly purified CD34+ hematopoietic progenitor cells from human umbilical cord blood. MKE did not affect the total number of colonies of erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM) or granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-Mix/CFU-GEMM) in a methylcellulose culture with exogenous recombinant human granulocyte colony-stimulating factor, granulocytemacrophage colony-stimulating factor, interleukin-3, stem cell factor and erythropoietin. MKE significantly increased the proportion of BFU-E- or CFU-Mix-derived colonies, and suppressed the formation CFU-GM-derived colonies depending on the MKE dose. However, because of an increase in small megakaryocyte colonies derived from mature CFU-Meg MKE increased by approximately 40% the growth of megakaryocyte colony-forming units (CFU-Meg) in plasma clot culture stimulated by recombinant human thrombopoietin. Also MKE promoted an increase in hyperploid megakaryocytes, suggesting that the active factor(s) in MKE acts on the mature CFU-Meg and promotes the maturation of megakaryocytes. Gel-filtration high performance liquid chromatography of MKE showed that the promoting factor(s) in MKE was approximately 45 kDa. These results indicate that the factor(s) detected in MKE influence human hematopoiesis in vitro, especially thrombopoiesis.

Dysfunctional regulation of the development of monocyte-derived dendritic cells in cancer patients.

Dendritic cells (DCs) are highly effective antigen (Ag)-presenting cells (APCs) that are required for the initiation of the immune response. DCs derived from cancer patients have been shown to be defective in several phenotypic and functional properties. However, little is known about the capacity of monocytes derived from cancer patients to differentiate into DCs. Herein, we examined the differentiation of monocyte-derived DCs in cancer patients. Flow cytometric analysis revealed that monocytes derived from cancer patients cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) exhibited lower levels of CD11c, CD40, CD86, and HLA-DR expression as compared with those of monocyte-derived DCs from healthy volunteers. Furthermore, the capacities of DCs derived from cancer patients' monocytes to stimulate allogeneic T cell responses and to migrate in response to regulated-on-activation normal T cells expressed and secreted (RANTES) were impaired in comparison with those of monocyte-derived DCs from healthy volunteers. However, the two cell types had similar pinocytotic capacities for fluorescein isothiocyanate labeled-dextran (FITC-DX) and lucifer yellow (LY). These results suggest that monocytes from cancer patients may be defective in the capacity to develop into DCs.

Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan.

To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O2-) from opsonized symosan (OZ)--stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37 degrees C. Then a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30 sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals (.OH), DMPO-CH3 adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O2- were produced inside phagosomes of OZ-stimulated PMNs and .OH were produced outside them by spontaneous decomposition of the DMPO-OOH adducts.

IL-12 responsiveness and expression of IL-12 receptor in human peripheral blood monocyte-derived dendritic cells.

We analyzed the expression of IL-12Rbeta1 and IL-12Rbeta2 and the role of IL-12 in the activation of monocyte-derived dendritic cells (DCs) via IL-12Rbeta1-mediated signaling events. Flow cytometric analysis revealed that IL-12Rbeta1 was expressed in T cells, Con A blasts, and monocyte-derived DCs, but not in monocytes, while its transcript was detected in all of these cell types. Transcriptional expression of IL-12Rbeta2 was observed in T cells, Con A blasts, and monocyte-derived DCs, but not monocytes. The ligation of DCs as well as Con A blasts by IL-12 induced the production of GM-CSF, IL-1beta, IL-6, TNF-alpha, and IFN-gamma at the transcription levels. Furthermore, stimulation of DCs with IL-12 induced IL-12p40 transcript, but not IL-12p35 transcript, whereas this stimulation caused the expressions of both transcripts in Con A blasts. Stimulation of DCs with IL-12 caused a tyrosine phosphorylation of several intracellular proteins, and the pattern of these events were distinct from those of IL-12-stimulated Con A blasts. IL-12 also induced tyrosine phosphorylation of IL-12Rbeta1 as well as recruitment of several tyrosine-phosphorylated proteins to IL-12Rbeta1 in DCs and Con A blasts. Receptor engagement of DCs as well as Con A blasts by IL-12 resulted in activation of Janus kinase 2 and Tyk2 kinases and Stat3 and Stat4 transcription factors and the association of these proteins to IL-12Rbeta1. Stimulation with IL-12 caused a tyrosine phosphorylation and enzymatic activity of a family of mitogen-activated protein kinases, p38mapk. These results suggest that IL-12 acts directly on DCs to induce their functional activation via IL-12Rbeta1-mediated signaling events.

Exposure of normal monocyte-derived dendritic cells to human immunodeficiency virus type-1 particles leads to the induction of apoptosis in co-cultured CD4+ as well as CD8+ T cells.

The depletion of immune T cells by human immunodeficiency virus type-1 (HIV-1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte-derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs exposed to HIV-1 particles were co-cultured with healthy donor-derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV-1 particles.