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American journal of transplantation 22(11), 2529-2547 (2022). doi:10.1111/ajt.17154, Published by Elsevier, [Amsterdam]

Long-term results following solid organ transplantation do not mirror the excellent short-term results achieved in recent decades. It is therefore clear that current immunosuppressive maintenance protocols primarily addressing the adaptive immune system no longer meet the required clinical need. Identification of novel targets addressing this shortcoming is urgently needed. There is a growing interest in better understanding the role of the innate immune system in this context. In this review, we focus on macrophages, which are known to prominently infiltrate allografts and, during allograft rejection, to be involved in the surge of the adaptive immune response by expression of pro-inflammatory cytokines and direct cytotoxicity. However, this active participation is janus-faced and unspecific targeting of macrophages may not consider the different subtypes involved. Under this premise, we give an overview on macrophages, including their origins, plasticity, and important markers. We then briefly describe their role in acute allograft rejection, which ranges from sustaining injury to promoting tolerance, as well as the impact of maintenance immunosuppressants on macrophages. Finally, we discuss the observed immunosuppressive role of the vitamin-like compound tetrahydrobiopterin and the recent findings that suggest the innate immune system, particularly macrophages, as its target.

Summary Background and Objectives We examined retrospectively whether the combination of standard dacarbazine (DTIC) and/or fotemustine chemotherapy and autologous peptide‐loaded dendritic cell (DC) vaccination may improve survival of stage IV melanoma patients. Furthermore, a small cohort of long‐term survivors was studied in more detail. Patients and methods Between 1998 and 2008, 41 patients were vaccinated at least three times with DCs while receiving chemotherapy and compared to all other 168 patients in our database who only received chemotherapy (1993–2008). Results Median life expectancy of patients receiving additional DC‐vaccination was 18 months, compared to eleven months for patients under standard chemotherapy alone. In contrast to patients with other haplotypes, the HLA‐A1/A1 subset of DC‐treated patients showed significantly lower median survival (12 vs. 25 months). Autoantibodies were frequently detected in serum of both vaccinated and non‐vaccinated patients, and there was no correlation between titers, loss or appearance of autoantibodies and survival. Additionally, phenotyping of DCs and PBMCs also did not reveal any conspicuous correlation with survival. Conclusions Combining standard chemotherapy and DC vaccination appears superior to chemotherapy alone. The impact of HLA haplotypes on survival emphasizes the importance of a careful selection of patients with specific, well‐defined HLA haplotypes for future vaccination trials using peptide‐pulsed DCs, possibly combined with checkpoint inhibitors.

Study question Within this prospective study, we aim to differentiate immune cell subpopulations in recurrent implantation failure (RIF) patients and fertile controls. Summary answer A misbalanced immune profile of NK cell subpopulations is present in RIF patients and might be a potential risk factor that requires further detailed analysis. What is known already So far, there is no conclusive opinion on the prognostic value of testing immune cell populations in women with RIF. Increased numbers of cytotoxic (CD56dimCD16bright) peripheral natural killer (pNK)-cells and CD56brightCD16dim mainly in the uterus occurring NK cells (uNK) seemed to be more prevalent in RIF patients. NK cell cytotoxicity is regulated by a complex interaction of activating and inhibiting receptors, such as the NKGD2 and natural cytotoxicity receptors including NKp46, NKp30 and NKp44. Dysregulated pNK cells could affect the adhesion and implantation of the embryo thereby contributing to RIF. Study design, size, duration Within this prospective study between March 2018 and August 2020 immune diagnostics of pNK cells and subpopulations as well as regulatory T-cells in RIF patients (defined as ≥ 3 failed fresh or frozen embryo transfers of good quality embryos (Istanbul criteria) and non-pregnant controls (nulli- and multipara) were performed using flow cytometry analysis. Participants/materials, setting, methods In total, n = 42 RIF and n = 85 controls were included. Absolute numbers and percentages of total lymphocytes of CD56dimCD16bright, CD56brightCD16dim NK cells, CD45+CD25+FoxP3+-regulatory T-cells and activation markers (CD57+, CD62L+, NKGD2+, NKp46+) were measured in patients and controls (n = 60 nulligravida, n = 25 para) in the mid-luteal phase. Statistical analysis was performed using SPSS Version 26 considering p Main results and the role of chance RIF patients showed significantly lower numbers and percentages of CD56dimCD16bright pNK cells (mean±SD per µl: 187,5±113,3 vs. 281,9±163,4 p = 0.001;%: 87,4±8,8 vs. 90,6±6,0 p = 0.017) and higher levels of CD56brightCD16dim pNK cells (mean±SD per%: 10,5±8,3 vs. 7,6±5,5 p = 0.021) compared to controls. Further, lower percentages of CD56dimCD16brightCD62L + (mean±SD per%: 23,5±11,1 vs. 32,0±14,0 p = 0.001), CD56dimCD16brightNKGD2 + (mean±SD per%: 94,0±6,8 vs. 96,4±4,2 p = 0.014) and CD56dimCD16brightNKp46 + (mean±SD per%: 65,8±19,5 vs. 76,1±14,0 p = 0.001) were observed in RIF patients (p Limitations, reasons for caution As controls composed out of not only nulli- but also multipara, higher levels of pNK cells in controls, could be induced by fetal microchimerism in multiparas, however, results remained significant after removing multipara from statistical analysis. Wider implications of the findings: These findings condense into the assumption of a non-linear association between NK cytotoxicity and successful pregnancy. A lower NK cytotoxicity in RIF patients could potentially lead to an altered immune environment impeding a successful implantation process. Trial registration number Drks00020803

Rigosertib, via reactive oxygen species (ROS), stimulates cJun N-terminal kinases 1/2 (JNK1/2), which inactivate RAS/RAF signaling and thereby inhibit growth and survival of tumor cells. JNK1/2 are not only regulated by ROS&mdash, they in turn can also control ROS production. The prooxidant and cell death function of p66Shc requires phosphorylation by JNK1/2. Here, we provide evidence that establishes p66Shc, an oxidoreductase, as a JNK1/2 effector downstream of Rigosertib-induced ROS production, DNA damage, and cell death. This may provide a common pathway for suppression of tumor cell growth by Rigosertib.

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co-stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC-disparate BALB/c-derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1-/- ). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46+ NK cells in Klrk1-/- recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4+ , but a lesser infiltration of CD8+ T cell frequencies. Contrary to published observations, co-stimulation blockade with CTLA4-Ig resulted in a significant acceleration of cardiac rejection by Klrk1-/- recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1-/- recipients were characterized by significantly higher levels of interferon-γ mRNA, and both CD4+ and CD8+ T cells displayed a greater capacity for degranulation and interferon-γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation. Electronic supplementary material The online version of this article (10.1007/s00018-017-2700-0) contains supplementary material, which is available to authorized users.

Background Clinical data suggest that iron disturbances deleteriously affect graft survival after heart transplantation (HTx), but immunological mechanisms underlying this phenomenon have not yet been elucidated. Methods To identify the mechanistic influence of iron in a murine model of HTx, fully allogeneic BALB/c donor organs were transplanted into iron-overloaded or iron-deficient C57BL/6 mice, and recipients were analyzed for functional and immunological parameters. Results After HTx, iron overload accelerated acute rejection as observed by shortened graft survival (HTx vs HTx + iron; p = 0.01), elevated rejection score ( p p + T cells ( p − NKp46 + natural killer cells ( p p p = 0.0051), which was rescued after treatment with the iron chelator deferoxamine. Iron deficiency (ID) also resulted in enhanced intragraft infiltration of inflammatory cells and accelerated rejection in the acute setting (HTx vs HTx + ID; p = 0.02) and after co-stimulation blockade ( p = 0.0059). Conclusions We provide novel insights into the understanding of disturbances in iron homeostasis and their consequences after HTX, allowing novel insights regarding improvements in personalized immunosuppression to prolong allograft survival.

The definition of biological donor organ age rather than chronological age seems obvious for the establishment of a valid pre-transplant risk assessment. Therefore, we studied gene expression for candidate markers in 60 zero-hour kidney biopsies. Compared with 29 younger donors under age 55, 31 elderly donors age 55 and older had significant mRNA expression for immunoproteasome subunits (PSMB8, PSMB9 and PSMB10), HLA-DRB, and transcripts of the activating cytotoxicity receptor NKG2D. Gene expression was validated in an independent donor cohort consisting of 37 kidneys from donors 30 years and under (Group I), 75 kidneys from donors age 31-54 years (Group II) and 75 kidneys from donors age 55 and older (Group III). Significant gene induction was confirmed in kidneys from Group III for PSMB9 and PSMB10. Strikingly, transcripts of NKG2D had the significantly highest gene induction in Group III versus Group II and Group I. Similar results were obtained for CDKN2A, but not for telomere length. Both NKG2D and CDKN2A mRNA expression were significantly correlated with creatinine levels at 24 months after transplantation. Univariate regression analysis showed significant predictive power regarding graft function at 6 and 12 months for NKG2D and CDKN2A. However, only NKG2D remained significantly predictive in the multivariate model at 12 months. Thus, our results reveal novel candidate markers in aged renal allografts, which could be helpful in the assessment of organ quality.

OBJECTIVES Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic–polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3−/− mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3−/− animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antibody-mediated rejection (AMR) has been identified among the most important factors limiting long-term outcome in cardiac and renal transplantation. Therapeutic management remains challenging and the development of effective treatment modalities is hampered by insufficient understanding of the underlying pathophysiology. However, recent findings indicate that in addition to AMR-triggered activation of the classical complement pathway, antibody-dependent cellular cytotoxicity by innate immune cell subsets also promotes vascular graft injury. This review summarizes the accumulating evidence for the contribution of natural killer cells, the key mediators of antibody-dependent cellular cytotoxicity, to human AMR in allotransplantation and xenotransplantation and illustrates the current mechanistic conceptions drawn from animal models.

Thymic stromal lymphopoietin (TSLP) endows human blood-derived CD11c+ dendritic cells (DCs) and Langerhans cells (LCs) obtained from human epidermis with the capacity to induce pro-allergic T cells. In this study, we investigated the effect of TSLP on umbilical cord blood CD34+-derived LC-like cells. These cells are often used as model cells for LCs obtained from epidermis. Under the influence of TSLP, both cell types differed in several ways. As defined by CD83, CD80 and CD86, TSLP did not increase maturation of LC-like cells when compared with freshly isolated LCs and epidermal émigrés. Differences were also found in the production of chemokine (C-C motif) ligand (CCL)17. LCs made this chemokine only when primed by TSLP and further stimulated by CD40 ligation. In contrast, LC-like cells released CCL17 in response to CD40 ligation, irrespective of a prior treatment with TSLP. Moreover, the CCL17 levels secreted by LC-like cells were at least five times higher than those from migratory LCs. After maturation with a cytokine cocktail consisting of tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and prostaglandin (PG)E2 LC-like cells released IL-12p70 in response to CD40 ligation. Most importantly and in contrast to LC, TSLP-treated LC-like cells did not induce a pro-allergic cytokine pattern in helper T cells. Due to their different cytokine secretion and the different cytokine production they induce in naïve T cells, we conclude that one has to be cautious to take LC-like cells as a paradigm for ‘real’ LCs from the epidermis.

The pregnane X receptor (PXR) is a ligand-activated transcription factor regulating genes central to drug and hormone metabolism in the liver. Previous reports indicated that PXR is expressed in PBMC, but the role of PXR in immune cells remains unknown. In this paper, we report increased PXR expression in mouse and human T lymphocytes upon immune activation. Furthermore, pharmacologic activation of PXR inhibits T lymphocyte proliferation and anergizes T lymphocytes by decreasing the expression of CD25 and IFN-γ and decreasing phosphorylated NF-κB and MEK1/2. Although these effects are preceded by an increase of suppressor of cytokine signaling 1, a master switch for IFN-γ expression, in a PXR-dependent manner, T-bet expression remains unchanged. Conversely, PXR-deficient mice exhibit an exaggerated T lymphocyte proliferation and increased CD25 expression. Furthermore, PXR-deficient lymphocytes produce more IFN-γ and less of the anti-inflammatory cytokine IL-10. In summary, these results reveal a novel immune-regulatory role of PXR in T lymphocytes and identify suppressor of cytokine signaling 1 as an early signal in PXR-mediated T lymphocyte suppression.

HIV infection is characterized by progressive immunodeficiency: HIV-infected peripheral blood mononuclear cells (PBMCs) cannot properly react to stimulation with allo-antigens and mitogens. In this study, we examined interferon-γ (IFN-γ)-mediated pathways and the proliferative response of mitogen-stimulated HIV-infected PBMCsin vitro. PBMCs of 30 HIV-infected patients were stimulated with the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Mitogen stimulation induced expression of IFN-γ, GTP cyclohydrolase I (GCH-I), and indoleamine (2,3)-dioxygenase (IDO) resulting in enhanced neopterin formation and tryptophan degradation by HIV-infected and control PBMCs. IFN-γ concentrations correlated with neopterin levels and tryptophan degradation. Proliferative responses to PHA and PWM cytokine were lower in HIV patients, with IFN-γ formation predicting proliferative responses. Higher mRNA expression of IFN-γ, GCH-I and IDO after 6 h was related to better proliferative responses in HIV-infected PBMCs. In conclusion, induction of IFN-γ and subsequent enzymes appears to importantly influence the proliferative response of HIV-infected PBMCsin vitro, suggesting a prominent role of the cytokine in the development of immunodeficiency.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx.

BACKGROUND A daily injection of low-molecular-weight heparin (LMWH) is often prescribed to women with unexplained recurrent pregnancy loss (RPL), although evidence suggesting a benefit is questionable. OBJECTIVE To determine whether LMWH increases ongoing pregnancy and live-birth rates in women with unexplained RPL. DESIGN Controlled, multicenter trial with randomization using minimization conducted from 2006 to 2013. (ClinicalTrials.gov: NCT00400387). SETTING 14 university hospitals and perinatal care centers in Germany and Austria. PATIENTS 449 women with at least 2 consecutive early miscarriages or 1 late miscarriage were included during 5 to 8 weeks' gestation after a viable pregnancy was confirmed by ultrasonography. INTERVENTION Women in the control group received multivitamin pills, and the intervention group received vitamins and 5000 IU of dalteparin-sodium for up to 24 weeks' gestation. MEASUREMENTS Primary outcome was ongoing pregnancy at 24 weeks' gestation. Secondary outcomes included the live-birth rate and late pregnancy complications. RESULTS At 24 weeks' gestation, 191 of 220 pregnancies (86.8%) and 188 of 214 pregnancies (87.9%) were intact in the intervention and control groups, respectively (absolute difference, -1.1 percentage points [95% CI, -7.4 to 5.3 percentage points]). The live-birth rates were 86.0% (185 of 215 women) and 86.7% (183 of 211 women) in the intervention and control groups, respectively (absolute difference, -0.7 percentage point [CI, -7.3 to 5.9 percentage points]). There were 3 intrauterine fetal deaths (1 woman had used LMWH); 9 cases of preeclampsia or the hemolysis, elevated liver enzyme level, and low platelet count (HELLP) syndrome (3 women had used LMWH); and 11 cases of intrauterine growth restriction or placental insufficiency (5 women had used LMWH). LIMITATION Placebo injections were not used, and neither trial staff nor patients were blinded. CONCLUSION Daily LMWH injections do not increase ongoing pregnancy or live-birth rates in women with unexplained RPL. Given the burden of the injections, they are not recommended for preventing miscarriage. PRIMARY FUNDING SOURCE Pfizer Pharma.

When T cells are primed by dendritic cells (DCs) to initiate antigen-specific immune responses screening for matching antigen receptor-MHC/peptide pairs takes place in DC-T-cell conjugates. For an immune response DC-T-cell conjugates formed during priming events need to dissolve. Although detailed knowledge on molecules involved in the conjugate formation is available, dissolving of them has not been considered to be an active process. Here, we identify CYTIP (cytohesin-interacting protein) to mediate DC-T-cell deattachment. CYTIP, which is induced during maturation of DCs, shortly accumulates to the contact zones with T cells within the first hour of coculture. Specific silencing of CYTIP results in stronger adhesion of DCs to T cells and to fibronectin. When a need for deattachment is created in a T-cell priming assay by only partially loading DCs with antigen, CYTIP silencing causes reduced priming capacity. Thus, CYTIP allows DCs to actively control DC-T-cell interactions.

CD34(+) progenitor cells carrying human herpesvirus-8, Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), have been described in the peripheral blood of AIDS patients suffering from Kaposi's sarcoma (KS). In this study, we investigated the influence of HHV-8 on the differentiation of CD34(+) progenitor cells. Native CD34(+) cells derived from cord blood could be infected by a laboratory strain of HHV-8, as shown by immunofluorescence staining and polymerase chain reaction, but no significant initial maturation/differentiation effects were observed. In addition, these infected cells were differentiated into immature and mature dendritic cells (DCs) using cytokine induction with recombinant human granulocyte-macrophage colony-stimulating factor (rhGm-CSF), recombinant human tumor necrosis factor (rhTNF-alpha) and recombinant human stem cell factor (rhSCF). Double immunofluorescence and flow cytometry studies demonstrated that virus infection did not impair the development of immature and mature DC populations. Subsequently, the immunostimulating capacity of DC populations was tested in a mixed lymphocyte reaction using allogeneic T-cells. The HHV-8-infected CD34(+) progenitor cell-derived mature DC population showed a significantly enhanced antigen-presenting capacity, compared to non-infected DCs, which was not observed with the immature DCs. This suggests stimulation of DC function by HHV-8 infection. Because there are only a small percentage of HHV-8-positive DCs in the preparations and because it is not clear whether infection is abortive or productive to some extent, this seems to be most likely due to an indirect viral effect.

C-type lectins are cell surface receptors that recognize carbohydrate structures which are often part of microbial pathogens. Several of these molecules are expressed on dendritic cells and are involved in antigen uptake. Expression of C-type lectins on dendritic cells of the human skin, i.e. Langerhans cells of the epidermis and dermal dendritic cells, has been incompletely studied to date. We therefore investigated C-type lectins in situ and on dendritic cells obtained by migration from skin explants by immunofluorescence and flow cytometry. Emphasis was laid on expression patterns of DEC-205/CD205 and BDCA-2, a marker for plasmacytoid dendritic cells. Langerhans cells in situ expressed low levels of DEC-205. Expression was upregulated upon maturation in skin explant organ culture. Most dermal dendritic cells were found to be positive for DEC-205 and DC-SIGN/CD209. Few BDCA-2-expressing cells were found in most skin samples. They were located in small groups in the dermis close beneath the basement membrane. The vast majority of all types of dendritic cells in normal human skin was of immature phenotype, i.e. did not express DC-LAMP/CD208. It is concluded that normal appearing human skin harbors different subsets of dendritic cells including few scattered BDCA-2-expressing cells, presumably plasmacytoid dendritic cells, expressing variable sets of C-type lectin receptors. This may critically contribute to the capacity of the skin immune system to flexibly respond to the world of microbial pathogens.

Background High doses (10(-6)-10(-8) M) of tacrolimus (FK506) were reported to induce a type-2 T-helper cell (Th2)-promoting function in developing dendritic cells (DC). We used a therapeutic dose (2.4 x 10(-9 )M) of tacrolimus to investigate its effect on human monocyte-derived DC. Methods Using untreated and treated immature and mature DC we compared T cell-activating capacity, surface marker expression, T cell and DC cytokine profile and transcription of genes coding for a panel of DC function-related molecules. Results Tacrolimus-treated mature DC had reduced T-cell stimulatory capacity. Although interleukin (IL)-12 production of DC was impaired, they did not promote Th2 development as T cells activated by tacrolimus-treated DC produced less interferon (IFN)-gamma, IL-4 and IL-10. The up-regulation of the T-cell activation marker CD69 and the production of IL-2 were impaired. In addition, tacrolimus-treated DC produced less IP-10 (CXCL10), which is known to be involved in allograft rejection. Other molecules related to DC function remained unchanged. Conclusions Tacrolimus treatment reduces the ability of DC to stimulate T cells and the impaired production of DC-derived IP-10 (CXCL10) and IL-12 might play a role in the immunosuppressive action of tacrolimus.

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA-matched normal T-cell clones for use in vivo.

In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis.

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-alpha. The other method makes use of the more abundant CD34- precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day 'maturation culture' to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes ('veils'), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig. cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8-3.3 x 10(6) mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Vaccinations in medicine are typically administered into the muscle beneath the skin or into the subcutaneous fat. As a consequence, the vaccine is immunologically processed by antigen-presenting cells of the skin or the muscle. Recent evidence suggests that the clinically seldom used intradermal route is effective and possibly even superior to the conventional subcutaneous or intramuscular route. Several types of professional antigen-presenting cells inhabit the healthy skin. Epidermal Langerhans cells (CD207/langerin(+)), dermal langerin(neg), and dermal langerin(+) dendritic cells (DC) have been described, the latter subset so far only in mouse skin. In human skin langerin(neg) dermal DC can be further classified based on their reciprocal expression of CD1a and CD14. The relative contributions of these subsets to the generation of immunity or tolerance are still unclear. Yet, specializations of these different populations have become apparent. Langerhans cells in human skin appear to be specialized for induction of cytotoxic T lymphocytes; human CD14(+) dermal DC can promote antibody production by B cells. It is currently attempted to rationally devise and improve vaccines by harnessing such specific properties of skin DC. This could be achieved by specifically targeting functionally diverse skin DC subsets. We discuss here advances in our knowledge on the immunological properties of skin DC and strategies to significantly improve the outcome of vaccinations by applying this knowledge.

The manifestation of atopic dermatitis (AD) results from a complex interaction of environmental factors, skin barrier defects and immunological phenomena. The initial events in AD remain unclear while much progress has been made in identifying the mechanisms leading to the manifestation and maintenance of allergic inflammation. AD pathogenesis can be approached from many different points of view. In this review, we describe findings in mouse models and in humans separately. We focus on recent findings that underscore the importance of Langerhans cells in initial mechanisms of skin inflammation in AD.

The efficiency of immunotherapy using tumor-antigen-loaded dendritic cells (DCs) is severely limited by the impaired migration of injected cells from the application site to the draining lymph nodes. As described earlier, pretreatment of the injection site with inflammatory cytokines enhances DC migration. We wanted to test whether toll-like receptor (TLR) ligands can improve migration of murine bone marrow-derived DC (BMDC) and the subsequent T-cell responses. For this purpose, we established an experimental setup closely resembling human vaccination protocols that served to investigate DC migration from the skin to the draining lymph nodes. We observed that BMDC, matured with a cytokine cocktail (tumor necrosis factor-alpha, interleukin-beta, interleukin-6, prostaglandin E2), strongly expressed CCR7. The migration efficiency of adoptively transferred mature BMDCs was determined by the number of cells injected and the application site. We decided to inject DC intradermally into the ear skin and investigated the effects of pretreatment of the injection site with various TLR ligands. Conditioning of the skin site with the TLR ligands CpG and Peptidoglycan increased the number of DCs arriving in the lymph node. Mechanical stress applied to the skin, such as tape stripping of the skin was equally effective. Importantly, only pretreatment with CpG enhanced responses of endogenous CD8 T cells. Thus, conditioning of the injection site with the TLR ligand CpG could be a new promising way to improve the outcome of DC immunotherapy.

Dendritic cells (DC) are crucial for the induction of immune responses and populate various tissues to fulfil their special role. The skin harbours different DC subsets, the Langerhans cells (LC) in the epidermis and the dermal DC in the dermis. The investigation of skin DC is cumbersome since these cells are rare in the skin. As a consequence, it is laborious to receive enough cells from the tissue for experiments. Several approaches have been developed to isolate skin DC based on either enzymatic digestion of the tissue or skin explant culture. Immature LC can be obtained by trypsinization of epidermis, cultured in vitro and be highly enriched with gradient centrifugation and magnetic bead sorting. Mature skin DC can be easily received from skin explant culture. For this purpose skin pieces are cultured for several days and migratory DC emigrate from epidermis and dermis. Both techniques are described for human and mouse skin in the following chapter of the book.

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4(+) T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4(+) T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

OBJECTIVE: In vitro peripheral blood mononuclear cells (PBMCs) of HIV-infected subjects show defective immune responses including impaired proliferative responses after stimulation with antigens and mitogens. The cytokine interferon-gamma, which seems to play an important role in HIV infection, induces neopterin formation and in parallel also tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase. DESIGN: : In this study, interferon-gamma-mediated pathways were examined in the plasma of patients and evaluated for associations with the proliferative responses of PBMC to mitogens in vitro. METHODS: To assess the actual status of immune system activation in patients, plasma concentrations of interferon-gamma, neopterin, tryptophan, and kynurenine were measured by enzyme-linked immunosorbent assay or by high-performance liquid chromatography; the ratio of kynurenine to tryptophan was calculated. Viral load, CD4 counts, and the in vivo immune activation status were compared with the in vitro responses of PBMC isolated from the same patients. PBMCs were stimulated with the mitogens concanavalin A, phytohemagglutinin, and pokeweed mitogen, and their proliferation was assessed by H-thymidine incorporation. RESULTS: The in vitro proliferative capacity of PBMC was associated with viral load, CD4 counts, and also tryptophan degradation. Particularly, high neopterin concentrations were observed to be the best predictor of impaired proliferative response in logistic regression analysis. CONCLUSIONS: The higher the degree of immune activation in vivo, the lower is the proliferative capacity of PBMC in vitro.

Background. Suppressive dendritic cells (DCs) are a promising tool for tolerance induction in transplantation. A human DCs subpopulation, which constitutively expresses indoleamine-2,3-dioxygenase (IDO), a molecule shown to prevent the rejection of fetus during pregnancy, has recently been described. This subset, characterized by nonadherence and CD123/CCR6 expression, exhibited sustained IDO production if exposed to interleukin (IL)-10. In the present work, we generated human nonadherent, CD123 + /CCR6 + DCs secreting IL-10. Methods. Monocytes were separated by plastic adherence and differentiated to DCs in the presecence of IL-3 and IL-4. Expression of IDO was determined by reverse-transcriptase polymerase chain reaction and enzyme activity by reverse-phased high-performance liquid chromatography. Mixed lymphocyte cultures were performed with allogeneic nylon wool-purified T-cells. Results. Contradicting previous findings, CD123+/CCR6+ DCs did not express IDO. Maturation of these cells with inducer-cytokines up-regulated IDO, but the allogeneic T-cell stimulatory capacity of these DCs was even stronger than that of immature IDO - DCs, and chemical abrogation of IDO activity did not increase T-cell proliferation. In parallel, we generated mature IDO - DCs, but these cells also did not induce stronger T-cell stimulation than their IDO + counterpart. Conclusions. In conclusion, CD123 + /CCR6 + DCs do not constitutively express IDO and "induced" IDO + DCs, even coexpressing anti-inflammatory IL-10, do not suppress allogeneic T-cell responses.

New experimental models and methods have rendered the field of Langerhans cells very lively. An interesting and productive scientific debate as to the functions of Langerhans cells in vivo is currently going on. We have not yet reached the point where the "pros" would weigh out the "cons", or vice versa. There is good evidence for a lack of Langerhans cell function and for down-regulatory Langerhans cell function in some models. On the other hand, there is also evidence for an active immunogenic and tolerogenic role of Langerhans cells. These recent developments will be discussed.

Background Thymic stromal lymphopoietin (TSLP) endows human CD11c + dendritic cells (DCs) from peripheral blood with the capacity to induce proallergic T cells. TSLP is present at high levels in the epidermis of atopic dermatitis where it appears to trigger emigration of epidermal Langerhans cells (LCs); however, nothing else is known about the influence of TSLP on LCs. Objective Effects of TSLP on human epidermal LCs were investigated. Methods LCs were isolated by trypsinization from healthy human skin, highly enriched by immunomagnetic techniques (via CD1a) and cultured for 2 days. Additionally, migratory LCs were obtained by emigration from epidermal sheets for 3 days. Results The addition of TSLP promoted survival and maturation of LCs obtained by trypsinization, as indicated by their increased expression of CD83, CD86, and high levels of MHC II. TSLP markedly increased numbers of migratory LCs. Allogeneic naive CD4 + T cells, cocultured with migratory TSLP-LCs produced less IFN-γ and IL-10 and more IL-4, IL-5, IL-13, and TNF-α. Finally, TSLP-LCs secreted markedly more of the T H 2 T-cell–attracting chemokine CCL17/thymus and activation-regulated chemokine. Conclusion These cytokine patterns correspond to those described for TSLP-treated blood DCs. They highlight a direct effect of TSLP on epidermal LCs. Clinical implications Our data emphasize a critical role for LCs in the triggering of atopic dermatitis. Furthermore, they underscore the interest in TSLP as a potential therapeutic target in atopic diseases.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Con sequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein–protein interactions.

DC are sentinels of the immune system. In order to reach the skin, bone-marrow-derived DC precursors need to bind and migrate through microvascular endothelial cells. Binding of DC toprimary endothelial cells of the skin has not been investigated. We therefore determined adhesion of DC at different stages of development to human dermal microvascular endothelial cells (HDMEC). DC were derived from CD34+ progenitors in cord blood. To enhance DC maturation, a defined cocktail of IL-1beta+IL-6+TNF-alpha+PGE2 was applied. Adhesion was quantified by fluorimetric and phase-contrast microscopical assays. Significantly more DC precursors (tested on day 5 after isolation) than mature DC (spontaneously matured or cytokine-cocktail-matured and tested on day 13) bound to unstimulated HDMEC. In contrast, the maturation stage of DC had no influence on their binding to human umbilical vein endothelial cells. Pretreatment of HDMEC with TNF-alpha and IFN-gamma resulted in an enhanced attachment of both DC precursors and mature DC. Mature DC lacked expression of CD31, CD36, CD45RA and CLA, and expressed lower levels of CD11a, CD11b and CD49d as compared with precursors tested on day 5. mAb against CD18, CD11a, CD11b, and CD36 markedly inhibited DC binding, whereas anti-CLA, anti-DC-SIGN, anti-CD29 and anti-CD49 mAb did not. Our data support the hypothesis of immunosurveillance with selective recruitment of blood DC precursors to resting and, more so, to inflamed skin. The data have potential relevance for anti-cancer immunotherapy strategies favoring the intracutaneous application of mature DC.

Increased neopterin concentrations in body fluids are found in diseases associated with activated, cell-mediated immunity including infections, autoimmune diseases, and certain malignancies. Monocytes/macrophages are known to secrete large amounts of neopterin upon stimulation with interferon-γ (IFN-γ). Ontogenetically, the major part of dendritic cells (DC) belongs to the myeloid lineage. Therefore, we investigated whether cultured monocyte-derived DC can elaborate neopterin. Cells were treated with cytokines in the presence or absence of monocyte-conditioned medium as a maturation stimulus. DC secreted an average 3.5 nmol/l neopterin. In response to IFN-γ, cells significantly increased their output of neopterin. In distinction to monocytes/macrophages, neopterin production in DC was highly sensitive to IFN-α and IFN-β. Further, lipopolysaccharides (LPS) enhanced neopterin synthesis, whereas tumor necrosis factor α, interleukin (IL)-1β, IL-2, IL-10, and IL-18 were ineffective. Simultaneously, tryptophan degradation by induction of indoleamine (2,3)-dioxygenase (IDO) was tested in stimulated cells. Our results showed that IFN-γ as well as LPS are inducers of IDO in DC.

Background: IL-12 is a crucial factor in the development and course of allergic diseases. By virtue of their IL-12 production, dendritic cells (DCs) are potent inducers of T H 1 responses. However, distinct subsets of DCs have also been shown to induce T H 2 differentiation. Objective: We hypothesized that DCs from atopic and nonatopic individuals might differ in their propensity to skew T-cell responses to either the T H 1 type or the T H 2 type. To this end, we investigated the cytokine patterns produced by DCs from atopic and nonatopic individuals, and we attempted to clarify whether this could be due to different DC lineages or, alternatively, to different microenvironmental factors. Methods: DCs were generated from lymphocyte-depleted PBMCs from atopic and nonatopic donors and fully matured with monocyte-conditioned medium. Production of IL-4, IL-5, IL-10, IL-12, and IL-13 in response to CD40 ligation was measured with ELISA. DC subsets were identified in PBMCs from freshly drawn blood by 3-color flow cytometry. Results: Compared with DCs from healthy donors, monocyte-derived DCs from atopic patients produced less bioactive IL-12 and IL-10. DC production of IL-4, IL-13, and IL-5 was not detected. Relatively more CD123 + DCs, corresponding to T H 2-inducing "DC2s," were found in PBMCs from atopic patients. Conclusion: The data suggest that in addition to the described abnormalities in the patients' T-cell populations, DCs might also critically contribute to the atopic/allergic T H 1 outcome in the patient and thus to the disease. (J Allergy Clin Immunol 2002;109:89-95.)

Many blood banks now use whole blood inline filtration to produce leukocyte-depleted blood products. As a result, a common source of large numbers of human dendritic cells (DC) for research purposes, namely standard buffy coats, has been lost. Therefore, we have adapted our conventional method for growing DC from CD14 + precursors in order to make use of these filter units. A dextran solution containing human serum albumin was used to flush back the filters. After pelleting, mononuclear cells were obtained by standard density gradient centrifugation (Lymphoprep™). To eliminate T cells, we used rosetting with sheep red blood cells. In addition to the classical PBMC, the cell population obtained after Lymphoprep™ centrifugation was found to contain high numbers of CD14 + granulocytes which could be depleted by separation on an additional Percoll gradient. At this stage, FACS analysis revealed a cell population that resembled the CD14 + monocyte-enriched population, obtained from traditional buffy coat preparations after Lymphoprep™ centrifugation and T cell elimination. Culture of the cells and the induction of maturation was identical to the previously described procedures, except that the culture time was reduced from 7 to 5 days and the maturation time from 3 to 2 days. Analyses of the major molecules indicative of DC maturation (CD83, CD86, CD208/DC-LAMP) and functional analyses of the T cell-stimulatory capacity of the DC population (using the MLR assay with normal peripheral T cells and naive T cells) revealed no major differences from buffy coat-derived DC preparations.