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Animal models have long been used to investigate human mental disorders, including depression, anxiety, and schizophrenia. This practice is usually justified in terms of the benefits (to humans) outweighing the costs (to the animals). The author argues on utility maximization grounds that we should phase out animal models in neuropsychiatric research. The leading theories of how human minds and behavior evolved invoke sociocultural factors whose relation to nonhuman minds, societies, and behavior has not been homologized. Thus, it is not at all clear that we are gaining the epistemic or clinical benefits we want from this animal-based research.

We evaluated the immunological responses of lymph-node involved (stage III) melanoma patients to adjuvant dendritic cell vaccination with subsets of naturally occurring dendritic cells (nDCs). Fifteen patients with completely resected stage III melanoma were randomized to receive adjuvant dendritic cell vaccination with CD1c + myeloid dendritic cells (cDC2s), plasmacytoid dendritic cells (pDCs) or the combination. Immunological response was the primary endpoint and secondary endpoints included safety and survival. In 80% of the patients, antigen-specific CD8 + T cells were detected in skin test-derived T cells and in 55% of patients, antigen-specific CD8 + T cells were detectable in peripheral blood. Functional interferon-γ-producing T cells were found in the skin test of 64% of the patients. Production of nDC vaccines meeting release criteria was feasible for all patients. Vaccination only induced grade 1-2 adverse events, mainly consisting of fatigue. In conclusion, adjuvant dendritic cell vaccination with cDC2s and/or pDCs is feasible, safe and induced immunological responses in the majority of stage III melanoma patients., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14 + DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system.

Mounting an effective immune response relies critically on the coordinated interactions between adaptive and innate compartments. How and where immune cells from these different compartments interact is still poorly understood. Here, we demonstrate that the cross-talk between invariant natural killer T cells (iNKT) and CD8 + T cells in the spleen, essential for initiating productive immune responses, is biphasic and occurs at 2 distinct sites. Codelivery of antigen and adjuvant to antigen-presenting cells results in: 1) initial short-lived interactions (0 to 6 h), between CD8 + T cells, dendritic cells (DCs), and iNKT cells recruited outside the white pulp; 2) followed by long-lasting contacts (12 to 24 h) between iNKT cells, DCs, and CD8 + T cells occurring in a 3-way interaction profile within the white pulp. Both CXCR3 and CCR4 are essential to orchestrate this highly dynamic process and play nonredundant in T cell memory generation. While CXCR3 promotes memory T cells, CCR4 supports short-lived effector cell generation. We believe our work provides insights into the initiation of T cell responses in the spleen and their consequences for T cell differentiation., Competing Interests: The authors declare no competing interest.

Background: Health-related quality of life (HRQoL) is an important issue in the rapidly evolving field of adjuvant treatment for stage III melanoma. Dendritic cell vaccination is one of the adjuvant forms of therapy currently investigated., Methods: We enrolled adults with stage III melanoma to receive adjuvant dendritic cell vaccination after a complete radical lymph node dissection. HRQoL assessment was one of the secondary endpoints of this trial and investigated with the EORTC-QLQ-C30 questionnaire at baseline and week 26., Results: Fifteen patients with a median age of 50 years were included in the study, with twelve evaluable patients on study at time of the second questionnaire. Global health status and role functioning improved clinically relevant with a mean difference of 15 (p = 0.010) and 26 points (p = 0.005), respectively., Discussion: Despite the small number of patients, we found a clinically relevant improved global health status. Besides, compared to the other investigated therapies, toxicity of dendritic cell vaccination is low, which supports our finding., Conclusion: This is the first description of HRQoL in melanoma patients receiving dendritic cell vaccination. We show the expected improvement in global health status after surgical treatment of stage III melanoma. Thus, adjuvant dendritic cell vaccination does not seem to hamper this improvement, as shown in our small explorative study.

The knowledge of in vitro and in vivo stability of polymeric nanoparticles is vital for the development of clinical formulations for drug delivery and cell labeling applications. Förster resonance energy transfer (FRET)-based fluorescence labeling approaches are promising tools to study nanoparticle stability under different physiological conditions. Here, we present the FRET-based stability assessment of poly(lactic- co -glycolic acid) (PLGA) nanoparticles encapsulating BODIPY-FL12 and Nile Red as the donor and acceptor, respectively. The stability of PLGA nanoparticles is studied via monitoring the variations of fluorescence emission characteristics along with colloidal characterization. Accordingly, PLGA nanoparticles are colloidally stable for more than 2 weeks when incubated in aqueous buffers in situ, whereas in vitro particle degradation starts in between 24 and 48 h, reaching a complete loss of FRET at 72 h as shown with fluorescence microscopy imaging and flow cytometry analysis. PLGA nanoparticles systemically administered to mice predominantly accumulate in the liver, in which FRET no longer takes place at time points as early as 24 h postadministration as determined by ex vivo organ imaging and flow cytometry analysis. The results of this study expand our knowledge on drug release and degradation behavior of PLGA nanoparticles under different physiological conditions, which will prove useful for the rational design of PLGA-based formulations for various applications that can be translated into clinical practice., Competing Interests: The authors declare no competing financial interest.

Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell-substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, αMβ2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current "core-ring" model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.

Tissue-resident dendritic cells patrol for foreign antigens while undergoing slow mesenchymal migration. Using actomyosin-based structures called podosomes, dendritic cells probe and remodel extracellular matrix topographical cues. Podosomes comprise an actin-rich protrusive core surrounded by an adhesive ring of integrins, cytoskeletal adaptor proteins and actin network filaments. Here we reveal how the integrity and dynamics of protrusive cores and adhesive rings are coordinated by the actomyosin apparatus. Core growth by actin polymerization induces podosome protrusion and provides tension within the actin network filaments. The tension transmitted to the ring recruits vinculin and zyxin and preserves overall podosome integrity. Conversely, myosin IIA contracts the actin network filaments and applies tension to the vinculin molecules bound, counterbalancing core growth and eventually reducing podosome size and protrusion. We demonstrate a previously unrecognized interplay between actin and myosin IIA in podosomes, providing novel mechanistic insights into how actomyosin-based structures allow dendritic cells to sense the extracellular environment.

Adoptive transfer of cells for therapeutic purposes requires efficient and precise delivery to the target organ whilst preserving cell function. Therefore, therapeutically applied cells need to migrate and integrate within their target tissues after delivery, e.g. dendritic cells (DCs) need to migrate to lymph nodes to elicit an antigen-specific immune response. Previous studies have shown that inappropriate cell delivery can hinder DC migration and result in insufficient immune induction. As migration can be extremely difficult to study quantitatively in vivo, we propose an in vitro assay that reproduces key in vivo conditions to optimize cell delivery and migration in vivo. Using DC migration along a chemokine gradient, we describe here a novel (19)F MR-based, large-scale, quantitative assay to measure cell migration in a three-dimensional collagen scaffold. Unlike conventional migration assays, this set-up is amenable to both large and small cell numbers, as well as opaque tissue samples and the inclusion of chemokines or other factors. We labeled primary human DCs with a (19)F label suitable for clinical use; (0.5-15) × 10(6) cells in the scaffolds were imaged sequentially, and migration was assessed using two independent methods. We found no migration with larger numbers of cells, but up to 3% with less than one million cells. Hence, we show that the cell density in cell bolus injections has a decisive impact on migration, and this may explain the limited migration observed using large cell numbers in the clinic., (Copyright © 2012 John Wiley & Sons, Ltd.)

Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and chemical properties using a hybrid magnetic chemical biosensor. We coupled a fluorescent dye, which serves as a chemical sensor, to a magnetic particle that is used for measurement of the viscoelastic environment by studying the response of the particle to magnetic force pulses. As a demonstration of the potential of this approach, we applied the method to study the process of phagocytosis, wherein cytoskeletal reorganization occurs in parallel with acidification of the phagosome. During this process, we measured the shear modulus and viscosity of the phagosomal environment concurrently with the phagosomal pH. We found that it is possible to manipulate phagocytosis by stalling the centripetal movement of the phagosome using magnetic force. Our results suggest that preventing centripetal phagosomal transport delays the onset of acidification. To our knowledge, this is the first report of manipulation of intracellular phagosomal transport without interfering with the underlying motor proteins or cytoskeletal network through biochemical methods., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The induction of tumor-specific immune responses is largely dependent on the ability of dendritic cells (DCs) to present tumor-associated antigens to T lymphocytes. Therefore, we investigated the use of DC-associated promoter-driven genetic vaccines to specifically target DC in vivo. Restricted expression of vaccine-encoding genes in DC should enhance specificity and improves their safety for clinical applications. Hereto, 3-5 kb upstream sequences of the murine genes encoding CD11c, DC-SIGN, DC-STAMP and Langerin were isolated, characterized and subcloned into enhanced green fluorescent protein (EGFP) reporter constructs. Upon electroporation, EGFP was expressed in DC cell lines, but not in other cell lines, confirming DC-restricted promoter activity. When these promoters were cloned into a construct upstream of the gene for ovalbumin (OVA), it appeared that DC-STAMP promoter-driven expression of OVA (pDCSTAMP/OVA) in DC yielded the most efficient OVA-specific CD4+ and CD8+ T-cell responses in vitro. Administration of pDC-STAMP/OVA in vivo, using the tattoo gun vaccination system, evoked specific immune responses as evidenced in a mouse tumor model. Adoptively transferred pDC-STAMP/OVA-transfected DCs induced strong CD8+ T-cell proliferation in vivo. These experiments demonstrate that our DC-directed promoter constructs are potential tools to restrict antigen expression in DC and could be implemented to modulate DC function by the introduction of relevant proteins.

Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

Context: The first therapeutic cancer vaccine demonstrating effectiveness in a phase 3 study was approved by the US Food and Drug Administration on 29 April 2010. The pivotal trial demonstrated overall survival (OS) benefit in patients treated with antigen-loaded leukapheresis cells compared with a control infusion. Results of other prostate cancer (PCa) vaccination strategies are awaited, as this approach may herald a new era in the care for patients with advanced PCa., Objective: Consider effectiveness and safety of vaccination strategies in the treatment of PCa., Evidence Acquisition: We searched three bibliographic databases (January 1995 through October 2010) for randomised phase 2 and 3 studies of vaccination strategies for PCa based on predetermined relevant Medical Subject Heading terms and free text terms., Evidence Synthesis: Data from 3 randomised phase 3 and 10 randomised phase 2 vaccination trials are discussed with respect to clinical outcome in terms of progression-free survival and OS, toxicity, prostate-specific antigen (PSA) response, and immunologic response. Three phase 3 trials (D9901, D9902A, and D9902B) that enrolled a total of 737 patients, all controlled and double-blinded, tested the efficacy of sipuleucel-T. The largest of these three trials, called Immunotherapy for Prostate Adenocarcinoma Treatment (IMPACT), has demonstrated safety and effectiveness of sipuleucel-T (now marketed as Provenge) as measured by prolonged survival of 512 asymptomatic patients with metastatic castration-resistant PCa (mCRPC). The study showed a 4.1-mo median survival benefit in the sipuleucel-T vaccine-treated group compared with the control group (25.8 vs 21.7 mo; hazard ratio [HR]: 0.78; 95% confidence interval [CI], 0.62-0.98; p=0.032) and extended 3-yr survival (31.7% vs 23.0%). In contrast, two phase 3 vaccination trials with a whole-tumour-cell mixture of two PCa cell lines (GVAX) and testing GVAX either alone or in combination with chemotherapy versus chemotherapy alone (VITAL1 and 2) were terminated prematurely based on futility and increased deaths. Other phase 2 vaccination trials testing different types of vaccines in castration-resistant PCa patients have been reported with variable outcomes. Notably, a controlled, double-blind, randomised phase 2 vaccine trial of PROSTVAC-VF, a recombinant viral vector containing complementary DNA encoding PSA, in 125 patients with chemotherapy-naïve, minimally symptomatic mCRPC also demonstrated safety but no significant effect on the time to disease progression. In comparison with controls (n=40), PROSTVAC-VF-treated patients (n=82) experienced longer median survival of 8.5 mo (25.1 vs 16.6 mo; HR: 0.56; 95% CI, 0.37-0.85; p=0.0061) and extended 3-yr survival (30% vs 17%). In general, PCa vaccines are perceived to have less toxicity compared with current cytotoxic or targeted therapies. Evaluation of clinical efficacy of different vaccination strategies (eg, protein-, peptide- and DNA-based vaccines) in the context of properly designed and controlled phase 3 studies is warranted., Conclusions: Cancer vaccines represent a new paradigm in the treatment of PCa. The IMPACT trial showed improved survival but no difference in time to disease progression in mCRPC patients with minimal tumour burden. Observations in phase 2 and 3 trials pave the way for other vaccination approaches for this disease, raise questions regarding the most appropriate clinical trial designs, and underscore the importance of identifying biomarkers for antitumour effect to better implement such therapies., (Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

This article clarifies three principles that should guide the development of any cognitive ontology. First, that an adequate cognitive ontology depends essentially on an adequate task ontology; second, that the goal of developing a cognitive ontology is independent of the goal of finding neural implementations of the processes referred to in the ontology; and third, that cognitive ontologies are neutral regarding the metaphysical relationship between cognitive and neural processes., (Copyright © 2011 Cognitive Science Society, Inc.)

Background: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system., Methods: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccine-induced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)-peptide pulsed DCs., Results: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished., Conclusion: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatin-based chemotherapy.

Cellular therapy promises to revolutionize medicine, by restoring tissue and organ function, and combating key disorders including cancer. As with all major developments, new tools must be introduced to allow optimization. For cell therapy, the key tool is in vivo imaging for real time assessment of parameters such as cell localization, numbers and viability. Such data is critical to modulate and tailor the therapy for each patient. In this review, we discuss recent work in the field of imaging cell therapies in the clinic, including preclinical work where clinical examples are not yet available. Clinical trials in which transferred cells were imaged using magnetic resonance imaging (MRI), nuclear scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography (PET) are evaluated from an imaging perspective. Preclinical cell tracking studies that focus on fluorescence and bioluminescence imaging are excluded, as these modalities are generally not applicable to clinical cell tracking. In this review, we assess the advantages and drawbacks of the various imaging techniques available, focusing on immune cells, particularly dendritic cells. Both strategies of prelabeling cells before transplant and the use of an injectable label to target cells in situ are covered. Finally, we discuss future developments, including the emergence of multimodal imaging technology for cell tracking from the preclinical to the clinical realm., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: Chemokine (C-X-C motif) ligand 16 (CXCL16) is secreted by macrophages and dendritic cells (DCs) to attract memory type T cells. CXCL16 expression is increased in arthritic joints of patients with rheumatoid arthritis (RA) and a role for CXCL16 has been suggested in the pathogenesis of RA. To date, little is known about the regulation of CXCL16 on monocytes/macrophages and DCs. The aim of this study was to elucidate how CXCL16 expression is regulated in healthy donors and patients with RA., Methods: CD14+cells were isolated from the peripheral blood or synovial fluid of patients with RA and healthy controls, differentiated into different types of dendritic cells or macrophages and stimulated with various cytokines or lipopolysaccharide (LPS). Cell surface proteins, including surface CXCL16, were measured by flow cytometry and soluble CXCL16 was measured by ELISA., Results: Distinct types of dendritic cells constitutively express and secrete CXCL16, which is not affected by maturation. Monocytes rapidly upregulate membrane-bound CXCL16 expression and release soluble CXCL16 upon culture. CXCL16 expression by monocytes is transiently inhibited by the Toll-like receptor (TLR)4 ligand LPS. Th2 type cytokines inhibit soluble CXCL16, whereas T helper (Th)1 cell stimulus enhances its release. In RA monocytes/macrophages, neither CXCL16 expression, nor CXCL16 regulation is different from healthy controls., Conclusions: Culture of monocytes is the main trigger for CXCL16 surface expression in vitro, which is not altered in RA. Together our data suggest that the increased CXCL16 expression in patients with RA is likely to be caused by increased influx of monocytes rather than intrinsic differences in CXCL16 regulation.

The potential role of antibodies and T lymphocytes in the eradication of cancer has been demonstrated in numerous animal models and clinical trials. In the last decennia new strategies have been developed for the use of tumor-specific T cells and antibodies in cancer therapy. Effective anti-tumor immunotherapy requires the identification of suitable target antigens. The expression of tumor-specific antigens has been extensively studied for most types of adult tumors. Pediatric patients should be excellent candidates for immunotherapy since their immune system is more potent and flexible as compared to that of adults. So far, these patients do not benefit enough from the progresses in cancer immunotherapy, and one of the reasons is the paucity of tumor-specific antigens identified on pediatric tumors. In this review we discuss the current status of cancer immunotherapy in children, focusing on the identification of tumor-specific antigens on pediatric solid tumors.

Recent studies have shown that C-type lectins, a family of surface receptors known to recognize microbial carbohydrate moieties, also sense products from dying cells and transduce inflammatory signals that modulate the immune system.

The dynamics of cell surface membrane proteins plays an important role in cell-cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where optical tweezers are used to trap cells and precisely control in space and time the initiation of interactions between a cell and a functionalized surface. This approach is combined with total internal reflection fluorescence microscopy to monitor dynamics of membrane bound proteins. We demonstrate an accuracy of approximately 2 s in determining the onset of the interaction. Furthermore, we developed a data analysis method to determine the dynamics of cell adhesion and the organization of membrane molecules at the contact area. We demonstrate and validate this approach by studying the dynamics of the green fluorescent protein tagged membrane protein activated leukocyte cell adhesion molecule expressed in K562 cells upon interaction with its ligand CD6 immobilized on a coated substrate. The measured cell spreading is in excellent agreement with existing theoretical models. Active redistribution of activated leukocyte cell adhesion molecule is observed from a clustered to a more homogenous distribution upon contact initiation. This redistribution follows exponential decay behaviour with a characteristic time of 35 s.

We exploited dendritic cells (DC) to vaccinate melanoma patients. We recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine-related tumor antigen-specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, favorable clinical outcome is only observed in a minority of the treated patients. Therefore, it is obvious that current DC-based protocols need to be improved. For this reason, we study in small proof of principle trials the fate, interactions and effectiveness of the injected DC.

Dendritic cells (DC) are the directors of the immune system, capable of inducing tumour antigen-specific T- and B-cell responses. As such, they are currently applied in clinical studies in cancer patients. Early small clinical trials showed promising results, with frequent induction of anti-cancer immune reactivity and clinical responses. In recent years, additional trials have been carried out in melanoma patients, and although immunological responses are often reported, objective clinical responses remain anecdotal with objective response rates not exceeding 5-10%. Thus, DC vaccination research has now entered a stage in between 'proof of principle' and 'proof of efficacy' trials. Crucial questions to answer at this moment are why the clinical responses remain scarce and what can be done to improve the efficacy of vaccination. The answers to these questions probably lie in the preparation and administration of the DC vaccines. Predominantly, cytokine-matured DC are used in clinical studies, while from preclinical studies it is evident that DC that are activated by pathogen-associated molecules are much more potent T cell activators. For sake of easy accessibility monocyte-derived DC are often used, but are these cells also the most potent type of DC? Other yet unsettled issues include the optimal antigen-loading strategy and route of administration. In addition, trials are needed to investigate the value of manipulating tolerizing mechanisms, such as depletion of regulatory T cells or blockade of the inhibitory T cell molecule CTLA-4. These issues need to be addressed in well-designed comparative clinical studies with biological endpoints in order to determine the optimal vaccine characteristics. DC vaccination can then be put to the ultimate test of randomized clinical trials. Here, we review the immunobiology of DC with emphasis on the different aspects that are most relevant for the induction of anti-tumour responses in vivo. The different variables in preparing and administering DC vaccines are discussed in this context and the immunological and clinical results of studies with DC vaccines in melanoma patients are summarized.

Application of tetrameric MHC class I-peptide complexes has significantly improved the monitoring of antigen-specific T cell immune responses in mouse models as well as in clinical studies. Especially MHC class I tetramer analysis of tumor-specific T cells in suspension or on thick vibratome sections from viable tissue has been proven extremely useful. Using the well-characterized mouse tyrosinase-related-protein-2 specific cytotoxic T cell (CTL) clone LP9, we now developed a method that allows for specific identification of T cells with MHC class I tetramers in 8 mum thick, chemically fixed cryosections. The protocol was validated in a murine influenza virus-infection model. Moreover, analysis of delayed type hypersensitivity (DTH) skin biopsies from melanoma patients vaccinated with peptide-loaded mature dendritic cells, revealed the presence and location of anti-tumor CTLs. The specificity of the CTLs detected in situ correlated with both the DTH challenge specificity and reactivity of cell suspensions derived from the same biopsies. Collectively, our data demonstrate that in situ MHC class I tetramer staining provides a valuable tool to reveal the presence and anatomical location of specific CTLs in frozen tissue following immune-based treatment strategies in cancer patients.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.

Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivo-generated, tumour antigen-loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We formerly showed in murine models that radiofrequency-mediated tumour destruction can provide an antigen source for the in vivo induction of anti-tumour immunity, and we explored the role of DC herein. In this paper we evaluate radiofrequency and cryo ablation for their ability to provide an antigen source for DC and compare this with an ex vivo-loaded DC vaccine. The data obtained with model antigens demonstrate that upon tumour destruction by radiofrequency ablation, up to 7% of the total draining lymph node (LN) DC contained antigen, whereas only few DC from the conventional vaccine reached the LN. Interestingly, following cryo ablation the amount of antigen-loaded DC is almost doubled. Analysis of surface markers revealed that both destruction methods were able to induce DC maturation. Finally, we show that in situ tumour ablation can be efficiently combined with immune modulation by anti-CTLA-4 antibodies or regulatory T-cell depletion. These combination treatments protected mice from the outgrowth of tumour challenges, and led to in vivo enhancement of tumour-specific T-cell numbers, which produced more IFN-gamma upon activation. Therefore, in situ tumour destruction in combination with immune modulation creates a unique, 'in situ DC-vaccine' that is readily applicable in the clinic without prior knowledge of tumour antigens.

Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. As such they are currently used in clinical vaccination protocols in cancer patients., Patients and Methods: We evaluated the ability of mature DCs pulsed with carcinoembryonic antigen (CEA)-peptide to induce CEA-specific T cell responses in patients with resectable liver metastases from colorectal cancer. CEA-specific T cell reactivity was monitored in peripheral blood, biopsies of vaccination sites and post-treatment DTH skin tests, and when available also in resected abdominal lymph nodes and tumor tissue., Results: Ten patients were vaccinated intradermally and intravenously with CEA-peptide pulsed mature DCs three times prior to resection of liver metastases. High numbers of CEA-specific T cells were detected in post-treatment DTH biopsies in seven out of 10 patients, which produced high amounts of interferon (IFN)-gamma upon stimulation with CEA-loaded target cells. These responses were not found in biopsies of first vaccination sites, indicating a de novo T cell induction or at least a strong potentiation by the vaccine. In addition, CEA-specific T cells were detected in a resected lymph node in one patient, but not in peripheral blood or tumor tissue., Conclusions: Vaccination with CEA-peptide loaded mature DCs induced potent CEA-specific T cell responses in advanced colorectal cancer patients. In this study, antigen-specific T cell responses were readily detected in DTH skin tests, much less in abdominal lymph nodes, and not in peripheral blood and tumor tissue.

The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.

Background: Dendritic cells (DC) have a role in the regulation of immunity and tolerance, attracting inflammatory cells by the production of various chemokines (CK). Fc gamma receptors (Fc gamma R) may be involved in regulation of the DC function., Objective: To assess the expression of CK by immature (iDC) and mature DC (mDC) and its regulation by Fc gamma R in patients with RA and healthy donors (HC)., Methods: Expression of CK by DC from patients with RA and from HC was determined by real time quantitative PCR and ELISA. DC were derived from monocytes following standardised protocols. To study the potential regulation by Fc gamma R, iDC were stimulated with immune complexes (IC) during lipopolysaccharide (LPS) induced maturation. The presence of CK was studied in synovial tissue from patients with RA, osteoarthritis, and healthy subjects by RT-PCR and immunohistochemistry., Results: iDC from patients with RA had markedly increased mRNA levels of the CK CCL18 and CXCL8. Upon maturation with LPS, expression of CCL18, CCL19, CXCL8, CCL3, and CCL17 increased dramatically, reaching significantly higher levels in patients with RA. Monocytes failed to express these CK, except for CXCL8 and CCL3. IC-mediated triggering of the Fc gamma R on DC from patients with highly active RA down regulated all CK, whereas the reverse was seen when DC from patients with low disease activity and healthy donors were stimulated. CCL18 was significantly increased in RA synovial tissue., Conclusion: Increased CK expression by DC was found in patients with RA. This expression is partly regulated by Fc gamma R triggering and results in an inhibitory DC subtype in RA upon Fc gamma R-mediated triggering.

Objectives: To investigate potential differences in phenotype and behaviour of immature (iDC) and mature dendritic cells (mDC) from patients with RA and healthy subjects., Methods: iDC and mDC were derived from blood monocytes of patients with RA and healthy controls following standardised protocols. FACS was used to analyse expression of FcgammaRI, II, and III and molecules to characterise DC. Discrimination between FcgammaRIIa and FcgammaRIIb was achieved by RT-PCR. Immunohistochemistry was performed on synovial biopsy specimens of three patients with RA and three healthy controls. TNFalpha production by iDC and mDC upon FcgammaR dependent stimulation was compared between patients with RA and controls by ELISA., Results: iDC from patients with active RA but not from patients with inactive RA or healthy controls markedly up regulated FcgammaRII. mDC from patients with active RA also lacked the physiological down regulation of FcgammaRII that occurs upon maturation in both control groups. RT-PCR analysis confirmed the increased expression of FcgammaRII in RA-especially marked for FcgammaRIIb. FcgammaR dependent stimulation of DC using antigen-IgG immune complexes (IC) significantly increased TNFalpha production by DC from healthy subjects, but significantly decreased TNFalpha by DC from patients with RA. Overlapping expression patterns between FcgammaRII and DC-LAMP in the synovial tissue of patients with RA imply that in vivo, also, mature DC express increased levels of FcgammaRIIb., Conclusion: The presence and altered characteristics of DC during active RA suggest that DC help to modulate autoimmunity in RA. Further studies should elucidate the role of local factors in altering the function of DC in RA and in increasing expression of FcgammaRII.

Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon gamma (IFN-gamma) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-gamma secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses.

Dendritic cells (DC) function as professional phagocytes to kill Candida albicans and subsequently present it to the adaptive immune system. Monocytes, macrophages and DC were generated from five individual donors and their Candida-killing capacity and cytokine release were assessed. Compared to monocytes and macrophages, DC from healthy volunteers were significantly less effective in C. albicans--stimulated cytokine release, killing of C. albicans blastoconidia and damaging of C. albicans hyphae. In conclusion, while important as antigen-presenting cells and initiators of the adaptive immune system, DC are poor in both intracellular killing and damaging of C. albicans hyphae. Effective handling of large numbers of C. albicans is the prime task of the innate immune system consisting of large numbers of neutrophils and monocytes.

Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

DC-based immunotherapy has shown early promise in clinical cancer trials, and efforts are being made to determine the optimal method of delivery of cells to achieve the best outcome. While it is accepted that mature DCs are required for stimulation of tumor-specific immunity, the route and frequency of injection and the optimal number of cells needed for clinical success are issues that are still being debated. To solve these questions controlled clinical trials are needed.

Dendritic Cells (DC) are natural adjuvants able to elicit specific cellular interactions and priming of naive T cells at a mature stage of their differentiation. Recent genomic approaches helped defining DC or Langherans Cells (LC) in more molecular terms. DC-SIGN, the DC specific ICAM-3 grabbing non integrin is a C-type lectin, absent on LC but expressed on dermal, lymph node and tonsils DC. DC-SIGN is defined as an ICAM-3 receptor supporting DC mediated-T cell proliferation. Moreover, DC-SIGN plays an important role in binding and presentation of HIV virions, because DC-SIGN specifically binds the gp120 coat protein of HIV.DC-SIGN also plays a part in DC trafficking since it not only binds ICAM-3 but also ICAM-2 expressed by many endothelial cells, supporting tethering and rolling of DC on endothelium and chemokine induced-transmigration of DC across both resting and activated endothelium in vitro. ALCAM (Activated Leukocyte Cell Adhesion Molecule) is another cell surface protein expressed by DC upon differentiation from monocytes. ALCAM appears to be expressed on activated leukocytes and might be involved in inflammatory processes. ALCAM belongs to the IgG superfamily of proteins and mediate heterotypic (T cell antigen ligand CD6) or homotypic interactions. ALCAM is linked to the cytoskeleton and might play a role in DC migration. Measurements of cell/cell contacts at single molecular levels using optical traps is a useful tool to investigate intercellular interactions.

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.

A virus uses surface molecules to enter the cell and subsequently multiplies using the cell's machinery. Vaccination induces the whole immunological capability to get rid of the virus. However, viruses found several ways to escape from immunological elimination. Blocking viral entry is another way to prevent viral spread. Here, we describe a method to block HIV entry by inhibiting the association of the two viral proteins that are involved in viral entry., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

CD83 is an immunoglobulin (Ig) superfamily member that is upregulated during the maturation of dendritic cells (DCs). It has been widely used as a marker for mature DCs, but its function is still unknown. To approach its potential functional role, we have expressed the extracellular Ig domain of human CD83 (hCD83ext) as a soluble protein. Using this tool we could show that immature as well as mature DCs bind to CD83. Since CD83 binds a ligand also expressed on immature DCs, which do not express CD83, indicates that binding is not a homophilic interaction. In addition we demonstrate that hCD83ext interferes with DC maturation downmodulating the expression of CD80 and CD83, while no phenotypical effects were observed on T cells. Finally, we show that hCD83ext inhibits DC-dependent allogeneic and peptide-specific T cell proliferation in a concentration dependent manner in vitro. This is the first report regarding functional aspects of CD83 and the binding of CD83 to DCs.

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution--approximately 250 nm--dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the release of large amounts of heme. Recently, it was demonstrated that heme oxygenase (HO), the heme-degrading enzyme, and heme are able to modulate adhesion molecule expression in vitro. In the present study, the effects of heme and HO on inflammation in mice were analyzed by monitoring the biodistribution of radiolabeled liposomes and leukocytes in conjunction with immunohistochemistry. Small liposomes accumulate in inflamed tissues by diffusion because of locally enhanced vascular permeability, whereas leukocytes actively migrate into inflammatory areas through specific adhesive interactions with the endothelium and chemotaxis. Exposure to heme resulted in a dramatic increase in liposome accumulation in the pancreas, but also intestines, liver, and spleen exhibited significantly increased vascular permeability. Similarly, intravenously administered heme caused an enhanced influx of radiolabeled leukocytes into these organs. Immunohistochemical analysis showed differential up-regulation of the adhesion molecules ICAM-1, P-selectin, and fibronectin in liver and pancreas in heme-treated animals. Heme-induced adhesive properties were accompanied by a massive influx of granulocytes into these inflamed tissues, suggesting an important contribution to the pathogenesis of inflammatory processes. Moreover, inhibition of HO activity exacerbated heme-induced granulocyte infiltration. Here it is demonstrated for the first time that heme induces increased vascular permeability, adhesion molecule expression, and leukocyte recruitment in vivo, whereas HO antagonizes heme-induced inflammation possibly through the down-modulation of adhesion molecules.

Although endothelial cell seeding in small-diameter vascular prostheses significantly improves graft survival, the detachment of adherent endothelial cells after the restoration of circulation remains one of the major obstacles. Because in vivo experiments indicate that leukocyte infiltration is involved in endothelial cell loss, we hypothesize that seeded endothelial cells become activated and express leukocyte adhesion molecules and cytokines because of an interaction with the underlying polymer surface. The aim of this study was to investigate the expression of the leukocyte adhesion molecules ICAM-1, VCAM-1, PECAM-1, and E-selectin by cultured human umbilical vein endothelial cells (HUVECs) and human adipose microvascular endothelial cells (HAMVECs). The cells were seeded on tissue culture poly(styrene) and the vascular graft materials Dacron and Teflon. The results of this study indicate that the expression of leukocyte adhesion molecules by cultured endothelial cells is mainly affected by the endothelial cell origin, that is, umbilical vein or adipose tissue. Expressions of both ICAM-1 and E-selectin by HUVECs and HAMVECs are characterized by the presence of two cell populations with distinct levels of expression. With respect to endothelial cell seeding in vascular prostheses, the increased expression of E-selectin by microvascular endothelial cells deserves further attention., (Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 376-381, 2001)

The intercellular adhesion molecules (ICAMs) play a prominent role in regulating the migration and activation of both dendritic cells (DCs) and T lymphocytes in the immune system. Recent observations have demonstrated that both leukocyte function-associated molecule 1 (LFA-1) and DC-specific ICAM-grabbing nonintegrin (DC-SIGN), two structurally unrelated adhesion receptors, regulate the function of leukocytes and DCs by binding to the same ICAMs. Here, we focus on the structure-function relationships of DC-SIGN and LFA-1 to obtain an insight into their role in the migration and activation of DCs and T cells in the control of immunity.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.

Dendritic cells (DC) attract both T and B lymphocytes to induce an efficient antigen-specific immune response. Recently, it was shown that naïve T cells are attracted to DC by dendritic cell chemokine 1 (DC-CK1, CCL18). The potent B lymphocyte chemoattractant BLC (CXCL13) was previously shown to be essential for homing of lymphocytes into secondary lymphoid organs and for the development of B cell follicles. As the cells that produce BLC are largely unknown and BLC could be a candidate chemokine for the recruitment of B cells to DC, we analyzed different DC subsets for expression of BLC. Here we demonstrate that monocyte-derived DC as well as activated blood DC indeed express and secrete BLC. Interestingly, ligation of the CD40 molecule down-regulated BLC expression in monocyte-derived DC. Staining of tonsilar sections indicated that BLC is expressed by follicular dendritic cells and germinal center dendritic cells (GCDC) in vivo. Real-time quantitative PCR confirmed the expression of BLC in isolated GCDC. Since both B cells and activated T cells express the receptor for BLC, our findings implicate an important role for BLC in establishing the interaction of DC with T cells and B cells. Furthermore, CD40/CD40 ligand interactions could modulate this process by down-regulating the expression of BLC.

Upon maturation, dendritic cells (DCs) have to adjust their chemokine expression to sequentially attract different leukocyte subsets. We used real-time quantitative polymerase chain reaction analysis to study in detail the expression of 12 chemokines involved in the recruitment of leukocytes into and inside secondary lymphoid organs, by DCs in distinct differentiation stages, both in vitro and in vivo. Monocyte-derived immature DCs expressed high levels of DC chemokine 1 (DC-CK1), EBI1-ligand chemokine (ELC), macrophage-derived chemokine (MDC), macrophage-inflammatory protein (MIP)-1alpha, and thymus and activation-regulated chemokine (TARC). Upon maturation, DCs up-regulated the expression of DC-CK1 (60-fold), ELC (7-fold), and TARC (10-fold). Activation of DCs by CD40 ligand further up-regulated the expression of ELC (25-fold). We found that freshly isolated blood DCs expressed only low levels of interleukin-8, lymphotactin, and MIP-1alpha. It is interesting that the chemokine profile expressed by activated CD11c(-) lymphoid-like as well as CD11c(+) myeloid blood DCs mimics that of monocyte-derived DCS: Additionally, purified Langerhans cells that had migrated out of the epidermis expressed a similar chemokine pattern. These data indicate that different DC subsets in vitro and in vivo can express the same chemokines to attract leukocytes.

Cytotoxic T lymphocytes (CTL) are thought to play an important role in the graft-versus-leukemia (GVL) response. Unfortunately, GVL reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Characterization of CTL that selectively attack leukemic cells but not normal cells may lead to the development of adjuvant immunotherapy that separates GVL from GVHD. Here, we describe TCR gamma delta (V gamma 9/V delta 1) CTL, isolated from the peripheral blood of an AML patient after stem cell transplantation (SCT), that very efficiently lysed freshly isolated acute myeloid leukemia (AML) cells and AML cell lines. Interestingly, HLA-matched non-malignant hematopoietic cells were not killed. We revealed that the killer cell-inhibitory receptor (KIR) p58.2 (CD158b) specific for group 2 HLA-C molecules negatively regulates the cytotoxic effector function displayed by these TCR gamma delta CTL. First, an antibody against HLA-C enhances lysis of non-malignant cells. Secondly, stable transfection of HLA-Cw*0304 into the class I-negative cell line 721.221 inhibited lysis. Finally, engagement of p58.2 by antibodies immobilized on Fc gamma R-expressing murine P815 cells inhibits CD3- and TCR gamma delta-directed lysis. Compared to non-malignant hematopoietic cells, AML cells express much lower levels of MHC class I molecules making them susceptible to lysis by p58.2(+) TCR gamma delta CTL. Such KIR-regulated CTL reactivity may have a role in the GVL response without affecting normal tissues of the host and leading to GVHD.

Dendritic cells (DC) capture micro-organisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present in antigenic form to resting T cells and thus initiate adaptive immune responses. Here we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC, but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. The interaction of DC-SIGN with HIV gp120 may be an important target for therapeutic intervention and vaccine development., (Copyright 2001 IFPA and Harcourt Publishers Ltd.)

The leukocyte-specific beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)/beta(2)) mediates activation-dependent adhesion to intercellular adhesion molecule (ICAM)-1. In leukocytes, LFA-1 requires activation by intracellular messengers to bind ICAM-1. We observed malfunctioning of LFA-1 activation in leukemic T cells and K562-transfected cells. This defective inside-out integrin activation is only restricted to beta(2) integrins, since beta(1) integrins expressed in K562 readily respond to activation signals, such as phorbol 12-myristate 13-acetate. To unravel these differences in inside-out signaling between beta(1) and beta(2) integrins, we searched for amino acids in the beta(2) cytoplasmic domain that are critical in the activation of LFA-1. We provide evidence that substitution of a single amino acid (L732R) in the beta(2) cytoplasmic DLRE motif, creating the DRRE motif, is sufficient to completely restore PMA responsiveness of LFA-1 expressed in K562. In addition, an intact TTT motif in the C-terminal domain is necessary for the acquired PMA responsiveness. We observed that restoration of the PMA response altered neither LFA-1 affinity nor the phosphorylation status of LFA-1. In contrast, strong differences were observed in the capacity of LFA-1 to form clusters, which indicates that inside-out activation of LFA-1 strongly depends on cytoskeletal induced receptor reorganization that was induced by activation of the Ca(2+)-dependent protease calpain.