An Experimental Study Comparing the Expansion of Peripheral Blood Natural Killer (NK) Cells Cultured with Artificial Antigen-Presenting Cells, in the Presence or Absence of Bone Marrow Mesenchymal Stem Cells (MSCs).

NK cells have been seen as potential agents in adoptive immunotherapy for cancer. The main challenge for the success of this approach is to obtain a great quantity of activated NK cells for adoptive transfer. The present study had aimed to evaluate the effect of a feeder layer of irradiated MSCs in the in vitro expansion of NK cells. MSCs were obtained from the bone marrow (BM) cells remaining in the bag and filter used in the transplantation of hematopoietic stem cells. NK cells were obtained from peripheral blood (PB) of healthy volunteers. NK expansion and activation were stimulated by culture with artificial antigen-presenting cells (aAPCs) and IL-2, in the presence or absence of BM-MSCs. NK cell proliferation, phenotypic expression and cytotoxic activity were evaluated. Both culture conditions showed high NK purity with predominance of NK CD56brightCD16+ subset post expansion. However, cultures without the presence of MSCs showed higher NK proliferation, expression of activation markers (CD16 and NKG2D) and related cytotoxic activity. In this experimental study, the presence of a feeder layer of irradiated BM-MSCs interfered negatively in the expansion of PB-NKs, limiting their growth and activation. Further investigation is needed to understand the mechanisms of NK-MSC interaction and its implications.

[Regulation of immune responses by exosomes derived from antigen presenting cells]. / Los exosomas de las células presentadoras de antígeno y su papel en la regulación de las respuestas inmunológicas.

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Las células liberan biomoléculas de diversa naturaleza a su entorno para comunicarse con las células vecinas. Además de dichas moléculas, secretan también elementos más complejos como las vesículas; estructuras compuestas por bicapas lipídicas con proteínas transmembranales que encierran un contenido hidrofílico. Los exosomas son un subtipo pequeño de estas vesículas (de 30 a 150 nm), producidos por una amplia variedad de tipos celulares incluyendo las neuronas, células tumorales, células epiteliales y células del sistema inmunológico. De entre estas últimas, las células presentadoras de antígeno se han caracterizado como productoras de exosomas con contenido variable, tanto en condiciones de reposo como en aquellas que derivan de su estimulación o maduración. En los últimos años, el estudio de los exosomas ha aumentado debido a que se ha demostrado que dichas vesículas poseen propiedades inmunomoduladoras, razón por la que ostentan un gran potencial en aplicaciones de diagnóstico y desarrollo de terapias en diferentes patologías con componentes inflamatorios.

A rapid alternative method to evaluate T-cell hybridoma activation using an improved cytokine (IL-2) secretion assay.

T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of detectable CD4+IL-2+ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly (~18h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay (~48h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and evaluating their antigen-specific activation.

Characterization of antigen-presenting cells from the porcine respiratory system.

Antigen-presenting cells (APCs) are strategically placed in all anatomic sites with high antigen exposure such as the respiratory system. The aim of this study was to evaluate phenotypic and functional properties of APCs from the lung (L-Cs), mediastinal lymph node (LN-Cs) and bronchoalveolar lavage cells (BAL-Cs). The APCs were first analyzed based on forward scatter and side scatter profiles and the selection of MHC-II(high)CD172a(+) cells (referred to as APCs); then the expression of CD1a, CD163, CD206, CD16 and CD11R3 was evaluated in the APCs. The results showed that CD1a, CD163 and CD206 were differentially expressed among L-Cs, LN-Cs and BAL-Cs, suggesting the phenotype MHC-II(high)CD172a(+)CD1a(low/-)CD163(low)CD206(-) for L-Cs and MHC-II(high)CD172a(+)CD1a(+)CD163(low/-)CD206(+) for LN-Cs. BAL-Cs were MHC-II(high)CD172a(+)CD1a(-)CD163(high)CD206(+/-). The functional characteristics of L-Cs and LN-Cs were different from those of BAL-Cs, confirming that L-Cs and LN-Cs resemble specialized APCs. In conclusion, we present the characterization of APCs from L-Cs, LN-Cs and BAL-Cs of the porcine respiratory system.

Statins as modulators of regulatory T-cell biology.

Statins are pharmacological inhibitors of the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), an enzyme responsible for the synthesis of cholesterol. Some recent experimental studies have shown that besides their effects on the primary and secondary prevention of cardiovascular diseases, statins may also have beneficial anti-inflammatory effects through diverse mechanisms. On the other hand, the induction and activity of regulatory T cells (Treg) are key processes in the prevention of pathology during chronic inflammatory and autoimmune diseases. Hence, strategies oriented towards the therapeutic expansion of Tregs are gaining special attention among biomedical researchers. The potential effects of statins on the biology of Treg are of particular importance because of their eventual application as in vivo inducers of Treg in the treatment of multiple conditions. In this paper we review the experimental evidence pointing out to a potential effect of statins on the role of regulatory T cells in different conditions and discuss its potential clinical significance.

Impact of photodynamic therapy on inflammatory cells during human chronic periodontitis.

The aim of this study was to evaluate the effects of the photodynamic therapy (PDT) on the inflammatory infiltrate and on the collagen network organization in human advanced chronic periodontitis. Two different drug delivery systems (DDS) were tested (liposomes and nanoemulsions) to determine if the effects of PDT could differ according to the DDS used. Sixteen patients presenting two teeth with chronic advanced periodontitis and important tooth mobility with clinical indication of extraction were included in the group liposomes (group L, n=8) or in the group nanoemulsions (group N, n=8) in order to compare the effects of each DDS. Seven days before extractions one tooth of each patient was treated with PDT using phthalocyanine derivatives as photosensitizers and the contralateral tooth was taken as control. In group L the density of gingival collagen fibers (66±19%) was significantly increased (p<0.02) when compared to controls (35±21%). Concerning the antigen-presenting cells, PDT had differential effects depending on the drug delivery system; the number of macrophages was significantly decreased (p<0.05) in group L while the number of Langerhans cells was significantly decreased in group N (p<0.02). These findings demonstrate that PDT presents an impact on gingival inflammatory phenomenon during chronic periodontitis and leads to a specific decrease of antigen-presenting cells populations according to the drug delivery system used.

Dendritic cells and B cells cooperate in the generation of CD4(+)CD25(+)FOXP3(+) allogeneic T cells.

BACKGROUND: CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an essential role in immune tolerance, suppressing responses against self-antigens. Additionally, Treg play an important role in maintaining immunosuppression to alloantigens as well as to other antigens. It is well known that in the gut, a subset of dendritic cells produces retinoic acid (RA), which together with transforming growth factor (TGF-beta) is able to differentiate naïve T cells into Treg. The aim of this study was to establish the role of antigen-presenting cells (APC) in the differentiation of allogeneic Tregs under the effect of RA and TGF-beta. METHODS: Splenic CD4(+)CD25(-) naïve T cells from C57BL/6 mice were co-cultured with splenic CD11c-enriched APC from Balb/c mice in the presence of TGF-beta, RA, and interleukin (IL-2). After 6 days of culture, cells were analyzed for the expression of Foxp3 by flow cytometry. Additionally, we investigated the role of B cells and dendritic cells (DCs) and their stimulatory capacity in the generation of Tregs. RESULTS: Our results showed that co-culture of naive T cells with the appropriate level of stimulation by APC in the presence of TGF-beta, RA, and IL-2 provided a new powerful approach to generate allogeneic Treg cells. We demonstrated that although B cells and DCs can generate Tregs by themselves, a mixure of both APC improved their capacity to efficiently generate Tregs. Also, we observed that although the addition of IL-2 to the cultures was not crucial to generate Tregs, it was required to optimize their expansion and cell survival.

T-cell antagonism by short half-life pMHC ligands can be mediated by an efficient trapping of T-cell polarization toward the APC.

T-cell activation results from productive T-cell receptor (TCR) engagement by a cognate peptide-MHC (pMHC) complex on the antigen presenting cell (APC) surface, a process leading to the polarization of the T-cell secretory machinery toward the APC interface. We have previously shown that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters determining T-cell activation. However, whether the half-life of the TCR/pMHC interaction can modulate the efficiency of T-cell secretory machinery polarization toward an APC still remains unclear. Here, by using altered peptide ligands conferring different half-lives to the TCR/pMHC interaction, we have tested how this parameter can control T-cell polarization. We observed that only TCR/pMHC interactions with intermediate half-lives can promote the assembly of synapses that lead to T-cell activation. Strikingly, intermediate half-life interactions can be competed out by short half-life interactions, which can efficiently promote T-cell polarization and antagonize T-cell activation that was induced by activating intermediate half-life interactions. However, short TCR/pMHC interactions fail at promoting phosphorylation of signaling molecules at the T-cell-APC contact interface, which are needed for T-cell activation. Our data suggest that although intermediate half-life pMHC ligands promote assembly of activating synapses, this process can be inhibited by short half-life antagonistic pMHC ligands, which promote the assembly of non activating synapses.

Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes.

Antigen presenting cells (APC) are among the most important cells of the immune system since they link the innate and the adaptative immune responses, directing the type of immune response to be elicited. To modulate the immune response in immune preventing or treating therapies, gene delivery into immunocompetent cells could be used. However, APC are very resistant to transfection. To increase the efficiency of APC transfection, we have used liposome-based lipoplexes additionally modified with cell-penetrating TAT peptide (TATp) for better intracellular delivery of a model plasmid encoding for the enhanced-green fluorescent protein (pEGFP). pEGFP-bearing lipoplexes made of a mixture of PC:Chol:DOTAP (60:30:10 molar ratio) with the addition of 2% mol of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate (plain-L) or TATp-PEG-PE (TATp-L) were shown to effectively protect the incorporated DNA from degradation. Uptake assays of rhodamine-labeled lipoplexes and transfections with the EGFP reporter gene were performed with APC derived from the mouse spleen. TATp-L-based lipoplexes allowed for significantly enhanced both, the uptake and transfection in APC. Such a tool could be used for the APC transfection as a first step in immune therapy.

A pilot study with a therapeutic vaccine based on hydroxyapatite ceramic particles and self-antigens in cancer patients.

We describe an approach to produce an autologous therapeutic antitumor vaccine using hydroxyapatite (HA) for vaccinating cancer patients. The novel approach involved (1) the purification of part of the self-tumor antigens/ adjuvants using column chromatography with HA, (2) the employ of HA as a medium to attract antigen-presenting cells (APCs) to the vaccination site, and (3) the use of HA as a vector to present in vivo the tumor antigens and adjuvants to the patient's APCs. The vaccine was prepared using and combining HA particles, with at least 3 heat shock proteins (gp96 was one of them possibly with chaperoned proteins/peptides as shown in the slot blots) and with proteins from the cell membrane system (including Hsp70, Hsp27, and membrane proteins). The timing of HA degradation was tested in rats; the HA particles administered under the skin attracted macrophages and were degraded into smaller particles, and they were totally phagocytized within 1 week. In patients (n = 20), the vaccine was then administered weekly and showed very low toxicity, causing minor and tolerable local inflammation (erythema, papule, or local pain); only 1 patient who received a larger dose presented hot flashes, and there were no systemic manifestations of toxicity or autoimmune diseases attributed to the vaccine. Our study suggests that this therapeutic vaccine has shown some efficacy producing a positive response in certain patients. Stable disease was noted in 25% of the patients (renal carcinoma, breast carcinoma, and astrocytoma), and a partial response was noted in 15% of the patients (breast carcinoma and astrocytoma). The most encouraging results were seen in patients with recurrent disease; 4 patients in these conditions (20%) are disease free following the vaccine administration. However, we do not want to overstate the clinical efficacy in this small number of patients. The therapeutic vaccine tested in our study is working by activating the T-cell response as was shown in the comparative histological and immunohistochemical study performed in the pre- and postvaccine biopsy taken from a patient with inflammatory breast carcinoma. However, we cannot ruled out that the vaccine could also be producing an antibody(ies)-mediated response. In conclusion, this therapeutic vaccine based on HA ceramic particles and self-antigens can be safely administered and is showing some encouraging clinical results in cancer patients.

Regulation of T helper cell differentiation in vivo by GP43 from Paracoccidioides brasiliensis provided by different antigen-presenting cells.

Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen-presenting cells (APCs) can influence the Th1/Th2 balance in vivo. It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro. It was seen that DCs from resistant mice stimulated predominantly interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas macrophages activated IL-10, IL-4 and IFN-gamma-secreting T cells and B cells IL-4 and IL-10 only. Results presented here clearly demonstrate that DC drives the development of cells secreting Th1-derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.