Specific transcriptional programs differentiate ICOS from CD28 costimulatory signaling in human Naïve CD4+ T cells.

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.

Characterization of M1 and M2 polarization phenotypes in peritoneal macrophages after treatment with graphene oxide nanosheets.

Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes indentified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO+ and GO- cells, respectively). The CD80 expression diminished in GO+ macrophages after 48 h of GO treatment but the CD206 expression in GO+ population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-γ), a significant decrease of CD80+ cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206+ and CD163+ cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO- and GO+ cells than in control macrophages. However, higher CD163 expression was obtained in both GO- and GO+ cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets.

Graphene oxide nanosheets increase Candida albicans killing by pro-inflammatory and reparative peritoneal macrophages.

Graphene oxide (GO) is a new nanomaterial with different potential biomedical applications due to its excellent physicochemical properties and ease of surface functionalization. Macrophages play key roles in the control of fungal infections preventing invasive candidiasis by both limiting the growth of the opportunistic fungal pathogen Candida albicans and activating other immune effector cells. In order to know if macrophages maintain their immunocompetence against this microorganism after GO uptake, we have evaluated the interactions at the interface of GO nanosheets, macrophages and Candida albicans. Poly (ethylene glycol-amine)-derivatized GO nanosheets labelled with fluorescein isothiocyanate (FITC-PEG-GO), were efficiently taken up by peritoneal macrophages inducing a significant increase of C. albicans phagocytosis by both pro-inflammatory macrophages (M1/stimulated with LPS/IFN-γ) and reparative macrophages (M2/stimulated with IL-4). On the other hand, after FITC-PEG-GO treatment and C. albicans infection, the percentages of GO+ macrophages diminished when Candida uptake increased in every condition (macrophages with no stimuli, M1 and M2 macrophages), thus suggesting the exocytosis of this nanomaterial as a dynamic mechanism favoring fungal phagocytosis. For the first time, we have analyzed the effects of PEG-GO nanosheets on Candida albicans killing by unstimulated, M1 and M2 macrophages, evidencing that intracellular GO modulates the macrophage candidacidal activity in a multiplicity of infection (MOI) dependent manner. At MOI 1, the high intracellular GO levels increase the fungicidal activity of basal and stimulated macrophages. At MOI 5, as intracellular GO decreases, the previous pro-inflammatory or reparative stimulus predefines the killing ability of macrophages. In summary, GO treatment enhances classical M1 macrophage activation, important for pathogen eradication, and diminishes alternative activation of M2 macrophages, thus decreasing fungal persistence and avoiding chronic infectious diseases.

B7h triggering inhibits the migration of tumor cell lines.

Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of ß-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2Rγnull mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h-ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10-metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response.

Class I phosphoinositide 3-kinases in immunity: Effect of inhibitors in immune and autoimmune reactions / Las fosfatidilinositol 3-cinasas de clase I en la inmunidad: Efecto de inhibidores en respuestas inmunes y autoinmunes

Las fosfatidilinositol 3-cinasas (PI3K) de clase I dan lugar a fosfolípidos trifosforilados (PtdIns (3,4,5)P3) que son clave en las señales de crecimiento, diferenciación y la supervivencia de las células y son esenciales para el funcionamiento de la inmunidad innata y adaptativa. Los nuevos inhibidores de PI3K generados para el tratamiento de tumores pueden ser útiles en inmunoterapia, especialmente en enfermedades autoinmunes, y ha de investigarse su impacto en la inmunidad anti tumoral. Se revisa el papel de las PI3K de clase I en las respuestas inmunes adaptativas, y los datos conocidos relativos al efecto de inhibidores en respuestas inmunes adaptativas

Class I phosphoinositide-3 kinases (PI3Ks) generate PtdIns (3,4,5)P3 to activate cell signaling cascades essential to cell growth, differentiation and survival, and are essential to the function of innate and adaptive immunity. The generation of a vast array of newly developed PI3K inhibitors to treat cancer poses the question of their use in the modulation of pathological immune reactions like autoimmune diseases, or the effect of these drugs in the anti-tumor immune reactions. Here, the role of PI3K in adaptive immune reactions and data concerning the use of inhibitors to control immune responses are reviewed

Early in vitro response of macrophages and T lymphocytes to nanocrystalline hydroxyapatites.

HYPOTHESIS: Synthetic hydroxyapatite (HA) and Si substituted hydroxyapatite (SiHA) are calcium phosphate ceramics currently used in the field of dentistry and orthopaedic surgery. The preparation of both biomaterials as polycrystalline solid pieces or grains formed by nanocrystallites has awakened a great interest to enhance the bioactive behavior due to the microstructural defects and the higher surface area. The study of the macrophage and lymphocyte behavior in contact with nanocrystalline HA and SiHA will allow to elucidate the immune response which conditions the success or rejection of these biomaterials. EXPERIMENTS: HA and SiHA granules (with sizes of tens of microns) have been prepared by controlled aqueous precipitation avoiding subsequent high temperature sintering. HA and SiHA granules were constituted by crystallites smaller than 50 nm. The effects of both nanocrystalline materials on immune system have been evaluated with macrophages (main components of innate immune system) and T lymphocytes (specific cells of adaptive response) after short-term culture as in vitro models of the early immune response. FINDINGS: Significant decreases of macrophage proliferation and phagocytic activity, increased production of inflammatory cytokines (IL-6, TNF-α) and T lymphocyte apoptosis, were induced by these nanocrystalline ceramics suggesting that, after in vivo implantation, they induce significant effects on immune responses, including an early activation of the innate immune system.

Triggering of B7h by the ICOS modulates maturation and migration of monocyte-derived dendritic cells.

B7h, expressed by several cell types, binds ICOS expressed by activated T cells. We have previously shown that B7h triggering by ICOS-Fc inhibits human endothelial cell adhesiveness. This work investigated the effect of ICOS-Fc on human monocyte-derived dendritic cells (DCs). We found that DCs matured with LPS in the presence of ICOS-Fc (mDCs(ICOS)) produced greater amounts of IL-23 and IL-10, and promoted a higher secretion of IL-17A and IL-17F in MLCs than did those DCs matured with LPS alone (mDCs). Moreover, mDCs(ICOS) pulsed with the keyhole limpet hemocyanin Ag during the maturation phase were better stimulators of Ag-specific MHC class I-, but not class II-restricted T cells than mDCs. This was probably due to promotion of cross-presentation because it was not detected when the Flu-MA(58-66) Ag was directly loaded on already matured DCs and mDCs(ICOS). Finally, ICOS-Fc inhibited the adhesion of both immature DCs and mDCs to vascular and lymphoid endothelial cells, their migratory activity, and the expression of the Rac-1 activator ß-Pix involved in cell motility. These data suggest that B7h stimulation modulates DC function with effects on their maturation and recruitment into tissues. This opens a novel view on the use of interactors of the ICOS:B7h system as immunomodulatory drugs.

B7h triggering inhibits umbilical vascular endothelial cell adhesiveness to tumor cell lines and polymorphonuclear cells.

Vascular endothelial cells (ECs) are key players in leukocyte recruitment into tissues and metastatic dissemination of tumor cells. ECs express B7h, which is the ligand of the ICOS T cell costimulatory molecule. The aim of this work was to assess the effect of B7h triggering by a soluble form of ICOS (ICOS-Fc) on the adhesion of colon carcinoma cell lines to HUVECs. We found that B7h triggering inhibited HUVEC adhesiveness to HT29 and DLD1 cells (by 50 and 35%, respectively) but not to HCT116 cells. The effect was dependent on the ICOS-Fc dose and was detectable as early as 30 min after treatment and was still present after 24 h. It was inhibited by soluble anti-ICOS reagents (mAb and B7h-Fc) and silencing of B7h on HUVECs, and it was not displayed by an F119S mutated form of ICOS-Fc that does not bind B7h. HUVEC treatment with ICOS-Fc did not modulate expression of adhesion molecules and cytokines, but it substantially downmodulated ERK phosphorylation induced by E-selectin triggering or osteopontin, which may influence HUVEC adhesiveness. Moreover, HUVEC treatment with ICOS-Fc also inhibited adhesion of polymorphonuclear cells and several tumor cell lines from different origins. Therefore, the B7h-ICOS interaction may modulate spreading of cancer metastases and recruitment of polymorphonuclear cells in inflammatory sites, which opens a view on the use of ICOS-Fc as an immunomodulatory drug.

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El 35 Aniversario de la Sociedad Española de Inmunología(29 de Abril, Día Global de la Inmunología) / The 35th anniversary of the spanish society of immunology. On the occassion of april 29th, world immunology day

El pasado día 13 de Diciembre se cumplieron treinta y cinco años de la Reunión Científica celebrada en 1975, en la sede del Colegio de Médicos de Barcelona, que sería el acto fundacional de la Sociedad Española de Inmunología y su Primer Congreso. En este escrito, al hilo de los recuerdos personales de diversas personas que asistieron a aquel acto, y también de la comparación de los contenidos de ese Primer Congreso con los del 35 Congresocelebrado en Palma de Mallorca en 2008, se realizan diversas consideraciones sobre el desarrollo de la Inmunología en España en estos treinta y cinco años (AU)

LLast December 13th was the 35th anniversary of the celebration in1975, in the facilities of the Medical College of Barcelona, of a ScientificMeeting which would become the foundational act of the Spanish Societyof Immunology and its First Congress. Here, we present a few reflectionson the development of Immunology in Spain during this time, throughthe personal memories of some of the people attending that meeting, andcomparing the contents of the 1st Congress with those of the 35th Congress held in Palma de Mallorca in 2008 (AU)

ICOS gene haplotypes correlate with IL10 secretion and multiple sclerosis evolution.

Human ICOS is a T cell costimulatory molecule supporting IL10 secretion. A pilot study investigating variations of the ICOS gene 3'UTR detected 8 polymorphisms forming three haplotypes (A, B, C). Haplotype-A and -C displayed the highest difference. Activated T cells from healthy AA homozygotes expressed significantly less ICOS and secreted more IL10 than AC heterozygotes, whereas AB heterozygotes displayed intermediate levels. Analysis of 441 multiple sclerosis patients and 793 controls showed that frequency of AA homozygosity was significantly lower in MS patients with relapsing-remitting onset (N=416) than in controls (OR=0.70). Moreover, AA patients with relapsing-remitting onset had lower relapse rate and multiple sclerosis severity score than non-AA patients.

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells.

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4+ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti-CD3 mAb. ICOS strikingly potentiated secretion of IL-2, IFN-gamma, IL-10, and TNF-alpha, but not IL-4, promoted by optimal stimulation of CD3+CD28, and it was the key switching-factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL-2. In these conditions, blockade of IL-2 and IFN-gamma showed that ICOS builds up a positive feedback loop with IFN-gamma, which required IL-2 and was inhibited by IL-4. By contrast, in the absence of CD28 triggering or exogenous IL-2, ICOS-induced costimulation mainly supported expression of TGF-beta1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4+ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL-2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts.

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.

C3 promotes clearance of Klebsiella pneumoniae by A549 epithelial cells.

The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

[The canine model in the gene therapy of glioblastoma]. / El modelo canino en la terapia génica del glioblastoma.

The group of research directed by Marta Izquierdo (in which I colaborate close to Dra. García-Escudero and other colleagues) has used in the last three years the linamarase/linamarin system for treatment of brain tumors in the rat. This is a killer/suicide method based upon the plant linamarase gene, that hydrolyses the innocuous substrate linamarin to acetone cyanohydrin and glucose. The acetone cyanohydrin spontaneously breaks down to acetone and CNH, which kills the tumor cell. We try this system in dogs, but we could only induce tumors large enough to be treated by gene therapy in 2 out of 5 adult dogs. One of those dogs was treated with adenovirus carrying linamarase gene, and after with linamarine, observing the remission of the tumor. The other, used as control, died as a consecuence of the tumor. The W&W canine cell line seems to behave as allogeneic when injected into the brain of Beagle adult dogs, and is not, therefore an excellent model for this type.

[A remembrance of Clarín's illnesses in his centenary]. / Las enfermedades de Clarín. Un recuerdo en su centenario.

The Spanish famous writer Leopoldo Alas, also known by the pseudonymous of "Clarín" suffered from two main kind of illnesses: nervous and digestives. Both began early, when he was only thirty two. At that moment, and during all his thirties, nervous ailments were conspicuous, but digestive problems were growing up slow but steadily and became the most serious and even menacing during his forties. Nervous pathology was double: on the one hand, some attacks of migraine with visual disturbances (scotoma), dysphasia and other "indescribable nervous oddities", which happened about 4-5 times per year; on the other, several emotional and vague symptoms, such as melancholia, dejected mood, anxiety, nervousness, etc. Digestive symptomatology, consisting of atonic constipation, intestinal dyspepsia, febricula, and a feeling of being "as a blocked drain", is due--no doubt--to tuberculous peritonitis diagnosed by his young nephew Dr. Martínez. This serious illness was the cause of his death on the 13th June 1901, when he was only 49.