Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development.

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies.

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.

Fast and efficient microfluidic cell filter for isolation of circulating tumor cells from unprocessed whole blood of colorectal cancer patients.

Liquid biopsy offers unique opportunities for low invasive diagnosis, real-time patient monitoring and treatment selection. The phenotypic and molecular profile of circulating tumor cells (CTCs) can provide key information about the biology of tumor cells, contributing to personalized therapy. CTC isolation is still challenging, mainly due to their heterogeneity and rarity. To overcome this limitation, a microfluidic chip for label-free isolation of CTCs from peripheral blood was developed. This device, the CROSS chip, captures CTCs based on their size and deformability with an efficiency of 70%. Using 2 chips, 7.5 ml of whole blood are processed in 47 minutes with high purity, as compared to similar technologies and assessed by in situ immunofluorescence. The CROSS chip performance was compared to the CellSearch system in a set of metastatic colorectal cancer patients, resulting in higher capture of DAPI+/CK+/CD45- CTCs in all individuals tested. Importantly, CTC enumeration by CROSS chip enabled stratification of patients with different prognosis. Lastly, cells isolated in the CROSS chip were lysed and further subjected to molecular characterization by droplet digital PCR, which revealed a mutation in the APC gene for most patient samples analyzed, confirming their colorectal origin and the versatility of the technology for downstream applications.

Exploring sialyl-Tn expression in microfluidic-isolated circulating tumour cells: A novel biomarker and an analytical tool for precision oncology applications.

Circulating tumour cells (CTCs) originating from a primary tumour, lymph nodes and distant metastases hold great potential for liquid biopsies by providing a molecular fingerprint for disease dissemination and its temporal evolution through the course of disease management. CTC enumeration, classically defined on the basis of surface expression of Epithelial Cell Adhesion Molecule (EpCAM) and absence of the pan-leukocyte marker CD45, has been shown to correlate with clinical outcome. However, existing approaches introduce bias into the subsets of captured CTCs, which may exclude biologically and clinically relevant subpopulations. Here we explore the overexpression of the membrane protein O-glycan sialyl-Tn (STn) antigen in advanced bladder and colorectal tumours, but not in blood cells, to propose a novel CTC isolation technology. Using a size-based microfluidic device, we show that the majority (>90%) of CTCs isolated from the blood of patients with metastatic bladder and colorectal cancers express the STn antigen, supporting a link with metastasis. STn+ CTC counts were significantly higher than EpCAM-based detection in colorectal cancer, providing a more efficient cell-surface biomarker for CTC isolation. Exploring this concept, we constructed a glycan affinity-based microfluidic device for selective isolation of STn+ CTCs and propose an enzyme-based strategy for the recovery of viable cancer cells for downstream investigations. Finally, clinically relevant cancer biomarkers (transcripts and mutations) in bladder and colorectal tumours, were identified in cells isolated by microfluidics, confirming their malignant origin and highlighting the potential of this technology in the context of precision oncology.

Sialyl-Tn identifies muscle-invasive bladder cancer basal and luminal subtypes facing decreased survival, being expressed by circulating tumor cells and metastases.

OBJECTIVES: To evaluate the potential of sialyl-Tn (STn), a cancer-associated glycan antigen present in membrane glycoproteins, to improve a recent molecular model for stratification and prognostication of advanced stage bladder tumors based on keratins (KRT14, 5, and 20) expression. In addition, determine the association between STn and disease dissemination based on the evaluation of circulating tumor cells (CTCs) and the metastasis, which is a critical matter to improve patient management. PATIENTS AND METHODS: A retrospective series of 80 muscle-invasive primary bladder tumors and associated metastasis were screened for KRT14, 5, and 20 and STn by real-time polymerase chain reaction and immunohistochemistry. Peripheral blood was collected in a patients' subset, CTCs were isolated through a size-based microfluidic chip and screened for KRTs and STn. RESULTS: Basal-like lesions presented worse cancer-specific and disease-free survival compared to luminal tumors. STn antigen inclusion discriminated patients with worst survival in each subgroup (P = 0.047 for luminal; P = 0.027 for basal-like tumors). STn expression in CTCs and distant metastasis was also demonstrated. CONCLUSION: This work reinforces the potential of the KRT-based model for bladder cancer management and the association of STn with aggressiveness, supporting its inclusion in predictive molecular models toward patient-tailored precision medicine. Moreover, we describe for the first time that CTCs and the metastasis present a basal phenotype and express the STn antigen, highlighting its link with disease dissemination. Future studies should focus on determining the biological and clinical significance of these observations in the context of liquid biopsies. Given the membrane nature of STn, highly specific targeted therapeutics may also be envisaged.

Exosomes as Reconfigurable Therapeutic Systems.

Historically, small molecules, including steroid hormones and cytokines, have been attributed a role in paracrine and endocrine signaling, and now include a new player: biological nanoparticles, or 'exosomes'. Generated intracellularly, and defined simply as nanoparticulate packages of signaling moieties, exosomes have emerged as vehicles for highly specialized local and distant intercellular communication. Exosomes are increasingly being recognized as contributing factors in many diseases, and their potential as biomarkers and in therapeutics is rapidly emerging. This review highlights recent advances in the exploitation of exosomes in diagnostic and therapeutic applications. We discuss various facets of nanoparticles, namely the isolation and manipulation of exosomes, the construction of synthetic exosome-like particles in vivo, and their potential use in the treatment of various diseases.

Rapid and specific detection of cell-derived microvesicles using a magnetoresistive biochip.

Microvesicles (MVs) are a promising source of diagnostic biomarkers which have gained a wide interest in the biomedical and biosensing field. They can be interpreted as a "fingerprint" of various diseases. Nonetheless, MVs implementation into clinical settings has been hampered by the lack of technologies to accurately characterize, detect and quantify them. Here, we report the specific sensing and quantification of MVs from endothelial cells using a portable magnetoresistive (MR) biochip platform, in less than one hour and within physiologically relevant concentrations (1 × 108 MVs per ml). MVs were isolated from both endothelial and epithelial cells undergoing apoptosis, and characterized by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA), which revealed similar MV sizes. Importantly, our results showed that the two distinct MV populations could be discriminated with the MR biochip platform, with over a 5-fold capture efficiency of endothelial MVs in comparison to the control (epithelial MVs). Also, unspecific binding of MVs to BSA was less than 1% of the specific signal. The detection strategy was based on a sandwich immunoassay, where MVs were labelled with magnetic nanoparticles (MNPs) functionalized with Annexin V and then captured by anti-CD31 antibodies previously immobilized on the surface of the sensor. Results suggest that this approach allows the detection of specific MVs from complex samples such as serum, and highlight the potential of this technology to become a suitable tool for MVs detection as a complementary method of diagnosis.

Adsorbed Fibrinogen stimulates TLR-4 on monocytes and induces BMP-2 expression.

Modulation of inflammatory responses to implanted biomaterials towards tissue regeneration has gained prominence as an innovative tissue engineering strategy. Recent in vitro and in vivo studies showed that Fibrinogen (Fg) adsorbed to Chitosan (Ch) substrates modulates immune cell responses, enhances the production of osteogenic factors by monocytes/macrophages and promotes bone regeneration, but the mechanisms involved remain poorly understood. Thus, the present work was conducted to clarify the molecular mechanisms of interaction between primary human monocytes and the above substrates. Cell surface expression of TLR-4 was significantly downregulated in the presence of pre-adsorbed Fg, when compared to Ch control, indicating an interaction via this receptor. The same substrate triggered MAPK activation, specifically the ERK 1/2 and JNK pathways. Importantly, both ERK 1/2 and JNK phosphorylation were reduced when TLR-4 signalling was blocked using a specific pharmacological inhibitor. Functionally, adsorbed Fg induced production of the potent osteogenic mediator BMP-2 by monocytes, while TLR-4 inhibition resulted in a significant decrease of BMP-2 mRNA and protein levels, in response to Fg stimulation. Overall, our data reveals that adsorbed Fg exerts a pro-osteogenic effect on human monocytes through its interaction with TLR-4 and subsequent production of BMP-2, elucidating two key aspects of the immunomodulatory action of adsorbed Fg in bone regeneration. STATEMENT OF SIGNIFICANCE: Recent studies showed that when Fibrinogen (Fg) is used to modify Chitosan (Ch) substrates, it modulates the immune response, enhances production of osteogenic factors by monocytes/macrophages, and promotes bone regeneration. However, the mechanisms involved in monocyte-Fg interaction, were only partially known. Current work addresses the interaction between primary human monocytes and Ch surfaces modified by Fg adsorption (Ch-Fg) at the molecular level. Results show that monocytes interact specifically with Ch-Fg via TLR-4, triggering particular intracellular signalling pathways (ERK and JNK, but not p38), downstream of TLR-4. Functionally, Ch-Fg induced monocytes to produce the osteogenic mediator BMP-2. Thus, we clarify herein two essential aspects of the interaction between adsorbed Fg and monocytes, with impact on immunomodulation and regeneration, upon biomaterial implantation.

Fibrinogen scaffolds with immunomodulatory properties promote in vivo bone regeneration.

The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac-1+ (CD18+/CD11b+) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1ß and increased TGF-ß1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials.

Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour.

BACKGROUND: The interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms. METHODS: Following exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy. RESULTS: Our results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was essential for macrophage-mediated cancer cell invasion and angiogenesis. CONCLUSIONS: We propose that IL-10- and LPS-stimulated macrophages distinctly modulate gastric and colorectal cancer cell behaviour, as result of distinct proteolytic profiles that impact cell invasion and angiogenesis.

Resveratrol as a natural anti-tumor necrosis factor-α molecule: implications to dendritic cells and their crosstalk with mesenchymal stromal cells.

Dendritic cells (DC) are promising targets for inducing tolerance in inflammatory conditions. Thus, this study aims to investigate the effects of the natural anti-inflammatory molecule resveratrol on human DC at phenotypic and functional levels, including their capacity to recruit mesenchymal stem/stromal cells (MSC). Primary human monocyte-derived DC and bone marrow MSC were used. DC immunophenotyping revealed that small doses of resveratrol (10 µM) reduce cell activation in response to tumor necrosis factor (TNF)-α, significantly decreasing surface expression of CD83 and CD86. Functionally, IL-12/IL-23 secretion induced by TNF-α was significantly reduced by resveratrol, while IL-10 levels increased. Resveratrol also inhibited T cell proliferation, in response to TNF-α-stimulated DC. The underlying mechanism was investigated by Western blot and imaging flow cytometry (ImageStreamX), and likely involves impairment of nuclear translocation of the p65 NF-κB subunit. Importantly, results obtained demonstrate that DC are able to recruit MSC through extracellular matrix components, and that TNF-α impairs DC-mediated recruitment. Matrix metalloproteinases (MMP) produced by both cell populations were visualized by gelatin zymography. Finally, time-lapse microscopy analysis revealed a significant decrease on DC and MSC motility in co-cultures, indicating cell interaction, and TNF-α further decreased MSC motility, while resveratrol recovered it. Thus, the current study points out the potential of resveratrol as a natural anti-TNF-α drug, capable of modulating DC phenotype and function, as well as DC-mediated MSC recruitment.

Impact of 3-D printed PLA- and chitosan-based scaffolds on human monocyte/macrophage responses: unraveling the effect of 3-D structures on inflammation.

Recent studies have pointed towards a decisive role of inflammation in triggering tissue repair and regeneration, while at the same time it is accepted that an exacerbated inflammatory response may lead to rejection of an implant. Within this context, understanding and having the capacity to regulate the inflammatory response elicited by 3-D scaffolds aimed for tissue regeneration is crucial. This work reports on the analysis of the cytokine profile of human monocytes/macrophages in contact with biodegradable 3-D scaffolds with different surface properties, architecture and controlled pore geometry, fabricated by 3-D printing technology. Fabrication processes were optimized to create four different 3-D platforms based on polylactic acid (PLA), PLA/calcium phosphate glass or chitosan. Cytokine secretion and cell morphology of human peripheral blood monocytes allowed to differentiate on the different matrices were analyzed. While all scaffolds supported monocyte/macrophage adhesion and stimulated cytokine production, striking differences between PLA-based and chitosan scaffolds were found, with chitosan eliciting increased secretion of tumor necrosis factor (TNF)-α, while PLA-based scaffolds induced higher production of interleukin (IL)-6, IL-12/23 and IL-10. Even though the material itself induced the biggest differences, the scaffold geometry also impacted on TNF-α and IL-12/23 production, with chitosan scaffolds having larger pores and wider angles leading to a higher secretion of these pro-inflammatory cytokines. These findings strengthen the appropriateness of these 3-D platforms to study modulation of macrophage responses by specific parameters (chemistry, topography, scaffold architecture).

Adsorbed fibrinogen enhances production of bone- and angiogenic-related factors by monocytes/macrophages.

Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-ß3, and insulin-like growth factor-binding proteins, as well as several angiogenic mediators, including endocrine gland-derived vascular endothelial factor, fibroblast growth factor-7, and placental growth factor, were significantly promoted by Fg. This work provides a new perspective on the inflammatory response in the context of bone repair/regeneration mediated by a pro-inflammatory protein (Fg) adsorbed onto a biomaterial (Ch) that does not otherwise exhibit osteogenic properties.

Chitosan drives anti-inflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation.

Macrophages and dendritic cells (DC) share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch), with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-ß1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1ß levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.

The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase.

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

Molecular cloning and analysis of SSc5D, a new member of the scavenger receptor cysteine-rich superfamily.

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30kb of genomic DNA, and contains fourteen exons producing a 4.8kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.

Extracellular isoforms of CD6 generated by alternative splicing regulate targeting of CD6 to the immunological synapse.

The great majority of mammalian genes yield multiple transcripts arising from differential mRNA processing, but in very few instances have alternative forms been assigned distinct functional properties. We have cloned and characterized a new isoform of the accessory molecule CD6 that lacks the CD166 binding domain and is expressed in rat and human primary cells. The novel isoform, CD6Deltad3, results from exon 5 skipping and consequently lacks the third scavenger receptor cysteine-rich (SRCR) domain of CD6. Differential expression of the SRCR domain 3 resulted in a remarkable functional difference: whereas full-length CD6 targeted to the immunological synapse, CD6Deltad3 was unable to localize at the T cell:APC interface during Ag presentation. Analysis of expression of CD6 variants showed that, while being more frequent in coexpression with full-length CD6, the CD6Deltad3 isoform constituted the sole species in a small percentage of T cells. In the rat thymus, CD6Deltad3 is less represented in double-positive thymocytes but is detectable in nearly 50% of single-positive CD4 or CD8 thymocytes, suggesting that CD6 switching between full-length and Deltad3 isoforms may be involved in thymic selection. Strikingly, CD6Deltad3 is markedly up-regulated upon activation of T lymphocytes, partially substituting full-length CD6, as evaluated by RT-PCR analysis at the single-cell level, by immunoblotting, and by flow cytometry using Abs recognizing SRCR domains 1 and 3 of human CD6. This elegant mechanism controlling the expression of the CD166 binding domain may help regulate signaling delivered by CD6, through different types of extracellular engagement.

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.