Insulin and insulin-like growth factor I receptors utilize different G protein signaling components.

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

The ubiquitin-like protein LC3 regulates the Rho-GEF activity of AKAP-Lbc.

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells.

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.

Gas-filled microbubble-mediated delivery of antigen and the induction of immune responses.

The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.

Bystander-Activated Memory CD8 T Cells Control Early Pathogen Load in an Innate-like, NKG2D-Dependent Manner.

During an infection the antigen-nonspecific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T cell response.

The phagocytosis of gas-filled microbubbles by human and murine antigen-presenting cells.

This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c(+)DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates that MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.

Demyelination induced in aggregating brain cell cultures by a monoclonal antibody against myelin/oligodendrocyte glycoprotein.

A monoclonal antibody (8-18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentration decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase. In the absence of complement, anti-MOG antibody did not induce detectable demyelination. In contrast to the effect of anti-MOG antibody and as expected, anti-MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody-induced demyelination. Indeed, they show that a purified anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of anti-MOG antibodies in demyelinating diseases.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors.

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression. Cancer Res; 73(12); 3591-603. ©2013 AACR.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9.Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS).Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.

Hepatitis B virus infection is associated with impaired immunological recovery during antiretroviral therapy in the Swiss HIV cohort study.

Hepatitis B virus (HBV) infection is a major cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected patients worldwide. It is unclear whether HIV-related outcomes are affected by HBV coinfection. We compared virological suppression and immunological recovery during antiretroviral therapy (ART) of patients of different HBV serological status in the Swiss HIV Cohort Study. CD4 cell recovery during ART was significantly impaired in hepatitis B surface antigen-positive patients and in those with anti-hepatitis B core antigen alone compared with HBV-uninfected patients, despite similar virological efficacy of ART. CD4 increase in patients with resolved HBV infection was similar to that in HBV-uninfected individuals.

Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF.

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.