Cross-linking Of Surface Receptors Research Articles

Inhibition of Immunosuppressive Tumors by Polymer-Assisted Inductions of Immunogenic Cell Death and Multivalent PD-L1 Crosslinking.

Checkpoint blockade immunotherapies harness the host's own immune system to fight cancer, but only work against tumors infiltrated by swarms of pre-existing T cells. Unfortunately, most cancers to date are immune-deserted. Here, we report a polymer-assisted combination of immunogenic chemotherapy and PD-L1 degradation for efficacious treatment in originally non-immunogenic cancer. "Priming" tumors with backbone-degradable polymer-epirubicin conjugates elicits immunogenic cell death and fosters tumor-specific CD8+ T cell response. Sequential treatment with a multivalent polymer-peptide antagonist to PD-L1 overcomes adaptive PD-L1 enrichment following chemotherapy, biases the recycling of PD-L1 to lysosome degradation via surface receptor crosslinking, and produces prolonged elimination of PD-L1 rather than the transient blocking afforded by standard anti-PD-L1 antibodies. Together, these findings established the polymer-facilitated tumor targeting of immunogenic drugs and surface crosslinking of PD-L1 as a potential new therapeutic strategy to propagate a long-term antitumor immunity, which might broaden the application of immunotherapy to immunosuppressive cancers.

Drug-free albumin-triggered sensitization of cancer cells to anticancer drugs

Chemosensitization strategies have been used to sensitize cancer cells to conventional drugs, but their utility is often obstructed by additional off-target toxicity, limited access to intracellular targets and heterogeneous tumor pathogenesis. To address these challenges, we rationally developed a drug-free human serum albumin (HSA)-based therapeutic (KH-1) that functions extracellularly and exhibits pleiotropic effect on multiple intracellular signaling pathways. It is a two-step touch-trigger system that consists of a pretargeting anchor on surface receptor CD20 (anti-CD20 Fab’ conjugated with a morpholino oligonucleotide 1) and a CD20 clustering actuator (HSA grafted with multiple copies of complementary morpholino oligonucleotide 2). The extracellular actuation by surface CD20 crosslinking boosts robust activations of numerous intracellular responses, and promotes cancer cell susceptibility to various anticancer drugs, including docetaxel (microtubule stabilizer), gemcitabine (nucleoside analogue) and GDC-0980 (PI3K/mTOR inhibitor). The broad applicability of KH-1 is demonstrated to result from simultaneous inhibition of survival pathways and augmentation of apoptotic pathways. In addition, KH-1 covalently conjugated with anthracycline anticancer agent, epirubicin, integrates the advantages of both chemosensitization function and improved intracellular drug delivery in a single system and takes effect on the same cell. Therefore, in the present study, we have provided mechanistic demonstration that crosslinking of surface receptors can be leveraged to elicit chemosensitization.

Roles of Ca(2+)/calmodulin-dependent protein kinase II in subcellular expression of striatal N-methyl-D-aspartate receptors in l-3, 4-dihydroxyphenylalanine-induced dyskinetic rats.

BackgroundThe role of N-Methyl-D-aspartate (NMDA) receptors is critical to the development of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) in Parkinson’s disease (PD). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to regulate the expression and activation of NMDA receptors in LID, but the interaction between LID and CaMKII-modulated NMDA receptor activity is not clear so far.MethodsWe used 6-hydroxydopamine-lesioned rats to create PD rat model, and at least 21 days of l-DOPA was administrated followed with or without microinjection of CaMKII inhibitor KN-93 into the lesioned striatum of all the PD rats and sham rats. A surface receptor cross-linking assay was used to distinguish expression of striatal NMDA receptors in surface and intracellular compartments.Resultsl-DOPA treatment enhanced surface levels of GluN1 expression and reduced its intracellular expression, but did not change total levels of GluN1 protein in the lesioned striatum. In contrast, l-DOPA decreased GluN2A surface expression but increased its intracellular expression. l-DOPA increased GluN2B expression preferentially in the surface compartment. We also found that l-DOPA increased CaMKII autophosphorylation at T286 in striatal neurons. The inhibition of CaMKII by microinjecting CaMKII inhibitor KN-93 into the lesioned striatum largely reversed the l-DOPA-induced changes in three subunits. In addition, dyskinetic behaviors of animals were observed alleviated after treatment of KN-93.ConclusionOur research indicates that long-term l-DOPA administration activates CaMKII in striatal neurons. Activated CaMKII is involved at least in part in mediating l-DOPA-induced changes of NMDA receptors surface/intracellular expression.

Changes in surface expression of N-methyl-D-aspartate receptors in the striatum in a rat model of Parkinson's disease

BackgroundN-methyl-D-aspartate (NMDA) receptors play a central role in glutamatergic synaptic transmission in the mammalian brain and are linked to the pathophysiology and symptomatology of Parkinson’s disease (PD). However, changes in NMDA receptor expression in distinct subcellular compartments in PD have not been elucidated. In this study, we investigated changes in subcellular expression of NMDA receptors in striatal neurons in a rodent PD model.MethodsIntracranial injection of the neurotoxin 6-hydroxydopamine (6-OHDA) was selectively lesioned into the nigrostriatal dopaminergic pathway in adult Sprague Dawley rats, which is a common rat model of PD. A surface receptor crosslinking assay was conducted to examine the response of individual NMDA receptor subunits to dopamine depletion in isolated and confined surface and intracellular compartments of striatal neurons.ResultsIn PD rats where 6-OHDA was selectively lesioned, surface expression of NMDA receptor GluN1 subunits as detected by surface protein crosslinking assays was increased in the striatum. In contrast, intracellular levels of GluN1 were decreased in the lesioned region. The NMDA receptor GluN2B subunit was elevated in its abundance in the surface pool of the lesioned striatum, while intracellular GluN2B levels were not altered. GluN2A subunits in both surface and intracellular fractions remained stable. In addition, total cellular levels of striatal GluN1 and GluN2A were not changed in lesioned tissue, while total GluN2B proteins showed an increase.ConclusionThese results demonstrate the differential sensitivity of principal NMDA receptor subunits to dopamine depletion. GluN1 and GluN2B expression in the distinct surface compartment underwent upregulation in striatal neurons after selective lesions of the dopaminergic pathway by 6-OHDA.

A role for lipid rafts in immune cell signaling.

Cross-linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src-family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.

Receptor mediated subversion of macrophage cytokine production by intracellular pathogens

Appropriately regulated immune responses depend on the controlled production of cytokines from antigen presenting cells. IL-12 synthesis is tightly regulated by several redundant mechanisms. One mechanism of IL-12 regulation involves the cross-linking of surface receptors on macrophages. This pathway may be exploited by intracellular pathogens of macrophages to inhibit IL-12 production and delay or prevent the development of cell mediated immunity.

Death-effector filaments: novel cytoplasmic structures that recruit caspases and trigger apoptosis.

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.

Quantitation of AMPA receptor surface expression in cultured hippocampal neurons

Protein and messenger RNA levels of the AMPA-type glutamate receptor subunits 1–3 are high in many brain regions, but it is not known how much of the glutamate receptor protein is expressed on the surface of neurons in the form of functional receptors. To provide insight into this matter, western blot immunoreactivities for glutamate receptors 1 and 2/3, as well as binding of the specific ligand [ 3H]AMPA, were quantified following three independent treatments modifying surface receptors in intact primary hippocampal cultures: (i) proteolysis of surface receptors by chymotrypsin, (ii) cross-linking of surface receptors with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate, and (iii) biotinylation of surface receptors with the membrane-impermeant reagent sulfosuccinimidyl-2(biotinamido)ethyl-1,3-dithiopropionate. All three of these methods demonstrated that 60–70% of total glutamate receptor subunit 1 protein and 40–50% of total glutamate receptor 2/3 protein are expressed on the surface of hippocampal neurons. Parallel studies revealed that 52% of total [ 3H]AMPA binding sites could be precipitated with avidin beads following biotinylation of intact cultures, providing an estimate of [ 3H]AMPA binding site surface expression in accord with the estimates of the surface expression of glutamate receptor subunits 1–3. Experiments examining the surface expression of 32P-labeled glutamate receptor subunit 1 demonstrated that approximately 65% of the phosphorylated form of the subunit is located in the plasma membrane, an estimate similar to the that derived via western blot for the entire glutamate receptor subunit 1 population in the same samples. Moreover, no significant change in the surface expression profile of the glutamate receptor subunits 1–3 was observed following stimulatory treatments known to increase glutamate receptor phosphorylation. These data indicate that slightly more than half of the AMPA receptors in cultured hippocampal neurons are located in the plasma membrane, and that AMPA receptor surface expression is not rapidly altered by glutamate receptor phosphorylation.

Tumor Necrosis Factor Beta Is a Major Source of Antibody-Activated Effector Cells in the Killing of Virally Infected Target Cells

We have extended our previous work on cross-linking CD4 molecules using specific monoclonal-antibody-induced antigen-nonspecific major-histocompatibility-complex unrestricted killing by CD4+ T cells of virally infected target cells. The killing activity of antibody-activated CD4+ T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylmaleimide, a protein kinase C inhibitor. Herbimycin-A-treated human or bovine peripheral blood CD4+ T cells lacked PTK activity and failed to kill virally infected target cells even after cross-killing of CD4 molecules. The cross-linking of CD4 molecules failed to induce effector cell proliferation or the transcription of TNF-beta. The upregulation of TNF-beta was induced by incubating antibody-activated effector cells with bovine herpesvirus type 1 (BHV-1)-infected D17 target cells for 10 h. Anti-TNF-beta antibody partially abolished (13-44%) direct effector-cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70-100% of antiviral activity of effector and target cell culture supernatants against BHV-1-infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector:target cell ratio. These results support the hypothesis that increased p56lck enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines were produced that are known to participate in target cell killing. A better understanding of the killing activity displayed by CD4+ T lymphocytes following surface receptor cross-linking activity displayed by CD4+ T lymphocytes following surface receptor cross-linking will give insight into the mechanisms of cytotoxic activity directed toward virally infected cells.

Phosphatidylinositol hydrolysis in freshly isolated human T lymphocytes

Antigen receptor-mediated activation of T and B lymphocytes results in activation of phospholipase C-γ isozymes with subsequent hydrolysis of membrane inositol phospholipids. As a method of screening autoimmune or immunodeficient patients for early receptor signaling defects, we have developed a rapid technique for studying phosphatidylinositol (PI) hydrolysis in cultured cells and fresh clinical specimens resulting from surface receptor crosslinking. Using staphylococcal α-toxin, we permeabilized freshly isolated, purified human T lymphocytes to facilitate incorporation of [ 3H]myoinositol into membrane phospholipids. Aggregation of surface antigen receptors (TCR-CD3 complex and CD28 on T cells) with specific antibodies produced extensive ATP and Mg 2+-dependent hydrolysis of the membrane inositol phospholipids as measured by release of water soluble inositol phosphates. Anti-human CD3 antibody produced 18.5 ± 1.6 net % PI hydrolysis and anti-human CD28 antibody produced 4.6 ± 0.2 net % PI hydrolysis. Simultaneous anti CD3 CD28 crosslinking produced 30.8 ± 1.2 net % PI hydrolysis, an increase over either stimulus alone ( p = 0.0013 two tailed t test). Isotype matched control antibodies produced 11.6 ± 0.4% PI hydrolysis. The tyrosine phosphatase inhibitor orthovanadate (Na 3VO 4) was used as a positive control because it induces maximal protein tyrosine kinase-dependent PI hydrolysis in permeabilized cells. Na 3VO 4 consistently induced hydrolysis of > 50% of the membrane inositol phospholipid pool. These data indicate that costimulation of T cells with antibodies to CD3 and CD28 is synergistic and reinforces the importance of CD28 as an accessory T cell stimulus. This easy technique allows quick evaluation of the integrity of the early signaling cascade in lymphocytes as a screen for autoimmune and immunodeficiency diseases.

Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells.

Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP-dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation.

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment.

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross-linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.

Cytoskeleton of living, unstained cells imaged by scanning force microscopy

Subsurface cytoskeletal structure can be visualized in either fixed or living mammalian cells in aqueous medium with approximately 50 nm resolution using the Scanning Force Microscope (SFM). In living cells, changes in cell topography, or subsurface cytoskeleton caused by the introduction of drugs (colchicine) or cross-linking of surface receptors (by antibodies against IgE bound to the IgE receptor) can be followed in time. Contrast in SFM images of cell surfaces result from both topographic features of the cell and from variations in cell surface "stiffness". The SFM is therefore capable of measuring local compliance and stress in living cells, and so should make it possible to map the cytoskeletal forces used to generate cell motions and changes in cell shape.

Co-engagement of CD8 with the T cell receptor is required for negative selection

Although it is established that the CD8 and CD4 co-receptors are involved in T-lymphocyte recognition and activation in the periphery, it is less clear whether these molecules participate in thymic selection events. Analysis of thymic selection in mice transgenic for T cell-receptor genes or for major histocompatibility complex (MHC) genes, or mice injected with antibodies against CD8, CD4 or MHC molecules, is consistent with the participation of CD8 and CD4 in thymic selection. But antibody-mediated crosslinking of surface receptors in thymic organ cultures has indicated that CD8 is not involved in thymic deletion. We show here that mice transgenic for a mutant MHC class I molecule that cannot interact with CD8 do not delete CD8-dependent T cells reactive with the wild-type molecule. This finding unequivocally establishes that for negative selection in the thymus, CD8 must interact with the same MHC class I molecule as the T cell receptor.

Effect of concanavalin A on the formation of the mouse blastocyst

Culture of precompact zona-free mouse embryos in the plant lectin concanavalin A (Con A) at 20 μg/ml inhibits blastocyst formation. Cell division is retarded compared with control embryos, compaction of the morula is prevented, and fluid accumulation occurs within but not between cells. As little as two hours of incubation in Con A can cause developmental delay and an eventual increased incidence of lysis, and although embryos can recover from more prolonged pulses of Con A, the blastocysts formed are often abnormal. Blastocyst formation occurs in some embryos, but does not do so unless at least some cells have undergone a fifth round of cell division before incubation in the lectin. However, not all embryos that contained more than 32 cells compacted and showed intercellular fluid accumulation in Con A. Thus, blastocyst formation is not restricted solely by an inhibition of cell division or a reduction in cell number, but is also impaired by interference of the lectin with the cell surface and its interactions. Morulae bind divalent Con A and monovalent succinyl-Con A similarly, but developmental interference is absent with the latter, suggesting that divalent Con A acts through a cross-linking of surface receptors.

Induction of serotonin secretion by cross-linking of surface receptors of a derivative of wheat germ agglutinin on human platelets

A nonagglutinating derivative of wheat germ agglutinin has been prepared that binds to platelets and precipitates an antibody to the lectin. Platelets treated with this inactive derivative released serotonin when exposed to bivalent F(ab′) 2, but not monovalent Fab, fragments of the lectin antibody. Bridging of platelet-bound Fab by an antibody again induced secretion. The F(ab′) 2 or Fab fragments plus IgG, without the derivative, did not induce secretion. This secretion was not affected by indomethacin showing a direct activation of platelets. Platelets treated with con A followed by F(ab′) 2 to con A did not secrete. In addition, lentil lectin failed to release platelet serotonin. The receptors of the lectin derivative are mobile on the platelet surface and their redistribution may lead to secretion.

Determination of cell form in cultured fibroblasts: Role of surface components and cytokinetic elements

Functions involved in the determination of the morphology of mouse embryo fibroblasts have been studied using as a system the cell rounding and reflattening related to the presence or absence of a protein component of the cell surface [18]. Cells that had undergone rounding following the removal of the surface protein gradually reflattened if their protein synthesis was not impaired but could rapidly revert to the flattened state if a fraction containing the surface protein was returned to the cells. Studies with sodium azide and potassium cyanide showed that both the rounding and reflattening were energy-dependent. When the rounded state was induced the organised appearance of the microfilaments present in the cortical layer adjacent to the plasma membrane was lost. However, microfilament structures reappeared when rounded cells respread over the glass substrate. On the other hand no reflattening occurred if microfilament function was impaired by treatment with cytochalasin B (CB), showing a requirement of microfilament integrity for the flattened form to be manifest. Microtubules also were shown to be involved in shape determination as their integrity was necessary for cells to progress from the flattened form to the rounded state, but they did not appear to be primarily involved in the process of reflattening. The distribution of macromolecular components of the plasma membrane was also shown to affect changes of cell shape as cross-linking of surface receptors by concanavalin A (ConA) or specific anti-cell immunoglobulins prevented cells from acquiring the rounded form and treatment of cells with media of low pH, which causes aggregation of intra-membrane particles, also prevented rounding. A comparison between quiescent cells and cells in S phase showed the changes of morphology to be similar in nature, but more prompt and marked in the S population. The indication of the studies presented is that determination of cell form requires the cooperative interaction between the protein component of the cell surface, integral constituents of the plasma membrane and cytokinetic organelles.