scholarly journals Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody.

1992 ◽  
Vol 175 (1) ◽  
pp. 257-266 ◽  
Author(s):  
J Lesley ◽  
Q He ◽  
K Miyake ◽  
A Hamann ◽  
R Hyman ◽  
...  
Keyword(s):  
T Cells ◽  
Hyaluronic Acid ◽  
Cell Surface ◽  
Cell Sorting ◽  
Cytoplasmic Domain ◽  
Binding Activity ◽  
Wild Type ◽  
T Lymphoma ◽  
Mutant Construct ◽  
Hyaluronic Acid Binding

The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

scholarly journals 231 Molecular insights on safety and anti-tumor activity of a non-irAE-inducing anti-CTLA-4 monoclonal antibody ONC-392

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A246-A246
Author(s):  
Yang Liu ◽  
Yan Zhang ◽  
Xuexiang Du ◽  
Mingyue Liu ◽  
Xianfeng Fang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cell Surface ◽  
Cancer Immunotherapy ◽  
Immune Tolerance ◽  
Ph Sensitive ◽  
Clinical Testing ◽  
Wild Type ◽  
Treg Depletion ◽  
Anti Tumor Activity

BackgroundAnti-CTLA-4 antibodies have brought about limited clinical benefit because severe toxicity limits dosing levels and/or duration. We used CTLA-4 knockin mice to screen for antibodies with higher anti-tumor activity but lower autoimmunity. We have revealed that the key for better safety and preclinical efficacy is preservation of CTLA-4 for immune tolerance and intratumorial Treg depletion. Our work established that, independent of blocking activities, mAbs that preserve CTLA-4 recycling maintain the physiological immune tolerance checkpoint function while allowing more efficient and selective elimination of tumor-infiltrating regulatory T cells, resulting in highest efficacy and lowest toxicity and was thus developed for clinical testing of all antibodies tested.1–6 The antibody with best safety and efficacy profile, ONC-392 was developed for clinical testing. The first-in human studies showed that ONC-392 is safe and well tolerated. Remarkably, no irAE has been reported among patients who has received repeated dosing of 3.0 mg/kg and 10.0 mg/kg of ONC-392. The molecular and cellular characterization of ONC-392 will be presented.MethodsIn vitro binding and disassociation assay were determined between pH 4.0–7.0. The intracellular traffic of both antibodies and CTLA-4 molecules were visualized by confocal microscopy. The binding to human and mouse FcgRI, IIA, IIB, and III (A), FcRn as well as mouse FcgRIV were evaluated by surface plasmon resonance (SPR). Depletion of regulatory T cells in tumor and lymphoid tissues were determined by flow cytometry.ResultsONC-392 is a pH-sensitive antibody that preserves CTLA-4 recycling. By preserving cell surface CTLA-4, Onco-392 preserves immune tolerance. Preserving CTLA-4 on tumor-infiltrating Treg contribute to more effective immunotherapy. In addition to its unique feature of pH sensitive binding, OncoC4 also have several important features in Fc. ONC-392 shown comparable binding to human FcgRI and IIIA as wild-type IgG1s. As expected from the mutations introduced, ONC-392 show about 6 fold higher affinity for FcRn than wild-type IgG1. Interestingly, ONC-392 has shown 7–10-fold reduction to FcgRIIB, which is generally considered to be a negative signaling FcR. ONC-392 binding to mouse FcgRI-IV was lower that WT IgG1.ConclusionsUnlike other clinical anti-CTLA-4 antibodies, ONC-392 preserves CTLA-4 recycling and thus Treg function in the peripheral tissues. The higher cell surface CTLA-4 allows more efficient Treg depletion in the tumor microenvironment. In addition, despite reduced binding to mouse activating Fc?RI, III/IV, ONC-392 was more effective in intratumor Treg depletion in the mice. Therefore, lacking negative signaling from Fc?RIIB may also contribute to its anti-tumor activity.Trial RegistrationNCT04140526ReferencesDu X, et al. Uncoupling therapeutic from immunotherapy-related adverse effects for safer andeffective anti-CTLA-4 antibodies in CTLA4 humanized mice. Cell Res 2018;28:433–447.Du X, et al. A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Cell Res 2018;28:416–432.Liu Y, Zheng P. How does an anti-CTLA-4 antibody promote cancer immunity? Trends Immunol 2018;39:953–956.Zhang Y, et al. Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy. Cell Res 2019;29:609–627.Liu Y, Zheng P. Preserving the CTLA-4 checkpoint for safer and more effective cancer immunotherapy. Trends Pharmacol Sci 2020;41(1):4–12.Zhang P, et al. Mechanism- and immune landscape-based ranking of therapeutic responsiveness of 22 major human cancers to next generation anti-CTLA-4 antibodies. Cancers 2020;12:284.

scholarly journals GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

1993 ◽  
Vol 122 (6) ◽  
pp. 1253-1265 ◽  
Author(s):  
GW Kemble ◽  
YI Henis ◽  
JM White
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Binding Activity ◽  
Great Distance ◽  
Fusion Peptides ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Receptor Binding Activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.

Targeting MHC-linked wild type p53 with TCR mimic single chain diabody for cancer immunotherapy.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2524-2524
Author(s):  
Suman Paul ◽  
Jacqueline Douglass ◽  
Annika Schaefer ◽  
Emily Han-Chung Hsiue ◽  
Alexander Pearlman ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cancer Immunotherapy ◽  
Cell Killing ◽  
Human Cells ◽  
Wild Type ◽  
Single Chain ◽  
Nsg Mice ◽  
Wild Type P53

2524 Background: Increased tumor suppressor protein p53 expression is observed in a wide range of human cancers. As a result there is intense interest in targeting p53 for cancer therapy. Intracellular p53 is inaccessible to therapeutic antibodies that bind cell surface proteins. However, intracellular proteins including p53 are degraded into peptides that are presented on cell surface in association with HLA class I molecules. Thus p53 peptide-HLA (p53-HLA) complexes can be antibody targets. Methods: Using phage display we identified a novel anti-p53-HLA single chain variable fragment (scFv) clone-43 that recognizes a wild-type p53 10-mer epitope bound to HLA-A*2402. By coupling our clone-43 scFv with an anti-CD3 scFv, we generated a single chain diabody (scDb) designed to activate T-cells against p53-expressing target cells. Results: In-vitro co-culture of clone-43 scDb with donor human T-cells and p53 expressing SIG-M5 cancer cells results in SIG-M5 cell killing and concomitant T-cell interferon gamma (IFNγ) release. In contrast, similar co-culture with SIG-M5 p53-knock out (KO) cells showed no cell killing and minimal IFNγ release demonstrating specificity of clone-43 to p53 expressing cells. Additionally, in-vivo growth of p53 expressing SW480 cancer cell xenografts in NSG mice was completely terminated by clone-43 scDb injections. A major concern for wild-type p53 epitope targeting is potential on-target off-tumor effect on non-cancerous tissue. We observed significant in-vitro clone-43 scDb mediated killing of human HLA-A*24:02 peripheral blood mononuclear cells. To better evaluate effect of clone-43 scDb on non-neoplastic human cells, we engrafted HLA-A*24:02 human CD34+ hematopoietic stem cells into NSG mice to generate a humanized mouse model with circulating mature human CD45+ cells. Clone-43 scDb treatment resulted in selective depletion of circulating human cells while the same cells persisted in mice treated with unrelated control scDb. Conclusions: Our observation that immune targeting of wild-type p53 epitope results in significant off-tumor hematopoietic cell death is contrary to previously published reports and carries important implications for future anti-p53 antibody and vaccine design for cancer immunotherapy.

scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon γ production

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2899-2907 ◽  
Author(s):  
Duncan Howie ◽  
Susumo Okamoto ◽  
Svend Rietdijk ◽  
Kareem Clarke ◽  
Ninghai Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Dna Synthesis ◽  
Interferon Γ ◽  
T Cell Proliferation ◽  
Wild Type ◽  
Ifn Γ

CD150 (signaling lymphocyte activation molecule [SLAM]) is a self-ligand cell surface glycoprotein expressed on T cells, B cells, macrophages, and dendritic cells. To further explore the role of CD150 signaling in costimulation and TH1 priming we have generated a panel of rat antimouse CD150 monoclonal antibodies. CD150 cell surface expression is up-regulated with rapid kinetics in activated T cells and lipopolysaccharide/interferon γ (IFN-γ)–activated macrophages. Anti-CD150 triggering induces strong costimulation of T cells triggered through CD3. DNA synthesis of murine T cells induced by anti-CD150 is not dependent on SLAM-associated protein (SAP, SH2D1A), because anti-CD150 induces similar levels of DNA synthesis in SAP−/− T cells. Antibodies to CD150 also enhance IFN-γ production both in wild-type and SAP−/− T cells during primary stimulation. The level of IFN-γ production is higher in SAP−/− T cells than in wild-type T cells. Anti-CD150 antibodies also synergize with interleukin 12 (IL-12) treatment in up-regulation of IL-12 receptor β2 mRNA during TH1 priming, and inhibit primary TH2 polarization in an IFN-γ–dependent fashion. Cross-linking CD150 on CD4 T cells induces rapid serine phosphorylation of Akt/PKB. We speculate that this is an important pathway contributing to CD150-mediated T-cell proliferation.

scholarly journals Variants of vaccinia virus hemagglutinin altered in intracellular transport.

1986 ◽  
Vol 6 (11) ◽  
pp. 3734-3745 ◽  
Author(s):  
H Shida
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Slow Movement ◽  
Wild Type ◽  
Sequence Analyses ◽  
In The Wild ◽  
Virus Mutant

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.

scholarly journals Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor.

1988 ◽  
Vol 168 (3) ◽  
pp. 1145-1156 ◽  
Author(s):  
B E Bierer ◽  
A Peterson ◽  
J C Gorga ◽  
S H Herrmann ◽  
S J Burakoff
Keyword(s):  
T Cells ◽  
Cell Adhesion ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
Hla Dr

T cells may be activated either by the antigen-specific T cell receptor (TCR)-CD3 complex or the cell surface receptor CD2. A natural ligand for CD2 has been found to be lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glycoprotein. To investigate the interaction of these two pathways, we have expressed the cDNA encoding the human CD2 molecule in a murine T cell hybridoma that produces IL-2 in response to HLA-DR antigens. Expression of the CD2 molecule markedly enhances IL-2 production in response to LFA-3+ antigen-bearing stimulator cells, and this stimulation is inhibited by anti-CD2 and anti-LFA-3 mAb. To further define the role of LFA-3 in antigen-dependent T cell activation, we have studied the ability of the purified ligands of CD2 and the TCR to stimulate the hybridoma. Neither liposomes containing purified HLA-DR antigens nor liposomes containing purified LFA-3 were able to stimulate the parent or the CD2+ hybridoma. However, liposomes containing both purified LFA-3 and HLA-DR, the physiological ligands for CD2 and the TCR, respectively, stimulate IL-2 production by the CD2+ but not the parent hybridoma, suggesting that complementary interactions between the TCR-CD3 complex and the CD2 pathway may regulate lymphocyte activation. To determine whether the CD2/LFA-3 interaction participates in cell-cell adhesion and provides an activation signal, we have constructed a cytoplasmic deletion mutant of CD2, CD2 delta B, in which the COOH-terminal 100 amino acids of CD2 have been replaced with a serine. Hybridomas expressing the CD2 delta B molecule were examined. Deletion of the cytoplasmic domain of CD2 did not alter binding of LFA-3 but eliminated the ability of CD2 to increase the response of the hybridoma to liposomes containing both HLA-DR and LFA-3, demonstrating that adhesion of LFA-3 to CD2 alone was insufficient for activation, and that the cytoplasmic domain was required for LFA-3 stimulation through the CD2 molecule. T cells may be activated by purified LFA-3 binding to CD2 and the TCR interacting with its ligand, and these signals appear to be synergistic for the T cell. These results suggest that the CD2/LFA-3 interaction not only plays a role in cell-cell adhesion but provides a stimulatory signal for T cell activation.

scholarly journals Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells

2020 ◽  
Author(s):  
Gabor Tajti ◽  
Tibor Gabor Szanto ◽  
Agota Csoti ◽  
Greta Racz ◽  
César Evaristo ◽  
...  
Keyword(s):  
T Cells ◽  
Ion Channels ◽  
Patch Clamp ◽  
Cell Surface ◽  
Ion Channel ◽  
Immune Cells ◽  
Cell Sorting ◽  
Membrane Capacitance ◽  
Suitable Method ◽  
Kinetics Of

Abstract Background:Ion channels play emerging role in the physiological and pathological function in many cell types, including the non-excitable immune cells. As immune cells represent a heterogeneous population with considerable functional diversity, subtype-specific functional studies, such as single-cell electrophysiology of ion channels (patch-clamp), require proper subset identification and separation. Magnetic-activated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS on patch-clamp experiments were not studied yet. Among others, the presence of MACS-related beads on the cell surface may alter the biophysical and pharmacological parameters of the ion channels expressed in the membrane. This motivated our experiments to describe the feasibility of MACS for electrophysiology studies on immune cells. We examined the biophysical and pharmacological parameters of the voltage gated Kv1.3 K+ channel in activated CD4+ T-cells as well as the membrane capacitance following immunomagnetic positive separation, using the REAlease® kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads and the label-free configuration following removal of the REAlease® complex congaing the antibody fragment that recognizes CD4. As controls we used FACS separation as well as immunomagnetic negative selection.Results:We found similar purity and cell viability using FACS or MACS-based positive (REAlease®) and negative selection approaches. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were oblivious to the presence of the beads or any components of the REAlease® kit on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current in cells obtained using various cell isolation configurations that could not be explained by the presence of MACS-related compounds on the cells. Neither the equilibrium block of Kv1.3 by TEA or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Conclusions:Taken together our results support, that MACS is a suitable method for studying ion channel in non-excitable cells, such as T-lymphocytes with the benefit of easy application and relatively low instrumentation costs.

scholarly journals Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae of Brugia pahangi

2002 ◽  
Vol 70 (11) ◽  
pp. 5997-6004 ◽  
Author(s):  
Richard A. O'Connor ◽  
Eileen Devaney
Keyword(s):  
Nitric Oxide ◽  
T Cells ◽  
Cell Surface ◽  
Proliferative Response ◽  
No Synthase ◽  
Wild Type ◽  
Activated T Cells ◽  
Brugia Pahangi ◽  
Interferon Receptor ◽  
Almost All

ABSTRACT Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematode Brugia pahangi results in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4hi). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4hi cells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4hi cells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-γR−/−) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4+ T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4+ T cells from IFN-γR−/− mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO.

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