Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Competition-Based Cell Assay Employing Soluble T Cell Receptors to Assess MHC Class II Antigen Processing and Presentation.

ABSTRACT: Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time and labor-intensive while in silico methodologies have limitations. Here, we propose a novel cell-based assay capable of investigating an antigen's endosomal processing and MHC class II presentation capabilities. This novel assay relies on competition between epitopes for MHC class II binding and employs labeled soluble T cell receptors (sTCRs) as detectors of epitope presentation.

SUBMITTER: Tasker C

PROVIDER: S-EPMC7812987 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Competition-Based Cell Assay Employing Soluble T Cell Receptors to Assess MHC Class II Antigen Processing and Presentation.

Tasker Carley C Patel Jenny J Jawa Vibha V Maamary Jad J

The AAPS journal 20210118 2

Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time ...[more]

PMID: 33459871

Similar Datasets

Redirecting soluble antigen for MHC class I cross-presentation during phagocytosis.

Project description:Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.

| S-EPMC4324331 | biostudies-literature

Suppression of the macrophage proteasome by ethanol impairs MHC class I antigen processing and presentation.

Project description:Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

| S-EPMC3581560 | biostudies-literature

Enterovirus Competition Assay to Assess Replication Fitness.

Project description:In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.

| S-EPMC6561778 | biostudies-literature

CD8+ T-cell cross-competition is governed by peptide-MHC class I stability.

Project description:A major contributing factor to the final magnitude and breadth of CD8(+) T-cell responses to complex antigens is immunodomination, where CD8(+) T cells recognizing their cognate ligand inhibit the proliferation of other CD8(+) T cells engaged with the same APC. In this study, we examined how the half-life of cell surface peptide-MHC class I complexes influences this phenomenon. We found that primary CD8(+) T-cell responses to DNA vaccines in mice are shaped by competition among responding CD8(+) T cells for nonspecific stimuli early after activation and prior to cell division. The susceptibility of CD8(+) T cells to 'domination' was a direct correlate of higher kinetic stability of the competing CD8(+) T-cell cognate ligand. When high affinity competitive CD8(+) T cells were deleted by self-antigen expression, competition was abrogated. These findings show, for the first time to our knowledge, the existence of regulatory mechanisms that direct the responding CD8(+) T-cell repertoire toward epitopes with high-stability interactions with MHC class I molecules. They also provide an insight into factors that facilitate CD8(+) T-cell coexistence, with important implications for vaccine design and delivery.

| S-EPMC3744744 | biostudies-literature

Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation.

Project description:Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell's own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors-tapasin for class I and HLA-DM for class II-contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity.

| S-EPMC5355494 | biostudies-literature

B Cell-Mediated Antigen Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression.

Project description:Depletion of B cells attenuates plaque development and modulates T cell responses in mouse models of atherosclerosis, suggesting that Ag presentation by B cells may promote disease progression. Thus, we set out to determine the role of B cell-mediated MHC class II (MHC II) Ag presentation during atherosclerotic plaque development. We developed murine conditional MHC II deletion and expression systems under control of the B cell-restricted CD19 promoter in an experimental model of atherosclerosis. Mice lacking MHC II expression only on B cells exhibited systemic shifts in germinal center and marginal zone B cell populations, leading to a reduced Ab response compared with littermate control animals. However, all populations were present and normal cholesterol uptake was detected in the plasma following high-fat diet treatment. In a second model, in which conditional expression of MHC II is limited only to B cells, showed similar overall cellularity characteristics compared with mice with complete MHC II deficiency. High-fat diet feeding showed no major changes in atherosclerotic plaque size or plaque cellular content in either conditional deletion or conditional expression approaches, compared with control animals. By testing the necessity and sufficiency of MHC II on B cells in the progression of atherosclerosis, we determine that MHC II on B cells does not directly regulate lesion development in murine models.

| S-EPMC6999615 | biostudies-literature

Dendritic cell preactivation impairs MHC class II presentation of vaccines and endogenous viral antigens.

Project description:When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at least in vitro. Such impairment of MHC class II (MHC II) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs matured in vivo could still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activation in vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II-peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections.

| S-EPMC2077019 | biostudies-literature

A CD74-dependent MHC class I endolysosomal cross-presentation pathway.

Project description:Immune responses are initiated and primed by dendritic cells (DCs) that cross-present exogenous antigen. The chaperone CD74 (invariant chain) is thought to promote DC priming exclusively in the context of major histocompatibility complex (MHC) class II. However, we demonstrate here a CD74-dependent MHC class I cross-presentation pathway in DCs that had a major role in the generation of MHC class I-restricted, cytolytic T lymphocyte (CTL) responses to viral protein- and cell-associated antigens. CD74 associated with MHC class I in the endoplasmic reticulum of DCs and mediated the trafficking of MHC class I to endolysosomal compartments for loading with exogenous peptides. We conclude that CD74 has a previously undiscovered physiological function in endolysosomal DC cross-presentation for priming MHC class I-mediated CTL responses.

| S-EPMC4933585 | biostudies-literature

MHC Class II-restricted antigen presentation by plasmacytoid dendritic cells drives proatherogenic T cell immunity.

Project description:Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive.Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4(-/flox) bone marrow transplanted into Ldlr(-/-) mice. Compared with control Ldlr(-/-) chimeric mice, CD11c-Cre × Tcf4(-/flox) mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr(-/-) mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100-specific CD4(+) T cells in response to native low-density lipoprotein, whereas production of interferon-? was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell-derived interferon-? and lesional T cell infiltration, and was abrogated in CD4(+) T cell-depleted animals.This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity.

| S-EPMC4428652 | biostudies-literature

Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.

Project description:Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.

| S-EPMC3458372 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data