scholarly journals High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

2019 ◽  
Author(s):  
Sarah A. Overall ◽  
Jugmohit S. Toor ◽  
Stephanie Hao ◽  
Mark Yarmarkovich ◽  
Son Nguyen ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput ◽  
Molecular Chaperone ◽  
Robust Method ◽  
Single Experiment ◽  
Mhc I ◽  
Combined Analysis ◽  
Transcription Profiles ◽  
Peptide Exchange ◽  
High Throughput Manner

ABSTRACTPeptide exchange technologies are essential for the generation of pMHC-multimer libraries, used to probe highly diverse, polyclonal TCR repertoires. Using the molecular chaperone TAPBPR, we present a robust method for the capture of stable, empty MHC-I molecules which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Combined with tetramer barcoding using multi-modal cellular indexing technology (ECCITE-seq), our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment.

scholarly journals HypercubeME: two hundred million combinatorially complete datasets from a single experiment

2019 ◽  
Author(s):  
Laura Avino Esteban ◽  
Lyubov R. Lonishin ◽  
Daniil Bobrovskiy ◽  
Gregory Leleytner ◽  
Natalya S. Bogatyreva ◽  
...  
Keyword(s):  
Experimental Data ◽  
High Throughput ◽  
Recursive Algorithm ◽  
Random Mutagenesis ◽  
Higher Order ◽  
Single Experiment ◽  
Manual Curation ◽  
Complete Dataset ◽  
Genotype Space ◽  
High Throughput Manner

AbstractMotivationEpistasis, the context-dependence of the contribution of an amino acid substitution to fitness, is common in evolution. To detect epistasis, fitness must be measured for at least four genotypes: the reference genotype, two different single mutants and a double mutant with both of the single mutations. For higher-order epistasis of the order n, fitness has to be measured for all 2n genotypes of an n-dimensional hypercube in genotype space forming a “combinatorially complete dataset”. So far, only a handful of such datasets have been produced by manual curation. Concurrently, random mutagenesis experiments have produced measurements of fitness and other phenotypes in a high-throughput manner, potentially containing a number of combinatorially complete datasets.ResultsWe present an effective recursive algorithm for finding all hypercube structures in random mutagenesis experimental data. To test the algorithm, we applied it to the data from a recent HIS3 protein dataset and found all 199,847,053 unique combinatorially complete genotype combinations of dimensionality ranging from two to twelve. The algorithm may be useful for researchers looking for higher-order epistasis in their high-throughput experimental data.Availabilityhttps://github.com/ivankovlab/HypercubeME.git.

scholarly journals Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules

2018 ◽  
Vol 115 (40) ◽  
pp. E9353-E9361 ◽  
Author(s):  
F. Tudor Ilca ◽  
Andreas Neerincx ◽  
Mark R. Wills ◽  
Maike de la Roche ◽  
Louise H. Boyle
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Receptor ◽  
Target Cells ◽  
Intact Cells ◽  
Mhc I ◽  
Immunogenic Peptides ◽  
Presentation Pathway ◽  
Peptide Exchange

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.

scholarly journals Identification of the targets of T cell receptor therapeutic agents and cells by use of a high throughput genetic platform

2018 ◽  
Author(s):  
Ron S. Gejman ◽  
Heather F. Jones ◽  
Martin G. Klatt ◽  
Aaron Y. Chang ◽  
Claire Y. Oh ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Cancer Therapeutics ◽  
Cell Receptor ◽  
Therapeutic Agents ◽  
Target Cells ◽  
Mhc I ◽  
Immunological Assays

T cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapeutics. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on major histocompatibility complex receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high throughput genetic platform (termed “PresentER”) that encodes MHC-I peptide minigenes for functional immunological assays as well as for determining the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this report, we demonstrate that PresentER can be used to identify the on-and-off targets of T cells and TCR mimic antibodies using in vitro co-culture assays or binding assays. We find dozens of MHC-I ligands that are cross-reactive with two TCR mimic antibodies and two native TCRs and that are not easily predictable by other methods.

scholarly journals Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system

2020 ◽  
Author(s):  
Hyeonseob Lim ◽  
Soyeong Jun ◽  
Minjeong Park ◽  
Junghak Lim ◽  
Jaehwan Jeong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Functional Screening ◽  
Single Experiment ◽  
Substitution Mutations ◽  
Genome Scale ◽  
High Throughput Manner ◽  
Substitution Mutation

ABSTRACTWe developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by co-utilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.

High-Throughput T-Cell Epitope Discovery Through MHC Peptide Exchange

2009 ◽  
pp. 383-405 ◽  
Author(s):  
Sine Reker Hadrup ◽  
Mireille Toebes ◽  
Boris Rodenko ◽  
Arnold H. Bakker ◽  
David A. Egan ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput ◽  
Cell Epitope ◽  
T Cell Epitope ◽  
Epitope Discovery ◽  
Peptide Exchange

scholarly journals P05.01 Organoid-specific optimization of killing assays to test novel immunotherapies in a high-throughput system

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A18-A18
Author(s):  
F van den Ham ◽  
WM Kholosy ◽  
K Ober ◽  
AM Cornel ◽  
S Nierkens ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Seeding Density ◽  
Mhc I ◽  
Car T Cells ◽  
Culture Time ◽  
Car T

BackgroundThe immunotherapeutic drug dinutuximab, which binds to disialoganglioside (GD2) and activates natural killer (NK) cells, is part of the standard regimen in high-risk neuroblastoma (NB) patients. However, dinutuximab only results in tumor reduction in a subset of patients, and survival rates of high-risk neuroblastoma patients are below 60%. Novel immunotherapies are therefore needed. Current in vitro models lack the ability to study novel immunotherapies with high-throughput screening (HTS). We aimed to optimize NB organoid-lymphocyte cocultures for HTS, and possibly personalized testing, of novel antibody-mediated and cellular immunotherapies.Materials and MethodsTwo patient-derived organoids (691B: GD2+MHC-I- and 691T: GD2-MHC-I+) were transduced with an endogenous luciferase construct to use D-luciferin-induced bioluminescence as readout for cell growth. The growth rate, optimal seeding density and optimal pre-culture time per organoid were determined by density curves, and the number of needed cells was downscaled to facilitate HTS. After pre-culture, the luciferase-transduced organoids were co-cultured with primary PBMCs from healthy donors, PRAME-TCR transduced T cells or CAR-T cells.1 Several effector:target (E:T) ratios and timepoints were tested to identify the optimal window for read-out of dinutuximab-induced antibody-dependent cytotoxicity (ADCC) and T-cell mediated cytotoxicity. The required number of immune cells per ratio was calculated based on the expansion rate of organoid cells after 48 and 72 hours.ResultsThe density screens showed an optimal seeding density of 5000-10.000 organoid cells per well, yielding a high luminescence signal while minimizing the number of cells needed. Already at the lowest E:T ratio (1:3), we observed killing of the MHC-I expressing 691T organoid, likely based on allogeneic recognition of the organoids by T cells. The killing efficacy increased with higher E:T ratios and co-culture time. Pre-culturing of organoids for 72 hours before addition of effector cells resulted in formation of larger 3D spheres, which reduced the killing efficacy for all E:T ratios. ADCC effects of dinutuximab were studied in GD2+MHC-I- 691B organoids. Addition of dinutuximab resulted in 25% increase of killing after 24 hours and reached up to 70% increase after 72 hours for 10:1 and 20:1 E:T ratios. Higher E:T ratios were likely needed because NK cells make up a smaller proportion of PBMCs than T cells. Dinutuximab did not increase killing of the GD2- organoid, confirming specificity of the antibody. T cell mediated killing was almost 100% for MHC-I+691T organoids after 24 hours of culturing with PRAME-TCR transduced T cells and CAR-T cells at a 1:3 E:T ratio, showing the high anti-tumor cytotoxicity of these cells and potential for HTS at very low E:T ratios.ConclusionsWe have developed a robust in vitro bioluminescence-based organoid/lymphocyte co-culture assay with a low cell input, to facilitate high-throughput screening of novel antibody-based or cellular immunotherapies, possibly combined with chemotherapeutic or targeted compounds. In the future this method may be applied for personalized drug screens.ReferenceAvital L Amir, et al. PRAME-Specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer. Clin Cancer Res 2011.Disclosure InformationF. van den Ham: None. W.M. Kholosy: None. K. Ober: None. A.M. Cornel: None. S. Nierkens: None. J. Anderson: None. J.J. Molenaar: None. J. Wienke: None.

Kinetic Solvent Effects in Organic Reactions

2017 ◽  
Author(s):  
Belinda Slakman ◽  
Richard West
Keyword(s):  
Solvent Effect ◽  
Reaction Kinetics ◽  
Computational Methods ◽  
High Throughput ◽  
Kinetic Modeling ◽  
Solvent Effects ◽  
Reaction Rates ◽  
Prior Work ◽  
Solvent Phase ◽  
High Throughput Manner

<div> <div> <div> <p>This article reviews prior work studying reaction kinetics in solution, with the goal of using this information to improve detailed kinetic modeling in the solvent phase. Both experimental and computational methods for calculating reaction rates in liquids are reviewed. Previous studies, which used such methods to determine solvent effects, are then analyzed based on reaction family. Many of these studies correlate kinetic solvent effect with one or more solvent parameters or properties of reacting species, but it is not always possible, and investigations are usually done on too few reactions and solvents to truly generalize. From these studies, we present suggestions on how best to use data to generalize solvent effects for many different reaction types in a high throughput manner. </p> </div> </div> </div>

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