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

Overall, Sarah A.; Toor, Jugmohit; Hao, Stephanie; Yarmarkovich, Mark; Nguyen, Son; Japp, Alberto Sada; Moschidi, Danai; Betts, Michael R.; Maris, John M.; Smibert, Peter; Sgourakis, Nikolaos G.

doi:10.1101/653477

Cited by 6 publications

(9 citation statements)

References 33 publications

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“…However, while these platforms utilize different methods to assure single-cell resolution, and use different approaches to label the cells, they all use high-throughput sequencing to count signal from a variety of oligo-conjugated probes (such as antibodies, MHC-peptide multimers, B-cell receptor antigens etc.) [1,2,5,[12][13][14]. Most of the observations, results and conclusions from this study will thus also be applicable to a variety of platforms where improving oligo-conjugated probe signal is critical for ensuring their broad utility and economic feasibility.…”

Section: Discussionmentioning

confidence: 99%

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Buus

Herrera

Ivanova

et al. 2020

Preprint

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Simultaneous measurement of surface proteins and gene expression within single cells offers highresolution snapshots of complex cell populations. These methods rely on staining cells with oligoconjugated antibodies analogous to staining for flow-and mass cytometry. Unlike flow-and mass cytometry, signal from oligo-conjugated antibodies is not hampered by spectral overlap or limited by the number of metal isotopes, making it a highly sensitive and scalable approach. Signal from oligoconjugated antibodies is quantified by counting reads from high-throughput sequencing. Consequently, cost of sequencing is strictly dependent on the signal intensities and background from the pool of antibodies used in analysis. Thus, considering the "cost-of-signal" as well as optimizing "signal-tonoise", makes titration of oligo-conjugated antibody panels more complex and even more important than for flow-and mass cytometry. In this study, we investigated the titration response of a panel of oligo-conjugated antibodies towards four variables: Antibody concentration, staining volume, cell number at staining, and tissue of origin. We find that staining with high antibody concentrations recommended by published protocols and commercial vendors cause unnecessarily high background signal and that concentrations of many antibodies can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting highly abundant epitopes used at low concentrations and can be counteracted by reducing cell numbers at staining. We find that background signal from empty droplets can account for a major fraction of the total sequencing reads and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background signal of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved.

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Section: Discussionmentioning

confidence: 99%

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Buus

Herrera

Ivanova

et al. 2020

Preprint

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“…However, due to substantial sequence variability in surface-exposed residues at the P2-P8 positions, we observe a range of electrostatic features ranging from negative (epitope TMADLVYAL), to neutral (NLIDSYFVV) or positively charged (KLWAQCVQL). Further classification and ranking of the top binders in our set on the basis of their molecular surface features would enable the selection of the most diverse panel of peptides for high-throughput pMHC tetramer library generation (23–25). Tetramer screening of T cells from COVID-19 patients, recovered individuals and healthy donors can be used to identify critical gaps in the T cell repertoire of high-risk groups, and to design epitope DNA strings for vaccine development.…”

Section: Resultsmentioning

confidence: 99%

“…The computed binding energies of our models can be used as an additional validation layer to select high-affinity epitopes from large peptide sets. As detailed epitope mapping data from high-throughput tetramer staining (23–25) and T cell functional screens (26) become available, the models presented here can provide a toehold for understanding links between pMHC-I antigen structure and immunogenicity, with actionable value for the development of peptide vaccines to combat the disease.…”

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confidence: 99%

Structure-based modeling of SARS-CoV-2 peptide/HLA-A02 antigens

Nerli

Sgourakis

2020

Preprint

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11As a first step toward the development of diagnostic and therapeutic tools to fight the Coronavirus 12 disease , it is important to characterize CD8+ T cell epitopes in the SARS-CoV-2 13 peptidome that can trigger adaptive immune responses. Here, we use RosettaMHC, a comparative 14 modeling approach which leverages existing high-resolution X-ray structures from peptide/MHC 15 complexes available in the Protein Data Bank, to derive physically realistic 3D models for high- 16affinity SARS-CoV-2 epitopes. We outline an application of our method to model 439 9mer and 17 279 10mer predicted epitopes displayed by the common allele HLA-A*02:01, and we make our 18 models publicly available through an online database (https://rosettamhc.chemistry.ucsc.edu). As 19 more detailed studies on antigen-specific T cell recognition become available, RosettaMHC 20 models of antigens from different strains and HLA alleles can be used as a basis to understand the 21 link between peptide/HLA complex structure and surface chemistry with immunogenicity, in the 22 context of SARS-CoV-2 infection. 24An ongoing pandemic caused by the novel SARS coronavirus (SARS-CoV-2) has become the 25 focus of extensive efforts to develop vaccines and antiviral therapies (1). Immune modulatory 26 interferons, which promote a widespread antiviral reaction in infected cells, and inhibition of pro-27 inflammatory cytokine function through anti-IL-6/IL-6R antibodies, have been proposed as 28 possible COVID-19 therapies (2, 3). However, stimulating a targeted T cell response against 29 specific viral antigens is hampered by a lack of detailed knowledge of the immunodominant 30 epitopes displayed by common Human Leukocyte Antigen (HLA) alleles across individuals 31 (public epitopes). The molecules of the class I major histocompatibility complex (MHC-I, or HLA 32 in humans) display on the cell surface a diverse pool of 8 to 15 amino acid peptides derived from 33 the endogenous processing of proteins expressed inside the cell (4). This MHC-I restriction of 34 peptide antigens provides jawed vertebrates with an essential mechanism for adaptive immunity: 35 surveillance of the displayed peptide/MHC-I (pMHC-I) molecules by CD8+ cytotoxic T-36 lymphocytes allows detection of aberrant protein expression patterns, which signify viral infection 37 and can trigger an adaptive immune response (5). A recent study has shown important changes in 38 T cell compartments during the acute phase of SARS-CoV-2 infection (6), suggesting that the 39 ability to quantify antigen-specific T cells would provide new avenues for understanding the 40 (which was not certified by peer review) : bioRxiv preprint 2 expansion and contraction of the TCR repertoire in different disease cohorts and clinical settings. 41Given the reduction in breadth and functionality of the naïve T cell repertoire during aging (7), 42 identifying a minimal set of viral antigens that can elicit a protective response will enable the 43 design of diagnostic tools to monitor critical gaps in the T cell repertoir...

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“…To enable real biophysical characterization, different pMHC-I molecules have to be produced using recombinant methods [1, [47][48][49][50][51]. Stable pMHC-I molecules are traditionally used in cytometry for probing CD8 + T cells but unsuitable reagents for peptide-exchange.…”

Section: Favoring Biophysical Characterization With Easier Pmhc-i Promentioning

confidence: 99%

“…Thus Bakker et al used a photolabile peptide cleavable by UV irradiation to make empty MHC-I molecules to enable peptide exchange [51]. Similarly, advances in peptide-exchange technologies include peptide deficient MHC-I/TAPasin binding protein related complexes and thermal exchangeable pMHC-I molecules to overcome possible photodamage using UV cleavable pMHC-I molecules [49,50]. More importantly, to The SCT is a single assembly of the peptide, β2-microglobulin and the α-chain, each separated by a linker.…”

Section: Favoring Biophysical Characterization With Easier Pmhc-i Promentioning

confidence: 99%

Expanding the Cancer Neoantigen Peptide Repertoire beyond In silico Tools

2021

J Cancer Immunol

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

Cited by 6 publications

References 33 publications

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Structure-based modeling of SARS-CoV-2 peptide/HLA-A02 antigens

Expanding the Cancer Neoantigen Peptide Repertoire beyond In silico Tools

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