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1. Abstract 5376: Protein structure-based modeling to improve MHC class I epitope predictions

   https://doi.org/10.1158/1538-7445.am2023-5376  

   Wilson, Eric A ; Cava, John Kevin ; Chowell, Diego ; Singharoy, Abhishek ; Anderson, Karen S ( April 2023 , Cancer Research)

   The ability to accurately identify peptide ligands for a given major histocompatibility complex class I (MHC-I) molecule has immense value for targeted anticancer therapeutics. However, the highly polymorphic nature of the MHC-I protein makes universal prediction of peptide ligands challenging due to lack of experimental data describing most MHC-I variants. To address this challenge, we have developed a deep convolutional neural network, HLA-Inception, capable of predicting MHC-I peptide binding motifs using electrostatic properties of the MHC-I binding pocket. By approaching this immunological issue using molecular biophysics, we measure the impact of sidechain arrangement and topology on peptide binding, feature not captured by sequence-based MHC-I prediction methods. Through a combination of molecular modeling and simulation, 5821 MHC-I alleles were modeled, providing extensive coverage across human populations. Predicted peptide binding motifs fell into distinct clusters, each defined with different degrees of submotif heterogeneity. Peptide binding scores generated by HLA-Inception are strongly correlated with quantitative MHC-I binding data, indicating predicted peptides can be ranked, both within and between alleles. HLA-inception also showed high precision when predicting naturally presented peptides and can be used for rapid proteome-scale MHC-I peptide binding predictions. Finally, we show that the binding pocket diversity measured by HLA inception predicts response to checkpoint blockade.

   Citation Format: Eric A. Wilson, John Kevin Cava, Diego Chowell, Abhishek Singharoy, Karen S. Anderson. Protein structure-based modeling to improve MHC class I epitope predictions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5376.

    

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2. Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents

   https://doi.org/10.1002/cpz1.36  

   Willis, Richard A. ; Ramachandiran, Vasanthi ; Shires, John C. ; Bai, Ge ; Jeter, Kelly ; Bell, Donielle L. ; Han, Lixia ; Kazarian, Tamara ; Ugwu, Kyla C. ; Laur, Oskar ; et al ( February 2021 , Current Protocols)

   Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Lentivirus production and expression line creation

   Support Protocol 1: Six‐well assay for estimation of production cell line yield

   Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

   Basic Protocol 2: Cultures for production of Class II MHC proteins

   Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

   Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

   Support Protocol 3: Protein concentration measurements and adjustments

   Support Protocol 4: Polishing purification by anion‐exchange chromatography

   Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

   Basic Protocol 4: Peptide exchange

   Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

   Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

   Basic Protocol 6: Multimerization

   Basic Protocol 7: Staining cells with Class II MHC tetramers

    

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3. Rapid microsphere‐assisted peptide screening (MAPS) of promiscuous MHCII‐binding peptides in Zika virus envelope protein

   https://doi.org/10.1002/aic.16697  

   Smith, Mason R. ; Bugada, Luke F. ; Wen, Fei ( July 2019 , AIChE Journal)

   Despite promising developments in computational tools, peptide‐class II MHC (MHCII) binding predictors continue to lag behind their peptide‐class I MHC counterparts. Consequently, peptide–MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high‐throughput semiquantitative peptide–MHCII screening strategy termed microsphere‐assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4⁺T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.

    

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4. Systematic genetic analysis of the MHC region reveals mechanistic underpinnings of HLA type associations with disease

   https://doi.org/10.7554/eLife.48476  

   D'Antonio, Matteo ; Reyna, Joaquin ; Jakubosky, David ; Donovan, Margaret KR ; Bonder, Marc-Jan ; Matsui, Hiroko ; Stegle, Oliver ; Nariai, Naoki ; D'Antonio-Chronowska, Agnieszka ; Frazer, Kelly A ( November 2019 , eLife)

   The MHC region is highly associated with autoimmune and infectious diseases. Here we conduct an in-depth interrogation of associations between genetic variation, gene expression and disease. We create a comprehensive map of regulatory variation in the MHC region using WGS from 419 individuals to call eight-digit HLA types and RNA-seq data from matched iPSCs. Building on this regulatory map, we explored GWAS signals for 4083 traits, detecting colocalization for 180 disease loci with eQTLs. We show that eQTL analyses taking HLA type haplotypes into account have substantially greater power compared with only using single variants. We examined the association between the 8.1 ancestral haplotype and delayed colonization in Cystic Fibrosis, postulating that downregulation of RNF5 expression is the likely causal mechanism. Our study provides insights into the genetic architecture of the MHC region and pinpoints disease associations that are due to differential expression of HLA genes and non-HLA genes. 

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5. Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

   https://doi.org/10.1002/eji.201948126  

   Becerra‐Artiles, Aniuska ; Cruz, John ; Leszyk, John D. ; Sidney, John ; Sette, Alessandro ; Shaffer, Scott A. ; Stern, Lawrence J. ( May 2019 , European Journal of Immunology)

   Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3⁺CD4⁺, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.

    

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