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T Cells + Agonists for Memory Human CD8 Single-Chain Trimers Are Amino-Terminal Extended Peptide Single-Chain Trimers Are Potent Synthetic Agonists for Memory Human CD8+ T Cells This information is current as Beatriz M. Carreno, Michelle Becker-Hapak, Megan Chan, of September 26, 2021. Wen-Rong Lie, Xiaoli Wang, Ted H. Hansen and Gerald P. Linette J Immunol 2012; 188:5839-5849; Prepublished online 9 May 2012; doi: 10.4049/jimmunol.1103647 Downloaded from http://www.jimmunol.org/content/188/12/5839 Supplementary http://www.jimmunol.org/content/suppl/2012/05/09/jimmunol.110364 Material 7.DC1 http://www.jimmunol.org/ References This article cites 43 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/188/12/5839.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Amino-Terminal Extended Peptide Single-Chain Trimers Are Potent Synthetic Agonists for Memory Human CD8+ T Cells Beatriz M. Carreno,* Michelle Becker-Hapak,* Megan Chan,* Wen-Rong Lie,† Xiaoli Wang,‡ Ted H. Hansen,‡ and Gerald P. Linette* Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8+ T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8+ T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8+ T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC Downloaded from class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT- SCT– and SCT-activated T cells. Probing of SCT and AT-SCT peptide–MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide–MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8+ T cells. The Journal of Immunology, 2012, 188: 5839–5849. http://www.jimmunol.org/ he specificity of T cell activation is largely dictated by for activation (13, 14) and are more sensitive to cell death com- the interaction of the TCR with peptide–MHC (p-MHC) pared with naive resting T cells upon Ag exposure (15–17). T (1). TCR–p-MHC interactions can lead to a spectrum of MHC class I single-chain trimers (SCT) are fusion proteins, responses in mature T cells ranging from full activation, resulting which consist of an antigenic peptide-flexible linker–b2-micro- in clonal expansion (or cell death), to a state of anergy, resulting in globulin (b2m)-flexible linker-class I H chain polypeptide (18). unresponsiveness (2–4). These distinct outcomes have been at- The essential properties of SCT are multifold and include pres- tributed, in part, to differences in binding affinity or off-rates of ervation of the native structure of peptide/b2m/MHC class I, as the interaction between TCR and p-MHC (5–8). Single amino acid detected by CD8+ T cells and conformational-dependent mAbs, by guest on September 26, 2021 substitutions can create structural variants of antigenic peptide such as 25-D1.16 (19). Crystallographic data with the SIINFEKL/ b ligands, referred to as altered peptide ligands (APL), which allow b2m/K SCT confirm structural equivalence with free peptide bound b b manipulation of the strength of the TCR–p-MHC interaction. APL to K (20) and, importantly, SCT encoding SIINFEKL/b2m/K can induce a complete range of T cell functions (agonist) or a subset stimulates IL-2 secretion and vigorous proliferation of primed of T cell functions (partial agonist) (9, 10); in a few instances, it can CD8+ OT-1 transgenic T cells (21). Additionally, several indepen- be more potent (superagonist) than the native ligand (11, 12). It is dent studies demonstrated that, as DNA vaccines, SCT can elicit ro- also well established that memory T cells display a lower threshold bust CD8+ T cell responses to well-defined Ags in murine models (19). Recently, we reported on the biochemical stability of HLA *Division of Oncology, Department of Medicine, Washington University School class I-containing SCT and their serological recognition by of Medicine, St. Louis, MO 63110; †EMD Millipore, Billerica, MA 01822; and conformational-dependent Abs, as well as demonstrated their ‡ Department of Pathology and Immunology, Washington University School of Med- recognition by Ag-specific T cells (22). In the current study, we icine, St. Louis, MO 63110 examine whether SCT, expressed in the MHC class I/II-null cell Received for publication December 15, 2011. Accepted for publication April 5, 2012. line K562, may represent a p-MHC entity for ex vivo stimulation This work was supported in part by a grant from Washington University Bear Cub + Fund (to G.P.L.), a grant from the Barnes-Jewish Hospital Foundation (to G.P.L.), and of memory (Ag-primed) human CD8 T cells. From a series of National Institutes of Health Grant AI055849 (to T.H.H.). The Siteman Cancer Cen- SCT constructs generated, a new p-MHC entity, the amino-ter- ter High-Speed Cell Sorter Core Facility is supported in part by National Cancer minal extended peptide MHC class I single-chain trimer (AT-SCT), Institute Cancer Center Support Grant P30 CA91842. was identified as a potent inducer of human CD8+ T cell expansion Address correspondence and reprint requests to Dr. Beatriz M. Carreno, Division of Oncology, Washington University School of Medicine, 660 South Euclid Avenue, and growth. AT-SCT for two well-characterized Ags are described Box 8007, St. Louis, MO 63110. E-mail address: [email protected] in detail: the CMV pp65 antigenic peptide (NLVPMVATV) and the The online version of this article contains supplemental material. melanoma gp100 G209-2M antigenic peptide (IMDQVPFSV). AT- Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; APL, altered SCT delivers a TCR signal distinct from conventional native SCT, peptide ligand; AT-SCT, amino-terminal extended peptide MHC class I single- resulting in .400-fold expansion of Ag-specific human CD8+ chain trimer; AT-SCT dt, disulfide trap amino-terminal extended peptide MHC class T cells from healthy donors, as well as melanoma patients. I single-chain trimer; DC, dendritic cell; FSC, forward light scatter; b2m, b2-micro- globulin; mDC, mature dendritic cell; p-MHC, peptide–MHC; RICD, restimulation- induced cell death; RPMI-5, RPMI 1640 supplemented with 5% pooled human sera; SCD, single-chain dimer; SCT, MHC class I single-chain trimer; SCT dt, disulfide Materials and Methods trap MHC class I single-chain trimer; SSC, side scatter; Stemline-5, Stemline media Generation of SCT-expressing K562 transfectants supplemented with 5% pooled human sera. HLA-A*0201 SCT were generated, as previously described (22). For AT- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 SCT generation, synthetic DNA oligonucleotides (Integrated DNA Tech- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103647 5840 SYNTHETIC AGONISTS FOR CD8+ T CELLS nologies, Coralville, IA) encoding 10-mer antigenic peptides extended at of cytolytic granule content, cells were fix/permeabilized with Cytofix/ the N terminus were ligated into the SCT vectors at restriction sites spe- Cytoperm (BD), according to the manufacturer’s instructions, and stain- cifically designated for peptide sequence shuffling. Multiple amino acid ed with Abs to perforin, granzyme A (BioLegend), and granzyme B residues were introduced at the N terminus of optimal 9-mer peptide to (Invitrogen). Polyfunctionality of expanded Ag-specific T cells was de- generate AT-SCT. For NLV (CMV pp65), constructs were made containing termined as follows: 14 d after SCT or AT-SCT stimulation, T cells were the naturally occurring amino acid residue (ANLV, underline identifies restimulated with SCT at 1:1 ratio in RPMI 1640 supplemented with 5% amino acid residue at N terminus of peptide in AT-SCT) or amino acids pooled human sera (RPMI-5) and brefeldin A (10 mg/ml; eBioscience). As with bulky nonpolar (YNLV) and uncharged polar (NNLV) R groups, as controls, cells were cultured in media in the absence of stimulation. Six- well as nonbulky uncharged polar (GNLV) R group. The ANLV construct teen hours after stimulation, cells were harvested, washed, fix/perme- was poorly expressed on the cell surface and was not studied further; all abilized with Cytofix/Cytoperm, and stained with pretitered Abs specific other AT-SCT constructs were expressed on the cell surface at levels for IFN-g (BioLegend), TNF-a, IL-2 (eBioscience), and MIP-1b (BD).
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