High affinity soluble ILT2 receptor: a potent inhibitor of CD8<sup>+</sup> T cell activation

doi:10.1007/s13238-010-0144-5

Prot Cell

2010, Vol. 1

Issue (12) : 1118-1127 https://doi.org/10.1007/s13238-010-0144-5 PMID: 21213105

RESEARCH ARTICLE

High affinity soluble ILT2 receptor: a potent inhibitor of CD8⁺ T cell activation

Ruth K. Moysey^1,2, Yi Li¹, Samantha J. Paston¹, Emma E. Baston¹, Malkit S. Sami¹, Brian J. Cameron¹, Jessie Gavarret¹, Penio Todorov¹, Annelise Vuidepot¹, Steven M. Dunn^1,3, Nicholas J. Pumphrey^1,4, Katherine J. Adams¹, Fang Yuan¹, Rebecca E. Dennis¹, Deborah H. Sutton¹, Andy D. Johnson¹, Joanna E. Brewer^1,5, Rebecca Ashfield¹, Nikolai M. Lissin¹, Bent K. Jakobsen¹(

)

1. Immunocore Limited, 57c Milton Park, Abingdon, Oxon, OX14 4RX, UK; 2. Current address: Oxford Nanopore Technologies, UK; 3. Current address: Geneva Merck Serono S.A., Switzerland; 4. Current address: Adaptimmune Limited, UK; 5. Current address: Adaptimmune Limited, UK

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Abstract

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t_1/2) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8⁺ cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with sub-nanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8⁺ CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

Keywords CD8⁺ T cells cellular activation autoimmunity cell surface molecules binding affinity phage display

Corresponding Author(s): Jakobsen Bent K.,Email:bent.jakobsen@immunocore.com

Issue Date: 01 December 2010

Cite this article:

Yi Li,Ruth K. Moysey,Samantha J. Paston, et al. High affinity soluble ILT2 receptor: a potent inhibitor of CD8⁺ T cell activation[J]. Prot Cell, 2010, 1(12): 1118-1127.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-010-0144-5
https://academic.hep.com.cn/pac/EN/Y2010/V1/I12/1118

Fig.1 Engineering of ILT2-based antagonist of CD8 co-receptor.
(A) Concept sketch. Soluble ILT2 binds to conserved region of major histocompatibility complex I (MHC I), blocking its contact with CD8. Binding of T cell receptor (TCR) alone to the antigenic peptide presented on MHC I is insufficient for cytotoxic T lymphocyte (CTL) activation. (B) Crystal structure-based model of soluble ILT2 (immunoglobulin-like transcript 2) bound to peptide-MHC I complex. The MHC heavy chain is shown in blue, β2m in cyan, and antigenic peptide in pink. ILT2 domains, D1 (N-terminal) and D2, are shown in red, and N- and C- termini are indicated. Parts of the ILT2 chain, which have been subjected to mutagenesis, are shown in yellow. Numbers indicate the areas wherein mutation contributed to increased affinity. (Part of the ILT2 structure unresolved by X-ray crystallography is shown as a dotted loop.) On the corresponding wild type amino acid pane (bottom) the mutated areas are highlighted and the positions of affinity-enhancing mutations underlined. For bacterial expression a methionine-encoding start codon was added and the C-terminal sequence LVL was changed to DVDG to improve folding and stability of the protein.

Tab.1 Selected ILT2 variants and their HLA-A2 binding properties

Tab.2 Binding properties of two ILT2 variants toward a range of classical and non-classical MHC I complexes

Fig.2 Design and the ligand binding properties of soluble ILT2 dimers.
(A) Dimeric ILT2-based compounds: schematic representation of chemically cross-linked with PEG (left) genetically fused with the immunoglobulin Fc-domain (center). A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of a c138-Fc dimer sample under reducing and non-reducing conditions is also shown (right). (B) The binding of dimeric monomeric ILT2 c50 to MHC I (HLA-A2). Dimerization resulted in a dramatic enhancement of the ligand binding strength, with the increase from 7 s in c50 monomer to about 80 min in c50-PEG dimer. RU, response unit. (C) High affinity dimeric ILT2 prevents the interaction of CD8 with MHC I. CD8 binds with low affinity to MHC I. Injected over one flow cell, high affinity dimeric ILT2 (c138-Fc) binds to MHC I very strongly, cancelling the subsequent binding of CD8, but with no effect on the binding of the peptide-specific TCR. (D) High affinity dimeric ILT2 (c138-Fc) does not affect the binding affinity of TCR toward peptide-MHC I. Biacore trace illustrates the binding of a TCR (HTLV-1 Tax-specific) to its relevant peptide-MHC I, before and after the binding of c138-Fc. values, derived from equilibrium binding (not shown on the trace), were 2.7 μM (free peptide-MHC I as ligand) 3.0 μM (c138-Fc bound ligand), which is not statistically different. (E) Visualization, using fluorescence microscopy, of the high affinity dimeric ILT2 (c138-Fc) on the surface of MHC I expressing cells. MHC I-negative cell line, K562, served as control. (F) Time course of dissociation from cells of high affinity dimeric ILT2 (c138-Fc) measured by flow cytometry (>24 h). ILT2, immunoglobulin-like transcript 2; MHC, MHC, major histocompatibility complex; HLA, human leukocyte antigen; TCR, T cell receptor.

Fig.3 Inhibition of CTL responses by high affinity ILT2.
(A) Inhibition by ILT2 compounds of interferon γ (IFNγ) release from a CTL clone in the presence of target cells: monomeric dimeric tetrameric ILT2 c50. (B) Inhibition by ILT2 dimers of IFNγ release by a CTL clone in the presence of the target cells: affinity effect on potency. (C) Inhibition by c138-Fc of the release of inflammatory cytokines IL-2, tumor necrosis factor alpha (TNFα), IFNγ and MIP-1β. (D) Inhibition by ILT2 compounds of the killing of target cells by a CTL clone. MIP-1β, macrophage inflammatory protein-1β. The other abbreviations are the same as in Fig. 1 and 2.

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