CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors

Abstract

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vβ11 TCRβ chain, where complementarity-determining region (CDR)3β is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3β sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3β sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease.

Key Residues at Third CDR3β Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs

“…Among the 54 SupT1 iNKT TCR transfectants (24), 2 clones, Cl.3007 and Cl.2133, exhibited noticeably lower staining by the pan anti-Vα24 mAb (clone C15), despite expressing comparable levels of CD3 (Supplementary Fig. 1).…”

“…Full-length cDNAs encoding the Vα24i, Vβ11, and CD1d genes were molecularly cloned via RT-PCR using gene-specific primers into the pMX vector (24, 27–29). Nucleotide sequencing was performed at the Centre for Applied Genomics, The Hospital for Sick Children (Toronto, Canada).…”

“…The Jurkat 76.clone 6 (Cl6) expressing Vα24i and low levels of CD1d was established by the limiting dilution method. Cl.6 cells were further transduced with various clonotypic TCR Vβ11 genes encoded by the 293GPG virus (24, 31). Jurkat 76 cells transduced with the HLA-A2/TAX-restricted TCR clone A6 was used as a control (32).…”

“…Then, CD1d-expressing K562-based artificial APCs (aAPCs) pulsed with 500 ng/mL of α-GalCer were irradiated (200 Gray) and added to the responder cells at a responder to stimulator ratio of 20:1 (day 0), as previously described (24). The T cells were restimulated every 7 days and supplemented with 100 IU/ml of IL-2 every three days.…”

“…Since CDR3β does not directly contact the ligand, previous studies have highlighted a ligand-non-selective role in its control of the overall affinity of iNKT TCRs for CD1d-lipid complexes (20, 22, 23). We have previously identified three CDR3β amino acid sequence motifs that are associated with greater human iNKT TCR auto-reactivity strength, regardless of the lipid presented by CD1d (24). Interestingly, studies of human iNKT TCR crystal structures have suggested an additional role for CDR3β in influencing antigen recognition indirectly via interactions with the CDR3α loop (21, 25).…”