Endogenous Ligands Selectively Stimulate Highly Avid Autoreactive Human Invariant Natural Killer T Cells with Distinctive T-Cell Receptor Vβ11 CDR3 Sequence Motifs
Authors: Imataki Osamu , Tanaka Makito , Berezovskaya Alla , Butler Marcus O. , Nadler Lee M. , Hirano Naoto 
Published: October 2019

Abstract
Abstract 999
Human CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of innate T-cells that carry an invariant T-cell receptor (TCR) Vα24 chain paired with a TCR Vβ11 chain. They constitutively express a variety of activation and memory markers and possess the capacity to rapidly produce a variety of cytokines including IFN-γ and IL-4 upon TCR engagement. As an immune response launches, iNKT cells can induce innate immune responses and serve as a bridge between innate and adaptive immunity. In addition, it has also been shown that iNKT cells themselves have anti-tumor and anti-infectious effects in mice. However, to translate these findings and develop iNKT cell-mediated immunotherapy, iNKT cells must recognize target cells while sparing normal cells. Autoreactivity is primarily dictated by 1) the specificity and avidity of the TCR on iNKT cells; 2) the expression level of CD1d on target cells; and 3) the density and stability of endogenous ligand(s) presented by CD1d on target cells.
In this study, we sought to determine the TCR Vβ11 CDR3 sequence motifs that distinguish high avidity and low avidity iNKT cells. We developed an artificial antigen-presenting cell (aAPC) to expand iNKT cells by transducing K562 with CD1d, CD80, and CD83. Using this CD1d+aAPC loaded with or without α-galactosylceramide (αGC), we expanded CD1d-restricted iNKT cells by stimulating primary CD3+ T cells expressing canonical iNKT TCR Vα24 chain. Expanded iNKT cells were stained with αGC-loaded CD1d tetramers and were found to express TCR Vβ11 chain in conjunction with transgenic canonical TCR Vα24 chain. Sequence analysis of cloned Vβ11 CDR3 revealed that the clonality of iNKT cells generated using unloaded aAPC was significantly lower than that of iNKT cells generated using loaded aAPC. Surprisingly, when cotransfected with canonical TCR Vα24 chain, some TCR Vβ11 chains isolated from iNKT cells generated using unloaded aAPC were stained with unloaded CD1d tetramers produced in HEK293 cells. This result suggests that these reconstituted iNKT TCR recognized endogenous ligand(s) derived from HEK293 cells in the context of CD1d. A comprehensive analysis of the structural avidity demonstrated that these TCR Vβ11 chains reconstituted TCR with significantly higher structural avidity than those cloned from NKT cells generated using loaded aAPC. However, this significant difference was only observed when the structural avidity was measured using unloaded tetramers but not αGC-loaded tetramers. A univariate analysis found that structural avidity was significantly higher when 1) Vβ11 CDR3 used J2-5; 2) Vβ11 CDR3 consisted of exactly 23 amino acids; and 3) Vβ11 CDR3 encoded 3 or more acidic amino acids (asparagic and glutamic acids). A multivariate analysis confirmed that all three variables were independent predictors of higher structural avidity. Furthermore, each variable is sufficient to increase the structural avidity of an iNKT TCR with low structural avidity. Intriguingly, unloaded mouse CD1d tetramers produced in HEK293 cells also stained a human iNKT TCR with high structural avidity reconstituted on both human and mouse T cells. This result suggests that the human iNKT TCR with high structural avidity possessed a cross-species reactivity to mouse CD1d in conjunction with endogenous ligand(s) derived from HEK293 cells.
We next studied the autoreactivity of cloned human iNKT TCR with high structural avidity. A human T cell line, Jurkat, reconstituted with high avidity iNKT TCR, was able to recognize cells expressing CD1d endogenously (itself, SUP-T1, and primary human monocytes) and ectopically (K562, C1R, Hela). Furthermore, mouse T cells expressing human iNKT TCR with high structural avidity recognized mouse cell lines, B16, EL4, and 58, all endogenously expressing CD1d. Finally, human primary T cells transduced with the iNKT TCR with high structural avidity were autoreactive, secreting both IFN-γ and IL-4 in response to CD1d+ target cells.
Using our CD1d+ aAPC-based system, we successfully isolated human iNKT cells with high structural avidity and identified the TCR Vβ11 CDR3 sequence motifs that dictate the structural avidity of iNKT cells. To develop clinically effective iNKT cell-mediated immunotherapy without unwanted autoimmunity, it will be critically important to define the range of iNKT TCR avidity that enables reactivity to pathologic but not normal cells.

Disclosures:
No relevant conflicts of interest to declare.