Structural basis of NKT cell inhibition using the T-cell receptor-blocking anti-CD1d antibody 1B1

Ge Ying,Jing Wang,Thierry Mallevaey,Serge Van Calenbergh,Dirk M Zajonc

doi:10.1074/jbc.ra119.009403

Abstract

Natural killer T (NKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the CD1d molecule (CD1d). They rapidly respond to antigen challenge and can activate both innate and adaptive immune cells. To study the role of antigen presentation in NKT cell activation, previous studies have developed several anti-CD1d antibodies that block CD1d binding to T-cell receptors (TCRs). Antibodies that are specific to both CD1d and the presented antigen can only be used to study the function of only a limited number of antigens. In contrast, antibodies that bind CD1d and block TCR binding regardless of the presented antigen can be widely used to assess the role of TCR-mediated NKT cell activation in various disease models. Here, we report the crystal structure of the widely used anti-mouse CD1d antibody 1B1 bound to CD1d at a resolution of 2.45 Å and characterized its binding to CD1d-presented glycolipids. We observed that 1B1 uses a long hydrophobic H3 loop that is inserted deep into the binding groove of CD1d where it makes intimate nonpolar contacts with the lipid backbone of an incorporated spacer lipid. Using an NKT cell agonist that has a modified sphingosine moiety, we further demonstrate that 1B1 in its monovalent form cannot block TCR-mediated NKT cell activation, because 1B1 fails to bind with high affinity to mCD1d. Our results suggest potential limitations of using 1B1 to assess antigen recognition by NKT cells, especially when investigating antigens that do not follow the canonical two alkyl-chain rule.

Highlights

Natural killer T (NKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the CD1d molecule (CD1d)
Some antibodies are specific for the major histocompatibility complex (MHC) molecule and the antigen, such as the natural killer T (NKT) cell antigen receptor-blocking antibody L363 that recognizes mouse (m)CD1d presenting ␣-galactosylceramide (␣GalCer), the anti-mouse CD1d (mCD1d) antibody 1B1 binds to mCD1d regardless of the presented antigen [9]
In this study we have determined the crystal structure of the 1B1 Fab bound to mouse CD1d. 1B1 binds to mCD1d in a perpendicular binding orientation across the FЈ pocket, directly overlapping with the binding site of the T-cell receptors (TCRs) of iNKT cells but with minimal overlap with the binding site of the Type II NK TCR clone XV19 that binds above the AЈ pocket of CD1d (31, 34 –37)

Summary

To whom correspondence should be addressed

Some antibodies are specific for the MHC molecule and the antigen, such as the natural killer T (NKT) cell antigen receptor-blocking antibody L363 that recognizes mouse (m)CD1d presenting ␣-galactosylceramide (␣GalCer), the anti-mCD1d antibody 1B1 binds to mCD1d regardless of the presented antigen [9]. Type I NKT cells maintain a conserved binding footprint centered over the FЈ pocket of CD1d, whereas the sulfatide-restricted Type II NKT cell clone XV19 binds over the AЈ pocket (29 –33). The anti-mCD1d antibody 1B1 is able to block both TCR-mediated activation of Type I and Type II NKT cells, suggesting it has an overlapping binding site with the TCRs from both NKT cell subsets. To understand how 1B1 binds to mCD1d and blocks both Type I and Type II NKT cell activation, we assessed the binding of 1B1 to CD1d presenting different glycolipids using surface plasmon resonance studies. We have further assessed the ability of 1B1 Fab and 1B1 IgG in blocking Type I and Type II NKT cell responses toward different lipid antigens and determined the crystal structure of 1B1 Fab bound to mCD1d at a resolution of 2.45 Å

Results

Discussion

Experimental procedures