University of Birmingham Butyrophilin-2A1 directly binds germline-encoded regions of the V9V2 TCR and is essential for phosphoantigen sensing

V g 9V d 2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufﬁcient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1’s P-Ag sensing function. These experiments identiﬁed butyrophilin-2A1 (BTN2A1) as essential to V g 9V d 2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for V g 9V d 2 TCR-mediated responses. Surface plasmon resonance experiments established V g 9V d 2 TCRs used germline-encoded V g 9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to V g 9V d 2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the V g 9V d 2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner. overexpressed, puriﬁed refolded as described et al., 2019). BTN2A1 BTN2A2 IgV domains L-Arginine-HCl, 2 mM EDTA, mM cystamine, 2.7 mM cysteamine, mM PMSF, and puriﬁed exclusion chromatography on a Superdex-200 (GE Healthcare) pre-equilibrated with mM Tris, mM NaCl, pH or mM Na3PO4 pH BTN3A1 IgV refolded, gd nickel chromatography previously SDS-PAGE gels (BioRad). Proteins were transferred to PVDF using the BioRad TransBlot Turbo system, blocked in 3% BSA, then incubated with streptavidin-HRP (Thermo). To investigate potential association of BTN2A1 and BTN3A1 at the cell surface, CHO cells overexpressing BTN2A1-HA, FLAG-BTN3A1, or both, were treated with the soluble, membrane-impermeable crosslinker sulfo-EGS (ThermoFisher) at 0.5mM in PBS, at 4 (cid:4) C for 2 h. Following this, the reaction was quenched by addition of Tris pH 7.5 to 20mM. Cells were washed in TBS and lysed in 1% NP40 lysis buffer. After centrifugation to remove insoluble material, immunoprecipitation was carried out using anti-HA resin or 20.1 antibody bound to protein A Sepharose (GE Healthcare). Immunoprecipitates were run on duplicate 4%–20% gels (BioRad) and blotted with anti-BTN2A1 or anti-FLAG antibodies. residue (V58) is likely to be energetically unfavorable for binding in this region. By substituting an Ala (ie a non-polar residue) at this position, the S72A mutation is likely to introduce hydrophobic interactions with V58, consistent with enhanced binding compared to wild-type (11-15 m M (S72A) versus 50 m M (Wild-type)). K79A leads to reduced binding to TCR (100 m M). K79 forms a salt bridge interaction with E76 (TCR). Change to Ala will result in loss of this interaction consistent with a reduction in binding afﬁnity. The fact that binding is not totally abolished suggests that this interaction is a not a major contributor to the binding energy. Note however that E76 also contacts R65 (see above). R124A mutation in BTN2A1 abolishes binding to TCR. The HADDOCK model suggests that R124 forms a salt bridge interaction 9-IgV TCR) bonding hydroxyl group of T83. These interactions will be lost when