Payneet al.,Science 369 , 942–949 (2020) 21 August 2020 3of8
Fig. 2. BTN3A1-specific antibodies relieveabT cell inhibition and activate
Vg9Vd2Tcells.(A) Proliferation and IFN-grelease of CD4+and CD8+T cells
cultured with OKT3-loaded (500 ng/ml) BTN3A1-K32 or mock-K32 cells at a 10:1ab
T cell:K32 ratio after 6 days. (B) Proliferation and IFN-grelease of NY-ESO-1 TCR+
T cells cultured with HLA-A2+BTN3A1-K32 cells, or HLA-A2+mock-K32 cells, loaded
with 1mM SLLMWITQV peptide at a 10:1 T cell:K32 ratio after 6 days. (C)
Proliferation and IFN-grelease after 6 days of NY-ESO-1 TCR+T cells cultured at a
10:1 T cell:K32 ratio with HLA-A2+BTN3A1-K32 cells previously treated, or not, with
zoledronate (Zol; 1mM), loaded with 1mM SLLMWITQV peptide, and preincubated
with 1mg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or 103.2). (D)Specific
killing of NY-ESO-1 TCR+T cells cocultured with OVCAR3Lucicells pretreated
with 1mM zoledronate, or not, and preincubated with 1mg/ml isotype control or anti-
CD277 (CTX-2026, 20.1, or 103.2) at a 10:1abT cell:target cell ratio for 6 hours.
Data are presented as the fold change relative to isotype control. (E)Cytotoxicityof
immunopurified tumor-infiltrating CD3+abT cells from three HGSOC samples
after coculturing with matched tumor cells pretreated with 1mM zoledronate, or not,
andpreincubatedwith1mg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or
103.2) at a 5:1abT cell:target cell ratio for 12 hours. (F, left) Crystal structure of the
CTX-2026 interaction with CD277 homodimer in space-fill and ribbon diagram.
(Middle and right) Interface highlighting hydrogen bonds between CD277 and
CDRH2, CDRH3, and CDRL2 of CTX-2026 (middle) or CDRH1 of CTX-2026 (right).
(Bottom) Epitopes of CTX-2026 and 20.1 are shown, with common residues
highlighted in red (bottom). (G) Similar to the experiment shown in (D), except
immunopurifiedgdT cells were used at a 5:1gdT cell:target cell ratio for 24 hours.
(H) Similar to the experiment in (E), except immunopurifiedgdTcellswereused
at a 5:1gdT cell:target cell ratio for 24 hours. (I) Relative quantity ofBTN2A1mRNA in
BTN-K32 cells electroporated with CRISPR RNA targetingBTN2A1,ornot.(J)IFN-g
release from immunopurifiedgdT cells cocultured with BTN3A1-K32 cells, or
BTN3A1-K32BTN2A1-ablatedcells previously treated, or not, with zoledronate (1mM)
and preincubated with 1mg/ml isotype control or anti-CD277 (CTX-2026, 20.1, or
103.2) at a 5:1gdT cell:K32 ratio after 72 hours. (K) Proliferation and IFN-grelease of
immunopurifiedabT cells cultured with OKT3-loaded (500 ng/ml) BTN3A1-K32
or BTN3A1-K32BTN2A1-ablatedcells at a 10:1abT cell:K32 ratio after 6 days. For
all histogram panels, data are representative of three independent experiments with
similar results, and data represent means ± SEM. Statistical analysis was performed
as follows: for (B) and (I), two-tailed Student’sttest; for (A), (J), and (K), two-way
analysis of variance (ANOVA); for (C), (D), (E), (G), (H), and (K), one-way ANOVA.
ns, not significant; *P<0.05;**P≤0.01; ***P≤0.001.RESEARCH | RESEARCH ARTICLE