B35-Q65 and TCR55a-A98H (fig. S3H). BFP
showed that B35-Q65A–HIV formed catch bonds
with TCR55a-A98H but exhibited shorter peak
bond lifetimes than the B35-HIV–TCR55a-A98H
interaction (fig. S3I).Calibrating TCR55 signaling strength by
bond lifetime
The acquisition of T cell activation by B35-
HIV(Pol) coincident with catch bond formation
by a single-point mutant of TCR55 provided an
opportunity to investigate structure-function
relationships between amino acid substitu-
tions and activation strength. We mutated
the TCR55a-A98 to 12 different amino acids to
investigate how residue identity at this posi-
tion affected the strength of TCR signaling. In
addition to histidine, mutations to aspartate,
glutamate, phenylalanine, glutamine, and tyro-
sine also enabled TCR55 signaling through B35-
HIV(Pol) engagement for lymphocyte activation,
albeit to different extents (Fig. 2E). By contrast,
mutations to cysteine, lysine, asparagine, argi-
nine, serine, threonine, and tryptophan did not
activate TCR55 (fig. S5A). Therefore, only select
polar, aromatic, and charged amino acids re-
placing residue TCR55a-A98 enabled effective
signaling in response to B35-HIV. To inves-
tigate whether there was a correlation between
signaling capacity and binding strength, we
measured the 3D affinity by SPR for each of
the different TCR55a-A98 mutants binding
to B35-HIV pMHC. Most mutants have a 3D
affinity in a narrow range betweenKD=3mM
andKD= 20mM (fig. S6 and table S1). Neither
the maximum CD69 MFI [coefficient of deter-
mination (R^2 ) = 0.1893] nor the median effective
concentration (EC 50 )(R^2 =0.02855)ofstimula-
tory mutants was correlated to the SPR affinity
of stimulatory mutants, which suggests that
3D affinity could not explain the gain of func-
tion exhibited by the stimulatory mutants
(Fig. 2F and fig. S5B). TCR55a-A98W (KD=
6.5mM), a variant that exhibited higher affinity
than WT-TCR55 (KD= 19mM), did not enable
TCR-dependent activation in response to B35-
HIV(Pol). Furthermore, the most ligand-sensitive
of the TCR mutants, TCR55a-A98H (KD=
5.9mM), did not have the highest affinity (Fig.
2F and table S1). Based on BFP measurements
for two B35-HIV responsive mutants—TCR55a-
A98E and TCR55a-A98Q (Fig. 2G)—we found
that the maximal effect (Emax) was correlated
with the peak bond lifetime (R^2 = 0.996) rather
than affinity (Fig. 2H). Thus, the strength of the
catch bonds is a key parameter for the discrim-
ination between agonist and nonagonist TCR-
pMHC interactions.
We carried out a parallel screen on the
TCR55bCDR library (diversity: 20,736) and
identified a TCR55 variant, clone 36, that ex-
hibited a high level of T cell activation by B35-
HIV(Pol) (fig. S7, A and B). Clone 36 contained
two mutations: a CDR1 mutation, TCR55b-N28Q,Zhaoet al.,Science 376 , eabl5282 (2022) 8 April 2022 3 of 14
Fig. 2. A hotspot on the TCR can tune TCR signaling strength.(A) B35-HIV tetramer staining and anti-CD69
staining of cells transduced with library clones in each round of selection. The gate is based on the staining
of WT TCR55. (B) A stimulatory clone, TCR55a-A98H, was selected from the library and was stimulated by
KG-1 cells pulsed with titrated HIV peptides for 14 hours. Anti-CD69 staining was performed on the transduced
SKW3 T cells and analyzed by flow cytometry. (C) SPR experiments of TCR55a-A98HproteinbindingtoB35-HIV.
Biotinylated B35-HIV monomer was immobilized on the streptavidin chip, and the TCR55a-A98H protein was
flowed through the chip. (D) BFP experiments to measure bond lifetime force curves for TCR55a-A98H or
TCR55 WT binding to B35-HIV. (E) TCR55a-A98 was mutated to D, E, F, Q, Y, and H and used to transduce SKW3
T cells with WT TCR55b. The transfectants were stimulated by KG-1 cells pulsed with titrated HIV peptides
for 14 hours. Anti-CD69 staining was performed on the transduced SKW3 T cells and analyzed by flow cytometry.
(F) Mean value of maximal anti-CD69 MFI versus 3D binding affinityKDof TCR55a-A98 mutants transfectants.
The linear correlation analysis was performed for stimulatory mutants and TCR55 WT. (G) BFP experiments
to measure bond lifetime force curves for TCR55a-A98H, TCR55a-A98E, or TCR55a-A98Q T cell transfectants
binding to B35-HIV. (H) Mean value of maximal anti-CD69 MFI versus peak bond lifetime of TCR55a-A98 mutants
transfectants. [(B) and (E)] Data are representative of three independent experiments. Data are shown as
means ± SDs of technical triplicates. [(D) and (G)] Data are shown as means ± SEMs of 500+ individual bond
lifetimes per force curve. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys;
D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;
W, Trp; and Y, Tyr.
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