Fig. 2. TCR clonal dynamics and mutation-associated neoantigen
recognition in patients responding to PD-1 blockade. (A) TCR
sequencing was performed on serial peripheral Tcell samples obtained
before and after PD-1 blockade. Tumor tissue with mismatch repair
deficiency was obtained from three responding patients. Shown for each
patient are 15 TCR clones with the highest relative change in frequency
after treatment (left) that were also found in the original tumor (right
panels). (B) Whole-exome sequencing was performed on tumor and
matched normal tissue from patient 19. Somatic alterations were analyzed
using a neoantigen prediction pipeline to identify putative MANAs.
Reactivity to 15 candidate MANAs was tested in a 10-day cultured IFN-g
ELISpot assay. Data are shown as the mean number of spot-forming cells
(SFC) per 106 T cells (left) or mean cytokine activity (right) of triplicate
wells ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Seven candidate
MANAs were selected for TCR analysis on the basis of ELISpot reactivity.
(D) MANA-specific Tcell responses were identified against three of seven
candidate MANAs (MANA1, MANA2, and MANA4) after 10 days of in vitro
stimulation (left panels). MANA-specific clones were identified by significant
expansion in response to the relevant peptide and no significant expansion in
response to any other peptide tested (fig. S3). Data are shown as the relative
change in TCR clone frequency compared to the frequency of that clone after
identical culture without peptide. These Tcell clones were also found in the
original tumor biopsy (right panels). (E) Frequency of MANA-specific clones,
carcinoembryonic antigen (CEA), and radiographic response in the tumor
[from (D)] were tracked in the peripheral blood before treatment and at various
times after pembrolizumab treatment. Time is shown in weeks after the first
pembrolizumab dose. (F) In vitro binding and stability assays demonstrate the
affinity kinetics of each relevant MANA and the corresponding wild-type peptide
(when applicable) for their restricting HLA class I allele. The A*02:01-restricted
influenza M GILGFVTL epitope was used as a negative control for each assay;
known HLA-matched epitopes were used as positive controls when available.
Data are shown as counts per second with increasing peptide concentration for
binding assays (top) or counts per minute over time for stability assays (bottom).
Data points indicate the mean of two independent experiments ± SD. Amino
acid abbreviations: 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; Y, Tyr.