Supplemental Data:
Fig. S1. The binding specificity of consensus sequence CWCWR to
MDA-MB-435 and MDA-MB-231 cells. The binding ability of control
peptide to MDA-MB-231 (A), consensus sequence to MDA-MB-231 (B),
control peptide to MDA-MB-435 (C) and consensus sequence to
MDA-MB-435 (D).
Fig. S2. A, The schematic illustration of construction the
focused library. B, Ten clones were randomly selected for
sequencing to examine the diversity of the focused library.
Fig. S3. Binding properties of peptides displayed on the
bacteria surface. A, The binding ability of the clone 1, clone 2,
clone 3 and a random library clone to PD-L1 at 2 nM (n=3). The
binding ability with different PD-L1 concentrations (B) (n=3),
under different washing procedures(C) (n=3). D. Binding specificity
of peptides to PD-L1 (n=3). All the experiments were examined by
flow cytometry. Data were presented as mean ± SEM, *P<0.05,
unpaired t-test.
Fig. S4. The binding properties of TPP-1 and SPP-1 to PD-L1 were
determined by the ELISA (n=3).
Fig. S5. A, The PD-L1 expression of CHO-K1/PD-L1, MDA-MB-231 and
MDA-MB-435 cell lines were determined by anti-PD-L1 antibody and
flow cytometer. B, The binding properties of TPP-1 to MDA-MB-231
and MDA-MB-435 cell lines.
Fig. S6. The bright field (A) and fluorescence microscopic image
(B) of TPP-1 to CHO-K1. The bright field (C) and fluorescence
microscopic image (D) of TPP-1 to CHO-K1/PD-L1.
Fig. S7. A, The ED50 of PD-L1 to block the activated T cells
(n=3). B, The TPP-1 peptide or PD-L1 plus TPP-1 peptide did not
activate the T cells without the CD3 antibody. C, TPP-1 could
effectively restore T cells proliferation which were inhibited by
PD-L1 evaluated by T cell activation assay (n=3). Data were
presented as mean ± SEM, *P<0.05, **P<0.01, unpaired
t-test.
Fig. S8. A, The PD-L1 expression in matured DCs, the dotted line
represented the isotype mAb and filled line represented the
anti-PD-L1 mAb. B, The proliferation of T cells in MLR assay
(n=3).
Fig. S9. A, The bioluminescence signals of H460-Luc cells were
determined by Cytation 3. B, The expression of PD-L1 on H460 was
detected with flow cytometry. The dotted line represented the
isotype mAb and filled line represented the anti-PD-L1 mAb. Data
were presented as mean ± SEM, **P<0.01, unpaired t-test.
Fig. S10. A, In vivo fluorescence images of mice taken at
different time points post s.c. injection of FITC labeled TPP-1. B,
The statistical result of the relative fluorescence unit.
Fig. S11. A, The location of CWCWR (yellow) in TPP-1 peptide. B,
The possible binding location of TPP-1 (blue) peptide to PD-L1 (PDB
ID of PD-L1: 3BIS). C, The crystal structures of PD-1 (light sea
green) and PD-L1 (dark khaki) complex (PDB ID of the complex:
3BIK).
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