PD-1 blockade restores impaired function of ex vivo expanded CD8⁺ T cells and enhances apoptosis in mismatch repair deficient EpCAM⁺PD-L1⁺ cancer cells

Figures & data

Table 1 Disease characteristics of five colorectal patients selected for TEC isolation

Characteristics	Total
Age	62
Sex
Male	3
Female	2
AJCC stage
II	3
III	2
Differentiation
T3N0 M0	4
T3N1 M0	1
Chemotherapy	–
Radiotherapy	–
Biomarkers
MMR (MSI-H)	5
Cytokeratin 20 (CK20)	5

Abbreviations: TECs, tumor epithelial cells; AJCC, American Joint Committee on Cancer; MMR, mismatch repair; MSI-H, high microsatellite instability.

Figure 1 The PD-1 expression on ex vivo expanded CD8⁺ T cells obtained from patient’s blood before surgery.

Notes: (A) Mean PD-1 level of CD4 and CD8 cells on day 0 and day 14 (n=11). (B) Representative dot plots of flow cytometry on day 14. (C) Percent normalized PD-1 expression after 48 hrs treatment with PD-1 mAb (n=5). (D) Different clones (MIH4 and J105) used for PD-1 staining on CD8⁺ T cells after treatment by flow cytometry. **P<0.005; ***P<0.0005.
Abbreviations: mAb, monoclonal antibody; ns, not significant.

Figure 2 Apoptotic effect of CD8⁺ T cells on EpCAM⁺ tumor cells after PD-1 blockade for 48 hrs.

Notes: (A) Different concentrations of PD-1 mAb used to optimize the dose (n=3). (B) Apoptosis measured at different effector-to-target ratios, ranging from 1:1 to 50:1. (C) Representative dot plot of apoptosis in EpCAM⁺ TECs (D) Differential change of apoptosis in tumor cells; TECs (MSI-H), DLD-1, SW480, and SW620 after PD-1 mAb treatment (n=5). (E) Cell death induced in dose-dependent manner after PD-L1 epitope blockade of PD-1 protein (left) at 1.0 μg/mL, and PD-1 mAb neutralized (right) in presence of nPD-1 antibody (n=3). **P<0.005; ***P<0.0005.
Abbreviations: mAb, monoclonal antibody; TECs, tumor epithelial cells; MSI-H, high microsatellite instability.

Figure 3 Effect of PD-1 mAb on cytolytic IFN-γ, GrB, and CD107a.

Notes: Mobilization after 48 hrs. Representative spots of (A) IFN-γ and (B) GrB from CD8⁺ T cells co-cultured with TECs and DLD-1 respectively. Histogram of (C) IFN-γ in CD8⁺ T cells + TECs and CD8⁺ T cells + DLD-1 and (D) GrB in CD8⁺ T cells + TECs and CD8⁺ T cells + DLD-1 (n=5). (E) Dot plot of CD107a mobilization and (F) histogram of CD8⁺ T cells + TECs and CD8⁺ T cells + DLD-1 (n=4). *P<0.05; **P<0.005.
Abbreviations: mAb, monoclonal antibody; TECs, tumor epithelial cells.

Figure 4 Cellular interactions, cytosolic uptake, and CD8⁺ T cells’ migration after 48 hrs.

Notes: (A) Cytosolic uptake by CD8⁺ T cells from TECs and DLD-1 cells validated by flow cytometry (n=3). (B) Chemotactic attraction increased after pre-treatment with PD-1 mAb; conditioned medium from TECs and DLD-1 cells was used in the lower chamber of the transwell plate (n=3). *P<0.05; **P<0.005; ***P<0.0005.
Abbreviations: mAb, monoclonal antibody; TECs, tumor epithelial cells.

Figure S1 Information on primary tumor.

Notes: (A) PD-L1 staining on 5 μM tumor sections utilized for TECs’ isolation. (B) MHC-I/II staining of isolated TECs shown by flow cytometry, and (C) apoptotic effect of CD8⁺ T cells on TECs (MSS).

Abbreviations: TECs, tumor epithelial cells; Pt, patient; MSS, MMR proficient; MMR, mismatch repair.

Figure S2 The representative dot plot of flow cytometry staining of tumor antigens, and average level on TECs, DLD-1, SW480, and SW620 along with K562 as a negative control (n=3).

Note: (A) Extracellular EpCAM, (B) intracellular CK20, and (C) intracellular PD-L1.

Abbreviations: TECs, tumor epithelial cells; CK20, cytokeratin 20; SS lin, side scatter linear; FS lin, forward scatter linear.

Figure S3 Percentages of (A) killer immune cells, and (B) intracellular CTLA4 before and after ex vivo T cell expansion by flow cytometry (n=11).