Fig 2: Overexpression of soluble PD-L1 variants did not help the tumor growth alone.(A) Mice were injected with 1 × 106 MC38 cells transduced with empty vector (n = 6). (B) MC38_PD-L1–KO cells transduced with empty vector (n = 6). (C) MC38_PD-L1–KO cells transduced with murine WT PD-L1 (n = 6). (D) MC38_PD-L1–KO cells transduced with mh #1 (n = 6). (E) MC38_PD-L1–KO cells transduced with mh #2 (n = 6). (F) MC38_PD-L1–KO cells transduced with mm (n = 6). Each line represents the tumor volumes for each mouse. mh #1 and #2 are different clones. Each group was compared with 2-sided Mann-Whitney U test.

Fig 3: PD-L1–vInt4 expression was found in various types of cancer.(A) The percentage of cases expressing PD-L1–vInt4–specific sequence in intron 4 with FPKM ≥ 1, ≥ 2, ≥ 5, and ≥ 10 in each TCGA cancer type. (B) Structural changes associated with PD-L1–vInt4 expression in a case of HNSCC (TCGA-CV-5443-01) and STAD (TCGA-BR-8361-01). Aberrant transcripts are colored in red. (C) PD-L1–vInt4 was detected in some of the clinical samples. Data were obtained from CCLE.

Fig 4: PD-L1–vInt4 demonstrated its function as a decoy in vitro.(A) Evaluation of NFAT activity by measuring luminescence when PD-1 effector cells preincubated with αPD-L1 and secreting PD-L1 variants were cocultured with K1_PD-L1 WT cells. PD-L1–v242 and αPD-L1 were added in 3:1 molar ratio, whereas the ratio was 6:1 with PD-L1–vInt4 and αPD-L1. n = 3. **P < 0.01 by paired 2-tail Student’s t test. (B) Comparison of NFAT activity by measuring luminescence — this time using PD-1 effector cells preincubated with nivolumab and secreting PD-L1 variants. Secreted variants do not significantly suppress the signaling; paired 2-tail Student’s t test. Each condition was compared with the third bar counted from the left end in A and B. (C) Evaluation of NFAT activity by measuring luminescence. PD-1 effector cells were cocultured with K1 or K1_PD-L1 WT cells. PD-L1–vInt4 was added at a physiologically plausible concentration and at a 10-fold concentration. Secreted variants do not significantly suppress the signaling; paired 2-tail Student’s t test. Each condition was compared with the bar in the left end in C. Data represent mean ± SEM in A–C. (D) Jurkat–PD-1 cells incubated with or without Fc-tagged PD-L1–vInt4 were evaluated with FACS. As for nivolumab, it was added 30 minutes before PD-L1–vInt4.

Fig 5: Secreted PD-L1 variants were not stainable with SP142 (the intracellular domain–recognizing antibody), which shows that they can be differentiated with IHC in clinical specimens.(A) Immunofluorescence staining of PD-L1 expressed in PC9 cells. The nuclei of the cells are stained with Hoechst 33342. This shows that secreted variants are detectable by comparing staining patterns, with 2 or more different antibodies recognizing different epitopes. Scale bars: 20 μm. The exposure time was 40 ms for 28-8 and 60 ms for E1J2J. (B) Secreted PD-L1 variants were visually identifiable in IHC. Scale bars: 20 μm. The numbers represent each case number, in accordance with Table 1. A PD-L1 WT–expressed case #5 is shown as a positive control for SP142. (C) The structure of full-length PD-L1 and recognition sites of each antibody.