Fig 2: PD-1H expression in peripheral T cells and monocytes. (A) PD-1H expression on the surface of CD4+ T cells, CD8+ T cells, and CD14 + monocytes was analyzed by FCM at 6, 24, and 48 h after blood collection. The expression levels of PD-1H cells are shown in a representative FCM histogram (upper graph), and the mean fluorescence intensity (MFI) of PD-1H positive staining was pooled from two independent experiments (lower graph). (B) T cells were treated with different concentrations of PHA (0, 5, and 10 μg/ml), and then PD-1H mRNA and PD-1H cell surface expression was analyzed by qRT-PCR (left graph) and FCM (right graph), respectively. (C) Membrane PD-1H expression was analyzed 24 h after T cells were treated with PMA (50 ng/ml), LPS (200 ng/ml), poly I:C (2 μg/ml), or anti-CD3/anti-CD28 beads. (D,E) Membrane PD-1H expression was analyzed on the cell surface of purified monocytes and PBMCs after the cells were stimulated with PMA, LPS, and poly (I:C) for 24 h. The data are representative of at least two independent experiments. ns = non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001. FCM, flow cytometry; qRT-PCR, real-time quantitative reverse transcription PCR; PBMC, peripheral blood mononuclear cells; Mon, monocytes; NC, negative control; PHA, polyhydroxyalkanoates; LPS, lipopolysaccharide; poly (I:C), polyinosinic:polycytidylic acid; PMA, Phorbol 12-myristate 13-acetate.

Fig 3: PD-1H expression in ESCC analyzed by immunohistochemistry. (A–D) Immunohistochemistry staining of PD-1H in ESCC tumor tissues was performed and calculated using immunoreactive score (IRS), which was categorized as 0–1 (negative), >1–4 (mild), >4–8 (moderate), or >8–12 (strong). Representative images of four categories of classification of PD-1H expression in ESCC tumor tissues are shown. (E–F) Two representative cases with strong positive PD-1H staining demonstrated that PD-1H protein was predominantly located in the membrane and cytoplasm of tumor cells and immune cells. Scale bars: 50 μm, original magnification × 200; 25 μm, original magnification ×400. PD-1H: Programmed Ddeath-1 Homolog; ESCC: esophageal squamous cell carcinoma. Scale bar: 25 μm, original magnification × 400.

Fig 4: The association between PD-1H expression levels and overall survival in ESCC. (A) The association of PD-1H mRNA expression and overall survival of patients was assessed in 41 ESCC patients and 44 EAC patients from the TCGA database. (B–D) With the applied scoring system, a score of 7.2 as the optimal cutoff point was identified by the “survMisc” package in R. The correlation between PD-1H protein expression and prolonged overall survival in 114 ESCC patients (B), in the G1 and G2/3 subgroups (C) and in the T1/T2 and T3/T4 tumor stage subgroups (D) was analyzed by using log-rank (Mantel-Cox) test in GraphPad 8.0. ESCC, esophageal squamous cell carcinoma; EAC, esophageal adenocarcinoma; G (G2/G3), Grades of differentiation; T (T1/T2, T3/T4), Tumor stage.

Fig 5: PD-1H expression in tumor-infiltrating immune cells assessed by multiplex immunohistochemistry (mIHC). (A) Representative images of mIHC staining of PD-1H, CD4, CD8, CD68, and DAPI in ESCC tumor tissues. Scale bar: 25 μm, original magnification × 400. (B) The cell numbers of PD-1H+CD68+ myeloid cells were significantly higher than cell numbers of PD-1H+CD4+ and PD-1H+CD8+ cells. (C) The percentage of PD-1H positive cells among CD4+ T cells, CD8+ T cells, and CD68+ cells was estimated. (D) The expression score of PD-1H was calculated and classified into high-expression (PD-1H high) or low-expression (PD-1H low) groups by using the R package of “survMisc”. (E) The absolute counts of immune cells and CD4+ T cells in tumor sites were significantly higher in patients with high PD-1H expression than in patients with low PD-1H expression. ns = non-significant, *p < 0.05, **p < 0.01.