Programmed death-ligand 1 and its soluble form are highly expressed in nasal natural killer/T-cell lymphoma: a potential rationale for immunotherapy

Abstract

Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive neoplasm with poor therapeutic responses and prognosis. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays an important role in immune evasion of tumor cells through T-cell exhaustion. The aim of the present study was to examine the expression of PD-L1 and PD-1 molecules in NNKTL. We detected the expression of PD-L1 in biopsy samples from all of the NNKTL patients studied. PD-L1 was found on both malignant cells and tumor-infiltrating macrophages, while PD-1-positive mononuclear cells infiltrated the tumor tissues in 36% of patients. Most significantly, soluble PD-L1 (sPD-L1) was present in sera of NNKTL patients at higher levels as compared to healthy individuals and the levels of serum sPD-L1 in patients positively correlated with the expression of PD-L1 in lymphoma cells of tumor tissues. In addition, the high-sPD-L1 group of patients showed significantly worse prognosis than the low-sPD-L1 group. Furthermore, we confirmed that membrane and soluble PD-L1 was expressed on the surface and in the culture supernatant, respectively, of NNKTL cell lines. The expression of PD-L1 was observed in tumor tissues and sera from a murine xenograft model inoculated with an NNKTL cell line. Our results suggest that sPD-L1 could be a prognostic predictor for NNKTL and open up the possibility of immunotherapy of this lymphoma using PD-1/PD-L1 axis inhibitors.

Keywords: Immunotherapy; Nasal NK/T-cell lymphoma; PD-1; PD-L1; Soluble form.

Fig. 1

Expression of PD-L1 on lymphoma cells in biopsy tissues from NNKTL patients. a–d Representative immunohistological features of FFPE samples (Patient 17). a Nasal mucosa is infiltrated by atypical lymphoid cells (H&E staining). b ISH for EBER. Nuclei of EBER-positive cells are stained (brown). c Staining for CD56. d Staining for PD-L1. Serial sections were used for c and d. Scale bar in a–d is 100 µm. e Double immunofluorescent staining of CD56 (green) and PD-L1 (red), counterstained with DAPI (blue). Arrows indicate colocalization of CD56 and PD-L1. Scale bar is 50 µm

Fig. 2

Expression of PD-L1 on tumor-infiltrating macrophages in biopsy tissues from NNKTL patients. Representative immunohistological features of FFPE samples (Patient 16) stained for CD68 (a) and PD-L1 (b). Serial sections were used for a and b. Scale bar in a and b is 100 µm. c Double immunofluorescent staining of CD68 (green) and PD-L1 (red), counterstained with DAPI (blue). Arrows indicate colocalization of CD68 and PD-L1. Scale bar is 50 µm

Fig. 3

Localization of PD-1 and PD-L1-positive cells in NNKTL tissues. Representative immunohistological features of NNKTL FFPE samples. a Staining for PD-1 (brown) (Patient 15). Scale bar is 100 µm. Original magnification, ×400. b, c Staining for PD-1 (b) and PD-L1 (c) was performed using serial slides (Patient 17). Right panels show enlargement of the boxed areas that are positive for PD-1 or PD-L1 staining. PD-1 or PD-L1-positive cells are stained brown. Scale bar is 100 µm. Original magnification, ×100 (left) and ×400 (right)

Fig. 4

Levels of sPD-L1 in sera and correlation with overall survival. a sPD-L1 levels in the sera from 16 NNKTL patients and 23 healthy controls were measured using ELISA. The horizontal lines indicate mean values. Statistical significance was determined using the Mann–Whitney U test. b Correlation between the levels of serum sPD-L1 and the expression levels of PD-L1 in CD56-positive cells of tumor tissues in NNKTL patients. Statistical significance was determined using the Spearman rank correlation. c Kaplan–Meier curves for overall survival of the 16 NNKTL patients. The high-sPD-L1 group (n = 7) showed significantly worse prognosis than the low-sPD-L1 group (n = 9). Statistical significance was determined using the log-rank test

Fig. 5

Expression of PD-L1 in NNKTL cell lines and in tumor tissue and sera from a murine xenograft model inoculated with SNK-6 cells. a Flow cytometric analysis of the surface expression of PD-L1 or PD-1 in the indicated cell lines. Cells were stained with a PE-conjugated anti-PD-L1 or anti-PD-1 mAb (red lines). Black lines, cells stained with isotype control Ab. b PD-L1 or PD-1 expression in cell lines as assessed by western blotting. β-Actin was used to verify the amount of loading. c Supernatants from the indicated cell cultures (5 × 10⁵/mL) in 96-well round-bottomed plates were collected after 24 h, and sPD-L1 production was assessed using ELISA. Columns means of triplicate determinations; bars SEM. In a–c, HDLM-2 and Raji cells were used as positive and negative controls, respectively, for the expression of PD-L1. Molt-4 cells were used as a positive control for the expression of PD-1. d–g Representative immunohistological features of FFPE tumor samples from a murine xenograft model. d H&E-stained section showing tumor tissue formed by highly atypical lymphoid cells. e ISH for EBER. Nuclei of EBER-positive cells are stained brown. f Staining for CD56. g Staining for PD-L1. Scale bar is 100 µm. h sPD-L1 levels in the sera from NOG mice inoculated with SNK-6 cells (NOG-SNK-6, n = 5) and normal NOG mice (NOG, n = 4) were measured using ELISA. Columns means; bars SEM