PD-L1-expressing natural killer cells predict favorable prognosis and response to PD-1/PD-L1 blockade in neuroblastoma

Figures & data

Figure 1. Spatial distribution of CD8⁺ T cells, NCR1⁺ NK cells, and PD-L1⁺ cells in NB patients.

a-b. Representative photomicrographs of IHC staining for intratumoral and peritumoral CD8, intratumoral and peritumoral NCR1, and tumor and immune cell PD-L1 protein in NB tissues from cohort_1 (a) and cohort_2 (b); Scale bars, 100μm.

c. Representative images of immunofluorescent staining for the colocalization of CD8 (red), NCR1 (cyan), and PD-L1 (green) in NB tissues from cohort_1 (top) and cohort_2 (bottom). DAPI, blue. The figure panel pairs the representative images taken with different zooming options. Scale bars, 50μm.

d. Flow cytometry analyses for the percentage of CD8⁺ T and NK cells in tumor-infiltrating leukocytes and the proportion of PD-L1⁺ cells in CD8⁺ T, CD56⁺ NK, and tumor cells isolated from human NB tumors (n = 9). CD8⁺ T cells were identified as CD45⁺ CD3⁺ CD8⁺ cells, NK cells were identified as CD45⁺ CD3⁻ CD56⁺ cells, tumor-infiltrating leukocytes were identified as CD45⁺ cells, and tumor cells were identified as CD45⁻ NCAM⁺ cells.

E-F. Flow cytometry analyses for the expression of cytotoxic markers IFN-γ, granzyme B, and TNF-α (n = 7) (e) and activation markers CD25 and CD69 (n = 9) (f) by PD-L1⁺ and PD-L1⁻ subsets in CD8⁺ T and NK cells isolated from human NB tumors.

Intra, intratumoral; peri, peritumoral; TC, tumor cell; IC, immune cell; ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. ScRNA-seq analysis reveals that PD-L1-positive NKT and NK cells have an activated status in NB tumor.

a. PD-L1-negative or -positive NKT cell type annotation by the expression of marker gene signatures in human NB tumors from GSE154037 dataset based on t-Uniform Manifold Approximation and Projection (UMAP). Color key from gray to red indicates relative expression levels from low to high.

b. This functional enrichment analysis of differentially expressed genes between PD-L1-negative and PD-L1-positive NKT cells. Bar plot ranking of the top GO terms.

c. Gene Set Variation Analysis on gene sets related to Cytotoxic_Signature (left), T_Cell_Activation (middle), and NK_Cell_Activation (right) to obtain the corresponding gene set scores in PD-L1-positive and PD-L1-negative NKT cells. The difference of the gene sets between the two groups were analyzed.

d. PD-L1-negative or -positive NK cell type annotation by the expression of marker gene signatures in human NB tumors from GSE147766 dataset based on UMAP. Color key from gray to red indicates relative expression levels from low to high.

e. This functional enrichment analysis of differentially expressed genes between PD-L1-negative and PD-L1-positive NK cells. Bar plot ranking of the top GO terms.

f. Gene Set Variation Analysis on gene sets related to Cytotoxic_Signature (left), NK_Cell_Activation (middle), and Chemokines (right) to obtain the corresponding gene set scores in PD-L1-positive and PD-L1-negative NK cells. The difference of the gene sets between the two groups were analyzed. ***P < 0.001.

Table 1. Univariate and multivariate analysis of overall survival and event-free survival in neuroblastoma patients.

	Cohort_1 (n = 96)								Cohort_2 (n = 89)
Variable	Overall survival				Event-free survival				Overall survival
	Univariate cox		Multivariate cox		Univariate cox		Multivariate cox		Univariate cox		Multivariate cox
	P-value	HR (95%CI)	P-value	HR（95%CI）	P-value	HR（95%CI）	P-value	HR（95%CI）	P-value	HR（95%CI）	P-value	HR（95%CI）
Age
≥18 months or <18 months	0.836	1.10 (0.45–2.71)			0.053	2.11 (0.99–4.51)			0.324	1.63 (0.62–4.28)
Gender
Male or female	0.258	1.63 (0.701–3.79)			0.938	0.98 (0.55–1.74)			0.160	1.96 (0.77–4.98)
INSS stage
4 or 1/2/3/4S	0.030	2.43 (1.09–5.41)	0.168	1.90 (0.76–4.73)	0.001	2.77 (1.49–5.16)	0.345	1.49 (0.65–3.40)	0.041	2.64 (1.04–6.72)	0.152	0.30 (0.06–1.56)
MYCN amplified
Yes or no	0.010	3.44 (1.34–8.82)	0.433	1.51 (0.54–4.19)	0.014	0.42 (0.21–0.84)	0.769	1.13 (0.50–2.54)	0.005	4.37 (1.58–12.1)	0.198	1.97 (0.70–5.52)
Risk group
High or low/intermediate	0.053	2.40 (0.99–5.85)			0.001	3.68 (1.78–7.58)	0.021	3.26 (1.20–8.90)	0.006	3.52 (1.43–8.67)	0.139	3.45 (0.67–17.8)
Total CD8 expression
High or low	0.219	0.65 (0.32–1.29)			0.043	0.57 (0.33–0.98)	0.758	0.90 (0.45–1.80)	0.186	0.53 (0.21–1.36)
Peritumoral CD8 expression
High or low	0.796	0.91 (0.46–1.83)			0.735	0.91 (0.53–1.56)			0.643	0.81 (0.33–1.99)
Intratumoral CD8 expression
High or low	0.003	0.30 (0.14–0.67)	0.410	0.67 (0.253–1.75)	0.009	0.47 (0.27–0.83)	0.677	0.85 (0.38–1.87)	0.011	0.24 (0.08–0.72)	0.642	0.76 (0.24–2.42)
Total NCR1 expression
High or low	0.236	0.66 (0.33–1.32)			0.100	0.633 (0.37–1.09)			0.068	0.41 (0.15–1.07)
Peritumoral NCR1 expression
High or low	0.601	1.20 (0.60–2.40)			0.705	1.11 (0.65–1.90)			0.541	1.33 (0.53–3.30)
Intratumoral NCR1 expression
High or low	<0.001	0.153 (0.06–0.40)	0.019	0.25 (0.08–0.80)	<0.001	0.26 (0.14–0.47)	0.003	0.29 (0.12–0.66)	0.003	0.05 (0.01–0.36)	0.007	0.05 (0.06–0.45)
Total PD-L1 expression
High or low	0.032	0.43 (0.20–0.93)	0.641	1.30 (0.43–3.91)	0.015	0.51 (0.29–0.88)	0.997	1.00 (0.47–2.16)	0.213	0.55 (0.22–1.41)
Tumor cell-PD-L1 expression
High or low	0.394	0.73 (0.35–1.51)			0.056	0.59 (0.34–1.02)			0.517	0.74 (0.30–1.84)
Immune cell-PD-L1 expression
High or low	0.003	0.28 (0.12–0.65)	0.301	0.56 (0.19–1.68)	0.002	0.406 (0.23–0.72)	0.920	1.04 (0.50–2.19)	0.080	0.42 (0.16–1.11)

Figure 3. Expression levels of intratumoral CD8 and intratumoral NCR1 correlate with increased survival in NB patients regardless of immune cell-PD-L1 expression.

a-b. Kaplan–Meier curves of EFS and OS in patients from cohort_1 (left) and OS in those from cohort_2 (right) based on intratumoral CD8 (a) or intratumoral NCR1 (b) combined with immune-cell PD-L1 expression in IHC staining. IC, immune cell.

C-D. Kaplan–Meier curves of EFS and OS in patients from cohort_1 (left) and OS in those from cohort_2 (right) based on the levels of intratumoral PD-L1⁺ CD8⁺ T cell (c) or intratumoral PD-L1⁺ NCR1⁺ NK cell (d) in mIF staining.

Figure 4. Intratumoral NCR1 expression levels correlate with increased survival in NB patients regardless of PD-1 expression and the density and spatial distribution of CD8⁺ T cells.

a. Representative photomicrographs of IHC staining for PD-1 in NB tissues from cohort_1 (left) and cohort_2 (right); Scale bars, 100μm.

b. Correlation analyses of PD-1 with total CD8, intratumoral CD8, peritumoral CD8, total NCR1, intratumoral NCR1, peritumoral NCR1, total PD-L1, tumor cell-PD-L1, and immune cell-PD-L1 based on IHC staining score.

c. Representative images of immunofluorescent staining for the colocalization of CD8 (red), NCR1 (cyan), and PD-1 (green) in NB tissues from cohort_1 (top) and cohort_2 (bottom) cohorts. DAPI, blue. The figure panel pairs the representative images taken with different zooming options. Scale bars, 50μm. Number of CD8⁺, NCR1⁺, CD8⁺ PD-1⁺, NCR1⁺ PD-1⁺, peritumoral CD8⁺ PD-1⁺, peritumoral NCR1⁺ PD-1⁺, intratumoral CD8⁺ PD-1⁺, and intratumoral NCR1⁺ PD-1⁺ cells was quantified.

d. Flow cytometry analyses for the expression of PD-L1 in CD8⁺ T and NK cells isolated from human NB tumors (n = 9).

e-g. Kaplan–Meier curves of OS according to PD-1 alone (e), PD-1 combined with intratumoral CD8 (f), or intratumoral NCR1 (g) expression in patients from cohort_1.

Intra, intratumoral; peri, peritumoral; TC, tumor cell; IC, immune cell; ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Cytotoxicity of CD8⁺ T or NK cell combined with anti-PD-1/PD-L1 antibodies to NB cells in vitro.

a. The expression of MYCN protein in five NB cell lines was detected by western blot.

b. The surface expression of PD-L1 by five NB cell lines was assessed by flow cytometry.

c-f. CD8⁺ T and NK cells from the PBMCs of healthy donors were co-cultured with or without MYCN amplified (SK-N-BE.²) and non-amplified (SK-N-SH) NB cells in Transwell system for 6 h. The expression of PD-1 (c) and PD-L1 (d) by CD8⁺ T and NK cells were examined by flow cytometry. The expression of IFN-γ in PD-1⁻ or PD-1⁺ subsets in CD8⁺ T and NK cells was assessed by flow cytometry (e). The expression of PD-L1 by SK-N-BE.² and SK-N-SH cells was examined by flow cytometry (f). n = 4.

g-i. In vitro induced CD8⁺ T and NK cells blocked with or without anti-PD-1 or anti-PD-L1 antibodies were co-cultured SK-N-BE.² and SK-N-SH cells for 6 h with an effector: target ratio of 1:1. The lysis of SK-N-BE.² and SK-N-SH cells was assessed by cytotoxicity assay (g). The percentage of IFN-γ (h) or PD-L1 (i) positive cells in CD8⁺ T and NK cells activated with or without anti-PD-1 or anti-PD-L1 antibodies was assessed by flow cytometry. n = 3.

ns, no significance. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Antitumor activity of CD8⁺ T or NK cell combined with PD-1/PD-L1 blockade in xenograft models of NB.

a. Experimental scheme for the subcutaneous xenograft models of NB in NCG mice. Mice are inoculated with MYCN amplified (SK-N-BE.²) and non-amplified (SK-N-SH) NB cells subcutaneously. Then, mice were intravenously injected with in vitro induced human CD8⁺ T and NK cells that were blocked with or without anti-PD-1 or anti-PD-L1 antibodies; n = 5 for each group.

b. Tumor volumes for mice in different treatment groups in A were recorded on the indicated days; n = 5 for each group.

c-p. Tumor volume of mice in each group as indicated were recorded on the indicated days and plotted individually.

q. Kaplan–Meier survival curves for the mice in different treatment groups in A. Survival curves were compared using the log-rank test (two-tailed). n = 5 for each group.

ns, no significance. Data are representative of three independent experiments. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369(9579):2106–2120. doi:10.1016/S0140-6736(07)60983-0.

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