TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer

Figures & data

Table 1. Ovarian cancer patient characteristics and DNAM-1/TIGIT expression profile

Patient Characteristics							CD56dim NK cell population
Patient no.	EOC Stage	CA125 (U/mL)	Treatment	Complete	PFS (months)	OS (months)	DNAM-1 (%gated)	DNAM-1 (∆MFI)	TIGIT (%gated)	TIGIT (∆MFI)	Responder (≥10% CD107a)	CD107a Control	CD107a TIGIT blockade	IFNγ control	IFNγ TIGIT blockade
1	3 c	1304	Interval debulking	Complete	8.4	29.4	NA	NA	NA	NA	Yes	14	22	4	7
2	4	1800	Interval debulking	Complete	0	8.6	79	0.90	38	1.8	Yes	10	21	3	8
3	3 c	358	Primary debulking	Optimal	0	28.8	46	0.40	48	2.7	Yes	26	34	8	9
4	3 c	1242	Primary debulking	Optimal	0	13.7	43	0.30	33	1	No	4	5	1	2
5	4	1400	chemotherapy	Treatment not continued	0	1.7	55	0.40	37	2.6	No	3	5	1	0
6	3 c	2380	Primary debulking	Optimal	20.5	55.5	15	0.10	78	4.8	Yes	22	27	3	4
7	3 c	928	Interval debulking	Complete	23.9	41.3	23	0.30	69	4.8	No	1	1	0	0
8	4	1223	Interval debulking	Optimal	4.1	9.4	81	1.17	29	1.79	Yes	37	51	34	46
9	4	1430	Interval debulking	Optimal	12.2	54.6	59	0.64	16	0.88	Yes	38	51	27	35
10	3 c	2528	Primary debulking	Complete	17.2	54	61	0.60	34	1.3	NA	NA	NA	NA	NA

Characteristics of ovarian cancer patients from whom NK cells were phenotyped and functionally assessed. Patient samples were obtained at diagnosis. EOC, Epithelial Ovarian Cancer; PFS, Progression-Free Survival; OS, Overall Survival; MFI, Median Fluorescence Intensity; NA, not applicable.

Figure 1. NK cell phenotype of the DNAM-1/TIGIT/CD96 pathway in healthy donors and ovarian cancer patients. (a) DNAM-1, TIGIT and CD96 expression of healthy donor (HD) peripheral blood and OC patient ascites-derived CD56^dim and CD56^bright NK cells of a representative HD and OC patient, respectively. In grey the isotype control and in black the marker of interest are depicted. Numbers in the plot represent the median fluorescence intensity. (b) DNAM-1, TIGIT and CD96 expression on CD56^dim and CD56^bright NK cells of 10 HDs and 9 OC patients. Data is shown as mean+SEM. Kruskal-Wallis with Dunn’s Multiple Comparison Test was used for statistical analysis, * p < 0.05, ** p < 0.01 and *** p < 0.001. (c) DNAM-1, TIGIT and CD96 expression in 5 NK subsets, defined by characteristic CD56 and CD16 expression patterns, of 11 benign peritoneal fluids (bottom three panels) and 9 malignant ascites (top three panels). Datapoints with less than 100 events are excluded from the graphs. For a detailed number of cells per datapoint refer to Supplemental fig 1G. Kruskal-Wallis with Dunn’s Multiple Comparison Test was used for statistical analysis, * p < 0.05, ** p < 0.01*** and p < 0.001. (d) Co-expression patterns were determined with SPICE software. The pies depict the average proportion of cells expressing DNAM-1, TIGIT and CD96, and the arcs indicate which receptors are expressed by the corresponding pies. Statistical differences were analyzed using the built-in statistical tool. *p < .05; n = 10 for HD and n=9 for OC patients.

Figure 2. DNAM-1, TIGIT and CD96 are dose-dependently upregulated by rhIL-15 and DNAM-1 is downregulated by OC tumor cells. (a) DNAM-1 (n = 8), TIGIT (n = 6) and CD96 (n = 5) expression on NK cells of healthy donors co-cultured with SKOV-3, IGROV-1 or OVCAR-3 spheroids and increasing rhIL-15 concentrations for 7 days. Data is shown as mean+SEM. One-Way ANOVA with Bonferroni correction was used for statistical analysis, * p < .05, ** p < .01 and *** p < .001. (b) DNAM-1, TIGIT and CD96 expression on healthy donor NK cells co-cultured with a patient-derived primary tumor cell line and increasing rhIL-15 concentrations for 7 days cultured in duplicate. (c) DNAM-1, TIGIT and CD96 expression on healthy donor NK cells co-cultured with patient-derived tumor cells and increasing rhIL-15 concentrations for 7 days cultured in duplicate

Figure 3. SKOV-3 tumor-bearing mice have significantly reduced DNAM-1 expression in CD56^dim and CD56^bright NK cells. (a) Schematic overview of the mouse experiment. (b) Bioluminescence imaging (BLI) signal of the SKOV-3 tumor-bearing mice over time (n = 5). (c) Expression of DNAM-1, TIGIT and CD96 on CD56^dim (left) and CD56^bright (right) NK cells by flow cytometric measurement on day 0. The top graphs depict percentage positive cells and the bottom graphs depict ΔMFI. (d) Expression of DNAM-1, TIGIT and CD96 on intraperitoneal NK cells harvested 14 days after adoptive transfer in SKOV-3 tumor-bearing NSG mice and control mice. Day 0 data is represented by dotted lines as a reference. The top graphs depict percentage positive cells and the bottom graphs depict ΔMFI. Cumulative data are shown (lines indicate mean, n = 5 per group). A One-Way ANOVA with Bonferroni correction was used for statistical analysis, * p < .05 and *** p < .001. (e) Representative plots are shown of CD107a and IFNγ expression by CD56^dim NK cells after 4 h co-culture with SKOV-3 target cells, low dose rhIL-15 and TIGIT blockade or matching isotype control. (f) The percentage of CD107a and IFNγ expressing CD56^dim NK cells are shown on day 0 and day 14. Day 14 results are pooled NK cells from 5 different mice from either tumor-bearing or control mice

Figure 4. CD56^dim NK cell degranulation toward OC cell lines is boosted by TIGIT blockade and inhibited by DNAM-1 blockade. (a) Healthy donor CD56^dim NK cells fold change for CD107a and IFNγ after 4 h stimulation with SKOV-3 target cells, low dose rhIL-15, and TIGIT, DNAM-1 and/or CD96 blockade or matching isotype controls. Fold change in CD107a and IFNγ expression on CD56^dim cells following antibody treatment is calculated relatively to the condition with low dose rhIL-15 and 4 h SKOV-3 stimulation only. Cumulative data are shown as mean+SEM (n = 6) (b) IGROV-1 and OVCAR-3 stimulation for 4 h in the presence of TIGIT and/or DNAM-1 blockade or matching isotype controls. Fold change in CD107a and IFNγ expression on CD56^dim cells following antibody treatment is calculated relatively to the condition with low dose rhIL-15 and 4 h IGROV-1 or OVCAR-3 stimulation only −3 (n = 6 for IGROV-1 and n = 6 for OVCAR). A One-Way repeated measure ANOVA with Bonferroni correction was used for statistical analysis, * p < .05, ** p < .01 and *** p < .001

Table 2. Ovarian cancer (primary) cell line characteristics

	Cell line	CD112 Expression (∆MFI±SD)	CD155 Expression (∆MFI±SD)
	SKOV-3	49.7 (±8.0)	1040 (±197)
	IGROV-1	31.3 (±15.5)	1050 (±350)
	OVCAR-3	24.7 (±15.3)	783 (±215)
	ASC009	22.7 (±4.9)	1567 (±116)

CD112 and CD155 expression of ovarian cancer cell lines. SKOV-3, OVCAR-3, IGROV-1 and ASC009 were all acquired on three different time points. Data shown is the average ∆MFI with standard deviation.

Figure 5. TIGIT blockade boosts functional responsiveness of CD56^dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. (a) Schematic overview of the mouse experiment. (b) Bioluminescence imaging (BLI) signal of SKOV-3 tumor-bearing mice over time in “control”, “NK” only and “NK+TIGIT” blockade groups (n = 15 per group). (c) Total number of peritoneal NK cells harvested from “NK” only and “NK+TIGIT” blockade groups as measured by flow cytometry (average of bead and volume count cytoflex). (d) Percent CD107a (top graph) and IFNγ (bottom graph) from peritoneal NK cells harvested from “NK” only and “NK+TIGIT” groups which were re-stimulated ex-vivo with SKOV-3 for 4 h in the presence of low dose rhIL-15. (e) Percent CD107a (top graph) and IFNγ (bottom graph) from peritoneal NK cells harvested from “NK” only group re-stimulated with SKOV-3 in the presence of low dose rhIL-15 in combination with TIGIT, DNAM-1, TIGIT/DNAM-1 co-blockade or corresponding isotype control(s). (f-g) Percentage CD107a⁺ and IFNγ⁺ CD56^dim NK cells upon overnight treatment with low dose rhIL-15 (1 nM) and subsequent 4 h stimulation with K562 (f) or SKOV-3 (g). Based on a cutoff of 10% CD107a expression on CD56^dim NK cells co-cultured with SKOV-3 and rhIL-15 (without additional treatment) patients were subdivided in responder (≥10%) and non-responder (<10%) cohorts. Cumulative data of healthy donors (HD; n = 10) and ovarian cancer patients (Pt; n = 9) are shown with median. The Mann-Whitney test was used for statistical analysis, * p < .05. (h) CD56^dim NK cells positive for CD107a and IFNγ after 4 h stimulation with SKOV-3 target cells, low dose rhIL-15 (1 nM), and TIGIT and/or DNAM-1 blockade or matching isotype controls. Cumulative data shown for SKOV-3 responders only (lines indicate mean, n = 6). A One-Way ANOVA with Bonferroni correction was used for statistical analysis, * p < .05 and *** p < .001