CD25bright NK cells display superior function and metabolic activity under regulatory T cell-mediated suppression

Abstract

Infusion of natural killer (NK) cells is an attractive therapeutic modality in patients with cancer. However, the activity of NK cells is regulated by several mechanisms operating within solid tumors. Regulatory T (Treg) cells suppress NK cell activity through various mechanisms including deprivation of IL-2 via the IL-2 receptor alpha (CD25). Here, we investigate CD25 expression on NK cells to confer persistence in Treg cells containing solid tumor models of renal cell carcinoma (RCC). Compared with IL-2, stimulation with IL-15 increases the expression of CD25 resulting in enhanced response to IL-2 as evidenced by increased phosphorylation of STAT5. Compared with CD25^dim NK cells, CD25^bright NK cells isolated from IL-15 primed NK cells display increased proliferative and metabolic activity as well as increased ability to persist in Treg cells containing RCC tumor spheroids. These results support strategies to enrich for or selectively expand CD25^bright NK cells for adoptive cellular therapy of NK cells.

Keywords: CD25; IL-15; Natural killer cells; regulatory T cells; tumor microenvironment.

Figure 1.

IL-15 primed NK cells show increased pSTAT5 levels and are less susceptible to Treg-mediated suppression (a) Kaplan–Meier survival curves showing disease specific survival in patients with clear cell renal cell carcinoma partitioned by transcript abundance of Treg or NK gene signature from KIRC-TCGA cohort. Survival curve differences were analyzed using Kaplan–Meier log-rank test. (b) Experimental setup of IL-2 re-stimulation. (c) Spice chart showing phosphorylation markers of NK cells primed with IL-2 or IL-15 analyzed 15 minutes after re-stimulation with 100 U/ml IL-2 (n = 3). (d) Frequency of pSTAT5+ NK cells responding to 100 U/ml IL-2 (n = 3). (e) Flow cytometry quantification of pSTAT5 positive NK cells primed with IL-2 or IL-15 (n = 3) following re-stimulation at different doses of IL-2. (f) Representative flow chart showing the phosphorylation of STAT5 (Y694) at different doses of IL-2 re-stimulation comparing IL-2 and IL-15 activated NK cells. (g) Experimental setup of NK and Treg co-culture. Isolated and activated Treg were added at 1:1 ratio to NK cells. (h) CD25 expression of NK cells and Treg in co-cultures (n = 3). (i) NK/Treg ratio of surface IL-2 expression of after 4-hour co-culture (n = 4). (j) Frequency of Ki-67 positive NK cells in the absence or presence of Treg (n = 4).

Figure 2.

IL-15 primed NK cells show increased proliferative capacity and anti-tumor activity under Treg suppression. (a) Experimental setup of NK-Treg-K562 co-culture. Two-day activated NK cells and Tregs were co-cultured at a 1:1 ratio (25,000 NK cells to 25, 000 Treg) for two days. Thereafter, K562 cells (25,000) were added to NK:Treg cultures on day four for four hours for analysis of IFNγ production and proliferation. (b and c) Proliferation (Ki-67) of NK cells and Treg performed after two days Treg-NK cell co-culture and upon 4 hours exposure to K562 cells (n = 6). (d) Representative flow cytometry plots showing proliferation (Ki-67) of NK cells and Treg. (e) NK/Treg ratio on day 4 after 4 hours culture with K562 cells (n = 6). (f) IFN γ production by NK cells upon 4 hours exposure to K562 cells (n = 6). (g) Experimental setup of NK cell infiltration in NK-Treg-RCC spheroids. On day 0, tumor cells were seeded at either 5,000 or 10,000 cells. On day 5 and 7, activated Treg and cytokine-activated NK cells were added to RCC cell line spheroids respectively at a 1:1 ratio of either 25,000 or 10,000 cells. NK cell infiltration and proliferation were analyzed by flow cytometry on day 8. (h) Flow cytometry analysis of tumor-infiltrating NK cells and Treg into 786-O and A498 spheroids (n = 3 to 5). (i) Representative Incucyte image of NK cell infiltration (Red) into 786-O tumor spheroids on day 8. (j) Proliferation (Ki-67) of NK and Treg in 786-O RCC spheroids (n = 5). Results (h-j) are derived from experiments with seeding of 10,000 tumor cells on day 0, and addition of 10,000 Treg and 10,000 NK cells on days 5 and 7 respectively. Paired Student’s t-test was used to determine significance.

Figure 3.

CD25 expression is associated with improved NK cell response to IL-2 re-stimulation and metabolic activity. (a) Phosphorylation of STAT5 and (b) proliferation among CD25 NK cell subsets (n = 3). Sorted NK cells were co-cultured with Treg for two days (100 U/ml IL-2) before flow cytometry analysis (n = 3). (c) Quantification of mitochondria membrane potential per cells via microscopy, each dot represents a single cell. (d) Oxygen consumption rate (OCR) was measured after injection of oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and Rotenone & antimycin A (n = 4). (e and f) Frequencies and proliferation of live NK cells in 786-O RCC spheroids as detected by flow cytometry (n = 6). Tumor cells (10,000) were seeded and day 0, followed by addition of Treg (10,000) on day 5, and NK cells (10,000) on day 7. NK cell infiltration (E, n = 6) and proliferation (F, n = 3) were analyzed by flow cytometry at day 8.