Fig 2: LAG3 expressed on DNTs was upregulated when cocultured with the breast cancer cell, and LAG3Ab can enhance the cytotoxic abilities of DNTs to breast cancer. (A–C) Expression of LAG3, PD1, NKG2D on DNTs when cocultured with MDA-MB-231. *p < .05, **p < .01, ***p < .001. The results represent three independent experiments. (D–F) Expression of LAG3, PD1, NKG2D on DNTs with or without stimulation with LAG3Ab. *p < .05, **p < .01, ***p < .001. The results represent three independent experiments. (G) LAG3Ab blocked the interaction between LAG3 on DNTs and its ligands on tumor cells. (H) LAG3Ab enhances DNT cell-mediated cytotoxicity to the breast cancer cells. %AV and specific killing of tumor cells are shown. The results represent three independent experiments. AV, Annexin V; DNT, double negative T; LAG3, lymphocyte-associated gene 3; LAG3Ab, LAG3 monoclonal antibody; PD1, programmed cell death protein 1

Fig 3: LAG3, PD1, and NKG2D expression in breast cancer from TCGA data. (A, B) Expression of LAG3, PD1, and NKG2D on DNT cells and its ligands on MDA-MB-231 and SK-BR-3 cells. Experiments were repeated twice with similar results. (C) Box-whisker plots showing the expression of LAG3 (lag3) in subgroups of breast cancer samples from the TCGA database, which was tested by UALCAN. (D) Dataset analyzed by TIMER 2.0 revealed the correlation of lag3 expression with the overall survival of different breast cancer subtype patients. (E) Spearman's correlation of LAG3 (lag3), PD1 (pdcd1), and NKG2D (klrk1) with immune cell infiltrations across breast cancer (TIMER). p < .05 was considered statistically significant. DNT, double negative T; LAG3:, lymphocyte-associated gene 3; PD1, programmed cell death protein 1; TIMER, tumor immune estimation resource

Fig 4: NK cells maintain the expression of activating receptors after cryopreservation. PM21-NK cells expanded from T-cell-depleted PBMCs were obtained from multiple donors (N = 3–7). Cells were cryopreserved while donor-matched fresh PM21-NK cells (black squares) were maintained in culture. Frozen PM21-NK cells were thawed and rested 16 h the day before analysis (blue triangles) or thawed and rested for 1 h on the day of analysis (gray circles). Expression of NK cell activating receptors CD16, NKG2D, DNAM-1, NKp30, NKp46, NKp44, as well as CD69, granzyme B, and perforin was determined by flow cytometry. No statistically significant difference was seen in the expression of activating receptors either 1 h immediately post-thaw or after a 16-h rest in donor and expansion-matched PM21-NK cells (N = 5–7 donors). Data are presented as a scatter plot with each point representing the average of two technical replicates with donor-pair lines. No statistical significance was determined by multiple paired t-tests with a threshold for significance at p < 0.05.

Fig 5: MMP-9 cleaves NKG2D in TILs and MICA/B in HPMCs. Purified STILs were stimulated with rhMMP-9 (5 µg mL-1) in the presence or absence of 20 µm SB-3CT for 24 h. A) NKG2D expression of STILs were analyzed by FACS. B) Fold changes of flow cytometry mean fluorescence intensity (MFI) values were quantified. HPMCs were stimulated with rhMMP-9 in the presence or absence of 20 µm SB-3CT for 24 h. C) MICA/B expression levels in HPMCs were analyzed by FACS. D) Fold changes of flow cytometry MFI values were quantified. All experiments were repeated five times. Data are mean determinants ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 versus controls; NS, not significant, Unpaired Student's t-test.