The PD-L1 t-haNK and haNK cells were provided by NantKwest through a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI), National Institutes of Health (NIH). haNK cells were cultured as described9 10 and were irradiated (10 Gy) 24 hours before being used in in vitro tests. PD-L1 t-haNK cells were provided cryopreserved and irradiated (15 Gy) and were thawed on the same day as the experiment. Human cancer cell lines MDA-MB-231, BT549, T47D, SUM149, MCF7, H460, H441, HCC4006, SW480, SW620, DU145, HTB1, IOMM, CaSki, AGS, SNU-1, SNU16, SNU-5 and KATO III were purchased from American Type Culture Collection. CH22 was a kind gift from the Chordoma Foundation. Mouse oral cancer 1 (MOC1) was obtained from R. Uppaluri (Boston, MA) and cultured as described.18 19 PD-L1 null MDA-MB-231 cells were generated by Synthego via CRISPR/Cas9 technology and sorted to >98% knockout purity via fluorescence-activated cell sorting (FACS) using BD FACSAria (BD Biosciences) at the LGI Flow Cytometry Core, NCI, NIH. All cultures were mycoplasma-free and were maintained in the recommended culture media. Human MDSCs were expanded from human PBMCs as previously described20 and were isolated using CD33 magnetic beads (Miltenyi Biotec).

Total RNA was extracted from PD-L1 t-haNK and haNK cells using the RNeasy Mini kit (Qiagen). NanoString analysis of the isolated RNA was completed with the nCounter Pan-Cancer Immune Profiling Panel (NanoString Technologies), run by the Genomics Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland. Raw data files and reporter library files were uploaded and analyzed using the nSolver analysis software V.4.0.70 (NanoString) with the mRNA counts normalized to the most consistently expressed housekeeping genes and with the haNK cell sample as the categorical reference values. Genes that were deregulated by log2 fold >3 were selected and uploaded into Ingenuity Pathway Analysis (IPA) software V.01–14 (Qiagen). An IPA Core analysis focused on NK cell-related pathways was performed.

CAR expression was detected using biotinylated soluble human PD-L1 protein (Biosystems Acro) as a primary stain followed by PE-conjugated streptavidin (BD Biosciences) as the secondary stain. The following antibodies and stains were also used: CD56-APC (BD Biosciences), CD16-BV510 (BD), granzyme B-PE (BD Biosciences), perforin-BV421 (BD Biosciences), PD-L1-PE (BD Biosciences), and LIVE/DEAD fixable blue dead stain (Thermo Fisher). To identify the 135 immune subsets in the PBMCs (online supplementary table S1), methodology and gating strategies that have been previously described21 22 were used with slight modifications. Antibody panels were slightly modified (online supplementary table S2). Flow cytometry was performed on BD LSRFortessa or BD FACSVerse (BD Biosciences) and analyzed using FlowJo V.9.7.6 or V.10.5.3 (TreeStar). FACS of PD-L1^high and PD-L1^low MDA-MB-231 was performed using the MA900 (Sony) sorter.

PDL1 t-haNK, MDA-MB-231 and PD-L1 null MDA-MB-231 cells were stained as described.9 The following were used as primary antibodies: NKG2d (Abcam) and perforin (Abcam), PD-L1 (Abcam). The following were used as secondary antibodies: goat anti-rabbit-AlexFLuor647 (Thermo Fisher), goat anti-mouse-AlexFLuor488 (Thermo Fisher), and goat-anti-rabbit AlexaFluor488 (Thermo Fisher). The samples were mounted using ProLong gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher). The samples were imaged on a Leica DMI4000B microscope (Leica Microsystems).

¹¹¹In-release assays were performed as previously described.9 For experiments with interferon (IFN)-γ pretreatment, tumor cell lines were incubated with 20 ng/mL IFN-γ for 24 hours prior to ¹¹¹In-labeling. For ADCC experiments, 1 µg/mL each of cetuximab (Eli Lilly), anti-PD-L1 (provided by NantKwest through a CRADA) and anti-hen egg lysozyme IgG1 isotype control were added to the appropriate samples. For the blocking experiments, PD-L1 t-haNK cells were preincubated for 2 hours with either 50 µg/mL anti-CD16 antibody (eBioscience) or 200 nM concanamycin A (CMA) before being used in lysis assays. For assays evaluating the indirect effect of N-803 on PD-L1 t-haNK cells, C57BL/6 splenocytes were cultured with 0.5, 1.0, or 2.0 µg immobilized N-803 for 18 hours. Supernatants from the cultures were quantified for IFN-γ using a mouse ELISA kit (Life Technologies). MOC1 cells were incubated for 18 hours in supernatant harvested from a splenocyte culture incubated overnight with 1 µg immobilized N-803. Afterwards, the MOC1 cells were evaluated for PD-L1 expression and were used as PD-L1 t-haNK cell target.

For the PDL1^high vs PDL1^low coculture flow-based killing assay, MDA-MB-231, IFN-γ-treated MDA-MB-231 and PD-L1^high flow-sorted MDA-MB-231 cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (BioLegend). BT549, MCF7, T47D, MDA-MB-231, and PD-L1 null MDA-MB-231 were labeled with CellTrace Violet Proliferation dye (Thermo Fisher). Equal numbers of CFSE- and violet-labeled cells were mixed and cocultured with PD-L1 t-haNK cells at 37°C for 18 hours. The samples were then analyzed by flow cytometry. Per cent killing was determined based on the formula 100%×[1–(percentage of viable tumor cells in the presence of NK cells/percentage of viable tumor cells in the absence of NK cells)].

For the killing assays targeting PBMCs, cryopreserved or fresh PBMCs were cocultured with PD-L1 t-haNK cells. For some experiments, PBMC-PD-L1 t-haNK cell contact was blocked using a 3 μm transwell membrane. Following the 16 hours coculture incubation, cells were harvested and examined by flow cytometry. Immune subsets with a potentially biologically relevant change following coincubation with PD-L1 t-haNK cells were defined as those with a p value <0.05, the majority of donors having a >25% change, difference in medians of >0.05% of PBMCs, and a median frequency of >1% of total PBMCs.

PD-L1 t-haNK cells were labeled with CellTracker Violet BMQC (Thermo Fisher), and MDA-MB-231 cells were labeled with CellTracker CM-Dil (Thermo Fisher) and were cocultured together in an eight-well glass bottom μ-slide (Ibidi) at 1:2 effector to target (E:T) ratio. Immediately, CellEvent Caspase 3/7 Green (Thermo Fisher) was added to the coculture. The cells were imaged using the Zeiss LSM 780 at the NCI Confocal Microscopy Core Facility.

Wild-type (WT) MDA-MB-231 and PD-L1 null MDA-MB-231 lysates (30 µg each) were resolved by SDS-PAGE before electro-transfer onto nitrocellulose membranes before probing with anti-PD-L1 (Abcam) and anti-vinculin (Sigma-Aldrich) antibodies followed by goat anti-rabbit-IRDye 680RD (LI-COR) and goat anti-mouse-IRDye 800CW (LI-COR) antibodies. The probed proteins were visualized using the Odyssey system (LI-COR).

Female NSG mice 10–16 week old (originally obtained from Jackson Laboratory and bred in-house at NCI, Bethesda, Maryland) were inoculated with a suspension of MDA-MB-231, PD-L1 null MDA-MB-231, H460 or HTB1 cells (5.0×10⁶ per mouse) in 100 µL of phosphate-buffered saline (PBS) admixed with Matrigel 50% (v/v). Tumors grew for 9–10 days before randomizing into untreated control (PBS) or PD-L1 t-haNK cell-treated groups of 8–10 mice per group with an average initial tumor volume of ∼100 mm³. The treated cohorts were injected with 1×10⁷ cells PD-L1 t-haNK cells intraperitoneally one or two times per week. For the combination study, C67BL/6 mice were inoculated with MOC1 cells (5×10⁶ cells/mouse, subcutaneously). Tumors grew for 10 days (such that the average tumor volume was ~80 to 100 mm³) before randomizing into groups of eight. The tumor-bearing mice were administered with PD-1 (200 mg, intraperitoneally), N-803 (1 µg, subcutaneously) or PD-L1 t-haNK cells (10⁷ cells, intraperitoneally) as monotherapy, doublets or triplets on days 10, 17, and 24. For all tumor studies, tumor growth was monitored. The animal studies were terminated within a week after the last treatment or when the tumors reached the ethical limit. Liver and lungs were collected and metastatic tumor nodules were manually counted. Lungs were perfused with India ink to increase contrast.

Student’s t-test was used to determine the significance of two-group comparisons. One-way analysis of variance (ANOVA) was used to compare more than two groups. Two-way ANOVA was used to determine the significance of multiple comparisons between two or more groups. Tukey’s or Sidak’s post hoc test was used to correct both one-way and two-way ANOVAs. Pearson’s correlation coefficient was calculated to determine the correlation between two factors, and linear regression was used to calculate the best-fit lines. P values less than 0.05 were considered significant. Error bars represent means±SEM.

We have previously demonstrated that irradiated haNK cells (10 Gy) continued to express NK markers such as NKG2D and CD56 and maintained high levels of granzyme B and perforin.9 10 Here, we characterized PD-L1 t-haNK cells, which are haNK cells that have been engineered to express a CAR receptor composed of an extracellular single-chain variable fragment that binds to PD-L1, a transmembrane domain, and an FcεRIγ intracellular domain for downstream signaling. RNA analysis of irradiated PD-L1 t-haNK and irradiated haNK cells using the nCounter PanCancer Immune Profiling Panel (NanoString) revealed that the two cell types have comparable gene expression of common NK markers, perforin, and granzyme B (figure 1A). Of the genes investigated, 15.4% were deregulated by threefold or more (44 genes upregulated and 72 genes downregulated) in PD-L1 t-haNK cells compared with haNK cells (online supplementary figure S1). IPA focused on NK cell-related pathways revealed that this set of genes is involved in NK activation and migration (online supplementary table S3). Flow cytometric analysis verified that PD-L1 t-haNK and haNK cells have comparable expression of CD16, perforin, and granzyme (figure 1B). More importantly, it demonstrated that PD-L1 t-haNK cells maintained the expression of CAR molecules that are capable of binding to PD-L1 post-irradiation and post-thaw (figure 1B). NKG2D and perforin expression of PD-L1 t-haNK cells was confirmed via immunofluorescence microscopy (figure 1C).

Next, we examined the cytolytic function of PD-L1 t-haNK cells. The triple negative breast cancer (TNBC) cell line MDA-MB-231 was used as target because it has been shown to express high levels of PD-L1,23 and we have previously shown that it is susceptible to haNK cell ADCC-mediated lysis.9 10 We observed that anti-PD-L1 antibody (1 µg/mL) augmented haNK cell lytic activity through ADCC mechanisms (p<0.05, figure 1D). PD-L1 t-haNK cells mediated MDA-MB-231 cell lysis at a higher rate compared with haNK cells with anti-PD-L1 antibody (p<0.01). The difference may be due to the complex interactions involved in ADCC versus the integrated extracellular recognition and intracellular signaling with CAR that can accelerate the activation of PD-L1 t-haNK cells.17 Indeed, real-time tracking of cell lysis revealed that PD-L1 t-haNK cells initially killed MDA-MB-231 at a faster rate, but after an extended incubation, the overall killing was comparable between the two NK cell lines (online supplementary figure S2). Anti-PD-L1 antibody decreased the activity of PD-L1 t-haNK cells (figure 1D) likely through competition for PD-L1 binding, implying that this combination may not be a viable option for therapy.

The anti-EGFR antibody cetuximab has previously been shown to elicit ADCC-mediated killing of MDA-MB-231 by haNK cells.9 Although PD-L1 t-haNK cells express high-affinity CD16, their cytolytic activity against MDA-MB-231 was not enhanced by cetuximab nor was it diminished by CD16 blockade (figure 1E). This suggests that PD-L1 t-haNK cell cytotoxicity is more dependent on the CAR pathway than ADCC in the context of PD-L1^high cells such as MDA-MB-231. On the other hand, CMA effectively mitigated PD-L1 t-haNK cell-mediated tumor cell lysis (p<0.005; figure 1F), demonstrating perforin and granzyme B release as a critical mechanism of action of PD-L1 t-haNK cell killing.

We employed live cell imaging to observe PD-L1 t-haNK cell-mediated killing of MDA-MB-231 in real time (figure 1F). When a PD-L1 t-haNK cell associated with an MDA-MB-231 cell, it resulted in the activation of the caspase 3/7 pathway in the tumor cell, ultimately triggering the target’s apoptotic cell death. Overall, these data demonstrated that PD-L1 t-haNK cells are specialized haNK cells engineered to target PD-L1 expressing tumor cells via CAR.

Through In¹¹¹-release assays, we found that PD-L1 t-haNK cells efficiently lysed different human cancer cell lines, including breast (n=5, 3 of which are TNBC), lung (n=3), colon (n=2), urogenital (n=2), ovarian (n=1), chordoma (n=1), meningioma (n=1), and gastric (n=5) cancer cell lines at varying degrees (figure 2A and online supplementary figure S3). To determine the relationship between PD-L1 t-haNK cell-mediated cell lysis and PD-L1 expression, we calculated the Pearson correlation coefficient of % cell lysis with % PD-L1 score, which was calculated by scoring each cell line for % positive cells and for mean fluorescence intensity (MFI) on a quartile scale ranging from 1 to 4. The sum of the two values was considered the PD-L1 score.24 The degree of cell lysis of a cancer cell line as a result of PD-L1 t-haNK cell activity strongly corresponded to its PD-L1 score (figure 2B).

As in figure 1D, we also found that at 50:1 E:T ratio, PD-L1 t-haNK cells induced a greater tumor cell killing than haNK cells on all cell lines tested, except SW620, which expressed low PD-L1 (figure 2A). The addition of anti-PD-L1 antibody generally improved haNK cell activity through ADCC mechanisms.9 10 The increased haNK cell-mediated lysis on anti-PD-L1 antibody addition was particularly noticeable in cell lines with high PD-L1 scores.

After establishing the correlation between PD-L1 expression and the % cell lysis induced by PD-L1 t-haNK cells, we investigated whether modulating the cell surface expression of PD-L1 affects PD-L1 t-haNK cell-mediated tumor cell lysis. We pretreated four breast cancer cell lines (MDA-MB-231, BT549, T47D, and MCF7) with IFN-γ, which has been reported to regulate PD-L1 expression,25 and then used these cells as PD-L1 t-haNK cell targets. We observed that IFN-γ treatment upregulated % PD-L1 and/or MFI in the four breast cancer cell lines, which corresponded to a significant improvement in cell lysis induced by PD-L1 t-haNK cells (figure 3).

After determining that cell lysis by PD-L1 t-haNK cells is correlated to the level of PD-L1 expression on the tumor cell surface, we examined the specificity of PD-L1 t-haNK cells against PD-L1⁺ cells. To address this, cocultures of CFSE-labeled PD-L1^high (MDA-MB-231) and CellTrace Violet-labeled PD-L1^low (BT549, MCF7, and T47D) breast cancer cells were coincubated with PD-L1 t-haNK cells at different E:T ratios overnight (figure 4A–F). The numbers of viable residual PD-L1^high and PD-L1^low breast cancer cells were then analyzed by flow cytometry. The results showed a greater decrease in the PD-L1^high population, demonstrating that PD-L1 t-haNK cells preferentially lysed the cells expressing the CAR target.

To ensure that the difference in targeting is not due to the difference in cell type, we treated MDA-MB-231 cells with IFN-γ, which we have shown to increase PD-L1 expression (figure 3), and labeled these cells with CFSE prior to coculture with untreated CellTrace Violet-labeled MDA-MB-231 (figure 4G). We also flow-sorted for PD-L1^high and PD-L1^low MDA-MB-231 cells and labeled the two cell populations with CFSE and CellTrace Violet, respectively (figure 4I). After coincubation with PD-L1 t-haNK cells, we observed that these effector cells preferentially killed the MDA-MB-231 populations that have higher PD-L1 expression. This confirms that the PD-L1 t-haNK cell activity is robust against tumor cells that express its target.

Next, PD-L1 was knocked out of the MDA-MB-231 via CRISPR/Cas9 (PD-L1 null MDA-MB-231). The PD-L1 knockout was confirmed via flow cytometry (figure 5A), western blot (figure 5B), and immunofluorescence staining (figure 5C). Predictably, the removal of PD-L1 cell surface expression resulted in the markedly diminished ability of PD-L1 t-haNK cells to lyse the PD-L1 null MDA-MB-231 compared with the WT (p<0.005, figure 5D). We also observed that PD-L1 t-haNK cell cytotoxicity against PD-L1 null MDA-MB-231 cells is comparable to the cytotoxicity of haNK cells against WT and PD-L1 null MDA-MB-231 cells (figure 5E), again illustrating the importance of the CAR activity of PD-L1 t-haNK cells. Furthermore, the PD-L1 t-haNK cells preferentially killed the WT MDA-MB-231 over the PD-L1 null MDA-MB-231 at the lower E:T:T ratios (figure 5F). Overall, the data demonstrate that the PD-L1 t-haNK cells preferentially target the PD-L1-expressing tumor cells.

We first determined whether PD-L1 t-haNK cells traffic into the tumors. Female NSG mice bearing WT or PD-L1 null MDA-MB-231 tumors were injected with 10^{7 111}In-labeled irradiated PD-L1 t-haNK cells intravenously or intraperitoneally. ¹¹¹In signal was found in the WT MDA-MB-231 tumors 24 and 72 hours after injection, indicating successful trafficking of PD-L1 t-haNK cells into the PD-L1-expressing tumor (online supplementary figure S4). Furthermore, PD-L1 t-haNK cell tumor infiltration was greater in the WT MDA-MB-231-bearing mice than in the PD-L1 null MDA-MB-231-bearing cohort 24 hours after treatment.

Next, we investigated the antitumor therapeutic effect of these effector cells. Female NSG mice (n=10/group) bearing WT or PD-L1 null MDA-MB-231 tumors were treated with irradiated PD-L1 t-haNK cells (10⁷ cells/animal intraperitoneally) two times a week for four consecutive weeks. Significant tumor growth control was achieved in PD-L1 t-haNK cell-treated WT MDA-MB-231-bearing mice on day 17 (figure 6A). Meanwhile, significant tumor growth inhibition was achieved in the PD-L1 null MDA-MB-231-bearing mice much later on day 28 (figure 6B). The delayed ability of PD-L1 t-haNK cells to inhibit PD-L1 null MDA-MB-231 in vivo corresponded with the decreased PD-L1 t-haNK cell-mediated cell lysis observed in vitro (figure 5D) and reduced PD-L1 t-haNK cell trafficking (online supplementary figure S4).

The aggressive nature of MDA-MB-231 can result in the dissemination of the cancer cells leading to the development of metastases in multiple sites.26 27 To investigate the metastatic disease burden, organs were harvested from the WT MDA-MB-231 cohort 2 days after the last treatment and examined.

Significantly more macroscopic metastatic lesions were found in the liver and lungs of the control animals than in the corresponding tissues of the PD-L1 t-haNK cell-treated mice (figure 6C).

Next, we investigated whether PD-L1 t-haNK cells could provide therapeutic effect when given less frequently. Female NSG mice (n=10/group) were implanted with MDA-MB-231 and were injected with PD-L1 t-haNK cells (10⁷ cells/mouse intraperitoneally) once a week (figure 6E). Compared with the control group, tumor growth inhibition was observed in the PD-L1 t-haNK cell-treated animals starting on day 14 (p<0.05). Also, the anti-tumor response was abrogated when the PD-L1 t-haNK cells were pretreated with CMA, indicating the importance of perforin and granzyme B in the activity of PD-L1 t-haNK cells in vivo.

Lastly, we examined whether PD-L1 t-haNK cells could also be effective against other solid tumor xenografts. HTB1 and H460, which are human bladder and lung tumor models, respectively, were established onto female NSG mice (n=7–10/group, figure 6F). PD-L1 t-haNK cells (10⁷ cells/mouse intraperitoneally) were administered to the animals once a week for four consecutive weeks and tumor growth was monitored. For both models, PD-L1 t-haNK cell-treated animals had significantly smaller tumor volumes compared with their control counterparts. Due to the accelerated growth of H460, the animals were only given PD-L1 t-haNK cells two times, and the experiment had to be aborted early before the ethical tumor size limit was reached. Nevertheless, we were able to show the efficacy of PD-L1 t-haNK cells in MDA-MB-231, HTB1, and H460 xenograft models.

Other studies have reported that N-803 (formerly ALT-803), an IL-15 superagonist complexed with IL-15RαSushi-Fc fusion protein, significantly expanded and activated NK cells in mice.28 29 We investigated whether N-803 could improve the cytolytic activity of PD-L1 t-haNK cells by coincubating the two immunotherapeutic agents with MDA-MB-231 tumor cells (figure 7A). We observed that N-803 had no synergistic effect with PD-L1 t-haNK cells; however, it should be noted that the activity of PD-L1 t-haNK cells alone was already high. In addition, PD-L1 t-haNK cells do not express the IL-15 receptor (not shown). On the other hand, we found that N-803 was able to increase the activity of healthy donor NK cells (figure 7B); thus, while N-803 did not directly enhance PD-L1 t-haNK cells, it is capable of activating endogenous NK cells that can potentially contribute an additional antitumor effect.

Next, we explored whether N-803 could have an indirect effect on PD-L1 t-haNK cell–cell activity. N-803 has been demonstrated to stimulate IFN-γ production in mouse and human immune cells.30 Indeed, we have detected significant IFN-γ levels in the supernatant of murine splenocytes cultured with immobilized N-803 (approximately 2 ng/mL IFN-γ for all N-803 concentrations tested, figure 7C). When MOC1 cells were incubated in the supernatant harvested from N-803-treated splenocyte cultures, PD-L1 t-haNK cell killing of the MOC1 cells was improved and the improvement corresponded with the increased PD-L1 expression on the tumor cells (figure 7D). Therefore, we were able to demonstrate that N-803 could potentially increase PD-L1 t-haNK cell cytolytic activity by inducing other immune cells to produce IFN-γ, which results in elevated PD-L1 expression on tumor cells, thereby making them more targetable by PD-L1 t-haNK cells (figure 7E).

We tested the therapeutic efficacy of PD-L1 t-haNK cells in combination with N-803 and anti-PD-1 antibody in a MOC1 murine tumor model (figure 7F). C57BL/6 mice (n=8/group) were implanted with MOC1 cells and treatment was commenced when the average tumor size reached 80–100 mm³, wherein PD-L1 t-haNK cells (10⁷ cells, intraperitoneally), N-803 (1 µg, subcutaneously on the contralateral flank), and anti-PD-1 antibody (200 mg, intraperitoneally) were administered weekly for 3 weeks. Monotherapy with N-803 or anti-PD-1 antibody was not effective in inhibiting MOC1 tumor growth. On the other hand, weekly administration of PD-L1 t-haNK cells significantly reduced the tumor burden in the mice. Contrary to our in vitro data, the PD-L1 t-haNK cells+N-803 combination did not result in better tumor growth control when compared with PD-L1 t-haNK cell treatment alone. Likewise, the combination of PD-L1 t-haNK cells and anti-PD-1 antibody did not result in improved therapeutic benefit over PD-L1 t-haNK cell treatment alone. However, when PD-L1 t-haNK cells, N-803, and anti-PD-1 were all coadministered, tumor growth was significantly impeded with five out of eight animals completely rejecting the tumor. N-803 and anti-PD-1 each exerts an effect on T cells: N-803 expands and activates T cells while anti-PD-1 maintains T-cell function by minimizing T-cell exhaustion.28 29 31 A phase Ib trial employing N-803 with nivolumab has shown that this combination resulted in increased frequency and activity of CD8⁺ T cells32; this may explain the modest antitumor activity we observed in our N-803+anti-PD-1 group. Our animal data imply that superior antitumor effect could be achieved by having robust NK and T-cell compartments, and this could be attained through the combination of PD-L1 t-haNK cells with immunomodulators that expand, enhance, and enable the T cells.

A previous report demonstrated that different immune cell types in PBMCs from patients with cancer have varied PD-L1 expressions.22 We investigated whether PD-L1 t-haNK cells have any adverse effects on human peripheral immune cells. PBMCs from human healthy donors were coincubated with PD-L1 t-haNK cells at different E:T ratios for 24 hours before flow cytometric analysis for different immune subsets (figure 8A). We observed that PD-L1 t-haNK cells had no effect on NK, NK-T, CD4⁺ T and CD8⁺ T cells but promoted the expansion of the regulatory T cell (Treg) (p<0.05) and B-cell populations (p<0.001). However, we observed that the peripheral blood MDSC population was drastically decreased by PD-L1 t-haNK cells by up to 90% (p=0.001 for all E:T ratios tested, figure 8A). Furthermore, PD-L1 t-haNK cells were capable of killing both monocytic (in two out of two healthy donors) and granulocytic (in one out of two healthy donors) MDSCs (figure 8B). In addition, killing assays performed using transwell chambers demonstrated that PD-L1 t-haNK cell-mediated killing of MDSCs was contact dependent (figure 8B).

Among the different immune cells in the peripheral blood of patients with cancer, the MDSCs express high levels of PD-L1.22 We sought to determine whether the PD-L1 t-haNK cell-mediated anti-MDSC activity we observed in healthy donor PBMCs also holds true in patients with cancer PBMCs. PBMCs from patients with prostate cancer (n=3, figure 8C) and head and neck squamous cell carcinoma (n=3, figure 8D) were obtained and cocultured with PD-L1 t-haNK cells for 24 hours before the frequency of MDSCs was assessed via flow cytometry. Coincubation of patients with cancer PBMCs with PD-L1 t-haNK cells resulted in the reduction of peripheral MDSCs by more than 60% in both cancer types. To determine whether the MDSC killing was a direct effect of PD-L1 t-haNK cells, we isolated MDSCs from healthy donor PBMCs and performed an ¹¹¹In-release assay. Figure 8E validates that MDSC lysis by PD-L1 t-haNK cells did not rely on other immune cells but is a direct consequence of the activity of PD-L1 t-haNK cells itself.