Modulation of intrinsic inhibitory checkpoints using nano‐carriers to unleash NK cell activity
2021; Springer Nature; Volume: 14; Issue: 1 Linguagem: Inglês
10.15252/emmm.202114073
ISSN1757-4684
AutoresGuy Biber, Batel Sabag, Anat Raiff, Aviad Ben‐Shmuel, Abhishek Puthenveetil, Jennifer I. C. Benichou, Tammir Jubany, Moria Levy, Shiran Killner, Mira Barda‐Saad,
Tópico(s)CAR-T cell therapy research
ResumoArticle2 November 2021Open Access Source DataTransparent process Modulation of intrinsic inhibitory checkpoints using nano-carriers to unleash NK cell activity Guy Biber Guy Biber The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel These authors contributed equally to this work Search for more papers by this author Batel Sabag Batel Sabag orcid.org/0000-0002-3003-8080 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel These authors contributed equally to this work Search for more papers by this author Anat Raiff Anat Raiff orcid.org/0000-0003-1670-5892 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Aviad Ben-Shmuel Aviad Ben-Shmuel The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Abhishek Puthenveetil Abhishek Puthenveetil orcid.org/0000-0002-6310-3328 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Jennifer I C Benichou Jennifer I C Benichou The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Tammir Jubany Tammir Jubany orcid.org/0000-0001-9736-5062 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Moria Levy Moria Levy The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Shiran Killner Shiran Killner The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Mira Barda-Saad Corresponding Author Mira Barda-Saad [email protected] orcid.org/0000-0002-0305-7438 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Guy Biber Guy Biber The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel These authors contributed equally to this work Search for more papers by this author Batel Sabag Batel Sabag orcid.org/0000-0002-3003-8080 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel These authors contributed equally to this work Search for more papers by this author Anat Raiff Anat Raiff orcid.org/0000-0003-1670-5892 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Aviad Ben-Shmuel Aviad Ben-Shmuel The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Abhishek Puthenveetil Abhishek Puthenveetil orcid.org/0000-0002-6310-3328 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Jennifer I C Benichou Jennifer I C Benichou The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Tammir Jubany Tammir Jubany orcid.org/0000-0001-9736-5062 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Moria Levy Moria Levy The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Shiran Killner Shiran Killner The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Mira Barda-Saad Corresponding Author Mira Barda-Saad [email protected] orcid.org/0000-0002-0305-7438 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Search for more papers by this author Author Information Guy Biber1, Batel Sabag1, Anat Raiff1, Aviad Ben-Shmuel1, Abhishek Puthenveetil1, Jennifer I C Benichou1, Tammir Jubany1, Moria Levy1, Shiran Killner1 and Mira Barda-Saad *,1 1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel *Corresponding author. Tel: +972 3 5317311; Fax: +972 3 7384058; E-mail: [email protected] EMBO Mol Med (2022)14:e14073https://doi.org/10.15252/emmm.202114073 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Natural killer (NK) cells provide a powerful weapon mediating immune defense against viral infections, tumor growth, and metastatic spread. NK cells demonstrate great potential for cancer immunotherapy; they can rapidly and directly kill cancer cells in the absence of MHC-dependent antigen presentation and can initiate a robust immune response in the tumor microenvironment (TME). Nevertheless, current NK cell-based immunotherapies have several drawbacks, such as the requirement for ex vivo expansion of modified NK cells, and low transduction efficiency. Furthermore, to date, no clinical trial has demonstrated a significant benefit for NK-based therapies in patients with advanced solid tumors, mainly due to the suppressive TME. To overcome current obstacles in NK cell-based immunotherapies, we describe here a non-viral lipid nanoparticle-based delivery system that encapsulates small interfering RNAs (siRNAs) to gene silence the key intrinsic inhibitory NK cell molecules, SHP-1, Cbl-b, and c-Cbl. The nanoparticles (NPs) target NK cells in vivo, silence inhibitory checkpoint signaling molecules, and unleash NK cell activity to eliminate tumors. Thus, the novel NP-based system developed here may serve as a powerful tool for future NK cell-based therapeutic approaches. Synopsis Natural Killer (NK) cells serve as a first line of immune defense against tumor growth and viral infections. This study demonstrates a nanobiology-based drug delivery system to enhance NK cytotoxicity by suppressing intracellular inhibitory checkpoints in the tumor microenvironment (TME). NK cytotoxicity was enhanced by nano-carriers encapsulating siRNAs that target the negative regulatory genes SHP-1 and Cbls. Tumor growth was suppressed by these molecularly modified NK cells in-vivo. The paper explained Problem Current Natural Killer (NK) cell-based immunotherapy relies heavily on adoptive transfer and ex vivo manufacture of NK cells, which has major limitations to achieve therapeutic impact. Moreover, NK cells express multiple inhibitory checkpoint receptors. Therefore, even if a given receptor is effectively blocked, NK cells may still be inhibited via alternative pathways, compromising the efficiency of this approach. These current limitations call for a novel approach for targeting prevailing intracellular inhibitory signaling cascades shared by multiple surface inhibitory receptors, to unleash NK cells against cancer. Results In this study, we developed a novel non-viral lipid nanoparticle-based delivery system encapsulating small interfering RNAs (siRNAs) targeting three key negative regulatory genes (i) SHP-1, (ii) Cbl-b, and (iii) c-Cbl. We demonstrate that these nano-carriers effectively enhance NK cell activity against HLA-matched cancer cells. These nanoparticles (NPs) also provide an effective in vivo delivery system to enhance NK cytotoxicity in the tumor microenvironment (TME). Targeting NK cells in vivo bypasses the need for ex vivo isolation of NK cells. Furthermore, this technology provides an innovative and broad therapeutic approach that includes both the active-modulating compounds and the systemic delivery platform. Impact The nano-based delivery system that targets key intracellular inhibitory checkpoints represents a promising immunotherapy for improving NK cells killing activity against cancer in the TME, in vivo. Introduction Natural killer (NK) cells are cytolytic effector lymphocytes that participate in the surveillance of stressed, infected, or cancerous cells (Orange et al, 2006; Orange, 2008; Vivier et al, 2011). NK cells exert their effector functions through secretion of cytolytic granules, which induce target cell apoptosis, and secretion of cytokines, which can activate the adaptive immune response (Vivier et al, 2008). Downregulation or lack of major histocompatibility complex (MHC) class-I molecules increases NK cell activity by diminishing inhibitory signals transduced through inhibitory killer-cell immunoglobulin-like receptor (KIR): MHC interactions (Ljunggren et al, 2007; Dahlberg et al, 2015). Moreover, NK cell responses depend on a balance between activating and inhibitory signaling cascades, derived from activating and inhibiting surface receptors. Common NK cell-activating receptors include CD16, natural cytotoxicity receptors (NCRs) NKp46, NKp44, NKp30, and NKp80, 2B4, and natural killer group 2 member D (NKG2D) (Moretta et al, 2001; Welte et al, 2006; Amand et al, 2017). The critical human inhibitory NK cell receptors, KIRs, have long cytoplasmic tails (KIR-L) containing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Each KIR recognizes a subgroup of human leukocyte antigen (HLA) class I allotypes and displays two (KIR2DL) or three (KIR3DL) extracellular Ig-domains, conferring specificity for HLA-C or HLA-A/B allotypes, respectively (Thielens et al, 2012). Engagement of KIR receptors with cognate MHC ligands induces the recruitment of Src homology 2 (SH2) domain-containing protein tyrosine phosphatase–1 (SHP-1) to the KIR ITIM domains (Poole et al, 2005). SHP-1 was previously shown to inhibit NK cell activity by dephosphorylating the Guanine nucleotide exchange factor, VAV1, and as we previously demonstrated, the Linker for activation of T cells (LAT), phospholipase C-γ1 (PLC-γ1), and PLC-γ2 (Stebbins et al, 2003; Campbell, 2016b; Matalon et al, 2016). In addition to SHP-1, the E3 ubiquitin ligases Cbl-b and c-Cbl serve as key negative regulators of NK cell activity (Paolino et al, 2014). We previously showed that during NK cell inhibition, LAT is ubiquitylated by Cbl-b and c-Cbl (Campbell & Bennett, 2016; Matalon et al, 2016), leading to LAT degradation and thereby abolishing NK cell activation and cytotoxicity. Furthermore, a previous study in NK cells showed that genetic deletion of the E3 ubiquitin ligase, Cbl-b, or targeted inactivation of its E3 ligase activity activates NK cells to spontaneously reject metastatic tumors (Paolino et al, 2014). Furthermore, c-Cbl inhibits VAV1, resulting in the suppression of NF-κB-driven cytotoxic programs (Kwon et al, 2017). Thus, it is evident that both SHP-1 and Cbls play critical roles in dictating the NK cell activation threshold, and are therefore attractive targets for NK cell-based immunotherapy. Cancer immunotherapy is designed to enhance immune cell activity against cancer cells while leaving normal cells unharmed (Wang et al, 2016; Ott et al, 2017). Most immunotherapeutic approaches focus on the adaptive immune system for tumor immune surveillance, although increasing evidence supports a significant role for innate immune effector cells, such as NK cells, in the elimination of tumors (Marcus et al, 2014). NK cell-based immunotherapy offers significant advantages over current immune-based treatments, including the ability of NK cells to kill tumor cells directly without relying on recognition of specific tumor antigens, and reduced non-specific toxicity compared to T cell-based therapies (Vivier et al, 2011; Paolino et al, 2014; Guillerey et al, 2016; Esin et al, 2017; Martín-Antonio et al, 2017; Bugide et al, 2018). NK cells can also complement T-cell immune surveillance by targeting MHC-I-deficient tumors, which decrease β2-microglobulin expression to escape T-cell activity (Rodríguez, 2017). Furthermore, NK cells can initiate cross-talk with dendritic cells and T cells in the tumor microenvironment (TME), resulting in the recruitment of potent adaptive anti-tumor responses (Ferlazzo et al, 2014). Current NK cell-based treatments rely heavily on adoptive transfer; however, ex vivo manufacture of NK cell-based therapeutics has major limitations, including the need for extensive ex vivo expansion procedures subject to a high risk of contamination, lack of sufficient NK cell numbers to attain therapeutic impact, and the reduction of the NK cell cytolytic phenotype (Davies et al, 2014; Hu et al, 2019). Furthermore, transduction protocols for the generation of chimeric antigen receptor (CAR) expressing NK cells may cause undesired genomic effects, including oncogenic transformation (Hu et al, 2018). Therefore, there is increasing interest in developing immunotherapies that harness NK cells in vivo. In contrast to adoptive cell transfer, immune checkpoint inhibition (ICI) therapy aims to block immune checkpoint molecules such as PD-1 and CLTA-4 or their ligands, to unleash the immune response in vivo (Parry et al, 2005; Conniot et al, 2019). For NK cells, these inhibitory checkpoints include cell surface receptors such as KIRs and CD94/NKG2A, and may also include PD-1, TIM-3, TIGIT, CEACAM1, CD96, and LAG-3, expressed on both NK and T cells (Topalian et al, 2015). Recent evidence demonstrates that ICI targeting NK cell surface receptors could be a potential therapeutic avenue for cancer immunotherapy (Souza-Fonseca-Guimaraes et al, 2019; Sun et al, 2019), yet ICI is beneficial only for a subset of patients and cancer types (Seidel et al, 2018). Often, immune editing of the tumor niche provides resistance to conventional checkpoint blockade (Jenkins et al, 2018). In the context of NK cell immune surveillance, tumors can often upregulate particular HLA molecules such as HLA-E/G to repress NK cell activity (Freund-Brown et al, 2018). Moreover, the suppressive cellular milieu of the TME (e.g., stromal cells, fibroblasts, regulatory T cells, and myeloid suppressor cells), in addition to inhibitory cytokines (e.g., TGF-β, IL-10), can induce T/NK cell dysfunction (Vasievich et al, 2011). A major caveat limiting the efficacy of immune checkpoint blockade therapy is the fact that multiple inhibitory checkpoint receptors are expressed on the surface of T and NK cells; therefore, even if a given receptor is effectively blocked, NK cells may still be inhibited via alternative pathways, compromising the efficiency of this approach. Simultaneously blocking every inhibitory checkpoint receptor to overcome exhaustion in the TME is not a feasible option. These current limitations in immunotherapy call for a novel approach for targeting prevailing inhibitory signaling cascades shared by multiple inhibitory receptors, to unleash NK cells against cancer. Here, we engineered a non-viral delivery system, lipid-based nano-carriers encapsulating small interfering RNAs (siRNAs), to target common intrinsic inhibitory NK cell signaling pathways. The nano-carriers encapsulate siRNAs targeting three genes critical for suppression of NK cell activity, namely, SHP-1, Cbl-b, and c-Cbl. We employed a target cell system in which NK cell activity is repressed through the common immune-editing pathway of high expression of the ligands for the KIR inhibitory checkpoint receptor. We demonstrate that nano-carriers effectively and safely silence SHP-1 and Cbls in NK cells in vitro and in vivo, unleashing NK cell activity against HLA-matched cancer cells, and prolong survival in humanized murine models. Our study demonstrates that nanoparticles (NPs) encapsulating small molecules provide an effective systemic in vivo delivery strategy to enhance NK cytotoxicity in the TME. Results Gene silencing of SHP-1 and Cbls enhances NK cell activity To suppress the key inhibitors of NK cell cytotoxicity, we designed siRNAs targeting Cbls and SHP-1. For this purpose, YTS KIR2DL1 (henceforth referred to as YTS-2DL1) cells were transfected with 250 or 500 pmol of Cbl-b (Fig EV1A), c-Cbl (Fig EV1B), or SHP-1 (Fig EV1C) siRNA and monitored for gene silencing efficiency after 48 h. A significant decrease in all the three proteins was detected relative to non-specific (N.S) siRNA control. More efficient gene silencing was obtained by using 500 pmol of siRNA for Cbl-b and c-Cbl (Cbl-b siRNA: 250 pmol vs and 500 pmol P = 0.05; c-Cbl siRNA: 250 pmol vs and 500 pmol P = 0.03), but the efficiency of SHP-1 gene silencing was not affected by the siRNA concentration. A concentration of 250 pmol was selected for use in further experiments for each of these three proteins whenever they were targeted without being encapsulated in NPs. To determine the effect of our designed SHP-1 and Cbls siRNAs on the NK cell activation threshold, we used siRNAs to target each of the proteins individually or in combination. In an attempt to combine the three siRNAs in lipid-based NPs and to avoid potential off-target effects and cell damage (Jackson et al, 2010), we decided to use 250 pmol/each siRNA. YTS-2DL1 cells were transfected with Cbl-b, c-Cbl, and SHP-1 siRNAs. Gene silencing was determined by Western blot analysis after 48 h. A significant decrease was observed in the expression of Cbl-b, c-Cbl, and SHP-1 relative to the control, non-specific siRNA-treated cells (Fig 1A). Click here to expand this figure. Figure EV1. Gene silencing of SHP-1 and Cbls enhances NK cell function YTS-2DL1 cells were either mock-transfected or transfected with 250 or 500 pmol of (A) Cbl-b siRNA, (B) c-Cbl siRNA, or (C) SHP-1 siRNA using Amaxa electroporation. After 48 h, cells were lysed, and the nitrocellulose membranes were blotted with anti-Cbl-b, anti-c-Cbl, or anti-SHP-1 antibodies. GAPDH served as a loading control. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values. Relative expression of the three proteins compared to the mock control group is presented within the graph. Analysis by ImageJ densitometry revealed a decrease of Cbl-b siRNA: 48 ± 3% and 77 ± 4%, P ≤ 0.03 for Cbl-b, 45 ± 6% and 68 ± 4%, P ≤ 0.03 for c-Cbl, and 79 ± 3% and 79 ± 5%, P ≤ 0.03 for SHP-1 following siRNA gene silencing concentrations of 250 pmol and 500 pmol, respectively (shown in bar graphs underneath each blot). The data represent three independent experiments (n = 3). Data are shown as mean ± SEM. P values were calculated vs mock-treated control cells by one-sample t-tests and independent t-test. P values are indicated by asterisks. *P ≤ 0.05. YTS KIR2DL1 cells were gene silenced for either SHP-1, Cbl-b, c-Cbl or a combination of both Cbls proteins or of Cbls and Shp-1. NK cells treated with N.S siRNA served as control. After 48 h, YTS KIR2DL1 were incubated with 721.221 Cw4 target cells for 2 h and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was compared by mean fluorescence intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. The data represent three independent experiments (n = 3). Data are shown as mean ± SEM. P values were calculated by one-sample t-test and are indicated by asterisks. **P ≤ 0.001. Data information: Exact P values are shown in Appendix Table S1. Download figure Download PowerPoint Figure 1. Inhibition of Cbl-b, c-Cbl, and SHP-1 enhance NK cell function YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or Cbl-b siRNA, c-Cbl siRNA and SHP-1 siRNA, using Amaxa electroporation. After 48 h, cells were lysed and subjected to Western blot with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of three independent experiments (n = 3). P values were calculated by one-sample t-test and are indicated by asterisks *P < 0.05. YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 h, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 100 s. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments. YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 h and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments (n = 5). P values were calculated by one-sample t-tests and one-way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P < 0.0001. Granzyme release. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 h, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified using standard recombinant granzyme B concentrations. Data are means ± SEM of eight independent experiments (n = 8); P values were calculated by one-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05. Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 h, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 h of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments (n= 3). P values were calculated by one-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01. Data information: Exact P values are shown in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 1 [emmm202114073-sup-0004-SDataFig1.zip] Download figure Download PowerPoint Next, we used functional assays to determine the effect of SHP-1 and Cbls gene silencing in enhancing NK cell activation following inhibitory interactions. To determine the impact of SHP-1 and Cbl repression of NK cell activity, we simulated a common immune-editing pathway that inhibits NK cells: high surface expression of HLA ligands for KIR receptors. To this end, YTS-2DL1 cells were incubated with 721.221 target cells that overexpress the HLA–Cw4, a cognate MHC Class I ligand for the KIR2DL1 inhibitory receptor (221-Cw4 cells), which induces NK cell inhibition, or with 721.221 targets expressing the irrelevant HLA-Cw7 (221-Cw7 cells) molecule. This allotype is not recognized by the KIR2DL1 receptor and therefore target cells expressing it are recognized and lysed by NK cells. As can be seen in Fig EV1D, NK cell degranulation (LAMP1, CD107a expression) was higher in YTS-2DL1 that were transfected with a combination of SHP-1 and Cbl siRNAs and interacted with 221-Cw4 cells, relative to the levels observed in YTS-2DL1 transfected with N.S siRNA, or siRNA targeting SHP-1, Cbl-b, and c-Cbl, individually (Fig EV1D). These results demonstrate that Cbl-b, c-Cbl, and SHP-1 tune the NK cell activation threshold. Therefore, these intrinsic inhibitory checkpoint molecules are attractive targets for increasing NK cell function. Combined inhibition of SHP-1 and Cbls controls the balance of NK cell activation After validating an optimal concentration for efficient combined knockdown of Cbls and SHP-1 (Fig 1A), we next determined whether gene silencing of these intrinsic inhibitory checkpoints reduces the NK cell activation threshold and increases NK cell functional activities. Accordingly, YTS-2DL1 were incubated with 221-Cw4 target cells, resulting in NK cell inhibition. As seen in Fig 1B, intracellular calcium levels following inhibitory interactions with 221-Cw4 target cells were markedly elevated in YTS-2DL1 cells gene silenced for SHP-1 and Cbls, relative to the levels observed in YTS-2DL1 transfected with N.S siRNA. In addition, YTS-2DL1 transfected with SHP-1 and Cbls siRNA exhibited a significant increase in their ability to secrete cytolytic granules following inhibitory interactions (721-Cw4 mCherry) compared to mock-transfected cells or cells transfected with NS siRNA that were subjected to inhibitory interactions (1.83 ± 0.3 relative CD107a levels, P ≤ 0.04; Figs 1C and EV2A). The increase in degranulation of NK cells silenced for SHP-1 and Cbls resembled the degranulation levels during an activating NK cell response (SHP-1 and Cbls siRNA/Cw4 1.83 ± 0.3; Mock/Cw7 1.72 ± 0.1, P = 0.1). These results were also verified by measuring granzyme B release (Fig 1D). YTS-2DL1 cells gene silenced for SHP-1 and Cbls that were incubated with inhibitory 221-Cw4 cells exhibited a significant increase of granzyme B secretion compared to YTS-2DL1 pretreated with N.S siRNA (SHP-1 and Cbls siRNA/Cw4 287.9 ± 26.1 pg/ml; N.S siRNA/Cw4 165.4 ± 16.3 pg/ml, P = 0.003). Finally, we established the role of SHP-1 and Cbls in downregulating NK cell cytotoxicity by direct measurement of NK cell killing of target cells. To this end, we utilized a standard radioactive [35S] Met release killing assay (Fig 1E). NK cells that were gene silenced for SHP-1 and Cbls and incubated with 221-Cw4 cells demonstrated superior killing of target cells relative to N.S siRNA-transfected cells (SHP-1 and Cbls siRNA/Cw4 46.02 ± 5.69%; N.S siRNA/Cw4 24.67 ± 4.1%, P = 0.01). Interestingly, NK cells that were gene silenced for SHP-1 and Cbls and incubated with inhibitory 221-Cw4 cells showed no significant difference relative to NK cells incubated with activating 221-Cw7 cells (Mock/Cw7 50.76 ± 5.44%,P = 0.91). Altogether, these data demonstrate that Cbl-b, c-Cbl, and SHP-1 serve as negative key checkpoints suppressing NK cell function, and thus, inhibition of these key intrinsic checkpoints overcomes inhibitory signaling. Click here to expand this figure. Figure EV2. Gating strategies for the identification of NK cells Gating strategy to distinguish between YTS-2DL1 cells and 221-Cw4/7-expressing mCherry cells, using side scatter (SSC) and mCherry. Cells that were negative for mCherry were analyzed for CD107a expression. Imaging of fluorescently labeled NPs using confocal microscopy following sonication. The data shown are representative of three independent experiments. Gating strategy to define YTS-2DL1 incorporated NKp46 antibody-coated NPs population, using anti-KIR2DL1 to define NK cells. Cells that were positive for both rhodamine-labeled NPs and KIR2DL1 antibody staining were analyzed for CD107a expression. Primary NK cells were stained with PE-Cy5-CD3 and FITC-CD56 antibodies followed by staining with PE-KIR2DL1/S1. The pNK-expressing KIR2DL1 subset was then enriched by FACS sorting according to the PE signal. This subset was then incubated with target cells. Gating strategy to define the pNK-KIR2DL1+ using forward scatter (FSC) and side scatter (SSC). Cells that were positive for rhodamine-labeled NPs were analyzed for CD107a expression. Download figure Download PowerPoint Lipid-based nanoparticles for SHP-1 and Cbl siRNA delivery Liposomal NPs are pharmaceutically proven delivery vehicles that can encapsulate a therapeutic agent, and can also display ligands that target cell surface receptors (Torchilin, 2005). Leukocytes are challenging targets for delivery due to their dispersion within the body, making it difficult to successfully localize or passively deliver molecules via systemic administration (Bitko et al, 2005). To circumvent these obstacles, we engineered NK cell-targeting NPs. Liposome-based NPs are efficient in transporting RNAi molecules for the treatment of different malignancies (Peer, 2013). These particles have a lipid bilayer with an internal hydrophilic space—maintaining the payload segregated from the bloodstream. This bilayer formation protects the encapsulated particle content from being degraded by immune cells and enzymes in the bloodstream. Furthermore, the external liposome layer, which enables the coating of its polymeric components, does not induce an immune system response and is not toxic, preventing unwanted side effects (Daka et al, 2012). To enable the targeting of NK cells, NPs were coated with a monoclonal anti-NKp46 antibody (Yossef et al, 2015), which can recognize NK cells via their NKp46 activating receptor (Fig 2A). This receptor serves as a useful marker for NK cell targeting, since it is expressed on all mature NK cells (Glasner et al, 2012). Furthermore, recent work demonstrated that NKp46 expression in tumor-associated NK cells of multiple cancer types is relatively stable compared to that of other activating NK receptors in cancers, such as CD16, NKG2D, and NKp30, which can undergo proteolytic cleavage (Zingoni et al, 2016; Gauthier et al, 2019). Therefore, NKp46 is a prominent marker for NK cells and provides a stable target for NP delivery. The size and the charge of the NPs during the preparatory stages were assessed using dynamic light scattering (DLS; Fig 2B). To monitor NP's distribution and to verify they do not form aggregates, DPPE labeled with rhodamine red (DPPE-PE) was incorporated into the lipid mixture, allowing NP visualization as demonstrated by confocal microscopy (Figs 2C and EV2B). Figure 2. Structure and characterization of liposomal NPs Schematic presentation of liposomal NP layers and composition. Characterization of the diameter and zeta potential (Z-potential
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