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Natural killer (NK) cells are innate lymphocytes with cytotoxic activity. Understanding the factors regulating cytotoxicity is crucial for improving NK-cell adoptive therapies. Here, we studied a previously unknown role of p35 (CDK5R1), a coactivator of cyclin-dependent kinase 5 (CDK5) in NK-cell function. p35 expression was thought to be neuronal-specific and the majority of studies are still focused on neuronal cells. Here, we show that CDK5 and p35 are expressed in NK cells and are kinase-active. NK cells from p35 knockout mice were analyzed and showed significantly increased cytotoxicity against murine cancer cells, while they did not show any differences in cell numbers or maturation stages. We confirmed this using human NK cells transduced with p35 short hairpin RNA (shRNA), showing similar increase in cytotoxicity against human cancer cells. Overexpression of p35 in NK cells resulted in moderate decrease in cytotoxicity, while expressing a kinase-dead mutant of CDK5 displayed increased cytotoxicity. Together, these data suggest that p35 negatively regulates NK-cell cytotoxicity. Surprisingly, we found that TGFβ, a known negative regulator of NK-cell cytotoxicity, induces p35 expression in NK cells. NK cells cultured with TGFβ exhibit reduced cytotoxicity, while NK cells transduced with p35 shRNA or mutant CDK5 expression exhibited partial reversal of this inhibitory effect pointing to an interesting hypothesis that p35 plays an important role in TGFβ-mediated NK-cell exhaustion. Significance: This study reports a role for p35 in NK-cell cytotoxicity and this might help to improve NK-cell adoptive therapy.

Overexpression of kinase-dead CDK5 increases NK92 cytotoxicity while overexpression of p35 decreases cytotoxicity. A, Western blot analysis of protein from NK92 cells overexpressing CDK5 (OE CDK5), kinase-dead mutant CDK5 (OE CDK5-K33T), p35 (OE p35), or empty vector control (OE Ctrl). B, NK92 cells were plated in complete media with a starting count of 250,000 cells. Live cell count was obtained after 48 hours cell culture via trypan blue staining and manual counting on a hemocytometer. ns, not significant. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. C, NK92 cells were stained with Annexin V and PI and analyzed via flow cytometry. Percentage of cells staining positive for Annexin V and/or PI were graphed. Graphs display mean ± SD, n = 3 biological cultures. D, NK92 cells were fixed in 70% ethanol and stained with PI, then analyzed via flow cytometry. G0–G1, S, and G2–M phases of the cell cycle are indicated. E, NK92 cells (OE Ctrl, OE CDK5, OE CDK5-K33T) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at different E:T ratios, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. ****, P < 0.0001. Graph displays mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells (OE Ctrl, OE p35) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at 5:1 E:T ratio, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. *, P < 0.05. Graph displays mean ± SD, n = 3 biological cocultures, unpaired two-tailed t test.

Natural killer (NK) cells are innate lymphocytes with cytotoxic activity. Understanding the factors regulating cytotoxicity is crucial for improving NK-cell adoptive therapies. Here, we studied a previously unknown role of p35 (CDK5R1), a coactivator of cyclin-dependent kinase 5 (CDK5) in NK-cell function. p35 expression was thought to be neuronal-specific and the majority of studies are still focused on neuronal cells. Here, we show that CDK5 and p35 are expressed in NK cells and are kinase-active. NK cells from p35 knockout mice were analyzed and showed significantly increased cytotoxicity against murine cancer cells, while they did not show any differences in cell numbers or maturation stages. We confirmed this using human NK cells transduced with p35 short hairpin RNA (shRNA), showing similar increase in cytotoxicity against human cancer cells. Overexpression of p35 in NK cells resulted in moderate decrease in cytotoxicity, while expressing a kinase-dead mutant of CDK5 displayed increased cytotoxicity. Together, these data suggest that p35 negatively regulates NK-cell cytotoxicity. Surprisingly, we found that TGFβ, a known negative regulator of NK-cell cytotoxicity, induces p35 expression in NK cells. NK cells cultured with TGFβ exhibit reduced cytotoxicity, while NK cells transduced with p35 shRNA or mutant CDK5 expression exhibited partial reversal of this inhibitory effect pointing to an interesting hypothesis that p35 plays an important role in TGFβ-mediated NK-cell exhaustion.Significance:This study reports a role for p35 in NK-cell cytotoxicity and this might help to improve NK-cell adoptive therapy.

TGFβ induces p35 expression in a dose- and time-dependent manner. A, NK92 cells were incubated with fluorescently-labeled Jeko-1 cells and different concentrations of TGFβ for 16 hours. Cells were stained with PI and gated on labeled cancer cells via flow cytometry to measure cancer cell death. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, one-way ANOVA with Dunnett multiple comparisons test. B, NK92 cells were incubated with different concentrations of TGFβ in complete media for 48 hours. Protein was collected and analyzed via Western blot analysis. C, NK92 cells were incubated with 5 ng/mL TGFβ in complete media for 24 hours with or without the TGFβ receptor inhibitor EW-7197 (5 ng/mL). Protein was collected and analyzed via Western blot analysis. D, 1° NK cells were incubated in complete media with different concentrations of TGFβ and/or 5 ng/mL EW-7197 for 48 hours. Protein was collected and analyzed via Western blot analysis. E, 1° NK cells were cultured in cytokine-free media for 48 hours, followed by culture with either 200 U/mL IL2 or 5 ng/mL TGFβ. Protein lysate was collected at the indicated timepoints.

NK92 cells with p35 knockdown or CDK5-K33T overexpression mitigate TGFβ-induced inhibition of cytotoxicity. A, NK92 cells (shGFP or shp35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. B, Supernatant from NK92 cell culture (shGFP or shp35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. C, NK92 cells (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test. D, Supernatant from NK92 cell culture (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test.

Murine NK cells from p35 k/o mice display heightened cytotoxicity. A, Murine splenocytes or bone marrow cells from WT or p35 k/o mice were stained for CD3 and NK1.1 and mixed with counting beads, then analyzed via flow cytometry. Gating on NK1.1+CD3− cells, as well as the counting beads, allowed determination of mNK concentration and the total mNK cell count in each compartment. ns, not significant. Graphs display mean ± SD, n = 3 mice, unpaired two-tailed t test. B, mNK cells were isolated from WT, p35 k/o, or p35 Het mice and cocultured with calcein AM-labeled B16F10 cells at different E:T ratios for 4 hours. Fluorescence intensity of supernatant corresponding to calcein release from dead cells was measured to calculate NK-mediated cytotoxicity. **, P < 0.01. Graphs display mean ± SD, n = 3 biological cocultures (n = 2 for WT 1:1 ratio, p35 k/o 1:1 ratio, and p35 k/o in WT versus p35 Het versus p35 k/o experiment), and multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method (WT vs. p35 k/o) or one-way ANOVA with Tukey multiple comparisons test (WT vs. p35 Het vs. p35 k/o). C, Murine splenocytes or bone marrow cells were stained for CD11b, CD27, CD3, and NK1.1 and analyzed via flow cytometry after gating on NK1.1+CD3− cells. Representative flow plots are shown. Percentages of mNK cells in each subset (CD11b+CD27−, CD11b−CD27+, and CD11b+CD27+) for each mouse were recorded and graphed. ns, not significant. Graphs display mean ± SD, n = 8 mice, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. D, Murine splenocytes were stained for CD3, NK1.1, and NKG2D, NKp46, NKG2A, 2B4, LY6C, or LY49C/F/I/H. Representative histograms of activating/inhibitory receptor expression are shown, gated on live NK1.1+CD3− cells. E, Isolated mNK cells pooled from multiple mice were seeded at a concentration of 2e6 cells/mL and cultured ex vivo in RPMI media supplemented with 1,000 U/mL IL2. An additional 1,000 U/mL IL2 was added 2 days later without changing the media, followed by collection of supernatant after culture for an additional 2 days. Cytokine concentration was determined using a flow-based, multiplex murine cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01. Graphs display mean ± SD, n = 3 biological cultures, unpaired two-tailed t tests.

CDK5 knockdown inhibits NK92 survival while p35 knockdown increases NK92 cytotoxicity. A, qRT-PCR results using RNA isolated from NK92 cells transduced with p35 shRNA (shp35) or CDK5 shRNA (shCdk5) compared with shGFP control. GAPDH was used as the housekeeping gene. Graphs display mean ± SD, n = 3 technical replicates. B, Western blot analysis of protein from NK92 cells transduced with p35 shRNA (shp35) or negative control GFP shRNA (shGFP). C, Phase contrast microscopy of NK92 cells transduced with CDK5 shRNA or one of two p35 shRNA clones, compared with shGFP control. Images are 10x magnification; scale bar, 100 μm. D, NK92 cells were plated in complete media with a starting count of 250,000 cells. Live cell count was obtained after 48 hours cell culture via trypan blue staining and manual counting on a hemocytometer. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. E, NK92 cells were stained with Annexin V and PI and analyzed via flow cytometry. Percentage of cells staining positive for Annexin V and/or PI were graphed. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours to measure the effect of p35 knockdown on cytotoxicity. Cells were stained with PI and gated on the labeled cancer cells to measure cancer cell death. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test.

Supplemental Figures. This file contains supplemental figures concerning analysis of megakaryocyte progenitor and erythroid progenitor analysis in the leukemia marrow, platelet and megakaryocyte progenitor analysis after DBZ treatment of leukemia mice, as well as CXCL12 promoter CHIP analysis

Supplemental Figure Legends

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients. Cancer Res; 76(6); 1641–52. ©2016 AACR.

Supplemental Materials and Methods This file contains detailed methods of IHC, WB, CHIP assay, luciferase reporter assay, qRT-PCR and 2-photon imaging analysis and etc.

Arterial and venous thrombosis constitutes a major source of morbidity and mortality worldwide. Long considered as distinct entities, accumulating evidence indicates that arterial and venous thrombosis can occur in the same populations, suggesting that common mechanisms are likely operative. Although hyperactivation of the immune system is a common forerunner to the genesis of thrombotic events in both vascular systems, the key molecular control points remain poorly understood. Consequently, antithrombotic therapies targeting the immune system for therapeutics gain are lacking. Here, we show that neutrophils are key effectors of both arterial and venous thrombosis and can be targeted through immunoregulatory nanoparticles. Using antiphospholipid antibody syndrome (APS) as a model for arterial and venous thrombosis, we identified the transcription factor Krüppel-like factor 2 (KLF2) as a key regulator of neutrophil activation. Upon activation through genetic loss of KLF2 or administration of antiphospholipid antibodies, neutrophils clustered P-selectin glycoprotein ligand 1 (PSGL-1) by cortical actin remodeling, thereby increasing adhesion potential at sites of thrombosis. Targeting clustered PSGL-1 using nanoparticles attenuated neutrophil-mediated thrombosis in APS and KLF2 knockout models, illustrating the importance and feasibility of targeting activated neutrophils to prevent pathological thrombosis. Together, our results demonstrate a role for activated neutrophils in both arterial and venous thrombosis and identify key molecular events that serve as potential targets for therapeutics against diverse causes of immunothrombosis.

Background: This study sought to evaluate the current services and delivery models of adolescent and young adult oncology (AYAO)–specific programs at NCI-designated Cancer Centers (NCI-CCs). Patients and Methods: NCI, academic, and community cancer centers were electronically sent surveys from October to December 2020 and administered via REDCap. Results: Survey responses were received from 50 of 64 (78%) NCI-CCs, primarily completed by pediatric oncologists (53%), adult oncologists (11%), and social workers (11%). Half (51%) reported an existing AYAO program, with most (66%) started within the past 5 years. Although most programs combined medical and pediatric oncology (59%), 24% were embedded within pediatrics alone. Most programs saw patients aged 15 (55%) to 39 years (66%) mainly via outpatient clinic consultation (93%). Most centers reported access to a range of medical oncology and supportive services, but dedicated services specifically for adolescent and young adults (AYAs) were available at a much lower extent, such as social work (98% vs 58%) and psychology (95% vs 54%). Although fertility preservation was offered by all programs (100%), only two-thirds of NCI centers (64%) reported providing sexual health services to AYAs. Most NCI-CCs (98%) were affiliated with a research consortium, and a lesser extent (73%) reported collaboration between adult and pediatric researchers. Nearly two-thirds (60%) reported that AYA oncology care was important/very important to their respective institution and reported providing good/excellent care to AYAs with cancer (59%), but to a lesser extent reported good/excellent research (36%), sexual health (23%), and education of staff (21%). Conclusions: Results of this first-ever national survey to assess AYAO programs showed that only half of NCI-CCs report having a dedicated AYAO program, and that areas of improvement include staff education, research, and sexual health services for patients.

Response to mechanical signals is an important mechanism by which T lymphocytes drive inflammation. A potential contributor in this process is Piezo1, a mechanosensitive cation channel which is abundantly expressed in T lymphocytes. Previously, our lab has shown that Piezo1 colocalizes with high affinity LFA-1 on activated human T lymphocytes during chemokine-activated crawling, suggesting a role for Piezo1 in the adhesion cascade. However, the mechanism by which Piezo1 contributes to the regulation of the adhesion cascade is unknown. We hypothesize that Piezo1 mediates T lymphocyte migration through direct contact with integrin to regulate its function. We investigate the role of Piezo1 in the expression, binding affinity, and turnover of LFA-1 and VLA-4. Co-immunoprecipitation revealed a preferential association of Piezo1 with the alpha subunit of integrins, specifically LFA-1, MAC-1, and VLA-4 in the active forms. Moreover, microfluidic devices were used to study the contribution of Piezo1 on T lymphocytes under laminar flow-induced shear stress. We found that activated T lymphocytes with CRISPR/Cas9-mediated Piezo1 ablation resulted in a reduced chemokine-induced crawling speed that correlates to the efficiency of Piezo1 gene ablation. Finally, in studies of lymph node homing where fluorescently labeled T lymphocytes were tracked in vivo, Piezo1 deficient cells exhibit increased ingress and decreased egress through the lymph node, suggesting an intricate role of Piezo1 on T lymphocyte migration. Taken together, our data support the hypothesis that Piezo1 modulates inflammatory processes through complex integrin interactions and mediates T lymphocyte migration from the lymph node to the site of inflammation. Case Postbaccalaureate Research Education Program grant NIH/NIGMS 5R25GM075207-15; St. Baldrick’s Foundation; Hyundai Hope-on-Wheels Program; The I’m Not Done Yet Foundation; and Pediatric Cancer Research Foundation