YAC-1 Target Research Articles

Jak2V617F Bone Marrow Exposure Leads to Dysfunction of Normal NK Cells

Introduction: We have previously demonstrated a functional impairment of NK cells in the Jak2V617F murine model and peripheral blood cells from patients with Myeloproliferative Neoplasms, with emphasis on the increased frequency of immature NK cells and reduced cytotoxicity of the more mature CD27-CD11b+ fraction. It is not known whether MPN initiating cells are intrinsically resistant to tumor immunity or whether they can modify NK cells of the leukemic microenvironment, leading to dysfunction. Aims: To clarify these mechanisms, the present work aimed to model the leukemic exposure of normal NK cells and verify their modifications using a Jak2V617F chimeric transplant model. Methods: For transplantation, a non-myeloablative conditioning (single sublethal irradiation of 400cGy) was performed to partially preserve the recipient's hematopoiesis (50-60% of donor engraftment). 5 x 10 6 total bone marrow (BM) cells from Jak2VF or Jak2WT control animals (both C57BL/6, CD45.2) were injected retro-orbitally into wild-type mice (C57BL/6, CD45.1, N=6 per group). Engraftment was evaluated by flow cytometry according to CD45.2/CD45.1 differential ratio. After engraftment was confirmed, frequency, receptors, and functional subtypes of exposed NK cells were evaluated. NK cytotoxicity against YAC-1 target cells was evaluated after culture with serum and supernatant of whole BM from chimeric animals. Results: The frequency of host NK cells (CD45.1) exposed to Jak2VF BM was lower than that of NK cells exposed to Jak2WT BM (Jak2WT 44.05% vs Jak2VF 19.76%, p<0.0001). Evaluation of functional subtypes showed that host NK cells had a lower frequency of immature secretory (Jak2WT 44.32% vs Jak2VF 30.70%, p=0,0407) and higher frequency of tolerant (Jak2WT 12.43% vs Jak2VF 24.34%, p=0.0016) and cytotoxic (Jak2WT 7.15% vs Jak2VF 19.30%, p<0.0001) NK cells when exposed to leukemia than when exposed to normal BM. In comparison, secretory immature NK cells from the leukemic BM (CD45.2) were low numbered as compared to Jak2WT NK cells (Jak2WT 73.30% vs Jak2VF 35.74%, p<0.0001) and cytotoxic NK cells were more frequent (Jak2WT 7.28% vs Jak2VF 19.16%, p=0.0003). In addition, host NK cells presented decreased expression of the activating receptor DNAM-1 when exposed to leukemia than when exposed to normal BM (Jak2WT 51.20% vs Jak2VF 46.10%, p=0.0006), whereas CD244 and NKG2D activating receptors were not differentially expressed. In contrast, the expression of TIGIT (but not NKG2A) inhibitory receptor was increased after leukemia exposure (Jak2WT 14.83% vs 46.10%, p=0.0003). Regarding the effect of the leukemic medium on NK cells, cytotoxicity was not altered when WT NK cells were exposed to serum or BM supernatant from chimeric transplanted animals. However, NK cells exposed to Jak2VF chimeric serum were activated according to CD69 expression (Jak2WT 0.060% vs Jak2VF 0.30%, p=0.0114) but presented lower expression of the CD107a degranulation marker (Jak2WT 91.92% vs Jak2VF 62.34%, p=0.0075) than NK cells exposed to Jak2WT cells. Conclusions: Our findings demonstrated that leukemia exposure led to altered NK cell frequency and maturation distribution as demonstrated by reduced immature and an increased tolerant and cytotoxic fraction. Changes in the expression profile of activating and inhibitory receptors were also observed. Therefore, we concluded that the leukemic environment leads to NK dysfunction and that strategies that aim to control NK maturation and the interaction between stem cell ligands and functional NK receptors can be explored to restore immunosurveillance in JAK2V671F Myeloproliferative Neoplasms.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

Late Effects of Heavy-Ion Space Radiation on Splenocyte Subpopulations and NK Cytotoxic Function.

10 month old male CBA/CaJ mice were whole-body irradiated: sham (control); 150-cGy gamma-rays (delivered in 1 fraction); 40-cGy 1-GeV/nu 28Si14+ ions (delivered in 3 fractions); 40-cGy 1-GeV/nu 16O8+ ions (1 fraction); and 40-cGy 1-GeV/nu 16O8+ ions (3 fractions). The mice were sacrificed 1-1.5 yr post-exposure, and the spleens harvested. Splenocyte effector (E) cells were harvested and added to 51Cr-labeled Yac-1 target (T) cells in E:T ratios of 12:1, 25:1, 50:1, and 100:1. NK cytotoxicity was measured with 51Cr release. In addition, 2 million splenocytes were aliquoted and stained with a seven-antibody cocktail, and flow cytometry was used to determine the percentage of NK, B lymphocytes, and T lymphocytes in the splenocyte population. Mice exposed to either a single fraction of 150-cGy gamma rays or 40-cGy 16O8+ ions in 3 fractions were found to have significant decreases in NK cytotoxicity of approximately 30% and 25%, respectively. No significant differences were observed in NK cytotoxicity for 40-cGy 16O8+ ions delivered in 1 fraction, or 40-cGy 28Si14+ ions delivered in 3 fractions. No significant differences were observed in the percentage of spleen cells that were NK (%NK) amongst the groups. Fractionated HZE ion exposure has the potential to affect the innate arm of the immune system long after exposure, leading to decreases in NK cell function. Therefore, protective countermeasures may need to be considered to decrease the risk of reduced long-term immune function in elderly retired astronauts that undertook deep space missions.

Radium-223 dichloride causes transient changes in natural killer cell population and cytotoxic function

Rationale Natural killer (NK) cells play an important role in both the innate and adaptive arms of the immune system. While previous studies have demonstrated the effects of ionizing radiation on cytotoxic function of NK cells, little is known about how a chronic exposure to high LET alpha particles emitted by radionuclides will affect both NK population size and function. This study was conducted to determine the effects of 223RaCl2 on splenic NK cell population size and function in Swiss Webster mice. Methods Swiss Webster mice were administered intravenously with 0, 50, or 600 kBq/kg 223RaCl2. Spleens were harvested at 5, 12, and 19 days post-administration. The numbers of splenocytes per spleen were enumerated and flow cytometry was used to assess changes in the distribution of splenocyte subpopulations of B, CD4 and CD8 T lymphocytes, and NK cells. NK functional activity was quantified using YAC-1 target cells and the 51Cr-release assay. Results The total number of splenocytes was unaffected by 223RaCl2. However, significant changes in the distribution of splenocyte subpopulations were observed for NK cells and CD8 T lymphocytes. NK functional activity was enhanced substantially relative to controls at 12 days post-administration, but decreased markedly by day 19. Conclusion NK functional activity is both diminished and enhanced by 223RaCl2 depending on both administered activity and time post-administration. These results suggest that there may be an optimum window of time to combine the 223RaCl2-induced antitumor NK cell response with other cancer therapies that elicit immune activation.

Abstract 3333: EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity

Abstract Purpose: Breg (B regulatory) cells have been implicated in suppression of anti-tumor response in murine and human solid tumors. Prior studies in our laboratory demonstrated that murine EMT-6 breast carcinoma is rejected in B cell deficient mice (BCDM), but not in wild type (WT) mice, due to an enhanced immune response. Tumor infiltrating B cells (TIL-B), and normal splenic B cells co-cultured with EMT-6 (EMT6-B), express LAP/TGF-β, PD-L1, and suppress CD8+ and NK cell proliferation in vitro. Increased CD8+ and NK cell infiltration into the tumor bed is seen in BCDM. BCDM tumor rejection is abrogated by NK depletion. We studied EMT6-B effects on NK cell phenotype, proliferation and cytokine expression in vitro and in vivo. Experimental design: Normal splenic B cells were co-cultured with mitomycin C treated EMT-6, and Breg phenotype, and effects on NK cell phenotype and proliferation and cytokine function were studied in vitro and in vivo. Results: EMT6-B but not WT splenic B cells inhibited CFSE labeled CD49b+ NK cell proliferation in response to IL-15 in dose dependent fashion. EMT-6-B suppressed expression of CD226 and NKG2D activating receptors on NK cells, and increased expression of PD-1 and PD-L1 respectively. EMT6-B suppressed the induction of IFNγ+/CD49b+ NK cells following PMA/ION stimulation as assayed by intracellular flow cytometry (10%±1.2 to 2%±0.5) and also suppressed IL-2 induced induction of IFNγ+/CD49b+ NK cells (70%±5 to 20%±7.8). EMT-6 and CT26 colon carcinoma conditioned B cells, but not C166 endothelial cell conditioned B cells suppressed NK degranulation (IFNγ+/CD107a+ NK cells). EMT6-B inhibited YAC-1 cell induced NK cell degranulation, and killing of YAC-1 targets, more effectively than naïve B cells (40%±6.2 to 18%±5.1) (P=0.05). NK suppression was inhibited by anti-PD-L1, and anti-TGF-β antibody. EMT6-B expressed increased FasL compared to splenic B cells, and induced NK apoptosis as measured by 7AAD/Annexin-V staining. Blocking antibody to FasL decreased NK apoptosis, and restored NK cell proliferation. Infiltrating NK cells in the EMT6 tumor bed demonstrated increased PD-1 compared to splenic and TDLN NK cells, and higher levels of IFN-γ, CD107a, and Granzyme B in BCDM vs. WT mice. EMT6 expressed high levels of CD155 NK inhibitory ligand compared to C166 endothelial cells. The EMT-6 tumor bed contained higher levels of CD19+CD155+ B cells (22% / tumor bed vs 6% / TDLN, vs 9%/ spleen). BCDM expressed higher levels of CD226+/Ki67+ NK cells in spleen compared to WT splenic NK cells. Antibodies to Fas-L, TGF-beta, and PD-L1 partially restored anti-tumor response in WT mice and decreased tumor growth. Conclusions: EMT-6 TIL-B or normal B cells conditioned by EMT-6 coculture acquire an NK suppressive phenotype which inhibits NK cell activation, proliferation, degranulation and cytotoxic function. Targeting of Breg-NK interactions may improve anti-tumor immune response. Citation Format: Yu Zhang, Muhammad Husnain, Zhifen Luo, Mahmood Al Bayati, Joseph D. Rosenblatt. EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3333.

A Novel Bifunctional Fusion Protein Comprising of TGF-βRII trap and IL15/IL-15Rα as an Immunotherapeutic against Cancer

Abstract Interleukin-15 (IL-15) is a promising cytokine for cancer therapy. However, it has shown limited antitumor efficacy as monotherapy in clinical trials to date. Removal of immunosuppression of the tumor microenvironment is considered a promising approach to enhance IL-15-mediated immune responses. TGF-β is a key component for creating an immunosuppressive tumor microenvironment. Therefore, we constructed a novel bifunctional fusion protein complex, designated HCW9218, comprising a soluble fusion of two TGFβRII, human tissue factor, and human IL-15, and a second soluble fusion of two TGFβRII and a sushi domain of IL-15Rα. HCW9218 activates IL-15R signaling and the dimeric TGFβRII functions as “trap” for all the three TGF-β isoforms. In healthy and tumor-bearing C57/BL6 mice, subcutaneously administrated HCW9218 was well tolerated with a long serum half-life and was able to stimulate and induce proliferation of CD8+ T cells and NK cells. Splenocytes from HCW9218-treated mice showed enhanced metabolic activity and cytotoxicity against Yac-1 target cells compared to untreated mouse splenocytes. In the syngeneic B16F10 melanoma mouse model, a single-dose of HCW9218 exhibited strong antitumor activity in combination with chemotherapy and TA99 antibody. HCW9218 also significantly increased immune cell infiltration into tumors. Mutagenesis studies demonstrated that both the TGFβRII and IL-15/IL-15Rα domains are essential for antitumor efficacy of HCW9218. Collectively, our preclinical data demonstrate that HCW9218 elicits potent immune responses with a remarkable safety profile and serves as a novel immunotherapeutic against cancer alone or in combination with therapeutic antibodies.

Abstract LB-A19: Intratumoral immunosuppression is reversed by blocking adenosine production with an oral inhibitor of CD73

Abstract Introduction: CD73 mediates the final dephosphorylation in the conversion of extracellular ATP to adenosine, a metabolite which signals through the A2 family of receptors. Like PD-1, adenosine appears to be part of a negative feedback loop to limit immune activation and prevent excessive inflammation in the context of tissue damage. However, intratumoral hypoxia likely drives excessive CD73-mediated adenosine generation and prevents optimal anti-tumor immune responses. We show that intratumoral adenosine levels are sufficient to drive pervasive suppression of multiple immune subsets in vitro and an orally bioavailable small molecule can prevent adenosine generation in vitro and within the tumor in vivo. Methods We examined the effects of adenosine signaling on multiple immune cell subsets through in vitro functional assays. We interrogated T cell activation, proliferation, and cytolytic activity, as well as NK cell function in vitro. Furthermore, we investigated and identified functional targets of adenosine signaling within T cells using transcriptomic and protein expression analyses. In addition, we performed in vitro myeloid differentiation assays to determine the effect of adenosine signaling on myeloid lineage maturation. Finally, we explored the activity of a novel, orally bioavailable inhibitor of CD73 in vitro and assessed its ability to rescue T cell activation and suppress AMP conversion to adenosine within the tumor microenvironment. Results and Conclusions We found that T cell-expressed CD73 was sufficient to drive adenosine generation from AMP in vitro, resulting in suppression of anti-CD3/CD28-induced T cell activation and proliferation and a reduced capacity to kill cognate antigen-expressing tumor cells. Adenosine-exposed T cells displayed decreased induction of markers associated with activation at both the mRNA and protein level, including Ki67, ICOS, and PD-1, while showing higher expression of naïve-associated CD73. Interestingly, we also observed a third subset of markers which were uniquely induced by adenosine. In addition to its activity on T cells, adenosine signaling caused functional suppression of NK cells in the presence of target Yac-1 cells. In the myeloid compartment, NECA, a stable analog of adenosine, prevented in vitro differentiation of CD103+ cross-presenting dendritic cells, a population which provides a critical stimulus to tumor-infiltrating T cells. Additionally, NECA impaired macrophage expression of CD80, a canonical M1 marker and mediator of T cell costimulation, in both the presence and absence of M1-polarizing IFNγ and LPS. Finally, we showed that a novel, orally bioavailable CD73 inhibitor was able to effectively inhibit AMP to adenosine conversion both in vitro and in vivo, while an anti-CD73 antibody had incomplete effects. Taken together, an orally bioavailable small molecule inhibitor of CD73 represents a potential therapeutic approach to reverse immunosuppression within the tumor microenvironment. Citation Format: Todd C Metzger, Brian R Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank L Duong, Valeria R. Fantin, Lori S Friedman, Jared T Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Qiuping Ye, Natalie Yuen, Tatiana Zavorotinskaya. Intratumoral immunosuppression is reversed by blocking adenosine production with an oral inhibitor of CD73 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A19. doi:10.1158/1535-7163.TARG-19-LB-A19

Abstract 3949: Characterization of MP1115; a new natural killer cell activator

Abstract MP1115 is in development as a new immuno-oncology therapeutic lead comprised of bioactive phospholipid containing nanoparticles with activities unique to phospholipid active pharmaceutical ingredients. MP1115 demonstrated potent activation of NK cells in vitro and in vivo and had a secondary anti-tumor cell proliferation mechanism of action. Methods: MP1115 was formulated as ~100 nM diameter nanoparticles formulated in buffered sucrose and stably stored frozen. MP1115 nanoparticles were also loaded with a CDK8 inhibitor test compound at a 30 percent loading efficiency. MP1115 was tested for anti-tumor cell proliferation in 2D and 3D in vitro assays, NK cell activation in ex vivo splenocyte and NK cell activation assays, and anti-tumor efficacy and NK cell activation in vivo in immunocompetent and immunodeficient mice. MP1115-CDK8 inhibitor loaded nanoparticles were tested in vitro for increased anti-tumor cell proliferation compared to non-loaded MP1115 nanoparticles and non-loaded CDK8 inhibitor controls. Results: MP1115 demonstrated increased NK cell activation compared to a CDK8 test compound as measured by IFN-γ release in an ex vivo mouse splenocytes-Yac-1 mouse lymphoma assay (p<0.0001). This activity was maintained with splenocytes from immunocompetent BALB/c mice, immunodeficient BALB/c scid mice, and with purified NK cells from each strain background. MP1115 demonstrated increased NK cell activation of mouse splenocytes in vivo in immunocompetent BALB/c mice compared to controls (p<0.0001), as seen by increased CD69 and CD107a signal, as well as the significant killing of target Yac-1 cells ex vivo (p<0.001). The levels of activation and killing observed were similar to that achieved by a CDK8 inhibitor test compound (p>0.9999). MP1115 also successfully demonstrated the ability to deliver a CDK8 test compound payload to human non-Hodgkin’s lymphoma cells in vitro with increased anti-tumor cell proliferation compared to controls (p<0.0001). MP1115 achieved increased anti-tumor cell proliferation efficacy in acute myeloid leukemia, multiple myeloma, non-Hodgkin’s lymphoma, non-small cell lung cancer, KRAS mutant colorectal cancer models in vitro, and in pancreatic cancer tumor models in vivo. MP1115 demonstrated high tolerability in rodent models when dosed at over 200 mg/kg daily for multiple doses. MP1115 is currently being validated in additional in vivo models including human immune system mice with human xenografts. Conclusions: MP1115 is being advanced to IND-enabling studies based on demonstrated low toxicity, high systemic delivery, potent NK cell activation, and anti-tumor cell proliferation activities. Citation Format: Pitchaimani Kandasamy, Jason E. Duex, Greg Miknis, Julie Lang, Ana Chauca-Diaz, Chris Johnson, Colleen Hudson, Jakub Staszak-Jirkovsky, Neal C. Goodwin. Characterization of MP1115; a new natural killer cell activator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3949.

Chemo-immunotherapy improves long-term survival in a preclinical model of MMR-D-related cancer

BackgroundMismatch Repair Deficiency (MMR-D)-related tumors are highly immunogenic and constitute ideal vaccination targets. In a proof-of-concept study delayed tumorigenesis and prolonged survival has been shown in a clinically-relevant mouse model for MMR-D-related diseases (=MLH1 knock out mice). To refine this approach, vaccination was combined with immune modulatory low-dose chemotherapy to polarize immune regulatory subtypes.MethodsMice (prophylactic: 8–10 weeks; therapeutic: > 36 weeks) received a single injection of cyclophosphamide (CPX, 120 mg/kg bw, i.p.) or gemcitabine (GEM, 100 mg/kg bw, i.p.) prior to vaccination (lysate of a gastrointestinal tumor allograft, 10 mg/kg bw, n = 9 mice/group). The vaccine was given repetitively (10 mg/kg bw, s.c., 4 x / once a week, followed by monthly boosts) until tumor formation or progression. Tumor growth ([18F] FDG PET/CT imaging) and immune responses were monitored (flow cytometry, IFNγ ELISpot). The microenvironment was analyzed by immunofluorescence.ResultsProphylactic application of GEM + lysate delayed tumorigenesis compared to lysate monotherapy and CPX-pre-treatment (median time of onset: 53 vs. 47 vs. 48 weeks). 33% of mice even remained tumor-free until the experimental endpoint (= 65 weeks). This was accompanied by long-term effect on cytokine plasma levels; splenic myeloid derived suppressor cells (MDSC) as well as regulatory T cell numbers. Assessment of tumor microenvironment from GEM + lysate treated mice revealed low numbers of MDSCs, but enhanced T cell infiltration, in some cases co-expressing PD-L1. Therapeutic chemo-immunotherapy (GEM + lysate) had minor impact on overall survival (median time: 12 (GEM + lysate) vs. 11.5 (lysate) vs. 3 weeks (control)), but induced complete remission in one case. Dendritic and T cell infiltrates increased in both treatment groups. Reactive T cells specifically recognized MLH1−/− tumor cells in IFNγ ELISpot, but lacked response towards NK cell targets YAC-1.ConclusionsCombined chemo-immunotherapy impairs tumor onset and growth likely attributable to modulation of immune responses. Depleting or ‘re-educating’ immunosuppressive cell types, such as MDSC, may help moving a step closer to combat cancer.

C/Ebpg (CCAAT/Enhancer Binding Protein Gamma) Balances Cytotoxic and Secretory Potential of Natural Killer Cells

C/EBPs (CCAAT/enhance-binding proteins) are a family of transcription factors involved in a variety of hematopoietic processes, regulating both terminal differentiation and cellular proliferation. Among these, it was previously reported that C/EBP gamma (C/EBPg) has a role in the development of Natural Killer (NK) cells. However, the mechanisms of such regulation are unknown. NK cells are lymphocytes with effector functions of cytotoxicity and production of cytokines, both dependent on a dynamic equilibrium between the expression of activating and inhibitory receptors as well as cytokine receptors. The two functions (cytotoxic and secretory) make NK cells important components of hematopoiesis, able to eliminate susceptible targets as well as recruit other cells to amplify inflammatory responses. With the aim of studying the regulation of NK cells by C/EBPg, we isolated NK cells from transgenic Cebpg knockout (KO) mice and controls to analyze their function. To characterize NK cells, we analyzed their frequency (Lineage-/CD3-/NK1.1+ cells) and the expression of the receptors NKG2D, Ly49D and NKG2A by flow cytometry of splenocytes. Both analyses showed no difference between control or Cebpg KO NK cells. Although the numbers of NK cells and their receptors were similar between Cebpg WT and KO animals, a functional assay that measured NK cell degranulation by CD107a expression after co-incubation with YAC-1 target cells showed that the expression of this marker was 5-times lower in Cebpg KO splenocytes than in controls (CT = 12.44 ± 2.50%; KO = 2.255 ± 0.67%, p=0.007), suggesting that Cebpg deficient NK cells are not fully activated after target cell recognition. In addition, a cytotoxicity assay by flow cytometry was performed using a fluorescent probe (Cell Tracker Orange) that was incorporated to YAC-1 cells upon exposure to sorted and IL-2 activated NK cells in culture. In the 10:1 NK:target cells ratio, Cebpg KO cells were significantly less cytotoxic than NK control cells (CT = 23.36 ± 8.67%; KO = 10.60 ± 1.66%, p=0.038). The other NK:target cells ratios of 5:1 and 1:1 showed the same tendency. In addition, the functional subtypes of these cells were characterized according to the expression of CD27 and CD11b, which allowed the identification of NK subpopulations as immature secretory, mature secretory, cytotoxic or tolerant. The KO animals showed higher percentages of secretory cells (CT = 10.77 ± 5.38%; KO = 12.98 ± 13.63%, p=0.0002) and a reduction of cytotoxic cells in comparison to the NK control cells (CT = 12.22 ± 11.08%; KO = 10.65 ± 3.82% p=0.013). Cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17α, TNFα and IFNγ, obtained from NK culture supernatants, were measured by flow cytometry, after IL-2 activation. Among these cytokines, the production of IFNγ by Cebpg-deficient NK cells was reduced (CT = 37.68 ± 0.51 pg/mL; KO = 22.34 ± 0.14 pg/mL, p=0.023). Together, these experiments indicate that C/EBPg regulates NK cell cytotoxicity. This may be explained, at least in part, by the reduced frequency of the mature cytotoxic NK subpopulation as compared to the secretory subtypes. Moreover, IFNγ may be an important target for the regulation of NK cell function. Finally, C/EBPg seems to be critical to mediate NK cell functions and not only for their development from the ontogenetic point of view. DisclosuresNo relevant conflicts of interest to declare.

Rehmannia glutinosa polysaccharide induced an anti-cancer effect by activating natural killer cells

Rehmannia glutinosa polysaccharide (RGP) has exhibited an immune stimulatory effect, such as dendritic cell activation, but it has not been studied in terms of the activation capacity of natural killer (NK) cells in mice in vivo. In this study, we examined the effect of RGP on the proliferation and activation of NK cells in the spleen, mesenteric lymph node (mLN), and blood in vivo and compared that function with that of Escherichia coli lipopolysaccharide. The administration of RGP to C57BL/6 mice induced the increase in circulating blood NK cell numbers and in the proliferation of NK cells in the spleen, mLN, and blood. Moreover, RGP treatment promoted the toll-like receptor-4-dependent production of interferon-gamma and the up-regulation of CD69 expression in spleen NK cells. RGP-treated NK cells enhanced the cytotoxic activity with type I IFN production against target Yac-1 cells. Finally, RGP treatment inhibited the CT26 tumor growth in the lung. These findings demonstrated that RGP promoted the activation of NK cells and inhibited tumor growth in mice in vivo.

Involvement of NFAT Transcription Factors in NK Cell Reactivity

NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells.We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance.Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. DisclosuresNo relevant conflicts of interest to declare.

Porphyromonas gingivalis Lipopolysaccharide Induced Proliferation and Activation of Natural Killer Cells in Vivo.

Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) promoted different innate immune activation than that promoted by Escherichia coli (E. coli) LPS. In this study, we examined the effect of P. gingivalis LPS on the proliferation and activation of natural killer (NK) cells in vivo and compared that function with that of E. coli LPS. Administration of P. gingivalis LPS to C57BL/6 mice induced stronger proliferation of NK cells in the spleen and submandibular lymph nodes (sLNs) and increased the number of circulating NK cells in blood compared to those treated with E. coli LPS. However, P. gingivalis LPS did not induce interferon-gamma (IFN-γ) production and CD69 expression in the spleen and sLN NK cells in vivo, and this was attributed to the minimal activation of the spleen and sLN dendritic cells (DCs), including low levels of co-stimulatory molecule expression and pro-inflammatory cytokine production. Furthermore, P. gingivalis LPS-treated NK cells showed less cytotoxic activity against Yac-1 target cells than E. coli LPS-treated NK cells. Hence, these data demonstrated that P. gingivalis LPS promoted limited activation of spleen and sLN NK cells in vivo, and this may play a role in the chronic inflammatory state observed in periodontal disease.

Augmentation of natural killer cell activity in vitro and in vivo by sericin-derived oligopeptides

This study investigated the effects of sericin-derived oligopeptides on natural killer (NK) activity. In vitro exposure of human peripheral blood mononuclear cells with sericin-derived oligopeptides resulted in an augmentation of NK cell activity against K562 target cells and the effects appeared to be dose-related. Experiments designed to examine whether enhanced NK activity was due to direct or indirect activation of NK cells revealed that sericin oligopeptides did not induce activity of purified NK cells, and that sericin oligopeptides augmented NK activity indirectly by inducing the production of IL-2 and IFN-γ cytokines. In in vivo experimentation where mice were orally administered with sericin oligopeptides and splenic mononuclear cells tested against YAC-1 target cells, significant increase in NK activity was obtained compared to control mice. Elevated levels of IL-2 were also evident in all oligopeptides-treated groups. As demonstrated both in vitro and in vivo, these results indicate that sericin-derived oligopeptides have efficient NK-enhancing activity and suggest the potential therapeutic applications of such oligopeptides for functional improvement of NK cells, and possibly for treatment of tumor and infectious diseases in which NK activity contributes to host defense.

Abstract 2567: Enhancement of antitumor activity of DSP-6434, a novel TLR7 agonist through reduction of TLR tolerance

Abstract Introduction: Systemic administration of the small molecule Toll-like receptor (TLR)-7 agonist DSR-6434 leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, clinical responses with systemically administered TLR7 agonists have been underwhelming, in part because activation of TLRs with small-molecule agonists can induce TLR tolerance; defined as a state of hyporesponsiveness to subsequent agonism. This study is undertaken to identify conditions to overcome TLR tolerance. Experimental procedures: To confirm the anti-tumor effect of DSR-6434, mice were inoculated with the mouse renal cell carcinoma cell line, Renca, at day 0, and then administrated DSR-6434 weekly from day 1, or twice weekly from day 6. To examine tolerance, wild-type or IFN-α/β receptor knockout (IFN-AR KO) mice were intravenously administrated DSR-6434 at intervals of 3, 7 or 10 days. Plasma samples were taken 2 hours after the second administration of DSR-6434 and IFN-α levels were measured. Splenocytes were isolated 24 hours following either single or sequential i.v. doses. To measure lymphocyte activation, CD69 expression was assessed and splenocyte-mediated cytotoxicity against YAC-1 target cells was determined. Bone-marrow derived pDC (BM-pDC) were also treated with DSR-6434 for 5 or 48 hours. Expression of TLR7 signaling-related genes was determined by real-time quantitative RT-PCR. Results: Weekly administration of DSR-6434 significantly reduced tumor burden when compared to vehicle-treated mice. By contrast, pre-inoculation and 2qw administration of DSR-6434 completely abolished anti-tumor activity. IFN-α induction was completely impaired following the second administration of DSR-6434 after 3 days, partially impaired after 7 days, and fully functional when the dosing interval was extended to 10 days. Sequential dosing of DSR-6434 reduced the frequency of activated splenocytes (defined as CD69+) and the level of cytotoxicity, compared with a single administration of DSR-6434. TLR7 tolerance was also observed in IFN-AR KO mice, suggesting this effect was independent of IFN signaling. Interestingly, TLR7 down-regulation was only observed after DSR-6434 dosing, while the expression of other TLR7-signaling related genes was unaltered. Conclusion: The dosing schedule of systemically administered TLR7 agonists significantly affect TLR tolerance and antitumor activity, offering a potential solution to overcome TLR tolerance . Furthermore, TLR7 expression on BM-pDC cells may serve as a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists. Citation Format: Erina Koga-Yamakawa, Masashi Murata, Simon J. Dovedi, Robert W. Wilkinson, Hiroki Umehara, Eiji Sugaru, Yuko Hirose, Hideyuki Harada, David T. Robinson, Philip J. Jewsbury, Setsuko Yamamoto, Chiang J. Li. Enhancement of antitumor activity of DSP-6434, a novel TLR7 agonist through reduction of TLR tolerance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2567. doi:10.1158/1538-7445.AM2014-2567

HPV16 E2 protein promotes innate immunity by modulating immunosuppressive status

The balance between active immune responses against human papillomavirus (HPV) and HPV-induced immune escape regulates viral clearance and carcinogenesis. To understand the role of the early viral protein HPV16 E2 in host innate immune responses, the HPV16 E2-transfected murine squamous cell carcinoma cell line SCCVII (SCC/E2) was generated and anti-tumor responses in T-cell-depleted mice were evaluated. Tumor growth of SCC/E2 was markedly reduced. Cytotoxicity against the NK-sensitive targets YAC-1 and SCCVII was clearly enhanced in SCC/E2-inoculated mice. Despite the comparable ratio of NK cells, the proportion of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) was significantly decreased in SCC/E2-inoculated mice. The transcription of MDSC-related mediators such as inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, and heme oxygenase-1 was significantly impaired in the SCC/E2-inoculated tumor tissues on day 3. Our results suggest that HPV16 E2 promotes anti-tumor innate effector function by modulating immunoregulatory events mediated by MDSCs and their mediators. This report describes a new role for HPV16 E2 as a local immunomodulator at infected sites.

The Antinociceptive Effect of Dexmedetomidine Modulates Spleen Cell Immunity in Mice

Background: Pain plays roles in both the nervous system and immune system. Changes in the neuroendocrine pathway under pain conditions give rise to sympathetic outflow with increased plasma catecholamines and activate immune reactions. Dexmedetomidine exerts sedative, analgesic, and anesthetic-sparing effects and is known to diminish pro-inflammatory processes by central sympatholytic effects. To investigate the influence of the analgesic effect of dexmedetomidine on immunomodulation under pain conditions, splenic natural killer (NK) tumoricidal cytotoxic activity, proliferative ability of T lymphocytes, and cytokine changes were assessed.Methods: After evaluation of the analgesic efficacy of dexmedetomidine in C57BL mice that were subjected to formalin-induced pain, dexmedetomidine (30 µg/kg) or saline was injected intraperitoneally (ip) 30 min before formalin (20 µL of 2% formalin in 0.9% saline) injection. NK cell activity against NK-sensitive YAC-1 lymphoma cells was evaluated by the percentage of specific lactate dehydrogenase (LDH) release. Various numbers of effector cells (NK cells) were added to the wells of a microtiter plate containing 2 × 104 target YAC-1 cells in 100 μL, to achieve final effector-to-target cell ratios of 80:1, 40:1, and 20:1. The level of lymphocyte proliferation in response to phytohemagglutinin (PHA) was detected by bromodeoxyuridine (BrdU) incorporation assay. TNF-α, IL-1β, and IL-10 levels were determined in blood samples and supernatants of splenocyte preparations.Results: IP administration of dexmedetomidine significantly decreased the time of licking and biting during the first and second phases of the formalin test (p <0.001). Formalin-induced pain led to higher activity of NK cells than in sham-treated mice (p <0.05), but NK activity was not increased significantly by ip dexmedetomidine treatment. Formalin-induced pain significantly increased splenic lymphocyte proliferation (p <0.05), but dexmedetomidine did not alter this response. There was a significant increase in plasma TNF-α (p = 0.048) and IL-6 (p = 0.014) levels after formalin-induced pain. However, the differences between the responses after ip dexmedetomidine did not change significantly.Conclusions: Dexmedetomidine showed antinociceptive effect on both of acute pain phase 1 and hyperalgesic phase 2 of formalin pain model. Formalin-induced pain alters cellular immunity of spleen in mice. Dexmedetomidine attenuates the activation of NK cells under pain condition, but neither the proliferative response of the splenic lymphocytes nor the cytokine production was affected by dexmedetomidine.

The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics

To test whether marginating-pulmonary (MP) leukocytes in mice have a unique potential to identify and destroy aberrant circulating cells, we compared MP to circulating leukocytes with respect to natural killer (NK) cytotoxicity, proinflammatory characteristics, molecular determinants of activation, and response to IL-12 immunostimulation. Cytotoxicity was assessed employing the YAC-1, B16F10, and 3LL target lines. C57BL/6 mice were injected with either saline or murine IL-12 (0.1 or 0.5 µg/mouse), either once or three times 48-h apart. Twenty-four hours after last injection, cardiac blood was withdrawn and MP leukocytes were collected by forced lung perfusion. NK cytotoxicity, cellular composition, and surface molecular markers were studied. MP leukocytes exhibited greater NK cytotoxicity than circulating leukocytes against the syngeneic B16F10 and 3LL tumor lines, but not against the allogeneic YAC-1 line. NKG2D and IL-12 receptor expression predicted NK cytotoxicity in circulating leukocytes, but not in MP leukocytes. IFNγ-receptor, IL-12-receptor, CD69, CD11a, and CD11b showed different patterns of expression in the two leukocyte populations, suggesting pro-inflammatory characteristics of the MP compartment. IL-12 stimulation caused differential effects on these markers and also elevated cytotoxicity in both compartments, but in different effector: target ratio-dependent patterns. MP leukocytes may play a critical role in eliminating aberrant circulating cells due to their enhanced NK cytotoxicity and given their strategic location in the lungs vasculature, which forces physical interactions with all circulating aberrant cells. MP-NK cells are unique in their cytotoxic mechanisms against syngeneic targets and in their activation profile and response to immunostimulatory agents.

Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

To investigate the stress-induced apoptosis of natural killer (NK) cells and the changes in their killing activity in mouse livers. A restraint stress model was established in mice. Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver. The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay. The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen, which decreased the cell number. The number and percentage of NK cells in the spleen decreased. However, the number of NK cells in the liver decreased, whereas the percentage of NK cells was significantly increased. The apoptosis of NK cells increased gradually with prolonged stress time, and the macrophage-1 (Mac-1)(+) NK cells were more susceptible to apoptosis than Mac-1(-) NK cells. Large numbers of Mac-1(-) NK cells in the liver, which are more resistant to stress-induced apoptosis, were observed than the Mac-1(-) NK cells in the spleen. The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased, but the retention of numerous Mac-1(-) NK cells in the liver maintained the killing ability. Significant stress-induced apoptosis was observed among Mac-1(+) NK cells, but not Mac-1(-) NK cells in the mouse liver. Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.