Murine Natural Killer Cells Research Articles

Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells

Natural killer (NK) cells are a promising tool for cell therapy due to their capacity to lyse tumor cells without prior activation and their influence on the innate as well as the adaptive immunity. To characterize distinct NK cell populations, it is important to find a reliable isolation method. We investigated separation methods for NK cell isolation by magnetic bead labeling. There are three commonly used different MACS protocols to isolate NK cells from murine spleen: CD49b (DX5) MicroBeads, the NK Cell Isolation Kit and a separation method which is based on a positive selection for NKp46 expressing cells.Interestingly, we found a significant difference of NK cell purities depending on the mouse strains and the purification protocol used. We observed a significantly higher level of purity and yield of NK cells by preparations from Balb/c splenocytes. In this study, we modified the negative selection protocol and adapted it to C57Bl/6 mice to obtain equal purity, viability and yields of NK cells in the different mouse strains. Moreover, we optimized the NKp46 NK cell selection method by addition of a B cell depletion step. To our knowledge, this is the first report that has directly compared and essentially modified the different NK cell purification strategies in parallel, both in C57Bl/6 and Balb/c mouse strains. Altogether, these results are a basic prerequisite for the further development of NK cell therapy protocols in murine in vivo models.

Low level of FcγRIII expression on murine natural killer cells

Cellular Fcγ-receptors are crucial for mediating the functions of therapeutic antibodies. Antibody dependent cellular cytotoxicity (ADCC) is an important mechanism by which Fcγ-receptor expressing cells of the innate immune system including natural killer (NK) cells can kill opsonized target cells. During FACS analysis, however, binding of the Fc-fragment of staining antibodies specific for cell type specific receptors can lead to false positive results and wrong interpretation of the data. Current strategies to block such unwanted binding largely target FcγRIIB and FcγRIII but not the recently identified mouse FcγRIV. In this study we demonstrate that Fc-dependent binding of the NK cell specific antibody NK1.1 by FcγRIV on monocytes results in a large overestimation of FcγRIII expression on murine NK cells. These results highlight the importance of blocking unwanted binding of FACS antibodies to FcγRIV and shed new light on the expression level of FcγRIII on NK cells in mice during the steady state.

Allogeneic Cytokine-Induced Killer Cells Kill Dendritic Cells Efficiently Resulting in Less Graft-Versus-Host Disease

Abstract 4308Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. The mechanism of suppression of GVHD after allogeneic bone marrow transplantation is, in part, due to the abundant production of IFN-gamma from the CIK cells, which has protective effect against GVHD. We have also demonstrated that allogeneic CIK cells displayed a significant lower acquisition of homing molecules, required for the entry of inflamed and GVHD target organs (a4b7, CCR9, E-selectin, CXCR3, and CCR5) and a higher susceptibility to apoptosis compared to allogeneic splenocytes. There also might be some causes other than we mentioned above. Host residual dendritic cells (DCs) have a crucial role for initiating GVHD reaction, because they present recipient alloantigens to donor T cells, which finally attack on recipient tissues. Murine alloreactive NK cells, even when infused in large numbers, do not cause GVHD in the mouse by killing recipient DCs. Similarly, it remains a possibility that the reduced GVHD in CIK cells receiving mice was due to the elimination of residual host DCs by CIK cells. To test this, DCs generated from bone marrow cells with the addition of GM-CSF were used for 51Cr release cytotoxicity assays as target cells. Although autologous CIK cells (Balb/c) had relatively strong killing activity against DCs (Balb/c), allogeneic CIK cells (B6) had much more killing activity even from at a 5:1 effector-target ratio as shown below. Allogeneic CD8+ cells did not show any killing activity against DCs. In addition, killing activity against DCs did not changed with/without adding NKG2D blocking antibody, suggesting that other mechanisms to undergo cell lysis should exist in CIK cells in addition to NKG2D/NKG2D ligand system, which are mainly involved in tumor eradication. To further evaluate whether allogeneic CIK cells kill host DCs in vivo, lethally irradiated Balb/c recipients were given BM (B6) with CIK cells or splenocytes to compare the percent of residual host-typed DCs in the spleen five days after bone marrow transplantation. Percent of host-typed DCs tended to be lower in the mice receiving alllogeneic CIK cells compared with those receiving fresh splenocytes or BM alone. In conclusion, allogeneic CIK cells caused less GVHD due in part to elimination of host DCs. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Lenalidomide Augments NKG2D-Mediated Cytokine Production but Not Cytotoxicity From NK Cells

Abstract 1918The ability to use natural killer (NK) cells as adoptive immunotherapy to treat malignancies is under intense clinical investigation. Cytokine activation is commonly used to enhance NK cell proliferation (Miller Blood 2005; Laport BBMT 2011). Drawbacks include long-term culture conditions to generate adequate numbers of NK cells and cytokine injections to patients post-infusion to maintain activity. Lenalidomide is an immunomodulatory drug widely used to induce tumor remission in multiple myeloma and myelodysplastic syndrome and has been shown to reduce the incidence of relapse after allogeneic hematopoietic cell transplantation. Lenalidomide has also been reported to augment T and NK cell activation and, coupled with its anti-proliferative and pro-apoptotic activity, could be a novel tool to optimize NK cell adoptive immunotherapy for malignancies. However, the precise molecular mechanisms that are influenced by lenalidomide have yet to be determined. In this study, we analyzed the role of lenalidomide on NKG2D-mediated murine NK cell activation and effector functions. NK cells collected from C57BL/6 mouse spleens were activated with plate-bound anti-NKG2D MAb (A10). After 18 hours, supernatants were collected and assayed using a Bioplex kit to evaluate cytokine and chemokine generation. To assess the ability of NK cells in mediating cytotoxicity through the NKG2D receptor, we tested EL4 murine thymoma cell lines that stably express differing levels of a ligand for NKG2D known as H60 (identified as either H60hi or H60lo depending on level of expression), in 4 hour 51Cr-release assays. Our results show that the NKG2D-mediated production of inflammatory cytokines (IFN-g, GM-CSF, TNF-a, IL-10) and chemokine (MIP-1a) were significantly augmented after the pre-incubation of NK cells for three hours with 5 mM lenalidomide. Specifically, we found at least threefold increases of MIP-1a and TNF-a in lenalidomide-treated IL-2-cultured NK cells compared to non-treated NK cells and twofold increases in IFN-g, GM-CSF, and IL-10 (Fig 1). However, NK cell recognition and cytotoxicity against H60hi- positive tumor cells were unaltered after culturing with lenalidomide (p>0.5). Neither increased drug doses (0.5 to 20 mM) with varied pre-incubation periods (1–18 hours) nor the addition of growth cytokines such as IL-2, IL-12 and IL-18 altered these outcomes. Furthermore, incubating H60hi-positive tumor cells alone with lenalidomide and co-culturing NK cells with targets or antibodies to induce NK cell activation during lenalidomide exposure did not appear to augment NK cell killing (p>0.5). To decipher whether the lack of increased cytotoxicity was a reflection of an already maximized level of NKG2D engagement in H60hi-positive tumor cells, we similarly tested H60lo thymoma cell lines in concurrent experiments and found comparable lack of increased cytotoxicity (p>0.5). To define the mechanism of lenalidomide action, we are currently investigating the phosphorylation status of multiple signaling molecules downstream of the NKG2D receptor in NK cells, with specific focus of its effects on cytokine/chemokine production and its recruitment of other effector populations in both in vitro and in vivo systems. Additionally, we are evaluating other tumor cell lines and fresh tumor samples as targets. Collectively, our in vitro studies demonstrate that lenalidomide-treated NK cells significantly immunomodulate cytokine and chemokine production but have little or no effect on NK cell-mediated thymoma tumor killing. Our preclinical findings may provide insight when designing NK-based adoptive immunotherapy strategies using lenalidomide in the clinical setting. [Display omitted] Disclosures:Bartlett:Celgene Corporation: Employment.

Changes in Cytokine Levels and NK Cell Activation Associated with Influenza

Several studies have highlighted the important role played by murine natural killer (NK) cells in the control of influenza infection. However, human NK cell responses in acute influenza infection, including infection with the 2009 pandemic H1N1 influenza virus, are poorly documented. Here, we examined changes in NK cell phenotype and function and plasma cytokine levels associated with influenza infection and vaccination. We show that absolute numbers of peripheral blood NK cells, and particularly those of CD56bright NK cells, decreased upon acute influenza infection while this NK cell subset expanded following intramuscular influenza vaccination. NK cells exposed to influenza antigens were activated, with higher proportions of NK cells expressing CD69 in study subjects infected with seasonal influenza strains. Vaccination led to increased levels of CD25+ NK cells, and notably CD56bright CD25+ NK cells, whereas decreased amounts of this subset were present in the peripheral blood of influenza infected individuals, and predominantly in study subjects infected with the 2009 pandemic H1N1 influenza virus. Finally, acute influenza infection was associated with low plasma concentrations of inflammatory cytokines, including IFN-γ, MIP-1β, IL-2 and IL-15, and high levels of the anti-inflammatory cytokines IL-10 and IL-1ra. Altogether, these data suggest a role for the CD56bright NK cell subset in the response to influenza, potentially involving their recruitment to infected tissues and a local production and/or uptake of inflammatory cytokines.

Abstract A68: An IL-12/Shh-C domain fusion protein based IL-12 autocrine loop for sustained natural killer cell activation

Abstract Background: It has been known that resting natural killer (NK) cells do not possess high levels of cytotoxicity or inflammatory cytokines that are needed for efficient tumor cell killings. Ex vivo activated NK cells, when transferred back in vivo, need a large dose of IL-2 or IL-12 to maintain such an activated status, which is a main concern for systemic toxicity. The aim of the present study is to establish an NK cell self-activation system by employing the autoprocessing domain of mouse sonic hedgehog gene to form an IL-12/Shh-C fusion gene. This fusion protein can go through autocatalytic processing to form cholesterol-modified IL-12 molecules, which would in turn bind back to the NK cells in which it is produced in an autocrine fashion. Methods: EGFP and IL-12 sequences were fused in frame with mouse sonic hedgehog C-terminal domain (Shh-C) to form eGFP/shh-C and IL-12/shh-C fusions. IL-12 without shh-C tail was used as control. Pseudo-lentiviral particles were produced and used to transduce purified murine NK cells. Subsequently, cell surface markers, cell proliferation rates, and IFN-Y secretion of NK cells transduced with different viral particles were analyzed with flow cytometry, alamar blue cell proliferation assay, and cytokine ELISA respectively. For in vivo studies, C57BL/6 mice received transduced NK cells intravenously without IL-2 support 3 days after receiving tail vein injection of B16 melanoma tumor cells and lung tissues were analyzed to determine the infiltration of modified NK cells into the tumor microenvironment. Results: The transduction rates of the three (eGFP/shh-C, IL-12, IL-12/shh-C) lentivirus-transduced NKcell populations 5 days after infection are ranging from 20% to 32%, and were maintained within this range at least for up to 10 days after transduction. CD25 (IL-2 receptor) was found to be substantially higher in eGFP/shh-C infected (2.59-fold), IL-12 infected (2.67-fold), and IL-12/shh-C (2.04-fold) infected NK cells respectively, compared to untreated NK cells. EGFP/shh-C infected, IL-12 infected, and IL-12/shh-C infected NK cells were also observed to have significantly increased Mac-1 expressions (25%, 27%, and 18% respectively). Perforin production in IL-12/shh-C infected NK cells was doubled compared to that in untreated NK cells. In the presence of 60 lU/mL and 6000 lU/mL IL-2, IL-12/shh-C infected NK cells have 63.9% (p<0.05) and 216.7% increase of proliferation rate respectively when compared to eGFP/shh-C infected NK cells, suggesting that the fusion protein decreased the requirement of NK cells for IL-2. The amounts of IFN-Y in the supernatant of IL-12/shh-C virus transduced NK cells 5 days and 7 days after transduction were 40% (p=0.008) and 48% (p=0.00045) higher respectively compared to the untreated group. Results from the cytotoxicity assay indicated that lentivirus transduction did not alter NK cell-associated cytotoxicity. Immunofluorescent staining in the lung tissue slides from B16-bearing mice suggested that donor NK cells successfully infiltrated into tumor nodules in lung tissues. Conclusion: The IL-12/Shh-C domain fusion protein could function, once autoprocessed to form cholesterol anchored IL-12, to maintain the activated status of NK cells in vitro, as evidenced by the greatly reduced amount of IL-2 required as well as the greatly increased secretion of IFN-Y by NK cells in vitro. In addition, IL-12/shh-C virus transduced NK cells successfully infiltrated into B16 tumor nodules in lung tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A68.

Hydrodynamic Delivery of Human IL-15 cDNA Increases Murine Natural Killer Cell Recovery after Syngeneic Bone Marrow Transplantation

Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets invitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes

AbstractThe engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αβ CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I–guided education process. These data suggest that education is an NK cell–specific function of inhibitory MHC-I receptors.

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Interleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18. In the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells. There was a 4.8-fold significant increase in NK-cell population (CD3(-)/16(+)/56(+); P < 0.03), 21-fold significant increase in CD3(-)/56(+)/158a(+) (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3(-)/56(+)/158b(+) (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3(-)/56(+)/NKB1(+) (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively. We have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.

Prostaglandin E2 (PGE2) suppresses natural killer cell function primarily through the PGE2 receptor EP4

The COX-2 product prostaglandin E(2) (PGE(2)) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE(2) production or PGE(2)-mediated signaling through the PGE(2) receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE(2), but very little is known about the mechanism by which PGE(2) affects NK effector activity. We now report the direct effects of PGE(2) on the NK cell. Endogenous murine splenic NK cells express all four PGE(2) receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE(2), the EP4 agonist PGE(1)-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE(2), the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE(1)-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE(2) production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.

CD34+ Cord Blood DC-induced Antitumor Lymphoid Cells Have Efficacy in a Murine Xenograft Model of Human ALL

Acute lymphocytic leukemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We postulate that cytotoxic T lymphocytes (CTLs) generated from total RNA loaded cord blood CD34+-derived dendritic cells can control the kinetics of leukemic growth in a nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse model of human ALL. CD34+-derived dendritic cells electroporated with total RNA from an ALL xenograft generate antileukemic CTL with specificity for the ALL xenograft while sparing autologous cord blood mononuclear cells. The CD3+ T-cell compartment of the CTL was dominated by CD4+ T cells, although CD8+ T cells accounted for an average of 30% of the CD3+ T cells present. Expansion of both CD4+ and CD8+ memory and terminal effector memory subsets from predominantly naive cells was evident. Natural killer (NK) cells accounted for an average of 13% of the final antitumor lymphoid cells produced. Blocking experiments confirmed that the CD8+ T-cell compartment was responsible for the antileukemic activity of the polyclonal CTL pool. Administration of antileukemic CTL to NOD-SCID mice bearing ALL xenograft cells was able to delay, but not prevent the growth of ALL in vivo. Coadministration of antigen-loaded antigen-presenting cells did not further improve upon the delay in ALL engraftment kinetics observed with CTL alone. The efficacy of adoptively transferred polyclonal CTL can be improved with coadministration of recombinant human interleukin-2. However, in NOD-SCID mice, the efficacy of these adoptively transferred cells is masked by interleukin-2 stimulation of murine NK cells, which facilitate killing of ALL cells. Our data highlights the role for NK cells in antileukemic responses posttransplant. Collectively, our results support the notion that ALL-specific adoptive immunotherapy could be used clinically and provide an alternative strategy for preventing and treating disease relapse posttransplant and that the success of this therapy is likely to be maximized if given in the setting of minimal residual disease.

Expansion of 2B4+ natural killer (NK) cells and decrease in NKp46+ NK cells in response to influenza

Several studies have highlighted the importance of murine natural killer (NK) cells in the control of influenza virus infection, notably through the natural cytotoxicity receptor NKp46. However, little is known about the involvement of NK cells in human influenza infection. Here, we show that upon in vitro exposure to influenza, NKp46 expression on NK cells decreases, whereas expression of 2B4, an activating receptor that can enhance natural cytotoxicity in synergy with NKp46, is up-regulated. Consistent with these observations, NKp46(dull) and 2B4(bright) NK cells had a higher functional activity in response to influenza than NK cells expressing high levels of NKp46 or low levels of 2B4, respectively. Importantly, we assessed whether the expression of these receptors was also modified in vivo in response to influenza antigens and showed that an increase in 2B4-expressing NK cells and a decrease in NKp46(+) NK cells occurred following intramuscular influenza vaccination. Altogether, our results further suggest that NKp46 may play an important role in the innate immune response to human influenza and reveal that exposure to influenza antigens is associated with a previously unrecognized increase in 2B4 expression that can impact NK cell activity against the virus.

Cho-kyung-jong-ok-tang, a traditional Korean herbal formula induces type 2 shift in murine natural killer cell cytokine production

Cho-kyung-jong-ok-tang (CKJOT) is a traditional Korean herbal formula specifically used for female infertility including unexplained recurrent pregnancy loss (RPL). The present study aims to evaluate the effects of CKJOT on mouse natural killer (NK) cells to address the possible immunological basis of protective effects of this herbal medicine on unexplained RPL. NK cells isolated from spleens of 6-week-old C57BL/6 mice were differentiated into NK0, NK1, and NK2 cells in the presence of various concentrations of CKJOT-extract. Apoptotic cell number, level of intracellular cytokines, and expression of cytokine-related transcription factors were measured. CKJOT had little effect in improving viability of NK0, NK1, and NK2 cells. However, CKJOT addition during NK cell differentiation suppressed the production of interferon-gamma (IFN-γ), and enhanced that of interleukin-5, in the NK1 and NK2 subsets, respectively. T-bet, a transcription factor associated with IFN-γ expression was down-regulated; while Th2 linked transcription factors (STAT6 and GATA3) were up-regulated especially with 100 μg/mL treatment of CKJOT. The type 2 shift in NK cell-secreted cytokines induced by CKJOT in mouse NK cells may explain the protective effect associated with its traditional use in unexplained RPL.

4-1BBL-Expressing aAPCs Attenuate IL-15-Induced NK Cell Expansion and Cytokine Production In Vitro but Induce NK Cell-Mediated Gvhd In Vivo

AbstractAbstract 2536Methods to expand natural killer (NK) cells ex vivo for adoptive cell therapy are being explored to improve outcomes after allogeneic blood and marrow transplant (alloBMT). Artificial antigen presenting cells (aAPCs) can present cytokines and/or co-stimulatory molecules that can potentially improve expansion and activity. 4-1BBL (CD137L) has demonstrated mixed results on murine and human NK cells, but the impact on murine NK cell biology after alloBMT has not been explored. NK cells were harvested from either C57BL/6 (B6) or CB6F1 spleens and cultured ex vivo with a recombinant interleukin (IL)-15/IL-15 receptor alpha (Ra) complex in the presence or absence of a CD137L+ aAPC. Because IL-15 is typically presented in trans by IL-15Ra, the complex was utilized to potently increase agonist bioactivity. NK cells cultured with IL-15/IL-15Ra alone showed a peak of 20-fold expansion, but this expansion was decreased with the addition of CD137L+ aAPCs if the ratio of aAPC to NK cells was greater than 1:1. In the presence of IL-15/IL-15Ra, the impact of CD137L+ aAPCs on expression of the inhibitory receptors, Ly49C+I and activating receptor Ly49H was variable and strain dependent, with increased expression in B6 NK cells, but decreased expression in CB6F1 NK cells. The expression of major histocompatibility complex (MHC) class I was not affected in NK cells from either strain by the presence of CD137L+ aAPCs. The production of gamma interferon and tumor necrosis factor-a was robust in NK cells expanded by IL-15/IL-15Ra alone, but attenuated with the addition of CD137L+ aAPCs. Animal experiments showed that administration of NK cells expanded ex vivo with IL-15/IL-15Ra alone was well tolerated after T cell depleted MHC-mismatched alloBMT (CB6F1–>B6), but surprisingly the addition of CD137L+ aAPCs to cultures caused NK cells to induce GVHD-associated weight loss. In summary, IL-15/IL-15Ra expanded murine NK cells demonstrate increased cytokine production and do not cause toxicity when infused after alloBMT. The presence of CD137L+ aAPCs attenuated cytokine production and increased Ly49 receptor expression in NK cells from B6 mice. Remarkably, NK cells expanded by IL-15/IL-15Ra in the presence of CD137L+ aAPCs demonstrate increased propensity to cause GVHD. Ongoing studies are exploring the anti-tumor efficacy of IL-15/IL-15Ra expanded murine NK cells cultured in the presence and absence of CD137L. Disclosures:No relevant conflicts of interest to declare.

Natural killer cells are crucial for the efficacy of Icon (factor VII/human IgG1 Fc) immunotherapy in human tongue cancer

BackgroundIcon is a novel, dual neovascular- and cancer cell-targeting immunotherapeutic agent and has shown efficacy in the treatment of cancer, wet form macular degeneration and endometriosis. However, its underlying mechanism remains to be investigated. The objective of this study is to elucidate the mechanism of Icon immunotherapy in cancer using a squamous carcinoma human tongue cancer line TCA8113 in vitro and in vivo in severe combined immunodeficiency (SCID) mice.ResultsWe showed that Icon, as a chimeric factor VII and human IgG1 Fc immunoconjugate, could separately induce murine natural killer (NK) cells and activate complement to kill TCA8113 cancer cells in vitro via antibody dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, Icon-NK ADCC had a significantly stronger effect than that of Icon-CDC. Moreover, Icon could completely eradicate established human tongue tumour xenografts in vivo in the CB-17 strain of SCID mice that have functional NK cells at a normal level, whereas it was less effective in SCID/Beige mice that do not have functional NK cells.ConclusionsWe conclude that NK cells are crucial for the efficacy of Icon immunotherapy in the treatment of cancer. The results also suggest that impaired NK level/activity could contribute to the resistance to therapeutic antibodies that are currently under investigation in preclinical and clinical studies.

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3' untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology.

FimH Can Directly Activate Human and Murine Natural Killer Cells via TLR4

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH(+), but not FimH(-), bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections.

Murine trophoblast cells induce NK cell interferon-gamma production through KLRK1.

Murine models suggest that natural killer (NK) cells are important for normal implantation site development, in part, through the production of interferon gamma (IFNG). As KLRK1 (NKG2D) is expressed on human and murine uterine NK (uNK) cells, we examined the role of KLRK1 in the interaction between murine trophoblasts and NK cells. Flow cytometric analysis revealed that both murine trophoblast stem (TS) cells and differentiated trophoblast giant cells expressed the KLRK1 ligand retinoic acid early transcript 1, or RAET1. Coculture of activated NK cells with either TS cells or giant cells led to the production of IFNG, as measured by ELISA. In addition, coculture with TS cells led to the downregulation of KLRK1. Both responses were inhibited by soluble KLRK1 ligand, but not by irrelevant protein. Further studies demonstrated the presence of KLRK1 ligand on uterine cells derived from either virgin or pregnant mice, although uterine RAET1 protein expression was upregulated in vitro by progesterone, but not estradiol. We suggest that the interaction of KLRK1 and RAET1 may be involved in IFNG production by uNK cells, and thus, this receptor-ligand pair may contribute to successful murine implantation site development.

Adaptive immune responses mediated by natural killer cells

Adaptive immunity has traditionally been considered a unique feature of vertebrate physiology. Unlike innate immune responses, which remain essentially unchanged upon exposure to a recurrent challenge with the same stimulus, adaptive immune cells possess the ability to learn and remember. Thus, secondary adaptive responses to a previously encountered challenge are qualitatively and/or quantitatively distinct from those elicited by a primary encounter. Besides this capacity to acquire long-lived memory, the second cardinal feature of adaptive immunity is antigen specificity. It has been generally believed that only T and B cells can develop antigen-specific immunologic memory, because these lymphocytes uniquely express recombination-activating gene (RAG) proteins, which are necessary for somatic rearrangement of V(D)J gene segments to assemble diverse antigen-specific receptors. However, recent work has uncovered discrete subsets of murine natural killer (NK) cells capable of mediating long-lived, antigen-specific recall responses to a variety of hapten-based contact sensitizers. These NK cells appear to use distinct, RAG-independent mechanisms to generate antigen specificity. Murine NK cells have also recently been shown to develop memory upon viral infection. Here, we review recent evidence indicating that at least some NK cells are capable of mediating what appears to be adaptive immunity and discuss potential mechanisms that may contribute to RAG-independent generation of antigenic diversity and longevity.