Gut microbiota metabolite butyric acid alleviated Klebsiella Pneumoniae induced lung injury by regulating CX3CR1+NK via PI3K/AKT pathway

Interleukins (IL)-2 and IL-15 regulate natural killer (NK) cell proliferation, survival, and cytolytic activity. Ets1 is a transcription factor expressed early in NK cell differentiation. Because IL-2Rbeta, IL-2Rgamma, IL-15, and Ets1 knock-out mice similarly lack NK cells, we explored a molecular connection between IL-2R signaling and Ets1. Here we report the post-transcriptional regulation of Ets1 by IL-2R signaling in human NK cells. IL-2 and IL-15 stimulation leads to increased Ets1 protein levels with no significant change in mRNA levels. Pulse and pulse-chase experiments show that IL-2 stimulation results in both a marked increase in the nascent translation of Ets1 and an increased protein half-life. Pharmacological inhibition of MEK specifically blocks IL-2- and IL-15-induced translation, whereas p38, phosphatidylinositol 3-kinase, and mTOR inhibitors had no effect on Ets1 levels. Fli1, an Ets family member, exhibited a different mechanism of regulation, illustrating the specificity of IL-2R beta and gamma subunit signaling on the regulation of Ets1 expression. Expression of a dominant negative form of MNK1, a regulator of the translation initiation factor eIF4E, blocks the expression of Ets1 as do the dominant negative forms of the common IL-2R beta and gamma chains. Expression of Ets1 is regulated similarly in normal peripheral human NK cells. Taken together, our findings provide a direct link between IL-2R subunit signaling and Ets1 expression and helps to explain the interdependence of the IL-2R subunits and Ets1 for NK cell development and function.

Background: The prevalence of COVID-19 cases in Indonesia as of June 9, 2020, has been confirmed 32.076 positive cases, with 1.923 death cases. The total number of deaths reached 92,941 cases. There has been a recent update on stem cell-based biological, medical therapy as an optional treatment to handling COVID-19 due to its potential viability besides using the prevalent conventional chemical drug therapy. Methods: In this study, in vitro research was conducted to determine the potential of hematopoietic stem cells (HSCs) and natural killer cells (NK cells) against SARS-CoV-2 viruses, which virus isolates were collected in Indonesia. The SARS-CoV-2 virus was planted in rat kidney cells and Vero cells. The cells that had been planted with the virus were given HSCs and NK cells, followed by being evaluated at intervals of 24, 48, and 72 hours. The evaluation was done by collecting cells and supernatant from the cell plate and then determining the viral load using a Polymerase Chain Reaction (PCR) machine. Results: The results showed that the addition of HSCs and NK on cells that had been infected by SARS-CoV-2 resulted in a decrease in viral load within 24 to 72 hours in all variations of Multiples of Infection (MoI) values. Conclusions: The administration of HSCs and NK cells has the potential to eliminate the SARS-CoV-2 virus. Although this study is only an in vitro study, it could be the basis for the development of alternative stem cell-based therapies to tackle COVID-19 cases.

Natural Killer Cells in Hepatitis C Virus Infection: From Innate Immunity to Adaptive Immunity

p110γ and p110δ Phosphoinositide 3-Kinase Signaling Pathways Synergize to Control Development and Functions of Murine NK Cells

A View to Natural Killer Cells in Hepatitis C

A Critical Role for Type I IFN–dependent NK Cell Activation in Innate Immune Elimination of Adenoviral Vectors In Vivo

Natural killer (NK) cells can detect and kill tumor cells and infusion of NK cells to cancer
\npatients may be a promising option to treat cancer. In this context, ex vivo expansion is used to
\nproduce large quantities of activated NK cells, because sufficient numbers of these effector cells
\nare essential for successful NK cell based adoptive cancer immunotherapy. The development of
\nefficient NK cell expansion protocols and the transfer of these protocols to clinically applicable
\nmethods represent a major challenge. To overcome this issue, the aim of my project was to
\ndevelop a clinically applicable method that yields large numbers of highly functional NK cells.
\nFirst, a fully automated technical process was developed to activate and expand NK cells with
\n(interleukin) IL-2 and irradiated clinical-grade feeder cells (EBV-LCL). In comparison to the
\nmanual procedure, the automated process yielded similar NK cells in terms of cell numbers,
\nsurface marker profile, gene expression and in vitro effector functions. Upon expansion, NK
\ncells up-regulated functional surface molecules, such as TRAIL, FasL, NKG2D and DNAM-1,
\nthey increased the production of interferon (IFN)-g and tumor necrosis factor (TNF)-a and they
\nbecame more cytotoxic against tumor cell lines. Next, because in the used protocol NK cell
\nexpansion was restricted to a period of 2-4 weeks, a more efficient protocol for long-term
\nexpansion was developed. Manual NK cell expansion with EBV-LCL and IL-2 induced a 22–
\nfold mean NK cell expansion after one week that was significantly increased to 53–fold by
\naddition of IL-21. Furthermore, repeated stimulation with irradiated EBV-LCL and IL-2 and
\naddition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with
\n1011–fold NK cell expansion after six weeks, which is an unprecedented high expansion rate not
\nachieved by any other method so far. Most importantly, adoptive transfer of NK cells expanded
\nwith this optimized protocol led to significant inhibition of tumor growth in a melanoma
\nxenograft mouse model, proofing the therapeutic efficacy of the ex vivo generated NK cells.
\nThis anti-tumor efficacy was superior over that from conventionally IL-2 activated NK cells,
\ndemonstrating that the improved NK cell expansion method enhanced not only the quantity but
\nalso the therapeutic quality of NK cells.
\nIn conclusion, the outcome of this project is a fully automated process for ex vivo production of
\nNK cells and an optimized protocol for NK cell expansion with unparalleled efficacy. The
\nexpanded NK cells possess potent anti-tumor features and showed therapeutic efficacy in a
\npreclinical melanoma xenograft model. Thereby, the project serves clinical needs and makes it
\npossible to generate high cell doses of functional NK cells for the use in cancer immunotherapy.

Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Infection leads to heightened activation of natural killer (NK) cells, a process that likely involves direct cell-to-cell contact, but how this occurs in vivo is poorly understood. We have used two-photon laser-scanning microscopy in conjunction with Toxoplasma gondii mouse infection models to address this question. We found that after infection, NK cells accumulated in the subcapsular region of the lymph node, where they formed low-motility contacts with collagen fibers and CD169(+) macrophages. We provide evidence that interactions with collagen regulate NK cell migration, whereas CD169(+) macrophages increase the activation state of NK cells. Interestingly, a subset of CD169(+) macrophages that coexpress the inflammatory monocyte marker Ly6C had the most potent ability to activate NK cells. Our data reveal pathways through which NK cell migration and function are regulated after infection and identify an important accessory cell population for activation of NK cell responses in lymph nodes.

Mosquito Salivary Gland Extracts Induce EBV-Infected NK Cell Oncogenesis Via CD4+ T Cells in Patients with Hypersensitivity to Mosquito Bites

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon γ in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I–deficient tumor cells were ∼10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I–deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

Innate immunity and cardiac allograft rejection

Memory NKG2C+ nk Cells Undergo Robust Expansion and Exhibit Strong Effector Functions upon Re-Exposure to the HCMV gpUL40 Antigen Peptide