Enhanced Proliferation and Activation of Natural Killer Cells by Deer Antler Velvet Extract, YC-1101, and Its Synergistic Antitumor Effects in CT26 Syngeneic Mouse Model.

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

A View to Natural Killer Cells in Hepatitis C

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.

IL-21 Enhances Antitumor Responses without Stimulating Proliferation of Malignant T Cells of Patients with Sézary Syndrome

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.

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

3076 Background: NK cell cytotoxicity is tightly controlled through a balance of signals from the functionally opposing activating and inhibitory receptors found on each cell. Interleukin-15 (IL-15) has a major role in NK cell homeostasis. IL-15 induces the differentiation of NK cells from hematopoietic progenitors, stimulates the expansion of peripheral NK cells and supports their survival. Modulation of the relative frequency and intensity of expression of the NK cell receptors by IL-15 may increase NK mediated cytotoxicity in cancer patients. Methods: We investigated the receptor repertoire and measured NK cell activity in the peripheral blood of 21 patients with newly diagnosed AML prior to any treatment and 15 normal donors. Ex-vivo effects of IL-15 on the NK cell repertoire and NK cell cytotoxicity were investigated. Results: The expression of the activating natural cytotoxicity (NCR) receptors NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C were significantly decreased in the AML patients compared to the NK cells of healthy donors (p<0.0001–0.03). Furthermore, NK cytotoxicity was significantly lower (p<0.0003) in the AML patients at diagnosis compared to that of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly increased compared to pre-culture levels: NKp30, 84 vs 20% p<0.001; NKp44, 71 vs 4% p<0.0001; NKp46, 83 vs 32% p<0.0002; NKG2D 87 vs 43% p<0.01; NKG2C, 58 vs 18% p<0.005. Concomitantly, cytotoxic activity of NK cells sorted from AML patients increased following IL-15 stimulation (790 vs 2211 LU, p<0.05). This IL-15 induced increase in the activity was blocked by neutralizing antibodies against the NK cell activating receptors. Conclusions: We determined that IL-15, a homeostatic NK cell cytokine, can upregulate the expression of the NK activating receptors and concomitantly increase the NK lytic activity in patients with AML. These data support the use of IL-15 as a platform for NK- based therapies for AML patients. No significant financial relationships to disclose.

NK Cell Deficiency In Job Syndrome Patients With Dominant Negative STAT3 Mutations

Hepatitis C virus-induced natural killer cell proliferation involves monocyte-derived cells and the OX40/OX40L axis

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

This study was undertaken to investigate the natural killer (NK) and natural killer-like (NK-like) cell cytotoxic activity toward autologous thyrocytes of peripheral blood mononuclear cells (PB-MNC) and thyroid gland mononuclear cells (TG-MNC) from previously hyperthyroid patients with Graves' disease, and the effects of recombinant interleukin-2 on such cytotoxic activity. The average cytotoxicities of PB-MNC from Graves' patients toward K562 cells (NK-sensitive cells), Raji cells (NK-resistant cells), and autologous thyrocytes were 23.9 +/- 10.8 (+/- SD) lytic units (LU), 7.4 +/- 3.8 LU, and 11.7 +/- 4.4 LU, respectively. There were no differences in the NK and NK-like cell activity of PB-MNC between Graves' disease patients and normal subjects. In contrast to PB-MNC from patients with Graves' disease, NK and NK-like cell activity was markedly decreased in TG-MNC (NK cell activity, 2.1 +/- 2.3 LU; NK-like cell activity, 1.5 +/- 1.5 LU). TG-MNC from Graves' patients had no cytotoxic activity against autologous thyrocytes. Using the monoclonal anti-Leu 7 and anti-CD16 antibodies and a two-color immunofluorescence method, the NK cell subsets were examined in PB-MNC and TG-MNC from Graves' patients. The percentage of CD16+ cells was significantly decreased in TG-MNC compared to PB-MNC, whereas there was no significant difference in the percentage of Leu 7+ cells between PB-MNC and TG-MNC. Incubation of TG-MNC with medium only did not increase the NK and NK-like cell activity of these cells. Furthermore, incubation of autologous PB-MNC with supernatants of minced thyroid tissues did not alter their NK and NK-like cell activity. The decreased NK and NK-like cell activity of TG-MNC was augmented when these cells were incubated with recombinant interleukin-2. These results suggest that the reduction of NK cell activity in TG-MNC may allow perpetuation of B-cell activation and lead to excessive production of autoantibody in thyroid tissue.

INTRODUCTION: Understanding how Natural Killer (NK) cell activity is regulated by cancer cells is critical for developing NK-cell based immunotherapies. Our studies show that the deubiquitinating enzyme ubiquitin-specific protease-6 (USP6) can induce an immune-stimulatory hot tumor microenvironment in Ewing sarcoma (ES), the 2nd most common pediatric bone cancer. High USP6 expression in ES is associated with significantly improved overall and progression-free survival. We found that USP6 expression in ES cells inhibits xenograft growth in nude mice, but not NSG mice, implicating a role for the innate immune system, particularly cytotoxic NK cells. Notably, USP6 expression enhanced intra-tumoral abundance, activation, and proliferative capacity of NK cells. In vitro cytotoxicity assays confirm that USP6 can directly augment the ability of ES cells to activate NK cells. Based on these observations, we hypothesize that NK cells are essential for USP6-mediated tumor suppression and seek to determine the mechanism by which their cytolytic function is activated by USP6. METHOD: We used ES cell lines A673 and RD-ES expressing USP6 in a doxycycline (dox)-inducible manner, the immortalized human NK cell line NK-92, and primary human and mouse NK cells. Athymic nude or RAG2-/- mice were xenografted subcutaneously with ES cell lines and fed dox-infused water to induce USP6. Immune cell abundance and activation were monitored both in tumors and peripheral blood by flow cytometry. For in vivo depletion of NK cells, 300ug of anti-NK1.1 (PK136) ab was injected i.p. into RAG2-/- mice one day prior to ES xenografting, then once weekly thereafter. RESULTS: USP6 induces in ES cells the surface expression of multiple NK activating ligands, as well as receptors for the apoptotic factor TRAIL (DR5) and Type I/II interferons (IFNAR1 and IFNGR1), both in vitro and in vivo. This leads to enhanced NK activation and renders ES cells hypersensitive to NK-mediated cytotoxicity. IFNy released by activated NK cells feeds back on the USP6-expressing ES cells, inducing synergistic expression of multiple IFNy-inducible genes, including chemokines CXCL9/10 (chemoattractants for NK cells) and ICAM-1 (essential for immunological synapse formation between NK cells and their targets). A potent feed forward loop for NK activation/ES cell killing is thus generated. Concordantly, in RAG2-/- mice, USP6 induces increased intratumoral infiltration and activation of NK cells, and also, strikingly, systemic activation of NK cells. Depletion of NK cells using anti-NK1.1(PK136) completely abrogates USP6-mediated inhibition of ES xenograft growth. CONCLUSION: NK cells are essential for mediating the tumor inhibitory functions of USP6 in ES. We posit that USP6’s ability to activate NK cells both intratumorally and systemically underlies its association with improved ES patient prognosis, which we seek to exploit for therapeutic benefit in future studies. Citation Format: Kanika Jain, Ian C. Henrich, Laura Nicole Quick, Robert A. Young, Margaret M. Chou. Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3147.

Immunologic messenger molecules: Cytokines, interferons, and chemokines

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.

Activation and Expansion of Natural Killer (NK) Cells with Potent Cytotoxicity for Multiple Myeloma