Expression Of NKp46 Research Articles

Effect of the invivo application of granulocyte colony-stimulating factor on NK cells in bone marrow and peripheral blood.

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.

The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

SummaryTRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s) and a subset of natural killer (NK) cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells.

Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

Attenuation of NK cells facilitates mammary tumor growth in streptozotocin-induced diabetes in mice.

Diabetic patients have higher incidence and mortality of cancer. Recent study revealed that hyperglycemia-induced oxidative stress is involved in the acceleration of tumor metastasis. We used model of high-dose streptozotocin-induced diabetes to investigate its effect on tumor growth and modulation of antitumor immune response of 4T1 murine breast cancer in BALB/c mice. Diabetes accelerated tumor appearance, growth and weight, which was associated with decreased NK cells cytotoxicity against 4T1 tumor cells in vitro Diabetes reduced frequencies of systemic NKG2D+, perforin+, granzyme+, IFN-γ+ and IL-17+ NK cells, while increased level of PD-1 expression and production of IL-10 in NK cells. Diabetes decreased percentage of NKG2D+NK cells and increased percentage of PD-1+ NK cells also in primary tumor. Diabetes increased accumulation of IL-10+ Tregs and TGF-β+ myeloid-derived suppressor cells (MDSCs) in spleen and tumor. Diabetic sera in vitro significantly increased the percentage of KLRG-1+ and PD-1+ NK cells, decreased the percentage of IFN-γ+NK cells, expression of NKp46 and production of perforin, granzyme, CD107a and IL-17 per NK cell in comparison to glucose-added mouse sera and control sera. Significantly increased percentages of inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) producing MDSCs and dendritic cells (DC) were found in the spleens of diabetic mice prior to tumor induction. 1-methyl-DL-tryptophan, specific IDO inhibitor, almost completely restored phenotype of NK cells cultivated in diabetic sera. These findings indicate that diabetes promotes breast cancer growth at least in part through increased accumulation of immunosuppressive cells and IDO-mediated attenuation of NK cells.

Bu–Shen–Yi–Qi formula impairs cytotoxicity of NK cells by up-regulating IDO expression in trophoblasts

Recurrent spontaneous abortion (RSA) is a common health problem that affects about 5% of fertile women, when it occurs for unknown reasons, it is called unexplained recurrent spontaneous abortion (URSA). Traditional Chinese medicine, such as Bu–Shen–Yi–Qi formula which consists of Dangshen, Tusizi, Baizhu, Baishuo, Duzhong, Sangjisheng, Sugeng, and Tiaohuangqin, has played an invaluable role in the treatment of RSA since ancient times. However, the mechanism of how it takes effect is still not clear. To identify Bu–Shen–Yi–Qi formula could modulate immune condition at maternal-fetal interface via its effect on trophoblasts, HTR-8 of different treatment were co-cultured with peripheral or decidual natural killer (NK) cells, and the receptors such as NKP30 and NKP46 expression on NK cells were measured by flow cytometry (FCM). In this study, we found that herb medium could increase the IDO expression at appropriate concentrations. As an inhibitor of IDO, 1-MT could impair the inhibitory function of trophoblasts on NK cells. Furthermore, Bu–Shen–Yi–Qi formula could enhance the inhibitory function of trophoblasts on NK cells. In conclusion, Bu–Shen–Yi–Qi formula can inhibit NK cytotoxicity by up-regulating IDO expression in trophoblasts and play a role in the treatment of URSA patients.

Decitabine enhances targeting of AML cells by CD34+ progenitor-derived NK cells in NOD/SCID/IL2Rgnull mice

AbstractCombining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34+ hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rgnull mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1–activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML.

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

Modulation the expression of natural killer cell activating receptor (NKp44) in the peripheral blood of diffuse large B-cell lymphoma patients and the correlation with clinic pathological features

NK cell activation is one strategy to improve the immunotherapy of non-Hodgkin's lymphoma. So, we aimed to investigate expression of Natural killer cell activating receptor NKp44 in patients with diffuse large B-cell lymphoma (DLBCL) and its correlation with clinic pathological data. In this study, 30 new cases with DLBCL in addition to 20 healthy control were involved. All were submitted to full history, clinical examination, histopathology, Routine laboratory investigations including CBC, LDH, β2microgloubine and bone marrow examination. Cell culture of peripheral blood mononuclear cells and expression of CD56 and NKp44 by flowcytometry was done. We demonstrated increased NK cell populations (CD 56 +ve NKp44 –ve, CD 56 –veNKp44 +ve, total CD 56 +ve) and NKp44 MFI after in-vitro activation in both healthy control and DLBCL cases except for CD 56 +ve NKp44 +ve which significantly increased in patients not in healthy control (p=0.005, 0.601) respectively. No significant difference between the DLBCL and healthy control regarding all NK cell populations without PHA stimulation. However, the culture with PHA in DLBCL showed significant increase in NK cell populations than the healthy control (CD 56 +ve NKp44 +ve 12.37±7.52vs 6.80±4.07, p=0.008), (Total CD 56 +ve 18.80±8.74vs 12.66±5.17, p=0.017), (MFI of NKp44 10.95±6.18vs 5.58±1.70, p=0.001). Regarding the association with clinic pathologic features, increased expression of NKp44 was associated with lower values of LDH and earlier stages of DLBCL (p<0.05). So, activating receptor NKp44 can be modulated by in-vitro activation, hence improvement of its function as an approach of immunotherapy of DLBCL.

Proteus mirabilis inhibits cancer growth and pulmonary metastasis in a mouse breast cancer model.

A variety of bacteria have been used as agents and vectors for antineoplastic therapy. A series of mechanisms, including native bacterial toxicity, sensitization of the immune system and competition for nutrients, may contribute to antitumor effects. However, the antitumor effects of Proteus species have been minimally studied, and it is not clear if bacteria can alter tumor hypoxia as a component of their antineoplastic effect. In the present study, Proteus mirabilis bacteria were evaluated for the ability to proliferate and accumulate in murine tumors after intravenous injection. To further investigate the efficacy and safety of bacterial injection, mice bearing 4T1 tumors were treated with an intravenous dose of 5×107 CFU Proteus mirabilis bacteria via the tail vein weekly for three treatments. Histopathology, immunohistochemistry (IHC) and western analysis were then performed on excised tumors. The results suggested Proteus mirabilis localized preferentially to tumor tissues and remarkably suppressed the growth of primary breast cancer and pulmonary metastasis in murine 4T1 models. Results showed that the expression of NKp46 and CD11c was significantly increased after bacteria treatment. Furthermore, tumor expression of carbonic anhydrase IX (CA IX) and hypoxia inducible factor-1a (HIF-1a), surrogates for hypoxia, was significantly lower in the treated group than the control group mice as assessed by IHC and western analysis. These findings demonstrated that Proteus mirabilis may a promising bacterial strain for used against primary tumor growth and pulmonary metastasis, and the immune system and reduction of tumor hypoxia may contribute to the antineoplastic and antimetastatic effects observed.

Enhanced natural killer cell activity is found in exposed uninfected recipients of hepatitis C-contaminated blood.

A minority of injecting drug users, termed exposed uninfected, are resistant to hepatitis C (HCV) infection despite repeated low-dose exposures. We identify for the first time a cohort of blood recipients who remained uninfected despite large-dose exposure to HCV-contaminated blood and characterize immune factors that may confer protection. Of 1340 blood recipients from the English Look Back database who were transfused HCV-contaminated blood, we identified 8 who remained uninfected. In these 8 exposed uninfecteds, we characterized their natural killer (NK) cell populations and HCV-specific T-cell responses. Findings were compared with 10 spontaneous resolvers of HCV infection, 10 patients with chronic HCV infection and 10 healthy controls. Exposed uninfecteds had significantly greater numbers of NK cells with the activating receptor NKp30+ on CD56bright and CD56dim subsets compared with other groups (P<.05). Following interleukin-2 activation, NK cells of exposed uninfecteds had enhanced cytotoxicity that positively correlated with NKp30 expression (P=.02). Differences in NKp80 and KIR2DL3 expression were also observed. HCV-specific T-cell responses were observed in some exposed uninfecteds but of low amplitude. Exposure without infection following transfusion of HCV-contaminated blood is a very rare phenomenon and suggests a high level of resistance to infection. Enhanced NK cell activation and killing, with weak HCV-specific T-cell responses, were observed many years after exposure in uninfected recipients and may contribute to protection from HCV acquisition, although additional protective factors are being sought in this important cohort.

The forkhead transcription factor Foxo3 negatively regulates natural killer cell function and viral clearance in myocarditis.

Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis. Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3-/- mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3-/- mice at Day 3 while interferon-γ (IFNγ) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b+CD27+ effector NK cells and cytotoxicity of Foxo3-/- mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3-/- NK cells while its inhibition led to diminished IFNγ production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFNγ and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control. Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Chronic NKG2D Engagement In Vivo Differentially Impacts NK Cell Responsiveness by Activating NK Receptors.

Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.

Expression of NKp46 on NK cells in endometrium in women with endometriosis

Expression of NKp46 on NK cells in endometrium in women with endometriosis

Veritable antiviral capacity of natural killer cells in chronic HBV infection: an argument for an earlier anti-virus treatment

BackgroundThere is limited information on innate immunity, especially natural killer (NK) cell function, in different chronic hepatitis B (CHB) stages. Therefore, we examined whether the clinical staging strategy accurately reflects veritable NK cell immunity.MethodsA total of 237 eligible CHB patients and 22 healthy controls were enrolled in our study. Demographic and clinical data were collected, and the CHB phases (immune active-IA, immune tolerant phase-IT, inactive CHB-IC, and grey zone-GZ) were classified according to the latest American Association for the Study of Liver Disease guidelines. Peripheral blood mononuclear cells from patients and healthy controls were tested for NK cell frequency, phenotype and function using flow cytometry.ResultsA significant decrease in activating receptor NKp44 and NKp46 expression and significant increase of exhaustion molecule Tim-3 expression were observed in NK cells from CHB patients. Reduced cytokine secretion and preserved or elevated cytotoxic function were also observed. Patients in the IT group exhibited comparable cytokine secretion and cytolytic capacity as age-matched IA patients. NK cell anti-viral functions were preserved in GZ patients. Some of the NK cell function in patients who were excluded from treatment by the current treatment guidelines was less compromised than patients who qualified for treatment.ConclusionOur findings provide evidence of veritable NK cell immunity during different natural history phases in treatment-naïve patients with chronic HBV Infection. Chronic HBV infection hindered NK cell function in CHB patients. However, the presumed IT and GZ statuses of CHB patients based on the clinical parameters may not accurately reflect the inner immune status of these patients and should be reconsidered. Some patients excluded from treatment by the current treatment guidelines may be able to be selected as candidates for treatment.

CD335 (NKp46)+ T-Cell Recruitment to the Bovine Upper Respiratory Tract during a Primary Bovine Herpesvirus-1 Infection.

Bovine natural killer (NK) cells were originally defined by the NK activation receptor CD335 [natural killer cell p46-related protein (NKp46)], but following the discovery of NKp46 expression on human T-cells, the definition of conventional bovine NK cells was modified to CD335+CD3− cells. Recently, a bovine T-cell population co-expressing CD335 was identified and these non-conventional T-cells were shown to produce interferon (IFN)-γ and share functional properties with both conventional NK cells and T-cells. It is not known, however, if CD335+ bovine T-cells are recruited to mucosal surfaces and what chemokines play a role in recruiting this unique T-cell subpopulation. In this study, bovine herpesvirus-1 (BHV-1), which is closely related to herpes simplex virus-1, was used to investigate bovine lymphocyte cell populations recruited to the upper respiratory tract following a primary respiratory infection. Immunohistochemical staining with individual monoclonal antibodies revealed significant (P < 0.05) recruitment of CD335+, CD3+, and CD8+ lymphocyte populations to the nasal turbinates on day 5 following primary BHV-1 infection. Dual-color immunofluorescence revealed that cells recruited to nasal turbinates were primarily T-cells that co-expressed both CD335 and CD8. This non-conventional T-cell population represented 77.5% of CD355+ cells and 89.5% of CD8+ cells recruited to nasal turbinates on day 5 post-BHV-1 infection. However, due to diffuse IFN-γ staining of nasal turbinate tissue, it was not possible to directly link increased IFN-γ production following BHV-1 infection with the recruitment of non-conventional T-cells. Transcriptional analysis revealed CCL4, CCL5, and CXCL9 gene expression was significantly (P < 0.05) upregulated in nasal turbinate tissue following BHV-1 infection. Therefore, no single chemokine was associated with recruitment of non-conventional T-cells. In conclusion, the specific recruitment of CD335+ and CD8+ non-conventional T-cells to viral-infected tissue suggests that these cells may play an important role in either the clearance of a primary BHV-1 infection or regulating host responses during viral infection. The early recruitment of non-conventional T-cells following a primary viral infection may enable the host to recognize viral-infected cells through NKp46 while retaining the possibility of establishing T-cell immune memory.

Activating and inhibitory receptors on natural killer cells in patients with systemic lupus erythematosis-regulation with interleukin-15.

Natural killer (NK) cells may play an important role in the pathogenesis of SLE. Interleukin(IL)-15, an NK-enhancing cytokine, is over-expressed in SLE patients. In the present study, we examined the effect of IL-15 on NK cytotoxicity of SLE patients, and the expression of various activating and inhibitory NK receptors on NK cells from SLE patients in relation to disease activity. We also sought to determine how IL-15 would affect the NK receptor expression on NK cells from SLE patients. PBMCs were collected from 88 SLE patients with inactive disease activity (SLEDAI score<6) and active disease activity (SLEDAI score≥6), 26 age-matched healthy adults were used as controls. PBMC were incubated in the presence or absence of IL-15 (10ng/ml) for eighteen hours. CD3-CD56+ lymphoctes were gated using flow cytometry and further divided into CD56dim and CD56bright subsets according to the MFI of CD56. We observed that 1. Serum IL-15 was elevated in SLE patients, and higher in active disease than in inactive disease; 2. NK cytotoxicity of SLE patients was deficient compared to controls and showed an impaired response to IL-15 compared to controls; 3.CD69, CD94, NKG2A, NKp30, and CD158b on NK cells from SLE patients were higher than controls, and could be further enhanced by IL-15; 4. NKp46 expression from SLE patients was higher than controls, but down-regulated by IL-15; 5.Deficient NKG2D and NKAT-2 expression were found on NK cells from SLE patients, which were enhanced by IL-15; 6. A unique NKp46- subset and CD158b+ subsets were observed in NK cells from SLE patients but not controls. 7. Unlike controls, CD158k on NK cells from SLE patients failed to respond to IL-15. Taken together, we demonstrated the aberrant NCR and iNKR expression on NK cells and their distinct response to IL-15 in SLE patients. As IL-15 predominantly aggravates the aberrant NKR expression found in SLE, IL-15 antagonist may have therapeutic benefits in SLE patients.

Effects of total flavonoids of sea buckthorn ( Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells.

Sea buckthorn (Hippophae rhamnoides L.) has multifarious medicinal properties including immunoregulatory effect. The total flavonoids of Hippophae rhamnoides L. (TFH) are the main active components isolated from berries of sea buckthorn. The aim of this study was to evaluate the effects of TFH on the cytotoxicity of NK92-MI cells and its possible mechanisms. NK92-MI cells were treated with TFH (2.5 or 5.0 mg/L) or phosphate-buffered saline (PBS) for 24 h, the cytotoxicity against K562 was detected by measuring the release of lactate dehydrogenase (LDH), expression levels of NCRs (NKp30, NKp44, NKp46) and NKG2D were detected by flow cytometry, and expression levels of perforin and granzyme B were detected by western blot. Cytokine Antibody Arrays with 80 cytokine proteins were used to profile the effect of TFH on cytokines. Western blot was adopted to detect the effects of TFH on STAT1, STAT4, and STAT5 signal pathway. Compared with the normal control group, TFH could significantly enhance NK92-MI cell cytotoxicity against K562 cells, upregulate expressions of NKp44, NKp46, perforin, and granzyme B. TFH could upregulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-β and downregulate expressions of IL-16, MIP-1β, CX3CL-1, and MIF. TFH could increase expressions of phospho-STAT1 and phospho-STAT5. The results suggest that TFH stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells.

Large scale ex vivo expansion of clinical‑grade effector cells for adoptive immunotherapy

Cell-based adoptive immunotherapy for the treatment of various cancer types has attracted the attention of scientists. However, due to the absence of unitary standard protocols to produce large quantities of clinical-grade effector cells, it remains challenging to translate the experimental findings into clinical applications. The present study used methods complying with good manufacturing practice to induce effector cells from human peripheral blood mononuclear cells (PBMCs) of healthy donors by interleukin-2 and anti-Her-2 antibody with or without anti-CD3 antibodies (OKT3). The results indicated that the addition of OKT3 resulted in a greater expansion of the total cells and CD8+ T cells, and primarily induced the PBMCs to differentiate into CD3+ T cells. Regardless of the presence of OKT3, the expression of activating receptor of natural killer (NK) group 2, member D, and the inhibitory receptors of CD158a and CD158b on NK cells and NKT cells was increased, while the expression of NKp46 was inhibited on NK cells, but not on NKT cells. Furthermore, OKT3 did not affect the toxicity of the effector cells. Subgroup analysis indicated that although a variation of the composition of effector cells was present in different individuals under identical culture conditions, consistent marker expression on effector cells and target cell-killing effects were observed in different subgroups treated with or without OKT3. Furthermore, western blot analysis indicated that OKT3, apart from its involvement in cell cycle regulation, affects transcription and protein translation during processes of proliferation and differentiation. The present study provided experimental data regarding the production of effector cells for adoptive immunotherapy as a clinical application.

Expression of natural cytotoxicity receptors and cytokine production on endometrial natural killer cells in women with recurrent pregnancy loss or implantation failure, and the expression of natural cytotoxicity receptors on peripheral blood natural killer cells in pregnant women with a history of recurrent pregnancy loss.

Natural cytotoxicity receptors (NCR) are unique markers that regulate natural killer (NK) cell cytotoxicity and cytokine production. In this study, we investigated the expression of NCR (NKp46, NKp44, and NKp30) and cytokine production in NK cells derived from the uterine endometrium of women with recurrent pregnancy loss (RPL). We also investigated the expression of NCR in peripheral blood NK cells in pregnant women with and without a history of RPL. The expression of NCR (NKp46, NKp44, and NKp30) in NK cells (CD56dim and CD56bright ) in the uterine endometrium was analyzed using 3-color flow cytometry. Cytokine (tumor necrosis factor-α and interferon-γ) production was also analyzed. NK cells from the mid-secretory endometrium of 28 women with RPL, 34 women with implantation failure, and 74 controls were collected and mechanically dispersed using a tissue grinder. The expression of NCR in peripheral blood NK cells from pregnant women with (n = 17) and without (n = 91) a history of RPL was analyzed. The percentages of NKp46+ NK cells were significantly lower in both women with RPL and pregnant women with a history of RPL. The percentages of tumor necrosis factor-α- and/or interferon-γ-producing uterine endometrial NK cells were significantly lower in women with RPL compared with controls. The changes in NCR expression and cytokine production, especially decreased NKp46 expression in endometrial NK cells, suggests the presence of abnormal NK cell regulation in women with reproductive failures.

Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia

Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal–fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.