BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors.

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

NK-92 cells labeled with Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobes for the targeted treatment and noninvasive therapeutic evaluation of breast cancer.

Adoptive cellular immunotherapy as a promising and alternative cancer therapy platform is critical for future clinical applications. Natural killer (NK) cells have attracted attention as an important type of innate immune regulatory cells that can rapidly kill multiple adjacent cancer cells. However, these cells are significantly less effective in treating solid tumors than in treating hematological tumors. Herein, we report the synthesis of a Fe3O4-PEG-CD56/Avastin@Ce6 nanoprobe labeled with NK-92 cells that can be used for adoptive cellular immunotherapy, photodynamic therapy and dual-modality imaging-based in vivo fate tracking. The labeled NK-92 cells specifically target the tumor cells, which increases the amount of cancer cell apoptosis in vitro. Furthermore, the in vivo results indicate that the labeled NK-92 cells can be used for tumor magnetic resonance imaging and fluorescence imaging, adoptive cellular immunotherapy, and photodynamic therapy after tail vein injection. These data show that the developed multifunctional nanostructure is a promising platform for efficient innate immunotherapy, photodynamic treatment and noninvasive therapeutic evaluation of breast cancer.

HLA-DR Expression in Natural Killer Cells Marks Distinct Functional States, Depending on Cell Differentiation Stage.

HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.

The alteration of NK cells phenotypes related to the functions and dengue disease outcomes.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease.

Changes in subset distribution and impaired function of circulating natural killer cells in patients with colorectal cancer.

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

A specific natural killer cells phenotypic signature associated to long term elite control of HIV infection.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16dimCD56dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV.

[Clinical Characteristics of CD4-CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm].

OBJECTIVE: To explore the clinical manifestations, pathological features, immunophenotype, as well as diagnosis, treatment and prognosis of patients with CD4-CD56+ blastic plasmacytoid dendritic cell neoplasm (BPDCN), in order to further understand the rare disease. METHODS: The clinical data, laboratory examinations and treatment regimens of two patients with CD4-CD56+ BPDCN in the First Affiliated Hospital of Wannan Medical College were retrospectively analyzed. RESULTS: The two patients were both elderly males with tumor involved in skin, bone marrow, lymph nodes, etc. Immunohistochemical results of skin lesions showed that both CD56 and CD123 were positive, while CD4, CD34, TdT, CD3, CD20, MPO and EBER were negative. Flow cytometry of bone marrow demonstrated that CD56, CD123, and CD304 were all positive, while specific immune markers of myeloid and lymphoid were negative. Two patients were initially very sensitive to acute lymphoblastic leukemia or lymphomatoid chemotherapy regimens, but prone to rapid relapse. The overall survival of both patients was 36 months and 4 months, respectively. CONCLUSION: CD4-CD56+ BPDCN is very rare and easily misdiagnosed as other hematological tumors with poor prognosis. Acute lymphoblastic leukemia or lymphomatoid therapy should be used first to improve the poor prognosis.

Generation of an Inhibitory NK Cell Subset by TGF-ß1/IL-15 Polarization.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined in vitro conditions that could polarize human NK cells into an inhibitory subset. TGF-ß1 has been shown to induce regulatory T cells in vitro and in vivo; we therefore investigated if TGF-ß1 could also induce immunosuppressive NK-like cells. First, we found that TGF-ß1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells in vitro in a TGF-ß-dependent manner. Interestingly, TGF-ß1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells in vitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-ß1-rich environments.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.

Identification of potential biomarkers for neuromyelitis optica by quantitative proteomics.

OBJECTIVE: Neuromyelitis optica (NMO) was a serious autoimmune inflammatory condition affecting the central nervous system. Currently, there was a lack of diagnostic biomarkers for AQP4-IgG-negative NMO patients. METHODS: A comparative proteomic analysis was conducted on the CSF of 10 patients with NMO and 10 patients with non-inflammatory neurological disorders (NND) using tandem mass tagging technology. Differentially expressed proteins (DEPs) were analyzed using bioinformatic methods. The candidate proteins were then validated through ELISAs in a subsequent cohort of 160 samples, consisting of paired CSF and plasma samples from 50 NMO patients, CSF samples from 30 NND patients, and plasma samples from 30 healthy individuals. RESULTS: We identified 389 proteins via proteomics, screening 79 DEPs. NCAM1, SST and AHSG were selected as candidate molecules for further validation. Compared to NND patients, there were decreased levels of AHSG in CSF and increased levels of NCAM1 and SST in NMO patients. The ELISA results revealed significantly higher levels of AHSG, SST and NCAM1 in the CSF of the NMO group compared to the NND group. Similarly, the serum levels of these three proteins were also higher in the NMO group compared to the healthy control group. It was found that serum NCAM1 levels significantly decreased in patients with non-relapsed NMO compared to patients with relapsed NMO and CSF NCAM1 level increased in patients with bilateral NMO compared to patients with unilateral NMO. Furthermore, CSF SST levels increased in AQP4 antibody-positive NMO patients compared to AQP4 antibody-negative patients. INTERPRETATION: CSF NCAM1, serum NCAM1 and serum SST may serve as potential biomarkers for NMO patients and aid in the diagnosis of AQP4 antibody-negative NMO patients.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

Evaluation of the follicular patterned thyroid lesions based on the WHO 2022 criteria with an emphasis on the grey-zone lesions.

Follicular-patterned thyroid nodules (FPTN) are classified byWHO-2022 into benign, borderline and malignant categories. There are however, grey-zone lesions that pose a diagnostic challenge due to ambiguity in defining criteria and inter-observer variability. WHO-2022 has enumerated specific diagnostic criteria for these lesions. Accurate categorization of morphologically similar TNs is vital to reduce overtreatment of indolent lesions. In this study, we have reclassified FPTNs according to WHO-2022 criteria, emphasizing on grey-zone lesions. We studied the utility of immunohistochemistry (IHC)-CD56, HBME-1 and CK19 in distinguishing benign from malignant nodules and BRAFV600E IHC to better distinguish the (widely-invasive) encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) from infiltrative FVPTC. Only those cases with dominant nodule having follicular pattern histology were included and re-evaluated for following histopathological features-focality, encapsulation, circumscription, nuclear PTC features, capsular-invasion, angio-invasion, papillae and necrosis. IHC findings for above-mentioned markers were noted. Seventy-nine cases met the inclusion criteria. Amendment of original diagnosis was done in 19 % cases. BRAFV600E IHC was positive in the two cases of infiltrative FVPTC while it was negative in all nine IE (invasive encapsulated) FVPTCs. Diffuse HBME1 was noted in most malignant nodules (61 %) while CD56 was expressed more often in benign lesions (70 %). CK19 was positive in lesions displaying nuclear PTC features (86 %). Using WHO 2022 criteria, we were able to re-classify follicular thyroid lesions with greater confidence. Appropriate IHC panel in adjunct to histology aids in categorizing challenging cases.

Extranodal nasal-type NK/T-cell lymphoma with CD20 positive: A case report and review of literature.

ABSTRACT: Extranodal nasal-type natural killer (NK)/T-cell lymphoma is a type of non-Hodgkin lymphoma. Neoplastic lymphocytes are positive for CD4, CD56, and CD20, a specific B-cell marker. CD20 positive NK/T-cell lymphoma is rare, with only nine reported cases. This paper reports a case of nasal-type NK/T-cell lymphoma with CD20 positivity in a 47-year-old woman. The patient presented with bilateral nasal congestion and bloody nasal cavity secretions for 2 months. Computed tomography revealed thickening of the nasal mucosa and posterior wall of the nasopharyngeal crest, and the left and right cervical lymph nodes were enlarged. On histopathology, the lesion was composed of medium-sized atypical lymphoid cells and vascular infringement. Immunohistochemical staining showed that the tumor cells were positive for CD20, CD3, CD56, and Epstein-Barr virus (EBV)-encoded RNA in situ hybridization. The patient was treated with radiotherapy for 2 months and is currently well.

NK cells and the profile of inflammatory cytokines in the peripheral blood of patients with advanced carcinomas.

BACKGROUND: Natural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56brightCD16- (regulatory) and CD56dimCD16+ (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production. METHODS: Peripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56brightCD16- and CD56dimCD16+ NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum. RESULTS: Cancer patients had a lower frequency of the cytotoxic CD56dim CD16+ NK cells subset in comparison with healthy controls. Also, the regulatory CD56bright CD16- NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56brightCD16- NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56dimCD16+ was positively correlated with EGF and negatively correlated with MIP-1ß. CONCLUSIONS: Taken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches.

Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

[Interferon-α mediating the functional damage of CD56dimCD57+natural killer cells in peripheral blood of systemic lupus erythematosuss].

OBJECTIVE: To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism. METHODS: A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 µmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI. RESULTS: Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 µmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 µmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 µmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation. CONCLUSION: High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.

The Diagnostic Utility of Natural Killer Cell Subsets in Deep Vein Thrombosis.

Background: Natural killer (NK) cells are important components of adaptive and innate immune responses. NK cell subsets have different functions and may play a role in vascular disorders. This study aimed to evaluate the proportions of NK cells and their subsets to determine whether they can be used as markers of venous thrombosis and to identify whether there was a link between NK cell proportion and citrullinated histone (H3) levels. Patients and Methods: This study included 100 participants divided into Group I (n=50, patients with deep venous thrombosis (DVT)) and Group II (n=50, age- and sex-matched healthy controls). Group I was further categorized into Group Ia (n=25, patients with acute DVT) and Group Ib (n=25, patients with chronic DVT). The proportions of NK cells and their subsets were evaluated by flow cytometry using CD3/CD16/CD56. The levels of citrullinated histones (H3) were estimated using enzyme-linked immunosorbent assay (ELISA). Results: Compared to the control group, DVT patients had a significantly lower proportion of (CD56 dim/CD16+) NK cells, a significantly higher proportion of (CD56-/CD16+) NK cells and a high level of citrullinated histone (H3). Conclusion: NK cell subsets and citrullinated histone (H3) could be used as markers for DVT and as targets for therapeutic drugs to inhibit the formation or progression of thrombosis.

Immunohistochemical study of the expressed cluster differentiation markers proteins type 20 and 56 in breast tissues from a group of Iraqi patients with breast cancers.

BACKGROUND: Tumor-infiltrating lymphocytes (TIL) are important immunological components in response to cancers. Patients with higher numbers of TIL in breast cancerous tissues, comprising T- cytotoxic and T - helper cells along with B- and rare natural killer (NK) cells, have more favorable clinical outcomes. OBJECTIVE: To analyze the rate of the expressed surface biomarker proteins of CD20-B cells and CD56- NK cells on the infiltrative lymphocytic subpopulations in a group of breast tumorous tissues (invasive and benign) from female patients in Iraq and explore the relations to the grade of the invasive breast cancerous tissues. PATIENTS AND METHODS: One hundred and 75 archived breast tissues were enrolled in this retrospective research: 100 archived breast from female patients with invasive breast cancers (BC) [20 well differentiated BC tissues; 48 moderately differentiated BC and 32 poorly differentiated BC tissues]; 50 tissue biopsies from female patients with benign breast tumors and 25 apparently normal individuals with healthy breast tissues (included as the control group for this study). Immunohistochemistry was achieved for the detection of the expressed surface biomarker proteins related to B cell CD20 and NK cell CD56 present on the infiltrative lymphocytic subpopulations in breast tissues by using specific primary antibodies for these proteins via utilizing an immune-enzymatic antigen detection system. RESULTS: The detection of IHC reactions for the expressed B cell CD20 - cell surface ( CD) biomarker proteins were observed in 53 out of 100 (53.0%) BC tissues, and in 24 out of 50 (48.0%) benign breast tumorous tissues, while CD20- positive cell surface markers was detected in apparently healthy breast tissues of the control group in a percentage of  32.0% (8 out of 25 tissues). Statistical significant differences (P<0.05) between both groups of malignant and benign breast tumors and the control group were found. However, between breast malignant and benign tumor groups, no significant difference was found ( p >0.05). Detection of CD56- IHC reactions revealed in 14% (14  out of 100 BC tissues), in 16% (8 out of 50 benign breast tissues) and none of control breast tissues revealed CD56- IHC reactions. Among all the enrolled groups, no significant differences (P>0.05) were detected. CONCLUSIONS: The observed significant rates that showed highly significant differences  between both  studied groups of breast malignant and benign tumor in comparison to the control group indicate that the CD20- positive infiltrative B cell- lymphocytic subpopulations might contributed in the defense against these subsets of benign and malignant breast tumors. However, the observed rates of NK cell CD56 present on the lymphocytic subpopulations infiltrating the examined malignant and benign breast tumorous tissues  seeming to play  irrelevant roles in the defense against  these studied breast tumor groups.

The change of Siglec-9 expression in peripheral blood NK cells of SFTS patients can affect the function of NK cells.

OBJECTIVES: To investigate the changes and mechanism of Siglec-9 on NK cells in peripheral blood of patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: First, we used single-cell RNA sequencing to analyze the frequency of NK cells in Peripheral Blood Mononuclear Cells (PBMCs) of SFTS patients and healthy controls (HCs), as well as the differences in the genes on NK cells. Secondly, we analyzed the expression of Siglec-9 and other receptors on NK cells by flow cytometry. Thirdly, we analyzed the correlation between Siglec-9 on NK cells and DBV viral load in plasma. RESULTS: Compared with HCs, the frequency of NK cells in peripheral blood of SFTS patients was significantly decreased, and the activating receptors on NK cells were reduced. The expression of Siglec-9 on NK cells and the frequency of Siglec-9+NK cells decreased significantly in SFTS patients. The expression of Siglec-9 on CD16+CD56dim NK cells was negatively correlated with DBV viral load. In addition, Siglec-9+NK cells expressed higher levels of activating receptors and exhibited stronger effector functions than Siglec-9-NK cells. CONCLUSIONS: The decreased expression of Siglec-9 on NK cells predicts NK cell dysfunction in SFTS patients, suggesting that Siglec-9 may be a potential marker for functional NK cell subsets in SFTS patients.

The upregulation of peripheral CD3-CD56+CD16+ natural killer cells correlates with Th1/Th2 imbalance in asthma patients during acute upper respiratory viral infections.

PURPOSE: The aim of this study is to clarify the changes of peripheral CD3-CD56+CD16+ NK cells and their correlation with Th1/Th2 immunity profiles in asthma during the phase of acute upper respiratory viral infections (AURVIs). METHODS: Peripheral venous blood and induced sputum samples were collected from 56 mild asthma patients, 49 asthma patients with AURVIs and 50 healthy subjects. Peripheral CD3-CD56+CD16+ NK cells were monitored by flow cytometry during the course of acute viral infections. Meanwhile, the induced sputum Th2 cytokines IL-4 and IL-5, and Th1 cytokine IFN-γ were also detected by ELISA assay. RESULTS: The asthmatics had lower levels of peripheral CD3-CD56+CD16+ NK cells populations as well as higher induced sputum cytokines (IL-4, IL-5 and IFN-γ) compared to healthy controls at baseline. Upon upper respiratory viral infections, peripheral CD3-CD56+CD16+ NK cells numbers in asthma patients sharply elevated on day 3 and slowly decreased by day 14, in accordance with induced sputum IFN-γ changes. IL-4 and IL-5 levels spiked much later (day 8) and lasted until day 14. Compared with asthma alone group, the IFN-γ/IL-4 and IFN-γ/IL-5 ratios of the asthma patients with AURVIs on day 1 were higher and peaked on day 3. The changes of peripheral CD3-CD56+CD16+ NK cells proportions positively correlated with the IFN-γ/IL-4 and IFN-γ/IL-5 ratios on day 1 to day 3 in asthma subsequent to upper respiratory viral infections. CONCLUSIONS: Our findings showed an imbalanced Th1/Th2 immunity in airways of asthma with acute upper respiratory viral infections. Upregulated peripheral CD3-CD56+CD16+ NK cells play a crucial role in biased Th1 immunity of airways in asthma during the acute phase of viral infections. The anti-viral Th1 immunity by targeting NK cells may be a possible therapeutic option for virus-induced asthma exacerbation.