Natural Killer Cytotoxicity Research Articles

Single-cell RNA sequencing highlights the role of distinct natural killer subsets in sporadic amyotrophic lateral sclerosis

BackgroundNeuroinflammation plays a major role in amyotrophic lateral sclerosis (ALS), and cumulative evidence suggests that systemic inflammation and the infiltration of immune cells into the brain contribute to this process. However, no study has investigated the role of peripheral blood immune cells in ALS pathophysiology using single-cell RNA sequencing (scRNAseq).MethodsWe aimed to characterize immune cells from blood and identify ALS-related immune alterations at single-cell resolution. For this purpose, peripheral blood mononuclear cells (PBMC) were isolated from 14 ALS patients and 14 cognitively unimpaired healthy individuals (HC), matched by age and gender, and cryopreserved until library preparation and scRNAseq. We analyzed differences in the proportions of PBMC, gene expression, and cell-cell communication patterns between ALS patients and HC, as well as their association with plasma neurofilament light (NfL) concentrations, a surrogate biomarker for neurodegeneration. Flow cytometry was used to validate alterations in cell type proportions.ResultsWe identified the expansion of CD56dim natural killer (NK) cells in ALS (fold change = 2; adj. p-value = 0.0051), mainly driven by a specific subpopulation, NK_2 cells (fold change = 3.12; adj. p-value = 0.0001), which represent a mature and cytotoxic CD56dim NK subset. Our results revealed extensive gene expression alterations in NK_2 cells, pointing towards the activation of immune response (adj. p-value = 9.2 × 10− 11) and the regulation of lymphocyte proliferation (adj. p-value = 6.46 × 10− 6). We also identified gene expression changes in other immune cells, such as classical monocytes, and distinct CD8 + effector memory T cells which suggested enhanced antigen presentation via major histocompatibility class-II (adj. p-value = 1.23 × 10− 8) in ALS. The inference of cell-cell communication patterns demonstrated that the interaction between HLA-E and CD94:NKG2C from different lymphocytes to NK_2 cells is unique to ALS blood compared to HC. Finally, regression analysis revealed that the proportion of CD56bright NK cells along with the ALSFRS-r, disease duration, and gender, explained up to 76.4% of the variance in plasma NfL levels.ConclusionOur results reveal a signature of relevant changes occurring in peripheral blood immune cells in ALS and underscore alterations in the proportion, gene expression, and signaling patterns of a cytotoxic and terminally differentiated CD56dim NK subpopulation (NK_2), as well as a possible role of CD56bright NK cells in neurodegeneration.

P0150 A precision medicine approach for carriers of defined IL2RA polymorphism: JAK inhibition blocks exaggerated IL-2 signalling in vitro, with no difference in in vivo clinical response rates in a small clinical cohort

Abstract Background The rs61839660 (660) polymorphism lies within the IL2RA intronic enhancer and is a causal variant in Crohn’s disease, with a heterozygote frequency of 16.8%. IL-2Ra (CD25) is the high-affinity receptor for IL-2, with binding activating JAK-STAT signalling. We have demonstrated that risk (T) allele carriers exhibit higher CD4+ T cell and natural killer (NK) cell CD25 expression. In response to IL-2, ‘T’ allele carriers show enhanced JAK-STAT signalling, greater CD4+ effector T cell (Teff) pro-inflammatory cytokine release, and enhanced NK cytotoxicity compared to wild-type (CC) subjects.1 We hypothesised that this drives inflammation, and that ‘T’ allele carriers would be more likely to respond to Janus kinase inhibition (JAKi). Tofacitinib (TF) is a JAKi licensed for the treatment of UC. We examined the ability of JAKi to block IL-2-induced JAK-STAT signalling in 660 risk allele carriers in vitro, and explored clinical response to TF in a genotyped UC cohort, with the aim of understanding whether JAKi presents a personalised therapeutic approach in this population. Methods CD patients of each genotype were recruited in collaboration with the NIHR IBD Bioresource.2 We extracted PBMCs and flow-sorted CD4+ Teff and CD56dim NK cells. Cells were cultured for 18h in the presence/absence of 20nM TF, stimulated with IL-2, fixed, and stained for intracellular pSTAT5. TF-treated patients were identified, and provided a blood or saliva sample. Genotyping was performed by qPCR. Data were obtained from patient records, with clinical response defined as a reduction in the Simple Clinical Colitis Activity Index or Partial Mayo Score of >/= 3. Results pSTAT5 levels were elevated in TT subjects’ Teff compared to CC/CT subjects in response to 10IU/ml IL-2 (Fig 1a), with a reduction in Teff pSTAT5 MFI in the presence of TF. In NK cells, pSTAT5 MFI was increased in TT compared to CC donors in response to 100IU/ml IL-2 (Fig 1g). The pSTAT5 response to IL-2 was non-significantly reduced in the presence of TF in both CC and TT subjects’ NK cells. In the real-world UC cohort, there was no significant difference in clinical response to TF between genotypes (Fig 2a), nor in the proportion of patients who had a disease flare on dose reduction or requiring colectomy. There was no difference in TF treatment persistence between genotypes (Fig 2b). Conclusion TF diminishes the in vitro pSTAT5 response to IL-2 in CD4+ Teff and NK cells in carriers of the 660 risk allele. In a UC population, there was no difference in clinical response to TF between genotype cohorts. Our data are limited by the small study population, with larger studies needed to confirm whether the use of JAKi may represent a personalised therapeutic approach in 660 risk allele carriers.

A Review of Immunological Evaluation of Patients with Recurrent Spontaneous Abortion (RSA).

In approximately half of the recurrent spontaneous abortion (RSA) cases, the underlying cause is unknown. However, most unexplained miscarriages are thought to be linked to immune dysfunction. This review summarizes the current evidence regarding the immunological evaluations of patients with RSA, with potential implications for clinical research. The immune system plays a crucial role in the successful outcome of pregnancy, as it tolerates the semi-allogeneic fetus while offering protection to both the mother and fetus from pathogens. The maternal-fetal interface is the place where the crosstalk between various immune cells such as macrophages, dendritic cells, natural killer (NK) cells, and T cells takes place. An adequate balance is required between these immune cells for pregnancy to progress. In RSA, a dysregulation between these immune players is witnessed. For example, in RSA, NK cells are not increased but also undergo a change in their activity, manifested as cytotoxic decidual NK. Similarly, regulatory T cells, which are crucial for fostering a tolerant immune environment, are decreased in RSA women. Similarly, imbalances between T-helper (Th1, Th2, Th17) cell subsets have been implicated in RSA. Furthermore, the imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophage phenotypes has been documented, with studies indicating a predominance of M1 macrophages in RSA patients. Targeting immune imbalances with therapies such as immunoglobulin administration, TNF inhibitors, and anticoagulants may improve pregnancy outcomes in women with RSA.

The emergence of DNAM-1 as the facilitator of NK cell-mediated killing in ovarian cancer.

Ovarian cancer (OC) is the sixth most common malignancy in women and the poor 5-year survival emphasises the need for novel therapies. NK cells play an important role in the control of malignant disease but the nature of tumour-infiltrating and peripheral NK cells in OC remains unclear. Using flow cytometric analysis, we studied the phenotype and function of NK cells in blood, primary tumour and metastatic tissue in 80 women with OC. The cell type contexture of metastatic OC tissue was explored utilising scRNAseq analysis, with a focus on portraying an immunogenic tumour microenvironment and determining the characteristics of a dysfunctional NK cell population. The proportion of peripheral NK cells was markedly elevated with a highly activated profile and increased cytotoxicity. In contrast, NK cell numbers in primary tumour and metastasis were substantially reduced, with downregulation of activatory receptors together with elevated PD-1 expression. scRNA-Seq identified 5 NK cell subpopulations along with increased exhausted and immature NK cells within tumour tissue compared to normal tissue. These features were attenuated following chemotherapy where higher levels of activated and cytotoxic NK cells associated with improved disease-free survival. Correlation of NK cell phenotype with clinical outcomes revealed high levels of DNAM-1 expression on tissue-localised and peripheral NK cells to be associated with reduced survival. Expression of PVR, the DNAM-1 ligand, was significantly increased on tumours and DNAM-1 mediated NK cell lysis of primary tumour tissue was observed in vitro. These findings reveal profound modulation of the tumour tissue and systemic profile of NK cells which likely contributes to the high rates of local progression and metastasis seen with OC. Immunotherapeutic approaches that overcome local immune suppression and enhance DNAM-1-targeted lysis of OC offer the potential to improve disease control.

Obesity modulates NK cell activity via LDL and DUSP1 signaling for populations with adverse social determinants.

African American (AA) women are disproportionately affected by obesity and hyperlipidemia, particularly in the setting of adverse social determinants of health (aSDoH) that contribute to health disparities. Obesity, hyperlipidemia, and aSDoH appear to impair NK cells. As potential common underlying mechanisms are largely unknown, we sought to investigate common signaling pathways involved in NK cell dysfunction related to obesity and hyperlipidemia in AA women from underresourced neighborhoods. We determined in freshly isolated NK cells that obesity and measures of aSDoH were associated with a shift in NK cell subsets away from CD56dim/CD16+ cytotoxic NK cells. Using ex vivo data, we identified LDL as a marker related to NK cell function in an AA population from underresourced neighborhoods. Additionally, NK cells from AA women with obesity and LDL-treated NK cells displayed a loss in NK cell function. Comparative unbiased RNA-sequencing analysis revealed DUSP1 as a common factor. Subsequently, chemical inhibition of Dusp1 and Dusp1 overexpression in NK cells highlighted its significance in NK cell function and lysosome biogenesis in a mTOR/TFEB-related fashion. Our data demonstrate a pathway by which obesity and hyperlipidemia in the setting of aSDoH may relate to NK cell dysfunction, making DUSP1 an important target for further investigation of health disparities.

THE INFLUENCE OF BIFIDOBACTERIUM ANIMALIS AND LECTIN OF B. SUBTILIS IMV B-7724 ON THE ANTITUMOR IMMUNE RESPONSE OF MICE WITH EHRLICH ADENOCARCINOMA.

To investigate the effect of bacteria of the genus Bifidobacterium and the extracellular metabolite of B. subtilis IMV B-7724 on the antitumor immune response of mice with a model tumor. The study was conducted on Balb/c mice with transplanted solid Ehrlich adenocarcinoma (ACE). Starting from the 2nd day after the transplantation of tumor cells, the animals of the experimental groups were treated with lectin of B. subtilis IMV B-7724 (s/c, 1 mg/kg of weight), Bifidobacterium animalis (per os, 7 × 105 CFU/mouse) or their combination. The immunological studies were performed on the 21st and 28th days of tumor growth. The functional activity of natural killer cells (NK), cytotoxic T-lymphocytes (CTL), as well as the ability of lymphocytes from the peripheral lymph nodes (PLN) to transform into blast cells under the influence of T- (Con A) and B-cell (LPS) mitogens were determined. Administration of probiotic components to the mice with ACE led to the activation of innate immune responses, that is, to a significant increase in the cytotoxic activity of NK, especially in the case of their combined use. The NK cytotoxicity index was higher than that in the non-treated ACE-bearing mice and the intact control by 3.7 and 2.1 times, respectively (p < 0.05). Similarly, the highest specific cytotoxic activity of spleen lymphocytes was observed upon the combined use of the microbial preparations: the CTL cytotoxicity index was nearly 2.5-fold higher than in the non-treated ACE-bearing mice. The data on the ability of PLN lymphocytes to transform into blast cells under the influence of Con A and LPS indicated the preservation of the functional activity of lymphocytes in the animals of the experimental groups during ACE growth. Both B. animalis and lectin of B. subtilis IMV B-7724 have a significant influence on the effectors of the natural and adaptive immunity of mice with ACE. Their combined use was found to be the most effective.

Functional and phenotypic changes in natural killer cells expressing immune checkpoint receptors PD-1, CTLA-4, LAG-3, and TIGIT in non-small cell lung cancer: the comparative analysis of tumor microenvironment, peripheral venous blood, and tumor-draining veins.

Natural killer (NK) cells are a cytotoxic subset of innate lymphoid cells and have key roles in antitumoral immunity. This study evaluates the roles of immune checkpoint receptors on NK cell phenotype and functions both before and after circulation through tumor tissue. Twenty non-small cell lung cancer patients undergoing surgery and 21 healthy controls were included. Lymphocytes were isolated from peripheral venous blood, tumor-draining venous blood, and tumor tissue. Immune checkpoint receptor (ICR) expressions, intracellular cytokines, and cytotoxic capacity of NK cell subsets were analyzed by flow cytometry. Circulatory levels of sPD-1, sCTLA-4, sLAG-3, and sTIGIT were determined by ELISA. PD-1, CTLA-4, and LAG-3 expressions of both cytotoxic (CD56neg/dimCD16bright) and cytokine-producing (CD56bright/dimCD16neg) NK cells increased in tumor tissue compared to both peripheral and tumor-draining veins. NK cells expressing PD-1, CTLA-4, or LAG-3 had significantly lower IFN- and TNF- and increased IL-10 expressions in tumor tissue compared to peripheral venous blood. The cytotoxic activity (perforin and granzyme A expressions) of NK cells from tumor tissue was significantly reduced compared to peripheral blood. Soluble ICRs decreased in peripheral blood and tumor-draining vein of the patients compared to peripheral blood of healthy individuals. However, NK cell phenotype and functions were similar in peripheral blood and tumor-draining vein. NSCLC tumor microenvironment impacts ICR expressions in NK cells, and ICR-expressing NK cells have impaired inflammatory cytokine secretion and cytotoxic activities with a regulatory phenotype. However, tumor-draining venous blood did not reflect the immune status of the tumor tissue.

Long COVID is associated with lower percentages of mature, cytotoxic NK cell phenotypes.

Long COVID is associated with lower percentages of mature, cytotoxic NK cell phenotypes.

Sex-specific impact of obstructive sleep apnea on peripheral blood mononuclear cells.

Experimental sleep disruption in healthy adults is more deleterious to immune function in females relative to males; however, it remains unknown if this translates to patients with obstructive sleep apnea (OSA). Thus, this study explored sex differences in peripheral blood mononuclear cells (PBMCs) from patients with untreated OSA. Participants completed sleep studies to identify the presence of OSA via the apnea-hypopnea index (AHI). PBMCs were isolated, cryopreserved, and batch phenotyped via mass cytometry. Females with (n = 6, AHI = 25.9 ± 21.4 events/hr, age = 37 ± 14yrs, BMI = 30.5 ± 7.4kg/m2) and without (n = 9, AHI = 2.6 ± 1.6 events/hr, age = 35 ± 10yrs, BMI = 29.2 ± 6.3kg/m2) OSA were compared to males with (n = 7, AHI = 13.7 ± 8.5 events/hr, age = 33 ± 11yrs, BMI = 30.0 ± 4.8kg/m2) and without (n = 7, AHI = 2.6 ± 1.6 events/hr, age = 33 ± 10yrs, BMI = 28.9 ± 3.8kg/m2) OSA. No significant group-by-sex interactions were observed in CD3 T cells (p = 0.273), CD8 T cells (p = 0.656), B cells (p = 0.190), monocytes (p = 0.638), nor granulocytes (p = 0.267) expressed as a percent of their respective parent population. While the percentage of total NK cells did not differ between groups (group-by-sex p = 0.822), females with OSA had fewer CD57- (42.4 ± 14.7 vs. 62.4 ± 10.4%) and more CD57+ (57.6 ± 14.7 vs. 37.6 ± 10.4%) NK cells than females without OSA (p < 0.050). No differences in CD57- (53.6 ± 18.1 vs. 44.9 ± 16.8%) and CD57+ (46.4 ± 18.1 vs. 55.2 ± 19.8%) NK cells were observed between males (p = 0.283). Tregs were more prevalent in females with vs. females without OSA (2.17 ± 0.64 vs. 1.31 ± 0.41%, p = 0.006) with no difference between males (1.55 ± 0.50 vs. 1.71 ± 0.71%, p = 0.601). Our data suggest that OSA increases the prevalence of cytotoxic NK cells and Tregs in females. The causes and downstream effects of these changes remain undetermined.

Blocking CXCR3B Expression Increases Tumor Aggressiveness in Hepatocellular Carcinoma.

CXCR3B has been positively involved in the inhibition of cancer and angiogenesis. The present study investigated the role of CXCR3B in a cell model of hepatocellular carcinoma, SK-Hep1. The blockade of CXCR3B expression in SK-Hep1 was investigated in terms of cell viability, cell cycle, and cell apoptosis using MTT assay and flow cytometry. In addition, the effect of blocking CXCR3B expression on cell migration and invasion was examined using scratch motility, transwell migration, and invasion assays. Furthermore, the cytotoxic effect of NK-92 cells against CXCR3B blocked SK-Hep1 was analyzed using the CytoTox96 assay, and the expression of NKp30+, NKG2D+, and NKG2C+ on NK-92 cells in a co-culture with SK-Hep1 was measured using flow cytometry. Blocking CXCR3B expression had no effect on the viability, cell cycle or apoptosis of SK-Hep1 cells. However, blockade of CXCR3B expression significantly increased the migratory and invasive ability of SK-Hep1 along with increased protein expression of slug, vimentin, and N-cadherin. CXCR3B blockade reduced the cytotoxicity of NK-92 against SK-Hep1 and inhibited the expression of activating receptors, NKp30+, NKG2D+, and NKG2C+ in NK-92 cells. CXCR3B may play a positive role in suppressing HCC by attenuating natural killer cell cytotoxicity against HCC.

Abstract C020: Discovery &amp; characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors

Abstract While adoptive cellular therapies (ACT) such as CAR-T and NK therapy have shown clinical efficacy against hematologic malignancies, their use against solid tumors (such as ovarian or pancreatic cancer) has had limited success to date. In particular, CAR-T therapy for solid cancers has led to severe toxicities, including on-target, off-tumor killing of healthy cells and graft vs. host disease (GVHD) with allogeneic transplant. NK cell therapies, in contrast, have fewer associated toxicities due to specific and innate recognition/killing of cancer cells. This greater safety profile makes NK therapies an attractive treatment option for solid cancers. NK therapies are currently limited by insufficient in vivo tumor clearance, caused largely by their lower cytotoxicity and in vivo expansion compared to CAR-T cells. To address this, we have screened 300,000 small molecule compounds for their ability to enhance NK cytotoxicity against ovarian cancer (OVCAR) cells. Our primary screen identified 11 lead compounds which increase NK killing activity against OVCAR cells &amp;gt;= 10% over baseline (vehicle-treated control). Secondary in vitro assays have confirmed that at least 3 out of 11 lead compounds enhance NK cytotoxicity by at least 10% over baseline. As such, we hypothesize that our lead compounds will increase in vivo NK cytotoxicity and tumor clearance, through mechanisms which might include I) upregulation of NK activating receptors, II) downregulation of NK inhibitory receptors, and/or III) modulation of NK metabolism in the immunosuppressive tumor microenvironment. Immediate future studies include in vivo testing of our lead compounds’ ability to enhance NK cytotoxicity, using immunodeficient mouse models of subcutaneous OVCAR tumors. Moreover, we will utilize 2D and 3D in vitro culture techniques alongside flow cytometry and RNA sequencing to identify how lead compounds impact markers of NK activation and metabolism. This will include expression of activating/inhibitory receptors, exhaustion markers, and key metabolic factors. Finally, we will assess the effect of lead compounds on NK function within an immunosuppressive tumor environment. Our findings will contribute to effective future NK cell therapies for solid tumors. Citation Format: Indrani Das, Olubukola Abiona, Riufu Liu, Grace K. Lee, David N. Wald. Discovery &amp; characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C020.

Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer.

Bladder cancer (BC) is a common malignancy and its most prevalent type is urothelial carcinoma, which accounts for ~90% of all cases of BC. The current treatment options for BC are limited, which necessitates the development of alternative treatment strategies. Hyperthermia (HT), as an adjuvant cancer therapy, is known to improve the efficacy of chemotherapy or radiotherapy. The present study aimed to investigate the anti‑tumor effects of HT on cell survival, invasiveness, chemoresistance and immune evasion in human BC cell lines (5637, T24 and UMUC3). Calcein AM staining was performed to analyze the cytotoxicity of natural killer (NK) cells against human BC cells following HT treatment. Cell migration and invasion affected by HT were analyzed using Transwell migration and invasion assays. It was found that HT inhibited the proliferation of BC cells by downregulating the phosphorylation of protein kinase B. Moreover, HT effectively enhanced the sensitivity of BC cells to the chemotherapy drug cisplatin (DDP) and reduced the chemoresistance of DDP‑resistant cells by downregulating the expression of cadherin‑11. It was further demonstrated that HT inhibited the migration and invasion of BC cells and enhanced the cytotoxic effects of NK cells. In summary, the antineoplastic effects of HT were mediated through three main mechanisms: Enhancement of the chemosensitivity of BC cells and mitigation of DDP‑induced chemoresistance, suppression of the invasive potential of BC cells and reinforcement of the anticancer response of NK cells. Thus, HT appears to be a promising adjunctive therapy for human BC.

Co-Colorectal cancer stem cells employ the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia

Colorectal cancer (CRC) ranks as China’s second most common cancer and fifth top cancer death cause. The study highlights the role of Natural Killer (NK) cells in targeting cancer stem cells (CSCs) that evade immune responses in CRC. Colorectal cancer stem cells (CCSCs) were stem from HT-29 cells and co-cultured with NK cells under normoxic or hypoxic conditions. The impact of this co-culture was evaluated using CCK8 assays for NK cell viability, ELISA for cytokine level changes, and flow cytometry for assessing NK cell apoptosis and activation. Comprehensive metabolomic and transcriptomic analyses were also performed to identify key genes and metabolites involved in the interaction between CCSCs and NK cells Co-culture of CCSCs with NK cells under hypoxia reduced NK cytotoxicity, increased NK apoptosis, and altered cytokine secretion by decreasing IFN-γ and TNF-α levels while increasing IL-6. Transcriptomic and metabolomic analysis identified 4 genes (FADS1, ALDH3A2, GCSH, MTCL1) and 3 metabolites (glyoxylic acid, spermine, DDA) as significant. Interfering with FADS1 counteracted the suppression of IFN-γ and TNF-α induced by CSC cells. Curiously, this inhibition caused by si-FADS1 could be neutralized by the addition of exogenous DDA. Co-culturing with NK cells notably increased spermine levels. Exogenous spermine resulted in a significant reduction in HT-29 cell death rates at 32 µM, 64 µM, and 128 µM, compared to NK cells without spermine. Our research explored CCSCs employed the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia.

Atopic Dermatitis Immune Dysregulation as Dengue Predisposing Factor.

The immune response is important in dengue's clinical manifestation, and the immune dysregulation in Atopic Dermatitis (AD) can permit immune evasion by viruses. There have been many studies describing the immune response in AD and the pathomechanism of dengue, but AD as a predisposing factor for dengue and its severity have not been much discussed. This review investigates how immune dysregulation in AD may be a predisposing factor for Dengue and its severe outcomes. We conducted a comprehensive analysis of studies from the past decade, focusing on dendritic cells (DCs), macrophages, mast cells (MCs), Innate Lymphoid Cell 2 (ILC-2), Natural Killer (NK) cells, interferon (IFN), interleukin (IL) 4, IL-13, and T helper (Th) 2 in AD patients with healthy subject as a comparison, using databases PubMed, Science Direct, and Google Scholar. We got 44 articles that met inclusion criteria. From those articles, we resumed that moderate and severe AD patients' immune profiles showed increased DC, MCs, M2 macrophage, NK cells, and ILC2 in the lesional and non-lesional skin, decreased DC and NK cells in peripheral blood, alteration cytotoxicity of NK cells,Th2-skewed adaptive immune response in lesional and non-lesional skin, and peripheral blood. Increased DC, M2 macrophage, and MCs provide target cells for Dengue virus (DENV) replication. Alteration cytotoxicity of NK cells, ILC2, and Th2 skewed immune response facilitated immune evasion by DENV. The innate and adaptive immune dysregulation in moderate and severe AD provides DENV target cells and facilitates virus immune evasion that can be a predisposing factor for dengue and severe dengue. Further research is recommended to clarify the association between AD and the incidence of dengue and severe dengue because this can be a consideration in determining the prognosis and management of Dengue.

Integrated single-cell transcriptome and TCR profiles of hepatocellular carcinoma highlight the convergence on interferon signaling during immunotherapy

BackgroundDespite the success of immune checkpoint inhibitor (ICI)-based combination therapies in hepatocellular carcinoma (HCC), its effectiveness remains confined to a subset of patients. The development of reliable, predictive markers is...

Trastuzumab-Mediated Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Enhances Natural Killer Cell Cytotoxicity in HER2-Overexpressing Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer. Although human epidermal growth factor receptor-2 (HER2) overexpression, a poor prognostic molecular marker in ovarian cancer, is found in almost 30% of ovarian cancer cases, there are no established therapies for HER2-overexpressing ovarian cancer. In this study, we investigated the efficacy of combined samfenet, a biosimilar compound of trastuzumab, and natural killer (NK) cells in preclinical model of HER2-overexpressing ovarian cancer. Firstly, we screened the HER2 expression in three ovarian cancer cell lines and eight ovarian cancer patient-derived tumor xenograft (PDTX) samples. Then, immunohistochemistry and silver in situ hybridization (SISH) were performed following clinical criteria. HER2-overexpressing cells exhibited the highest sensitivity to samfenet compared with low-HER2-expressing cells. In addition, the combination of samfenet with natural killer (NK) cells resulted in significantly enhanced sensitivity to HER2-overexpressing cells and showed a significant antitumor effect on PDTX mice compared with monotherapy. It is known that anti-HER2-humanized IgG1 monoclonal antibodies, including trastuzumab, induce antibody-dependent cellular cytotoxicity (ADCC). Consequently, the combination of samfenet with NK cells demonstrated NK cell-mediated ADCC, as confirmed using an in vitro NK cytotoxicity assay and in vivo antitumor efficacy. A transferase dUTP nick end labeling (TUNEL) assay using xenografted tumors further supported the ADCC effects based on the increase in the number of apoptotic cells in the combination group. Furthermore, high HER2 expression was associated with shorter progression-free survival and overall survival based on public mRNA expression data. In this study, we demonstrated that the combination of samfenet and NK cell therapy could be a promising treatment strategy for patients with HER2-overexpressing ovarian cancer, through ADCC effects. Therefore, this study supports a rationale for further clinical studies of the combination of samfenet and NK cells as a therapy for patients with HER2-overexpressing ovarian cancer.

Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression

Patients present a wide range of clinical severities in response severe acute respiratory syndrome coronavirus 2 infection, but the underlying molecular and cellular reasons why clinical outcomes vary so greatly within the population remains unknown. Here, we report that negative clinical outcomes in severely ill patients were associated with divergent RNA transcriptome profiles in peripheral immune cells compared with mild cases during the first weeks after disease onset. Protein–protein interaction analysis indicated that early-responding cytotoxic natural killer cells were associated with an effective clearance of the virus and a less severe outcome. This innate immune response was associated with the activation of select cytokine–cytokine receptor pathways and robust Th1/Th2 cell differentiation profiles. In contrast, severely ill patients exhibited a dysregulation between innate and adaptive responses affiliated with divergent Th1/Th2 profiles and negative outcomes. This knowledge forms the basis of clinical triage that may be used to preemptively detect high-risk patients before life-threatening outcomes ensue.

Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression.

Patients present a wide range of clinical severities in response severe acute respiratory syndrome coronavirus 2 infection, but the underlying molecular and cellular reasons why clinical outcomes vary so greatly within the population remains unknown. Here, we report that negative clinical outcomes in severely ill patients were associated with divergent RNA transcriptome profiles in peripheral immune cells compared with mild cases during the first weeks after disease onset. Protein-protein interaction analysis indicated that early-responding cytotoxic natural killer cells were associated with an effective clearance of the virus and a less severe outcome. This innate immune response was associated with the activation of select cytokine-cytokine receptor pathways and robust Th1/Th2 cell differentiation profiles. In contrast, severely ill patients exhibited a dysregulation between innate and adaptive responses affiliated with divergent Th1/Th2 profiles and negative outcomes. This knowledge forms the basis of clinical triage that may be used to preemptively detect high-risk patients before life-threatening outcomes ensue.

CD56 does not contribute to the anti-tumor, tissue homing, and glycolytic capacity of human NK cells.

Natural Killer (NK) cells are critical innate immune cells involved in the clearance of virally infected and malignant cells. Human NK cells are distinguished by their surface expression of CD56 and a lack of CD3. While CD56 expression and cell surface density has long been used as the prototypic marker to characterize primary human NK cell functional subsets, the exact functional role of CD56 in primary human NK cells is still not fully understood. Here we eliminated the expression of CD56 in human ex vivo expanded NK cells (CD56bright) using CRISPR/Cas9 in order to assess the function of CD56 in this highly activated and cytotoxic NK cell population. We show that the expression of CD56 has no effect on NK cell proliferative capacity or expression of various activation and inhibitory markers. Further, CD56 does not contribute to NK cell-mediated cytotoxicity, inflammatory cytokine production, or the ability of NK cells to control tumor engraftment in vivo. We also found that while deletion of CD56 did not impact NK cell glycolytic metabolism it did increase NK cell reliance on oxidative phosphorylation. Lastly, CD56 does not alter expanded NK cell in vivo tissue trafficking. Our results indicate that while CD56 expression could be used to indicate a hyper-functional state of NK cells, it does not directly influence the anti-tumor functions of expanded NK cells.

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy

The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.