Natural Killer Cell Function Research Articles

Abstract B022: Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer (BC) cases worldwide, yet due to its aggressive phenotype and lack of targeted therapies it has the worst prognosis amongst BC subtypes. Up to 40% of TNBC patients will relapse after treatment and develop distant metastasis. Metastasis is the complex process by which disseminated tumor cells (DTCs) leave the primary tumor and grow at distal sites. Since most metastases occur within 3 years of diagnosis, the rapid onset and lack of effective treatments in metastatic TNBC (mTNBC) means less than 20% of patients survive after 4 years. Even though it is the most “immune-activated” BC subtype, and immune checkpoint blockade (ICB) has shown clinical benefits in early TNBC, there is a clear need for novel immunotherapeutic approaches for patients with mTNBC. Natural killer (NK) cells are innate lymphocytes that function as the body’s first line defense against metastatic cancer cells. NK cells eliminate DTCs in the blood or at distal sites via NK cell cytotoxicity (NKCC) and this process is known to be impaired in successful metastasis. We hypothesized that other cells within the tumor microenvironment (TME) can inhibit NKCC at the earliest stages of metastatic outgrowth. We have used Cherry-niche, an in vivo labelling tool, to characterize how TME cells change in early and established lung micro-metastases by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Our initial analyses showed that monocytes are highly enriched, not only in the entire metastatic lung, but specifically in the established niche surrounding DTCs. Although we detected a slight increase in total NK cells, there was a shift in maturation status with fewer mature NK cells (CD27-CD11b+) and more immature NK cells (CD27+/-CD11b-) in the metastatic lung. Furthermore, NK cells were largely absent from the metastatic niche and those that were present displayed a dysfunctional phenotype. CellChat and NicheNet analyses of our scRNA-seq data predicted that several receptor-ligand interactions, including the cytokine macrophage migration inhibitory factor (MIF), can mediate the crosstalk between NK cells and monocytes. Next, we developed an in vitro 3D co-culture system to study the effect of blood-derived monocytes on human NKCC against mTNBC cells. We found that monocytes can suppress NKCC against MDA-MB-231 cells and decrease the viability of mature NK cells. Furthermore, recombinant MIF was able to directly inhibit NKCC, whilst a selective MIF antagonist was able to rescue monocyte-induced impairment of NKCC. These data implicate MIF as a critical monocyte-derived factor in the suppression of NK cell function against mTNBC cells. Future work will aim to identify the exact mechanism by which MIF inhibits NKCC, whilst also evaluate the effect of monocyte depletion and MIF inhibition on metastatic outgrowth and NK cell activation/maturation in vivo. This study will reveal novel inter-cellular dynamics occurring in the metastatic lung, as well as immunotherapeutic targets that may restore NKCC in mTNBC. Citation Format: Christos Ermogenous, Luigi Ombrato, Gordon Beattie. Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer [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 B022.

A PSMA-targeted Tri-specific Killer Engager enhances NK cell cytotoxicity against prostate cancer.

Natural killer (NK) cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with co-activation may enhance NK cell function in mCRPC. We describe a Tri-specific Killer Engager (TriKE) construct that engages with the activating receptor CD16 on NK cells, prostate-specific membrane antigen (PSMA) on mCRPC cells, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation and cytokine production of NK cells derived from healthy donors or prostate cancer patients. Bystander killing of PSMA-negative was also achieved with PSMA TriKE treatment when co-cultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment (TME), NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or MDSCs maintained their potent function while IL-15 treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared to IL-15 and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their anti-tumor potential in prostate cancer, especially in the setting of a hostile TME.

Dichotomous outcomes of TNFR1 and TNFR2 signaling in NK cell-mediated immune responses during inflammation.

Natural killer (NK) cell function is regulated by a balance of activating and inhibitory signals. Tumor necrosis factor (TNF) is an inflammatory cytokine ubiquitous across homeostasis and disease, yet its role in regulation of NK cells remains unclear. Here, we find upregulation of the immune checkpoint protein, T cell immunoglobulin and mucin domain 3 (Tim3), is a biomarker of TNF signaling in NK cells during Salmonella Typhimurium infection. In mice with conditional deficiency of either TNF receptor 1 (TNFR1) or TNF receptor 2 (TNFR2) in NK cells, we find TNFR1 limits bacterial clearance whereas TNFR2 promotes it. Mechanistically, via single cell RNA sequencing we find that both TNFR1 and TNFR2 induce the upregulation of Tim3, while TNFR1 accelerates NK cell death but TNFR2 promotes NK cell accumulation and effector function. Our study thus highlights the complex interplay of TNF-based regulation of NK cells by the two TNF receptors during inflammation.

Impact of Psychological Stressors on Natural Killer Cell Function: A Comprehensive Analysis based on Stressor Type, Duration, Intensity, and Species.

Patients with natural killer (NK) cell deficiency or dysfunction are more susceptible to infections by Herpesviridae viruses, herpesvirus-related cancers, and macrophage activation syndromes. This review summarizes research on NK cell dysfunction following psychological stress, focusing on stressor type, duration, age of exposure, and species studied. Psychological stressors negatively affect NK cell activity (NKCA) across species. Prolonged stress leads to more significant decreases in NK cell number and function, with rehabilitation efforts proving ineffective in reversing these effects. Early life and prolonged stress exposure particularly increases the risk of infections and cancer due to impaired NKCA. The review also highlights that stress impacts males and females differently, with females exhibiting a more immunosuppressed NK cell phenotype. Notably, mice respond differently compared to humans and other animals, making them unsuitable for NK cell stress-related studies. Most studies measured NKCA using cytolytic assays against K-562 or YAC-1 cells. Although the exact mechanisms of NK cell dysfunction under stress remain unclear, potential causes include reduced release of secretory lysosomes with perforin or granzyme, impaired NK cell synapse formation, decreased expression of synapse-related molecules like CD2 or LFA-1 (CD11a), altered activating receptor expression, and dysregulated signaling pathways, such as decreased Erk1/2 phosphorylation and NFkB signaling. These mechanisms are not mutually exclusive, and future research is needed to clarify these pathways and develop therapeutic interventions for stress-induced immune dysregulation.

Natural killer cell biology and therapy in multiple myeloma: challenges and opportunities.

Despite therapeutic advancements, multiple myeloma (MM) remains incurable. NK cells have emerged as a promising option for the treatment of MM. NK cells are heterogenous and typically classified based on the relative expression of their surface markers (e.g., CD56 and CD16a). These cells elicit an antitumor response in the presence of low mutational burden and without neoantigen presentation via germline-encoded activating and inhibitory receptors that identify the markers of transformation present on the MM cells. Higher NK cell activity is associated with improved survival and prognosis, whereas lower activity is associated with advanced clinical stage and disease progression in MM. Moreover, not all NK cell phenotypes contribute equally toward the anti-MM effect; higher proportions of certain NK cell phenotypes result in better outcomes. In MM, the proportion, phenotype, and function of NK cells are drastically varied between different disease stages; this is further influenced by the bone marrow microenvironment, proportion of activating and inhibitory receptors on NK cells, expression of homing receptors, and bone marrow hypoxia. Antimyeloma therapies, such as autologous stem cell transplant, immunomodulation, proteasome inhibition, and checkpoint inhibition, further modulate the NK cell landscape in the patients. Thus, NK cells can naturally work in tandem with anti-MM therapies and be strategically modulated for improved anti-MM effect. This review article describes immunotypic and phenotypic differences in NK cells along with the functional changes in homeostatic and malignant states and provides expert insights on strategies to harness the potential of NK cells for improving outcomes in MM.

Advances in CAR NK Cell Therapy for Targeting and Eradicating Circulating Tumor Cells: Challenges and Solutions for Aging Patients Mini Review

Chimeric Antigen Receptor (CAR) Natural Killer (NK) cells represent a promising advancement in cancer immunotherapy, particularly for targeting circulating tumor cells (CTCs) and preventing metastasis. This review examines the latest developments in CAR NK cell therapy, including diverse NK cell sources, genetic engineering techniques, and dual mechanisms of action. Targeting CTCs with CAR NK cells shows significant potential in aggressive cancers like triple-negative breast cancer (TNBC) and pancreatic cancer. The impact of aging on NK cell function, especially regarding cytotoxicity, cytokine secretion, and persistence, poses challenges for elderly patients, but strategies such as interleukin-15 and metabolic interventions offer potential solutions. The review also addresses current limitations, such as poor persistence in immunosuppressive microenvironments and low solid tumor infiltration, while proposing combination therapies to enhance effectiveness. Although still in earlier clinical stages compared to CAR T cells, CAR NK cells’ safety profile and MHC-independent recognition mechanisms make them a versatile therapeutic option. Future directions include optimizing NK cell persistence, improving infiltration, and developing age-adapted therapies for elderly patients. _________________________________________________________________________________________ Keywords: CAR NK cells, CAR T cells, Circulating tumor cells (CTCs), Cell Therapy, Aging

IMMU-69. THERAPEUTIC WINDOW ENABLING ERADICATION OF RESIDUAL GLIOMA STEM CELLS BY INTRACRANIAL NK CELL THERAPY FOLLOWING TEMOZOLOMIDE-INDUCED TUMOUR DEPOPULATION – A POSSIBLE ROLE OF CYTOTOXIC EXTRACELLULAR VESICLES

Abstract Glioblastoma (GBM) is an incurable brain cancer, where dismal outcomes result from disease recurrence driven by tumour-initiating glioma stem cells (GSCs). GSCs survive and expand in the brain after surgery, radiation and temozolomide (TMZ) amidst weak immune and natural killer (NK) cell surveillance. Efficient NK cell-mediated killing of GBM cells occurs at high effector to target ratios precluding effective eradication of large tumour remnants, as enforced by clinical trials with autologous NK cells in an adjuvant setting. Here we explore in a human GSC xenograft model whether tumor depopulation using high dose Temozolomide (TMZ) could create a window of curative opportunity for endogenous or exogenous NK cells. We observed that while subcutaneous (sc) xenografts of patient derived GSCs are infiltrated by endogenous functional (NCR1+) NK cells in SCID mice, the corresponding intracranial (ic) tumours remained NK cell depleted. Notably, while TMZ caused near complete regression of intracranial GSC xenografts this was followed by inevitable recurrence of drug-resistant lesions. To explore whether direct delivery of NK cells into depopulated intracranial tumours would change the lethal course of the disease, mice were injected with GSCs intracranially, and upon tumour formation were treated with a sequence of TMZ (systemically), at 2, 7, 14, and 21 days later with irradiated NK92MI cells. Remarkably, this combined therapy completely obliterated recurrent disease at 2 and 7 days but not beyond 14 days. To assess whether NK92MI cells could be replaced by their derived extracellular vesicles (NK-EVs), the latter were injected i.c. post TMZ in GSC xenograft bearing mice. A single injection of NK-EVs resulted in tumour eradication in some but not all mice. Thus, chemotherapy-dependent tumour depopulation may create a unique window of opportunity for curative NK-mediated immunotherapy in GBM.

EXTH-42. GLIOMA-DERIVED CD200 PROMOTES TUMOR GROWTH AND SUPPRESSES NK CELL AND CD8 T CELL ANTI-GLIOMA ACTIVITY

Abstract Immune-checkpoint inhibitors, such as anti-PD1 and anti-CTLA4, have emerged as promising therapeutic options for several malignancies yet show little efficacy against malignant brain cancers. CD200 is a newly recognized immune-checkpoint which is expressed by a marid of cell types, modulating immune homeostasis through multiple receptors. There is currently limited information regarding CD200 effects in brain tumors. Furthermore, while it is well accepted that CD200 binding to its inhibitory receptor induces a pro-tumorigenic environment through its ability to suppress immune responses, there are increasing evidence that CD200 could also have anti-tumor characteristics. Here we evaluated the role of tumor-derived CD200 on anti-glioma immunity. We demonstrate that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in serum of patients undergoing immunotherapy. Transcriptomic analysis of CD200 knockout (KO) glioma models reveals that glioma-derived CD200 significantly modifies the glioma tumor microenvironment (TME), not only through immune regulation but also through immune-independent pathways, such as tumor metabolism. Furthermore, we show that CD200 KO gliomas have reduced proliferation and are rejected by their hosts. Downstream analysis revealed that rejection of CD200 KO gliomas relied on a functional adoptive immune system, while decreased proliferation was linked to reduced CXCL10 production by the CD200 KO gliomas. Additionally, we demonstrate that glioma-mediated CD200 strongly suppresses anti-glioma NK cell function within the tumor microenvironment (TME), while secreted CD200 inhibits the priming of antigen-specific CD8 T cells in the lymphatic. Notably, NK cells were essential for the initial rejection of CD200 KO gliomas, while CD8 T cells played a critical role in establishing durable anti-tumor responses. Our work provides new mechanistic insights on glioma-derived CD200-mediated immunosuppression and an untapped potential for targeting CD200 by immunotherapies for malignant gliomas.

Emerging Technologies for the Assessment of Natural Killer Cell Activity

Understanding natural killer (NK) cell functionality is essential in developing more effective immunotherapeutic strategies that can enhance patient outcomes, especially in the context of cancer treatment. This review provides a comprehensive overview of both traditional and novel techniques for evaluating NK cell functionality, focusing on multiparameter assays and spatial methods that illuminate NK cell interactions within their microenvironment. We discuss the significance of standardized assays for assessing NK cell function across various research and clinical settings, including cancer immunotherapy, infectious diseases, and transplantation. Key factors influencing NK cell functionality include the origin of the sample, target–effector ratios, the functional state of NK cells, and the impact of pre-treatment conditions and their natural aging effect on NK cell activity. By emphasizing the importance of selecting a suitable technique for reliable measurements, especially for longitudinal monitoring, this review aims to give an overview on techniques to measure NK cell functionality in vitro and show the interaction with their microenvironment cells by spatial imaging. Ultimately, our understanding of NK cell functionality could be critical to biomarker development, drug design, and understanding of disease progression in the field of oncology or infectious disease.

TLR3 activation enhances antitumor effects of sorafenib in hepatocellular carcinoma by activating NK cell functions through ERK and NF-κB pathways

Background Sorafenib is a standard therapeutic agent for advanced hepatocellular carcinoma (HCC). However, its efficacy is moderate, as the survival of patients is prolonged for only a few months, and the response rate is low. The mechanism of low efficacy remains unclear. In this study, we investigated the effect of Toll-like receptor 3 (TLR3) on the effects of sorafenib on HCC. Methods Polyinosinic-polycytidylic acid [poly(I: C)] was used as a double-stranded RNA analog and TLR3 agonist in subsequent experiments. After orthotopic implantation of HCC tumors in BALBc nu/nu or C57BL/6 mice, survival time, tumor growth, and metastasis in the abdomen and lungs were analyzed. Flow cytometry and cytotoxicity assays were used to analyze NK cells isolated from the spleen or peripheral blood. ELISA was used to detect the expression of plasma interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1. In addition, the expression of phosphorylated-extracellular regulated kinase 1/2 (pERK1/2), phosphorylated-protein kinase B (pAKT), ERK1/2 and AKT was analyzed by Western blotting. Results Sorafenib reduced the number and activity of NK cells in tumor-bearing mice and simultaneously decreased the levels of MCP-1 and IFN-γ in the plasma. The combination of sorafenib and poly(I: C) synergistically inhibited tumor growth and metastasis in tumor xenograft mice and prolonged survival. Poly(I: C) not only exerts a direct inhibitory effect on tumor growth and metastasis by targeting the TLR3 receptor on tumor cells but also facilitates the proliferation and activation of NK cells, indirectly impeding tumor progression. Mechanistically, poly(I: C) decreased the sorafenib-induced inhibition of ERK phosphorylation and increased the phosphorylation of IκB in NK cells, thereby enhancing NK cell function. Conclusion Activation of TLR3 can enhance the antitumor effect of sorafenib on HCC. The combination of a TLR3 activator and sorafenib may be a new strategy for the treatment of HCC.

Homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung natural killer cells

Natural killer (NK) cells are important innate immune effector cells for controlling tumor growth and metastasis. Differentiated mature NK cells preferentially reside in the peripheral tissues and express higher levels of self-major histocompatibility complex class I (MHC-I)-recognizing inhibitory receptors. MHC-I recognition by NK cells are known to be important for their development and maturation processes, however, the role of homeostatic MHC-I recognition in maintaining effector functions of mature NK cells in the peripheral tissues needs to be elucidated. In this study, we utilized a pan anti-MHC-I blocking monoclonal antibody (anti-MHC-I) to examine the role of homeostatic MHC-I recognition in the response of pulmonary mature NK cells in an experimental lung metastasis model of B16F10 melanoma. Anti-MHC-I treatment showed significant inhibition of the lung metastasis of B16F10 melanoma in NK cell- and IFN-γ-dependent mechanisms. The blockade of homeostatic MHC-I recognition increased mature lung NK cell responsiveness, such as direct cytotoxicity and IFN-γ production, rather than the number of lung NK cells. Mechanistically, the gene expression of activating receptors including DNAX accessory molecule-1 (DNAM-1) was upregulated in NK cells treated with anti-MHC-I, and further the enhanced NK cell cytotoxicity against B16F10 cells was DNAM-1-dependent. Collectively, homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung NK cells by restraining the expression of activating receptors.

Preclinical ex vivo IL2RG gene therapy using autologous hematopoietic stem cells as an effective and safe treatment for X-linked severe combined immunodeficiency disease

X-linked severe combined immunodeficiency disease (X-SCID) is a rare inherited disease caused by mutations in the interleukin 2 receptor subunit gamma gene (IL2RG), which encodes the common γ chain protein, a subunit of the receptor for lymphocytes. X-SCID is characterized by profound defects in T-cell, B-cell, and natural killer cell function. Here, we report a Chinese cohort of nine X-SCID patients with six novel IL2RG mutations. Among those, the two adolescent patients carrying missense mutation with an atypical immunotype were confirmed by further analyzing IL-2-JAK-STAT5 signaling, T cell proliferation, and T cell receptor excision circles (Trecs). Interestingly, Bacillus Calmette-Guérin (BCG) disease occurred commonly in this cohort. Although allogeneic hematopoietic stem-cell transplantation is curative for the disease, it is not available to all patients due to the lack of suitable matched donors. Autologous gene therapy using a self-inactivating lentiviral vector (SIN-LV) technology, developed in recent decades has provided an alternative therapy for treating such mono-genetic diseases. Here, we performed the pre-clinical studies to assess our SIN-LV carrying IL2RG on human ED7R cells deficient in IL2RG and CD34+ stem cells derived from the bone marrow of a healthy donor and a patient with X-SCID. This work is done complied with the established "Good Manufacturing Practice" (GMP) used in the clinical trials. In addition, a safety study is performed using the transduced CD34+ cells implanted into the axilla of nude mice in vivo. Overall, our studies have demonstrated the efficiency and safety of SIN-IL2RG-LV, which paves the way for conducting X-SCID gene therapy clinical trials in China in the near future.

Metabolic programs drive function of therapeutic NK cells in hypoxic tumor environments.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

Inhibition of Cbl-b restores effector functions of human intratumoral NK cells

BackgroundT cell-based immunotherapies including immune checkpoint blockade and chimeric antigen receptor T cells can induce durable responses in patients with cancer. However, clinical efficacy is limited due to the ability of cancer cells to evade immune surveillance. While T cells have been the primary focus of immunotherapy, recent research has highlighted the importance of natural killer (NK) cells in directly recognizing and eliminating tumor cells and playing a key role in the set-up of an effective adaptive immune response. The remarkable potential of NK cells for cancer immunotherapy is demonstrated by their ability to broadly identify stressed cells, irrespective of the presence of neoantigens, and their ability to fight tumors that have lost their major histocompatibility complex class I (MHC I) expression due to acquired resistance mechanisms.However, like T cells, NK cells can become dysfunctional within the tumor microenvironment. Strategies to enhance and reinvigorate NK cell activity hold potential for bolstering cancer immunotherapy.MethodsIn this study, we conducted a high-throughput screen to identify molecules that could enhance primary human NK cell function. After compound validation, we investigated the effect of the top performing compounds on dysfunctional NK cells that were generated by a newly developed in vitro platform. Functional activity of NK cells was investigated using compounds alone and in combination with checkpoint inhibitor blockade. The findings were validated on patient-derived intratumoral dysfunctional NK cells from different cancer types.ResultsThe screening approach led to the identification of a Casitas B-lineage lymphoma (Cbl-b) inhibitor enhancing the activity of primary human NK cells. Furthermore, the Cbl-b inhibitor was able to reinvigorate the activity of in vitro generated and patient-derived dysfunctional NK cells. Finally, Cbl-b inhibition combined with T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade further increased the cytotoxic potential and reinvigoration of both in vitro generated and patient-derived intratumoral dysfunctional NK cells.ConclusionsThese findings underscore the relevance of Cbl-b inhibition in overcoming NK cell dysfunctionality with the potential to complement existing immunotherapies and improve outcomes for patients with cancer.

The HLA-B -21 M/T dimorphism associates with disease severity in COVID-19.

Host genetics shape immune responses and influence severity of infectious diseases. The HLA-B -21 M/T dimorphism tunes the functionality of natural killer (NK) cells expressing the inhibitory receptor NKG2A. NKG2A+ NK cells have been reported to recognize SARS-CoV-2-infected cells, but it remains unclear whether the HLA-B -21 M/T dimorphism associates with COVID-19 severity. Here, we investigated the influence of the HLA-B -21 M/T dimorphism in a cohort of 230 unvaccinated patients hospitalized with COVID-19 and requiring respiratory support. We found that HLA-B -21 M/M genotypes were more prevalent in patients with moderate compared to severe COVID-19 (6.0% vs. 0.9%). Comparison of age- and sex-matched sub-groups revealed that patients with M/M genotypes required mechanical respiratory support less frequently (OR = 0.13, 95% CI = 0.01-0.76, P = 0.013). Furthermore, patients with M/M genotypes showed a coordinately shifted signature of clinical laboratory parameters, coinciding with elevated serum levels of the anti-viral cytokine IFN-γ. These findings demonstrate that HLA-B variants associate with COVID-19 severity and suggest that the robust functionality of NKG2A+ NK cells in patients carrying the M/M genotype may contribute to protection from severe disease.

Crucial immunological roles of the invasion front in innate and adaptive immunity in cervical cancer.

The immunostimulatory actions of innate and adaptive immune responses play a crucial role in the cancer-immunity cycle. Although cervical cancer (CC) exhibits a high recurrence rate, the relation with lymphocytes in the tumor tissue have not been analyzed. We analyzed NKT, NK, and T cells, not only in peripheral blood (PB), but also tumor tissue through histological analysis from 23 patients with CC collected before treatment. A correlation of them between PB and the tumor tissue were assessed. We detected functional NKT and NKG2Dhi NK cells and effector CD4+ Tregs in PB. In the tumor, we detected the infiltration of LAG-3+ TIM-3+ CD4+ and CD8+ T cells rather than NK cells particularly in the invasion front (IF) by fluorescent multiplex immunohistochemistry. The heatmap and correlation analysis revealed that LAG-3+ TIM-3+ CD8+ T cells are highly associated with CD69+ CD103- exhausted CD8+ T cells. We identified the statistical relationship between CD4+Tregs in PB and the number of LAG-3+ TIM-3+ CD4+ T cells in the IF, which may be related to recurrence in patients with CC. LAG-3+ TIM-3+ T cells located in the IF may play a key role in regulation of the tumor immune microenvironment.

Single-cell RNA sequencing uncovers molecular mechanisms of intravenous immunoglobulin plus methylprednisolone in Kawasaki disease: attenuated monocyte-driven inflammation and improved NK cell cytotoxicity.

Intravenous immunoglobulin (IVIG) plus methylprednisolone as initial intensive therapy or additional therapy in Kawasaki disease (KD) has been used in clinical practice. However, its molecular and cellular mechanism is unclear. We performed single-cell analysis on 14 peripheral blood mononuclear cell (PBMC) samples obtained from 7 KD patients who received either IVIG monotherapy or IVIG plus methylprednisolone therapy. This encompassed 4 samples from KD patients collected before and after IVIG treatment, as well as 3 samples from KD patients before and after IVIG plus methylprednisolone therapy. Both IVIG monotherapy and IVIG plus methylprednisolone therapy can increase lymphocyte counts (e.g. CD4+T, CD8+T, and gdT cells) to address lymphopenia. They can also decrease monocyte counts and repress the expression of S100A12, NLRP3, and genes associated with immune-cell migration in monocytes. IVIG combined with methylprednisolone downregulates more monocyte-driven inflammatory pathways than IVIG alone. Additionally, this combination uniquely enhances NK cell cytotoxicity by modulating receptor homeostasis, while significantly upregulating interferon-related genes in CD4+ T cells, CD8+ T cells, and B cells, particularly type I interferons. The combination of IVIG with methylprednisolone attenuated monocyte-driven inflammation and improved NK cell cytotoxicity which might provide clues for pediatricians to consider treatment options for children with KD. Whether the monocyte-driven hyperinflammatory state and NK cell function can be indicators for the clinical choice of IVIG with methylprednisolone therapy in KD needs further investigation.

Intra- and Extrahepatic Cholangiocarcinomas Display Differing Sensitivities to NK Cell Lysis and Modulate NK Cell Function Through Shared and Distinct Pathways.

Cholangiocarcinoma (CCA) is a rare cancer that arises from the bile duct and is broadly classified by the location of the tumor as either intrahepatic (iCCA) or extrahepatic (eCCA). Immunotherapy has revolutionized cancer treatment, yet its utility in CCA has been limited as the tumor microenvironment (TME) in CCA is poorly understood compared to other common cancers. Utilizing previously published transcriptome data, our re-analysis has revealed that CCA has one of the highest relative levels of natural killer (NK) cells, a potent cytotoxic immune cell, compared to other cancers. However, despite iCCA and eCCA having comparable relative levels of NK infiltration, NK cell infiltration only correlated with survival in eCCA patients. Our subsequent investigation revealed that while iCCA and eCCA profoundly altered NK activity, eCCA had a significantly reduced impact on NK functionality. Whereas iCCA was resistant to long-term NK co-culture, eCCA was markedly more sensitive. Moreover, while both iCCA and eCCA dysregulated key NK activating receptors, eCCA co-culture did not impact NKp30 nor NKp44 expression. Furthermore, tumor transcriptome analysis of NKHigh CCA samples revealed modulation of multiple immune and non-immune cell types within the TME. Implications: These studies are the first to investigate how iCCA and eCCA impact NK cell functionality through shared and distinct mechanisms and how elevated NK cell infiltration could shape the CCA TME in a subtype-dependent manner.

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.

Biologically-informed killer cell immunoglobulin-like receptor gene annotation tool.

Natural killer (NK) cells are essential components of the innate immune system, with their activity significantly regulated by Killer cell Immunoglobulin-like Receptors (KIRs). The diversity and structural complexity of KIR genes present significant challenges for accurate genotyping, essential for understanding NK cell functions and their implications in health and disease. Traditional genotyping methods struggle with the variable nature of KIR genes, leading to inaccuracies that can impede immunogenetic research. These challenges extend to high-quality phased assemblies, which have been recently popularized by the Human Pangenome Consortium. This article introduces BAKIR (Biologically informed Annotator for KIR locus), a tailored computational tool designed to overcome the challenges of KIR genotyping and annotation on high-quality, phased genome assemblies. BAKIR aims to enhance the accuracy of KIR gene annotations by structuring its annotation pipeline around identifying key functional mutations, thereby improving the identification and subsequent relevance of gene and allele calls. It uses a multi-stage mapping, alignment, and variant calling process to ensure high-precision gene and allele identification, while also maintaining high recall for sequences that are significantly mutated or truncated relative to the known allele database. BAKIR has been evaluated on a subset of the HPRC assemblies, where BAKIR was able to improve many of the associated annotations and call novel variants. BAKIR is freely available on GitHub, offering ease of access and use through multiple installation methods, including pip, conda, and singularity container, and is equipped with a user-friendly command-line interface, thereby promoting its adoption in the scientific community. BAKIR is available at github.com/algo-cancer/bakir.