NK Cell Expression Research Articles

Biologically informed machine learning modeling of immune cells to reveal physiological and pathological aging process

The immune system undergoes progressive functional remodeling from neonatal stages to old age. Therefore, understanding how aging shapes immune cell function is vital for precise treatment of patients at different life stages. Here, we constructed the first transcriptomic atlas of immune cells encompassing human lifespan, ranging from newborns to supercentenarians, and comprehensively examined gene expression signatures involving cell signaling, metabolism, differentiation, and functions in all cell types to investigate immune aging changes. By comparing immune cell composition among different age groups, HLA highly expressing NK cells and CD83 positive B cells were identified with high percentages exclusively in the teenager (Tg) group, whereas unknown_T cells were exclusively enriched in the supercentenarian (Sc) group. Notably, we found that the biological age (BA) of pediatric COVID-19 patients with multisystem inflammatory syndrome accelerated aging according to their chronological age (CA). Besides, we proved that inflammatory shift- myeloid abundance and signature correlate with the progression of complications in Kawasaki disease (KD). The shift- myeloid signature was also found to be associated with KD treatment resistance, and effective therapies improve treatment outcomes by reducing this signaling. Finally, based on those age-related immune cell compositions, we developed a novel BA prediction model PHARE (https://xiazlab.org/phare/), which can apply to both scRNA-seq and bulk RNA-seq data. Using this model, we found patients with coronary artery disease (CAD) also exhibit accelerated aging compared to healthy individuals. Overall, our study revealed changes in immune cell proportions and function associated with aging, both in health and disease, and provided a novel tool for successfully capturing features that accelerate or delay aging.

PP2A negatively regulates NK cell T-bet expression and anti-tumor effector function.

The transcription factor T-bet is essential for the anti-tumor effector function of NK cells, but the mechanism regulating its expression in NK cells remains unclear. In this study, we aimed to identify an NK cell intrinsic regulator that controls T-bet expression. Using T-bet-luciferase reporter assay screening, we identified a protein phosphatase inhibitor as a potential activator of T-bet expression. A series of PP2A-specific inhibitors (PP2Ai) or PP2A siRNA induced the expression of T-bet. In PP2Ai-treated mice, the expressions of T-bet and its downstream effector molecules, granzyme B and IFN-γ, were also upregulated in NK cells. Mechanistically, PP2Ai increased the phosphorylation of mTOR and ribosomal protein S6 in NK cells, and mTOR inhibitor canceled the effects of PP2Ai in NK cells. Importantly, NK cells isolated from PP2Ai-treated mice showed higher cytotoxicity and IFN-γ production; therefore, they increased the anti-tumor effector function of NK cells. Accordingly, PP2Ai treatment inhibited lung metastasis of B16 melanoma by NK cell- and mTOR-dependent mechanisms. These results suggest that PP2A negatively regulates NK cell T-bet expression and effector function by an mTOR-dependent mechanism.

Soluble MICA concentrations and genetic variability of MICA and its NKG2D receptor as factors affecting Graft-versus-Host Disease development after allogeneic haematopoietic stem cell transplantation

Despite new treatment strategies, graft-versus-host disease (GvHD) remains a formidable complication after allogeneic hematopoietic stem cell transplantation (HSCT). This study aimed to investigate the impact of polymorphisms and expression of MICA and NKG2D receptor on the development of GvHD in allogeneic HSCT recipients. Soluble MICA (sMICA) concentration was measured in serum collected 30 days after transplantation and the genetic variability of MICA and NKG2D genes was evaluated. The frequency of NKG2D+NK cells was determined by flow cytometry before and (21, 30, 60 and 90 days) after transplantation. Recipients with acute GvHD grades II-IV carried the NKG2D rs1049174 C allele more frequently than controls or patients with no or mild disease. Patients with chronic GvHD had higher frequency of NKG2D expressing NK cells posttransplant, reflecting increased activity of their NK cells. Although no direct relationship between MICA SNPs and GvHD were observed, the presence of MICA rs1051792 GG genotype correlated with elevated sMICA levels and increased serum level of sMICA was associated with higher risk of chronic GvHD. Our findings suggest that sMICA concentration may serve as a potential biomarker for chronic GvHD and emphasize the impact of genetic variability of NKG2D and its surface expression on the HSCT outcome.

Proteasome inhibition enhances the anti-leukemic efficacy of chimeric antigen receptor (CAR) expressing NK cells against acute myeloid leukemia

BackgroundRelapsed and refractory acute myeloid leukemia (AML) carries a dismal prognosis. CAR T cells have shown limited efficacy in AML, partially due to dysfunctional autologous T cells and the extended time for generation of patient specific CAR T cells. Allogeneic NK cell therapy is a promising alternative, but strategies to enhance efficacy and persistence may be necessary. Proteasome inhibitors (PI) induce changes in the surface proteome which may render malignant cells more vulnerable to NK mediated cytotoxicity. Here, we investigated the potential benefit of combining PIs with CAR-expressing allogeneic NK cells against AML.MethodsWe established the IC50 concentrations for Bortezomib and Carfilzomib against several AML cell lines. Surface expression of class-I HLA molecules and stress-associated proteins upon treatment with proteasome inhibitors was determined by multiparameter flow cytometry. Using functional in vitro assays, we explored the therapeutic synergy between pre-treatment with PIs and the anti-leukemic efficacy of NK cells with or without expression of AML-specific CAR constructs against AML cell lines and primary patient samples. Also, we investigated the tolerability and efficacy of a single PI application strategy followed by (CAR-) NK cell infusion in two different murine xenograft models of AML.ResultsAML cell lines and primary AML patient samples were susceptible to Bortezomib and Carfilzomib mediated cytotoxicity. Conditioned resistance to Azacitidine/Venetoclax did not confer primary resistance to PIs. Treating AML cells with PIs reduced the surface expression of class-I HLA molecules on AML cells in a time-and-dose dependent manner. Stress-associated proteins were upregulated on the transcriptional level and on the cell surface. NK cell mediated killing of AML cells was enhanced in a synergistic manner. PI pre-treatment increased effector-target cell conjugate formation and Interferon-γ secretion, resulting in enhanced NK cell activity against AML cell lines and primary samples in vitro. Expression of CD33- and CD70-specific CARs further improved the antileukemic efficacy. In vivo, Bortezomib pre-treatment followed by CAR-NK cell infusion reduced AML growth, leading to prolonged overall survival.ConclusionsPIs enhance the anti-leukemic efficacy of CAR-expressing allogeneic NK cells against AML in vitro and in vivo, warranting further exploration of this combinatorial treatment within early phase clinical trials.

Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease

X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations invitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ Tcells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

Potential role of B- and NK-cells in the pathogenesis of pediatric aplastic anemia through deep phenotyping.

Pediatric patients with unexplained bone marrow failure (BMF) are often categorized as aplastic anemia (AA). Based on the accepted hypothesis of an auto-immune mechanism underlying AA, immune suppressive therapy (IST) might be effective. However, due to the lack of diagnostic tools to identify immune AA and prognostic markers to predict IST response together with the unequaled curative potential of hematopoietic stem cell transplantation (HSCT), most pediatric severe AA patients are momentarily treated by HSCT if available. Although several studies indicate oligoclonal T-cells with cytotoxic activities towards the hematopoietic stem cells, increasing evidence points towards defective inhibitory mechanisms failing to inhibit auto-reactive T-cells. We aimed to investigate the role of NK- and B-cells in seven pediatric AA patients through a comprehensive analysis of paired bone marrow and peripheral blood samples with spectral flow cytometry in comparison to healthy age-matched bone marrow donors. We observed a reduced absolute number of NK-cells in peripheral blood of AA patients with a skewed distribution towards CD56bright NK-cells in a subgroup of patients. The enriched CD56bright NK-cells had a lower expression of CD45RA and TIGIT and a higher expression of CD16, compared to healthy donors. Functional analysis revealed no differences in degranulation. However, IFN-γ production and perforin expression of NK-cells were reduced in the CD56bright-enriched patient group. The diminished NK-cell function in this subgroup might underly the auto-immunity. Importantly, NK-function of AA patients with reduced CD56bright NK-cells was comparable to healthy donors. Also, B-cell counts were lower in AA patients. Subset analysis revealed a trend towards reduction of transitional B-cells in both absolute and relative numbers compared to healthy controls. As these cells were previously hypothesized as regulatory cells in AA, decreased numbers might be involved in defective inhibition of auto-reactive T-cells. Interestingly, even in patients with normal distribution of precursor B-cells, the transitional compartment was reduced, indicating partial differentiation failure from immature to transitional B-cells or a selective loss. Our findings provide a base for future studies to unravel the role of transitional B-cells and CD56bright NK-cells in larger cohorts of pediatric AA patients as diagnostic markers for immune AA and targets for therapeutic interventions.

Immune profiling and functional analysis of NK and T cells in ataxia telangiectasia.

Ataxia telangiectasia (AT) is a rare autosomal-recessive disorder characterized by profound neurodegeneration, combined immunodeficiency, and an increased risk for malignant diseases. Treatment options for AT are limited, and the long-term survival prognosis for patients remains grim, primarily due to the emergence of chronic respiratory pathologies, malignancies, and neurological complications. Understanding the dysregulation of the immune system in AT is fundamental for the development of novel treatment strategies. In this context, we performed a retrospective longitudinal immunemonitoring of lymphocyte subset distribution in a cohort of AT patients (n = 65). Furthermore, we performed FACS analyses of peripheral blood mononuclear cells from a subgroup of 12 AT patients to examine NK and T cells for the expression of activating and functional markers. We observed reduced levels of peripheral blood CD3+CD8+ cytotoxic T cells, CD3+CD4+ T helper cells, and CD19+ B cells, whereas the amount of CD3--CD56+ NK cells and CD3+CD56+ NKT-like cells was similar compared with age-matched controls. Notably, there was no association between the age-dependent kinetic of T-, B-, or NK-cell counts and the occurrence of malignancy in AT patients. Additionally, our results indicate an altered NK- and T-cell response to cytokine stimulation in AT with increased levels of TRAIL, FasL, and CD16 expression in NK cells, as well as an elevated activation level of T cells in AT with notably higher expression levels of IFN-γ, CD107a, TRAIL, and FasL. Together, these findings imply function alterations in AT lymphocytes, specifically in T and NK cells, shedding light on potential pathways for innovative therapies.

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

Comprehensive genomic and transcriptomic profiling of pulmonary nodules in synchronous multiple primary lung cancer.

e20022 Background: Synchronous Multiple Primary Lung Cancer (sMPLC) exhibits distinct histopathological characteristics among pulmonary nodules. However, a comprehensive understanding of the somatic mutation landscape and transcriptome heterogeneity is lacking. Therefore, our study aims to meticulously investigate genomic distinctions among multiple pulmonary nodules within individual patients. Methods: We performed targeted sequencing on tumor specimens and conducted bulk RNA transcriptome analysis on 53 multiple nodules originating from 26 lung cancer patients. The multiple nodules from the same patient was determined as major nodule and minor nodule. Additionally, the tumor tissues underwent histopathological evaluation through H&E staining, complemented by a comprehensive series of immunohistochemistry (IHC) analyses to detect protein expression. The detected protein markers encompassed PD-L1, Ki67, and others. Results: For the 53 nodule samples from 26 MPLCs patients, EGFR was the mostly mutated genes, and the TP53 mutation frequency demonstrated notably different between major and minor nodules. Furthermore, pathway enrichment analysis based on the differentially expressed genes (DEGs) between major and minor nodules revealed the significantly active cell cycle and p53 pathways in the major nodules. Additionally, both major and minor nodules demonstrated mostly similar immune microenvironment and PD-L1 protein expression, and a significantly higher expression of Ki67. A noteworthy suppression was observed in the immune microenvironment in nodules, revealed by the expression of macrophage, neutrophils, and NK cells. Furthermore, minor nodules exhibited a modestly elevated expression of macrophages compared to major nodules. Additionally, among the significantly up-regulated cell cycle related genes in the major nodules when compared with minor nodules, CCNE1 mRNA expression demonstrated significant correlation with poor prognosis in the lung cancer. Conclusions: This study validated molecular distinctions between samples from major and minor nodules in patients with sMPLC at both genomic and transcriptomic levels. The major nodules exhibited heightened activity in tumor cell proliferation pathways and demonstrated malignancy-related biological characteristics, which correlated with pathological assessment results.

Genetic mutations and immune microenvironment: unveiling the connection to AML prognosis

ABSTRACT Background: This study aims to investigate the correlation between NK and NKT cell proportion disparities and prognosis in patients with acute myeloid leukemia (AML). Methods: Forty-four cases of acute myeloid leukemia patients were selected, and flow cytometry was utilized to evaluate the expression of bone marrow NK and NKT cells. Next-generation sequencing technology was employed to detect genetic mutations in these 44 AML patients, and the rates of first induction remission and overall survival were recorded. Comparisons were made to analyze the respective differences in NK and NKT cell proportions among AML patients with various genetic mutations and risk stratifications. Results: The FLT-3-ITD+ group exhibited a significant increase in the proportion of NK cells compared to the normal control group and FLT3-ITD+/NPM1+ group, whereas the proportion of NKT cells was significantly decreased. Additionally, the CEBPA+ group showed an increased proportion of NKT cells compared to the TP53+ group and ASXL1+ group. The high-risk group had a higher proportion of NK cells than the intermediate-risk group, while the proportion of NKT cells was lower in the high-risk group compared to the intermediate-risk group.Patients achieving first induction remission displayed a higher proportion of NKT cells at initial diagnosis compared to those who did not achieve remission. The distribution of NK cells show significant differences among AML patients in different survival periods. Conclusion: This results implies that distinct genetic mutations may play a role not only in tumor initiation but also in shaping the tumor microenvironment, consequently impacting prognosis.

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.

Immunological Similarities and Differences between Post-COVID-19 Lung Sequelae and Idiopathic Pulmonary Fibrosis.

Pulmonary fibrosis is an irreversible condition that may be caused by known (including viral triggers such as SARS-CoV-2) and unknown insults. The latter group includes idiopathic pulmonary fibrosis (IPF), which is a chronic, progressive fibrosing interstitial pneumonia of unknown cause. The longer the insult acts on lung tissue, the lower the probability of a complete resolution of the damage. An emerging clinical entity post-COVID-19 is pulmonary fibrosis (PCPF), which shares many pathological, clinical, and immunological features with IPF. The fibrotic response in both diseases-IPF and PCPF-is orchestrated in part by the immune system. An important role regarding the inhibitory or stimulatory effects on immune responses is exerted by the immune checkpoints (ICs). The aim of the present study was to analyse the similarities and differences between CD4+, CD8+, and NK cells in the peripheral blood of patients affected by fibrotic disease, IPF, and PCPF compared with sarcoidosis patients and healthy controls. The second aim was to evaluate the expression and co-expression of PD-1 and TIGIT on CD4, CD8, and NK cells from our patient cohort. One hundred and fifteen patients affected by IPF, PCPF, and sarcoidosis at the rare pulmonary disease centre of the University of Siena were enrolled. Forty-eight patients had an IPF diagnosis, 55 had PCPF, and 12 had sarcoidosis. Further, ten healthy controls were enrolled. PCPF patients were included between 6 and 9 months following hospital discharge for COVID-19. The peripheral blood samples were collected, and through flow cytometric analysis, we analysed the expression of CD4, CD8, NK cells, PD-1, and TIGIT. The results show a greater depletion of CD4 and NK cells in IPF patients compared to other groups (p = 0.003), in contrast with CD8 cells (p < 001). Correlation analysis demonstrated an indirect correlation between CD4 and CD8 cells in IPF and sarcoidosis patients (p < 0.001 = -0.87 and p = 0.042; r = -0.6, respectively). Conversely, PCPF patients revealed a direct correlation between CD4 and CD8 cells (p < 0.001; r = 0.90) accentuating an immune response restoration. The expression of PD-1 and TIGIT was abundant on T and NK cell subsets of the two lung fibrotic groups, IPF and PCPF. Analogously, the co-expression of PD-1 and TIGIT on the surfaces of CD4 and CD8 were increased in such diseases. Conclusions: Our study shines a spotlight on the immune responses involved in the development of pulmonary fibrosis, idiopathic and secondary to SARS-CoV-2 infection. We observed a significant imbalance not only in CD4, CD8, and NK blood percentages in IPF and PCPF patients but also in their functional phenotypes evaluated through the expression of ICs.

Lower NKG2D expression in hepatic natural killer cells predicts poorer prognosis for chronic hepatitis B patients with cirrhosis

ObjectiveNatural killer cells (NK) acts a central player of the immune system in liver cirrhosis. The aim of this study was to examine the expression of activating intra-hepatic NK cell group 2D (NKG2D) in patients with chronic hepatitis B (CHB) and analyzed the correlation between NKG2D expression and prognosis of liver cirrhosis in these patients. MethodsThis was a cross-section study. Subjects with liver biopsy or sponge hemangioma surgery were included. The primary outcome was the NKG2D expression on intra-hepatic NK cells and their subtype cells in patients with CHB-related liver cirrhosis. Subsequently, the correlation of expression of NKG2D and clinical characteristic indicators were assayed ResultsAmong 38 subjects, 11 (28.95%) normal liver sections adjacent the sponge hemangioma (healthy group) were collected during surgery, and 27 (71.05%) CHB-cirrhosis tissues (Cirrhosis group) were preserved after liver biopsy. Compared with healthy group, sections from cirrhosis group revealed more severe inflammation and collagen deposition and lower NKG2D expression in hepatic NK cells. The proportion of hepatic NK cells and the mean fluorescence intensity (MFI) of NKG2D on hepatic NK cells showed a positive correlation with serum albumin (Alb) level, platelet (Plt) count. Moreover, they had a significantly negative correlation with patient prothrombin time (PT), international standardized ratio (INR), the sirius red positive stained area and fibrosis stages. ConclusionsLower NKG2D expression in intra-hepatic NK cells may be predictive of poorer prognosis of CHB patients with cirrhosis.

Low Percentage of Perforin-Expressing NK Cells during Severe SARS-CoV-2 Infection: Consumption Rather than Primary Deficiency.

Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.

IL-15 Complex-Induced IL-10 Enhances Plasmodium-specific CD4+ T Follicular Helper Differentiation and Antibody Production.

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. Although humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure to Plasmodium parasites and correlates with the acquisition of humoral immunity. Despite the established role Abs play in protection from malaria disease, dysregulated inflammation is thought to contribute to the suboptimal immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4+ T cells, in this study we demonstrate that IL-15C treatment enhances T follicular helper (Tfh) differentiation and modulates cytokine production by CD4+ T cells. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG Ab levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation during Plasmodium infection to promote Tfh differentiation and Ab generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.

CS1 Expression Pattern in NK Cells and Correlated Factors in Plasma Cell dyscrasias: Implications for Elotuzumab Therapy and CAR-T Efficacy.

Treatment with elotuzumab alone has no discernible antitumor effect and progress in chimeric antigen receptor T cells (CAR-T) therapy targeting CS1 is relatively slow. A retrospective analysis was performed on 236 patients with multiple myeloma (MM) and 30 patients with other plasma cell dyscrasias (PCDs). CS1 expression in NK cells, lymphocytes, and monoclonal plasma cells was assessed using multiparameter flow cytometry. Furthermore, new explorations were undertaken regarding the antitumor applications of elotuzumab. Patients with MM had significantly higher CS1 expression levels in plasma cells than other patients with PCDs, with no significant differences between lymphocytes and NK cells. In both patients with MM and other PCDs, CS1 expression was significantly higher in plasma cells than in NK cells and lymphocytes. Univariate and multivariate analyses revealed a significant correlation between CS1 expression in plasma (r = 0.60; P < 0.001) and NK (r = 0.79; P < 0.001) cells. Factors such as cytogenetic abnormalities, disease progression, and survival were not associated with CS1 expression in NK cells. Moreover, this study showed that elotuzumab strongly increases the cytotoxicity of NK cells against non-plasma and plasma tumor cells independent of their CS1 expression level. This underscores the potential of elotuzumab in combination with NK cells as an effective therapeutic strategy against a broad spectrum of tumor types.

Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity

Background aimsNatural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. MethodsUsing wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry–based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. ResultsMean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. ConclusionsTogether, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

Patterns of CS1 Expression on Natural Killer Cells and Correlated Factors in Plasma Cell Dyscrasias

Abstract BackgroundCS1 is overexpressed on myeloma cells, making it a promising target for immunotherapy in plasma cell dyscrasias (PCDs). However, monoclonal antibody (mAb) targeting CS1 showed limited efficacy when used alone. In addition, few clinical data of anti-CS1 chimeric antigen receptor T cell (CAR-T) have been published to date, suggesting that there may be some barriers to CS1-targeted immunotherapy. From previous studies, CS1 is expressed on some normal lymphocytes and plays an immunomodulatory role, and NK cells in particular play an important role in CS1-mediated immunity against MM. However, there are few studies on CS1 expression by NK cells in patients with plasma cell disease. Therefore, we investigated the expression of CS1 on NK cells, normal lymphocytes, and plasma cells in plasma cell dyscrasias and examined factors related to the CS1 expression level of NK cells and its relationship to survival. Methods Between January 2018 and September 2019, a retrospective analysis was performed in 236 patients with multiple myeloma (MM) and 30 patients with other PCDs, including systemic light chain amyloidosis (AL), monoclonal gammopathy of undetermined significance (MGUS), POEMS syndrome, and monoclonal gammopathy of renal significance (MGRS). CS1 expression levels in NK cells, lymphocytes, and monoclonal plasma cells were assessed in patients with plasma cell dyscrasias. Membrane-bound CS1 of these three cell types was measured by multiparameter flow cytometry. Associations of CS1 expression with clinical and laboratory features were determined by univariate and multivariate analyses. Correlations between CS1 expression and survival were computed using the Cox proportional hazards regression and by plotting Kaplan-Meier survival plots. Results When subgroups of MM and other PCDs were compared, there were no significant differences in CS1 expression in lymphocytes and NK cells alone. MM Patients had significantly higher CS1 expression levels in monoclonal plasma cells than other PCDs patients. In both MM and other PCDs patients, CS1 expression was significantly higher on monoclonal plasma cells than on NK cells and lymphocytes. There was a significant correlation between CS1 expression of monoclonal plasma cells (r = 0.60; P &amp;lt; 0.001) and that of NK cells (r = 0.79; P &amp;lt; 0.001) on univariate and multivariate analysis(Figure 1). Factors such as cytogenetic abnormalities, disease progression, or survival were not associated with CS1 expression level on NK cells. Neither CS1 expression level of monoclonal plasma cells nor that of NK cells was an independent predictor of patient survival. Conclusion We demonstrated that CS1 expression level in NK cells is consistent in plasma cell dyscrasias. CS1 expression in NK cells positively correlates with that in clonal plasma cells. This positive correlation may have implications for the development of immunotherapy targeting CS1.

Targeting Aberrantly Activated Glycolysis in Natural Killer T Cell Lymphomas

Background and aims: With advances in the diagnosis and treatment of malignant lymphoma, improvements in long-term survival have been achieved for most common lymphoma types. However, natural killer/T-cell lymphoma (NKTL), which is rare in Europe and North America (0.3%) but relatively common in Asia and Latin America, has been left behind as a subtype of lymphoma with a high relapse rate and a poor prognosis. In particular, the outcome of patients with advanced or recurrent NKTL is dismal, with an overall survival of only a few months. This is mainly attributed to the intrinsic resistance of NKTL cells to anthracycline due to the high expression of P-glycoprotein. Molecular-targeted drugs used for other hematological malignancies, such as anti-CD30 and anti-CD52 monoclonal antibodies, have only limited effects on NKTL, and conversely, these drugs can cause severe immunodeficiency and severe autoimmune diseases, often with a fatal course. The development of NKTL-specific and effective molecular-target therapies is expected to improve the prognosis of NKTL. Methods: We performed a gene expression array targeting genes involved in glucose cell metabolism to compare the gene signatures of NK cell lines (NKL, NK92, YT, and KYHG1) and freshly isolated NK cells from three healthy donors (FNK). Results: The expression of various glucose metabolism genes was higher in NK cell lines than in FNK cells, and in particular, the PKM gene was highly enriched in NK cell lines. The PKM gene encodes the pyruvate kinase muscle isozyme 2 (PKM2), which is responsible for ATP production within the glycolytic sequence and plays an essential role in aerobic glycolysis (the Warburg effect), a dominant metabolic pathway used by cancer cells. Immunohistochemistry studies revealed high PKM2 protein expression in 17 out of 20 tumor tissues derived from patients with extranodal NK/T cell lymphoma. Treatment of NKL, NK92, YT, and KYHG1 cells with Benserazide or Shikonin, two known PKM2 inhibitors, robustly induced cell apoptosis in a time- and dose-dependent manner and inhibited colony formation in in vitro clonogenic assays. Western blotting studies showed a dose-dependent inhibition of STAT1 and STAT3 in NK cell lines exposed to PKM2 inhibitors. A colorimetric assay showed that PKM2 glycolytic activity correlated with the PKM2 gene expression in NK cells, with significantly higher glycolytic activity in malignant NK cell lines than in normal FNK cells. Further mechanistic studies showed that in normal FNK cells, PKM2 is mainly in the tetrameric form, where glucose is converted to pyruvate to generate ATP. In contrast, in malignant NK cell lines, PKM2 is mainly expressed in its dimeric form, with a low affinity for pyruvate and a higher affinity for glycolytic intermediates that promote the synthetic processes and thereby lead to persistent cell proliferation. Conclusions: These results substantiate the importance of aerobic glycolysis in malignat NK cells and indicate that targeting this pathway by blocking PKM2 activity has therapeutic potential for NKTL.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P &amp;lt; 0.05; BBz.IL21 vs. BBz, P &amp;lt; 0.05; BBz.IL15 vs. BBz, P &amp;gt; 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.