Defective NK Cell Research Articles

Infections in Disorders of Immune Regulation.

Primary immune regulatory disorders (PIRDs) constitute a spectrum of inborn errors of immunity (IEIs) that are primarily characterized by autoimmunity, lymphoproliferation, atopy, and malignancy. In PIRDs, infections are infrequent compared to other IEIs. While susceptibility to infection primarily stems from antibody deficiency, it is sometimes associated with additional innate immune and T or NK cell defects. The use of immunotherapy and chemotherapy further complicates the immune landscape, increasing the risk of diverse infections. Recurrent sinopulmonary infections, particularly bacterial infections such as those associated with staphylococcal and streptococcal organisms, are the most reported infectious manifestations. Predisposition to viral infections, especially Epstein-Barr virus (EBV)-inducing lymphoproliferation and malignancy, is also seen. Notably, mycobacterial and invasive fungal infections are rarely documented in these disorders. Knowledge about the spectrum of infections in these disorders would prevent diagnostic delays and prevent organ damage. This review delves into the infection profile specific to autoimmune lymphoproliferative syndrome (ALPS), Tregopathies, and syndromes with autoimmunity within the broader context of PIRD. Despite the critical importance of understanding the infectious aspects of these disorders, there remains a scarcity of comprehensive reports on this subject.

Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV‐1 pathogenesis

Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV‐1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL‐2, and IL‐15, and inhibited by TGFB1, a cytokine increased in people living with HIV‐1. In HIV‐1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL‐6. NK‐cell knockout of the TGFB1‐stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV‐1 viremic people, and anti‐inflammatory gene MYDGF was increased in an NK‐cell subset from HIV‐1‐infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV‐1 correlated inversely with ILC percentage and CD4+ T‐cell counts. CD4+ T cells and their production of IL‐2 prevented the loss of NK‐cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV‐1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.

NK cell defects: implication in acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is a complex disease with rapid progression and poor/unsatisfactory outcomes. In the past few years, the focus has been on developing newer therapies for AML; however, relapse remains a significant problem. Natural Killer cells have strong anti-tumor potential against AML. This NK-mediated cytotoxicity is often restricted by cellular defects caused by disease-associated mechanisms, which can lead to disease progression. A stark feature of AML is the low/no expression of the cognate HLA ligands for the activating KIR receptors, due to which these tumor cells evade NK-mediated lysis. Recently, different Natural Killer cell therapies have been implicated in treating AML, such as the adoptive NK cell transfer, Chimeric antigen receptor-modified NK (CAR-NK) cell therapy, antibodies, cytokine, and drug treatment. However, the data available is scarce, and the outcomes vary between different transplant settings and different types of leukemia. Moreover, remission achieved by some of these therapies is only for a short time. In this mini-review, we will discuss the role of NK cell defects in AML progression, particularly the expression of different cell surface markers, the available NK cell therapies, and the results from various preclinical and clinical trials.

Outcomes of PD-1 Inhibitors Treatment of Chronic Active Epstein Barr Virus Infection : A Single Center Retrospective Analysis

INTRODUCTION Chronic active Epstein-Barr virus (CAEBV) disease is a high-mortality disease, which is characterized by persistent infectious mononucleosis-like symptoms. There is no standard treatment of CAEBV and allogeneic hematopoietic stem cell transplantation (HSCT) was considered as an only potentially therapeutic approach. PD-1 inhibitors have achieved high response in many Epstein-Barr virus-related diseases. In this single-center retrospective analysis, we report the outcomes of PD-1 inhibitors treatment of CAEBV. METHODS Sixteen CAEBV patients were treated with PD-1 inhibitors between 6/1/2017 and 12/31/2021. Hemophagocytic lymphohistiocytosis (HLH) was excluded. Patients received Nivolumab or Sindilizumab therapy every 3 to 4 weeks, 100 to 200 mg intravenously. Clinical and laboratory assessments were performed on patients before each infusion during treatment. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. RESULTS Among the sixteen patients with a median age at onset of 33 years (range, 11-67 years), twelve patients responded to PD-1 inhibitors, and their median duration of relapse-free survival (RFS) was 10.3 months (range, 1.0-54.8 months). Three of them achieved clinical complete response (clinical CR), as well as molecular CR, and nine patients achieved partial response (PR) (Figure 1). Four of the nine PR patients converted from PR to no response (NR), and five remained in PR. The remaining four patients did not respond to PD-1 inhibitor therapy. One patient eventually died from a severe lung infection and one patient died of severe liver failure. The median time and cycles from the first application of PD-1 inhibitor to clinical CR in three patients with clinical CR were 43 days (range, 30-72 days) and 3 cycles (range, 2-4 cycles). Patient 14 suffered immune-related pancreatitis (Grade 2, CTCAE 5.0) after 6 cycles of PD-1 inhibitor therapy. Four patients developed persistent or intermittent high fever after the first infusion of PD-1 inhibitors. Other than that, no treatment-related adverse events have been observed. There was no correlation of treatment outcome with blood count, aspartate aminotransferase (AST) level, alanine aminotransferase (ALT) level, LDH level, cytokine level or ferritin level. Comparing the NK cell function of responders and non-responders, the test results showed higher numbers of patients with defective NK cell function among non-responders. The immunohistochemical analysis showed that patients with high PD-L1 expression in tumor tissue may have a more significant response to PD-1 inhibitors. Genetic testing indicated that, except for 1 patient without gene sequencing and 8 patients with no related gene mutation detected, the other 7 patients were detected with potentially pathogenic or susceptibility mutations. CONCLUSION In patients with CAEBV, PD-1 inhibitors have tolerable toxicity and comparable outcomes while improving quality of life and financial toxicity. Larger prospective studies and longer follow-up time is needed to be conducted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The RNA helicase DHX15 is a critical regulator of natural killer-cell homeostasis and functions.

The RNA helicase DHX15 is widely expressed in immune cells and traditionally thought to be an RNA splicing factor or a viral RNA sensor. However, the role of DHX15 in NK-cell activities has not been studied thus far. Here, we generated Dhx15-floxed mice and found that conditional deletion of Dhx15 in NK cells (Ncr1CreDhx15fl/fl mice) resulted in a marked reduction in NK cells in the periphery and that the remaining Dhx15-deleted NK cells failed to acquire a mature phenotype. As a result, Dhx15-deleted NK cells exhibited profound defects in their cytolytic functions. We also found that deletion of Dhx15 in NK cells abrogated their responsiveness to IL-15, which was associated with inhibition of IL-2/IL-15Rβ (CD122) expression and IL-15R signaling. The defects in Dhx15-deleted NK cells were rescued by ectopic expression of a constitutively active form of STAT5. Mechanistically, DHX15 did not affect CD122 mRNA splicing and stability in NK cells but instead facilitated the surface expression of CD122, likely through interaction with its 3'UTR, which was dependent on the ATPase domain of DHX15 rather than its splicing domain. Collectively, our data identify a key role for DHX15 in regulating NK-cell activities and provide novel mechanistic insights into how DHX15 regulates the IL-15 signaling pathway in NK cells.

Cytokine-induced natural killer cell training is dependent on cellular metabolism and is defective in obesity

AbstractNatural killer (NK) cells are a population of innate immune cells that can rapidly kill cancer cells and produce cytokines such as interferon-γ. A key feature of NK cells is their ability to respond without prior sensitization; however, it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be trained to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study, we investigated if cellular metabolism is required during the initial period of NK cell cytokine training and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and oxidative phosphorylation during the initial cytokine activation period is essential for NK cell training, as is the metabolic signaling factor Srebp. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

Defective NK cell expansion, cytotoxicity, and lack of ability to differentiate tumors from a pancreatic cancer patient in a long term follow-up: implication in the progression of cancer.

The majority of the previous reports on NK cells use cross-sectional studies to establish the status of patient NK cell function, however such studies fail to evaluate the immune status of the patients on a continuous basis from the disease-free stage to progression of cancer. In this study, we performed a prospective study of the immune function by continuously monitoring the NK numbers, expansion and function of a pancreatic cancer patient from 1/6/2016 to 2/14/2019. The results indicated that at initial stages of the disease where no overt disease was identified, the patient had consistently higher percentages of NK and B cells and lower percentages of CD3 + T cells in the peripheral blood. The percentages of CD14 + monocytes were similar at the initial stages of the disease, and at the later stages of the disease, it increased and remained higher in the patient when compared to those from healthy donors. The numbers of expanded NK cells and the cytotoxic function, as well as secretion of IFN-γ from primary and osteoclast expanded patient NK cells remained consistently low throughout the years of follow up. Similarly, the majority of cytokines in patient's serum remained lower with the exception of IL-6 which was higher. The IFN-γ secreted from the patients' NK cells had much lower ability to differentiate the poorly differentiated oral tumors as assessed by their lack of ability to upregulate differentiation antigens. Overall, before any evidence of overt disease, patient NK cells exhibited significant dysfunction. Intervention at the stage of no disease or minimal disease may be important for the prevention of pancreatic cancer progression.

Low Natural Killer (NK) Cell Counts and Functionality Are Associated With Molecular Relapse After Imatinib Discontinuation In Patients (pts) With Chronic Phase (CP)-Chronic Myeloid Leukemia (CML) With Undetectable BCR-ABL Transcripts For At Least 2 Years: Preliminary Results From Immunostim, On Behalf Of STIM Investigators

BackgroundIn pts with CP-CML, the notion that tyrosine kinase inhibitors (TKI) may never be stopped is currently challenged in the setting of clinical trials. In the pioneering French STIM trial, pts on imatinib for at least 3 years who had achieved and maintained undetectable BCR-ABL transcripts for at least 2 years had a 24-month molecular relapse-free survival of 39% with no relapses beyond 24 months. A low Sokal risk group and a long-lasting imatinib treatment were significantly associated with an increased freedom from molecular relapse but the role of other factors such as cellular immunity cannot be excluded. The aim of IMMUNOSTIM performed on a subset of 51 pts enrolled in the STIM trial was to analyze the potential implication of NK-cell and T-cell immunity in molecular relapse-free survival after imatinib discontinuation. MethodsPeripheral blood was drawn prior to and after imatinib cessation. All analyses were performed in a centralized fashion. Blood cell counts and differentials were determined using an automated blood cell count analyzer. Flow cytometry was performed on fresh cells to quantify CD56dim and CD56bright NK cells, CD3+ T cells, naïve and memory CD4+ and CD8+ T cells and CD4+ regulatory (CD25+CD127neg/low) T cells (TREGs). Expression of activating and inhibitory NK cell receptors was also analyzed. Functional analyses were performed using frozen mononuclear cells. The focus was put on NK cell function by means of a CD107a degranulation assay against the K562 cell line and an IFN-g secretion assay upon IL-12 and IL-18 stimulation. ResultsStudied pts' characteristics were comparable to that of the entire STIM study population. Median age was 63 years, 64.7% of pts were females and 52.9% has received prior IFN. Sokal risk group was low in 54.9%, intermediate in 35.3%, high in 7.8% and unknown in 2%. Median imatinib treatment duration was 57 months (36-94). Median follow-up after imatinib discontinuation was 42 months (24-54). A molecular relapse occurred in 28 pts after a median time off therapy of 2 months (1-20). Median BCR-ABL/ABL transcript ratio at relapse confirmation was 0.015% IS (0.006-1.546). Molecular relapse-free survival at 24 months was 47.1% (95% CI: 31.4-58.7). A significantly higher proportion of low low Sokal risk group and a trend toward longer imatinib treatment duration were found in pts who did not relapse compared to those who relapsed.At baseline, median leucocyte and lymphocyte counts were 4820/mm3 (2550-7860) and 1310/mm3 (720-2610), respectively and were comparable in pts who relapsed and those who did not. Median CD3+, CD4+, CD4+ TREGs, CD8+ T cells and CD4/CD8 ratio were 879/mm3 (315-1949), 534/mm3 (228-1248), 37/mm3 (4-94), 279/mm3 (72-998) and 2 (0.5-14.2), respectively, with no significant difference between pts who relapsed and those who did not. Analysis of CD4+ and CD8+ naïve, effector and central memory T cells did not reveal any differences between the 2 groups.In contrast, NK cell counts at imatinib cessation were significantly lower in pts who relapsed than in those who did not relapse (145/mm3 (67-450) versus 233/mm3 (70-727), p=0.0146). This decrease specifically affected the cytotoxic CD56dim population. NK cells numbers did not significantly increase in either group after imatinib discontinuation.NK receptor expression analysis did not show any significant defect in the expression of the activating NKG2D receptor and the CD94, NKG2A and KIR inhibitory receptors. However, the cytotoxic activity of CD56dim NK cells toward K562 and the IFN-g secretion potential of CD56bright NK cells upon IL-12 and IL-18 stimulation were significantly decreased when compared to those of healthy donors. Analyses are ongoing to further dissect NK cell function in pts who relapsed and those who did not. Updated results will be presented. ConclusionsOur stud shows a specific association between peripheral blood NK cells and clinical outcome after imatinib discontinuation in pts with long-lasting molecularly undetectable residual disease. These results suggest that NK cell-based immune surveillance may contribute to CML control after TKI cessation. Whether NK cells may be used as biomarkers among other factors to predict disease relapse after TKI cessation needs to be investigated. The understanding of NK cell defects may help in building strategies to restore an optimal NK cell-mediated anti-leukemic activity. Disclosures:Rea:BMS, Novartis, Pfizer, Ariad, Teva: Honoraria. Nicolini:Novartis, Ariad and Teva: Consultancy; Novartis & Bristol Myers Squibb: Research Funding; Novartis, BMS, Teva, Pfizer, Ariad: Honoraria; Novartis, BMS, Teva: Speakers Bureau; Novartis, Ariad, Teva, Pfizer: Membership on an entity's Board of Directors or advisory committees. Rousselot:BMS, Ariad, Pfizer: Honoraria from BMS, Ariad, Pfizer. Grants from BMS Other.

Epstein–Barr virus-associated smooth muscle tumors in patients with primary immunodeficiencies

Epstein–Barr virus-associated smooth muscle tumors (EBV-SMTs) are rare soft tissue neoplasms that typically occur in conditions of secondary immunodeficiency due to human immunodeficiency virus or immunosuppression after organ transplantation. Beyond that, EBV-SMTs occur in patients with primary immunodeficiencies (PIDs). EBV-SMTs pathogenesis is still unclear but it was shown that the key mechanism of the tumor development is T- and NK-cell defect. Treatment strategy depends not only on tumor localization and resectability, but also on immunodeficiency etiology and its correction possibility. Here we report literature review and two patients with combined PIDs (CARMIL2- and ATM-genes deficiency) who developed EBV-SMT. In each case, the parents gave consent to the use of their child's data, including photographs, for research purposes and in publications.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Notch signaling defects in NK cells in patients with cancer.

Altered expressions of proto-oncogenes have been reported during normal lymphocytes mitogenesis and in T and B lymphocytes in patients with autoimmune diseases. We have recently demonstrated a significantly decreased expression of c-kit and c-Myc in NK cells isolated from patients with cancer, which might be related to the functional deficiency of NK cells in the tumor environment. Here, focusing on the regulatory mechanisms of this new clinical phenomenon, we determined expression of c-Myc, Notch1, Notch2, p-53, Cdk6, Rb and phosphorylated Rb in NK cells isolated from the healthy donors and cancer patients. The results of our study revealed a significant down-regulation of expression of Notch receptors and up-regulation of Cdk6 expression in NK cells in cancer, while no significant changes in the expression of p53 and Rb proteins were seen. These data revealed novel signaling pathways altered in NK cells in the tumor environment and support further investigation of the origin of deregulated expression of proto-oncogenes in NK cells patients with different types of cancer.

P104 Defective metabolism mediates NK cell dysfunction in inflammatory bowel disease (IBD)

Abstract Background Cell metabolism, in addition to providing energy and biochemical building blocks, also regulates immune cell function. We have increased our understanding of how natural killer cells (NK cell) metabolism impacts human NK cell function; however, little is known about NK cell role and metabolic changes in autoimmune diseases. The dysfunctional NK cell phenotype observed in pathological settings linked to defective cell metabolism will give insights into the development of new therapies for inflammatory diseases. IBD is an autoimmune disease of unknown origin characterised by severe gut inflammation and increased risk of colorectal cancer. NK cells can augment autoimmune responses through their activation of adaptive immune response by interaction with macrophages, dendritic cells and T cells; therefore, it is essential to decipher NK cells function in IBD to propose novel therapeutic targets. In this study, we defined the phenotype and metabolic profile of NK cells isolated from peripheral blood of IBD patients. Methods Blood samples were obtained from healthy donors and IBD patients from whom written consent had been obtained. PBMC were isolated by Lymphoprep (Axis-Shield) gradient. PBMC were stained with a combination of cell surface receptors, intracellular cytokines, proteins and analysed by flow cytometry. Cells were either stained ex vivo or after overnight stimulation with cytokine combination IL-12 + IL-15 and in the presence or absence of mTORC1 inhibitor rapamycin. Changes in OxPhos or glycolysis were detected by XFp extracellular flux analyser (Agilent Technologies). Mitochondria mass and polarisation were measured by flow cytometry utilising MitoTracker Green and MitoTracker Red, respectively (ThermoFisher). Results NK cells from IBD patients have impaired mTORC1 activity (a key signalling pathway regulating cell metabolism), which is associated with increased expression of pro-inflammatory cytokines IL-17 and TNF-α. Patients NK cells have dysfunctional mitochondria characterised by reduced mitochondria mass, low membrane potential and limited OXPHOS and glycolysis. Furthermore, patients NK cells have limited IFNγ production, reduced TRAIL expression and defective capability of killing cancer cells. Conclusion Taken together, our results strongly imply that NK cells from IBD patients have a distinct bioenergetic programme that favours the increased production of pro-inflammatory cytokines. This might be an important unknown defect of NK cells in the regulation of inflammation in IBD that can be used for the development of new treatments. Further studies need to be conducted to identify the reason for the observed defects and determination of other key metabolic pathways involved in NK-cell function during IBD.

Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rβ and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.

NK cell defects in X-linked pigmentary reticulate disorder.

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3–CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.

ICOS deficiency hampers the homeostasis, development and function of NK cells.

Signaling through the inducible costimulator ICOS is required for the homeostasis and function of various immune cell populations, with an outstanding role in the generation and maintenance of germinal centers. Very recently, it has been suggested that the clinical phenotype of ICOS-deficient patients is much broader than initially anticipated and the innate immune response might be also affected. However, the role of the ICOS/ICOS-Ligand axis in the homeostasis and development of innate NK cells is not known, and reports on its participation in NK cell activation are scarce. NK cells may express low levels of ICOS that are markedly enhanced upon activation. We show here that ICOS-deficient (ICOS-KO) mice present low NK cell numbers and defects in the homeostasis of these cells, with delayed maturation and altered expression of the developmental NK cell markers CD122, NK1.1, CD11b or CD27. Our experiments in mixed bone marrow chimera mice indicate that, both, cell-intrinsic defects of ICOS-KO NK and deficiencies in the milieu of these mice contribute to the altered phenotype. ICOS-deficient NK cells show impaired production of IFN-γ and cytotoxicity, and a final outcome of defects in NK cell-mediated effector function during the response to poly(I:C) or vaccinia virus infection in vivo. Interestingly, we show that murine innate cells like IL-2-cultured NK and bone marrow-derived dendritic cells can simultaneously express ICOS and ICOS-Ligand; both molecules are functional in NK intracellular signaling, enhancing early phosphorylation of Akt and Erk, or IFN-γ secretion in IL-2-activated NK cells. Our study shows the functional importance of the ICOS/ICOS-L pair in NK cell homeostasis, differentiation and activity and suggests novel therapeutic targets for NK manipulation.

Synergistic activity between IL-15 signaling and checkpoint blockade drives enhanced NK cell expansion and tumor control in ovarian cancer

Abstract Survival in ovarian cancer patients has not improved in the past 40 years, stressing a need for new lines of therapy. NK cell immunotherapy in ovarian cancer has not been extensively explored due to limited data showing defective NK cell function in these patients. Our previous work shows that an IL-15 super-agonist (NANT N-803 (formerly ALT-803)) can bypass some of the deficiencies. However, we show here that though N-803 enhances NK cell activation, it also induces NK PD-1 expression and inflammatory cytokines secreted by the NK cells induce PD-L1 expression on ovarian tumors. To bypass this negative feedback we combined N-803 with an anti-PD-L1 antibody (Avelumab) and show a potent increase in NK cell activation and long-term real-time in vitro tumor killing. Furthermore, combined N-803/Avelumab induces robust killing of ovarian cancer tumor spheroids, not seen with individual treatments. To evaluate the benefits of this combined treatment in vivo we tested them in a xenogeneic model of ovarian cancer containing human ovarian cancer cells (MA148lucs or OVCAR8lucs) and human NK cells. Our data clearly shows reduced tumor burden, by bioluminescent imaging, and potently increased NK cell expansion with the combined treatment when compared to individual treatments. Since Avelumab is capable of driving antibody dependent cell-mediated cytotoxicity (ADCC) we wanted to evaluate the impact of checkpoint signals in the synergy excluding ADCC using an anti-PD-1 antibody (Pembrolizumab). Combination of N-803 with Pembrolizumab yielded similar tumor control and NK cell expansion in the in vivo model, indicating that the synergy lies in checkpoint signals and IL-15 signaling, and not just in amplification of ADCC via N-803.

Defective Localization With Impaired Tumor Cytotoxicity Contributes to the Immune Escape of NK Cells in Pancreatic Cancer Patients.

Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although majority of TILs consist of CD8+/CD4+ T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients' NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients.

Multiple defects in NK cells of surgically resectable pancreatic cancer patients can be reversed by ex-vivo stimulation

Abstract Tumor infiltrating lymphocytes (TILs) are found in advanced pancreatic ductal adenocarcinoma (PDAC) patients and shown to correlate with overall survival (OS) rate. Although majority of TILs consisted of CD8+/CD4+T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL and PBMC to explore the role of NK cells in the newly developed PDAC patients. Multiple defects in cell migration and effector function were observed in NK cells of patients’ peripheral blood. Extremely low frequencies of NK cells (&amp;lt;0.5%) were found in TILs of PDAC patients which was attributable not to low expression of tumor chemokines, but rather lack of the CXCR2 chemokine receptor. Forced expression of CXCR2 on patients’ NK cells rendered them capable of trafficking PDAC tumors. Furthermore, patients’ NK cells exhibited impaired cell-mediated killing against autologous PDAC tumor cells and failed to proliferate within hypoxic tumor microenvironment. Importantly, these defects can be overcome by ex-vivo activation of NK cells from PDAC patients. When the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, a clear difference in DFS and OS was observed. Patients whose NK cells proliferated &amp;lt;250 fold experienced significantly lower DFS and OS than those expanded ≥250 fold. Multiple defects are observed in NK cells of PDAC patients. Ex-vivo activation of NK cells restores tumor reactivity and provides a therapeutic modality while their fold expansion can be a potentially significant prognostic indicator of OS and DFS in PDAC patients.

Primary Antibody Deficiencies: Scientific Discoveries Leading to Challenging Paradigms in a Complex Disease Landscape

Primary antibody deficiencies (PADs) are the most common primary immunodeficiencies in humans that affect both children and adults. The patients display a decrease in IgG and/or IgA serum levels associated with lower, normal or higher IgM levels, which cause an increased susceptibility to bacterial but also viral infections. Main other complications are auto-immunity, lymphoproliferation and increased susceptibility to various types of cancers. PADs can be caused by B intrinsic and/or extrinsic defects leading to impaired early B cell development, B cell migration, B cell survival, B cell activation or defects in Immunoglobulin class switch recombination (CSR). Among the latter (B cell activation- and CSR-deficiencies), some PADs are linked to unbalanced signaling pathways, in which both gain of function and loss of function variations lead to different immune defects, pinpointing the requirement of a tightly controlled activity for proper immune response.1) Activated PI3K-d Syndromes (APDS) are due to dominant mutations in either the PIK3CD gene encoding the p110d protein, the catalytic subunit of phosphoinositide 3 kinase d (PI3Kd) or the PIK3R1 gene encoding the regulatory subunit of phosphoinositide 3 kinase p85a. Both gene defects lead to hyperactivation of PI3K through an increased p110δ function. A frequent hyper-IgM phenotype is observed, likely due to both a T and a B cell defect. Of note, impaired or loss of function of p110δ or p85α subunit of PI3K leads to a completely different phenotype characterized by an autosomal recessive agammaglobulinemia. The main life-threatening complication of APDS is B lymphoma occurrence. Targeted therapy aiming to decrease PI3K activation is currently on trial.2) The importance of the canonical NF-kB signaling for B cell function was firstly highlighted by the description that missense hemizygous mutations in the IKBKG gene, encoding NEMO, the NF-kB essential modulator, cause an X-linked CSR-deficiency associated with hyphohydrotic ectodermal dysplasia. Recently, heterozygous mutations in either the NFKB1 or the NFKB2 genes (encoding respectively for p105 processed to p50 and p100 processed to p52), have been reported to be causative for common variable immunodeficiency (CVID) due to haploinsufficiency of NF-êkB1 (p52) or NF-kB2 (p50), with less nuclear NF-kB accumulation and less transcriptional regulation. Some carriers are asymptomatic, pinpointing to incomplete penetrance, that suggests the role of gene modifyers or environmental factors. Although NF-κB1-deficiency affects mostly B cells, NF-kB2- deficiency combines a T, B and NK cell defect and is frequently associated to pituitary hormone deficiency.CARD11 (caspase recruitment domain containing protein 11)-deficiency (caused by loss of function bi-allelic mutations) also indicated the importance of CARD11 mediated canonical NF-kB1 signaling for B and T cell function since CARD11-deficient patients have a CVID phenotype. Interestingly, heterozygous gain of function mutations in CARD 11 lead to a different phenotype, the so-called BENTA syndrome (“B cell expansion with NF-kB and T cell anergy”) characterized by B cell lymphocytosis, lymphoproliferation and immunodeficiency.These examples of PADs show that gain of function, haploinsufficiencies and loss of function mutations can be deleterious by disturbing the delicate balance required for adequate signaling pathways during the immune response. DisclosuresNo relevant conflicts of interest to declare.

Effect of Myeloma-Derived Exosomes on Surface Activating Receptors of NK Cells

To investigate the effect of myeloma-derived exosomes on surface activating receptors of NK cells, and to explore the mechanism of the function defect of NK cells. The exosomes from the supernatant of multiple myeloma cell lines RPMI8226 and U266 were extracted by ultracentrifugation, and the size of them was identified under electron microscope; the human primary NK cells were extracted, and were co-cultured with the myeloma-derived exosomes (40 µg/ml), then the expression levels of surface activating receptors NKp46, NKp30 and NKG2D of NK cells at 0,1,4 and 24 hours were detected by flow cytometry. The exosomes showed small vesicular, sized 30-100 nm under electron microscope. The expression of surface activating receptors of NK cells declined at different degree after co-cultured with myeloma-derived exosomes. Myeloma-derived exosomes can inhibit the expression of surface activating receptors of NK cells.