Cytotoxic Function Of NK Cells Research Articles

Cancer-associated fibroblasts impair the cytotoxic function of NK cells in gastric cancer by inducing ferroptosis via iron regulation

As the predominant immunosuppressive component within the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) inhibit Natural Killer cell (NK cell) activity to promote tumor progression and immune escape; however, the mechanisms of cross-talk between CAFs and NK cells in gastric cancer (GC) remain poorly understood. In this study, we demonstrate that NK cell levels are inversely correlated with CAFs abundance in human GC. CAFs impair the anti-tumor capacity of NK cells by inducing ferroptosis, a cell death process characterized by the accumulation of iron-dependent lipid peroxides. CAFs induce ferroptosis in NK cells by promoting iron overload; conversely, decreased intracellular iron levels protect NK cells against CAF-induced ferroptosis. Mechanistically, CAFs increase the labile iron pool within NK cells via iron export into the TME, which is mediated by the upregulated expression of iron regulatory genes ferroportin1 and hephaestin in CAFs. Moreover, CAF-derived follistatin like protein 1(FSTL1) upregulates NCOA4 expression in NK cells via the DIP2A-P38 pathway, and NCOA4-mediated ferritinophagy is required for CAF-induced NK cell ferroptosis. In a human patient-derived organoid model, functional targeting of CAFs using a combination of deferoxamine and FSTL1-neutralizing antibody significantly alleviate CAF-induced NK cell ferroptosis and boost the cytotoxicity of NK cells against GC. This study demonstrates a novel mechanism of suppression of NK cell activity by CAFs in the TME and presents a potential therapeutic approach to augment the immune response against GC mediated by NK cells.

Acute Exercise Increases NK Cell Mitochondrial Respiration and Cytotoxicity against Triple-Negative Breast Cancer Cells under Hypoxic Conditions.

Triple-negative breast cancer (TNBC) is an aggressive, highly metastatic malignancy with high recurrence rates. Hypoxia is a hallmark of the TNBC tumor microenvironment, which promotes tumor growth while impairing natural killer (NK) cell cytotoxic functions. Although acute exercise improves NK cell function under normoxic conditions, the effect of exercise on NK cell cytotoxic functions under hypoxic conditions mimicking O 2 tensions observed in solid tumors is unknown. The cytotoxic functions of resting and postexercise NK cells isolated from thirteen young inactive healthy women were assessed against breast cancer cells expressing different levels of hormone receptors (MCF-7 and MDA-MB-231) under normoxic and hypoxic conditions. Mitochondrial respiration and H 2 O 2 efflux rates of the TNBC-activated NK cells were assessed via high-resolution respirometry. Under hypoxia, postexercise NK cells exhibited greater killing of TNBC than resting NK cells. Further, postexercise NK cells were more likely to kill TNBC under hypoxia than normoxic conditions. In addition, mitochondrial respiration associated with oxidative (OXPHOS) capacity of TNBC-activated NK cells was greater in postexercise cells than resting cells under normoxia, but not under hypoxia. Finally, acute exercise was associated with reduced mitochondrial H 2 O 2 efflux by NK cells in both conditions. Together, we present crucial interrelationships between hypoxia and exercise-induced changes in NK cell functions against TNBC cells. By modulating their mitochondrial bioenergetic functions, we postulate that acute exercise improves NK cell function under hypoxic conditions. Specifically, NK cell O 2 and H 2 O 2 flow (pmol·s -1 ·million NK cells -1 ) changes in response to 30-min cycling suggest that exercise primes NK cell tumor killing by reducing mitochondrial oxidative stress and, thus, rescuing their function when exposed to harsh hypoxic environments as observed in the microenvironment of breast solid tumors.

CDC45 haploinsufficiency as a novel cause of natural killer cell deficiency

Natural killer cell deficiency (NKD) is a primary immunodeficiency where natural killer cells are the primary lineage affected. Individuals with NKD present with malignancies and susceptibility to viral infection, especially from the Herpesviridae family. Though NKD is rare, four genes that are involved in the CDC45-MCM-GINS (CMG) helicase have been described to result in NKD, including MCM4, GINS1, MCM10, and GINS4. The CMG helicase is critical for eukaryotic replication and plays a critical role in maintaining genomic stability. To our knowledge, CDC45 has never been described as a monogenic cause of NKD. However, here we describe a single damaging heterozygous variant in CDC45 in an individual with consistent defects in the NK cell compartment. This individual has a disruption in the ratio of CD56bright to CD56dim cells, severely decreased frequency of adaptive NK cells, and decreased NK cell cytotoxic function. As previously reported for other helicase variants that cause NKD, we found cell cycle defects, increased DNA damage, and impaired DNA damage repair signaling in patient lymphocytes. The description of CDC45 as a cause of NKD contributes to our understanding of the role of the CMG helicase in NK cell maturation, function, and biology.

Cord blood stem cell-generated KIR+NK cells effectively target leukemia cell lines

Acute lymphoid (ALL) and myeloid leukemia (AML) are known to be invasive and highly lethal hematological malignancies. Because current treatments are insufficient and have a variety of side effects, researchers are looking for new and more effective therapeutic methods. Interestingly, ongoing efforts to find the best approach to optimize NK cell anti-leukemia potential shed light on the successful treatment of cancer. Mature KIR+NK cells ability to remove HLA Class-I deficient cells has been exploited in cancer immunotherapy. Here, we generated KIR+NK cells from cord blood stem cells using IL-2 and IL-15 cytokines. Our finding underlined the importance of KIR expression in the cytotoxic function of NK cells. Taken together, this study presented an effective in vitro method for the expansion and differentiation of KIR+NK cells using cytokines without any feeder cells. Furthermore, the presented culture condition could be useful for the generation of mature and pure NK cells from limited numbers of CD34+ cord blood cells and might be used as a novel method to improve the current state of cancer therapy.

Abstract 1744: STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor

Abstract Interleukin-2 (IL-2) is a potent stimulator of T and NK cell proliferation, survival, and cytotoxic function. High dose IL-2 induces complete responses as a single agent in certain cancers. Its use is limited due to toxicities such as severe hypotension and vascular leak syndrome (VLS). A better understanding of the mechanisms of IL-2 efficacy and toxicity and development of more selective therapies are needed to improve the use of IL-2. Next generation IL-2 therapeutics have been engineered to bias activity towards the dimeric form of the IL-2 receptor, which consists of IL-2Rβ (CD122) and IL-2Rγ (CD132), and away from the high affinity trimeric form of the receptor, which also includes IL-2Rα (CD25). This approach is designed to stimulate NK cells and naïve effector T-cells, which express the dimeric, and not the trimeric, form of the receptor. However, this approach largely misses antigen-activated and tumor-specific T-cells, which have high expression of both CD25 and CD122. In addition, this approach expands peripheral NK cells, which have been shown in mice to cause VLS (1-3).To specifically stimulate antigen-activated CD25+ effector T cells in cancer patients and avoid systemic NK and naïve T cell activation, we have developed a PEGylated, CD25/CD122-selective IL-2 mutein (STK-012) and its mouse surrogate (STK-014). Here we present efficacy and safety of STK-014 compared to wild type IL-2 (wtIL-2) and a CD122/CD132 biased IL-2 (βγIL-2). We also present safety of STK-012 in non-human primates (NHP). STK-012/STK-014 selectively induced STAT5 phosphorylation and proliferation in antigen activated T cells but not in NK cells and naïve T cells. In mice, STK-014 showed reduced toxicity compared to wtIL-2 and βγIL-2. In particular, wtIL-2 and βγIL-2 induced VLS while STK-014 did not. Moreover, STK-014 demonstrated complete responses both as single agent and in combination with a PD-1 antibody in syngeneic tumor models. In general, STK-014 demonstrated improved efficacy compared to wtIL-2 and βγIL-2. STK-014 dramatically increased intratumoral T cells and intratumoral cytotoxic activity compared wtIL-2 and βγIL-2 while avoiding T cell activation in the spleen. STK-014 treatment drastically increased the CD8+ T cell to Treg ratio within the tumor when compared to control tumors (25-fold), but also in comparison to wtIL-2 (2.75-fold) and to βγIL-2 (5.2-fold).In NHP, STK-012 was well tolerated at doses supra-efficacious in mice. Weekly dosing of STK-012 led to continuous elevated serum concentrations, demonstrating selectivity for CD25/122+ T cells. STK-012 selectively induced memory T cell expansion, including CD28+ CD95+ CD8 T cells. In summary, STK-012 avoids IL-2 mediated toxicity and may enable the specific expansion of antigen activated memory T cells in cancer patients, leading to durable tumor response. 1 Peace DJ and Cheever MA. JEM, 1989.2 Gatley MK et al. JI, 1988.3 Assier E et al. JI; 2004. Citation Format: Jan Emmerich, Michele Bauer, Marie Semana, Bhargavi Jayaraman, Sandro Vivona, Scott McCauley, Romina Riener, Rene De Waal Malefyt, Paul-Joseph Aspuria, Deepti Rokkam, Patrick Lupardus, Rob Kastelein, Martin Oft. STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1744.

Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Effects of Dasatinib on the Expansion, Subsets, Receptor Expression and Cytotoxic Function of NK Cells in Vitro

To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

Abstract 3333: EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity

Abstract Purpose: Breg (B regulatory) cells have been implicated in suppression of anti-tumor response in murine and human solid tumors. Prior studies in our laboratory demonstrated that murine EMT-6 breast carcinoma is rejected in B cell deficient mice (BCDM), but not in wild type (WT) mice, due to an enhanced immune response. Tumor infiltrating B cells (TIL-B), and normal splenic B cells co-cultured with EMT-6 (EMT6-B), express LAP/TGF-β, PD-L1, and suppress CD8+ and NK cell proliferation in vitro. Increased CD8+ and NK cell infiltration into the tumor bed is seen in BCDM. BCDM tumor rejection is abrogated by NK depletion. We studied EMT6-B effects on NK cell phenotype, proliferation and cytokine expression in vitro and in vivo. Experimental design: Normal splenic B cells were co-cultured with mitomycin C treated EMT-6, and Breg phenotype, and effects on NK cell phenotype and proliferation and cytokine function were studied in vitro and in vivo. Results: EMT6-B but not WT splenic B cells inhibited CFSE labeled CD49b+ NK cell proliferation in response to IL-15 in dose dependent fashion. EMT-6-B suppressed expression of CD226 and NKG2D activating receptors on NK cells, and increased expression of PD-1 and PD-L1 respectively. EMT6-B suppressed the induction of IFNγ+/CD49b+ NK cells following PMA/ION stimulation as assayed by intracellular flow cytometry (10%±1.2 to 2%±0.5) and also suppressed IL-2 induced induction of IFNγ+/CD49b+ NK cells (70%±5 to 20%±7.8). EMT-6 and CT26 colon carcinoma conditioned B cells, but not C166 endothelial cell conditioned B cells suppressed NK degranulation (IFNγ+/CD107a+ NK cells). EMT6-B inhibited YAC-1 cell induced NK cell degranulation, and killing of YAC-1 targets, more effectively than naïve B cells (40%±6.2 to 18%±5.1) (P=0.05). NK suppression was inhibited by anti-PD-L1, and anti-TGF-β antibody. EMT6-B expressed increased FasL compared to splenic B cells, and induced NK apoptosis as measured by 7AAD/Annexin-V staining. Blocking antibody to FasL decreased NK apoptosis, and restored NK cell proliferation. Infiltrating NK cells in the EMT6 tumor bed demonstrated increased PD-1 compared to splenic and TDLN NK cells, and higher levels of IFN-γ, CD107a, and Granzyme B in BCDM vs. WT mice. EMT6 expressed high levels of CD155 NK inhibitory ligand compared to C166 endothelial cells. The EMT-6 tumor bed contained higher levels of CD19+CD155+ B cells (22% / tumor bed vs 6% / TDLN, vs 9%/ spleen). BCDM expressed higher levels of CD226+/Ki67+ NK cells in spleen compared to WT splenic NK cells. Antibodies to Fas-L, TGF-beta, and PD-L1 partially restored anti-tumor response in WT mice and decreased tumor growth. Conclusions: EMT-6 TIL-B or normal B cells conditioned by EMT-6 coculture acquire an NK suppressive phenotype which inhibits NK cell activation, proliferation, degranulation and cytotoxic function. Targeting of Breg-NK interactions may improve anti-tumor immune response. Citation Format: Yu Zhang, Muhammad Husnain, Zhifen Luo, Mahmood Al Bayati, Joseph D. Rosenblatt. EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3333.

Abstract B96: Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients

Abstract In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns (DAMPs). Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin (CRT) on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). CRT exposure not only correlated with enhanced T cell-dependent antitumor immunity in this setting but also affected the number of circulating NK cells upon restoration of normal hematopoiesis. Here, we report that CRT exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of CRT to stimulate NK cells relies on CD11c+CD14high cells that, upon exposure to CRT, display higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR), and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferons (IFNs). This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed CRT favors NK cell activation in AML patients. Citation Format: Iva Truxova, Lenka Kasikova, Cyril Salek, Michal Hensler, Daniel Lysak, Peter Holicek, Pavla Bilkova, Monika Holubova, Xiufen Chen, Romana Mikyskova, Milan Reinis, Marek Kovar, Barbora Tomalova, Justin Kline, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B96.

Key Aspects of the Immunobiology of Haploidentical Hematopoietic Cell Transplantation.

Hematopoietic stem cell transplantation from a haploidentical donor is increasingly used and has become a standard donor option for patients lacking an appropriately matched sibling or unrelated donor. Historically, prohibitive immunological barriers resulting from the high degree of HLA-mismatch included graft-vs.-host disease (GVHD) and graft failure. These were overcome with increasingly sophisticated strategies to manipulate the sensitive balance between donor and recipient immune cells. Three different approaches are currently in clinical use: (a) ex vivo T-cell depletion resulting in grafts with defined immune cell content (b) extensive immunosuppression with a T-cell replete graft consisting of G-CSF primed bone marrow and PBSC (GIAC) (c) T-cell replete grafts with post-transplant cyclophosphamide (PTCy). Intriguing studies have recently elucidated the immunologic mechanisms by which PTCy prevents GVHD. Each approach uniquely affects post-transplant immune reconstitution which is critical for the control of post-transplant infections and relapse. NK-cells play a key role in haplo-HCT since they do not mediate GVHD but can successfully mediate a graft-vs.-leukemia effect. This effect is in part regulated by KIR receptors that inhibit NK cell cytotoxic function when binding to the appropriate HLA-class I ligands. In the context of an HLA-class I mismatch in haplo-HCT, lack of inhibition can contribute to NK-cell alloreactivity leading to enhanced anti-leukemic effect. Emerging work reveals immune evasion phenomena such as copy-neutral loss of heterozygosity of the incompatible HLA alleles as one of the major mechanisms of relapse. Relapse and infectious complications remain the leading causes impacting overall survival and are central to scientific advances seeking to improve haplo-HCT. Given that haploidentical donors can typically be readily approached to collect additional stem- or immune cells for the recipient, haplo-HCT represents a unique platform for cell- and immune-based therapies aimed at further reducing relapse and infections. The rapid advancements in our understanding of the immunobiology of haplo-HCT are therefore poised to lead to iterative innovations resulting in further improvement of outcomes with this compelling transplant modality.

Altered NK cell receptor repertoire and function of natural killer cells in patients with acute myocardial infarction: A three-month follow-up study.

NK cells are important in the onset of acute myocardial infarction (AMI) by their ability to secrete IFN-γ and other inflammatory cytokines. They also participate in regulating pathological cardiac remodeling after myocardial infarction. Mechanisms of regulation, however, are incompletely understood. Herein, the aim of this study is to explore the possible association between the expression pattern of different NK cell receptors (phenotype), as well as the cytotoxic function of NK cells from AMI patients with their myocardial function after three months follow-up. We analyzed the phenotype and function of both CD56dimCD16+ and CD56brightCD16- NK cells from twenty-one patients within the first 72 h after ST-elevation AMI and three-month follow-up, as well as fifteen healthy controls. Clinical characteristics and ventricular function determined by echocardiography were also evaluated. NK cells from AMI patients showed an activated phenotype, characterized by high TNF-α production and low percentages of the activating receptor NKG2D. Interestingly, AMI patients display higher levels of circulating IL-10+ NK cells. Three-month follow-up showed that NK cells exhibit a diminished cytotoxic function. These data show that NK cells may have a role mediating myocardial remodeling by regulating the inflammatory response, mainly by the production of IL-10. We also propose that NKG2D may have a role in the onset of the inflammatory response immediately after AMI. The precise regulation of NK cells function may represent an important step in recovery of myocardial function.

MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection.

Peste des petits ruminants virus (PPRV) has emerged as a significant threat to the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and induces in the hosts a transient but severe immunosuppression, especially innate immunity. However, it remains largely unknown how NK cells respond and are regulated at the earliest time points after an acute viral PPRV infection in goats. In this study, we revealed that multiple immune responses of goat peripheral NK cells were compromised during PPRV infection, including the cytolytic effector molecule expression and cytokine production. Importantly, we demonstrated that PPRV infection stimulated the expression of TWEAK, a negative regulator of cytotoxic function of NK cells, which may be involved in the suppression of cytotoxicity as well as cytokine production in infected goat NK cells. Furthermore, we found that PPRV infection induced TWEAK expression in goat NK cells involving post-transcription by suppressing miR-1, a novel negative miRNA directly targeting the TWEAK gene. Moreover, replication of virus is required for inhibition of miR-1 expression during PPRV infection, and the non-structural V protein of PPRV plays an important role in miR-1 mediated TWEAK upregulation. Additionally, we revealed that the regulation of NK cell immune responses by TWEAK is mediated by MyD88, SOCS1, and STAT3. Taken together, our results demonstrated that TWEAK may play a key role in regulating goat peripheral NK cell cytotoxicity and cytokine expression levels during PPRV infection.

Descriptive Study of Hemophagocytic Lymphohistiocytosis (HLH) Admissions in Adult Population: Data from Nationwide Inpatient Sample (NIS) Database

Background: Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune activation and dysregulation, characterized by hyperactive macrophages and lymphocytes, resulting in multi-organ damage and high mortality. Epidemiologic data of HLH in the adult population is limited. The aim of our study is to characterize HLH admissions in the US adult population. Methods: We performed a retrospective study using the Nationwide Inpatient Database for the year 2016. 330 admissions with principal diagnosis of HLH in adults were identified, using ICD code specific for HLH. Using ICD 10 codes for malignancies and rheumatologic conditions, we subdivided HLH admissions into malignancy-associated HLH, autoimmune disease-related HLH and non-malignancy, non-autoimmune HLH. Statistical analyses were performed using STATA. One-way ANOVA was used to compare means of continuous variables and chi-square test was used to compare proportions of categorical variables. Results: Mean age of our study group was 50.1, with women making up 44% of the population. 15% of the patients had an underlying malignancy, most common type being mature T/NK cell lymphoma. Also, 15% of the patients had a rheumatologic diagnosis, SLE constituting half of this sub-group. 47% of the total population under study underwent either bone marrow, liver or splenic biopsy. Mortality rate was 21.2% and average length of stay was 17 days. 25.8% received systemic chemotherapy during the admission. Within the subgroups, we did not find a statistically significant difference in the age, gender, race, percentage undergoing biopsy or inpatient chemotherapy. There was also no statistically significant difference in mortality or length of stay between the three groups. Discussion: HLH is a life-threatening condition of immune hyper-activation and may be divided into primary HLH, which is an autosomal recessive condition, caused by mutations impairing the cytotoxic function of NK cells and cytotoxic T lymphocytes. Secondary HLH is the more frequent presentation in adults, and frequently identified triggers include hematologic malignancies, infections and autoimmune conditions. Autoimmune-associated HLH is usually termed as macrophage activation syndrome (MAS). Mortality in secondary HLH varies based on underlying condition, around 8% when associated with JIA, compared to more than 80% in malignancy associated HLH in some studies. Our study sample was underpowered to detect notable differences between the study groups. This can be attributed to the rarity of this condition. HLH is primarily a pediatric disease. Incidence of HLH in the pediatric age group has been estimated to be around 1 in 100,000 children. Incidence in the adult population is less clear. In our study, we were able to represent the epidemiologic aspects of adults with HLH in the real world. We need large observational studies to further characterize HLH in the adult population. Disclosures No relevant conflicts of interest to declare.

NKTR-255, a Polymer-Conjugated IL-15 Enhances Antibody-Dependent Cellular Cytotoxicity Mediated By NK Cells in a B Cell Lymphoma Model

Background IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has great potential as an immunotherapeutic agent for the treatment of cancer. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and poor tolerability. NKTR-255 is a polymer-conjugated IL-15 designed to retain binding affinity to the IL-15 receptor alpha chain and have decreased clearance to provide a sustained pharmacodynamics response. NKTR-255 has an enhanced immunotherapeutic effect when combined with certain targeted monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we further characterized the effect of NKTR-255 on NK cell-mediated ADCC in a B cell lymphoma model. Methods For in vitro ADCC, NKTR-255 pre-treated human NK cells (effector cells) were co-cultured with RPMI-8226 (target cells) and with daratumumab for 3 hours. The ability of NK cells to lyse target cells was evaluated by detecting 7-AAD stained RPMI-8226. In the Daudi lymphoma model, SCID or SCID beige mice were inoculated IV with 1x107 Daudi cells on Day 0. NKTR-255 (0.3 mg/kg IV) was administered on Days 14, 21 and 28 and a single dose of antibody treatment was given on Day 14 (daratumumab 0.5 mg/kg IP). Survival rate was determined by onset of hind limb paralysis or moribundity as surrogate parameters. Concurrent vs staggered regimen assessment was conducted with NKTR-255 dosed on day 14 and daratumumab on Day 14, 17, or 21. For dose examination, high (0.5 mg/kg) and low (0.05 mg/kg) doses of daratumumab were combined with low (0.03 mg/kg) and high (0.3 mg/kg) doses of NKTR-255 in a concurrent manner. Daudi cells in the bone marrow were assessed 10 days after the NKTR-255 dose by flow cytometry. Results In vitro, NKTR-255 pre-treatment of human purified NK cells enhanced daratumumab-mediated ADCC against RPMI-8226. The ability of NKTR-255 to enhance in vitro ADCC was translated into an enhanced therapeutic efficacy of daratumumab in the Daudi lymphoma model with SCID mice. NKTR-255 combined with daratumumab synergistically provided long-term survival benefit in a NKTR-255 dose-dependent manner. To confirm the contribution of NK cells as effector cells for ADCC in vivo, the therapeutic effect of the combination treatment was assessed in the tumor model in SCID beige mice, which have a selective impairment of NK cell cytotoxic function. The long-term survival benefit by the combination treatment, which was observed in SCID mice (median survival of 27 days in control and 59.5 days in treatment), was attenuated in SCID beige mice (median survival of 23 days in control and 30 days in treatment). Concurrent vs staggered dose regimens and dose ranging for NKTR-255 and daratumumab were evaluated in the Daudi model with SCID mice to understand optimal therapeutic use of NKTR-255 in future clinical studies. Tumor depletion in bone marrow was most effective in concurrent treatment of NKTR-255 and daratumumab and three days-staggered treatment (NKTR-255 first and the antibody 3 days later). In contrast, a 7-day stagger showed no benefit of the combination in tumor depletion. Combination of low dose daratumumab and high dose NKTR-255 was equally effective in tumor depletion as a combination of high dose daratumumab and low dose NKTR-255. Conclusions NKTR-255 was shown to enhance in vivo therapeutic efficacy of daratumumab, a monoclonal antibody that mediates tumor killing by ADCC. The effect was mainly mediated by NK cells. The treatment scheduling experiments demonstrated that antibody should be present during NKTR-255-driven NK cell activation/expansion phase to efficiently deplete Daudi tumor cells in bone marrow. In addition, even low dose of daratumumab treatment showed effective tumor depletion when combined with high dose of NKTR-255. This suggests that NKTR-255 has a potential to expand the therapeutic window of existing tumor-targeted antibodies to patients with, for example, low antibody concentration in target tumor site or low expression of tumor antigen. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kivimäe:Nektar Therapeutics: Employment, Equity Ownership. Hennessy:Nektar Therapeutics: Employment, Equity Ownership. Pena:Nektar Therapeutics: Employment, Equity Ownership. Quach:Nektar Therapeutics: Employment, Equity Ownership. Moffet:Nektar Therapeutics: Employment, Equity Ownership. Nieves:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

A point mutation in the linker domain of mouse STAT5A is associated with impaired NK-cell regulation.

The transcription factor STAT5 is critical for peripheral NK-cell survival, proliferation, and cytotoxic function. STAT5 refers to two highly related proteins, STAT5A and STAT5B. In this study, we verified the importance of STAT5A isoform for NK cells. We characterized an incidental chemically induced W484G mutation in the Stat5a gene and found that this mutation was associated with a reduction of STAT5A protein expression. Closer examination of NK-cell subsets from Stat5a mutant mice showed marked reductions in NK-cell number and maturation. IL-15 treatment of Stat5a mutant NK cells exhibited defective induction of both STAT5 and mTOR signaling pathways and reduced expression of granzyme B and IFN-γ. Finally, we observed that Stat5a mutant mice revealed more tumor growth upon injection of RMA-S tumor cell line. Overall, our results demonstrate that the W484G mutation in the linker domain of STAT5A is sufficient to compromise STAT5A function in NK-cell homeostasis, responsiveness, and tumoricidal function.

Abstract 3265: NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model

Abstract Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model. Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter. Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model. Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers. Citation Format: Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3265.

Atypical chronic rhinosinusitis in Papillon syndrome -Lefevre: Presentation of 2 cases

Papillon-Lefebre syndrome (SPL) is an extremely rare autosomal recessive disorder characterized by palmoplantar keratoderma and periodontitis, with an estimated incidence of 1-4 cases per million. Patients with SPL are highly susceptible to infection. The etiology of this susceptibility is unknown; however, an association with defects in neurophilic dysfunction, insufficient response of lymphocytes, defects in monocyte functions and deterioration of cytotoxic function of NK cells has been suggested. The manifestations include oral cavity specifically at the dental level, however at the level of paranasal sinuses there is a singular manifestation little known, as our two cases. We propose the criteria of Lugo cols. for diagnosis

First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

IntroductionChronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue.The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored.MethodsPB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin.ResultsComparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB).We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile.Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment.After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment.ConclusionIn conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. [Display omitted] DisclosuresEldering:Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Pleiotropic Immunosuppressive Role of Osteoclasts in Multiple Myeloma

Skeletal related events impact both quality of life and overall survival of multiple myeloma (MM) patients. The underlying cause of osteolytic bone disease is an imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts. Bone cells are regulated by a complex milieu of bone marrow microenvironmental components including the immune system via the effect of cytokines, signaling molecules and regulatory proteins. For example, osteoclastogenesis is tightly regulated by T cells through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, the effect of osteoclasts (OCs) on the immune system is less well defined.Here we investigated the effect of osteoclasts on immune cells including T cells, Th17 cells, NK cells and myeloid-derived suppressor cells (MDSCs) in MM. To gain insight into the functional impact of OCs on the immune cells, each type of immune cell was isolated from peripheral blood mononuclear cells (PBMNCs) and examined further in co-cultures with OCs. Our preliminary data showed that the frequency of NK cells and MDSCs in PBMNCs derived from myeloma patients increased when co-cultured with autologous OCs. Activation of the inhibitory immune checkpoints suppresses T cell-mediated antitumor immunity. Up-regulation of several co-inhibitory molecules has also been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, LAG-3 and B7-H3, on T cells in co-culture with or without OCs by multi-color flow cytometry. OC co-culture significantly increased PD-1 expression in both CD4+and CD8+ T cell populations. The presence of OCs also enhanced PD-L1 protein expression on MM cells. Th17 cells, a newsubset of helper T cells, have recently been identified as immunosuppressive cells. Th17 cells are increased in myeloma; morevover they secrete IL-17 which promotes myeloma cell growth (Prabhala RH et al., Blood 2010). Interestingly, we found that naïve CD4 T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Furthermore, we also observed an increased expansion of Th17 cells in co-culture with OCs. NK cell cytotoxic function is severely impaired in myeloma (Fauriat C et al., Leukemia. 2006). In our experiments, NK cells co-cultured in the presence of OCs demonstrated significant decrease in NK cell cytotoxicity, despite increase in NK cell numbers.Our study demonstrated that OCs suppress the cytotoxic function of T cells and NK cells and induce the expansion of immunosuppressive Th17 cells and MDSCs through direct contact. These findings indicate that OCs play an important role in tumor progression in part by enhancing immune suppression. Targeting OCs represent an attractive therapeutic option not only for controlling osteolytic bone disease but also for restoring the impaired immune surveillance in MM. Ongoing studies will focus on understanding direct-contact-mediated interactions between OCs and immune cells. Furthermore, the effect of OC inhibition using anti-resorptive drugs on the immune system will be interrrogated. DisclosuresRaje:Medscape: Honoraria; Research to Practice: Honoraria; AstraZeneca: Research Funding; Takeda: Consultancy; Merck: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Amgen Inc.: Consultancy.

Haemophagocytic Lymphohistiocytosis: A Case Report and Short Overview

Haemophogocyticlymphohistiocytosis (HLH) is a rare but potentially fatal disease, which describes a clinical syndrome of hyper-inflammation resulting in uncontrolled and ineffective immune response. It appears commonly in infancy, although it has been seen in all age groups. A vast majority of cases are acquired due to secondary causes (infections, autoimmune, malignancy, metabolic disorders) but primary HLH (genetic) is also not uncommon which also gets triggered by infection as suggested by recent studies. “Hypercytokinemia” which is the hallmark of HLH can result in end organ damage and even death in some cases if there is delay in diagnosis. The pathological hallmark of this syndrome is uncontrolled activation of T lymphocytes and macrophages, together with an impaired cytotoxic function of NK cells and CD8+T lymphocytes resulting into massive cytokine release (e.g. interferon-ã, TNF-á, Interleukin-6, 8, 10, 12, 18) from this cells and overwhelming inflammation. Lymphocytes and macrophages sometimes with haemophagocytic activity accumulate in bone marrow, spleen, liver or lymph nodes. This disorder is characterized by fever, hepatosplenomegaly, lymphadenopathy, skin rash, cytopenias, hepatitis, coagulopathy, and neurological symptoms. We report a case of 55 yr. old male presenting with fever and high colored urine who developed clinical and laboratory findings consistent with diagnosis of HLH according to HLH-2004 guidelines. Unfortunately the patient died despite receiving chemotherapy. HLH has multifaceted clinical presentations with often non-specific signs and symptoms that are often found in other clinical conditions. Early recognition of HLH is critical in initiating therapy early and preventing high mortality resulting from multi-organ failure.J Shaheed Suhrawardy Med Coll, June 2018, Vol.10(1); 51-58