scholarly journals 171 Targeting the Immunomodulatory roles of T-cell immunoglobulin– and mucin domain–containing (TIM)–3 on natural killer cells in glioblastoma

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A182-A182
Author(s):  
Tram Dao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Glucose Metabolism ◽  
Nk Cells ◽  
Cytokine Production ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Metabolism ◽  
Knock Out ◽  
Cytotoxic Function

BackgroundNatural killer (NK) cells have emerged as a viable alternative to T cells in adoptive cell transfer for cancer treatment. NK cell activity is driven by the balance between inhibitory and activating receptors, many of which remain elusive. In addition, NK cell metabolism is also a driver of NK cell fitness in tumor settings, where changes in NK metabolic states with the tumor microenvironment in vivo, or with stimulants ex vivo, further confounds the NK cells’ cytotoxic function in cancer settings. One receptor that lies at the intersection between NK cell function and metabolism is TIM-3, with its expression having consequences on NK cell cytokine production and glucose metabolism. However, the contribution of TIM-3 to NK cell anti-tumor immunity is unclear and its role in driving NK cell function so far not fully defined.MethodsNK cells were isolated from healthy adult peripheral blood and expanded in feeder-cell media. NK cell metabolism and function were evaluated by different flow cytometric assays to measure glucose uptake, cytotoxicity, degranulation, and cytokine production. TIM-3 knock-out cells were generated using the CRISPR-Cas9 system. Patient samples, including whole blood and tumor, were also processed and phenotyped to compare expression level with healthy donor samples.ResultsPreviously, we discovered that TIM-3 downregulation was associated with decreased cytokine production and target cytotoxicity, and that maintenance of expression above a certain threshold was needed for NK cell function. As cytokine production reflects immune cell metabolic state, we hypothesized that TIM-3 participates in regulation of ex vivo-activated NK cell metabolism, which in turn affect the production of the cytokine IFN- γ to sensitize cancer targets to NK cell-mediated lysis. Here, we report the consequences of glucose starvation on TIM-3 expression, and how knock-out of TIM-3 on human NK cells affects NK cell metabolism and functionalities against glioblastoma targets. We also cross-reference TIM-3 expression level with glioblastoma patient samples, which provide clinical context for microenvironmental cues and nutrient deprivation.ConclusionsOur findings suggest that TIM-3 expression is associated with both ex vivo-activated NK cell glucose metabolism and cytotoxic function against glioblastoma. As ex vivo-activated NK cells are considered to be highly glycolytic, and as such associated with higher cytotoxicity, TIM-3’s involvement with glucose uptake could prove crucial in sustaining NK cytotoxic phenotype in the tumor microenvironment. This information is shedding further light on the immunomodulatory roles of TIM-3, and aiding in leveraging this receptor usage in future NK cell-based immunotherapies.Ethics ApprovalAll procedures performed in studies involving human participants were approved by Purdue University’s Institutional Review Board (IRB) in August 2018 (#1804020540). All institutional safety and biosecurity procedures were adhered to.

scholarly journals Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment

2021 ◽  
Vol 12 ◽  
Author(s):  
Natasha Mupeta Kaweme ◽  
Fuling Zhou
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Substantial Improvement ◽  
Effector Cells ◽  
Cell Sources

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

scholarly journals CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

Blood ◽  
2020 ◽  
Vol 136 (21) ◽  
pp. 2416-2427 ◽  
Author(s):  
Meisam Naeimi Kararoudi ◽  
Yuya Nagai ◽  
Ezgi Elmas ◽  
Marcelo de Souza Fernandes Pereira ◽  
Syed Abbas Ali ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Human Monoclonal Antibody ◽  
Metabolic Reprogramming ◽  
Target Cells ◽  
Plasma Cell Neoplasm ◽  
Cd38 Expression ◽  
The Impact

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

scholarly journals The Tumor Microenvironment—A Metabolic Obstacle to NK Cells’ Activity

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3542
Author(s):  
Joanna Domagala ◽  
Mieszko Lachota ◽  
Marta Klopotowska ◽  
Agnieszka Graczyk-Jarzynka ◽  
Antoni Domagala ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Nk Cell ◽  
Metabolic Changes ◽  
Immune Effector ◽  
Effector Cells ◽  
Immune Effector Cells ◽  
Almost All

NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells

2021 ◽  
Vol 118 (45) ◽  
pp. e2107507118
Author(s):  
Jiao Wang ◽  
Sandra Toregrosa-Allen ◽  
Bennett D. Elzey ◽  
Sagar Utturkar ◽  
Nadia Atallah Lanman ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Cell Function ◽  
Immune Cell ◽  
Nk Cell ◽  
Specific Activity ◽  
Antibody Fragment ◽  
Genetically Engineered ◽  
Local Concentration ◽  
Local Tumor

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site–specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell–based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

NK Education: Disassociation Between Recovery of Cytoxicity and Cytokine Production In NK Cells After Allogeneic Transplantation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1462-1462
Author(s):  
Bree Foley ◽  
Sarah Cooley ◽  
Julie Curtsinger ◽  
Michael Verneris ◽  
Daniel J. Weisdorf ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Immune Suppression ◽  
Cytokine Production ◽  
Cell Function ◽  
Nk Cell ◽  
Class I ◽  
Inhibitory Receptors ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Abstract 1462 NK cells are the first lymphocyte subset to reconstitute following hematopoietic stem cell transplantation (HSCT) and they play a pivotal role in mediation of the graft versus leukemia (GvL) effect in myeloid leukemia. We hypothesized that for NK cells to mediate GvL, they must be fully functional via a process termed “licensing” or “education”. Although it has been presumed that NK cell functions (cytotoxicity and cytokine production) develop in parallel through interactions with their class I recognizing inhibitory receptors, new data suggests that this may not be the case. To address this issues we developed a 9-color flow cytometric-based assay to simultaneously measure both CD107a expression and IFNy production by CD56+ NK cells in the context of expression of inhibitory receptors for self-class I human leukocyte antigen (HLA). We tested a cohort of 30 patients who received either unmanipulated (T cell replete) or potently T cell depleted (CD34+ selected) grafts from adult unrelated donors. Thawed peripheral blood mononuclear cells (PBMC) were rested overnight in cytokine free media and then incubated with K562 cells to trigger cytotoxicity and cytokine production. PBMC were stained with CD107a (a surrogate for cytotoxicity), IFNy, CD56, CD3, CD45, CD158a, CD158b, CD158e and CD159a simultaneously. The same normal volunteer and the actual transplant donor were used as positive controls in each assay. Cytotoxicity or IFNy production was calculated as a percentage of the normal positive control. Cytotoxicity was intact but modestly suppressed (∼35%) at 3 months after both T cell deplete and T cell replete HSCT with further recovery of killing at 6 months. By contrast, at 3 months after T cell replete HSCT there was potent and sustained suppression of IFNy production by CD56+ cells (57%±11% suppression, p=0.009). The cohort of patients receiving T cell deplete (CD34-selected) grafts also exhibited significant suppression of IFNy at 3 months after HSCT (73%±9.6%, p=0.018), suggesting that the use of post-transplant immune suppression medications did not explain the effect. Suppression of IFNy production when exposed to targets continued through 6 months post-transplant in both cohorts and was partially restored with low concentrations of IL-15. Cells stimulated overnight with IL-12 and IL-18 produced IFNy at 3 and 6 months. Thus the cells were not globally hyporesponsive, suggesting the defect was based on physiologic interactions with the target. NK cells become educated following engagement of inhibitory receptors (eg. Killer-immunoglobulin-like receptors [KIR]) with self class I HLA. Therefore we compared NK cells that expressed at least one KIR with KIR negative NK cells. At 3 months post transplant, KIR expression had no effect on cytotoxicity. In contrast, KIR positive cells produced significantly higher amounts of IFNy than KIR negative cells at 3 (Figure 1) and 6 (data not shown) months post-transplant. Therefore following HSCT, expression of KIR discriminates a population of NK cells that produce IFNy, but does not correlate with cytotoxicity. While NK cell cytotoxicity is only partially suppressed following HSCT, IFNy production is significantly reduced. Consistent with this we found that while all IFNy producing cells degranulate, only a small fraction of CD107a+ cells also make IFNy. This effect is not a result of post-transplant immune suppression or graft versus host disease, as patients receiving CD34+ selected grafts had neither. Perhaps NKG2A, highly expressed on almost all NK cells early after transplant, selectively mediates education for cytotoxcity. In conclusion, our data shows distinct defects in NK cell education for either cytotoxicity or cytokine production. This highlights the importance of analyzing both cytotoxicity and cytokine production when assessing NK cell function post HSCT. Because of their critical anti-tumor and infection protection roles, methods to enhance broad in vivo NK cell function, such as the use of post-transplant IL-15 administration, are warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical-Grade mRNA Electroporation of NK Cells: A Novel and Highly Efficient Method to Genetically Reprogram Human NK Cells for Cancer Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2153-2153 ◽  
Author(s):  
Mattias Carlsten ◽  
Linhong Li ◽  
Su Su ◽  
Maria Berg ◽  
Robert Reger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Nk Cell ◽  
Ex Vivo ◽  
Clinical Grade ◽  
Advisory Committees ◽  
Highly Efficient ◽  
Cytotoxic Function ◽  
Mrna Electroporation

Abstract Natural killer (NK) cells are immune cells involved in the defense against cancer. They have also been shown to induce strong anti-tumor responses in the setting of hematopoietic stem cell transplantation and in early clinical trials on adoptive NK cell transfer. Several methods to expand large numbers of clinical-grade NK cells have been developed for trials exploring adoptive NK cell immunotherapy for cancer. However, long-term culturing of NK cells often lead to undesirable phenotypic changes that may compromise their homing capacity and cytotoxic function, and can also lead to senescence compromising in vivo longevity. Introducing genes into NK cells that improve their in vivo viability, cytotoxicity, and ability to home to disease sites could improve the efficacy of NK cell-based immunotherapy. Previously, we and others have shown that genetic manipulation of NK cells through viral transduction is challenging, typically resulting in substantial reduction in NK cell viability and low transduction efficiency. mRNA transfection is an alternative strategy to genetically modify ex vivo expanded NK cells that may overcome limitations of viral transduction. Here we present data characterizing the transgene expression, viability, proliferative capacity, phenotype and cytotoxic function of clinical-grade ex vivo expanded human NK cells following mRNA electroporation using the GMP compliant MaxCyte system. Using unmodified mRNA coding for GFP and the cell surface marker CD34, we established that this technology resulted in rapid and highly efficient protein expression in NK cells without compromising their viability and cytotoxic function (Figure). NK cells electroporated with GFP mRNA rapidly became GFP positive and remained fluorescent for more than two weeks. Following transfection of CD34 mRNA, nearly 100% of NK cells expressed CD34 that remained detectable on the cell surface for up to five days, without affecting viability amongst transfected cells. With the exception of a slight reduction in proliferative capacity compared to controls, no negative impacts of mRNA electroporation using the MaxCyte platform were observed. Transfection of expanded NK cells did not alter expression of twenty cellular markers as assessed by flow cytometry, including activating and inhibitory NK cell receptors and death receptor ligands such as TRAIL. Further, electroporated NK cells maintained high cytotoxic function against K562 cells and multiple myeloma cells (Figure). In conclusion, mRNA electroporation of ex vivo expanded NK cells using the clinical-grade MaxCyte transfection system is highly efficient and opens numerous new possibilities to advancethe field of NK cell-based cancer immunotherapy. Figure 1 Figure 1. Disclosures Li: MaxCyte Inc.: Employment, Patents & Royalties. Peshwa:MaxCyte Inc.: Employment, Patents & Royalties; Indian Biomedical Association: Membership on an entity's Board of Directors or advisory committees; Epidarex Capital: Membership on an entity's Board of Directors or advisory committees; BioMetrx LLC: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Differential requirements for CD45 in NK-cell function reveal distinct roles for Syk-family kinases

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3087-3095 ◽  
Author(s):  
David G. T. Hesslein ◽  
Emil H. Palacios ◽  
Joseph C. Sun ◽  
Joshua N. Beilke ◽  
Susan R. Watson ◽  
...  
Keyword(s):  
Viral Infection ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Tyrosine Phosphatase ◽  
Functional Difference ◽  
Cell Functions ◽  
Activation Motif ◽  
The Impact

AbstractThe protein tyrosine phosphatase CD45 is an important regulator of Src-family kinase activity. We found that in the absence of CD45, natural killer (NK) cells are defective in protecting the host from mouse cytomegalovirus infection. We show that although CD45 is necessary for all immunoreceptor tyrosine–based activation motif (ITAM)–specific NK-cell functions and processes such as degranulation, cytokine production, and expansion during viral infection, the impact of CD45 deficiency on ITAM signaling differs depending on the downstream function. CD45-deficient NK cells are normal in their response to inflammatory cytokines when administered ex vivo and in the context of viral infection. Syk and ζ chain–associated protein kinase 70 (Zap70) are thought to play redundant roles in transmitting ITAM signals in NK cells. We show that Syk, but not Zap70, controls the remaining CD45-independent, ITAM-specific NK-cell functions, demonstrating a functional difference between these 2 Syk-kinase family members in primary NK cells.

4-1BBL-Expressing aAPCs Attenuate IL-15-Induced NK Cell Expansion and Cytokine Production In Vitro but Induce NK Cell-Mediated Gvhd In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2536-2536
Author(s):  
Christian M. Capitini ◽  
Joanna L. Meadors ◽  
Monica M. Cho ◽  
Rimas J. Orentas ◽  
Crystal L. Mackall ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Biology ◽  
Cytokine Production ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Receptor Expression ◽  
The Impact ◽  
Cells Cultured

Abstract Abstract 2536 Methods to expand natural killer (NK) cells ex vivo for adoptive cell therapy are being explored to improve outcomes after allogeneic blood and marrow transplant (alloBMT). Artificial antigen presenting cells (aAPCs) can present cytokines and/or co-stimulatory molecules that can potentially improve expansion and activity. 4-1BBL (CD137L) has demonstrated mixed results on murine and human NK cells, but the impact on murine NK cell biology after alloBMT has not been explored. NK cells were harvested from either C57BL/6 (B6) or CB6F1 spleens and cultured ex vivo with a recombinant interleukin (IL)-15/IL-15 receptor alpha (Ra) complex in the presence or absence of a CD137L+ aAPC. Because IL-15 is typically presented in trans by IL-15Ra, the complex was utilized to potently increase agonist bioactivity. NK cells cultured with IL-15/IL-15Ra alone showed a peak of 20-fold expansion, but this expansion was decreased with the addition of CD137L+ aAPCs if the ratio of aAPC to NK cells was greater than 1:1. In the presence of IL-15/IL-15Ra, the impact of CD137L+ aAPCs on expression of the inhibitory receptors, Ly49C+I and activating receptor Ly49H was variable and strain dependent, with increased expression in B6 NK cells, but decreased expression in CB6F1 NK cells. The expression of major histocompatibility complex (MHC) class I was not affected in NK cells from either strain by the presence of CD137L+ aAPCs. The production of gamma interferon and tumor necrosis factor-a was robust in NK cells expanded by IL-15/IL-15Ra alone, but attenuated with the addition of CD137L+ aAPCs. Animal experiments showed that administration of NK cells expanded ex vivo with IL-15/IL-15Ra alone was well tolerated after T cell depleted MHC-mismatched alloBMT (CB6F1–>B6), but surprisingly the addition of CD137L+ aAPCs to cultures caused NK cells to induce GVHD-associated weight loss. In summary, IL-15/IL-15Ra expanded murine NK cells demonstrate increased cytokine production and do not cause toxicity when infused after alloBMT. The presence of CD137L+ aAPCs attenuated cytokine production and increased Ly49 receptor expression in NK cells from B6 mice. Remarkably, NK cells expanded by IL-15/IL-15Ra in the presence of CD137L+ aAPCs demonstrate increased propensity to cause GVHD. Ongoing studies are exploring the anti-tumor efficacy of IL-15/IL-15Ra expanded murine NK cells cultured in the presence and absence of CD137L. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Aging on NK Cell Population and Their Proliferation at Ex Vivo Culture Condition

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Sellamuthu Subbanna Gounder ◽  
Basri Johan Jeet Abdullah ◽  
Nur Ezzati Izyan Binti Mohd Radzuanb ◽  
Farah Dalila Binti Mohd Zain ◽  
Nurhidayah Bt Mohamad Sait ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Population ◽  
Cytokine Production ◽  
Nk Cell ◽  
Ex Vivo ◽  
Age Groups ◽  
Proliferation Ability ◽  
Males And Females ◽  
Ex Vivo Culture ◽  
Proliferation Potential

Age-associated changes in natural killer (NK) cell population, phenotype, and functions are directly attributed to the risk of several diseases and infections. It is predicted to be the major cause of the increase in mortality. Based on the surface density of CD56, NK cells are subdivided into two types, such as CD56brightand CD56dimcells, which represent cytokine production and cytotoxicity. In our study, we have examined the age-associated changes in the NK cell population and their subsets at different age groups of males and females (at a range from 41 to 80 years). We found that the total lymphocyte count significantly dropped upon aging in both genders. Although, the level of total immune cells also dropped on aging, and surprisingly the total NK cell population was remarkably increased with the majority of NK cells being CD56dim. Subsequently, we evaluated the proliferation potential of NK cells and our results showed that the NK cell proliferation ability declines with age. Overall, our findings prove that there is an increase in the circulating NK cell population upon aging. However, the proliferation rate upon aging declines when compared to the young age group (<41 yrs).

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

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S187-S188
Author(s):  
V Zaiatz Bittencourt ◽  
E Ryan ◽  
F Jones ◽  
G Doherty
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Inflammatory Cytokines ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Metabolism ◽  
Building Blocks ◽  
Cell Phenotype ◽  
Increased Risk ◽  
Pro Inflammatory Cytokines

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.

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