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The identity of allogeneic peptide/major histocompatibility complex (MHC) complexes that elicit vigorous T cell responses has remained an interesting problem for both practical and theoretical reasons. Although a few abundant MHC class I-bound peptides have been purified and sequenced, identifying the unique T cell-stimulating peptides from among the thousands of existing peptides is still a very difficult undertaking. In this report, we identified the antigenic peptide that is recognized by an alloreactive bm1 anti-B6 T cell clone using a novel genetic strategy that is based upon measurement of T cell receptor occupancy in single T cells. Using lacZ-inducible T cells as a probe, we screened a splenic cDNA library in transiently transfected antigen-presenting cells (APCs) and isolated a cDNA clone that allowed expression of the appropriate peptide/Kb MHC complex in APC. The antigenic octapeptide (SVVEFSSL) exactly matched the consensus Kb MHC motif, but was surprisingly encoded by a non-ATG defined translation reading frame. Furthermore, the abundance of the naturally processed analog in untransfected cells was estimated to be

A study was conducted to assess the microbiological and sensory properties of yoghurt incorporated with condensed coconut water by partial replacement of MSNF with or without a stabiliser gelatin. The treatments were divided in to Control, T2 (addition of condensed coconut water by replacing MSNF at 25 per cent), T3 (addition of condensed coconut water by replacing MSNF at 25 per cent with gelatin), T4 (addition of condensed coconut water by replacing MSNF at 50 per cent) and T5 (addition of condensed coconut water by replacing MSNF at 50 per cent with gelatin). The yoghurt mixes for different treatments were formulated by using computer linear programming model. No significant difference between control and treatments with regard to appearance score and body and texture scores. A significant difference (P<0.01) was noticed for the flavour score as well as total sensory scores. Condensed coconut water incorporated at 25 per cent level in partial replacement of milk solids not fat (MSNF) with or without stabiliser gelatin in yoghurt preparation had a similar sensory properties as compared to control. [ABSTRACT FROM AUTHOR]

IQGAP1 is a multi-functional scaffold protein. However, its role in B cell development and function is unknown. Here, we show IQGAP1 as an essential scaffold that regulates early B cell development and function. Iqgap1 -/- mice contained significantly increased numbers of B220 + B, B220 + IgM - pro/pre-B, and B220 Low IgM + immature-B cells in the bone marrow. In the spleens of the Iqgap1 -/- mice, newly formed and follicular B cell numbers were increased, while the marginal zone B cell numbers were significantly reduced. Lack of IQGAP1 reduced T-dependent and T-independent humoral responses. Mechanistically, the lack of IQGAP1 considerably decreased the phosphorylation of Mek1/2, Erk1/2, and Jnk1/2. B cells from Iqgap1 -/- mice failed to suppress IL-7R-mediated activation of Stat5a/b, an essential step for cell-cycle exit and initiate light-chain recombination, reducing RS rearrangement frequency. Our study provides the first evidence that IQGAP1-based signalosome is necessary for the development and functions of B cells., Competing Interests: Declarations. Conflict of interest: The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval: All mice used in this study were utilized responsibly, and all protocols were approved by the institutional IACUC committee at the Medical College of Wisconsin (MCW), Milwaukee, WI. Author consent: All the authors fully agree to be part of the manuscript and its content. Both authors have participated in collecting, analyzing, and preparing this manuscript., (© 2024. The Author(s).)

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large patient cohorts, we identified increased numbers of CD56 bright Perforin - regulatory-like NK cells (NK reg -like) associated with cGvHD suppression. Thus, we hypothesized that NK reg -like cells may be a potential candidate for cGvHD cell therapy., Aim: To expand NK reg -like cells while maintaining regulatory phenotype and function., Methods: Total NK cells were first expanded with IL-2, which was then combined with rapamycin, Transforming Growth Factor Beta 1 (TGF-β1), NECA (Adenosine A2A receptor (A2AR) agonist), metformin, or dexamethasone, to prevent change in cell phenotype/function. The functional characteristics were evaluated via T cell suppression assays and the phenotype was measured using flow cytometry. The optimal expansion protocol was compared in terms of function and metabolism for three NK expansion media, and cells from cord vs. peripheral blood. Further, expanded NK reg -like cell gene expression was characterized using bulk RNA sequencing. Finally, NK reg -like cells were differentiated from CD34 + hematopoietic stem and progenitor cells (HSPCs) and compared in terms of proliferation and function., Results: The expansion of total NK cells found the addition of TGF-β1 and/or NECA with the pulsing of rapamycin in IL-2 containing media to prevent NK reg -like differentiation (up to 200-fold expansion). Expanded NK reg -like cells maintained a phenotype, transcriptome, and T cell suppression similar to freshly isolated NK reg -like cells. NK reg -like expansion was greatest in the Immunocult media (up to 300-fold), and NK reg -like cells from peripheral blood demonstrated significantly greater proliferation than cells isolated from cord blood (65-fold). The metabolic profile of NK reg -like and cytolytic NK cells appeared similar at baseline, though rapamycin induced a shift to oxidative over glycolytic metabolism. Further, we demonstrated that suppressive NK reg -like cells may alternatively be expanded from CD34 + cells isolated from cord blood, reaching an average 340-fold expansion., Conclusions: In conclusion, our studies have optimized two alternative expansion approaches for deriving functional NK reg -like cells. Additionally, evaluating the transcriptomic and metabolic characteristics provides useful information regarding NK reg -like cell function and differentiation. With further optimization and in vivo validation, we may work towards preparing these cells as a therapy for cGvHD., Competing Interests: Declaration of competing interest None of the co-authors have any conflicts of interest to declare for the project., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Visceral white adipose tissues (WAT) regulate systemic lipid metabolism and inflammation. Dysfunctional WAT drive chronic inflammation and facilitate atherosclerosis. Adipose tissue-associated macrophages (ATM) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATM crosstalk with other adipose tissue cells, driving chronic inflammation. Here, we combined the single-cell RNA sequencing technique with cell metabolic and functional assays on major WAT ATM subpopulations using a diet-induced atherosclerosis mouse model ( Apoe -null). We also examined the role of CD36 using Apoe / Cd36 double-null mice. Based on transcriptomics data and differential gene expression analysis, we identified a previously undefined group of ATM displaying low viability and high lipid metabolism and labeled them as "unhealthy macrophages". Their phenotypes suggest a subpopulation of ATM under lipid stress. We also identified lipid-associated macrophages (LAM), which were previously described in obesity. Interestingly, LAM increased 8.4-fold in Apoe / Cd36 double-null mice on an atherogenic diet, but not in Apoe -null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation. In conclusion, CD36 mediates a delicate balance between lipid metabolism and inflammation in visceral adipose tissues. Under atherogenic conditions, CD36 deficiency reduces inflammation and increases lipid metabolism in WAT by promoting LAM accumulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Chen, Zhang, Chang, Beg, Vick, Wang, Gupta, Wang, Zhang, Dai, Kim, Song, Pereira, Zheng, Sodhi, Shapiro, Silverstein, Malarkannan and Chen.)

Innate lymphoid cells (ILCs) are largely tissue-resident, mostly described within the mucosal tissues. However, their presence and functions in the human draining lymph nodes (LNs) are unknown. Our study unravels the tissue-specific transcriptional profiles of 47,287 CD127 + ILCs within the human abdominal and thoracic LNs. LNs contain a higher frequency of CD127 + ILCs than in BM or spleen. We define independent stages of ILC development, including EILP and pILC in the BM. These progenitors exist in LNs in addition to naïve ILCs (nILCs) that can differentiate into mature ILCs. We define three ILC1 and four ILC3 sub-clusters in the LNs. ILC1 and ILC3 subsets have clusters with high heat shock protein-encoding genes. We identify previously unrecognized regulons, including the BACH2 family for ILC1 and the ATF family for ILC3. Our study is the comprehensive characterization of ILCs in LNs, providing an in-depth understanding of ILC-mediated immunity in humans., (© 2024. The Author(s).)

Background & Aims: The complex tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) has hindered the development of reliable predictive biomarkers for targeted therapy and immunomodulatory strategies. A comprehensive characterization of the TME is necessary to advance precision therapeutics in PDAC., Methods: A transcriptomic profiling platform for TME classification based on functional gene signatures was applied to 14 publicly available PDAC datasets (n = 1657) and validated in a clinically annotated independent cohort of patients with PDAC (n = 79). Four distinct subtypes were identified using unsupervised clustering and assessed to evaluate predictive and prognostic utility., Results: TME classification using transcriptomic profiling identified 4 biologically distinct subtypes based on their TME immune composition: immune enriched (IE); immune enriched, fibrotic (IE/F); fibrotic (F); and immune depleted (D). The IE and IE/F subtypes demonstrated a more favorable prognosis and potential for response to immunotherapy compared with the F and D subtypes. Most lung metastases and liver metastases were subtypes IE and D, respectively, indicating the role of clonal phenotype and immune milieu in developing personalized therapeutic strategies. In addition, distinct TMEs with potential therapeutic implications were identified in treatment-naive primary tumors compared with tumors that underwent neoadjuvant therapy., Conclusions: This novel approach defines a distinct subgroup of PADC patients that may benefit from immunotherapeutic strategies based on their TME subtype and provides a framework to select patients for prospective clinical trials investigating precision immunotherapy in PDAC. Further, the predictive utility and real-world clinical applicability espoused by this transcriptomic-based TME classification approach will accelerate the advancement of precision medicine in PDAC., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The role of FasL in initiating death signals through Fas is well characterized. However, the reverse signaling pathway downstream of FasL in effector lymphocytes is poorly understood. Here, we identify that FasL functions as an independent activation receptor in NK cells. Activation via FasL results in the production of LFN-γ, GM-CSF, RANTES, MIP-1α, and MIP1-β. Proximal signaling of FasL requires Lck and Fyn. Upon activation, FasL facilitates the phosphorylation of PI(3)K-p85α/p55α subunits. A catalytically inactive PI(3)K-p110δD910A mutation significantly impairs the cytokine and chemokine production by FasL. Activation of ITK and LAT downstream of FasL plays a central role in recruiting and phosphorylating PLC-γ2. Importantly, Fyn-mediated recruitment of ADAP links FasL to the Carmal/ Bcl10/Tak1 signalosome. Lack of Carma1, CARD domain of Carma1, or Tak1 significantly reduces FasL-mediated cytokine and chemokine production. These findings, for the first time, provide a detailed molecular blueprint that defines FasL-mediated reverse signaling.

Introduction and Methods: In this study we report that sequential treatment of supercharged NK (sNK) cells with either chemotherapeutic drugs or check-point inhibitors eliminate both poorly differentiated and well differentiated tumors in-vivo in humanized-BLT mice., Background and Results: sNK cells were found to be a unique population of activated NK cells with genetic, proteomic, and functional attributes that are very different from primary untreated or IL-2 treated NK cells. Furthermore, NK-supernatant differentiated or well-differentiated oral or pancreatic tumor cell lines are not susceptible to IL-2 activated primary NK cell-mediated cytotoxicity; however, they are greatly killed by the CDDP and paclitaxel in in-vitro assays. Injection of one dose of sNK cells at 1 million cells per mouse to aggressive CSC-like/poorly differentiated oral tumor bearing mice, followed by an injection of CDDP, inhibited tumor weight and growth, and increased IFN-γ secretion as well as NK cell-mediated cytotoxicity substantially in bone marrow, spleen and peripheral blood derived immune cells. Similarly, the use of check point inhibitor anti-PD-1 antibody increased IFN-γ secretion and NK cell-mediated cytotoxicity, and decreased the tumor burden in-vivo, and tumor growth of resected minimal residual tumors from hu-BLT mice when used sequentially with sNK cells. The addition of anti-PDL1 antibody to poorly differentiated MP2, NK-differentiated MP2 or well-differentiated PL-12 pancreatic tumors had different effects on tumor cells depending on the differentiation status of the tumor cells, since differentiated tumors expressed PD-L1 and were susceptible to NK cell mediated ADCC, whereas poorly differentiated OSCSCs or MP2 did not express PD-L1 and were killed directly by the NK cells., Conclusions: Therefore, the ability to target combinatorially clones of tumors with NK cells and chemotherapeutic drugs or NK cells with checkpoint inhibitors at different stages of tumor differentiation may be crucial for successful eradication and cure of cancer. Furthermore, the success of check point inhibitor PD-L1 may relate to the levels of expression on tumor cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kaur, Chen, Ko, Mei, Senjor, Malarkannan, Kos and Jewett.)

Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by the progressive loss of motor neurons in the brain and spinal cord. No effective therapeutic strategies have been established thus far, and therefore there is a significant unmet need for effective therapeutics to arrest the disease and reverse the pathologies induced by it. Although the cause of ALS is not well-defined, it appears to be heterogenous. Currently over 20 genes have been found to be associated with ALS. Family history can only be found in 10% of ALS patients, but in the remaining 90% no association with family history is found. The most common genetic causes are expansion in the C9orf72 gene and mutations in superoxide dismutase 1, TDP-43, and FUS. In our recent study, we also found mutations in TDP43 and FUS in ALS patients. To understand the pathogenesis of the disease, we set ourselves the task of analyzing the phenotype and function of all key immune effectors in ALS patients, comparing them with either a genetically healthy twin or healthy individuals. Our study demonstrated a significant increase in functional activation of NK and CD8+ T cytotoxic immune effectors and release of significant IFN-γ not only by the effector cells but also in the serum of ALS patients. Longitudinal analysis of CD8+ T cell-mediated IFN-γ secretion from ALS patients demonstrated continued and sustained increase in IFN-γ secretion with periods of decrease which coincided with certain treatments; however, the effects were largely short-lived. N-acetyl cysteine (NAC), one of the treatments used, is known to block cell death; however, even though such treatment was able to block most of the proinflammatory cytokines, chemokines, and growth factor release, it was not able to block IFN-γ and TNF-α, the two cytokines we had demonstrated previously to induce differentiation of the cells. In this review, we discuss the contribution of cytotoxic effector cells, especially primary NK cells, supercharged NK cells (sNK), and the contribution of sNK cells in expansion and functional activation of CD8+ T cells to memory/effector T cells in the pathogenesis of ALS. Potential new targeted therapeutic strategies are also discussed.

The mechanisms that govern the development of adaptive-like NK cells are elusive. Shemesh et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20220551) report that the development of FcRγ-/low adaptive-like NK cells requires reduced mTOR activity and depends on TGF-β or IFN-α. These findings provide exciting new molecular blueprints explaining the development and functions of adaptive-like NK cells., (© 2022 Khalil and Malarkannan.)

Amyotrophic lateral sclerosis (ALS) is an auto-immune neurodegenerative disorder affecting the motor-neuron system. The causes of ALS are heterogeneous, and are only partially understood. We studied different aspects of immune pathogenesis in ALS and found several basic mechanisms which are potentially involved in the disease. Our findings demonstrated that ALS patients' peripheral blood contains higher proportions of NK and B cells in comparison to healthy individuals. Significantly increased IFN-γ secretion by anti-CD3/28 mAbs-treated peripheral blood mononuclear cells (PBMCs) were observed in ALS patients, suggesting that hyper-responsiveness of T cell compartment could be a potential mechanism for ALS progression. In addition, elevated granzyme B and perforin secretion at a single cell level, and increased cytotoxicity and secretion of IFN-γ by patients' NK cells under specific treatment conditions were also observed. Increased IFN-γ secretion by ALS patients' CD8+ T cells in the absence of IFN-γ receptor expression, and increased CD8+ T cell effector/memory phenotype as well as increased granzyme B at the single cell level points to the CD8+ T cells as potential cells in targeting motor neurons. Along with the hyper-responsiveness of cytotoxic immune cells, significantly higher levels of inflammatory cytokines including IFN-γ was observed in peripheral blood-derived serum of ALS patients. Supernatants obtained from ALS patients' CD8+ T cells induced augmented cell death and differentiation of the epithelial cells. Weekly N-acetyl cysteine (NAC) infusion in patients decreased the levels of many inflammatory cytokines in peripheral blood of ALS patient except IFN-γ, TNF-α, IL-17a and GMCSF which remained elevated. Findings of this study indicated that CD8+ T cells and NK cells are likely culprits in targeting motor neurons and therefore, strategies should be designed to decrease their function, and eliminate the aggressive nature of these cells. Analysis of genetic mutations in ALS patient in comparison to identical twin revealed a number of differences and similarities which may be important in the pathogenesis of the disease.

A recent study highlights the presence of a unique memory-like natural killer (NK) cell subset, which accumulates with aging and appears to associate withdisease severity in COVID-19 patients. While the clinical relevance of memory in NK cells is being debated, the molecular identity of this subset in the form of a single-cell transcriptome is essential to define their origin, longevity, functions, and disease relevance., (© 2022. The Author(s).)

Natural killer (NK) cells are innate lymphocytes that control tumors and microbial infections. Human NK cells are transcriptomically and phenotypically heterogeneous. The site where NK cells develop and reside determines their phenotype and effector functions. Our current knowledge about human NK cells is primarily from blood- and bone marrow-derived NK cells. The major limitation in formulating organ-specific clinical therapy is the knowledge gap on how tissue-resident NK cells develop, home, and function. Thus, it is crucial to define the transcriptomic profiles and the transcriptional regulation of tissue-resident NK cells. The major challenges in studying tissue-resident NK cells include their total number and the complexity of the tissue. Additionally, during isolation, keeping them viable and naïve without activation are challenging tasks. Here, we provide methods for isolating and performing transcriptomic analyses of NK cells at the individual cell level. Single-cell RNA sequencing provides a higher resolution of cellular heterogeneity and a better understanding of cell-cell interactions within the microenvironment. Using these methods, we can efficiently identify distinct populations of NK cells in tissues and define their unique transcriptomic profiles., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Development of novel cellular therapies based on primary human NK cells is under active investigation. Human NK cells are comprised of distinct subsets with high transcriptomic heterogeneity. Unique methodologies are being developed to determine the transcriptomic profiles of human NK cells. NK cells account for 10-20% of total lymphocytes in the human peripheral blood, which mediates anti-tumor and anti-viral effector functions. Therapeutic success in the clinic depends on a better understanding of the single-cell transcriptome of human NK cell subsets. Moreover, a better understanding of the transcriptional network that regulates NK cell development, subset specification, and terminal maturation is obligatory for their in vitro generation and expansion toward clinical utilization. Here, we describe the procedure for single-cell RNA-sequencing of human NK cells and strategies for bioinformatic analyses. This protocol provides a data analysis roadmap for investigators who work on the basic biology and therapeutic applications of human NK cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immunological memory is a fundamental feature of the adaptive immune system that protects the host from recurrent infections from pathogens. Natural killer (NK) cells are a predominant member of the innate immune system that lack clonotypic receptors, which are essential for memory formation. However, evidence demonstrates that a unique subpopulation of NK cells develops adaptive-like features using germline-encoded receptors. Recent studies have shown that infection of cytomegalovirus (CMV) leads to clonal expansion of NKG2C + and Ly49H + NK cells, in humans and mouse, respectively. These activation receptors have the capability to recognize CMV-encoded proteins and facilitate a recall response upon reinfection. Although NK cells do not rearrange genes encoding their activating receptors as seen in B and T cells, they possess a selective process to generate memory features and a long-lived progeny. Here, we describe an established in vivo protocol for infecting mice with mouse cytomegalovirus (MCMV) to study an adaptive NK cell response., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Natural killer (NK) cells are innate cytotoxic immune cells essential for mediating first-line defense against various environmental antigens. With the discoveries of other subsets of innate lymphocytes over the last decade, NK cells are categorized as innate lymphoid cells (ILC) and as the innate counterparts of cytotoxic T cells. Besides NK cells, ILCs are classified into three groups distinguished by their dependence on distinct transcription factors for development and unique effector functions. Subsets of ILCs share many surface proteins that, however, have initially been identified as NK cell markers, making them hard to be distinguished for detailed investigations. Here, we describe a method to identify and individually isolate subsets of innate lymphoid cells from gut lamina propria using cell surface markers., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1βR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88 fl/fl Ncr1 Cre/+ ). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H + . In contrast, Ly49H - NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H + NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The clinical use of natural killer (NK) cells is at the forefront of cellular therapy. NK cells possess exceptional antitumor cytotoxic potentials and can generate significant levels of proinflammatory cytokines. Multiple genetic manipulations are being tested to augment the anti-tumor functions of NK cells. One such method involves identifying and altering microRNAs (miRNAs) that play essential roles in the development and effector functions of NK cells. Unique miRNAs can bind and inactivate mRNAs that code for cytotoxic proteins. MicroRNAs, such as the members of the Mirc11 cistron, downmodulate ubiquitin ligases that are central to the activation of the obligatory transcription factors responsible for the production of inflammatory cytokines. These studies reveal potential opportunities to post-translationally enhance the effector functions of human NK cells while reducing unwanted outcomes. Here, we summarize the recent advances made on miRNAs in murine and human NK cells and their relevance to NK cell development and functions.

Innate and adaptive immune systems are evolutionarily divergent. Primary signaling in T and B cells depends on somatically rearranged clonotypic receptors. In contrast, NK cells use germline-encoded non-clonotypic receptors such as NCRs, NKG2D, and Ly49H. Proliferation and effector functions of T and B cells are dictated by unique peptide epitopes presented on MHC or soluble humoral antigens. However, in NK cells, the primary signals are mediated by self or viral proteins. Secondary signaling mediated by various cytokines is involved in metabolic reprogramming, proliferation, terminal maturation, or memory formation in both innate and adaptive lymphocytes. The family of common gamma (γc) cytokine receptors, including IL-2Rα/β/γ, IL-7Rα/γ, IL-15Rα/β/γ, and IL-21Rα/γ are the prime examples of these secondary signals. A distinct set of cytokine receptors mediate a 'third' set of signaling. These include IL-12Rβ1/β2, IL-18Rα/β, IL-23R, IL-27R (WSX-1/gp130), IL-35R (IL-12Rβ2/gp130), and IL-39R (IL-23Rα/gp130) that can prime, activate, and mediate effector functions in lymphocytes. The existence of the 'third' signal is known in both innate and adaptive lymphocytes. However, the necessity, context, and functional relevance of this 'third signal' in NK cells are elusive. Here, we define the current paradigm of the 'third' signal in NK cells and enumerate its clinical implications.

Natural killer (NK) cells are major innate lymphocytes. NK cells do not require prior antigen exposure to mediate antitumor cytotoxicity or proinflammatory cytokine production. Since they use only nonclonotypic receptors, they possess high clinical value in treatment against a broad spectrum of malignancies. Irrespective of this potential, however, the transcriptional regulation that governs human NK cell development remains far from fully defined. Various environmental cues initiate a complex network of transcription factors (TFs) during their early development, one of which is GATA2, a master regulator that drives the commitment of common lymphoid progenitors (CLPs) into immature NK progenitors (NKPs). GATA2 forms a core heptad complex with six other TFs (TAL1, FLI1, RUNX1, LYL1, LMO2, and ERG) to mediate its transcriptional regulation in various cell types. Patients with GATA2 haploinsufficiency specifically lose CD56bright NK cells, with or without a reduced number of CD56dlm NK cells. Here, we review the recent progress in understanding GATA2 and its role in human NK cell development and functions.

Fanconi anemia (FA) is an inherited disorder characterized by diverse congenital malformations, progressive pancytopenia, and predisposition to hematological malignancies and solid tumors. The role of the Fanconi anemia pathway in DNA repair mechanisms and genome instability is well studied. However, the consequences of inherited mutations in genes encoding the FA proteins and the acquired mutations due to impaired DNA repair complex in immune cells are far from understood. Patients with FA show bone marrow failure (BMF) and have a higher risk of developing myelodysplasia (MDS) or acute myeloid leukemia (AML) which are directly related to having chromosomal instability in hematopoietic stem cells and their subsequent progeny. However, immune dysregulation can also be seen in FA. As mature descendants of the common lymphoid progenitor line, NK cells taken from FA patients are dysfunctional in both NK cell-mediated cytotoxicity and cytokine production. The molecular bases for these defects are yet to be determined. However, recent studies have provided directions to define the cause and effect of inherited and acquired mutations in FA patients. Here, we summarize the recent studies in the hematopoietic dysfunction, focusing on the impairment in the development and functions of NK cells in FA patients, and discuss the possible mechanisms and future directions.

The mechanistic target of Rapamycin (mTOR) is essential for multiple cellular processes. The unique roles of mTOR complex 1 (mTORC1) or mTOR2 in regulating immune functions are emerging. NK cells are the major lymphocyte subset of innate immunity, and their development and effector functions require metabolic reprogramming. Recent studies demonstrate that in NK cells, conditionally disrupting the formation of mTORC1 or mTOR complex 2 (mTORC2) alters their development significantly. Transcriptomic profiling of NK cells at the single-cell level demonstrates that mTORC1 was critical for the early developmental progression, while mTORC2 regulated the terminal maturation. In this review, we summarize the essential roles of mTOR complexes in NK development and functions., (Copyright © 2020 Yang and Malarkannan.)

Effector lymphocytes, including NK and T cells, express FasL. Expression of Fas, the receptor for FasL in tumor cells, renders them susceptible to NK and T cell-mediated killing. The functional relevance of FasL in initiating death signals in tumor cells is well-characterized. However, the cytoplasmic interacting partners and the potential signaling pathways downstream of FasL are far from fully defined. FasL possesses an 81 amino acid long cytoplasmic tail with multiple unique recruitment motifs. We predict multiple interdependent signaling complexes form the core of the 'reverse signaling' downstream of FasL. A direct interaction between the proline-rich domain of FasL and the SH3 domain of PI(3)K-p85α initiates the first pathway. This cascade helps FasL to link to PLC-γ2 via PIP 3 or the Akt-dependent activation of mTOR complexes. Independently, a GRB2/GADs-binding PXXP cytoplasmic motif of FasL can initiate a Ras-GTP-dependent PAK1→C-Raf→MEK1/2→ERK1/2 activation. FasL can recruit Fyn via the proline-rich domain leading to the recruitment of ADAP. Through its ability to directly interact with Carma1 and TAK1, ADAP initiates the formation of the Carma1/Bcl10/Malt1-based CBM signalosome that is primarily responsible for inflammatory cytokine production. Here, we explore the conserved cytoplasmic domains of FasL, the potential signaling molecules that interact, and the functional downstream consequences within the effector lymphocytes to define the FasL-mediated 'reverse signaling'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

During an immune response, natural killer (NK) cells activate specific metabolic pathways to meet the increased energetic and biosynthetic demands associated with effector functions. Here, we found in vivo activation of NK cells during Listeria monocytogenes infection-augmented transcription of genes encoding mitochondria-associated proteins in a manner dependent on the transcriptional coactivator PGC-1α. Using an Ncr1 Cre -based conditional knockout mouse, we found that PGC-1α was crucial for optimal NK cell effector functions and bioenergetics, as the deletion of PGC-1α was associated with decreased cytotoxic potential and cytokine production along with altered ADP/ATP ratios. Lack of PGC-1α also significantly impaired the ability of NK cells to control B16F10 tumor growth in vivo , and subsequent gene expression analysis showed that PGC-1α mediates transcription required to maintain mitochondrial activity within the tumor microenvironment. Together, these data suggest that PGC-1α-dependent transcription of specific target genes is required for optimal NK cell function during the response to infection or tumor growth., Competing Interests: The authors declare that they have no competing interests., (© 2020 The Author(s).)

Natural killer (NK) cells are the major lymphocyte subset of the innate immune system. Their ability to mediate anti-tumor cytotoxicity and produce cytokines is well-established. However, the molecular mechanisms associated with the development of human or murine NK cells are not fully understood. Knowledge is being gained about the environmental cues, the receptors that sense the cues, signaling pathways, and the transcriptional programs responsible for the development of NK cells. Specifically, a complex network of transcription factors (TFs) following microenvironmental stimuli coordinate the development and maturation of NK cells. Multiple TFs are involved in the development of NK cells in a stage-specific manner. In this review, we summarize the recent advances in the understandings of TFs involved in the regulation of NK cell development, maturation, and effector function, in the aspects of their mechanisms, potential targets, and functions.

Natural killer (NK) cells belong to type 1 innate lymphoid cells (ILC1) and are essential in killing infected or transformed cells. NK cells mediate their effector functions using non-clonotypic germ-line-encoded activation receptors. The utilization of non-polymorphic and conserved activating receptors promoted the conceptual dogma that NK cells are homogeneous with limited but focused immune functions. However, emerging studies reveal that NK cells are highly heterogeneous with divergent immune functions. A distinct combination of several activation and inhibitory receptors form a diverse array of NK cell subsets in both humans and mice. Importantly, one of the central factors that determine NK cell heterogeneity and their divergent functions is their tissue residency. Decades of studies provided strong support that NK cells develop in the bone marrow. However, evolving evidence supports the notion that NK cells also develop and differentiate in tissues. Here, we summarize the molecular basis, phenotypic signatures, and functions of tissue-resident NK cells and compare them with conventional NK cells.

Histone deacetylase inhibitors (HDACi) are an emerging cancer therapy; however, their effect on natural killer (NK) cell-mediated anti-tumor responses remain unknown. Here, we evaluated the impact of a benzamide HDACi, entinostat, on human primary NK cells as well as tumor cell lines. Entinostat significantly upregulated the expression of NKG2D, an essential NK cell activating receptor. Independently, entinostat augmented the expression of ULBP1, HLA, and MICA/B on both rhabdomyosarcoma and Ewing sarcoma cell lines. Additionally, entinostat increased both cytotoxicity and IFN-γ production in human NK cells following coculture with these tumor cells. Mechanistically, entinostat treatment resulted in increased chromatin accessibility to the promoter region for interferon-induced protein with tetratricopeptide repeats 1 ( IFIT1 ) gene and thereby increasing the transcript and protein levels of IFIT1 that augmented the IFIT1-mediated IRF1, STAT4, and STING pathways. Corresponding transcriptome analysis revealed enrichment of IRF1 and STAT4 and gene sets responsible for NK cell-mediated IFN-γ production and cytotoxicity, respectively. Our results show a novel mechanism by which entinostat initiates an IFIT1-STING-mediated potentiation of STAT4 via IRF1 to augment NK cell-mediated anti-tumor responses., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through Akt S473 -FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-Akt S473 -FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells., Competing Interests: CY, JS, RB, YZ, AM, BB, DW, RU, MR, SR, KC, MT, SM No competing interests declared, (© 2020, Yang et al.)

Chimeric Antigen Receptor (CAR)-based therapies offer a promising, targeted approach to effectively treat relapsed or refractory B cell malignancies. However, the treatment-related toxicity defined as cytokine-release syndrome (CRS) often develops in patients, and if uncontrolled, can be fatal. Grading systems have now been developed to further characterize and objectify clinical findings in order to provide algorithm-based guidance on CRS-related treatment decisions. The pharmacological treatments associated with these algorithms suppress inflammation through a variety of mechanisms and are paramount to improving the safety profile of CAR-based therapies. However, fatalities are still occurring, and there are downsides to these treatments, including the possibility of disrupting CAR-T cell persistence. This review article will describe the clinical presentation and current management of CRS, and through our now deeper understanding of downstream signaling pathways, will provide a molecular framework to formulate new hypotheses regarding clinical applications to contain proinflammatory cytokines responsible for CRS., (Copyright © 2020 Thakar, Kearl and Malarkannan.)

NK cells are innate lymphocytes that are vital to clearance of virally infected or malignantly transformed cells. Assessment of the cytotoxic response is an important component of NK cell research and investigation of human disease. Standard assays of NK cell-mediated cytotoxicity of CD107a degranulation or 51 Cr release assay utilize cultured or freshly isolated NK cell populations in vitro. In addition to requirements to maintain multiple target cell lines and radioactivity precautions in the case of 51 Cr, these are in vitro evaluations of a complex in vivo function. Here, we describe the in vivo assessment of NK cell-mediated cytotoxicity through the adoptive transfer of splenocytes and their subsequent rejection. This protocol offers rapid, quantitative, and concurrent assessment of NK cell-mediated cytotoxicity against the prototypic NK stimulations of "missing-self" and "nonself."

Recent advances in cancer immunotherapy emphasize the need for an efficient method to genetically modify effector lymphocytes to express exogenous "synthetic" genes. NK cells represent 10-20% of total lymphocytes in the peripheral blood of humans and play an essential role in clearing infections and malignant cells. A significant number of NK cells express and utilize non-clonotypic receptors that recognize cognate ligands expressed on a broad spectrum of target cells. Thus, NK cells can be utilized as potent immunotherapeutic tools with fewer limitations. Considerable amount of progress in improving effector functions through genetic manipulations has been centered around T cells. However, a similar technological and translational exploration on NK cells is lacking. One major constrain is the significantly low efficiency of lentiviral-mediated gene transductions into primary human or mouse NK cells. We found that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible methodology offers an approach that can help to improve gene delivery into NK cells and thereby cancer immunotherapy.

Rationale: A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood., Objective: To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways., Methods and Results: Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-κB (nuclear factor-κB) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-κB activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C + monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages., Conclusions: These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

It is well established that extracellular proteins that negatively regulate T cell function, such as Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) and Programmed Cell Death protein 1 (PD-1), can be effectively targeted to enhance cancer immunotherapies and Chimeric Antigen Receptor T cells (CAR-T cells). Intracellular proteins that inhibit T cell receptor (TCR) signal transduction, though less well studied, are also potentially useful therapeutic targets to enhance T cell activity against tumor. Four major classes of enzymes that attenuate TCR signaling include E3 ubiquitin kinases such as the Casitas B-lineage lymphoma proteins (Cbl-b and c-Cbl), and Itchy (Itch), inhibitory tyrosine phosphatases, such as Src homology region 2 domain-containing phosphatases (SHP-1 and SHP-2), inhibitory protein kinases, such as C-terminal Src kinase (Csk), and inhibitory lipid kinases such as Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase (SHIP) and Diacylglycerol kinases (DGKs). This review describes the mechanism of action of eighteen intracellular inhibitory regulatory proteins in T cells within these four classes, and assesses their potential value as clinical targets to enhance the anti-tumor activity of endogenous T cells and CAR-T cells., Competing Interests: Riese has received research funding from Bristol-Myers Squibb (Princeton, NJ), GI Innovations (Seoul, South Korea), Incyte Corporation (Alopocas, DE), and consulting fees from Bristol-Myers Squibb, GI Innovations, Incyte, Forma Pharmaceuticals (Watertown, MA), Ono Pharmaceuticals (Osaka, Japan). Abbvie (North Chicago, IL), and Boehringer Ingelheim (Ingelheim, Germany). The additional authors declare no potential conflicts.

Background: Donor lymphocyte infusion (DLI) is often used to treat leukemic relapse after hematopoietic cell transplantation (HCT). However, the relationship between outcomes and distinct DLI cellular composition has not been previously reported. Additionally, there are limited published data on efficacy in pediatrics. We evaluated whether DLI cellular content and development of graft-versus-host disease (GVHD) impacted disease and influenced overall survival (OS) in children receiving DLI for recurrent leukemia., Methods: We performed an Institutional Review Board-approved, retrospective study investigating all consecutive DLIs given to patients at the Children's Hospital of Wisconsin between 1980 and 2018. Analyses were conducted using Mann-Whitney, Fisher exact, and chi-square tests., Results: Thirty patients ≤20 years old with hematologic malignancies (myeloid [AML/MDS/CML/JMML], n = 23; lymphoid [ALL], n = 7) received DLI to treat post-transplant relapse. We found no significant difference in OS or development of GVHD based on CD3, CD4, CD8, CD56, or CD19 DLI cellular composition. With a median follow-up of 0.69 (range, 0.04-16.61) years, OS at five years was 32% ± 9%.  The lymphoid group had a five-year survival rate at 71% ± 17% compared with the myeloid group at 22% ± 9%, although not statistically significant (P = 0.11).  The development of GVHD did not affect OS (P = 0.62)., Conclusion: Here, we report a single-center, long-term experience of pediatric DLIs. Surprisingly, many children with ALL were able to achieve durable remissions. Although cellular composition did not have a significant effect on GVHD or OS in our small study, engineering DLI products to maximize specific effector cell populations could be one strategy to improve efficacy., (© 2019 Wiley Periodicals, Inc.)

Natural killer (NK) cells generate proinflammatory cytokines that are required to contain infections and tumor growth. However, the posttranscriptional mechanisms that regulate NK cell functions are not fully understood. Here, we define the role of the microRNA cluster known as Mirc11 (which includes miRNA-23a, miRNA-24a , and miRNA-27a ) in NK cell-mediated proinflammatory responses. Absence of Mirc11 did not alter the development or the antitumor cytotoxicity of NK cells. However, loss of Mirc11 reduced generation of proinflammatory factors in vitro and interferon-γ-dependent clearance of Listeria monocytogenes or B16F10 melanoma in vivo by NK cells. These functional changes resulted from Mirc11 silencing ubiquitin modifiers A20, Cbl-b, and Itch, allowing TRAF6-dependent activation of NF-κB and AP-1. Lack of Mirc11 caused increased translation of A20, Cbl-b, and Itch proteins, resulting in deubiquitylation of scaffolding K63 and addition of degradative K48 moieties on TRAF6. Collectively, our results describe a function of Mirc11 that regulates generation of proinflammatory cytokines from effector lymphocytes., (©2019 American Association for Cancer Research.)