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Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here we tested the ability of cell penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low-endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Hepatocellular carcinoma (HCC) patients with reduced natural killer (NK)–cell numbers and function have been shown to have a poor disease outcome. Mechanisms underlying NK-cell deficiency and dysfunction in HCC patients remain largely unresolved. α-Fetoprotein (AFP) is an oncofetal antigen produced by HCC. Previous studies demonstrated that tumor-derived AFP (tAFP) can indirectly impair NK-cell activity by suppressing dendritic cell function. However, a direct tAFP effect on NK cells remains unexplored. The purpose of this study was to examine the ability of cord blood-derived AFP (nAFP) and that of tAFP to directly modulate human NK-cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique proinflammatory, IL2-hyperresponsive phenotype in NK cells as measured by IL1β, IL6, and TNF secretion, CD69 upregulation, and enhanced tumor cell killing. In contrast, extended coculture with tAFP, but not nAFP, negatively affected long-term NK-cell viability. NK-cell activation was directly mediated by the AFP protein itself, whereas their viability was affected by hydrophilic components within the low molecular mass cargo that copurified with tAFP. Identification of the distinct impact of circulating tAFP on NK-cell function and viability may be crucial to developing a strategy to ameliorate HCC patient NK-cell functional deficits. Cancer Immunol Res; 5(6); 493–502. ©2017 AACR.

Supplementary Table S1. Donor median secretion levels (pg/ml). Supplementary Figure S1. Evaluation of AFP internalization mechanism. Supplementary Figure S2. nAFP and tAFP enhance IL-2-induced NK cell activation in a dosedependent manner. Supplementary Figure S3. Intracellular IL-6 and IL-1β staining of NK cells. Supplementary Figure S4. IL-1β and IL-6 production by CD3+ and CD14+ cell contaminants. Supplementary Figure S5. nAFP and tAFP stimulations induce a pro-inflammatory, IL-2-hyperresponsive activation profile in NK-cells. Supplementary Figure S6. Confirmation of protein observations by NanoString single molecule imaging. Supplementary Figure S7. nAFP and tAFP stimulations induce a pro-inflammatory, IL-2-hyperresponsive activation profile in NK-cells. Supplementary Figure S8. nAFP and tAFP stimulations enhance NK cell-mediated tumor killing. Supplementary Figure S9. FACS analysis strategy for evaluating internalized AFP by NK cells. Supplementary Figure S10. Gating strategy used to evaluate NK cell viability in long-term cultures. Supplementary Figure S11. tAFP inhibits proliferation and promotes the death of NK cells in extended cultures. Supplementary Figure S12. RP-HPLC Fractionation of tAFP and its cargo.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

Macrophage accumulation and nitrosative stress are known mechanisms underlying age-related cardiovascular pathology and functional decline. The cardiac muscle microenvironment is known to change with age, yet the direct effects of these changes have yet to be studied in-depth. The present study sought to better elucidate the role that biochemical and biomechanical alterations in cardiac tissue have in the altered phenotype and functionality of cardiac resident macrophages observed with increasing age. To accomplish this, naive bone marrow derived macrophages from young mice were seeded onto either functionalized poly-dimethyl-siloxane hydrogels ranging in stiffness from 2kPA to 64kPA or onto tissue culture plastic, both of which were coated with either young or aged solubilized mouse cardiac extracellular matrix (cECM). Both biomechanical and biochemical alterations were found to have a significant effect on macrophage polarization and function. Increased substrate stiffness was found to promote macrophage morphologies associated with pro-inflammatory macrophage activation, increased expression of pro-inflammatory inducible nitric oxide synthase protein with increased nitric oxide secretion, and attenuated arginase activity and protein expression. Additionally, exposure to aged cECM promoted attenuated responsivity to both canonical pro-inflammatory and anti-inflammatory cytokine signaling cues when compared to young cECM treated cells. These results suggest that both biomechanical and biochemical changes in the cardiovascular system play a role in promoting the age-related shift towards pro-inflammatory macrophage populations associated with cardiovascular disease development.

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000’s of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei.Teaser SentenceCRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

The liver contains a mixture of hepatocytes with diploid or polyploid (tetraploid, octaploid, etc.) nuclear content. Polyploid hepatocytes are commonly found in adult mammals, representing ~90% of the entire hepatic pool in rodents. The cellular and molecular mechanisms that regulate polyploidization have been well characterized; however, it is unclear whether diploid and polyploid hepatocytes function similarly in multiple contexts. Answering this question has been challenging because proliferating hepatocytes can increase or decrease ploidy, and animal models with healthy diploid-only livers have not been available. Mice lacking E2f7 and E2f8 in the liver (liver-specific E2f7/E2f8 knockout; LKO) were recently reported to have a polyploidization defect, but were otherwise healthy. Herein, livers from LKO mice were rigorously characterized, demonstrating a 20-fold increase in diploid hepatocytes and maintenance of the diploid state even after extensive proliferation. Livers from LKO mice maintained normal function, but became highly tumorigenic when challenged with tumor-promoting stimuli, suggesting that tumors in LKO mice were driven, at least in part, by diploid hepatocytes capable of rapid proliferation. Indeed, hepatocytes from LKO mice proliferate faster and out-compete control hepatocytes, especially in competitive repopulation studies. In addition, diploid or polyploid hepatocytes from wild-type (WT) mice were examined to eliminate potentially confounding effects associated with E2f7/E2f8 deficiency. WT diploid cells also showed a proliferative advantage, entering and progressing through the cell cycle faster than polyploid cells, both in vitro and during liver regeneration (LR). Diploid and polyploid hepatocytes responded similarly to hepatic mitogens, indicating that proliferation kinetics are unrelated to differential response to growth stimuli. Conclusion: Diploid hepatocytes proliferate faster than polyploids, suggesting that the polyploid state functions as a growth suppressor to restrict proliferation by the majority of hepatocytes.

SummaryRegular surveillance testing of asymptomatic individuals for SARS-CoV-2 has played a vital role in SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants’ experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

The liver contains diploid and polyploid hepatocytes (tetraploid, octaploid, etc.), with polyploids comprising ≥90% of the hepatocyte population in adult mice. Moreover, polyploid hepatocytes form multipolar spindles in mitosis, leading to individual chromosome gains and/or losses and random aneuploidy. The molecular mechanisms that regulate polyploidization have been well-characterized; however, it is unclear if diploid and polyploid hepatocytes function similarly in multiple contexts. The effect of aneuploidy on liver function is also unknown and the degree of liver aneuploidy is debated, with reports showing aneuploidy affects 5-60% of hepatocytes. We used mice lacking E2f7 and E2f8 in the liver (LKO), which have a polyploidization defect. We found diploid hepatocytes were enriched 20-fold in LKO livers and nearly all LKO hepatocytes were euploid compared to control hepatocytes, which were mostly aneuploid, suggesting polyploids are required for production of aneuploid progeny. Livers from LKO mice maintained normal function but became highly tumorigenic when challenged with tumor-promoting stimuli, suggesting that tumors in LKO mice were driven, at least in part, by diploid hepatocytes capable of rapid proliferation. Indeed, LKO hepatocytes were more proliferative and out-competed control hepatocytes in competitive repopulation studies. To eliminate potentially confounding effects associated with E2f7/E2f8 deficiency, diploid and polyploid hepatocytes from wild-type mice were examined. The wild-type diploid cells also showed a proliferative advantage, entering and progressing through the cell cycle faster than polyploid cells, both in vitro and during liver regeneration. Finally, to investigate the role of diploids and polyploids in chronic injury we bred LKO mice onto a tyrosinemia background, a disease model where the liver can develop disease-resistant, regenerative nodules. Survival was significantly reduced in tyrosinemic LKO mice, compared to controls, but they maintained the ability to form regenerative nodules. Molecular analyses revealed that the nodules in the tyrosinemic livers were generated via aneuploidy and inactivating mutations. In summary we identified diverse, context-specific roles for diploids and polyploids in liver function, demonstrating diploid hepatocytes are the most proliferative and have the highest capacity for liver regeneration, and polyploid hepatocytes are required for the formation of aneuploid progeny and facilitate adaptation to chronic liver disease.

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

Declining function of the immune system, termed "immunosenescence," leads to a higher incidence of infection, cancer, and autoimmune disease related mortalities in the elderly population. (1) Increasing interest in the field of immunosenescence is well-timed, as 20% of the United States population is expected to surpass the age of 65 by the year 2030. (2) Our current understanding of immunosenescence involves a shift in function of both adaptive and innate immune cells, leading to a reduced capacity to recognize new antigens and widespread chronic inflammation. The present review focuses on changes that occur in haematopoietic stem cells, macrophages, and T-cells using knowledge gained from both rodent and human studies. The review will discuss emerging strategies to combat immunosenescence, focusing on cellular and genetic therapies, including bone marrow transplantation and genetic reprogramming. A better understanding of the mechanisms and implications of immunosenescence will be necessary to combat age-related mortalities in the future.

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Hepatocellular carcinoma (HCC) patients with reduced natural killer (NK)–cell numbers and function have been shown to have a poor disease outcome. Mechanisms underlying NK-cell deficiency and dysfunction in HCC patients remain largely unresolved. α-Fetoprotein (AFP) is an oncofetal antigen produced by HCC. Previous studies demonstrated that tumor-derived AFP (tAFP) can indirectly impair NK-cell activity by suppressing dendritic cell function. However, a direct tAFP effect on NK cells remains unexplored. The purpose of this study was to examine the ability of cord blood-derived AFP (nAFP) and that of tAFP to directly modulate human NK-cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique proinflammatory, IL2-hyperresponsive phenotype in NK cells as measured by IL1β, IL6, and TNF secretion, CD69 upregulation, and enhanced tumor cell killing. In contrast, extended coculture with tAFP, but not nAFP, negatively affected long-term NK-cell viability. NK-cell activation was directly mediated by the AFP protein itself, whereas their viability was affected by hydrophilic components within the low molecular mass cargo that copurified with tAFP. Identification of the distinct impact of circulating tAFP on NK-cell function and viability may be crucial to developing a strategy to ameliorate HCC patient NK-cell functional deficits. Cancer Immunol Res; 5(6); 493–502. ©2017 AACR.

Under normal physiological conditions, skeletal muscle has a robust potential for tissue regeneration, which requires the activation of muscle progenitor cells (MPCs). However, degenerative muscle disease triggers recurrent muscle fiber destruction and repair, requiring persistent activation of MPCs, and ultimately leading to depletion of the progenitor cell pool. Duchenne muscular dystrophy (DMD) is a classic example of degenerative muscle disease which results in a reduced and dysfunctional progenitor cell pool, a phenomenon known as MPC depletion. Although DMD is a congenital disease, patients do not exhibit symptoms until 3–5 years of age, which coincides with MPC depletion. Replenishment of the MPC pool through cell therapy has therefore been explored as a potential treatment for DMD and other related degenerative muscle diseases. Several preclinical and clinical studies are ongoing to evaluate the efficacy of stem cell transplantation in patients with DMD. Notably, these studies are investigating various cell sources, genetic manipulations, and administration routes to achieve optimal treatment. In the past, studies have been hindered by cell availability, poor engraftment, and limited myogenic differentiation ability. Recent findings have revealed the many complexities of the degenerative muscle microenvironment, which include chronic inflammation, ectopic ossification, fatty infiltration, and pathological fibrosis. This hostile environment likely influences progenitor cell survival, engraftment, and differentiation after stem cell transplantation. This chapter aims to describe several clinical and preclinical examples of cell transplantation, highlighting the advantages and disadvantages of various approaches, while emphasizing the importance of the cellular and molecular disease environment in order to afford successful cell therapy.

Our previous analysis using genome-wide microarray expression data revealed extreme overrepresentation of immune related genes belonging the Natural Killer (NK) Cell Mediated Cytotoxicity pathway (hsa04650) in human abdominal aortic aneurysm (AAA). We followed up the microarray studies by immunohistochemical analyses using antibodies against nine members of the NK pathway (VAV1, VAV3, PLCG1, PLCG2, HCST, TYROBP, PTK2B, TNFA, and GZMB) and aortic tissue samples from AAA repair operations (n = 6) and control aortae (n = 8) from age-, sex- and ethnicity-matched donors from autopsies. The results confirmed the microarray results. Two different members of the NK pathway, HCST and GRZB, which act at different steps in the NK-pathway, were actively transcribed and translated into proteins in the same cells in the AAA tissue demonstrated by double staining. Furthermore, double staining with antibodies against CD68 or CD8 together with HCST, TYROBP, PTK2B or PLCG2 revealed that CD68 and CD8 positive cells expressed proteins of the NK-pathway but were not the only inflammatory cells involved in the NK-pathway in the AAA tissue. The results provide strong evidence that the NK Cell Mediated Cytotoxicity Pathway is activated in human AAA and valuable insight for future studies to dissect the pathogenesis of human AAA.

The immune system, and in particular macrophages, are heavily implicated in wound healing, response to infection, and cancer progression, all of which are linked to mortality in the elderly population. Recently, it has been shown that tissue-resident macrophages from spleen and peritoneum become dysfunctional with aging, likely due to the aged microenvironment. The liver contains approximately 80% of total mammalian tissue-resident macrophages, known as Kupffer cells. Kupffer cells can be divided into two F4/80+ macrophage subsets: CD68+ and CD11b+ cells, which have different origins and functions. Currently, it is unclear how these macrophage subsets are affected by aging. We sought to characterize differences in the Kupffer cell compartment from young (8–10 week) and aged (18–20 month) C57/Bl6 mice using immunofluorescent staining on tissue sections and flow cytometry. Preliminary histological analyses showed that the area of F4/80+ staining did not differ between young and aged mice. Interestingly the area of CD68+ staining was significantly higher in the aged murine livers. In addition, the area of CD32+ staining, which marks both endothelial cells and macrophage precursors, was significantly greater in aged mice, consistent with previous reports of blood vessel thickening in aged livers. Flow cytometry analysis confirmed differences in the macrophage subsets between young and aged murine livers. In addition, functional changes in phagocytosis were noted with aging, suggesting dysfunction of innate abilities. Characterizing the differences in the subsets of tissue-resident macrophages during aging will increase our understanding of age-related pathologies that will aid in the design of therapies in the future.

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors which contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explantation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. The present study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Objectives: Abdominal aortic aneurysm (AAA), a dilatation of the infrarenal aorta, typically affects males >65 years. The pathobiological mechanisms of human AAA are poorly understood. The goal of this study was to identify novel pathways involved in the development of AAAs. Methods: A custom-designed ‘AAA-chip’ was used to assay 43 of the differentially expressed genes identified in a previously published microarray study between AAA (n = 15) and control (n = 15) infrarenal abdominal aorta. Protein analyses were performed on selected genes. Results: Altogether 38 of the 43 genes on the ‘AAA-chip’ showed significantly different expression. Novel validated genes in AAA pathobiology included ADCY7, ARL4C, BLNK, FOSB, GATM, LYZ, MFGE8, PRUNE2, PTPRC, SMTN, TMODI and TPM2. These genes represent a wide range of biological functions, such as calcium signaling, development and differentiation, as well as cell adhesion not previously implicated in AAA pathobiology. Protein analyses for GATM, CD4, CXCR4, BLNK, PLEK, LYZ, FOSB, DUSP6, ITGA5 and PTPRC confirmed the mRNA findings. Conclusion: The results provide new directions for future research into AAA pathogenesis to study the role of novel genes confirmed here. New treatments and diagnostic tools for AAA could potentially be identified by studying these novel pathways.

Alpha-fetoprotein (AFP) is an oncofetal antigen produced by hepatocellular carcinomas (HCC). Previous studies demonstrated that tumor-derived AFP (tAFP) is a glycoprotein that has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cells have been attributed to tAFP-mediated immunosuppression of DC. However, a direct tAFP effect on NK cells remains unexplored. Here we compared the ability of cord blood-derived AFP (nAFP) to that of tAFP to modulate human NK cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique pro-inflammatory, IL-2 hyperresponsive phenotype in healthy donor NK cells as measured by CD69 upregulation, IL-1β, IL-6 and TNF secretion, and enhanced tumor cell killing. In contrast, extended co-culture with tAFP, but not nAFP, inhibited NK cell proliferation and viability. NK cell activation was directly mediated by the AFP protein itself, while their viability was affected by the low molecular mass cargo that co-purified with tAFP. Overall, these data show that nAFP and tAFP induce similar yet distinct changes in NK cell function and viability, respectively. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients.

Alpha fetoprotein (AFP) is an oncofetal antigen commonly produced by hepatocellular carcinomas (HCC). Previous studies have shown that tumor-derived AFP (tAFP) has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cell frequency and function have partially been attributed to tAFP-mediated immunosuppression of DC function. However, a direct tAFP effect on NK cells remains unclear. Here we examine the ability of cord blood-derived AFP (nAFP) and tAFP to modulate human NK cell activity in vitro. We show that exposure to tAFP or, especially, nAFP proteins induces a unique pro-inflammatory NK cell activation profile as measured by CD69 upregulation, IL-1β and IL-6 secretion, and enhanced tumor cell killing. Interestingly, AFP-treated plus interleukin-2 (IL-2) stimulated cultures promote a degree of NK cell activation that is higher than that of NK cells activated with IL-2 alone by both phenotypic and functional measures, including elevated IFN-γ and GM-CSF secretion. To confirm that the observed effects are directly mediated by AFP protein, we confirmed that NK cells can readily bind to and take up nAFP and tAFP. The observed synergism between AFP and IL-2 may be mediated by the ability of AFP to modulate IL-2 receptor signaling, as shown by the ability of AFP to upregulate CD25, and downregulate CD122 and CD132 on NK cells. Overall, these data show that nAFP and tAFP induce a unique pro-inflammatory, IL-2 hyperresponive phenotype in NK cells. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients, and for the development of an effective HCC-targeting immunotherapy.