bluuebunny/crossref_metadata_2025 huggingface dataset:
Group 3 innate lymhoid cells are absent in DOCK8-defective HIES patients
Authors: Eken Ahmet , Okus Fatma Zehra , Patiroglu Turkan , Erdem Serife , Karakukcu Musa , Cansever Murat , Donmez-Altuntas Hamiyet , Canatan Halit , Oukka Mohamed , Topal Erdem , Kiyim Ayca , Karakoc Elif , Metin Ayse , Reisli Ismail , Uygun Vedat , Baris Safa , Keles Sevgi , Ozen Ahmet , Karasu Gulsun , Unal Ekrem
Published: January 2023

Innate lymphoid cell (ILC) subsets ILC1, ILC2 and ILC3 mirror image the functions helper CD4+ T cell subsets, Th1, Th2 and Th17, respectively; rely on similar transcription factors for their development, and produce similar effector cytokines. Thus, they have recently been shown to be crucial for protective immunity in mice. More importantly, in several chronic inflammatory diseases activated or increased frequency of ILCs have been reported in the circulation or affected tissues of patients. Our previous work with Dock8-defective Dock8pri/pri mice revealed that Dock8 is required for the development/function and survival of murine ILC3s. Thus Dock8pri/pri mice lacked ILC3s and was susceptible to Citrobacter rodentium infections. However to date, whether DOCK8 regulates ILC3 development or functions in humans has not been addressed. In the current study, using 11 DOCK8 mutant patients from across Turkey, we show, for the first time, that humans with DOKC8 deficiency lack peripheral ILC3s. Reduction in blood ILC3 could be verified by flow cytometry. Additionally, sorted total blood ILC population obtained from HIES patients with DOCK8 mutations shows diminished ILC3-specific Gm-csf, AhR and il23r gene expression in line with ILC3 loss. The defect is dramatic in ILC3s and less so in ILC2s in circulation. Flow cytometric examination of 11 patients revealed full restroration of ILC3s following hematopoietic stem cell transplantation. We also show that sorted peripheral ILC precursor population obtained from DOCK8 mutant patients has impaired capacity to proliferate in ILC1-ILC2-ILC3 polarizing conditions. This is the first report in the literature to show a selective deletion of ILC3s in HIES patients with DOCK8 mutations.
The Role of Glycogen Metabolism in Glycolytic Reprogramming Required for Dendritic Cell Immune Responses
Authors: Amiel Eyal , Thwe Phyu M.
Published: January 2023

Dendritic cells (DCs) are potent professional antigen presenting cells of the immune system and serve as a bridge between innate and adaptive immune responses. Activated DCs undergo a process of maturation defined by cytokine & chemokine secretion, co-stimulatory molecule expression, antigen processing and presentation, and the ability to activate T cells. DC maturation is coupled with an increase in energy demand, which is fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and cellular energy required for DC effector function. Inhibition of glycolysis impairs both the survival and effector function of activated DCs. According to the prevailing model, TLR-driven glycolysis in DCs is sustained primarily by increases in extracellular glucose uptake. While non-immune cells, specifically hepatocytes and muscle cells, can store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen metabolism in supporting DC immune responses has not previously been described. Our data show that DCs possess intracellular glycogen stores and express enzymes essential for glycogen metabolism. Furthermore, inhibition of glycogen metabolism significantly impairs key activation parameters and effector functions of these cells. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function.
Ly49H-dependent IFNγ protein production by NK cells requires cytokine signaling that induces IFNγ mRNA transcription.
Authors: Piersma Sytse J. , Pak-Wittel Melissa A. , Yokoyama Wayne M.
Published: January 2023

Natural killer (NK) cells recognize and attack target cells, including cancer and pathogen-infected cells, through a combination of activating and inhibitory signals that follow receptor–ligand interactions. NK cells play a critical role in controlling murine cytomegalovirus (MCMV) infection in C57BL/6 mice, which is dependent on interactions between the Ly49H NK cell activation receptor and its MCMV-encoded ligand m157. This interaction is not sufficient to activate NK cells and control MCMV infection. We previously reported that IL-12 and type I IFN produced early during MCMV infection induce granzyme B and perforin in NK cells, which allows them to efficiently kill virus-infected cells upon recognition of m157 through Ly49H. We now show that m157-dependent IFNγ production also requires cytokine signaling. Using quantitative PCR, the PrimeFlow RNA assay, and chemical inhibitors we found that cytokine signaling was required for IFNγ mRNA transcription, whereas signaling through Ly49H resulted in translation of IFNγ mRNA into protein. Furthermore, initial exposure of NK cell to cytokines enables them to produce IFNγ protein later in the absence of cytokines. We propose the following model: MCMV infection induces cytokine production including IFN-I and IL-12. These cytokines induce the production of cytolytic proteins and IFNγ mRNA. As a result, the NK cells are poised and ready for attack. Virus-infected cells are subsequently recognized by NK cells trough activating ligands such as m157. This activation signaling results in IFNγ production and the release of lytic granules, which results in elimination of the virus infected cells. Together these two pathways enable NK cells to swiftly attack and eradicate virus infections.
Role of telomerase expression in T cell differentiation and proliferation
Authors: Patrick Michael S. , Cheng Nai-Lin , Kim Jaekwan , Dong Fangyuan , An Jie , Weng Nan-Ping
Published: January 2023

To understand the role of telomerase in T cell differentiation and function, we analyzed human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity in six T cell subsets from 85 healthy human donors (aged 17–82 years old). We found that levels of hTERT mRNA and telomerase activity were higher in CD4 T cell subsets as compared to corresponding CD8 T cell subsets, and decreased from naïve (TN) to memory (central, TCM and effector, TEM). In all six subsets, in vitro activation with anti-CD3/CD28 antibody-coated beads led to increased amounts of hTERT mRNA (from 12 to 105-fold) as well as telomerase enzymatic activity, while the differences in hTERT amounts and telomerase activity among subsets remained in the same order observed prior to stimulation. We next analyzed the impact of different amounts of hTERT and induced telomerase activity on proliferation and lifespan of these subsets in vitro. We found that proliferation and survival were most robust in CD4 TN and poorest in CD8 TEM, correlating with their respective levels of hTERT mRNA and telomerase activity both pre-activation and over the course of a 15-day culture following a single stimulation. These findings suggest that greater hTERT expression and telomerase activity promote T cell viability and proliferative potential, and that differentiation is associated with loss of proliferative potential in T cells. We are currently analyzing whether inhibition of hTERT and telomerase activity will lead to reduced proliferation and viability in TN cells.

This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.
Sostdc1 regulates natural killer cell maturation and cytotoxicity
Authors: Millan Alberto Jovani , Elizaldi Sonny , Lee Eric , Murugesh Deepa , Loots Gabriela G. , Manilay Jennifer O.
Published: January 2023

Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified novel roles for sclerostin domain containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (KO) mice display a progressive accumulation of transitional NK cells (CD27+CD11b+, tNK) with age, indicating a partial developmental block. The Ly49 repertoire on NK cells in KO mice is also changed. Lower frequencies (%) of KO splenic tNKs express inhibitory Ly49G2, but higher % of activating Ly49H+ and D+ cells. However, the % of Ly49I+, G2+, H+ and D+ populations were universally decreased at the most mature (CD27− CD11b+, mNK) stage. We hypothesized that the Ly49 repertoire in Sostdc1-KO mice would correlate with NK killing ability. We observed that KO NK cells are hyporesponsive against MHC-I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-gamma expression to controls. Further investigation is required to determine Sostdc1’s role in NK cell cytotoxicity and self/non-self recognition. Sostdc1 is expressed in the bone periosteum, and we discovered Sostdc1 expression in NK cells. Consistent with this, reciprocal BM transplant experiments showed that Sostdc1 regulates NK cells through cell-extrinsic and cell-intrinsic manners. Transcriptomic profiling should identify if Sostdc1’s known roles in Wnt and BMP signaling are active, and reveal novel effects of Sostdc1 on NK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation, and NK cell cytotoxicity, and identify potential NK cell niches.
Antigen binding to B cells activates a metabolic program that in the absence of a second signal leads to mitochondrial dysfunction and cell death
Authors: Akkaya Munir , Roesler Alexander S. , Traba Javier , Miozzo Pietro , Akkaya Billur , Theall Brandon P. , Sohn Haewon , Pena Mirna , Sack Michael N. , Pierce Susan K.
Published: January 2023

Activation of B cells to proliferate and differentiate into antibody secreting cells is regulated to avoid unwanted antibody responses by two temporally distinct signals, the first provided by antigen binding to the B cell receptor (BCR) and the second by T helper cells. Despite the central role of these two signals in driving antibody responses, our understanding of the consequences of each signal is incomplete. Here using in vitro and in vivo models, we investigated the metabolic changes that accompany B cell activation and show that antigen binding to the BCR signals for rapid increases in both oxidative phosphorylation and glycolysis, however early events following antigen engagement including spreading and contraction of antigen bound BCR and internalization of the bound antigen depend solely on the energy produced by oxidative phosphorylation. Changes in gene expression, mitochondrial performance and glucose uptake follow the initial activation, preparing the cells to respond to a second signal. However, in the absence of a second signal B cells undergo progressive loss of mitochondria function and glycolytic capacity ultimately leading to apoptosis. Mitochondria dysfunction is a result of the gradual accumulation of intracellular calcium which leads to inefficient oxidative phosphorylation and increased ROS production and eventually swollen patches of mitochondria. This process can be avoided for approximately nine hours after B cell antigen binding by either T helper cells or by signaling through Toll-like receptor 9. Thus, antigen binding to B cells appears to activate a metabolic program that imposes a short time window in which B cells either receive a second signal and survive or alternatively face elimination.
Lack of TGFβR signaling confers more mature, activated, and educated phenotypes to NK cells leading to resistance to MCMV infection
Authors: Jang Youngsoon , Laouar Yasmina
Published: January 2023

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. Balb/c mice lacking LY49H are more susceptible to MCMV infection than B6 mice. Strikingly, CD11cdnR mice that lack TGFβR signaling in NK cells are protected from MCMV infection in both strains. It is established that unlicensed NK cells that are not inhibited by self-MHC class I can control viral infection more than licensed NK cells. We found that CD11cdnR mice have increased frequency of unlicensed (LY49G2+) NK cells and slightly decreased frequency of licensed (LY49C/I+) NK cells in B6 strain. Moreover, TGFβ-resistant NK cells showed more activated phenotypes such as increased granzyme B protein expression and more KLRG1+ expression than control NK cells in both licensed and unlicensed cells. Interestingly, in CD11cdnR mice even unlicensed NK cells have a significant capacity to produce IFN-γ when triggered by anti-NK1.1, but not in control mice. Our data suggest that inactivation of TGFβR signaling might have a critical role in the licensing of NK cell and lead to the resistance against MCMV infection.
CRTAM shapes the microbiota and enhances the severity of gut infection.
Authors: Perez-Lopez Araceli , Nuccio Sean-Paul , Ushach Irina , Zlotnik Albert , Raffatellu Manuela
Published: January 2023

Gut lymphocytes and the microbiota establish a reciprocal relationship that tunes the host immune response. For example, the gut microbiota is required for T helper (Th)-17 cell differentiation; Th17 cytokines, in turn, stimulate the intestinal epithelium to produce antimicrobial proteins that control the gut microbiota. Residency and maintenance of T cells in the gut is controlled by Class I-restricted T cell-associated molecule (CRTAM). We thus evaluated whether CRTAM influences the gut microbiota composition. We found that CRTAM expression caused a shift in several members of the gut microbiota under homeostatic conditions. As the gut microbiota and T cells contribute to host defense against intestinal pathogens, we tested whether CRTAM plays a role during infection with the enteric pathogen Salmonella. We found lower levels of Salmonella colonization in the gut of Crtam−/− mice, along with lower expression of the Th17 cytokines Il17 and Il22 in the cecum. Salmonella-infected Crtam−/− mice exhibited fewer gut T cells and IL-17-producing cells. To investigate whether the altered gut microbiota composition of Crtam−/− mice influenced the host response to infection, we transplanted the fecal microbiota from WT or Crtam−/− littermate mice into germ-free mice, and then infected the mice with Salmonella. Relative to mice transplanted with “WT” fecal microbiota, mice transplanted with “Crtam−/−” microbiota exhibited lower numbers of T cells and IL-17-producing cells in the gut upon infection. Our results thus suggest an interplay between CRTAM expression and the gut microbiota, which ultimately impacts the mucosal immune system and the host response to enteric pathogens.
Absence of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis
Authors: Sahoo Manoranjan , Katara Gajendra K. , Bilal Mahmood Y. , Kulshrestha Arpita , Ibrahim Safaa A. , Fleetwood Sara , Beaman Kenneth D.
Published: January 2023

Vacuolar ATPase (V-ATPase) is expressed on surface of most cancer cells where it acidifies the extracellular milieu and promotes metastasis. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells, plays a key role during breast tumor progression and metastasis. However, the role of a2V in hematopoietic stem cells (HSC) during breast tumor progression is not known. Therefore, to understand the role of a2V in HSC during breast tumor progression, we conditionally knocked out a2V (cKO) from the HSC of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the spleen of the a2V cKO mice compared to control mice. We hypothesized that the reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that lack of a2V leads to larger and faster growing breast tumors compared to controls. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of the tumor microenvironment demonstrated that out of 499 genes those were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and pro-apoptotic genes. Collectively, these results demonstrate that deletion of a2V from the HSC leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster growing and larger breast tumor.
Arginase-1 is neither constitutively expressed in nor require for myeloid-derived suppressor cell (MDSC)-mediated inhibition of T cell proliferation
Authors: Kidder Koby S. , Bian Zhen , Shi Lei , Liu Yuan
Published: January 2023

Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit T cell proliferation by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition of T cell proliferation. Employing Percoll density gradient centrifugation, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and exhibit potent inhibition on T cell proliferation induced via TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. These MDSC, though suppressive toward T cell proliferation, do not hinder TCR-induced IFNγ secretion or granzyme B expression. Despite effectively inhibiting T cell proliferation, both G- and M-MDSC exhibit no arginase-1 expression; however, arginase-1 can be induced by exposure to TCR-activated T cells or their culture medium, but not by T cells activated by other means or growing tumor cells. Western blot analysis revealed that TCR-activated T cells confer arginase-1 expression in MDSC by secreting cytokines that manifest as two distinct signaling-relay axes, IL-6-to-IL-4 or GM-CSF/IL-4-to-IL-10. Specifically, IL-6 signaling increases IL-4R on MDSC, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R on MDSC, allowing IL-10-mediated induction. Surprisingly, our data demonstrate that the induction of arginase-1 is not conducive, per se, for MDSC-mediated inhibition of T cell proliferation, as neither supplementation of L-arginine nor arginase-1 inhibitor rescue T cell proliferation. Instead, direct cell contact is required for the inhibition and is not dampened by PD-L1 blockade or SIRPα deficiency.
Tumor-resident macrophages modulate intestinal barrier function through sialylation of Mucin 1 in IBD and colitis-associated cancer.
Authors: Cascio Sandra , Kvorjak Michael , Hashash Jana Al , Sriram Raahul , Hartman Douglas , Binion David , Finn Olivera J.
Published: January 2023

Mucin 1 (MUC1) is a transmembrane glycoprotein overexpressed and hypoglycosylated in chronic inflammatory conditions and human adenocarcinoma compared to healthy tissues. We have reported that AOM/DSS-treated human MUC1.Tg mice showed higher tumor incidence and greater inflammation when compared to wild-type (WT) mice. Moreover, high expression levels of pro-inflammatory cytokines, including TNF-α and IL-6, were found in MUC1+ inflamed colon tissues and exogenous TNF-α promoted the transcriptional activity of MUC1 as well as over-expression of its hypoglycosylated form. As macrophages are the major source of TNF-α and IL-6, we analyzed infiltrating type 1 (M1) and type 2 (M2) macrophages into the inflamed and tumorigenic colon tissues. Immunofluorescence assay indicated increased presence of macrophages, and in particular CD206+ (M2), in inflamed colon tissues of MUC1.Tg mice compared to WT mice. In addition, immunostaining of macrophages in colon tissues from IBD patients showed expression of hypoglycosylated MUC1 and the presence of macrophages expressing CD163+, a human M2 marker. Interestingly, in vitro co-culture model system indicated that M1 and M2 polarized macrophages, induced different glycoforms of MUC1 in HT-29 colon cancer cells. In addition, macrophage-secreted factors modulated glycosyltrasferase enzymes, sush as ST6GalNAc1 and ST3GAL1, involved in O-glycan sialylation. We are currently dissecting the molecular basis by which macrophages induced hypoglycosylated and tumor forms of MUC1 during inflammation and colitis-associated cancer (CAC). Our findings will elucidate novel functions of tumor-associated macrophages in altering the intestinal barrier function in IBD and CAC.
A novel mechanism of innate immune dysfunction in untreated Chronic Lymphocytic Leukemia.
Authors: Manso Bryce A. , Zhang Henan , Mikkelson Molly G. , Gwin Kimberly A. , Secreto Charla R. , Ding Wei , Parikh Sameer A. , Kay Neil E. , Medina Kay L.
Published: January 2023

The consequences of immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The known immune abnormalities are not able to consistently explain these complications and it is not easy to correct their immune status. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced functional capacity of hematopoietic progenitors in BM using colony forming unit assays and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations. Consistent with the reduction in HSPCs, monocytes and natural killer cells in BM were reduced and the reduction in BM was recapitulated in blood. Finally, we report imbalances in protein levels of the transcriptional regulators HIF-1α, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients, adding to the complex immunodeficiency state of these patients and the reduced capacity of the innate immune system.
IL-36γ promotes a local immune response via the formation of tertiary lymphoid structures in colorectal carcinoma.
Authors: Weinstein Aliyah M. , Chen Lu , Giraldo Nicolas , Petitprez Florent , Lacroix Laetitia , Brzana Emily A. , Fridman Wolf H. , Sautes-Fridman Catherine , Storkus Walter J.
Published: January 2023

Tertiary lymphoid structures (TLS) form at sites of chronic inflammation, and locally prime the immune system independently of secondary lymphoid organs. The intratumoral presence of TLS has been associated with a positive prognostic outcome for patients with solid tumors, including colorectal carcinoma. Thus, the therapeutic induction of TLS would be hypothesized to confer clinical benefit to patients.

Indeed, the therapeutic introduction of the IL-1 family cytokine IL-36 gamma into the tumor microenvironment (TME) of murine MC38 colon carcinoma leads to delayed tumor progression and the development of TLS at 5 days post-treatment. The TLS present with characteristics of secondary lymphoid organs, including PNAd+ high endothelial venules (HEV) surrounded by CD3+ T cells and CD11c+ dendritic cells, but are considered as “non-classical” due to the absence of a defined B cell zone. The formation of TLS is dependent upon the adaptive immune response, as IL-36 gamma-treated tumors grown in Rag2−/− animals fail to present with PNAd+ vasculature within the tumor. Thus, crosstalk between the immune system and cells of the vasculature is key to the efficacy of the IL-36 gamma-induced anti-tumor immune response.

In human colorectal carcinoma, IL-36 gamma was also observed to promote the local anti-tumor immune response. Expression of IL-36 gamma by vascular endothelial cells of HEV was associated with an increased density of CD20+ B cells in TLS. Specific IL-36 gamma production by CD68+ macrophages was associated with an increased infiltration of CD4+ central memory T cells into the TME. Therefore, IL-36 gamma is a pro-inflammatory mediator involved in the anti-tumor immune response in both murine and human colorectal cancer.
Phytohemagglutinin activates CD8 T cells in a model of the tumor microenvironment.
Authors: Riggs James Edward , Lomakova Yelizavet , Goldman Naomi , Somerville John
Published: January 2023

Tumors often include a high proportion of immune modulatory cells and molecules that restrain the anti-cancer response. Activation of T cells to eliminate cancer cells within the immune suppressive tumor microenvironment (TME) remains a challenge. We have shown that C57BL/6J peritoneal cavity (PerC) cell culture models features of macrophage (Mθ)-rich TMEs as TCR ligation fails to activate T cells unless IFNγ is neutralized or iNOS is inhibited. We tested other forms of T cell activation and found phytohemagglutinin (PHA) distinctive in the ability to markedly expand CD8 T cells in the TME model. Neither IFNγ neutralization or iNOS inhibition were required for this response. IFNγR−/− PerC cells revealed that the PHA response is IFNg-dependent, yet this mitogen triggered less IFNγ production than TCR ligation. PHA-FITC staining revealed strong binding to Mfs and PerC cell addition to normally quiescent spleen (SP) cell cultures led to a marked increase in T cell expansion. The stimulatory capacity of PHA might reflect an ability to bind Mθs with CD8 T cells. Intriguingly, PerC Mf expression of the T cell inhibitory PD-L1 molecule was lower after PHA stimulation than that seen following TCR ligation. If PHA can increase CD8 T cell activation within the TME without increasing PD-L1 expression it might serve as an antitumor immune stimulant.
Brightfield and Fluorescence Imaging using 3D PrimeSurface® Ultra-Low Attachment Microplates
Authors: Dang Anju , Larson Brad
Published: January 2023

Culturing cells in three-dimensions (3D) has become a well established approach as it is more representative of the in vivo environment than traditional two-dimensional (2D) cultures. Allowing cells to interact with each other in a spheroid creates a micro-environment which mimics in vivo tissue and is a better model for examining the effect of drugs in cancer. Developing uniform spheroids becomes especially important as it forms the basis for robust and reliable assays. S-BIO PrimeSurface® cultureware are ultra low attachment (ULA) dishes and plates that promote scaffold free, self assembly spheroid formation. The plates are pre-coated with a proprietary hydrophilic polymer that enables spontaneous spheroid formation of uniform size. PrimeSurface 96 and 384 ULA plates have good optical clarity making them highly suitable for brightfield and fluorescent imaging. Imaging technologies such as the BioTek Cytation™ 5 allows researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. Incorporation of z-stacking and projection techniques in the Gen5™ Microplate Reader and Imager Software create in-focus images of spheroidal cells, allowing accurate, robust, and repeatable determination of the effect of test molecules or conditions. In this app note, we present data generated with BioTek Cytation 5 using PrimeSurface ULA plates to develop simple robust spheroid assays for brightfield and fluorescence imaging.
Tumor transcriptome analysis and immune infiltrate profiling suggests a role for the IFNL3/IFNL4 genomic locus in liver cancer
Authors: Onabajo Olusegun O. , Wang Fang , Banday Rouf , Prokunina-Olsson Ludmila
Published: January 2023

HCV infection and cirrhosis are among the strongest risk factors for developing liver cancer. Impaired HCV clearance is strongly associated with a dG allele of a genetic variant, rs368234815-TT/dG; this allele creates an open reading frame for interferon lambda 4 (IFNL4). The dG allele has also been linked to reduced risk of cirrhosis. Despite strong association of HCV and cirrhosis with liver cancer, no link between rs368234815-TT/dG and liver cancer has been reported. To evaluate potential association of rs368234815-TT/dG with liver cancer, we explored resources of The Cancer Genome Atlas (TCGA). We used a genetic variant rs12980275-A/G located downstream of IFNL3 gene as a TCGA-genotyped proxy for rs368234815-TT/dG. Using multivariate statistical models, we observed that liver cancer patients homozygous for rs12980275-G allele (corresponds to rs368234815-dG allele) had less cirrhosis but reduced survival. Tumor transcriptome analysis showed association of rs12980275-G allele with immunosuppressed tumor microenvironment in non-cirrhotic patients. By deconvoluting tumor infiltrating immune cell signatures, we observed lower levels of CD8+ and follicular helper T cells, but increased M1 macrophages in non-cirrhotic patients with rs12980275-G allele. Transcriptome analysis also identified metallothioneins (MTs), which are involved in removal of reactive oxygen species in the liver, to be differentially expressed according to rs12980275 genotypes. Using hepatoma cell lines, we show that MTs are induced by IFNL4. Our results suggest an association of IFNL3/IFNL4 locus with both an immunosuppressed tumor microenvironment in liver cancer, and increased expression of MTs which could contribute to reduced fibrosis.
Identification of novel α-fetoprotein-specific T cell receptors to redirect human T cells for hepatocellular carcinoma immunotherapy
Authors: He Yukai , Zhu Wei He/Wei , Peng Yibing , Wang Lan , Hong Yuan , Jiang Xiaotao , Li Qi , Liu Heping , Huang Lei , Wu Juan , Celis Esteban , Merchen Todd , Kruse Edward
Published: January 2023

Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T cell receptors (TCR) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, by using lentivector and peptide immunization, we identified a population of CD8-T cells in HLA-A2 transgenic AAD mice that recognized AFP158 epitope on human HCC cells. Adoptive transfer of the AFP158-specific CD8-T cells from immunized AAD mice eradicated HepG2 tumor xenografts as large as 2cm in diameter in immunocompromised NSG mice. We then established T cell hybridoma clones from the AFP158-specific mouse CD8-T cells and identified three sets of paired TCR genes out of 5 hybridomas. Expression of the murine TCR genes redirected primary human T cells to bind HLA-A2/AFP158 tetramer. The TCR gene-engineered human T cells (TCR-T) specifically recognized HLA-A2+AFP+ HepG2 tumor cells and produced effector cytokines. Importantly, the TCR-T cells could specifically kill HLA-A2+AFP+ HepG2 tumor cells without significant toxicity to normal hepatocytes in vitro. Adoptive transfer of the AFP-specific human TCR-T cells could eradicate HepG2 tumors in NSG mice.

Conclusion

We have identified novel AFP-specific murine TCR genes that can redirect human T cells to specifically recognize and kill HCC tumor cells, and those AFP158-specific TCRs have a great potential to engineer a patient’s autologous T cells to treat HCC tumors.
Pan-filovirus monoclonal antibody cocktail protects against lethal challenge with Marburg virus in non-human primates.
Authors: Prugar Laura I. , Dorosky Danielle , Holtsberg Frederick , Shulenin Sergey , Vu Hong , Howell Katy , Bakken Russell , Brannan Jennifer , Dye John M. , Aman M. Javad
Published: January 2023

While disregarded in the past, the use of antibody therapeutics in the treatment of filovirus infection has been gaining traction in recent years. Early studies in our lab with the treatment of non-human primates (NHPs) with polyclonal antibodies from convalescent NHPs demonstrated protection against challenges with a lethal dose of Ebola virus (EBOV). Successes with monoclonal antibody (mAb) cocktails such as ZMapp™ have encouraged the development of a cocktail that may be protective against multiple species of filovirus. This may ultimately allow for the prepositioning of a single cocktail in filovirus prone locations, rather than storing and maintaining multiple cocktails at each location. We have previously described three antibodies, FVM04 and CA45, which can neutralize multiple species of ebolavirus, and MR191, which can neutralize marburgviruses, and have shown efficacy in rodent and NHP models of filovirus disease. In this study, we evaluated the efficacy of a pan-ebolavirus cocktail (FVM04+CA45) in combination with the Marburg virus specific mAb MR191 in a pan-filovirus cocktail (FVM04+CA45+MR191), in the NHP model of Marburg virus disease. Both the pan-ebolavirus and pan-filovirus cocktails have been shown to be efficacious against lethal challenge with Sudan virus (SUDV) or EBOV in the rhesus macaque model. These data are the first to demonstrate protective efficacy of MR191 as part of a pan-filovirus cocktail against MARV in the NHP model of disease.
Comparing NKT cell therapy to oseltamivir phosphate (Tamiflu®) for controlling pandemic H1N1 influenza
Authors: Artiaga Bianca Libanori , Yang Guan , Loeb Julia , Richt Jürgen , Abbott Jeffrey , Lednicky John , Driver John
Published: January 2023

The continuing global burden of disease from influenza infections reveals an urgent need for new approaches that more efficiently protect and potentially avoid the emergence of drug-resistant strains over time. One strategy could be to therapeutically target natural killer T (NKT) cells, an innate-like lymphocyte subset capable of inducing potent antiviral immune responses. We previously reported that disease outcome is significantly improved when influenza-infected pigs are intranasally treated with the NKT cell superagonist α-galactosylceramide (αGC). Here, we compared the protective effects of αGC to 7DW8-5, an αGC derivative that promotes pro-inflammatory immune responses, as well as oseltamivir phosphate (Tamiflu®), a neuraminidase inhibitor used to treat and prevent influenza. A total of 20 pigs challenged with H1N1 A/California/04/2009 (CA04) (4 per group) were administered vehicle, 7DW8-5, or αGC and Tamiflu®, alone or in combination. Four other pigs were mock infected and vehicle treated. Symptoms, viral shedding and peripheral blood leukocytes were measured for 7 days post infection (dpi). Pigs were then euthanized to examine viral titers and immune responses in the airway and lymphoid organs. Pigs treated with αGC and 7DW8-5 but not Tamiflu restored weight gain of infected pigs to the level of un-infected animals. All treatments eliminated virus shedding by 2 dpi compared to infected and untreated pigs that continued to shed virus throughout the study. So far, our results suggest that NKT cell therapy is similar to Tamiflu® for reducing the severity and transmission of influenza infections in pigs. Thus, it may be possible to use NKT cell superagonists to limit the spread of influenza viruses amongst swine and humans.
Sequential and liver-targeting activation of T cells overcomes PDL1-mediated viral persistence
Authors: Liu Longchao , Peng Hua , Fu Yang-Xin
Published: January 2023

Breaking the immune tolerance induced by persistence of hepatitis B virus (HBV) to achieve viral clearance has been challenging due to the unique liver tolerant microenvironment. In this study, we explored whether liver-targeted therapeutic strategies are able to reverse liver tolerant microenvironment. Using an adenovirus vaccine expressing mouse LIGHT (CD258) and HBsAg (Ad-L-H) to target liver, we observed that immunization of Ad-L-H induced a robust antigen-specific T-cell response in naïve mice, especially accumulated in liver. By adoptively transferring primed splenocytes to carrier mice, we demonstrated that Ad-L-H but not conventional vaccine primed T cells were sufficient to control HBV in carrier mice lacking endogenous T cells. However, in immune competent carrier mice, adoptively transferred antigen-specific T cells expressed high level of PD1 and failed to clear HBV. Finally, sequential combination of liver targeted immunization with anti-PDL1 blockage resulted in protective immunity. In conclusion, targeting liver with sequential vaccine and checkpoint blockade is a novel strategy for controlling HBV in the tolerant host.
Development of novel C type lectin receptor agonists as Th17-inducing adjuvants for next generation TB vaccines
Authors: Smith Alyson J. , Buhl Cassandra , Child Robert , Ettenger George , Schoener Roman , Burkhart Daivd J. , Ryter Kendal , Evans Jay T.
Published: January 2023

Mycobacterium tuberculosis (Mtb), the causative pathogen of tuberculosis (TB), is one of the leading causes of death among adults. Extensively drug resistant TB is an emerging threat shown to be spreading as a result of transmission; as such it could benefit greatly from the development of a new, effective vaccine. Mtb is structurally composed of many immunostimulatory elements, including trehalose 6,6′-dimycolate (TDM). It has recently been shown that these cell wall lipids act to stimulate the immune system through engagement of the innate, C type lectin receptor Mincle. Because of their potent immune stimulating properties these compounds have come into focus as potential vaccine adjuvants but are limited due to toxicity concerns. Therefore, we created a library of synthetic trehalose-based compounds with varying structures and assessed their ability to modulate innate immunity in human and mouse immune cells. We optimized the acyl chain configuration for maximal immune stimulation in human cells and discovered divergent structure-activity-relationships between murine and human responses. We also assessed the ability of the compounds to induce a Th17-favorable cytokine profile from primary human cells. Finally, in vivo studies of the optimal in vitro determined compound demonstrated good Th17-inducing activity against a candidate Mtb recombinant antigen. Our data demonstrates the species and structure-specific activity of our novel compounds and clearly highlights the necessity of multi-species structure-activity evaluation for potential therapeutic molecules even at the earliest stages of investigation. As well this study demonstrates the clear potential for Mincle agonist compounds for use as vaccine adjuvants.
Use of GLA-nanoalum as an effective adjuvant for a therapeutic ID93 TB vaccine
Authors: Baldwin Susan L. , Larsen Sasha E. , Reese Valerie A. , Pecor Tiffany , Granger Brian , Khandhar Amit , Fox Christopher B. , Reed Steven G. , Coler Rhea N.
Published: January 2023

Tuberculosis (TB) caused by the intracellular bacterium Mycobacterium tuberculosis (Mtb) reportedly killed 1.3 million people in 2016 and is the leading cause of death caused by a single infectious organism. Increasingly worrisome is the ability of Mtb to develop extensive drug resistance. According to the 2017 WHO global TB report, there were 600,000 new rifampicin-resistant cases in 2016, and almost half a million cases with multiple drug resistant (MDR) TB. The development of new host-targeted therapeutic strategies that prevent the outgrowth of resistant mutants and/or modulate the immune response to combat Mtb infection and/or reduce disease pathology is one solution to prevent the generation of antibacterial resistance and treat drug resistant (DR)-TB. Here we evaluate a novel nanoalum adjuvant formulation containing a synthetic TLR4 agonist, glucopyranosyl lipid adjuvant (GLA), with our clinical ID93 protein as an immunotherapeutic vaccine. We show that immunotherapy with ID93+GLA-nanoalum is effective against Mtb when given as an adjunct to drug treatment in a mouse TB therapy model.
-Intranasal immunization with Bacillus anthracis spore antigens sensitizes mice to death from inhalational anthrax in a B cell-dependent manner
Authors: Weilhammer Dina R. , Dunkle Alexis D. , Fischer Nicholas O. , Gilmore Sean F. , Blanchette Craig , Peters Sandra , Boone Tyler , Lehmann Doerte , Hoeprich Paul , Driks Adam , Rasley Amy
Published: January 2023

There is considerable interest in developing an improved Bacillus anthracis (BA) vaccine that would provide broader, more rapid protection of naïve populations. Here, we investigate intranasal (i.n.) immunization as a method of inducing a mucosal immune response against BA spore antigens (BAAs) to facilitate early recognition and clearance. Nanolipoprotein particles (NLPs) were used as a nanoparticle platform for co-localized delivery of multiple spore proteins in conjunction with monophosphoryl lipid A (MPLA) adjuvant via i.n. immunization in mice. Immunization with BAA-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar lavage. Surprisingly, when challenged with fully virulent BA, mice immunized with BAA-MPLA-NLPs died significantly faster than unimmunized mice. This hastened time to death following i.n. immunization was not antigen or adjuvant specific, nor related to the NLP platform, as similar results were observed using cholera toxin and CpG as adjuvants co-administered with BAAs in the absence of NLPs. Previous work has shown that B cells can traffic BA spores from the lungs to the lung-draining lymph nodes. Therefore, we investigated a role for B cells in sensitizing immunized mice to BA challenge. B cells in the lung increased following i.n. immunization, and depletion of B cells from immunized mice prior to challenge reversed the sensitized phenotype. Spore binding to B cells was enhanced by pre-incubation of spores with immune sera in a manner that was dependent upon the exosporium protein BclA. These results suggest i.n. immunization is not tenable as a protective strategy against inhalational anthrax due to an unexpected consequence of B cell expansion following immunization.
Novel cyclin D1-based DC vaccine inhibits TNBC tumor growth
Authors: Gibb Matthew James , Kroll Mitchell , Su Qingtai , Zurawski Sandy , Zurawski Gerard , Igyarto Botond
Published: January 2023

Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis after the failure of standard chemotherapeutic regimens. Immunotherapies activating the patient’s own immune system to fight various types of cancer are emerging as a powerful alternative and complement to standard chemotherapy and radiation therapy. Since dendritic cells (DCs) regulate every aspect of adaptive immune responses, we sought to generate DC vaccines that target TNBC. Recently, we found that targeting CD40 on DCs induced superior anti-tumor cytotoxic T cell responses compared to targeting other DC receptors. In the quest to find tumor-specific (overexpressed) antigens, we recently identified the cell cycle protein, cyclin D1, as a potential antigen for a DC-targeting-based vaccine against TNBC. Cyclin D1 is overexpressed in many tumors, including TNBC, and we found that cyclin D1-specific effector T cells could be readily expanded from the blood of TNBC patients or normal human donors. To test whether targeting cyclin D1 to DCs through CD40 can control tumor growth, we implemented a novel TNBC mouse model in our laboratory. This consists of a transgenic mouse expressing human CD40 in a C57BL/6 background and a recently characterized syngeneic EO771-LMB metastatic breast cancer cell line with TNBC characteristics. We found that in a therapeutic setting, targeting cyclin D1 (highly conserved between mice and humans) to DCs through CD40 led to significant inhibition of tumor growth in mice.

Thus, these data suggest that cyclin D1-based DC-targeting vaccines could be a novel immunotherapeutic candidate for TNBC.
Augmenting vaccine immunogenicity through the use of natural human anti-Rha antibodies and monoclonal Fc domains
Authors: Hossain Md Kamal , Vartak Abhishek , Sucheck Steven , Wall Katherine Ann
Published: January 2023

In order to obtain an effective cellular and humoral immune response, a vaccine construct has to be captured, processed and presented by antigen presenting cells (APCs) through their major histocompatibility complex (MHC) molecules. One effective way to enhance antigen presentation is through an antibody-dependent antigen uptake mechanism by targeting FcγR receptors of APCs that can bind with the Fc portion of an immune complexed antibody. Anti-Rhamnose (Anti-Rha) antibody is among the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. Binding rhamnose of a rhamnose-containing vaccine with murine anti-Rha antibody has previously been proven effective for capturing and processing of antigen by APCs via stimulatory Fc receptors. We show that human anti-Rha antibodies enhance the uptake of rhamnose-containing antigen by murine APCs in vitro. In-vivo, passive transfer of human anti-Rha antibodies into mice improves the priming of T cells to Rha-antigen. We synthesized and tested an anti-cancer vaccine containing a lipoglycopeptide cancer antigen in a Rha-bearing liposome and demonstrated improved immunogenicity. Also, we showed that direct conjugation of the Fc domain of an antibody to a liposomal vaccine instead of rhamnose can be a good approach for efficient uptake and presentation, as we can bypass the need for anti-Rha antibodies. A liposomal vaccine bearing IgG3Fc has been found to be equally effective as the Rha-vaccine at priming CD4+ T cells. Priming of CD8+ T cells for IFN-γ release and tumor cytotoxicity was promising in comparison to anti-Rha antibody targeting.
Efficient generation of MCC oncoprotein-specific CD4+ T cells for potential adoptive immunotherapy
Authors: Davies Sarah I. , Chang Mark J. , Whitehill Gregory , Koklanaris Eleftheria , Ito Sawa , Brownell Isaac , Buck Christopher , Barrett John , Muranski Pawel
Published: January 2023

Merkel Cell Carcinoma (MCC) is an aggressive skin tumor caused by Merkel Cell Polyomavirus (MCV) in ~80% of cases that responds poorly to standard chemotherapies. Virus positive tumors have a low mutational burden, and are driven by alternatively spliced T-antigens (TAG), small T (sT) and truncated large T antigen (LTT). These intracellular proteins are constitutively expressed in MCC tumors and are required for tumor survival. We hypothesized that MCV TAG can be targeted by cytotoxic T lymphocytes for potential use as an adoptive T cell immunotherapy to treat MCC. Peripheral blood T cells were stimulated with autologous dendritic cells pulsed with overlapping peptide libraries derived from MCV sT and LTT in the presence of a pro-inflammatory cytokine cocktail (IL-1β, IL-6, IL-7, IL-15, IL-21, IL-23, and TGFβ). This culturing method increases yields by 6 fold of antigen-specific T cells that produce TNFα and IFNγ, from both MCC subjects and healthy donors. These expanded TAG-specific cells are predominately CD4+ cells of a memory phenotype that expresses low levels of PD-1, but no expression of IL-17A. Growing evidence suggests that CD4+ Th1 and Th17 cells can have potent anti-tumor activities, especially against MHC class II positive tumors. Three virus-positive and two virus-negative MCC cell lines were tested for HLA-DR expression by flow cytometry. We found that MCC cell lines, both virus positive and virus negative, can be induced to express HLA-DR in a dose-dependent fashion in response to IFNγ in vitro. These results suggest that MCC virus positive tumors can likely be directly recognized and destroyed by adoptively transferred Th1 or Th17 CD4+ T cells and offers a promising avenue for adoptive T cell immunotherapy as a MCC treatment.