bluuebunny/crossref_metadata_2025 huggingface dataset:
Tuft cell tropism enables norovirus evasion of CD8+ T cells and viral persistence
Authors: Strine Madison S , Filler Renata , Alfajaro Mia Madel , Damo Martina , Gonzalez David , Haberman Ann Marie , Joshi Nikhil , Wilen Craig
Published: August 2023

Human norovirus (HNV) is a leading cause of viral gastroenteritis. Infection fails to elicit durable immunity and can cause chronic asymptomatic shedding. It is not known how norovirus escapes the adaptive immune response, and rigorous studies of the host and viral determinants underlying immune evasion are impeded by the lack of scalable cell culture systems and reverse genetic tools. Murine norovirus (MNV) recapitulates many aspects of HNV and enables studying norovirus biology in vitro and in vivo. MNVCW3 causes non-persistent infection with long-lasting immune memory, whereas MNVCR6 causes persistent infection despite inducing functional MNV-specific CD8+ T cells. We recently identified that MNVCR6 and other persistent MNV strains require tuft cells for chronic enteric infection. Tuft cells are rare, chemosensory epithelial cells with roles in pathogen surveillance, epithelial repair, and tumorigenesis. To interrogate tuft cell interactions with CD8+ T cells, we adoptively transferred JEDI (Just EGFP Death Inducing) CD8+ T cells that detect EGFP200–208 presented on H2-Kd into tuft cell reporter mice (Gfi1b-GFP). Surprisingly, tuft cells at steady state can resist JEDI CD8+ T cell-mediated killing despite normal levels of surface MHC-I. In contrast, Lgr5+ intestinal stem cells and extraintestinal tuft cells are highly susceptible to clearance by JEDI CD8+ T cells. We further find that JEDI CD8+ T cells cannot clear nor prevent MNVCR6 infection of tuft cells, suggesting that tuft cells offer a replicative niche for immune escape and viral persistence. Whether HNV infects tuft cells during chronic asymptomatic infection and whether tuft cell-derived cancers can evade CD8+ T cells will warrant further exploration in the future.

Supported by grants from the NIH (K08 A1128043, R01 AI148467) and the NSF (DGE1752134)
Inhibition of SARS-CoV-2-mediated thromboinflammation by CLEC2.Fc
Authors: Hsieh Shie-Liang
Published: August 2023

Thromboinflammation is the major cause of morbidity and mortality in COVID-19 patients, and post-mortem examination demonstrates the presence of platelet-rich thrombi and microangiopathy in lung and other visceral organs. Moreover, persistent microclots were detected in both acute COVID-19 and long COVID plasma samples. However, the molecular mechanism of SARS-CoV-2-induced thromboinflammation is still unclear. We found that the spleen tyrosine kinase (Syk)-coupled C-type lectin member 2 (CLEC2), which was highly expressed in platelets, interacted with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD) directly. Unlike the thread-like NETs, SARS-CoV-2-induced aggregated NET formation in the presence of wild-type (WT), but not CLEC2-deficient platelets. Furthermore, SARS-CoV-2 spike pseudotyped lentivirus was able to induce NET formation via CLEC2, indicating SARS-CoV-2 RBD engaged CLEC2 to activate platelets to enhance NET formation. Administration of CLEC2.Fc inhibited SARS-CoV-2-induced NET formation and thromboinflammation in AAV-ACE2-infected mice. Thus, CLEC2 is a novel pattern recognition receptor for SARS-CoV-2, and CLEC2.Fc is a promising therapeutic agent to inhibit SARS-CoV-2-induced thromboinflammation and reduced the risk of post-acute sequelae of COVID (PASC) in the future.

Translational Medical Research Program (AS-TM-108-02-10), Biotechnology Research Park Translational Project (AS-BRPT-110-02), Academia Sinica
Th1-guided de novo Th17 response alleviates lung inflammation through IL-17-EGFR-TRAF4 signaling cascade in severe influenza
Authors: Dutta Avijit , Hung Chen-Yiu , Hsiao Sung-Han , Chen Tse-Ching , Chang Chia-Shiang , Huang Yu-Lin , Lin Yung-Chang , Lin Chun-Yen , Huang Ching-Tai
Published: August 2023

Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. The severe disease in older than younger adult 6.5 mice with aging-related thymic involution and waning Th17 response further supports the protective role of the de novo Th17 response in severe influenza. Our results suggest that viral neuraminidase-activated TGF-β of the Th1 cells guides the Th17 evolution. IL-17 up-regulates and phosphorylates the non-canonical IL-17 receptor EGFR, activates the scaffold protein TRAF4, and promotes the translocation of TRAF4 to the EGFR signaling complex. Taken together, Th1-guided de novo Th17 response alleviates lung inflammation through the IL-17-EGFR-TRAF4 signaling cascade in severe influenza.

The Research Center for Emerging Viral Infections receives support from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and NSTC 111-2634-F-182-001 from the National Science and Technology Council, Taiwan. This work was supported by Projects MOST 111-2314-B-182-029 (AD) and MOST-105-2321-B-182A-003-MY3 (AD) from the National Science and Technology Council, Taiwan, and by CMRPG3L1671 (CTH), CMRPVVJ0052 (CTH), and CMRPG3J1771 (CYH) from the Medical Research Project Fund, Chang Gung Memorial Hospital, Taiwan.
Host mRNA deadenylation machinery selectively targets interferon mRNAs, regulating antiviral immunity
Authors: Braun Rachel M , Ferretti Max , Miller Jesse , Castellana Lauren , Lee Jae Seung , Lynch Kristen W , Cherry Sara
Published: August 2023

Elucidating host-mediated regulation of viral infection is critical in the development of antivirals and in understanding viral pathogenesis. Here, through high-throughput RNA interference screening, we determine that CNOT2 promotes SARS-CoV-2 infection in human respiratory epithelial cells. CNOT2 is a non-enzymatic subunit of the CCR4-NOT complex, which plays conserved roles in the control of gene expression by deadenylating the poly(A) tails of host mRNAs, thus targeting them for decay. CCR4-NOT can selectively degrade cohorts of mRNAs that are delivered by RNA Binding Proteins. Tristetraprolin (TTP) is among the best characterized of such cargo adapters and is known to recruit mRNAs containing AU-rich elements in their 3′ UTR to CCR4-NOT for deadenylation and decay. We found that in addition to CNOT2, another non-catalytic subunit of CCR4-NOT, CNOT1, in addition to the selectivity factor, TTP, also promote SARS-CoV-2 infection. Global transcriptomics revealed that CNOT2, CNOT1, and TTP specifically regulate interferons (IFNs) and interferon stimulated genes (ISGs) and that depletion of these mRNA decay factors leads to increased levels of IFNs and ISGs. Moreover, the pro-viral activity of CCR4-NOT components and TTP is dependent on IFN signaling: the CCR4-NOT complex has no effect on SARS-CoV-2 infection when IFN signaling is blocked. This led us to discover that CNOT2 promotes the selective deadenylation of IFN mRNAs, and that depletion of CNOT2 leads to increased poly(A) tail lengths of IFN mRNAs. Therefore, we found that the basal levels of IFNs, and thus IFN signaling, are post-transcriptionally regulated by deadenylation machinery, which in turn impacts susceptibility to viral infections including SARS-CoV-2.

Supported by grants from NIH (T32-AI-007324)
NF-κB signaling promotes SARS-CoV2 infection in human lung epithelial cells
Authors: Etwebi Zienab , Ayyanathan Kasirajan , Miller Jesse , Kamalia Brinda , Li Minghua , Lee Jae Seung , Ferretti Max , Braun Rachel M , Srivastava Tamanna , Dohnalova Lenka , Cox Timothy , Thaiss Christoph , Schultz David , Cherry Sara
Published: August 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is responsible for over 660 million infections, and more than 6 million deaths worldwide. There is an increasing need for more treatment options that addresses SARS-CoV-2 infection and its replication. Innate immune pathways including interferon signaling and inflammatory NF-κB signaling are important players in regulating immune responses, these pathways are often targeted by viral pathogens to enhance viral replication, host immune evasion, and host cell survival. The respiratory tract is the major target for SARS-CoV-2; thus, we are exploring the role of innate immune signaling in regulating infection in these cells. We found that both interferon and NF-κB signaling are induced by SARS-CoV-2 infection as measured by increases in transcription factor localization and gene expression. Moreover, pharmacological inhibition of NF-κB signaling but not interferon signaling blocks SARS CoV-2 infection of these respiratory cells. We are exploring how NF-κB signaling impacts viral infection. Indeed, we found that the activity of the NF-κB pathway is independent of interferon signaling. Further mechanistic studies are underway to define the role of NF-κB in coronavirus infection and suggest that inhibitors of the NF-κB signaling pathway may be a novel pharmacological target for the treatment of SARS-CoV-2.
Investigating links between persistent symptoms after COVID-19 and longitudinal changes in memory T cell responses to EBV, CMV, HSV-1, and SARS-CoV-2.
Authors: Zhong Jiabao , Smith Erika L , McConney Christina S , Bean David J , Belkina Anna C , Snyder-Cappione Jennifer E
Published: August 2023

Post-acute infection syndromes (PAISs) have long been observed following acute infections with bacteria, viruses or other microbes. Overlapping core symptoms across different PAISs suggest underlying mechanism(s) that is putatively independent of the causative agents. During the COVID-19 pandemic, accumulating reports of post-acute sequelae of SARS-CoV-2 infection (PASC), also known as “long Covid”, have provided new clues for the pathogenesis of PAISs, but the mechanistic drivers of persistent symptoms are still unclear. Here, we evaluated temporal changes in the memory T cell repertoire, in both frequency and functional signatures, of twelve individuals initially infected with SARS-CoV-2 in the spring of 2020 (ten outpatient, two hospitalized), with four participants showing evidence of PASC. Longitudinal T cell responses were quantified as the frequencies of cytokine producing cells after overnight (ex vivo) stimulation of PBMCs with peptide pools from cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1) and SARS-CoV-2; cell readouts were obtained using intracellular cytokine staining (ICS). PBMCs were collected from multiple timepoints from acute illness to 12–25 months post-infection. Our unique dataset serves as an indirect readout of viral replication and provides insights to two possible explanations for PASC: the reactivation of latently infected viruses during and after the acute SARS-CoV-2 infection, and the persistence of SARS-CoV-2 antigens triggering chronic inflammation. For future studies, longitudinal T cell responses after acute infections with other microbes will be assessed in a larger cohort to determine the specificity of the findings to SARS-CoV-2 infection.

Supported by MassCPR PASC Subaward #FP-0034311
SARS-CoV-2 infection and Spike protein exposure alter iPSC-derived human brain organoid homeostasis.
Authors: Cappelletti Gioia , Brambilla Lorenzo , Melzi Valentina , Rizzuti Mafalda , Nizzardo Monica , Trabattoni Daria , Corti Stefania , Clerici Mario , Biasin Mara
Published: August 2023

COVID-19 typically causes respiratory disorders, but several COVID-19 patients (30–60%) manifest also a wide range of neurological conditions, advocating for detrimental effects of SARS-CoV-2 or Spike (S) protein on the central nervous system. However, the molecular mechanisms responsible for these disfunctions as well as the potential neurotropism of SARS-CoV-2, are still under investigation. To assess this issue, by using iPSC-derived-human brain organoids (HBO) we evaluated: the expression of SARS-CoV-2 main receptors: ACE2, CD147, NRP1, Furin, TMPRSS2 (qPCR, IF); their infectability by SARS-CoV-2 over-time (QPCR, TCID50, IF); and the effect of SARS-CoV-2-infection and S exposure on HBO transcriptome (qPCR) and secretome (Multiplex ELISA). All the main SARS-CoV-2 receptors are expressed by HBO with lower NRP1 levels compared to CD147 and ACE2. By analyzing viral N1 and N2 gene sequence over time, infectability of VeroE6 cells by SARS-CoV-2 infected HBO supernatants, and nucleocapsid expression by IF we confirmed that HBO may be productively infected by the virus. Furthermore, SARS-CoV-2 infection was accompanied by the activation of apoptotic and stress pathways (caspase3, caspase8, Bcl2, S100B), inflammatory process (CCL2, NLRP3) Interferon Stimulated Genes (IFITM1, IFITM3, STAT1, NFkB) and antigen presentation pathway (ERAP1, ERAP2, HLA-A, TAP). Notably, the trend of the different targets was comparable following SARS-CoV-2 infection and S-stimulation.

These results confirm that SARS-CoV-2 infect HBO probably by binding CD147 and ACE2 receptors and indicate that the exposure to the S protein can affect their homeostasis and exert neurotoxic effects presumably driving the so called long-COVID symptoms.

Partially supported by grants from Fondazione Alessandro and Vincenzo Negroni Prati Morosini and Fondazione Romeo and Erica Invernizzi.
Potentiation of infiltrating adaptive and innate immune cells and metabolic responses by environmental cadmium exposure following respiratory syncytial virus infection
Authors: Go Young-Mi , Kim Ki-Hye , Kang Sang-Moo
Published: August 2023

Cadmium (Cd) accumulates in humans from regular dietary and environmental exposure including cigarette smoke, and occupational sources, and has a twenty-year biologic half-life. Most individuals have pre-existing immunity from early life exposure to respiratory syncytial virus (RSV) which causes serious lung disease and hospitalizations among the high-risk infants and elderly. Little is known about the effect of low environmental levels of Cd on inflammatory immune cell dynamics causing lung pathology in humans with RSV infection at early life. In this study, we examined the effects of low dose long-term Cd exposure after infant age RSV infection on phenotypes of infiltrating immune cells in a mouse model. C57BL/6J mice at 8 days old were exposed to RSV. A subset of RSV-infected mice was subsequently treated with Cd at a low dose in drinking water (3.0 mg CdCl2/L) for 16 weeks. Flow cytometry and histology data showed that the levels of both T cells (CD4+, CD4+IFNg+, CD4+ IL4+, CD4+ IL13+) and innate immune cells (CD11b+, monocytes, neutrophil, eosinophil, dendritic cells, activated dendritic cells) appear to be linked to inflammatory lung pathology. In particular, the results showed that the levels of T cells and innate immune cells were substantially higher in the group of RSV infection followed by Cd treatment than the other groups (Cd or RSV alone), providing evidence that low dose Cd following early-life RSV infection enhanced lung inflammation. Consistently metabolic disruption was evident in the group of RSV infection followed by Cd treatment. Taken together, our study demonstrates that cumulative Cd burden following RSV infection has a significant impact on inflammatory immune cell infiltration, lung pathology, and metabolism.

NIEHS Grants R01 ES031980 NIAID grans R01 AI54656 R21 AI147042
Metabolic profiling of COVID-19 severity and mortality: A targeted metabolomics approach
Authors: Cyprian Farhan S , Doudin Asmaa , Khatib Malkan , Elrayess Mohamed , Yassine Hadi , Musa Abdallah
Published: August 2023

The analysis of metabolites in response to SARS-CoV-2 infection provides a snapshot of the endogenous host metabolism and its role in shaping the interaction with SARS-CoV-2. In this study, using a targeted metabolomics approach, the metabolic signatures of severity and mortality were studied in COVID-19 patients. Our analysis revealed survival and severity-associated shifts in the metabolome of COVID-19 patients, where amino acids, particularly tryptophan and arginine, and fatty acid metabolism were altered in severe patients as compared to mild and asymptomatic cases. Herein, we also report alterations in the sums and ratios of several essential amino acids as well as kynurenine to tryptophan ratio, which is an indicator of indoleamine-2,3-dioxygenase (IDO) activity. Our findings suggest that metabolites of creatine including symmetric dimethylarginine, asymmetric dimethylarginine, and 1-methylhistidine can be used as biomarkers to identify patients at risk of poor outcomes in COVID-19, and that lower levels of these metabolites are associated with higher survival probabilities. Additionally, elevated levels of short chain acylcarnitines and carnitine palmitoyltransferase 1 and 2 enzymes indicators have been observed in critically ill COVID-19 patients, indicating their potential usefulness as predictors of hospital mortality and poor survival outcome, likely due to increased demand for energy, inflammation, and mitochondrial dysfunction. The metabolome analysis of COVID-19 patients can improve our understanding of disease severity by providing insight into the relationship between metabolites and disease progression, which can lead to the development of potential therapeutics and clinical risk models.

This work was supported by Emergency Response Grant ID: 252
Brain Regional Dependence of Antiviral and Inflammatory Genes Following Simian Immunodeficiency Virus Infection and Antiretroviral Therapy
Authors: Ellison Alysha , Franco Jose Javier Rosado , Alvarez Jade , Williams Dionna
Published: August 2023

Human immunodeficiency virus (HIV) initiates a perpetual cycle of inflammation in the brain that results in neuronal damage and consequent neurologic dysfunction. Since HIV preferentially infects and damages specific brain regions, there is a need for study of antiviral and immune markers in a brain region specific manner. We have evaluated expression of antiviral (IFNa, IFNb, TRAIL, MX1) and immune markers (CCL2, IL-6, IL-1b, IL-10) across nine brain regions in our virally suppressed rhesus macaque model of simian immunodeficiency virus (SIV) infection. Rhesus macaques from four study groups (uninfected, SIV, SIV/ART, SIV/ART cessation) were perfused, brain obtained by region, and snap frozen in liquid nitrogen. RNA was extracted and qRT-PCR performed to quantify the genes of interest in each brain region. Antiviral markers (MX1, TRAIL) were expressed in all brain regions and equally expressed across all groups, with occasional increase of MX1 in SIV infected animals. Alternatively, common immune markers (IL-6, CCL2) were not expressed across the brain regions of any study group, excepting the thalamus. Other antiviral (IFN-a) and immune markers (IL-10) were consistently expressed across brain regions in uninfected animals. However, the effects of SIV and ART on expression of these and other pertinent markers (IFN-b, IL-1b) were brain region dependent. Altogether, these data suggest that antiviral and immune marker expression is dynamic and differs depending on the marker and the brain region under investigation. This knowledge has implications for the way these markers are studied in the brain and implies a need for brain region specific investigation.

Supported by grants from the NIH (5R01DA052859-02) and the NIH funded Johns Hopkins University Center for AIDS Research (P30AI094189-01A1)
Signatures of HIV-infected CD4+ T cell resistance to NK cell-mediated killing
Authors: Grasberger Paula , Kucukural Alper , Zapp Maria , Clayton Kiera
Published: August 2023

BACKGROUND

In vitro experiments in which HIV-infected CD4+ T cells are co-cultured with natural killer (NK) cells yield incomplete elimination of the infected cells. We therefore hypothesize that cell-intrinsic pathways in CD4+ T cells are differentially regulated in HIV-infected cells to mediate resistance to NK cell-mediated killing.

METHODS

Mock-infected and HIV-89.6-infected CD4+ T cells from 6 HIV− donors were co-cultured overnight +/− autologous NK cells. Following co-culture, cells were stained for the surface exposed HIV envelope protein using fluorescently conjugated HIV antibodies. Fluorescence activated cell sorting was then used to isolate infected and uninfected cells, followed by bulk RNA-sequencing. Transcripts from infected and uninfected cells co-cultured overnight with versus without NK cells were compared.

RESULTS

RNA-seq analysis of CD4+ T cells that survived co-culture with NK cells revealed dozens of differentially expressed genes in infected, but not uninfected cells. This included upregulation of interferon-stimulated genes, including PD-L1 (padj=3×10−6), and NF-kB-related genes. Additional upregulated genes in the surviving infected cells included c-Fos (padj=1.9×10−5), c-Jun (padj=4.9×10−8), and cathepsin L (padj=5×10−3).

CONCLUSIONS

NF-kB genes that are downregulated in infected cells, potentially through Vpu activity, are normalized in surviving infected cells. This may be due to NK cell targeting of Vpu-mediated HLA-C downregulation. Deconvolution of the relative contributions of specific genes/proteins to target cell resistance will reveal novel targets for the development of therapeutics to eliminate the HIV reservoir.
Perturbation of Invariant Natural Killer T cells by the Clonal Hematopoiesis-related inflammatory environment
Authors: Luca Francesca , Kuznetsova Valeriya , Franceski Alana , Goel Paran , Costa Amanda , Welner Robert
Published: August 2023

Invariant Natural Killer T (iNKT) cells are T cells characterized by a semi-invariant TCR and NK markers. They respond to glycolipid antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d. Our single-cell transcriptomic profiling of several tissues identified marrow iNKT as a unique subset, possibly modulating marrow homeostasis. However, little is known about their role in hematological malignancies and how the disease-related environment impacts iNKT cells. For example, clonal hematopoiesis of indeterminate potential (CHIP) corresponds to a condition in which mutated hematopoietic cells have an enhanced fitness and expansion, increasing the risk of leukemia and other diseases. Loss of TET2 in CHIP promotes myeloid skewing and inflammation. TET2 is a methycytosine dioxygenase catalyzing DNA demethylation of genes involved in proliferation, differentiation and oncogenesis. However, the impact that TET2 loss has on iNKT lineage output and function is yet to be defined. In our chimeric mouse model of CHIP, only 15% of TET2 mutants differentiate into iNKT. We also observe a decrease of non-transformed iNKT cells in the marrow of our mice, suggesting environmental inhibition. We expect these bystander iNKT cells to be functionally impaired where inflammation leads to their chromatin rewiring potentially contributing to disease. This is addressed by studying CHIP-exposed iNKT transcriptomes and chromatin accessibility. Understanding the molecular basis of the functional reprogramming by the disease-related inflammation will improve clinical outcomes for marrow transplantation and for the development of cellular therapies using iNKT cells.
Targeting lL-38 triggers γδ T cell-dependent anti-tumor immunity
Authors: Silva Priscila , Mora Javier , Ernst Andreas , You Xin , Brüne Bernhard , Weigert Andreas
Published: August 2023

The IL-1 family receptor antagonist IL-38 suppresses auto-inflammatory reactions in murine disease models. Given the connection between auto-immunity and anti-tumor immunity, we wondered if targeting IL-38 would affect the latter. The transgenic PyMT mammary carcinoma model was employed to target IL-38 genetically or with a neutralizing antibody. IL-38-deficient (KO) PyMT mice had a markedly delayed tumor growth compared to WT mice, accompanied by increased NK cells, CD8+, NKT, and γδ T cell infiltrates. Accordingly, in human mammary carcinoma, IL-38 expression negatively correlated with infiltrating T cell subsets. This pattern was largely recapitulated with a IL-38-neutralizing antibody, where antibody-treated PyMT WT mice showed a strong decrease in tumor growth compared to IgG control treated animals, accompanied by an increase in CD8+ and γδ T cells. In a therapeutic model of chemoresistance, IL-38 antibody in combination with doxorubicin prevented tumor relapse, which was accompanied by increased in NK cells, CD8+, CD4+, and γδ T cells. To analyze the involvement of γδ T cells, we used γδ-TCR blocking antibody compared to IgG control in IL-38 KO mice. The delay in growth of IL-38 KO PyMT tumors compared to WT PyMT tumors was abolished upon γδ-TCR blockade. This was accompanied by a strong decrease in CD8+ cells upon effective γδ-TCR blockade, suggesting a cross talking between CD8+ and γδ T cells. Whole transcriptome sequencing analysis of PyMT tumors upon IL-38 neutralization and γδ-TCR blockage suggested NOTCH signaling to mediate the link between CD8+ and γδ T cells. Taken together, these results provide evidence for a therapeutic potential of anti-IL-38 antibodies to activate anti-tumor immunity in mammary carcinoma.

Supported by a grant from Deutsche Krebshilfe (70114051)
Regulation of NK cell chemotaxis by KCa3.1 channels
Authors: Alshwimi Abdulaziz O , Chimote Ameet A , Wise-Draper Trisha , Conforti Laura
Published: August 2023

CD8+ T cells and NK cells are the primary line of defense against tumor cells. However, the tumor microenvironment impairs their anti-tumor capabilities. One of the key functions impaired is the ability of T and NK cells to infiltrate the tumor, essential to allow the direct contact with a target cell and its killing. Kv1.3 and KCa3.1 K+ channels have a multitude of essential functions that have been established in CD8+ T cells: Kv1.3 regulates cytotoxicity and KCa3.1 chemotaxis. However, less is known about K+ channels in NK cells. Herein, we performed electrophysiological and 3D chemotaxis experiments to study the activity and functional role of K+ channels in human NK cells. NK cells were isolated from healthy donors (HD) and head and neck cancer (HNC) patients and activated with IL-2 and IL-15 for 24–48 hr. Activation led to an increase in Kv1.3 currents of 49.6% at 24 hr and 434.1% at 48 hr . KCa3.1 activity increased by 42.8% at 24 hr and 54.2% at 48 hr. Chemotaxis experiment showed that activated NK cells move randomly in the collagen matrix and migrated towards the chemokine gradient in presence of CXCL10. Chemotaxis, but not random migration, was inhibited by the selective KCa3.1 blocker TRAM-34 in NK cells from HD and HNC patients. Shk-Dap22, a Kv1.3 blocker, did not alter either chemotaxis or random migration. Overall, TRAM-34 reduced the distance achieved by NK cells in the chemokine gradient by 157% in HD (4 donors) and 89% in HNC NK cells (1 donor). These data highlight the critical role of KCa3.1 channels in NK cell migration and open the possibility of targeting KCa3.1 to facilitate tumor infiltration by NK cells.
Immune checkpoint blockade unleashes Mucosal Associated Invariant T (MAIT) cells in tumor microenvironment of NSCLC patients
Authors: Birla Pakhi , Zhang Boyang , Zhang Jiajia , Gulati Ananya , Yang Lansaol , Johnson Ramona , Lee Alex , Smith Kellie , Sears Cynthia , Shaikh Fyza , Housseau Franck , Pardoll Drew
Published: August 2023

Mucosal associated invariant T (MAIT) cells are innate like T-cells that recognise non-peptide metabolite antigens presented on monomorphic MHC related-1 (MR1) making them an attractive off the shelf tool for cancer immunotherapy. In this study, we investigate the function of MAITs in the tumor microenvironment of neoadjuvant anti-PD1 treated lung cancer patients (NCT02259621). Paired single cell RNA/TCR sequencing analysis revealed an oligoclonal expansion of MAITs comprising of canonical TRAV1–2+ and non-canonical MAITs TRAV1–2−. TCR capture from canonical MAIT clonotypes confirmed their in vitro recognition of 5-OP-RU, a bacteria derived riboflavin derivative. However, the nature of antigens recognised by non-canonical MAITs remains poorly understood. On mining transcriptional profiles of MAITs, we observed a high clonal amplification of canonical MAITs whereas the non-canonical clonotypes expressed a strong activation/exhaustion gene signature. This suggests that both these MAIT populations recognise different types of antigens, are differentially activated, and respond to immune checkpoint blockade (ICB), the non-canonical MAITs being highly dysfunctional. Using bacterial 16S RNA amplicon and metagenomics sequencing, we seek to identify microbial and metabolomic signatures associated with MAIT activation and response to ICB. Our findings will provide further insights into the nature of ligands recognised by MAITs and the mechanisms associated with MR1 dependent recognition and killing of tumor. Investigating the role of unconventional T-cells in response to ICB has the potential to reveal new tumor-associated antigens which could open up avenues for innovative combination immunotherapies.
Targeting γδ T cells for the immunotherapy of endometrial cancer
Authors: Cazzetta Valentina , Portuesi Rosalba , Fiamengo Barbara , Vitobello Domenico , Mikulak Joanna , Mavilio Domenico
Published: August 2023

Endometrial cancer (EC) is the most common gynecological neoplasm and the only gynecologic cancer with increasing incidence and mortality. EC is generally diagnosed at an early stage and is well managed with surgery and/or chemotherapy. However, EC patients with advanced/recurrent disease have a poor outcome and respond poorly to current treatments. Thus, there is the need to better investigate novel approaches. In this context, understanding the biology of the immune system is essential for recognition of new targets as well as optimization of existing immunotherapies.

Growing evidences highlight an important role of both adaptive and innate immunity in generating a suppressive environment. However, little is known about the specific role of unconventional gamma delta (γδ) T cells. Therefore, the aim of this project is to characterize the frequency, phenotype and function of γδ T cells in EC. We used flow cytometry to characterize γδ T cells in peripheral blood (PB) and tumor tissue of EC patients and in PB of healthy donors and analyzed two public datasets of single cell RNA-sequencing to deepen the transcriptional profile of γδ T cells. These analyses revealed that the main subsets of γδ T cells, namely Vδ1 and Vδ2 T cells, infiltrate the tumor of EC patients. Moreover, they are characterized by the upregulation of immune checkpoints, such as PD-1. However, upon stimulation, they maintain their anti-tumor potential producing cytokines, e.g. IFN-γ and TNF-α.

Overall, γδ T cells infiltrate EC and express immune checkpoints that can be targeted to implement the current immunotherapeutic strategies. Thus, further investigations are needed to exploit γδ T cells for EC therapy.
NCG-hIL15 humanized mice - an ideal model for human immune reconstitution of NK cells
Authors: Chen Santi Suryani , Liu Xing , Wu Meirong , Wang Huiyi , Yu Weiwei , Sun Hongyan , Ju Cunxiang , Wang Hongyu , Li Zhiying , Moore Mark , Zhao Jing , Xiang Gao
Published: August 2023

Immunodeficient NCG mice were unable to reconsituted human NK cells, but after knockin of human IL-15 (hIL-15), the NCG-hIL15 mice could assist the reconstitution of human NK cells. Compared with NCG, the level of hIL-15 was significantly increased in heterozygous and homozygous NCG-hIL15 mice. After transplanting human PBMC (huPBMC-NCG-hIL15) or NK cells into NCG-hIL15 (huNK-NCG-IL15), the level of peripheral blood hCD56+ NK cells in huNK-NCG-hIL15 mice were much higher than that in huPBMC-NCG-hIL15 mice. However, the reconstituted proportion of human CD3+ T cells in huNK-NCG-hIL15 mice was not comparable in huPBMC-NCG-hIL15 mice. The level of NK cell reconstitution is also highly dependent on the donor NK cells. The expression of perforin in the peripheral blood of huNK-NCG-hIL15 mice was significantly higher than that in huPBMC-NCG-hIL15 mice. In vitro functional analysis of NK cells from huPBNK-NCG-hIL15 and huPBMC-NCG-hIL15 showed Granzyme B expression in peripheral blood were comparable and human NK cells purified from splenocytes of huPBNK-NCG-hIL15 mice were cytotoxic upon coculture with Raji cells in the presence of Rituximab. Based on these in vitro data, we established huNK-NCG-hIL15 mice subcutaneously engrafted with Raji cells, and the in vivo efficacy of rituximab was evaluated. Efficacy study data showed that rituximab significantly inhibited the Raji tumor cells growth in huNK-NCG-hIL15 mice. Both huPBMC-NCG-hIL15 and huNK-NCG-hIL15 mouse model can rapidly reconstitute functional human NK cells compared to CD34+ HSC engrafted NCG-hIL15 mice. The development of huNK-NCG-hIL15 is an ideal mouse model to specifically evaluate the anti-tumor efficacy of drugs targeting human NK cells.
Microparticle-delivered stimulator of interferon genes (STING) agonist suppresses experimental autoimmune encephalomyelitis (EAE) via expansion of FoxP3+ CD4+ Tregs
Authors: Elzoheiry Manal Ali , Wang Yan , Kasimoglu Ezgi , Seyedsadr Maryamsadat , Batty Cole , Bachelder Eric , Kristy Ainslie , Markovic-Plese Silva
Published: August 2023

Background

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of intracellular STING receptors induces a powerful IFN-I response. Microparticles (MPs), synthesized biodegradable polymer, passively target the APCs, resulting in intracellular delivery of immuno-regulatory treatment with dose sparing and better tolerability. cGAMP MP treatment of EAE induced both IFN-I-dependent and -independent immunosuppressive responses through induction of IL-27 and IL-10.

Results

We show that cGAMP MP treatment reduced clinical disease scores of EAE after 3 doses. By flow cytometry, cGAMP MPs decreased absolute number of mononuclear cells in the CNS infiltrates, and the percentages of CD3− CD45high CD11+b Ly6C+ monocytes, CD11c+ and CD11c+ CD11b+ DCs, as well as their expression of CD80 and CD86, suggestive of tolerogenic phenotype. Moreover, we detected an increased percentage of FOXP3+CD4+ Tregs in CNS infiltrates, with increased expression of IL-10, IL-27R, and Granzyme B, and decreased percentage of IL-17A+CD4+ cells in cGAMP MP-treated mice. Furthermore, the supernatants of the in vitro co-cultured cGAMP MP-treated monocytes with CD3+CD4+, isolated from EAE spleenocytes, show increased IL-10 and IL-27 levels. Additionally, the in vitro D3+CD4+ cells show increased FOXP3+ expression with higher CTLA-4, and GITR in cGAMP treated group. Moreover, FOXP3cre+/+ IL-27RA fl/fl mice showed more aggressive disease compared to wild-type mice in attempt to test whether IL-27 induced effect of cGAMP MPs is mediated via Tregs.

Conclusion

cGAMP MPs suppress RREAE through induction of tolerogenic APCs and increase secretion of IL-10 and IL-27, which induce Treg expansion indicating its potential use as a tolerogenic therapy in patients with RRMS.
Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope
Authors: DiLisio James E , Reyes Kaitlin , Beard K Scott , Neef Tobias , Miller Stephen D , Baker Rocky L , Haskins Kathryn
Published: August 2023

An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX3CR1+ effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction.

NIH - T32 5T32DK120520-03, R01 2R01DK081166-11)

JDRF 2-SRA-2020-907-S-B
Artemotil mediates protection in experimental colitis through the induction of Type 1 regulatory T cells
Authors: Madan Upasna , Verma Bhawna , Binayke Akshay , Awasthi Amit
Published: August 2023

Artemotil (β-arteether) is a semi-synthetic derivative of artemisinin - a natural compound isolated from the plant Artemisia annua and used worldwide as a front-line anti-malarial drug. Besides its anti-malarial effect, several studies have reported its potential use as an anti-tumor and anti-inflammatory agent. Artemotil was found to suppress the generation and function of Th1 and Th17 while promoting the generation of Foxp3+ regulatory T cells (Tregs). The role of Artemotil in modulating the generation and functions of Type 1 regulatory T cells is not yet identified. Tr1 cells are one of the key cell types that are essential for inhibiting inflammatory response through IL-10. In this study, we report an additional role of Artemotil by selectively inducing the generation of Tr1 cells induced by IL-27 by upregulating the signature of Tr1 cells genes such as c-maf and Prdm1, IRF-1, and Batf. We found that co-administer of Artemotil with anti-CD3 antibody increases the induction of IL-10 and frequency of Tr1 cells while suppressing Th1 and Th17 cells in vivo associated with less severe T-cell-induced enteropathy and intestinal inflammation. We further show the anti-inflammatory role of Artemotil in the chronic T-cell-induced colitis model in which Artemotil treatment alleviates the signs of colitis associated with the increased frequencies of Tr1 cells. Taken together, these data suggest that Artemotil provides protection in T-cell-mediated colitis by increasing the expansion of Tr1 cells and inhibiting the generation of Th1 and Th17 cells.

This study was supported by grants from Science & Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (CRG/2018/002653)
Serum Albumin fused Flt3L expands a tolDC compartment and amplifies tolerogenic antigen immunotherapy for the prevention of pathogenic anti-drug antibody responses
Authors: Alpar Aaron T , Wallace Rachel P , Refvik Kirsten C , Hubbell Jeffrey
Published: August 2023

From antibodies to enzyme replacement therapies, biologics and protein drugs have become a major part of the medical market and revolutionized medicine. Repeated administration of many of these drugs, however, leads to humoral responses and potentially drug failure via neutralization, clearance, or immune related infusion reactions. Once pathogenic anti-drug antibodies are formed, patients must take broad immunosuppressive drugs, undergo continuous B cell depletion, or discontinue treatment entirely. Currently, no drugs are approved by the FDA to prevent the induction of anti-drug antibodies. Herein, we characterize the effect of our engineered cytokine, Flt3L-SA, in vivo and demonstrate how our molecule expands the dendritic cells while enhancing a tolerogenic, TGFβ expressing subpopulation. We then leverage these dendritic cells to attenuate the antibody response to the highly immunogenic drug Rasburicase and ultimately reduce antibodies by 100-fold when administered as part of a tolerogenic regime with low dose Rasburicase. We also show how this reduction in antibodies results in fewer and less severe infusion reactions upon final challenge, evidencing both a reduction in the antibody response as well as pathology. Future work will aim to determine the exact mechanism by which this tolerance is achieved as well as demonstrate efficacy in the context of a wide range of extremely immunogenic therapies, including enzyme replacement therapeutics for lysosomal storage disorders and AAV therapies.

Supported via the Chicago Immunoengineering Innovation Center and the Alper Family Foundation
Effects of BAMBI deficiency in the development of Systemic Lupus Erythematosus
Authors: Adrián Paula Pérez , Ruso Fulgencio , Cappitelli Vincenzo , Casado Victoria , Merino Jesús , Merino Ramón
Published: August 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and immune complexes deposition in various organs. Follicular CD4+ T cells play a crucial role in this autoimmune syndrome by activating autoreactive germinal center B cells. The transforming growth factor β (TGBβ) inhibitor BAMBI (Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor) is known to control the differentiation of CD4+ T lymphocytes into either tolerogenic Tregs or pathogenic Th17 cells. BAMBI absence in mutant mice or its pharmacological inhibition with the B101.37 monoclonal antibody promotes an increase in Treg differentiation and activity and at the same time, a reduction in Th17 cell differentiation, resulting in the inhibition of autoimmune disease development. Based on these findings, in the present study we have analysed the effects of BAMBI deficiency in the development of SLE. We have employed the chronic graft-versus-host disease lupus model induced after the transference of 40–60 × 106 lymphocytes from B6 or B6-BAMBI-KO mice into either Bm12 or Bm12-BAMBI-KO recipients. As reported previously, Bm12 mice receiving lymphocytes from B6 mice produce high levels of circulating IgG anti-DNA and anti-nucleosomal autoantibodies and exhibit linear glomerular IgG deposits in their kidneys. In contrast, both Bm12 mice injected with allogeneic B6-BAMBI-KO lymphocytes and Bm12-BAMBI-KO recipients receiving lymphocytes from B6 donors do not produce significant levels of circulating autoantibodies and do not reveal immune complexes deposits. Taken together, these results indicate that the absence of BAMBI in either donor or recipient lymphocytes protects mice against the development of SLE.
BAFF and APRIL signaling through B cell maturation antigen affects regulatory transitional B cells in multiple sclerosis
Authors: Kumar Gaurav , Massey Kaylea , Ko Rose M , Axtell Robert C
Published: August 2023

Multiple sclerosis (MS) is a debilitating neuro-autoimmune disease that can be stratified into relapsing remitting (RR) MS, secondary progressive (SP) MS, and primary progressive (PP) MS. B cells can have both inflammatory and regulatory functions in MS. Transitional B cells are considered to have regulatory functions in MS. BAFF and APRIL, two cytokines, signals through the receptors BAFFR, TACI and BCMA and are thought to drive the regulatory function of transitional B cells. Here, we assessed the frequencies and expression of BAFFR, TACI and BCMA on the surface of regulatory transitional B cells in the blood of RRMS, PPMS and healthy individuals along with soluble versions of these receptors in the plasma. We found that transitional B cells were decreased in both RRMS and PPMS patients. The surface expression of BCMA was significantly lower on transitional B cells from RRMS patients but not PPMS patients compared to healthy individuals with no change in the expression of TACI and BAFFR. In addition, we found elevated levels of soluble BCMA in blood of PPMS patients but not RRMS patients compared to healthy and no difference in soluble BAFFR and TACI. Our data suggests that reduced BCMA signaling is associated with reduced numbers of transitional B cells in RRMS and PPMS. In RRMS, expression of BCMA is reduced on regulatory transitional B cells. In PPMS patients, there are higher levels of soluble BCMA which could act as a blocker of APRIL signaling through BCMA on the surface of transitional B cells. Altogether, our data suggests that reduced BCMA signaling in transitional B cells is contributing to the MS disease and the dysregulation of the BCMA signaling in RRMS and PPMS may be through a different mechanism.

This study was supported by grants from the NMSS (RG-1602-07722), the NIH (R01AI137047 and R01EY027346) awarded to R.C. Axtell.
An albumin interleukin 33 fusion protein with enhanced lymph node retention suppresses murine models of multiple sclerosis
Authors: Budina Erica , Ishihara Jun , Solanki Aniruddhsingh , Nguyen Mindy , Hubbell Jeffrey A
Published: August 2023

Interleukin-33 (IL-33) is an immunoregulatory cytokine that suppresses pathogenic Th17 T cells. Recombinant IL-33 has been shown to prevent disease onset in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. However, the systemic administration of IL-33 has not been clinically translated, in part because the cytokine’s short half-life makes it difficult to achieve therapeutically effective concentrations in the secondary lymphoid organs. To overcome this challenge, we have recombinantly fused mouse serum albumin (SA) to IL-33. ST2(IL-33R)-expressing mouse group 2 innate lymphoid cells treated with serum albumin fused IL-33 (SA IL-33) increased CD25 expression and IL-13 production in a dose-dependent manner. SA fusion prolonged IL-33 half-life and increased its concentration in the lymph nodes of naïve C57BL/6 mice. Prophylactic administration of SA IL-33 prevented the onset MOG-induced EAE, demonstrating equal efficacy to the clinically approved MS drug fingolimod. In mice with already-established EAE, administration of SA IL-33 reduced disease score and improved weight gain. At the cellular level, SA-IL-33 treatment reduced the frequency of CD45+ cells, including disease causing Rorgt+ CD4+ T cells, in the spinal cord. EAE mice treated with SA-IL-33 displayed an increase in ST2+ FoxP3+ CD25+ Tregs and Th2 T cells in both the spleen and spinal cord draining lymph nodes, suggesting that SA-IL-33 treatment suppresses EAE by expanding the protective type 2 response.