IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

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Abstract

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T-cell receptor (TCR) signaling, while its role in CAR signaling is unknown. By electroporation of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.

Authors

Zheng Fu, Zineng Huang, Hao Xu, Qingbai Liu, Jing Li, Keqing Song, Yating Deng, Yujia Tao, Huifang Zhang, Peilong Wang, Heng Li, Yue Sheng, Aijun Zhou, Lianbin Han, Yan Fu, Chen-Zhi Wang, Saurav Kumar Choudhary, Kaixiong Ye, Gianluca Veggiani, Zhihong Li, Avery August, Weishan Huang, Qiang Shan, Hongling Peng

NEUROPILIN-1 INHIBITION SUPPRESSES NERVE-GROWTH FACTOR SIGNALING AND NOCICEPTION IN PAIN MODELS

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Abstract

Nerve growth factor (NGF) monoclonal antibodies inhibit chronic pain yet failed to gain approval due to worsened joint damage in osteoarthritis patients. We report that neuropilin-1 (NRP1) is a co-receptor for NGF and tropomyosin-related kinase A (TrkA) pain signaling. NRP1 was coexpressed with TrkA in human and mouse nociceptors. NRP1 inhibitors suppressed NGF-stimulated excitation of human and mouse nociceptors and NGF-evoked nociception in mice. NRP1 knockdown inhibited NGF/TrkA signaling, whereas NRP1 overexpression enhanced signaling. NGF bound NRP1 with high affinity and interacted with and chaperoned TrkA from the biosynthetic pathway to the plasma membrane and endosomes, enhancing TrkA signaling. Molecular modeling suggested that C-terminal R/KXXR/K NGF motif interacts with extracellular “b” NRP1 domain within a plasma membrane NGF/TrkA/NRP1 of 2:2:2 stoichiometry. G Alpha Interacting Protein C-terminus 1 (GIPC1), which scaffolds NRP1 and TrkA to myosin VI, colocalized in nociceptors with NRP1/TrkA. GIPC1 knockdown abrogated NGF-evoked excitation of nociceptors and pain-like behavior. Thus, NRP1 is a nociceptor-enriched co-receptor that facilitates NGF/TrkA pain signaling. NRP binds NGF and chaperones TrkA to the plasma membrane and signaling endosomes via GIPC1 adaptor. NRP1 and GIPC1 antagonism in nociceptors offers a long-awaited non-opioid alternative to systemic antibody NGF sequestration for the treatment of chronic pain.

Authors

Chloe J. Peach, Raquel Tonello, Elisa Damo, Kimberly Gomez, Aida Calderon-Rivera, Renato Bruni, Harsh Bansia, Laura Maile, Ana-Marie Manu, Hyunggu Hahn, Alex R.B. Thomsen, Brian L. Schmidt, Steve Davidson, Amedee des Georges, Rajesh Khanna, Nigel W. Bunnett

AAV9/SLC6A1 gene therapy rescues abnormal EEG patterns and cognitive behavioral deficiencies in Slc6a1-/- mice

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Abstract

The SLC6A1 gene encodes the gamma-aminobutyric acid (GABA) transporter GAT-1, the deficiency of which is associated with infantile encephalopathy with intellectual disability. We designed two AAV9 vectors, with either the JeT or MeP promoter, and conducted preclinical gene therapy studies using heterozygous and homozygous Slc6a1 KO mice at different developmental ages and various routes of administration. Neonatal intracerebroventricular administration of either vector resulted in significantly normalized EEG patterns in Slc6a1-/- or Slc6a1+/- mice, as well as improvement in several behavioral phenotypes of Slc6a1-/- mice. However, some mortality and adverse effects were observed in neonatal-treated mice. Intrathecal administration of either vector at postnatal day (PND) 5 normalized EEG patterns in Slc6a1+/- mice, but in Slc6a1-/- mice the treatment only rescued nest building without impact on EEG. Both vectors were well-tolerated in all mice treated at PND5 or later (including WT mice), up to 1 year post-injection. Overall, our data demonstrate compelling efficacy when mice are treated at an early development age. We also identified that outside of the neonatal treatment window, the severe homozygous KO model is more refractory to treatment, whereas our treatments in the heterozygous mice, which genotypically match human patients, have resulted in stronger benefits.

Authors

Weirui Guo, Matthew Rioux, Frances Shaffo, Yuhui Hu, Ze Yu, Chao Xing, Steven J. Gray

Endogenous antigens shape the transcriptome and TCR repertoire in an autoimmune arthritis model

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Abstract

The development of pathogenic autoreactive CD4+ T cells, particularly in the context of impaired signaling, remains poorly understood. Unraveling how defective signaling pathways contribute to their activation and persistence is crucial for identifying new therapeutic targets. We profiled a highly arthritogenic subset of naïve CD4+ T cells using bulk and single-cell RNA and TCR sequencing from SKG mice, which develop CD4+ T cell mediated autoimmune arthritis driven by a hypomorphic mutation in Zap70—a key TCR signaling kinase. Despite impaired signaling, these cells exhibit heightened expression of T cell activation and cytokine signaling genes, but diminished expression of a subset of tolerogenic markers (Izumo1r, Tnfrsf9, Cd5, S100a11) compared to wild-type cells. The arthritogenic cells show an enrichment for TCR variable beta (Vβ) chains targeting superantigens from the endogenous mouse mammary tumor virus (MMTV) but exhibit diminished induction of tolerogenic markers following peripheral antigen encounter, contrasting with the robust induction of negative regulators seen in wild-type cells. In arthritic joints, cells expressing superantigen-reactive Vβs expand alongside detectable MMTV proviruses. Antiretroviral treatment and superantigen-reactive T cell depletion curtail SKG arthritis, suggesting that endogenous retroviruses disrupt peripheral tolerance and promote the activation and differentiation of self-reactive CD4+ T cells into pathogenic effector cells.

Authors

Elizabeth E. McCarthy, Steven Yu, Noah Perlmutter, Yuka Nakao, Ryota Naito, Charles Lin, Vivienne Riekher, Joe DeRisi, Chun Jimmie Ye, Arthur Weiss, Judith F. Ashouri

Ferumoxytol nanozymes effectively target chronic biofilm infections in apical periodontitis

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Abstract

Bacterial biofilms are pervasive and recalcitrant to current antimicrobials, causing numerous infections. Iron oxide-nanozymes, including an FDA-approved formulation (ferumoxytol, FMX), show potential against biofilm infections via catalytic activation of hydrogen peroxide (H2O2). However, clinical evidence on its efficacy and therapeutic mechanisms is lacking. Here, we investigate whether FMX-nanozymes can treat chronic biofilm infections and compare their bioactivity to gold-standard sodium hypochlorite (NaOCl), a potent but caustic disinfectant. Clinical performance was assessed in patients with apical periodontitis, an intractable endodontic infection affecting half of the global adult population. Data show robust antibiofilm activity by a single application of FMX with H2O2 achieving results comparable to NaOCl without adverse effects. FMX binds efficiently to bacterial pathogens Enterococcus faecalis and Fusobacterium nucleatum and remains catalytically active without being affected by dental tissues. This allows for effective eradication of endodontic biofilms via on-site free-radical generation without inducing cytotoxicity. Unexpectedly, FMX promotes growth of stem cells of apical papilla (SCAP), with transcriptomic analyses revealing upregulation of proliferation-associated pathways and downregulation of cell-cycle suppressor genes. Notably, FMX activates SCAP pluripotency and WNT/NOTCH signaling that induces its osteogenic capacity. Together, we show FMX nanozymes are clinically effective against severe chronic biofilm infection with pathogen targeting and unique stem cell-stimulatory properties, offering a regenerative approach to antimicrobial therapy.

Authors

Alaa Babeer, Yuan Liu, Zhi Ren, Zhenting Xiang, Min Jun Oh, Nil Kanatha Pandey, Aurea Simon-Soro, Ranran Huang, Bekir Karabucak, David P. Cormode, Chider Chen, Hyun Koo

Beclin 1 prevents ISG15-mediated cytokine storms to secure fetal hematopoiesis and survival

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Abstract

Proper control of inflammatory responses is essential for embryonic development, but the underlying mechanism is poorly understood. Here, we show that under physiological conditions, inactivation of ISG15, an inflammation amplifier, is associated with the interaction of Beclin 1 (Becn1), via its ECD domain, with STAT3 in the major fetal hematopoietic organ of mice. Conditional loss of Becn1 caused sequential dysfunction and exhaustion of fetal liver hematopoietic stem cells, leading to lethal inflammatory cell-biased hematopoiesis in the fetus. Molecularly, the absence of Becn1 resulted in the release of STAT3 from Becn1 tethering and subsequent phosphorylation and translocation to the nucleus, which in turn directly activated the transcription of ISG15 in fetal liver hematopoietic cells, coupled with increased ISGylation and production of inflammatory cytokines, whereas inactivating STAT3 reduced ISG15 transcription and inflammation but improved hematopoiesis potential, and further silencing ISG15 mitigated the above collapse in the Becn1 null hematopoietic lineage. The Becn1-STAT3-ISG15 axis remains functional in autophagy-disrupted fetal hematopoietic organs. These results suggest that Becn1, in an autophagy-independent manner, secures hematopoiesis and survival of the fetus by directly inhibiting STAT3-ISG15 activation to prevent cytokine storms. Our findings highlight a previously undocumented role of Becn1 in governing ISG15 to safeguard the fetus.

Authors

Wen Wei, Xueqin Gao, Jiawei Qian, Lei Li, Chen Zhao, Li Xu, Yanfei Zhu, Zhenzhen Liu, Nengrong Liu, Xueqing Wang, Zhicong Jin, Bowen Liu, Lan Xu, Jin Dong, Suping Zhang, Jiarong Wang, Yumu Zhang, Yao Yu, Zhanjun Yan, Yanjun Yang, Jie Lu, Yixuan Fang, Na Yuan, Jianrong Wang

A large-scale population-based study reveals that gp42-IgG antibody is protective against Epstein-Barr virus-associated nasopharyngeal carcinoma

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Abstract

BACKGROUND. Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), but the existence of NPC protective antibody against EBV-associated antigens remains inconclusive. METHODS. NPC cases and matched controls were identified from prospective cohorts comprising 75,481 participants in southern China. ELISA and conditional logistic regression were applied to assess effects of gp42-IgG on NPC. The expression of HLA-II, the gp42 receptor, in nasopharyngeal atypical dysplasia and its impact on EBV infecting epithelial cells were evaluated. FINDINGS. gp42-IgG titers were significantly lower in NPC cases compared to controls across various follow-up years before NPC diagnosis (P<0.05). Individuals in the highest quartile of gp42-IgG titers had a 71% NPC risk reduction comparing to those in the lowest quartile (odds ratios [OR]Q4vsQ1=0.29, 95% confidence intervals [CIs]=0·15 to 0·55, P<0.001). Each unit antibody titer increase was associated with 34% lower risk of NPC (OR=0.66, 95% CI=0.54 to 0.81, Ptrend <0.001). Their protective effect was observed in cases diagnosed ≥5 years, 1-5 years and <1 year after blood collection (P<0.05). HLA-II expression was detected in 13 of 27 nasopharyngeal atypical dysplasia and its overexpression substantially promoted epithelial-cell-origin EBV infection. CONCLUSION. Elevated EBV gp42-IgG titers can reduce NPC risk, indicating gp42 as a potential EBV prophylactic vaccine design target. TRIAL REGISTRATION. NCT00941538, NCT02501980, ChiCTR2000028776, ChiCTR2100041628. FUNDING. Noncommunicable Chronic Diseases-National Science and Technology Major Project, National Natural Science Foundation of China, Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program, Central Financial Transfer Payment Projects of the Chinese Government, Cancer Research Grant of Zhongshan City.

Authors

Xiang-Wei Kong, Guo-Long Bu, Hua Chen, Yu-Hua Huang, Zhiwei Liu, Yin-Feng Kang, Yan-Cheng Li, Xia Yu, Biao-Hua Wu, Zi-Qian Li, Xin-Chun Chen, Shang-Hang Xie, Dong-Feng Lin, Tong Li, Shu-Mei Yan, Run-Kun Han, Nan Huang, Qian-Yu Wang, Yan Li, Ao Zhang, Qian Zhong, Xiao-Ming Huang, Weimin Ye, Ming-Fang Ji, Yong-Lin Cai, Su-Mei Cao, Mu-Sheng Zeng

Structural characterization of human monoclonal antibodies targeting uncommon antigenic sites on spike glycoprotein of SARS-CoV

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Abstract

The function of the spike protein N terminal domain (NTD) in coronavirus (CoV) infections is poorly understood. However, some rare antibodies that target the SARS-CoV-2 NTD potently neutralize the virus. This finding suggests the NTD may contribute in part to protective immunity. Pan-sarbecovirus antibodies are desirable for broad protection, but the NTD region of SARS-CoV and SARS-CoV-2 exhibit a high level of sequence divergence, and therefore, cross-reactive NTD-specific antibodies are unexpected, and there is no structure of a SARS-CoV NTD-specific antibody in complex with NTD. Here we report a monoclonal antibody COV1-65 encoded by the IGHV1-69 gene that recognizes the NTD of SARS-CoV S protein. A prophylaxis study showed the MAb COV1-65 prevented disease when administered before SARS-CoV challenge of BALB/c mice, an effect that requires intact Fc effector functions for optimal protection in vivo. The footprint on the S protein of COV1-65 is near to functional components of the S2 fusion machinery, and the selection of COV1-65 escape mutant viruses identified critical residues Y886H and Q974H, which likely affect the epitope through allosteric effects. Structural features of the mAb COV1-65-SARS-CoV antigen interaction suggest critical antigenic determinants that should be considered in the rational design of sarbecovirus vaccine candidates.

Authors

Naveenchandra Suryadevara, Nurgun Kose, Sandhya Bangaru, Elad Binshtein, Jennifer Munt, David R. Martinez, Alexandra Schäfer, Luke Myers, Trevor D. Scobey, Robert H. Carnahan, Andrew B. Ward, Ralph S. Baric, James E. Crowe Jr.

Elevated microRNA-187 causes cardiac endothelial dysplasia to promote congenital heart disease through inhibition of NIPBL

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Abstract

Cardiac endothelial cells are essential for heart development, and disruption of this process can lead to congenital heart disease (CHD). However, how miRNAs influence cardiac endothelial cells in CHD remains unclear. This study identified elevated miR-187 expression in embryonic heart endothelial cells from CHD fetuses. Using a conditional knock-in model, we showed that increased miR-187 levels in embryonic endothelial cells induce CHD in homozygous fetal mice, closely mirroring human CHD. Mechanistically, miR-187 targets NIPBL, which is responsible for recruiting the cohesin complex and facilitating chromatin accessibility. Consequently, the endothelial cell-specific upregulation of miR-187 inhibited NIPBL, leading to reduced chromatin accessibility and impaired gene expression, which hindered endothelial cell development and ultimately caused heart septal defects and reduced heart size both in vitro and in vivo. Importantly, exogenous miR-187 expression in human cardiac organoids mimicked developmental defects in the cardiac endothelial cells, reversible by NIPBL replenishment. Our findings establish the miR-187/NIPBL axis as a potent regulator that inhibits cardiac endothelial cell development by attenuating the transcription of numerous endothelial genes, with our mouse and human cardiac organoid models effectively replicating severe defects from minor perturbations. This discovery suggests that targeting the miR-187/NIPBL pathway could offer a promising therapeutic approach for CHD.

Authors

Chao Li, Zizheng Tan, Hongdou Li, Xiaoying Yao, Chuyue Peng, Yue Qi, Bo Wu, Tong-Jin Zhao, Chengtao Li, Jianfeng Shen, Hongyan Wang

Neisseria gonorrhoeae induces local secretion of IL-10 at the human cervix to promote colonization

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Abstract

Gonorrhea, caused by the human-restricted pathogen Neisseria gonorrhoeae, is a commonly reported sexually transmitted infection. Since most infections in women are asymptomatic, the true number of infections is likely much higher than reported. How gonococci (GC) colonize women’s cervixes without triggering symptoms remains elusive. Using a human cervical tissue explant model, we found that GC inoculation increased the local secretion of both pro- (IL-1β and TNF-α) and anti-inflammatory (IL-10) cytokines during the first 24-h. Cytokine induction required GC expression of Opa isoforms that bind the host receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). GC inoculation induced NF-κB activation in both cervical epithelial and subepithelial cells. However, inhibition of NF-κB activation, which reduced GC-induced IL-1β and TNF-α, did not affect GC colonization. Neutralizing IL-10 or blocking IL-10 receptors by antibodies reduced GC colonization by increasing epithelial shedding and epithelial cell-cell junction disassembly. Inhibition of the CEACAM downstream signaling molecule SHP1/2, which reduced GC colonization and increased epithelial shedding, decreased GC-induced IL-10 secretion. These results show that GC induce local secretion of IL-10, a potent anti-inflammatory cytokine, at the cervix by engaging the host CEACAMs to prevent GC-colonizing epithelial cells from shedding, providing a potential mechanism for GC asymptomatic colonization in women.

Authors

Yiwei Dai, Vonetta L. Edwards, Qian Yu, Hervé Tettelin, Daniel C. Stein, Wenxia Song