Tuberculosis, a chronic infectious disease caused by a single pathogen, holds the highest mortality rate worldwide. RNA-binding proteins (RBPs) are involved in autophagy — a key defense mechanism against Mycobacterium tuberculosis (Mtb) infection — by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during Mtb infection remain relatively unexplored. ZNFX1, a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in Mtb-infected macrophages. Here, we aimed to explore the immune regulatory functions of ZNFX1 during Mtb infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased Mtb burden, progressive lung tissue injury, and chronic inflammation in Mtb-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing Prkaa2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to Mtb growth suppression. These findings reveal a function of ZNFX1 in establishing anti-Mtb immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster anti-tuberculosis immunotherapy.
Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma
The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-β1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell–derived microglia, HSV-2–induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2–treated microglia exerted IKBKE-dependent type I IFN–mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.
Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi
Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by M-CSF or GM-CSF, which promote the generation of anti-inflammatory/immunosuppressive MAFB+ (M-MØ) or pro-inflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and demonstrated that MAFB regulates the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiates the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulates the expression of pro-fibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.
Miriam Simón-Fuentes, Israel Ríos, Cristina Herrero, Fátima Lasala, Nuria Labiod, Joanna Luczkowiak, Emilia Roy-Vallejo, Sara Fernández de Córdoba-Oñate, Pablo Delgado-Wicke, Matilde Bustos, Elena Fernández-Ruiz, Maria Colmenares, Amaya Puig-Kröger, Rafael Delgado, Miguel A. Vega, Angel L. Corbi, Angeles Domínguez-Soto
Regulatory T cells (Treg) have potential for the treatment of autoimmune diseases and graft rejection. Antigen-specificity and functional stability are considered to be critical for their therapeutic efficacy. In this study, expansion of human Treg in the presence of porcine PBMC (Xn-Treg) allowed the selection of a distinct Treg subset, co-expressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared to their unsorted and HLA-DA+CD27+ double positive (DP) cell depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-Enriched Xn-Treg expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal MLR. They also had less methylated Treg-specific demethylated region (TSDR) of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD-SCID IL2 receptor γ–/– (NSG) mice with HLA-DR+CD27+ DP-Enriched Xn-Treg in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Treg and upregulated expression of Treg functional genes, IL10 and CTLA4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-Enriched Xn-Treg expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Treg with potential as a Treg-based therapy.
Xiaoqian Ma, Lu Cao, Martina Raneri, Hannah Wang, Qi Cao, Yuanfei Zhao, Naiara G. Bediaga, Gaetano Naselli, Leonard C. Harrison, Wayne J. Hawthorne, Min Hu, Shounan Yi, Philip J. O’Connell
Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21–producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21–producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21–producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21–producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.
Hengcheng Zhang, Cecilia B. Cavazzoni, Manuel A. Podestà, Elsa D. Bechu, Garyfallia Ralli, Pragya Chandrakar, Jeong-Mi Lee, Ismail Sayin, Stefan G. Tullius, Reza Abdi, Anita S. Chong, Bruce R. Blazar, Peter T. Sage
The development of human prenatal adaptive immunity progresses faster than previously appreciated, with the emergence of memory CD4+ T cells alongside regulatory T (Treg) cells by mid-gestation. We previously identified a prenatal-specific population of PLZF+ CD4+ T cells with heightened effector potential that were enriched in the developing intestine and accumulated in the cord blood of infants exposed to prenatal inflammation. However, the signals that drive their tissue distribution and effector maturation are unknown. Here we define the transcriptional and functional heterogeneity of human prenatal PLZF+ CD4+ T cells and identify the compartmentalization of T helper (Th)-like effector function across the small intestine (SI) and mesenteric lymph nodes (MLN). IL-7 was more abundant in the SI relative to the MLN and drove the preferential expansion of naïve PLZF+ CD4+ T cells via enhanced STAT5 and MEK/ERK signaling. Exposure to IL-7 was sufficient to induce the acquisition of CD45RO expression and rapid effector function in a subset of PLZF+ CD4+ T cells, identifying a human analog of memory-phenotype CD4+ T cells. Further, IL-7 modulated the differentiation of Th1- and Th17-like PLZF+ CD4+ T cells, and thus likely contributes to the anatomic compartmentalization of human prenatal CD4+ T cell effector function.
Veronica Locher, Sara Park, Daniel G. Bunis, Stephanie Makredes, Margareta Mayer, Trevor D. Burt, Gabriela K. Fragiadakis, Joanna Halkias
Approximately 30% of breast cancer survivors deemed ‘free of disease’ will experience locoregional or metastatic recurrence even up to 30 years post initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state which is associated with immune dysfunction, and we find the tumor-derived stem/basal gene Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immune suppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.
Timothy N. Trotter, Carina E. Dagotto, Delila Serra, Tao Wang, Xiao Yang, Chaitanya R. Acharya, Junping Wei, Gangjun Lei, Herbert Kim Lyerly, Zachary C. Hartman
The penetration of allergens through the epithelial layer is the initial step in the development of allergic conjunctivitis. Although the pollinosis patients manifest symptoms in minutes after pollen exposure, the mechanisms of the rapid allergen transport remain unclear. In the present study, we found that the instillation of pollen shells rapidly induces a large number of goblet cell-associated antigen passages (GAPs) in the conjunctiva. Antigen acquisition by the stromal cells including macrophages and CD11b+ dendritic cells correlated with the surface GAP formation. Furthermore, a substantial amount of antigen was transported to the stroma during the first 10 minutes of the pollen exposure, which was sufficient for the full induction of an allergic conjunctivitis mouse model. This inducible rapid GAP formation and antigen acquisition was suppressed by topical lidocaine or trigeminal ablation, indicating that the sensory nervous system plays an essential role. Interestingly, pollen shell-stimulated GAP formation was not suppressed by topical atropine, suggesting that the conjunctival GAPs and intestinal GAPs are differentially regulated. These results identify pollen shell-induced GAP as a novel therapeutic target for allergic conjunctivitis.
Meiko Kimura, Tomoaki Ando, Yasuharu Kume, Saaya Fukase, Moe Matsuzawa, Kosuke Kashiwagi, Kumi Izawa, Ayako Kaitani, Nobuhiro Nakano, Keiko Maeda, Hideoki Ogawa, Ko Okumura, Shintaro Nakao, Akira Murakami, Nobuyuki Ebihara, Jiro Kitaura
Although SARS-CoV-2 evolution seeds a continuous stream of antibody-evasive viral variants, COVID-19 mRNA vaccines provide robust protection against severe disease and hospitalization. Here, we asked whether mRNA vaccine-induced memory T cells limits lung SARS-CoV-2 replication and severe disease. We show that mice and humans receiving booster BioNTech mRNA vaccine developed potent CD8 T-cell responses and show similar kinetics of expansion and contraction of granzyme B/perforin-expressing effector CD8 T cells. Both monovalent and bivalent mRNA vaccines elicited strong expansion of a heterogeneous pool of terminal effectors and memory precursor effector CD8 T cells in spleen, inguinal and mediastinal lymph nodes, pulmonary vasculature, and most surprisingly in the airways, suggestive of systemic and regional surveillance. Further, we document that: (1) CD8 T-cell memory persists in multiple tissues for > 200 days; (2) following challenge with pathogenic SARS-CoV-2, circulating memory CD8 T cells rapidly extravasate to the lungs and promote expeditious viral clearance, by mechanisms that require CD4 T cell help; (3) adoptively transferred splenic memory CD8 T cells traffic to the airways, and promote lung SARS-CoV-2 clearance. These findings provide new insights into the critical role of memory T cells in preventing severe lung disease following breakthrough infections with antibody-evasive SARS-CoV-2 variants.
Brock Kingstad-Bakke, Thomas Cleven, Hailey Bussan, Boyd L. Yount Jr., Ryuta Uraki, Kiyoko Iwatsuki-Horimoto, Michiko Koga, Shinya Yamamoto, Hiroshi Yotsuyanagi, Hongtae Park, Jay S. Mishra, Sathish Kumar, Ralph Baric, Peter J. Halfmann, Yoshihiro Kawaoka, M. Suresh
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