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Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1 -/- and Ifnar -/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients., Competing Interests: Declaration of interests The user committee for the RepliSAFE project included members from MSD Animal Health. MSD Animal Health provided TC-83 replicons and antibodies but otherwise provided no other resources or funding, had no input into the manuscript nor the decision to publish., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 ( NUDCD3 ) . Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T  - B - SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.

Ovarian cancer (OC) is the main cause of deaths worldwide in female reproductive system malignancies. Growing studies have indicated that eRNAs could regulate cellular activities in various tumors. Yet the potential roles of eRNAs in OC progression have not been elucidated. Thus, comprehensive assays were needed to screen the critical eRNAs and to explore their possible function in OC. We used Kaplan-Meier methods to identify survival-associated eRNAs in OC based on TCGA datasets. The levels of ZFHX4-AS1 were examined using TCGA datasets. Further exploration was carried out based on the following assays: clinical and survival assays, GO terms, and KEGG assays. TIMER was applied to delve into the relationships between ZFHX4-AS1 and tumor immune infiltration. In this research, we observed 71 survival-related eRNAs in OC patients. ZFHX4-AS1 was highly expressed in OC specimens and predicted a poor prognosis of OC patients. In addition, high ZFHX4-AS1 expression was positively related to the advanced stages of OC specimens. Multivariate assays revealed that ZFHX4-AS1 was an independent prognostic factor for overall survival of OC patients. KEGG analysis indicated that ZFHX4-AS1 may play a regulatory effect on TGF-beta signaling, PI3K-Akt signaling, and proteoglycans in cancer. The pan-cancer validation indicated that ZFHX4-AS1 was related to survival in eight tumors, namely, UCEC, STAD, SARC, OV, ACC, KICH, KIRC, and BLCA. The expression of ZFHX4-AS1 was correlated with the levels of B cells, T cell CD8+, neutrophil, macrophage, and myeloid dendritic cells. Simultaneously, ZFHX4-AS1 may be a prognostic biomarker and a distinctly immunotherapy-related eRNA in OC., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Changchang Huang et al.)

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur., Competing Interests: Disclosures: J. Cohen reported grants from National Institutes of Health outside the submitted work. E.J. Wherry is consulting or is an advisor for Merck, Marengo, Janssen, Related Sciences, Synthekine and Surface Oncology. E.J. Wherry is a founder of Surface Oncology, Danger Bio, and Arsenal Biosciences. E.J. Wherry reported personal fees from Merck, Marengo, Related Sciences, Synthekine, Surface Oncology, Danger Bio, and Arsenal Bioscience outside the submitted work. In addition, E.J. Wherry has a patent to US Patent (10,370,446) on the PD-1 pathway issued. P.M. Porrett has consulted for Janssen Research & Development within the last two years. No other disclosures were reported., (© 2021 Lewis et al.)

MicroRNAs (miRNAs) are important molecules that mediate virus-host interactions, mainly by regulating gene expression via gene silencing. Here, we demonstrated that HIV-1 infection upregulated miR-210-5p in HIV-1-inoculated cell lines and in the serum of HIV-1-infected individuals. Luciferase reporter assays and western blotting confirmed that a target protein of miR-210-5p, TGIF2, is regulated by HIV-1 infection. Furthermore, HIV-1 Vpr protein induced miR-210-5p expression. The use of a miR-210-5p inhibitor and TGIF2 overexpression showed that Vpr upregulated miR-210-5p and thereby downregulated TGIF2, which might be one of the mechanisms used by Vpr to induce G2 arrest. Moreover, we identified a transcription factor, NF-κB p50, which upregulated miR-210-5p in response to Vpr protein. In conclusion, we identified a mechanism whereby miR-210-5p, which is induced upon HIV-1 infection, targets TGIF2. This pathway was initiated by Vpr protein activating NF-κB p50, which promoted G2 arrest. These alterations orchestrated by miRNA provide new evidence on how HIV-1 interacts with its host during infection and increase our understanding of the mechanism by which Vpr regulates the cell cycle., Competing Interests: The authors have declared that no competing interests exist.

Background: The homeobox (HOX) gene family encodes highly conserved transcription factors, that play important roles in the morphogenesis and embryonic development of vertebrates. Mammals have four similar HOX gene clusters, HOXA, HOXB, HOXC, and HOXD, which are located on chromosomes 7, 17,12 and 2 and consist of 38 genes. Some of these genes were found to be significantly related to a variety of tumors; however, it remains unknown whether abnormal expression of the HOX gene family affects prognosis and the tumor microenvironment (TME) reshaping in colorectal cancer (CRC). Therefore, we conducted this systematic exploration to provide additional information for the above questions., Methods: RNA sequencing data from The Cancer Genome Atlas (TCGA) and mRNA expression data from Gene Expression Omnibus (GEO) combined with online tumor analysis databases (UALCAN, TIMER, PrognoScan) were utilized to explore the relationship among abnormal expression of HOX family genes, prognosis and the tumor immune microenvironment in CRC., Results: 1. Differential expression and prognosis analysis: 24 genes were significantly differentially expressed in CRC compared to adjacent normal tissues, and seven upregulated genes were significantly associated with poor survival. Among these seven genes, univariate and multivariate Cox regression analysis revealed that only high expression of HOXC6 significantly contributed to poor prognosis; 2. The influence of overexpressed HOXC6 on the pathway and TME: High HOXC6 expression was significantly related to the cytokine pathway and expression of T cell attraction chemokines, the infiltration ratio of immune cells, expression of immune checkpoint markers, tumor mutation burden (TMB) scores and microsatellite instability-high (MSI-H) scores; 3. Stratified analysis based on stages: In stage IV, HOXC6 overexpression had no significant impact on TMB, MSI-H, infiltration ratio of immune cells and response prediction of immune checkpoint blockers (ICBs), which contributed to significantly poor overall survival (OS)., Conclusion: Seven differentially expressed HOX family genes had significantly worse prognoses. Among them, overexpressed HOXC6 contributed the most to poor OS. High expression of HOXC6 was significantly associated with high immunogenicity in nonmetastatic CRC. Further research on HOXC6 is therefore worthwhile to provide potential alternatives in CRC immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qi, Ye, Zhang, Bai, Zheng, Hu and Yuan.)

Background: The homeobox A cluster (HOXA) gene family is participated in multiple biological functions in human cancers. To date, little is known about the expression profile and clinical significance of HOXA genes in cervical cancer., Methods: We downloaded RNASeq data of cervical cancer from The Cancer Genome Atlas (TCGA) database. The difference in HOXA family expression was analyzed using independent samples t test. Cox proportional hazard regression analysis was used to assess the effect of HOXA family expression on survival, and a nomogram predicting survival was generated. We assessed the infiltration difference in immune cells and expression difference of immunity biomarkers between two groups with different expression level of HOXA genes through Immune Cell Abundance Identifier (ImmuCellAI) and independent samples t test, respectively., Results: Our results showed that the HOXA1 gene was upregulated, while the HOXA10 and HOXA11 were downregulated in cervical cancer. Downregulation of HOXA1 was related to a poor outcome for cervical cancer patient. We also identified a significantly increased abundance of T helper 2 cells (Th2) and higher expression of PD-L1 in cervical cancer patients with lower expression of HOXA10 and HOXA11. The gene set enrichment analysis (GSEA) results indicated that HOXA1 and HOXA11 were involved in immune responses pathways and participated in the activation of a variety of classic signaling pathways related to the progression of human cancer., Conclusion: This study comprehensively analyzed different HOXA genes applying public database to determine their expression patterns, potential diagnostic, prognostic, and treatment values in cervical cancer., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes 1 . However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies 2,3 . Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks 4 . Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction., (© 2021. The Author(s).)

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type–specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology ( IL21 , IL21R , IL4R , and BCL6 ) and transcription factors regulating B cell differentiation ( POU2AF1 and HHEX ). Together, these analyses provide a powerful new cell type–resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Background: Asthma characterized by airway hyperresponsiveness, inflammation, fibrosis, and angiogenesis. SRY-related HMG-box 18 (SOX18) is an important transcription factor involved in angiogenesis, tissue injury, wound-healing, and in embryonic cardiovascular and lymphatic vessels development. The role of angiogenic transcription factors, SOX18 and the related, prospero homeobox 1 (PROX1) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), in asthma has had limited study., Objective: In this study, we aimed to elucidate the role of SOX18 in the pathogenesis of bronchial asthma., Methods: Plasma SOX18 protein was measured in control subjects, and subject with stable or exacerbated asthma. SOX18, PROX1, and COUP-TFII protein was measured by western blot, and immunohistochemistry in a murine model of ovalbumin-induced allergic asthma (OVA). SOX18, PROX1, and COUP-TFII protein was measured in lung human microvascular endothelial cells (HMVEC-L) and normal human bronchial epithelial (NHBE) cells treated with house dust mite ( Der p 1)., Results: Plasma SOX18 tended to be higher in subject with asthma compared to control subjects and increased more during exacerbation as compared to stable disease. In mice, OVA challenge lead to increased lung SOX18, PROX1, COUP-TFII, mucous gland hyperplasia and submucosal collagen. In NHBE cells, SOX18, PROX1 and COUP-TFII increased following Der p 1 treatment. SOX18 protein increased in HMVEC-L following Der p 1 treatment., Conclusion: These results suggest that SOX18 may be involved in asthma pathogenesis and be associated with asthma exacerbation.

Background: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine β-synthase/hydrogen sulfide (CBS/H 2 S) axis is involved in the regulation of inflammation. However, roles of CBS in HCC development and immune evasion have not been systematically investigated, and their underlying mechanisms remain elusive. Here, we investigated the roles of CBS in tumor cells and tumor microenvironment of HCC., Methods: 236 HCC samples were collected to detect the expression of CBS, cleaved Caspase-3 and paired related homeobox 2 (PRRX2) and the number of immune cells. HCC cell lines were employed to examine the effects of CBS on cellular viability, apoptosis and signaling in vitro. Cbs heterozygous knockout mice, C57BL/6 mice, nude mice and non-obese diabetic severe combined immunodeficiency mice were used to investigate the in vivo functions of CBS., Results: Downregulation of CBS was observed in HCC, and low expression of CBS predicted poor prognosis in HCC patients. CBS overexpression dramatically promoted cellular apoptosis in vitro and inhibited tumor growth in vivo. Activation of the Cbs/H 2 S axis also reduced the abundance of tumor-infiltrating Tregs, while Cbs deficiency promoted Tregs-mediated immune evasion and boosted tumor growth in Cbs heterozygous knockout mice. Mechanistically, CBS facilitated the expression cleaved Caspase-3 in tumor cells, and on the other hand, suppressed Foxp3 expression in Tregs via inactivating IL-6/STAT3 pathway. As a transcription factor of IL-6, PRRX2 was reduced by CBS. Additionally, miR-24-3p was proven to be an upstream suppressor of CBS in HCC., Conclusions: Our results indicate the antitumor function of CBS in HCC by inactivation of the PRRX2/IL-6/STAT3 pathway, which may serve as a potential target for HCC clinical immunotherapy., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The transcriptional repressor zinc finger homeobox 2 (ZHX2) is reported to regulate tumor progression in several human cancers, although little is known about its role in gastric cancer (GC). In the present study, we investigated the expression of ZHX2 and its relationship with the clinicopathological characteristics and prognosis of GC patients, and we also examined the effect of ZHX2 overexpression in GC cell lines. We used UALCAN (http://ualcan.path.uab.edu) and the Tumor Immune Estimation Resource (http://cistrome.org/TIMER) to examine ZHX2 mRNA expression, and also used Kaplan-Meier Plotter (https://kmplot.com) to determine whether ZHX2 expression was related to GC prognosis. Expression of ZHX2 protein was detected using immunohistochemical staining assays. Cell proliferation was evaluated using a cell counting kit-8 and colony formation assays, whereas apoptosis was examined by flow cytometry. Wound healing and transwell assays were used to detect cell migration and invasion. We also performed Gene Set Enrichment Analysis (https://www.gsea-msigdb.org) and used The Cancer Genome Atlas database (https://www.genome.gov/Funded-Programs-Projects/Cancer-Genome-Atlas) to examine the correlation of ZHX2 with immune infiltration. We report that ZHX2 is highly expressed in GC tissues and is significantly associated with clinical characteristics. Upregulation of ZHX2 predicted poor prognosis in GC. Furthermore, ZHX2 overexpression can promote the proliferation, invasion and migration, but inhibit apoptosis, of GC cells. High expression of ZHX2 in GC is correlated with the presence of infiltrating immune cells, including B cells, CD4 + T cells, macrophages and dendritic cells. Our data suggest that high expression of ZHX2 in GC predicts poor prognosis. In addition, ZHX2 may promote malignant behaviors of GC cells, and immune infiltration might be related to the oncogenic role of ZHX2 in GC., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA). However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the transcriptional and the clonal heterogeneity of synovial T lymphocytes in JIA patients by single-cell RNA sequencing combined with T cell receptor profiling on the same cells. We identify clonally expanded subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1 + TOX + EOMES + population of CD4 + T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1 + TOX + BHLHE40 + population of CD4 + , and a mirror population of CD8 + T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation in situ. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 ( Rag1/2 ) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Miyazaki and Miyazaki.)

Inflammatory bowel diseases (IBDs) are characterized by chronic, inflammatory gastrointestinal lesions and often require life-long treatment with immunosuppressants and repetitive surgical interventions. Despite progress in respect to the characterization of molecular mechanisms e.g. exerted by TNF-alpha, currently clinically approved therapeutics fail to provide long-term disease control for most patients. The transcription factor interferon regulatory factor 4 (IRF4) has been shown to play important developmental as well as functional roles within multiple immune cells. In the context of colitis, a T cell-intrinsic role of IRF4 in driving immune-mediated gut pathology is established. Here, we conversely addressed the impact of IRF4 inactivation in non-T cells on T cell driven colitis in vivo . Employing the CD4 + CD25 - naïve T cell transfer model, we found that T cells fail to elicit colitis in IRF4-deficient compared to IRF4-proficient Rag1 -/- mice. Reduced colitis activity in the absence of IRF4 was accompanied by hampered T cell expansion both within the mesenteric lymph node (MLN) and colonic lamina propria (cLP). Furthermore, the influx of various myeloids, presumably inflammation-promoting cells was abrogated overall leading to a less disrupted intestinal barrier. Mechanistically, gene profiling experiments revealed a Th17 response dominated molecular expression signature in colon tissues of IRF4-proficient, colitic Rag1 -/- but not in colitis-protected Rag1 -/- Irf4 -/- mice. Colitis mitigation in Rag1 -/- Irf4 -/- T cell recipients resulted in reduced frequencies and absolute numbers of IL-17a-producing T cell subsets in MLN and cLP possibly due to a regulation of conventional dendritic cell subset 2 (cDC2) known to impact Th17 differentiation. Together, extending the T cell-intrinsic role for IRF4 in the context of Th17 cell driven colitis, the provided data demonstrate a Th17-inducing and thereby colitis-promoting role of IRF4 through a T cell-extrinsic mechanism highlighting IRF4 as a putative molecular master switch among transcriptional regulators driving immune-mediated intestinal inflammation through both T cell-intrinsic and T cell-extrinsic mechanisms. Future studies need to further dissect IRF4 controlled pathways within distinct IRF4-expressing myeloid cell types, especially cDC2s, to elucidate the precise mechanisms accounting for hampered Th17 formation and, according to our data, the predominant mechanism of colitis protection in Rag1 -/- Irf4 -/- T cell receiving mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buchele, Konein, Vogler, Kunert, Enderle, Khan, Büttner-Herold, Lehmann, Amon, Wirtz, Dudziak, Neurath, Neufert and Hildner.)

Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent M. tuberculosis cells also evade the antimycobacterial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells. Independent mutations targeting the methionine and arginine biosynthetic pathways are bactericidal for M. tuberculosis in mice. However, in this study, we discovered that the addition of leucine and pantothenate auxotrophy altered the bactericidality of methionine auxotrophy. Whereas the leucine/pantothenate/methionine auxotrophic M. tuberculosis strain H37Rv Δ leuCD Δ panCD Δ metA was eliminated in immunocompetent mice, this strain persisted in multiple organs of immunodeficient Rag1 -/- mice for at least a year. In contrast, the leucine/pantothenate/arginine auxotroph H37Rv Δ leuCD Δ panCD Δ argB was eliminated in both immunocompetent and immunodeficient Rag1 -/- mice. Our results showed that leucine and pantothenate starvation metabolically blocked the sterilization mechanisms of methionine starvation but not those of arginine starvation. These triple-auxotrophic strains should be invaluable tools for unravelling the bacterial and host factors that enable persistence and for vaccine development studies to assess the efficacy of vaccines that boost immune recognition of M. tuberculosis in the persistent state. The sterilization of the Δ leuCD Δ panCD Δ metA auxotroph in immunocompetent mice, but not in mice lacking an adaptive immune response, could provide a new system for studying the antimycobacterial killing mechanisms of adaptive immunity. IMPORTANCE The bacterial pathogen Mycobacterium tuberculosis can enter into a persistent state in which M. tuberculosis can evade host immunity, thereby reducing the effectiveness of current tuberculosis vaccines. Understanding the factors that contribute to persistence would enable the rational design of vaccines effective against persisters. We previously generated two attenuated, triple-auxotrophic M. tuberculosis strains that are safe to use in a biosafety level 2 laboratory. Herein, we discovered that the triple-auxotrophic strain H37Rv Δ leuCD Δ panCD Δ metA persisted in immunodeficient Rag1 -/- mice, which lack adaptive immunity, but not in immunocompetent mice. The conditional persistence of this auxotrophic mutant, which is susceptible to the sterilizing effect of the adaptive immune response over time, provides an important tool to dissect the mycobactericidal effector mechanisms mediated by adaptive immunity. Furthermore, because of its remarkable safety attributes, this auxotrophic mutant can potentially be used to develop a practical human challenge model to facilitate vaccine development., (Copyright © 2021 Vilchèze et al.)

Background: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation., Objectives: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP)., Methods: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray., Results: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery., Conclusions: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis., (Published by Elsevier Inc.)

Neuroblastoma is an example of a difficult-to-treat tumor with high incidence of relapse. DNA vaccination could be applied as a relapse prophylactic option for patients with high-risk neuroblastoma. Its efficacy depends directly on a target antigen of choice and a delivery method. Three neuroblastoma-associated antigens (tyrosine hydroxylase, Survivin, PHOX2B) and two delivery methods were investigated. Our data suggest that antigen PHOX2B is a more immunogenic target that induces cellular immune response and tumor regression more effectively than tyrosine hydroxylase and Survivin. Immunogenicity testing revealed that the delivery of DNA vaccine by Salmonella enterica was accompanied by a stronger immune response (cytotoxicity and IFNγ production) than that by DNA-polyethylenimine conjugate. Nevertheless, intramuscular immunization with PEI led to higher decrease of tumor volume compared to that after oral gavage with Salmonella vaccine.

The basic helix-loop-helix transcription factor (TF) Bhlhe40 is emerging as a key regulator of immunity during infection, autoimmunity, and inflammatory conditions. We describe the roles of Bhlhe40 in the circulating and tissue-resident arms of the immune system, with emphasis on recent work on the regulation of cytokine production and proliferation. We explore the mechanisms behind these functions in mouse models and human cells, including interactions with other TFs, and propose that Bhlhe40 is a central mediator of both inflammation and pathogen control, as well as a crucial regulator of a growing number of tissue-resident leukocyte populations. Finally, we suggest areas for further study that may advance our understanding of immunity and disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cell type-specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor ( TCR ) and immunoglobulin ( Ig ) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage-specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage-specific enhancer ( R-TEn ) and the two B cell-specific elements, R1B and R2B By generating mice lacking either R-TEn or R1B and R2B , we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn , we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation., (Copyright © 2020 American Society for Microbiology.)

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Descending command neurons instruct spinal networks to execute basic locomotor functions, such as gait and speed. The command functions for gait and speed are symmetric, implying that a separate unknown system directs asymmetric movements, including the ability to move left or right. In the present study, we report that Chx10-lineage reticulospinal neurons act to control the direction of locomotor movements in mammals. Chx10 neurons exhibit mainly ipsilateral projection, and their selective unilateral activation causes ipsilateral turning movements in freely moving mice. Unilateral inhibition of Chx10 neurons causes contralateral turning movements. Paired left-right motor recordings identified distinct mechanisms for directional movements mediated via limb and axial spinal circuits. Finally, we identify sensorimotor brain regions that project on to Chx10 reticulospinal neurons, and demonstrate that their unilateral activation can impart left-right directional commands. Together these data identify the descending motor system that commands left-right locomotor asymmetries in mammals.

CD8 + T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8 + T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1 + progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy., Competing Interests: Declaration of Interests E.J.W. is a member of the Parker Institute for Cancer Immunotherapy which supported these studies. E.J.W. has consulting agreements with and/or is on the scientific advisory board for Merck, Roche, Pieris, Elstar, and Surface Oncology. E.J.W. has a patent licensing agreement on the PD-1 pathway with Roche/Genentech. E.J.W. is a founder of Arsenal Biosciences., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brucella spp. are facultative intracellular bacteria notorious for their ability to induce a chronic, and often lifelong, infection known as brucellosis. To date, no licensed vaccine exists for prevention of human disease, and mechanisms underlying chronic illness and immune evasion remain elusive. We and others have observed that B cell-deficient mice challenged with Brucella display reduced bacterial burden following infection, but the underlying mechanism has not been clearly defined. Here, we show that at 1 month postinfection, B cell deficiency alone enhanced resistance to splenic infection ∼100-fold; however, combined B and T cell deficiency did not impact bacterial burden, indicating that B cells only enhance susceptibility to infection when T cells are present. Therefore, we investigated whether B cells inhibit T cell-mediated protection against Brucella Using B and T cell-deficient Rag1 -/- animals as recipients, we demonstrate that adoptive transfer of CD4 + T cells alone confers marked protection against Brucella melitensis that is abrogated by cotransfer of B cells. Interestingly, depletion of CD4 + T cells from B cell-deficient, but not wild-type, mice enhanced susceptibility to infection, further confirming that CD4 + T cell-mediated immunity against Brucella is inhibited by B cells. In addition, we found that the ability of B cells to suppress CD4 + T cell-mediated immunity and modulate CD4 + T cell effector responses during infection was major histocompatibility complex class II (MHCII)-dependent. Collectively, these findings indicate that B cells modulate CD4 + T cell function through an MHCII-dependent mechanism which enhances susceptibility to Brucella infection., (Copyright © 2020 American Society for Microbiology.)

Background: Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described., Methods: We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line., Results: RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection., Conclusions: hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

Traditionally, primary immune deficiencies have been defined based on increased susceptibility to recurrent and/or severe infections. However, immune dysregulation, manifesting with autoimmunity or hyperinflammatory disease, has emerged as a common feature. This is especially true in patients affected by combined immune deficiency (CID), a group of disorders caused by genetic defects that impair, but do not completely abolish, T-cell function. Hypomorphic mutations in the recombination activating genes RAG1 and RAG2 represent the prototype of the broad spectrum of clinical and immunological phenotypes associated with CID. The study of patients with RAG deficiency and with other forms of CID has revealed distinct abnormalities in central and peripheral T- and B-cell tolerance as the key mechanisms involved in immune dysregulation. Understanding the pathophysiology of autoimmunity and hyperinflammation in these disorders may also permit more targeted therapeutic interventions.

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft-versus-host disease (GVHD), and pathological damage is largely attributable to inflammatory cytokine production. Recently, granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a cytokine that mediates inflammation in the GI tract, but the transcriptional program that governs GM-CSF production and the mechanism by which GM-CSF links adaptive to innate immunity within this tissue site have not been defined. In the current study, we identified Bhlhe40 as a key transcriptional regulator that governs GM-CSF production by CD4+ T cells and mediates pathological damage in the GI tract during GVHD. In addition, we observed that GM-CSF was not regulated by either interleukin 6 (IL-6) or IL-23, which are both potent inducers of GVHD-induced colonic pathology, indicating that GM-CSF constitutes a nonredundant inflammatory pathway in the GI tract. Mechanistically, GM-CSF had no adverse effect on regulatory T-cell reconstitution, but linked adaptive to innate immunity by enhancing the activation of donor-derived dendritic cells in the colon and subsequent accumulation of these cells in the mLNs. In addition, GM-CSF promoted indirect alloantigen presentation, resulting in the accumulation of donor-derived T cells with a proinflammatory cytokine phenotype in the colon. Thus, Bhlhe40+ GM-CSF+ CD4+ T cells constitute a colitogenic T-cell population that promotes indirect alloantigen presentation and pathological damage within the GI tract, positioning GM-CSF as a key regulator of GVHD in the colon and a potential therapeutic target for amelioration of this disease., (© 2020 by The American Society of Hematology.)

The control of cytoskeletal dynamics by dedicator of cytokinesis 2 (DOCK2), a hematopoietic cell-specific actin effector protein, has been implicated in TCR signaling and T cell migration. Biallelic mutations in Dock2 have been identified in patients with a recessive form of combined immunodeficiency with defects in T, B, and NK cell activation. Surprisingly, we show in this study that certain immune functions of CD8 + T cells are enhanced in the absence of DOCK2. Dock2 -deficient mice have a pronounced expansion of their memory T cell compartment. Bone marrow chimera and adoptive transfer studies indicate that these memory T cells develop in a cell-intrinsic manner following thymic egress. Transcriptional profiling, TCR repertoire analyses, and cell surface marker expression indicate that Dock2 -deficient naive CD8 + T cells directly convert into virtual memory cells without clonal effector T cell expansion. This direct conversion to memory is associated with a selective increase in TCR sensitivity to self-peptide MHC in vivo and an enhanced response to weak agonist peptides ex vivo. In contrast, the response to strong agonist peptides remains unaltered in Dock2 -deficient T cells. Collectively, these findings suggest that the regulation of the actin dynamics by DOCK2 enhances the threshold for entry into the virtual memory compartment by negatively regulating tonic TCR triggering in response to weak agonists., (Copyright © 2019 by The American Association of Immunologists, Inc.)

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intestinal innate lymphoid cells (ILCs) contribute to the protective immunity and homeostasis of the gut, and the microbiota are critically involved in shaping ILC function. However, the role of the gut microbiota in regulating ILC development and maintenance still remains elusive. Here, we identified opposing effects on ILCs by two Helicobacter species, Helicobacter apodemus and Helicobacter typhlonius , isolated from immunocompromised mice. We demonstrated that the introduction of both Helicobacter species activated ILCs and induced gut inflammation; however, these Helicobacter species negatively regulated RORγt + group 3 ILCs (ILC3s), especially T-bet + ILC3s, and diminished their proliferative capacity. Thus, these findings underscore a previously unknown dichotomous regulation of ILC3s by Helicobacter species, and may serve as a model for further investigations to elucidate the host-microbe interactions that critically sustain the maintenance of intestinal ILC3s., Competing Interests: The authors declare no competing interest.

Introduction: Complex regional pain syndrome (CRPS) is a complication following trauma or surgery and may be difficult to diagnose since biomarkers are lacking. Using protein array technology, we found antibodies binding to p29ING4, which we further characterized using ELISA., Methods: Thirty-six sera of early-stage type 1 CRPS, 66 sera of rheumatoid arthritis (RA), 53 sera of axial spondyloarthritis (axSpA), 29 sera of psoriatic arthritis (PsA), 22 sera of patients after radial fractures (trauma control), and 100 sera of blood donors (BD) were analyzed for anti-p29ING4. We established ELISAs with 7 different antigens and using different secondary antibodies binding to IgG, IgG1, IgG2, IgG3, IgG4, IgA, and IgM, and 2 different tests to detect immune complexes (IC) of p29ING4 and IgG or IgG1., Results: The highest likelihood ratios versus CRPS and trauma control were observed considering the A1-23 (sensitivity 19%, specificity 100%, LR > 19) using IgG as a secondary antibody, the A120-165 (sensitivity 17%, specificity 100%, LR = 17) using IgG as a secondary antibody and the A120-165 (sensitivity 31%, specificity 95%, LR = 6.2) using IgA as a secondary antibody. IC of p29ING4 and IgG were present in 11/36 (31%) CRPS sera, 17/64 (27%) RA sera, 13/53 (25%) SpA sera, 5/29 (17%) PsA sera, 1/22 (5%) trauma control sera, and 4/100 (4%) sera of BD. IC of p29ING4 and IgG1 were present in 14/36 (39%) CRPS sera, 19/64 (30%) RA sera, 13/53 (25%) SpA, 1/29 (3%) PsA, 2/22 (9%) trauma control, and 4/100 (4%) of the BD sera., Conclusion: Due to the lack of other biomarkers of type 1 CRPS, P29ING4 autoantibodies could be helpful in its diagnostic work-up.

Although immune checkpoint blockade therapies have demonstrated clinical efficacy in cancer treatment, harnessing this strategy is largely encumbered by resistance in multiple cancer settings. Here, we show that tumor-infiltrating T cells are severely exhausted in the microsatellite stable (MSS) colorectal cancer (CRC), a representative example of PD-1 blockade-resistant tumors. In MSS CRC, we found wound healing signature to be up-regulated and that T cell exhaustion is driven by vascular endothelial growth factor-A (VEGF-A). We report that VEGF-A induces the expression of transcription factor TOX in T cells to drive exhaustion-specific transcription program in T cells. Using a combination of in vitro, ex vivo, and in vivo mouse studies, we demonstrate that combined blockade of PD-1 and VEGF-A restores the antitumor functions of T cells, resulting in better control of MSS CRC tumors., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca 2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca 2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca 2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H 2 O 2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca 2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays., (© 2019 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease. MicroRNAs (miRNAs) are a group of endogenous small non‑coding RNAs that regulate target genes, and play a critical role in many biological processes. However, the underlying mechanism of specific miRNA, miR‑130a‑3p, in AS remains largely unknown. Therefore, the present study aimed to explore the underlying mechanism of miR‑130a‑3p in the development of AS. In the present study, it was revealed that miR‑130a‑3p was downregulated in T cells from HLA‑B27‑positive AS patients compared with the HLA‑B27‑negative healthy controls. Next, bioinformatics software TargetScan 7.2 was used to predict the target genes of miR‑130a‑3p, and a luciferase reporter assay indicated that HOXB1 was the direct target gene of miR‑130a‑3p. Furthermore, it was determined that HOXB1 expression was upregulated in T cells from HLA‑B27‑positive AS patients. In addition, the results of the present study indicated that miR‑130a‑3p inhibitor significantly inhibited cell proliferation ability and induced cell apoptosis of Jurkat T cells, while the miR‑130a‑3p mimic promoted proliferation ability and inhibited cell apoptosis of Jurkat T cells. Notably, all the effects of the miR‑130a‑3p mimic on Jurkat T cells were reversed by HOXB1‑plasmid. Collectively, our data indicated that miR‑130a‑3p was decreased in T cells from AS patients and it could regulate T‑cell survival by targeting HOXB1.

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1 -/- zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity- and apoptosis-related genes was increased in the rag1 -/- fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1 -/- zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1 -/- zebrafish compared to their wild-type (wt) siblings, rag1 -/- showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence-related genes and senescence-associated β-galactosidase, respectively, diminished telomere length, and abnormal self-renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1 -/- fish, as was the deficiency of the antioxidant metabolite l-acetylcarnitine (ALCAR) in rag1 -/- fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT-263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Background & Aims: The thymocyte selection-associated high mobility group box protein (TOX) plays a vital role in T cell development and differentiation, however, its role in T cell exhaustion was unexplored. Here, we aim to investigate the role of TOX in regulating the antitumor effect of CD8 + T cells in hepatocellular carcinoma., Methods: Fully functional, partially and severely exhausted tumor-infiltrating CD8 + T cells were sorted by flow cytometry and subjected to transcriptome sequencing analysis. Upregulated TOX expression was validated by flow cytometry. The antitumor function of CD8 + T cells with TOX downregulation or overexpression was studied in a mouse HCC model and HCC patient-derived xenograft mouse model. Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8 + T cells. The mechanism underlying the TOX-mediated regulation of PD1 expression was studied by laser confocal detection, immune co-precipitation and flow cytometer., Results: TOX was upregulated in exhausted CD8 + T cells in hepatocellular carcinoma. TOX downregulation in CD8 + T cells inhibited tumor growth, increased CD8 + T cell infiltration, alleviated CD8 + T cell exhaustion and improved the anti-PD1 response of CD8 + T cells. The mechanism behind this involved the binding of TOX to PD1 in the cytoplasm, which facilitated the endocytic recycling of PD1, thus maintaining abundant PD1 expression at the cell surface. High expression of TOX in peripheral CD8 + T cells correlated with poorer anti-PD1 responses and prognosis., Conclusions: TOX promotes CD8 + T cell exhaustion in hepatocellular carcinoma by regulating endocytic recycling of PD1. Downregulating TOX expression in CD8 + T cells exerts synergistic effects with anti-PD1 therapy, highlighting a promising strategy for cancer immunotherapy., Lay Summary: Abundant TOX expression in CD8 + T cells impairs their antitumor function in hepatocellular carcinoma. Mechanically, TOX reduces PD1 degradation and promotes PD1 translocation to the cell surface in CD8 + T cells, thus maintaining high PD1 expression at the cell surface. Downregulating TOX expression improves the antitumor function of CD8 + T cells, which shows the synergetic role of anti-PD1 therapy, highlighting a promising strategy for enhancement of cancer immunotherapy., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

A hallmark feature of schizophrenia is altered high frequency neural oscillations, including reduced auditory-evoked gamma oscillatory power, which is underpinned by parvalbumin (PV) interneuron dysfunction. Maternal immune activation (MIA) in rodents models an environmental risk factor for schizophrenia and recapitulates these PV interneuron changes. This study sought to link reduced PV expression in the MIA model with alterations to auditory-evoked gamma oscillations and transcript expression. We further aligned transcriptional findings from the animal model with human genome sequencing data. We show that MIA, induced by the viral mimetic, poly-I:C in C57Bl/6 mice, caused in adult offspring reduced auditory-evoked gamma and theta oscillatory power paralleled by reduced PV protein levels. We then showed the Arx gene, critical to healthy neurodevelopment of PV interneurons, is reduced in the forebrain of MIA exposed mice. Finally, in a whole-genome sequenced patient cohort, we identified a novel missense mutation of ARX in a patient with schizophrenia and in the Psychiatric Genomics Consortium 2 cohort, a nominal association of proximal ARX SNPs with the disorder. This suggests MIA, as a risk factor for schizophrenia, may be influencing Arx expression to induce the GABAergic dysfunction seen in schizophrenia and that the ARX gene may play a role in the prenatal origins of schizophrenia pathophysiology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tissue-resident memory CD8 + T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8 + T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a -/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD 50 ) of V. parahaemolyticus was significantly lower in notch1a -/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a -/- infected groups compared with notch1a -/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In order to better understand the development and function of γδ T cells in Xenopus frogs, it is necessary to determine where and when γδ T cells are found in Xenopus tissues. This study examined the expression of TCR genes, focused primarily on TCR γ, in tissues of adult and larval Xenopus laevis and provide new data about the expression pattern of these different TCR genes in this anuran amphibian. TCR gene expression was detected by RT-PCR in adult frog tissues including the thymus, spleen, skin, intestine, lung, and liver, but not the testes. TCR γ and β genes were detected in the larval (tadpole) tail and intestine. The absence of RAG-1 expression in these larval tissues is consistent with differentiation of the T cells in the thymus. Together, these data provide evidence that migration of these cells from the thymus likely occurs relatively early in larval development. These studies provide a necessary foundation for future studies of the functions of γδ T cells in amphibians, which are placed at an intermediate position flanked by fishes on one end and mammals and chickens on the other., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

B cells have been reported to have a suppressive function in autoimmune diseases, which appears to require an increase of CD11b expression on B cells. However, little is known how CD11b is induced in B cells to play the function. In this study, we found that the high expression of CD11b in B cells occurred not only in the mucosal immune organs, but also in systemically immune organs such as the spleen during dextran sulfate sodium (DSS)-induced colitis. Since the inflammatory lesions in mouse models of inflammatory bowel disease (IBD) were revealed to be significantly hypoxic or even anoxic, the B cells from colitic mice Peyer's patches (PP) were investigated to express higher levels of hypoxia-inducible factor-1α (HIF-1α) than naïve B cells from wildtype (WT) mice. HIF-1α siRNA transfection or HIF-1α protein inhibition led to decreased CD11b expression at both the mRNA and protein levels in vitro. B cells with HIF-1α specific knockdown were then adoptively transferred to Rag-1 -/- mice. The result displayed that CD11b expression was decreased in B cells and an exacerbated colitis occurred. The bio-informatics promoter analysis and ChIP assay showed that HIF-1α was the critical transcription factor for CD11b and cooperatively formed a complex with the p-STAT3 homodimers to bind onto hypoxia-responsive element (HRE) regions, which was guaranteed by MEK/ERK pathway activation and IL-10 secretion. In conclusion, our study demonstrated the key function of the hypoxia-associated transcription factor HIF-1α together with p-STAT3 in driving CD11b transcription in B cells and controlling B cell's protective activity in experimental inflammatory bowel disease (IBD)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis ., Competing Interests: AR, LP, GM, KC, DL, GG, GB, JC No competing interests declared, (© 2019, Roberts et al.)

Currently, inflammatory bowel disease (IBD) is a serious public health problem on the rise worldwide. In this work, we utilized the zebrafish to introduce a new model of intestinal inflammation triggered by food intake. Taking advantage of the translucency of the larvae and the availability of transgenic zebrafish lines with fluorescently labeled macrophages, neutrophils, or lymphocytes, we studied the behavior of these cell types in vivo during the course of inflammation. We established two feeding strategies, the first using fish that were not previously exposed to food (naïve strategy) and the second in which fish were initially exposed to normal food (developed strategy). In both strategies, we analyzed the effect of subsequent intake of a control or a soybean meal diet. Our results showed increased numbers of innate immune cells in the gut in both the naïve or developed protocols. Likewise, macrophages underwent drastic morphological changes after feeding, switching from a small and rounded contour to a larger and dendritic shape. Lymphocytes colonized the intestine as early as 5 days post fertilization and increased in numbers during the inflammatory process. Gene expression analysis indicated that lymphocytes present in the intestine correspond to T helper cells. Interestingly, control diet only induced a regulatory T cell profile in the developed model. On the contrary, soybean meal diet induced a Th17 response both in naïve and developed model. In addition, when feeding was performed in rag1 -deficient fish, intestinal inflammation was not induced indicating that inflammation induced by soybean meal is T cell-dependent.

The non-canonical NF-κB signalling cascade is essential for lymphoid organogenesis, B cell maturation, osteoclast differentiation, and inflammation in mammals 1,2 ; dysfunction of this system is associated with human diseases, including immunological disorders and cancer 3-6 . Although expression of NF-κB-inducing kinase (NIK, also known as MAP3K14) is the rate-limiting step in non-canonical NF-κB pathway activation 2,7 , the mechanisms by which transcriptional responses are regulated remain largely unknown. Here we show that the sine oculis homeobox (SIX) homologue family transcription factors SIX1 and SIX2 are integral components of the non-canonical NF-κB signalling cascade. The developmentally silenced SIX proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit the trans-activation function of the transcription factors RELA and RELB in a negative feedback circuit. In support of a physiologically pivotal role for SIX proteins in host immunity, a human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/P53-driven non-small-cell lung carcinomas from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents (small-molecule activators of the non-canonical NF-κB pathway). Our findings identify a NIK-SIX signalling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.

Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor.

The transcription factor Nkx2.3 regulates the vascular specification of Peyer patches in mice through determining endothelial addressin preference and may function as a susceptibility factor in inflammatory bowel diseases in humans. We wished to analyze the role of Nkx2.3 in colonic solitary intestinal lymphoid tissue composition and in colitis pathogenesis. We studied the colonic solitary intestinal lymphoid tissue of Nkx2.3-deficient mice with immunofluorescence and flow cytometry. Colitis was induced in mice using 2.5% dextran sodium sulfate, and severity was assessed with histology, flow cytometry, and quantitative PCR. We found that the lack of Nkx2.3 impairs maturation of isolated lymphoid follicles and attenuates dextran sodium sulfate-induced colitis independent of endothelial absence of mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which was also coupled with enhanced colonic epithelial regeneration. Although we observed increased numbers of group 3 innate lymphoid cells and Th17 cells and enhanced transcription of IL-22, Ab-mediated neutralization of IL-22 did not abolish the protection from colitis in Nkx2.3-deficient mice. Nkx2.3 -/- hematopoietic cells could not rescue wild-type mice from colitis. Using LacZ-Nkx2.3 reporter mice, we found that Nkx2.3 expression was restricted to VAP-1 + myofibroblast-like pericryptal cells. These results hint at a previously unknown stromal role of Nkx2.3 as driver of colitis and indicate that Nkx2.3 + stromal cells play a role in epithelial cell homeostasis., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The Recombination Activating Genes, RAG1 and RAG2 , are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo., (© 2019 by The American Society of Hematology.)