NUDCD3 deficiency disrupts V(D)J recombination to cause SCID and Omenn syndrome.

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.

An Integrative Analysis Revealing ZFHX4-AS1 as a Novel Prognostic Biomarker Correlated with Immune Infiltrates in Ovarian Cancer.

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.

Role of differentiated embryo-chondrocyte expressed gene 1 (DEC1) in immunity.

Differentiated embryo-chondrocyte expressed gene 1 (DEC1) belongs to the family of basic helix-loop-helix (bHLH)-type transcription factors. DEC1 is expressed in various mammalian cells, but early studies focused on its roles outside the immune system. In recent years, relevant studies have found that DEC1 plays an important role in the immunotherapy of tumors, the functional regulation of the immune system, and the onset of autoimmune diseases. DEC1 promotes interferon (IFN)-γand granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion through the production of CD4+ T cells, which promotes inflammatory defense responses and autoimmune diseases. Additionally, DEC1 can inhibit the expression of interleukin (IL)-10 to further strengthen the immune response. In this review, we summarized recent advances in our understanding of the roles of DEC1 in animal models and human cells, including regulating immune cell differentiation, controlling cytokine production, and maintaining the balance of pro- and anti-inflammatory signals.

NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy.

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.

HIV-1 Vpr protein upregulates microRNA-210-5p expression to induce G2 arrest by targeting TGIF2.

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.

The Effects of Differentially-Expressed Homeobox Family Genes on the Prognosis and HOXC6 on Immune Microenvironment Orchestration in Colorectal Cancer.

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.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. 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.

Expression of the HOXA gene family and its relationship to prognosis and immune infiltrates in cervical cancer.

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.

Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci.

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 type­specific 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 type­resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Cystathionine ß-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine ß-synthase/hydrogen sulfide (CBS/H2S) 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/H2S 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.

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression.

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.

Upregulation of ZHX2 predicts poor prognosis and is correlated with immune infiltration in gastric cancer.

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.

Sterilization by Adaptive Immunity of a Conditionally Persistent Mutant of Mycobacterium tuberculosis.

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 ΔleuCD ΔpanCD ΔmetA 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 ΔleuCD ΔpanCD ΔargB 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 ΔleuCD ΔpanCD ΔmetA 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 ΔleuCD ΔpanCD ΔmetA 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.

Antigen-driven PD-1+ TOX+ BHLHE40+ and PD-1+ TOX+ EOMES+ T lymphocytes regulate juvenile idiopathic arthritis in situ.

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.

Augmented angiogenic transcription factor, SOX18, is associated with asthma exacerbation.

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 p1). 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 p1 treatment. SOX18 protein increased in HMVEC-L following Der p1 treatment. CONCLUSION: These results suggest that SOX18 may be involved in asthma pathogenesis and be associated with asthma exacerbation.

Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency.

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.

Transcription Factor Bhlhe40 in Immunity and Autoimmunity.

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.

The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells.

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.

Multi-antigen DNA vaccine delivered by polyethylenimine and Salmonella enterica in neuroblastoma mouse model.

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.

Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport.

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.