bluuebunny/crossref_metadata_2025 huggingface dataset:
Unusual early onset of autoimmune thrombotic thrombocytopenic purpura in five children with Polynesian origin - in three combined with immunodeficiency (P4094)
Authors: Mansouri Taleghani Magnus , Bradbeer Peter , Cole Nyree , Chan George , Schaller Monica , Laemmle Bernhard , Kremer Hovinga Johanna A.
Published: January 2023

Autoimmune thrombotic thrombocytopenic purpura (aTTP) is a rare disease characterized by thrombocytopenia, microangiopathic hemolytic anemia and a variable degree of endorgan ischemia as well as an often relapsing course. Underlying is a severe ADAMTS13 deficiency due to inhibitory autoantibodies preventing normal processing of sticky, unusually large Von Willebrand factor multimers, to which platelets bind spontaneously. Acute episodes of aTTP typically occur in young women between 20-40 years of age and are rarely seen in children. Strikingly, over the last 7 years we observed aTTP with severe acquired ADAMTS13 deficiency in 5 children (aged 1-11 years) of Polynesian origin. The first 3 cases have hypogammaglobulinaemia requiring regular intravenous immunoglobulin (IVIG) infusions. At least 4 of the 5 cases have additional signs and symptoms indicative of a common variable immune deficiency syndrome (CVID), which in two cases (siblings) may have been demasked by rituximab used to treat their relapsing aTTP. Conclusions: Although cytopenias (mainly idiopathic thrombocytopenia, or Evans’ syndrome, both differential diagnosis of aTTP) and various autoimmune diseases have been reported in CVID the association with aTTP is novel (or underreported). Together with the common Polynesian ethnicity of the 5 cases and the unusual early aTTP onset this hints at new CVID underlying mechanisms. Further diagnostics and a search for mutual underlying defects are warranted.
Influence of background genes on Sjogren’s syndrome in interleukin 14 alpha transgenic mice (P4100)
Authors: Shen Long , Suresh Lakshmanan , Ambrus Julian
Published: January 2023

Interleukin 14 alpha transgenic mice (IL14aTG) mice on a C57BL/6 background mimic Sjogren’s syndrome (SS) seen in patients (Clin Immunol., 2009). Previous studies have demonstrated roles for lymphotoxin and type 1 interferon in the pathophysiology of the disorder (J.Immunol., 2006 and 2010). C57BL/6 mice respond to immunological challenges with TH1 >>TH2 responses whereas Balb/C mice respond to similar challenges with TH2 >> TH1 responses. We therefore examined the development of SS in IL14aTG mice on a Balb/c background. Histology of the salivary and lachrymal glands, spleen and lymph nodes were examined at 21 months of age. IL14aTG.Balb/C mice demonstrated only minimal infiltration of submandibular glands and minimal decrease in salivary gland secretions at 21 months of age. In contrast IL14aTG.C57BL/6 mice had dramatic infiltration of the submandibular and parotid glands by 12 months of age with loss of salivary gland secretions by 10 months of age. At 21 months of age 95% of IL14aTG.C57BL/6 mice had lymphoma while none of the IL14aTG.Balb/C mice had lymphoma. Evaluation of type 1 interferon, lymphotoxin and autoantibodies in the sera of IL14aTG.Balb/c mice are in progress. Development of SS in IL14aTG mice is markedly diminished with a Balb/c background compared to a C57BL/6 background. A TH1 program is central to the pathophysiology of SS. Further studies will elucidate particular genes that distinguish the development of SS in C57BL/6 mice compared to Balb/c mice.
Stage-specific binding profiles of cohesin in resting and switch-activated B cells at transcriptional control elements (P4396)
Authors: Günal Gamze , Paszkowski-Rogacz Maciej , Singaravelu Kalaimathy , Beyer Andreas , Buchholz Frank , Jessberger Rolf
Published: January 2023

The immunoglobulin (Ig) heavy chain locus comprises several higher order chromosomal interactions to facilitate stage-specific assembly of the Ig molecule. The cohesin complex acts together with CTCF to regulate long-range chromatin interactions important for transcriptional regulation during cell differentiation. We elucidated the binding profile of the cohesin complex in mature resting and switch-activated B lymphocytes by chromatin immuno-precipitation coupled with deep sequencing. Comparative genomic analysis revealed specific changes in patterns of cohesin binding to transcriptional control elements during Ig class switching. Specific binding changes of cohesin at B-cell specific gene loci suggest new cohesin-dependent regulatory pathways important for B cell maturation. Together with conserved cohesin/CTCF sites at the Igh locus, a prominent intronic cohesin/CTCF binding site at the Igh locus has been revealed. The functional mechanism of this mature B-cell specific regulatory site is yet to be elucidated by using chromosomal conformation capture assay. Our study shows that cohesin is involved in regulation of Ig class switching and will give rise to new insights of possible cohesin-dependent regulatory pathways of mature B cell development and class switching.
Recognition of CD47 on human hematopoietic stem cells results in improved human chimerism in xenografts and permits investigation of rare human progenitors in vivo. (P4469)
Authors: Huntington Nicholas , Di Santo James
Published: January 2023

Human immune system (HIS) mice represent an important biotechnology for fundamental human immunology research, disease models and drug development. The NOD mouse strain has been primarily used in the development of HIS mice as they possess several advantageous polymorphisms. NOD, but not Balb/c mice contain a polymorphism in Sirpα (an inhibitory receptor that prevents phagocytosis upon binding CD47) allowing NOD, but not Balb/c Sirpα to recognize human CD47 on engrafted cells. Despite this, Balb/c immunodeficient mice are still permissive to human engraftment and their use in human immune studies becoming more prevalent. To determine the importance of the CD47-Sirpα interactions in human hematopoietic reconstitution, we generated a congenic Balb/c Rag2-/-γC-/- HIS mouse expressing NOD Sirpα. A functional CD47-Sirpα interaction resulted in significantly higher human chimerism, with specific improvements in T cell, NK cell and progenitor reconstitution compared to HIS mice expressing only Balb/c Sirpα. Improved human reconstitution has allowed us to carefully study bone marrow resident human hematopoietic progenitors. We have identified 2 subsets of CD7+CD244.2+CD38+CD45RA+Lin-(CD56-) bone marrow precursors with either high or exclusive restriction to the NK cell lineage. Our results reveals the degree by which phagocytosis restricts xeno-engraftment and demonstrates a novel, highly sensitive xenograft model for human immunology studies.
Maternal methyl-donor supplementation protects against atherosclerosis in F1 ApoE-/- mice (P4424)
Authors: Yung Raymond , Delanaey Colin , Garg Sanjay , Fernandes Christopher , Allen Robert , Stabler Sally
Published: January 2023

T lymphocytes play an important role in the pathogenic inflammatory response that accelerates atherosclerosis. Previously, we showed that F1 mice born to dams fed a diet supplemented with methyl donors (MS diet) during pregnancy and lactation had decreased T cell-mediated pro-inflammatory cytokine and chemokine responses. Here, we report feeding Apolipoprotein E (ApoE-/-) deficient dams with the MS diet during pregnancy reduces atherosclerotic plaques in F1 mice that are subsequently fed a high fat diet (HFD) after weaning. F1 mice from dams on the MS diet (F1-MS) had increased global T cell DNA methylation and reduced Ccr2 mRNA and protein expression in CD3+ T cells, but not in CD11b+ monocytes, relative to F1-control mice. CCR2 plays an important role in the recruitment of inflammatory cells to atherosclerotic vascular lesions. F1 litter size, HFD consumption, body weight, body fat, and serum thiol metabolite levels were similar between F1-MS and F1-control mice. However, F1-MS mice had higher serum HDL and lower LDL+VLDL levels compared to F1-control mice. F1-MS mice had lower serum levels of inflammatory cytokines (IL-17, TNF-α, IL-6), and F1-MS T cells cultured in vitro expressed lower levels of those cytokines compared to F1-control mice. Together, these data suggest that the pre-natal MS diet ameliorates development of atherosclerosis by inhibiting T-cell CCR2 expression, reducing inflammatory cytokine production and increasing serum HDL:LDL ratio.
Loss of homeostatic granulopoiesis in IKKβ-deficient mice (P4472)
Authors: Huang Howard , Wang Xingan , Yamamoto Sumiharu , Ibrahim Mohsen , Qiu Chang-Shen , Zhu Jihong , Krupnick Alexander , Kreisel Daniel , Gelman Andrew
Published: January 2023

BACKGROUND IL-1β-mediated neutrophilia has been observed in mice with an inducible deletion of IKKβ under non-homeostatic conditions. We investigated the effects of a constitutive IKKβ deletion from the hematopoietic compartment. METHODS Mice carrying loxP-flanked IKKβ alleles (IKKβfl/fl) were crossed with Vav1-iCre mice to target IKKβ expression in hematopoietic cells (IKKβΔ/Δ). Bone marrow (BM), blood, spleen and peripheral tissues were analyzed by histopathology and FACS. Methylcellulose colony-forming cell (CFU) assays were performed using Lin-c-kit+ myeloid progenitors (MP) and splenocytes. MP proliferation was measured by EdU incorporation. RESULTS IKKβΔ/fl mice are phenotypically indistinguishable from IKKβfl/fl (WT) mice. In contrast, IKKβΔ/Δ mice are born in non-Mendelian ratios, develop patchy alopecia, osteopetrosis, splenomegaly and stunted growth. IKKβΔ/Δ mice also show massive neutrophilia in BM, spleen, blood and peripheral tissues as well as a sharp increase in BM and spleen granulocyte CFUs relative to IKKβΔ/fl and WT controls. Unlike in IKKβΔ/fl or WT granulocyte macrophage progenitors (GMP), IKKβΔ/Δ GMPs proliferate more in response to G-CSF than to GM-CSF or IL-3 stimulation. Moreover, G-CSF stimulated IKKβΔ/Δ MP produce more neutrophils relative to IKKβΔ/fl and WT MP. CONCLUSIONS Our data indicate that IKKβ controls homeostatic granulopoiesis through regulating sensitivity to G-CSF signals in MP.
Histone H2A deubiquitinase MYSM1 is essential for dendritic cell development (P4488)
Authors: Won Haejung , Chou Yuchia , Wang Tao , Jones Lindsey , Huang Xue , Chen Si-Yi
Published: January 2023

The mechanisms controlling differentiation of dendritic cells remain largely unknown. Using knockout mouse model, we identified Mysm1 as a critical regulator in dendritic cell differentiation. Mysm1 knockout mice showed a global reduction of both conventional dendritic cells and plasmacytoid dendritic cells in lymphoid organs and non-lymphoid organs whereas development of granulocyte and macrophage was not severely affected. Hematopoietic progenitors and dendritic cell precursors were significantly decreased in KO mice, and the precursor cells from Mysm1 knockout mice were unable to develop DC upon Flt3L stimulation in vitro. The developmental defect of Mysm1-/- progenitor cells was associated with decreased Flt3 expression. Molecular studies indicate that Mysm1 de-represses transcription of Flt3 gene by regulating histone modifications and mediating the recruitment of transcription factor Pu.1 at the promoter. In conclusion, we have identified a regulator of dendritic cell development, Mysm1, and provided novel mechanism of epigenetic control in steady-state dendritic cell development.
IRF8 regulates lymphoid-myeloid lineage priming and commitment defined by an IRF8-EGFP reporter mouse (P4374)
Authors: Wang Hongsheng , Jain Shweta , Sun Jiafang , Morse, III Herbert
Published: January 2023

IRF8 is a transcription factor critically involved in the differentiation of dendritic cells (DCs) and B cells. IRF8 is expressed in early hematopoietic progenitors, but it is not clear how IRF8 promotes these progenitors to commit to one lineage while preventing the development of alternative lineages. To better understand the functions of IRF8, we generated an IRF8-EGFP reporter mouse by replacing the stop codon of the Irf8 locus with the EGFP sequence. Flow cytometric analyses revealed progressive increases in expression of IRF8-EGFP from hematopoietic stem cells to progenitors destined to the granulocyte-macrophage pathway and to the common lymphoid progenitor pathway but not to the megakaryocytic-erythroid progenitor pathway. Cells committed to the B lineage expressed high levels of IRF8-EGFP, which is in sharp contrast with thymic T cells that expressed negligible levels of IRF8-EGFP. Unexpectedly, IRF8-EGFP expression in pre-pro-B cells was extremely heterogeneous, ranging from background to 4 logs above background. Furthermore, we identified an IRF8-EGFPbri population that expresses B220 with the potential to generate B cells, plasmacytoid DCs and myeloid cells in vivo. Finally, DC progenitors including common DC progenitors and pre-cDCs were marked by extremely high expression of IRF8-EGFP. Taken together, these data revealed a sequential expression pattern of IRF8-EGFP in early hematopoietic progenitors coinciding with lineage priming and commitment.
Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk (P2001)
Authors: Parker Katherine , Horn Lucas , Clements Virginia , Sinha Pratima , Yang Huan , Li Jianhua , Tracey Kevin , Ostrand-Rosenberg Suzanne
Published: January 2023

Tumor-induced immune suppression is driven by immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in most patients with cancer. They block T cell activation and drive type 2 immunity. Cross-talk with macrophages enhances MDSC secretion of pro-tumor molecules such as IL-10 and decreases macrophage production of IL-12. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin or Ethyl Pyruvate reduced lung metastases, while treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. BALB/c mice carrying 4T1 tumor cells down-regulated for HMGB1 by shRNA had an extended survival time relative to mice with irrelevant shRNA 4T1 tumor. Treatment of MC38 tumor-bearing mice with the anti-inflammatory A box domain of HMGB1 reduced primary tumor growth. In in vitro experiments, HMGB1 inhibitors decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages, and reduced MDSC suppressive potency for T cells. HMGB1 inhibitors also reduced MDSC differentiation from bone marrow progenitor cells. These findings indicate that full-length HMGB1 drives tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC accumulation.
Attenuated NK cell killing to hepatoma cells increases the mortality via decreased expression levels of MICA, CD112 and CD155 during UPR (P2023)
Authors: Chen Lihua , Gong Jiuyu , Fang Liang , Jin Boquan
Published: January 2023

Natural killer (NK) cells play important roles in anti-tumor immunity. Here, we demonstrate that the unfolded protein response (UPR) attenuated the sensitivity of hepatoma cells to NK cell cytotoxic activity by decreasing the expression levels of MHC class I-related chain A/B (MICA/B), CD112 and CD155 in human hepatocellular carcinoma (HCC) cells. The decreased expression levels of MICA/B, CD112 and CD155 were due to the involvement of the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1α (IRE1α) pathways. In addition, the IRE1α pathway contributed to the increased expression levels of the ER-associated degradation (ERAD)-related molecule HRD1 and facilitated the degradation of MICA/B, CD112 and CD155. Moreover, we found that low levels of MICA/B, CD112 and CD155 expression were significantly associated with poor prognosis in patients with HCC. Thus, our results provide molecular, cellular and clinical evidence demonstrating a novel NK cell-associated immune evasion mechanism, and indicate that targeting this immune evasion pathway may be meaningful in treating patients with HCC.
Tumor-induced STAT3 hyperactivation creates a dendritic cell differentiation block by significantly downregulating protein kinase C βII expression (P2003)
Authors: Farren Matthew , Spangler Haley , Netherby Colleen , Carlson Louise , Abrams Scott , Lee Kelvin
Published: January 2023

Tumor mediated blockade of dendritic cell (DC) differentiation is a large component of cancer induced immunosuppression, contributing to tumor outgrowth. Tumors impair DC differentiation via factors that hyperactivate STAT3 in DC progenitors, though the mechanisms by which this occurs are largely unknown. PKCβII is essential in DC differentiation, and we test here if tumor driven STAT3 hyperactivation downregulates PKCβII expression, and if this is the mechanism underlying impaired DC differentiation. Myeloid cells from tumor bearing mice have significantly decreased PKCβII expression. Culture in tumor conditioned media (TCM) significantly decreased PKCβII expression and significantly impaired phorbol ester driven DC differentiation in human monocytes and in a human cell line model (KG1). In KG1, this was dependent on PKCβII downregulation: enforced PKCβII expression preserved phorbol ester driven DC differentiation in the face of TCM. Interestingly, PKCβII overexpression and/or activation also antagonized TCM-driven STAT3 activation. STAT3 drives this decreased PKCβII expression: TCM induced STAT3 activation and drove 4.5 fold higher STAT3 binding to the PKCβ promoter (compared to media control, p<0.001). Mutation of a STAT3 consensus binding site in the promoter eliminates the ability of STAT3 to impair PKCβ promoter activity. Together, these observations argue that tumor driven STAT3 hyperactivation inhibits DC differentiation by downregulating PKCβII.
Anti-inflammatory effects of myeloid-derived suppressor cells and macrophage crosstalk contribute to tumor progression (P2043)
Authors: Beury Daniel , Parker Katherine , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid-Derived Suppressor Cells (MDSC) polarize macrophages (MΦ) to a Type II tumor-promoting phenotype via MDSC-MΦ crosstalk. However, the role of inflammation in MDSC-MΦ crosstalk is not well defined. To determine the role of inflammation, we utilized wild type, IL-6-/-, and IL-10-/- mice bearing syngeneic 4T1 mammary carcinoma. MΦ and 4T1-induced MDSC express IL-6 and IL-10 receptors, suggesting that they have the potential to respond to both cytokines. To determine if IL-6 and IL-10 contribute to tumor progression by modulating MDSC-MΦ crosstalk, MDSC from 4T1-bearing wild type or IL-10-/- mice were cultured with MΦ from wild type or IL-6-/- mice. MDSC were anti-inflammatory and decreased MΦ production of IL-6. IL-6 is important in vivo since 4T1-bearing IL-6-/- mice have extended survival and delayed primary tumor growth vs. wild type mice. Anti-inflammatory effects are exacerbated by MΦ themselves, since their production of IL-6 increases MDSC production of IL-10. However, MΦ-produced IL-6 affects MDSC indirectly, as exogenous IL-6 does not increase MDSC IL-10 production. Although IL-10 is classically considered as an anti-inflammatory cytokine, it contributes to tumor progression because 4T1-bearing IL-10-/- mice have delayed primary tumor growth and extended survival vs. wild type mice. These data demonstrate that MDSC have anti-inflammatory effects and that MDSC-MΦ cross-talk contributes to the overall milieu of IL-10 and IL-6 within the tumor microenvironment.
TNF signaling drives myeloid-derived suppressor cell accumulation (P2051)
Authors: Qin Zhihai
Published: January 2023

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.
Autophagy sustains the survival and pro-tumorigenic effects of neutrophils in human cancer (P2053)
Authors: Li Xue-Feng , Kuang Dong-Ming , Zheng Limin
Published: January 2023

Neutrophils are a common inflammatory infiltrate that are enriched predominantly in the peritumoral stroma of many human cancers. The underlying mechanisms, however, that allow tumor microenvironments to educate neutrophils in peritumoral stroma are largely unknown. Herein we showed that most neutrophils in peritumoral stroma areas, but not those within the blood vessels, expressed significant higher amount of autophagy-related LC3 protein. In consistence, exposure of neutrophils to culture supernatants from several types of solid tumor cells (TSN) resulted in a rapid up-regulation of autophagy in cells, an effect that could be inhibited by blocking the activation of Erk1/2, p38 and NF-κB signaling pathways. The increased autophagy in TSN-exposed neutrophils was associated with preserved mitochondria integrity, reduced production of mitochondrial reactive species and cleavage of Caspase 3, and attenuated apoptosis in these cells. Pretreatment with 3-methyladenine, a specific autophagy inhibitor, could effectively induce mitochondrial injury and subsequent apoptosis in neutrophils. Moreover, we provided evidence that the induction of autophagy markedly enhance the pro-tumorigenic activities of neutrophils by releasing higher amount of matrix metalloproteinase-9, hepatocyte growth factor, and oncostatin M. Thus, increased autophagy in tumor-associated neutrophils may represent a novel mechanism that links the inflammatory response to neoplastic metastasis.
Increased B7-1 (CD80) expression reduces overall tumorigenicity and metastatic potential of the murine pancreatic cancer cell model Pan02 (P2085)
Authors: Imasuen Imade , Bozeman Erica , He Sara , Patel Jaina , Selvaraj Periasamy
Published: January 2023

Pancreatic cancer is both difficult to detect and harbors survival rates as low as 5%, thus heightening the need for better and new therapies. Several molecules have been investigated to increase the immunogenicity of tumor cells including the costimulatory molecule B7-1. The murine pancreatic cancer cell line, Pan02, expresses low, endogenous levels of B7-1; however, the role of B7-1 in pancreatic cancer progression has not yet been elucidated. We hypothesized that increased cell surface expression of B7-1 in the Pan02 model would decrease tumor growth and metastasis. To test this, Pan02 cells were transfected with mB7-1 (Pan02-mB7-1). Flow cytometry analysis confirmed a 40-fold increase in B7-1 cell surface expression in transfected cells compared to WT. In order to assess the in vivo effects of increased B7-1 expression, C57BL/6 mice, were challenged with Pan02-mB7-1 and Pan02-WT cells. We observed that B7-1 transfected Pan02 cells showed an overall decrease in tumor kinetics compared to mice challenged with Pan02-WT. In addition, increased cell surface B7-1 expression prevented metastasis to the lung in tumor positive C57BL-6 mice. More interestingly, Pan02 cells that metastasized to the lung (Pan02-RG-M-Lung) from WT challenged mice showed lower B7-1 expression by flow cytometry compared to Pan02-WT cells. Taken together, increased cell surface B7-1 expression is successful in inhibiting tumor growth and preventing metastasis in the murine pancreatic cancer model.
Impairment of cytotoxic anti-tumor response by hypoxic stress (P2011)
Authors: Chouaib Salem , Hasmim Meriem , Noman Zaeem M
Published: January 2023

Hypoxia is a central hallmark of tumor microenvironment and a pivotal driving force of malignant progression. Although hypoxia has been reported to play a major role in the acquisition of tumor resistance to cell death, the molecular mechanisms that enable the survival of hypoxic cancer cells have not been fully elucidated. Recently, attention has focused on the mechanisms by which hypoxic tumour cells alter their transcriptional profiles allowing them to persist under conditions of hypoxic stress. Here we demonstrate that NANOG, a transcription factor associated with stem cell self renewal, is induced under hypoxic conditions at both transcriptional and translational levels and contributes to the decreased susceptibility of target cells to specific cytotoxic T lymphocyte (CTL)-mediated lysis. The precise mechanisms by which NANOG regulates tumor susceptibility to CTL-mediated lysis will be discussed. In addition since we have demonstrated that in vivo inhibition of hypoxia restores cytotoxic T lymphocyte activity and promotes in vivo tumor regression, we will also discuss the relationship between hypoxic stress and survival pathways associated with hypoxic stress in particular the emergence of cancer stem cells and autophagy induction.
Evaluation of the protective immunity of a novel subunit fusion vaccine in a murine model of systemic MRSA infection (P4254)
Authors: Zeng Hao
Published: January 2023

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on Hla and IsdB antigenic region. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). our results verified that the strategy of optimized chimeric bivalent antigens could be as an important approach for developing S. aureus mult-subunit vaccines against S. aureus infection.
Local and systemic immune responses predict clinical outcomes in influenza virus infection independently of age and viral titer (P3000)
Authors: Oshansky Christine , Gartland Andrew , Wong Sook-San , Jeevan Trushar , Wang David , Roddam Philippa , Caniza Miguela , Hertz Tomer , DeVincenzo John , Webby Richard , Thomas Paul
Published: January 2023

Influenza infection can result in mild or serious illness in some individuals, and studies suggest that increased inflammatory mediators have a role in disease severity. To investigate the role of inflammation in clinical disease, we performed a prospective, longitudinal study, sampling symptoms, blood, nasal lavages, and viral loads from participants during natural influenza infection. Surprisingly, clinical disease defined by symptoms or hospitalization did not correlate with viral dynamics, but consistent with other clinical reports, age was associated with hospitalization. There was no association between age and viral dynamics, though a strong inverse correlation between age and cytokine levels in the nasal lavage was maintained even when adjusted for prior immunity, indicating that children mount more aggressive inflammatory responses independently of pathogen dynamics. Correcting for this age-association, analysis of cytokine levels in nasal lavages revealed that MCP3 and IFNα were predictors of respiratory symptoms, while plasma MCP3 and IL-10 predicted symptom severity and hospitalization. In all cases, associations were not dependent on viral load. The association between IL-10 and symptom severity was confirmed in an independent cohort. In summary, children suffer significantly higher local inflammation, and a small subset of cytokines, including MCP3 and IL-10, serve as diagnostic markers for clinical outcome of influenza infection in all age groups.
Detection of antigen-specific B cells to Plasmodium falciparum apical membrane antigen-1 and circumsporozoite protein in humans and immunized mice (P3025)
Authors: Odumade Oludare , Frosch Anne , Taylor Justin , Dehnbostel Joanne , John Chandy
Published: January 2023

Worldwide, there are an estimated 216 million cases of malaria annually and of those affected 655,000 will succumb to the disease. Antibody responses to infection have been shown to be critical in limiting infection and disease, but the mechanisms by which these are acquired or lost are poorly understood. Various hypotheses have been proposed for dysfunctional humoral responses including B cell exhaustion, germinal center architectural disruption, parasite antigenic variation and inhibitory innate immune signaling. However, the relative infrequency of malaria specific B cells in the periphery has limited targeted detection of these cells. We present a strategy for phenotypic analyses of malaria specific B cells in peripheral blood using tetramerized biotinylated malaria antigen coupled to phycoerythrin labeled streptavidin, magnetic bead enrichment and cytometric flow analysis. Using this technique, we demonstrate the detection of an antigen specific germinal center reaction in B6 mice after immunization with P.pastoris expressing recombinant Plasmodium falciparum apical membrane antigen-1 and circumsporozoite protein. Preliminary results in humans identified low frequency B cells specific to these two antigens. Overall, our results suggest that this technique will be a valuable tool in studying naturally acquired and vaccine derived malaria specific B cell responses.
CD16+ expressing monocyte subsets and CD14+ HIV DNA levels predict HIV-associated neurocognitive disorders (HAND) in treatment-naïve HIV infected Thai subjects (P3043)
Authors: Byron Mary Margaret , Valcour Victor , Ananworanich Jintanat , Agsalda Melissa , Chalermchai Thep , Tipsuk Somporn , Sithinamsuwan Pasiri , Schuetz Alexandra , Hutchings Nicholas , Barbour Jason , Phanuphak Nittaya , Shikuma Cecilia , Shiramizu Bruce , Ndhlovu Lishomwa
Published: January 2023

Since the advent of anti-retroviral (ARV) therapy, HIV-dementia has significantly declined, however, milder forms of HIV-associated Neurocognitive Disorder (HAND) remain highly prevalent. HIV DNA reservoirs in CD14+ Monocytes (MO) have been associated with cognitive impairment. However, the role of MO subsets in the pathogenesis of HAND over time remains poorly characterized. We sought to analyze HIV DNA content and MO phenotypic properties in relation to cognitive impairment through a prospective study of 30 ARV therapy-naïve individuals enrolled by HIV DNA reservoir levels with subsequent cognitive characterization as normal (n=17), Asymptomatic Neurocognitive Impairment (n=6), Mild Neurocognitive Disorder (MND) (n=2), HIV-associated dementia (HAD) (n=5) and 44 HIV- matched controls. HIV DNA was extracted from CD14+ cells and quantified by PCR. MO subsets and cell surface chemokine receptors were measured by flow cytometry using an extensive panel to exclude non-MO. Our preliminary findings show that among 4 MO subsets, there was a statistically significant decline in frequency of CD14highCD16+ (p<0.001) and an increase in CD14lowCD16- (p<0.001) in HIV+ compared to HIV- individuals. Remarkably, we found that CD14lowCD16+ MO frequency was lower in cognitively impaired individuals (p<0.05) and frequency of CCR2+ and CCR5+ expressing MO was higher in symptomatic (MND and HAD) cognitively impaired HIV+ individuals (p<0.05, p<0.05).
RIP3 is involved in host defense against HSV-1 infection via enhancing STING mediated antiviral signaling (P1405)
Authors: Liang Yaoji , Wu Suqin , Han Jiahuai
Published: January 2023

Herpes simplex virus type 1 (HSV-1), a double stranded DNA virus, is a widespread virus that infects approximately 80% of adults worldwide. HSV-1 infection is also the major cause of fatal childhood HSV-1 encephalitis (HSE). The mechanisms of how host cells defend against HSV-1 are still not fully known. Here we report that receptor interacting protein 3 (RIP3) enhances type I interferon production in response to HSV-1 infection. In our study of the functions of RIP3, we have found that the HSV-1 replication in RIP3 knockout L929 was unrestrained, and that HSV-1 induced TBK1 and IRF3 activations were impaired in RIP3 deficient cells. Further study has shown that over-expression of wild type RIP3, but not RHIM-mutant RIP3, enhances type I interferon production, especially when co-expressed with STING, a central signaling molecule in the innate immune response to DNA virus infection. Upon treating the cell with HSV-1, TBK1 was recruited to RIP3. In addition, RIP3 can promote the interaction between STING and TBK1 in a RHIM dependent manner. We propose that upon HSV-1 stimulation, RIP3 promotes the recruitment of TBK1 to STING, which leads to TBK1-mediated IRF3 activation and subsequent type I IFN secretion. Our study revealed that, in addition to its function in necrosis, RIP3 plays a role in type I interferon production. These results may also provide a new perspective for the exploration of childhood HSE pathogenesis.
Unanchored Lysine48-linked polyubiquitin chains positively regulate the type I IFN-mediated antiviral response (P1391)
Authors: Rajsbaum Ricardo , Versteeg Gijs , Schmid Sonja , Maestre Ana , Belicha-Villanueva Alan , Fernandez-Sesma Ana , tenOever Benjamin , García-Sastre Adolfo
Published: January 2023

Type-I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of 100s of IFN-I stimulated genes (ISGs), many of which have known antiviral activity. The full breadth of ISG induction requires activation of a number of cellular factors including the IκB kinase epsilon (IKKϵ). However, the mechanism of IKKϵ activation upon viral infection or IFN receptor signaling remains elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacts with IKKϵ and promotes induction of IKKϵ-dependent ISGs. Some studies have suggested a role for unanchored K63-linked polyubiquitin chains, which are not conjugated to any protein, in regulation of kinase activity. However, no role has yet been established for unanchored K48-linked polyubiquitin chains in kinase activation. We show that TRIM6 and the E2-ubiquitin conjugase UbE2K cooperate in the synthesis of unanchored K48-linked polyubiquitin chains, which activate IKKϵ for subsequent STAT1 phosphorylation. Our work defines a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
The role of IFI16 in the innate immune response to cytosolic nucleic acids (P1395)
Authors: Thompson Mikayla , Jensen Soren , Sharma Shruti , Fitzgerald Katherine , Kurt-Jones Evelyn
Published: January 2023

Recently, the Interferon Gamma Inducible (IFI)16 was linked to cytoplasmic DNA induced type I interferon (IFN) responses. IFI16 was shown to associate with Stimulator of IFN genes (STING), leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I IFN genes. Here we created THP1 monocytic cell lines with stable knockdown of IFI16 by lentiviral transduction of shRNA and demonstrated that IFI16 knockdown leads to a severely attenuated type I IFN response following treatment of cells with either synthetic DNA ligands, or after infection with DNA viruses such as HSV-1. Surprisingly, these cells were also compromised in their ability to induce type I IFNs to other non-DNA ligands including synthetic 5’pppRNA and Sendai Virus, both of which signal via RIG-I. In contrast the NF-kB regulated cytokines IL-6 and IL-1β remain unaffected in IFI16 knockdown cells. Utilizing Nanostring technology, we also determined that IFI16 is capable of regulating DNA and RNA induced expression of a panel of Interferon Stimulated genes (ISGs), including RIG-I. We also monitored key steps in the type 1 IFN pathway and found that levels of total IRF3 were also reduced in IFI16 knockdown cells. These results suggest that IFI16 senses DNA viruses, eliciting a type I IFN response, but also acts more broadly in the regulation of ISGs in response to both DNA and RNA viruses.
Integrin α9 on lymphatic endothelial cell at draining lymph node regulates lymphocyte egress via controlling S1P secretion during inflammation (P5114)
Authors: Ito Koyu , Morimoto Junko , Kihara Akio , Simmons Szandor , Ishii Masaru , Uede Toshimitsu
Published: January 2023

Sphingosine 1-phosphate (S1P) plays a role in lymphocyte egress from lymphoid organs. However, it remains unclear how S1P production and secretion are regulated. We found that α9 integrin and its ligand, Tenascin-C (TN-C) were co-localized on lymphatic endothelial cell (LEC) of medullary sinus in the draining lymph nodes (dLN). To examine the role of α9 integrin in the dLN, mice that had been treated with Complete Freund Adjuvant (CFA) were treated with blocking anti-α9 integrin antibody (55A2C). Although CFA-induced inflammation was associated with appearance of migrating lymphocytes within medullary sinus at dLN, mice-treated with 55A2C showed vacant medullary sinus. In addition, adoptive T cell transfer experiments demonstrated that lymphocyte egress was significantly reduced by both FTY720, which was S1P receptor agonist, and 55A2C under inflamed condition. Stimulation of LEC by TN-C enhanced S1P secretion without affecting S1P secretion and / or degradation. We further show that blockade of α9 integrin-mediated signaling reduces lymphocyte egress from dLN in several models, including experimental autoimmune encephalomyelitis (EAE) where it improves clinical scores and pathology. Therefore, we propose that interaction of α9 integrin on LEC with TN-C induces S1P secretion, and subsequent lymphocyte egress, possibly by induction of S1P transporter and that manipulating α9 integrin function may offer a therapeutic strategy for treating various inflammatory disorders.
Conditional ablation of Langerhans cells exacerbates autoimmune corneal inflammation (P5134)
Authors: Gutierrez Miguel-Angel , Zhu Zejin , Philips Rachael , Eriksson Anna U , Halder Ramesh , Kim Peter , King Jennifer , Rao Narsing , Singh Ram Raj
Published: January 2023

Ocular surface inflammation is common and sometimes severe in autoimmune diseases. Little is known about the role of cornea-resident dendritic cells, including Langerhans cells (LC), in ocular surface autoimmunity. Here, we analyzed corneal LC in autoimmune-prone MRL mice. LC (CD11c+ CD207+) were more abundant and activated (CD86+ CD40+) in the corneal epithelium of MRL mice than of B6 mice. However, LC were ~5-fold lower in the corneal stroma of MRL mice than of B6 mice. LC in cervical lymph nodes were also lower in MRL mice than in B6 mice. Ongoing studies will examine whether the increase of activated LC in corneal epithelium, but their reduction in corneal stroma and eye-draining lymph nodes in autoimmune mice is due to a defect in the migration of LC from the cornea to eye-draining lymph nodes. Since LC are believed to carry antigens from tissues to their respective draining lymph nodes to maintain tolerance in a tissue-specific manner, the reduced LC in eye-draining lymph nodes may lead to the breakdown of tolerance to eye antigens. To directly test the role of LC in ocular autoimmunity, we introgressed the Lang-eGFP.DTR knockin mutation from the stock B6 mice that express diphtheria toxin receptor (DTR) driven by Langerin promoter (Malissen, 2005) onto the MRL background. Diphtheria toxin injections depleted LC in Lang-eGFP.DTR MRL mice and accelerated corneal inflammation. Taken together, these data suggest a protective role of LC in corneal inflammation.
Highly cytokinergic SPE-7 anti-dinitrophenyl-immunoglobulin E induces antigen-dependent thermal hyperalgesia and inflammatory cytokine production in the mouse hindpaw (P6000)
Authors: Chatterjea Devavani , Mack Madison , Wetzel Abigail , Tonc Elena , Balsells Evelyn , Engblom Camilla
Published: January 2023

SPE-7 anti-DNP-IgE initiates inflammatory cytokine production without antigen stimulation in vitro. In contrast, ϵ2624 anti-DNP IgE does not induce inflammatory cytokines independent of antigen-stimulation. However, their effects in vivo have not been described. We tested two well-characterized anti-mouse IgE antibody clones in our model of murine thermal hyperalgesia. We found pronounced hindpaw hyperalgesic responses lasting to at least 6 hours following antigen challenge after sensitization with SPE-7. These responses were accompanied by histamine release and neutrophil influx as we have previously shown in c48/80-mediated thermal hyperalgesia. In contrast, we found that antigen challenge following sensitization with clone ϵ2624 produced slight hyperalgesia at 1 hr that was resolved by 3 hours. We demonstrate a novel functional, behavioral difference between reactions mediated by these IgE antibody clones in vivo in mice. Also, SPE-7 does not lead to antigen-independent cytokine production in the mouse hind paw. After antigen challenge, both IgEs produce equivalent levels of TNF-α, IL-1, and IL-6, and cause equivalent histamine release and neutrophil influx. Thus tissue-resident sentinel mast cells may be critical initiators of allergen-evoked hyperalgesia in mice and differences in the nature and repertoire of IgE antibodies produced during an allergic response likely have pathophysiological implications for various health outcomes including pain.
Antibodies binding to discrete segments of CϵmX of mIgE on B lymphocytes (P6004)
Authors: Chan Yueh-Hsuan , Chu Hsing-Mao , Chen Jiun-Bo , Wu C. , Pan Chi-Chun , Chang Tse Wen
Published: January 2023

CϵmX is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ϵ heavy chain of membrane-bound IgE (mIgE) on human B cells. Among the monoclonal antibodies (mAbs) prepared for their ability to bind to CϵmX fragments or CϵmX-containing recombinant proteins in ELISA, those that can bind to native mIgE on B cells can cause ADCC, apoptosis, and other cytolytic mechanisms on the B cells and are potential useful for controlling IgE production for treating allergic diseases. The CϵmX-specific mAbs prepared previously appear to recognize discrete and overlapping peptide segments of CϵmX. A linear peptide epitope of an antibody is defined by a contiguous peptide segment of minimal stretch that confers maximum binding by the antibody. In some instances, while the peptide epitopes recognized by two mAbs may be adjacent to each other and overlap, the two mAbs do not bind to the adjacent epitopes by a substantial degree. This can be illustrated by the pair of humanized mAbs, h4B12 and h47H4. The mAb h4B12 binds to peptide P1-15 (GLAGGSAQSQRAPDR) and h47H4 binds to peptide P7-18 (AQSQRAPDRVLC). Although the two epitopes share a segment AQSQRAPDR, the mAb h4B12 does not bind to P7-18, and h47H4 does not bind to P1-15. A related presentation from our group (Hsing-Mao Chu et al.) will also reveal that the shared peptide segment AQSQRAPDR has two markedly different conformations when it is associated with h4B12 or with h47H4.
Inhibition of E protein activity is required for iNKT cell development and function (P1458)
Authors: Hu Taishan , Wang Hongcheng , Simmons Amie , Zhao Ying , Sun Xiao-hong , Alberola-Ila Jose
Published: January 2023

iNKT cells are a distinct subset of αβ T cells with diverse immunoregulatory functions. Like conventional T cells, iNKT cells arise in thymus from a common CD4+CD8+ double positive (DP) precursor, but these cells are positively selected by glycolipid-loaded CD1d. Our previous result showed that the RAS/MAPK cascade (RAS-Erk-Egr-ID3), a central player during conventional T cell positive selection, is also required for iNKT cell development. Since RAS/MAPK signaling cascade has been reported to promote T cell positive selection by antagonizing E2A activity, we hypothesize that the same mechanism might be applied to iNKT cell development. To test this hypothesis, we utilized a conditional knock-in mouse model expressing ET-2, a chimeric protein composed of the TAD domain of E47 and the bHLH domain of SCL/Tal. Functioning as a dominant negative mutant of ID, ET-2 dramatically impaired iNKT cell development and function. iNKT cells in ET-2 mice were blocked during transition of stage 2 to stage 3, and these cells were also defective in IFN-γ production. Further mechanistic dissection showed that ET-2 might block iNKT cell development through inhibition of T-bet, a transcriptional regulator required for iNKT cell terminal maturation and homeostasis by controlling CD122. Forced expression of T-bet rescued the defects in CD122 expression and IFN-γ production. These results further confirm the role of the RAS/MAPK cascade in iNKT cell development and function.
Using Zinc-finger technology to study transcriptional regulation of ThPOK identifies potential cis-acting elements and transcription factors' binding sites (P1460)
Authors: Zha Jikun , He Xi , Dai Zhongping , Hua Xiang , Kappes Dietmar
Published: January 2023

T cell maturation involves key branch points at which cells choose between commitment to different lineages, i.e. gd versus ab T cell lineages and CD4+ helper versus CD8+ killer lineages. We focussed on the characterization of a unique line of spontaneous mutant mice, HD-/- mice, which are blocked in the generation of CD4+ helper T cells. Eventually, we discovered of the Zn-finger transcription factor Th-POK. We and others further established that the transcription factor ThPOK is necessary and sufficient to trigger adoption of the CD4 lymphocyte fate. We start to investigate the regulation of ThPOK expression and its subsequent control of CD4+T cell commitment. We identified six separate DHS sites, of which two coincide with putative promoters and the others could represent potential enhancers and silencers. Functional analysis of these putative regulatory elements using a series of reporter transgenics revealed that the DHS site A functions as a lineage specific silencer (referred to as DRE, distal regulatory element).To further dissect the contribution of these DHS sites to the regulation of ThPOK expression, we employed the technology of Zinc finger nucleases (ZFN) mediated genome mutation. Analyzing these mutant mice lines indicates that these both these TF binding motifs work in concert to regulate ThPOK expression, which further affect the determination of thymocytes fate.