bluuebunny/crossref_metadata_2025 huggingface dataset:
Follicular B cells, not Marginal zone B cells, preferentially contribute to development of spontaneous autoimmune thyroiditis (SAT) and are targets of anti-CD20 therapy (56.12)
Authors: Hong So-Hee , Mullen Helen
Published: January 2023

SAT in NOD.H-2h4 mice is a chronic organ-specific autoimmune disease characterized by mononuclear cell infiltration of the thyroid and destruction of thyroid follicles by infiltrating inflammatory cells. Like other autoimmune diseases, B cells play a critical role in pathogenesis of SAT as important antigen presenting cells (APCs) for activation of CD4+ T cells. However it is not yet known which subsets of B cells are important for SAT development. In this study, we found that expression of the co-stimulatory molecules CD80 and CD86 was significantly increased on splenic CD23hiCD21low follicular (FO) B cells, but not marginal zone (MZ) B cells, after SAT development, but absolute numbers and percentages of splenic FO B cells were not altered. In draining lymph nodes, CD40 expression on FO B cells was also increased in mice with SAT. All thyroid infiltrating B cells in mice with SAT were FO B cells. Depletion of FO B cells by anti-CD20 antibody ameliorated established SAT, and anti-thyroglobulin antibody levels were reduced. Moreover, administering anti-CD20 to mice with established SAT results in depletion of B cells in the thyroid and depletion of thyroid B cells is longer lasting than depletion of splenic B cells. On the basis of these findings, we suggest the FO subset of B cells preferentially contributes to SAT development and persistence, and targeting FO B cells after disease develops provides an effective means to treat autoimmune diseases.
Rapid, column-free two-step procedure for the enrichment of human Th17 Cells from peripheral blood (56.13)
Authors: Fairhurst Maureen , Yu Jessie , Thomas Terry
Published: January 2023

Human Th17 cells play a critical role in the regulation of autoimmune disease and antimicrobial host defenses through production of pro-inflammatory cytokines IL17A and IL-17F. They can be characterized by expression of surface receptors including IL-23R, CCR6, CD161 and lineage-specific transcription factor RORC, but lack of CXCR3 expression. The major disadvantages of current isolation methods are the requirement for in vitro stimulation and the co-secretion of IFN-γ. We have developed a 2-step EasySepTM immunomagnetic column-free method for the enrichment of CD4+CXCR3-CCR6+ cells from fresh peripheral blood nucleated cells (PBNC). First, non-CD4 T cells and CXCR3+ cells are targeted for depletion using dextran-coated magnetic particles and a cocktail of antibody complexes. Labeled cells are separated using an EasySepTM magnet, and pre-enriched CD4 T-cells are poured off. Next, CCR6+ cells are positively selected from the pre-enriched fraction. The procedure can be automated using RoboSepTM. Starting with a frequency of 5 ± 2% CD4+CXCR3-CCR6+ cells, purities of 94 ± 3% (n=10) can be obtained. The resulting cells produce high levels of IL-17 with minimal IFN-γ when measured by ELISA and intracellular staining and increased RORC mRNA expression over total CD4 T cells and CD4+CXCR3+ T cells (Th1 cells). Enrichment of unstimulated human Th17 cells enables the investigation of adaptive immune responses and regulation mechanisms required for the development of future therapies.
Statins and rapamycin synergistically induce dendritic cell KLF2 expression and tolerogenic function in vivo (P1610) (56.8)
Authors: Bu De-xiu , Griffin Gabriel , Grabie Nir , Lederer James , Lichtman Andrew
Published: January 2023

The statins, a class of HMG CoA-reductase inhibitors display immunomodulatory properties, independent of their cholesterol-lowering effects. We recently demonstrated that statins modulate T cell function through upregulation of the transcription factor, kruppel-like factor 2 (KLF2). Dendritic cells are essential for initiating and regulating T cell responses. We therefore examined how statins might modulate DC function. We found that: 1) statins upregulate Klf2 gene expression in both splenic and bone marrow derived mouse DCs; 2) the combination of the mTORC1 inhibitor rapamycin and statin synergistically enhance DC Klf2 and chemokine receptor CCR7 expression at doses of rapamycin alone that have no effect on Klf2; 3) statin- plus rapamycin-treated DCs or DCs expressing Klf2 encoded by a retroviral vector skew T cell differentiation away from inflammatory pro-atherogenic Th1 and Th17 phenotypes and promote Th2 and regulatory T cell differentiation in vitro; 4) statins directly block mTOR activation in DCs as indicated by assays of phosphorylation of mTOR and its substrates; and 5) statin plus rapamycin treatment impaired the ability of myosin-peptide loaded DCs to induced experimental autoimmune myocarditis (EAM) when transferred to BALB/c mice. We conclude that pharmacologic targeting of KLF2 in T cells and DCs is a feasible goal for suppressing immune response in human disease.
Thymic stromal lymphopoietin is produced by dendritic cells. (57.3)
Authors: Kashyap Mohit , Yrina Rochman , Spolski Rosanne , Samsel Leigh , Leonard Warren
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.
Adrenergic regulation of p38 MAPK phosphorylation by hematopoietic protein tyrosine phosphatase in human B cells (57.4)
Authors: McMaken Sara , Gormley Matthew , Sanders Virginia
Published: January 2023

Asthma affects ~300 million people and is responsible for $14 billion in annual medical costs. Therapy utilizes agonists to stimulate the beta-2-adrenergic receptor (β2AR) on lung smooth muscle cells. However, β2AR stimulation on a B cell responding to an allergen in vitro or in vivo increases IgE levels without affecting isotype switching. Elucidating how the β2AR agonist increases IgE production is crucial in blocking this effect on the B cell while maintaining bronchodilation. We have shown that β2AR stimulation on B cells during CD40L/IL-4 priming in vitro induces a PKA-dependent phosphorylation of hematopoietic protein tyrosine phosphatase (HePTP). This releases bound p38 MAPK and allows for its phosphorylation. β2AR deficiency or depletion of the endogenous β2AR agonist norepinephrine in vivo decreases levels of IgE made per B cell and lung inflammation after antigen exposure. It is currently unknown if human HePTP functions in this capacity. We sought to determine if HePTP in human B cells contributed to the β2AR-mediated phosphorylation of p38 MAPK. In comparison to anti-CD40/IL-4-primed CL01 B cells, primed B cells exposed to β2AR agonist had increased HePTP phosphorylation and p38 MAPK phosphorylation. This finding suggests that stimulation of the β2AR on human B cells elevates p38 MAPK phosphorylation through a change in HePTP function to elevate IgE. Thus, HePTP may represent a novel target for regulating IgE levels in human B cells during beta-agonist therapy.
Evaluation of the effect of pollen lipid on iNKT cells from pollen-sensitized patients (57.6)
Authors: Fereidouni Mohammad , Jabbari Azad Farahzah , Faridhosseini Reza , Varateh Abdolreza
Published: January 2023

Introduction: iNKT cells are a small subset of T lymphocytes which express a invariant TCR and recognize lipid antigens. They are implicated in a range of immune responses but their role in allergy is controversial. Pollens are the most important trigger of allergic symptoms but their lipid contents may also have a role in allergy through interaction with iNKT cells. The aim of this study was to evaluate the in vitro effect of pollen lipid extract on iNKT cells. Subjects and methods: PBMCs from 18 pollen-sensitized subjects and 11 healthy controls were isolated and stimulated for 7 days with a mix crude lipid extract of five allergenic pollens prepared by Folch method. Alpha-galactosylceramide and medium were used as positive and negative controls respectively. Proportion of iNKT cells was measured by FACS using 6B11 and anti-CD3 monoclonal antibodies. Results: At day 0, the mean percentage of iNKT cells was 0.42 ± 0.3% and 0.44 ± 0.2% (P > 0.05) for sensitized and control groups respectively. After 7 days culture, iNKT cells from both groups expanded efficiently in response to alpha-galactosylceramide and iNKT proportion reaches to 8 ± 5.6% but lipid extract did not stimulate iNKT proliferation in any groups and the proportion of iNKT cells remained unchanged. Conclusion: The results of this study suggest that pollen lipids are not potent stimulator of iNKT proliferation but further studies need to evaluate the presence and frequency of pollen-specific iNKT cells.
Metagene projection strategies for discovering mechanisms of T cell differentiation in gene expression profiling data (58.13)
Authors: Alexe Gabriela , Tamayo Pablo , Mesirov Jill , Gupta Prakash , Wolski David , Lauer Georg , Haining William
Published: January 2023

Characterizing the phenotypic heterogeneity in memory T cells generated following antigen encounter is a major challenge in immunology. Tools such as CyTOF flow cytometry or gene expression profiling of T cells can capture increasingly large numbers of parameters. However analysis of high dimensional data with standard techniques such as principal component analysis or hierarchical clustering often fails to capture the biological basis for observed phenotypic heterogeneity. We address this problem by applying a metagene projection strategy based on independent component analysis and consensus non-negative matrix factorization clustering. This approach assumes that the gene expression profile of a population of CD8+ T cells is generated by groups of genes that are representative of different biological processes, and deconvolves the data into individual components (or metagenes) that together comprise some or all of the overall “signal” in the data-space. The metagene projection strategy is able to provide a robust low dimensional description of transcriptional (or other complex) data based on groups of genes that represent known and novel biological mechanisms. It is also able to provide sensitive clustering and classification solutions. We illustrate the ability of this metagene projection strategy to capture novel molecular patterns in T cell differentiation states common to antigen-specific CD8+ T cells responses to HIV, HCV and metastatic melanoma.
Dissolvable microneedle arrays deliver live adenovirus to the skin for genetic immunization. (58.16)
Authors: Erdos Geza , Donahue Cara , Zhang Jiying , Ozdoganlar Burak , Gambotto Andrea , Falo Louis
Published: January 2023

Recombinant DNA vaccines show incredible promise for the prevention of human disease by their capacity to effectively inducing both humoral and cellular immune responses. Among the available technologies adenovirus (Ad) vectored vaccines are among the most appealing. To date the clinical deployment of Ad-based vaccines has been limited by several considerations including the lack of delivery technology to enable safe, reproducible and effective vector delivery to patients. The skin is readily accessible and rich in antigen presenting cells, making it a preferred target site for immunization. To enable Ad-based clinical immunization, we developed microneedle arrays (MNAs) to deliver live Ad-vectors to the skin. The MNA design and fabrication process we developed results in MNAs that effectively preserve Ad viability, and enable efficient delivery of infectious vector to the skin. Specifically, MNA embedded live Ad5 virus retained infectivity for up to a year. Topical application of MNAs resulted in transduction of skin cells. Further, immunization of mice with an Ad5 expression vector encoding the SIVmac239 gag antigen resulted in the generation of potent CTL activity against the dominant gag 76-84 eptiope. Taken together, our results demonstrate the capacity of MNAs to preserve and deliver Ad5 virus to the skin for genetic immunization. This novel technology platform may enable a wide range of topical skin-targeted Ad vaccines.
Fusion of TDG and RHD for selective DNA demethylation and transcriptional enhancement of NOS2 (58.2)
Authors: Fedulov Alexey , Gregory David , Mikhaylova Lyudmila
Published: January 2023

DNA methylation is a key mechanism in epigenetic silencing of transcription. Removal of methylation marks in promoters and other key regulatory sequences is associated with increased gene transcription, which could be beneficial for immunomodulatory and other purposes. However there is no method to selectively demethylate DNA in living cells. We designed a molecular construct in which a putative demethylase thymine-DNA-glycosylase (TDG) is targeted to a specific sequence by fusion with a DNA binding domain of NF-κB, the Rel-homology domain (RHD). 3T3 murine fibroblasts were transduced with fusion TDG-RHD or control constructs. In these cells the promoter of inducible nitric oxide synthase (NOS2) gene is highly methylated and contains RHD-binding sequences. We observed decreased DNA methylation (by 5-10 %, p<0.05) in several C’s in the NOS2 promoter and nearest CpG island. We also observed decreased methylation in the vicinity of RHD-binding sites in LINE-1, suggesting that the effect was available across the genome. Stimulation with IFNγ (250 ug/ml, 6 hrs) led to increased NOS2 message (fold over unstimulated 97.7±27.3 vs. 18.9±6.3 in control, p<0.01) and protein, indicating increased gene function. The effect was not seen for control genes lacking either RHD-binding sites or high levels of methylation, nor in mock-transduced cells. Results suggest that direct targeting of TDG to specific DNA loci may be practical for selective re-activation of epigenetically silenced genes.
Quantitative phosphoproteomic analysis of Y3F SLP-76 mutant in T cell signaling pathway reveals new feedback pathways (58.20)
Authors: Ji Qinqin , Ding Yiyuan , Salomon Arthur
Published: January 2023

SRC homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the TCR mediated T-cell signaling pathway. SLP76 consists of three functional domains: an amino-terminal acidic domain with three TCR inducible tyrosine phosphorylation sites; a central proline-rich domain and a carboxyl-terminal SH2 domain. Previous studies indicated all three of these domains contribute to the function of SLP-76. In our work, phosphoproteomic methods in mass spectrometry were used to perform a quantitative phosphoproteomic analysis comparing SLP-76 reconstituted wild-type cells with cells in which Tyr113, Tyr128, and Tyr145 were mutated to phenylalanine (denoted as the Y3F mutant). The abundance of thousands of unique phosphorylation sites was determined in the presence of wild type or Y3F mutant SLP76 through a time course of receptor stimulation. Our data provides a highly detailed new glimpse into the network of phosphorylation sites regulated through tyrosine phosphorylation of SLP76 including feedback regulation of T cell receptor proximal signaling.
The role of peptide-MHC ligand density in stimulating T-cell receptor signaling (58.21)
Authors: Sykulev Yuri , Anikeeva Nadia , Gakamsky Dimitry
Published: January 2023

Cell surface MHC proteins form clusters on the cell membrane. The MHC density within clusters could affect recognition of noncognate (self) and cognate peptide-MHC (pMHC) ligands and influence the kinetics of TCR-mediate signaling and quality of T cell responses. To evaluate the role of density, orientation and valency of pMHC ligands we utilized different scaffolds to assemble pMHC oligomers modeling MHC clusters. We systematically investigated how these parameters influence the binding of the pMHC oligomers to live CD8+ cytotoxic T lymphocytes (CTL) and the kinetics of TCR-mediated signaling. We have found that the recognition of noncognate or self pMHC strongly depends on the pMHC density and architecture and size of the scaffolds. In contrast, the valency but not the pMHC density mostly determined the recognition of strong agonist pMHC ligands. In addition, the pMHC density significantly influences the ability of cognate and noncognate pMHC to cooperate and to induce robust and rapid TCR signaling. The latter regulates the kinetics of target cell destruction by CTL that is essential for successful anti-virus immunity.
Protein microarray analysis of serum factor-reactive autoantibodies reveals BAFF-binding autoantibodies in systemic lupus erythematosus (58.22)
Authors: Price Jordan , Kemmer Dodge , Balboni Imelda , Haddon David , Mandelbaum Gil , Delepine Guillaume , Sokolove Jeremy , Shum Anthony , Browne Sarah , Utz Paul
Published: January 2023

Autoantibodies to cytokines and chemokines have been shown to influence immune pathways in patients afflicted with a range of immune pathology. We hypothesized that a multiplex platform capable of detecting antibodies to cytokines, chemokines and other immune factors would allow us to identify novel biomarkers of immune diseases and uncover new mechanisms of disease pathogenesis. We created a planar microarray containing a library of recombinant human proteins deposited on nitrocellulose-coated slides. We resolved binding of commercially available antibodies to cytokines and chemokines, and detected previously described cytokine reactivity in samples from patients with non-tuberculosis mycobacterium infection and autoimmune polyendocrine syndrome type 1. We further used the arrays to profile samples from three cohorts of systemic lupus erythematosus (SLE) patients. Among several cytokine targets, the significance analysis of microarrays algorithm identified that B-cell activating factor (BAFF)-binding autoantibodies correlated with subgroups of SLE patients. Reactive sera inhibited the activity of BAFF in a reporter cell assay, indicating that BAFF autoantibodies may influence the activity of this critical cytokine in SLE. Our results demonstrate that serum factor protein microarrays can be used to detect novel and potentially relevant autoreactivity to cytokines in human patient samples, and that BAFF-reactive autoantibodies may play a role in the pathophysiology of SLE.
Cooperation of dendritic cells with naïve lymphocyte populations to induce the generation of antigen-specific antibodies in vitro (58.3)
Authors: Heilmann Katja , Wand Inga , Holzlöhner Pamela , Micheel Burkhard
Published: January 2023

The production of monoclonal antibodies by hybridoma technology is dependent on lymphocytes taken from vertebrates which have to be immunized against the corresponding antigen. We present here our first experiments which should allow the replacement of this in vivo immunization step by an in vitro immunization procedure. This work provides new possibilities for the specific activation of immune cells in order to use them for the generation of antibodies which are not of murine origin. Bone marrow-derived dendritic cells were loaded with antigen and co-cultured with naïve T and B lymphocytes of non-immunized mice. The interaction and activation of the different cell types were investigated by measuring the expression of specific cell surface markers, the release of activation-dependent interleukins and the secretion of antigen-specific antibodies. We could demonstrate that dendritic cells process and present antigen fragments and activate T cells, that T cells proliferate and release activation-induced interleukins, and that B cells maturate under the influence of activated T cells and secrete antigen-specific antibodies.
Quantifying selection in high-throughput Immunoglobulin sequencing datasets (58.4)
Authors: Yaari Gur , Uduman Mohamed , Kleinstein Steven
Published: January 2023

Quantifying antigen driven selection based on mutation patterns observed in Immunoglobulin (Ig) DNA sequences can provide insights into the basic biology that underlies the adaptive immune response, and may further serve as diagnostic and prognostic markers. High-throughput sequencing approaches make large-scale characterization of B cell Ig repertoires feasible. However, analyzing selection in these large datasets, presents fundamental challenges. Here I will present a new computational framework for Bayesian estimation of Antigen-driven SELectIoN (BASELINe) in Ig sequecnes. BASELINe provides a more intuitive means to analyze selection by shifting the problem from one of detecting selection to one of quantifying it. It allows, for the first time, comparative analysis between sequences derived from different germlines. We have made this method practical for complete repertoire analysis through an online tool (clip.med.yale.edu/baseline) and a highly optimized implementation that can analyze up to 10,000 sequences in under four minutes on a single processor. By testing this method on computer simulations as well as on real sequences from genetically engineered mice, we have shown that indeed there is a significant improvement in our ability to quantify selection strength. We then demonstrate how BASELINe can be applied to high throughput sequencing datasets to arrive at several novel non-trivial observations.
A high-sensitivity detection system for monitoring of rare antigen specific naïve and memory T helper cells (58.6)
Authors: Bacher Petra , Schink Christian , Teutschbein Janka , Kniemeyer Olaf , Assenmacher Mario , Brakhage Axel , Schmitz Jürgen , Scheffold Alexander
Published: January 2023

CD4+ T cells play a central role in the immune defense against pathogens or tumors but also in immunopathology, such as autoimmunity or allergy. However, due to technical limitations, comprehensive information about the frequency and phenotype of antigen-specific CD4+ T cells against many disease-relevant antigens is missing. In particular the nature of the human naïve repertoire is elusive. We have developed a new, sensitive detection system for antigen-specific T helper cells, based on a combination of magnetic pre-enrichment and multi-parametric flow cytometric analysis of CD154 (CD40L)-expressing CD4+ T cells upon in vitro activation with antigens. The high sensitivity of 1 cell out of 105 to 106 enables the direct enumeration of memory and naïve CD4+ T cells reactive against basically any recall or primary antigen. Importantly, our technology allowed us to rapidly analyze sufficient numbers of target cells to discriminate even small functional or phenotypic subpopulations within the total antigen-specific T cell pool, undetectable by conventional methods. We used this assay for a first characterization of tumor- and auto-antigen reactive CD4+ T cells from healthy subjects as well as for the in depth characterization of rare pathogen specific T cells. The possibility to visualize T cells reactive against any antigen of interest even in the naïve repertoire will open up new possibilities to advance human T cell research and to improve clinical diagnostics and prognosis.
Early detection of lymphangiogenesis by near-infrared imaging and responses to VEGF-C in a mouse full-thickness wound model (58.9)
Authors: Hall Mary , Aldrich Melissa , Robinson Holly , Kwon Sunkuk , Sevick-Muraca Eva
Published: January 2023

Wound healing is a multistep process involving interaction of various cell types, mediators such as cytokines, inflammation, and generation of new tissue. Both the blood and lymphatic vasculature aid in tissue repair, during which new vessels are generated by angiogenesis and lymphangiogenesis, respectively. We utilized a mouse full-thickness wound model to detect lymphangiogenesis near an incisional wound by near-infrared fluorescence (NIRF) imaging at multiple time points post-incision (PI). In addition, the systemic affects of locally administered vascular endothelial growth factor (VEGF)-C were investigated by measuring peripheral blood cytokine levels in treated and untreated animals. Lymphangiogenesis was detected at the wound site by NIRF imaging by day 7 or 8 PI and confirmed by intravital imaging, while angiogenesis was observed by day 2 or 3 PI. Interleukin (IL)-1α and IL-3 levels were significantly increased at day 7 and 21, respectively, relative to pre-incision levels found in the periphery of VEGF-C-treated mice. Also, regulated-upon-activation, normal T-cell expressed and secreted (RANTES) levels were significantly higher in VEGF-C-treated animals when compared to saline-treated mice at day 21 PI. Our findings demonstrate that NIRF imaging can be utilized to detect early lymphangiogenesis during wound healing, and local VEGF-C administration may help induce this process via increasing systemic levels of wound-healing mediators.
Neutrophil Gelatinase Associated Lipocalin (NGAL) deficiency accelerates the onset of serum autoantibodies in pristane induced lupus (60.10)
Authors: Pawar Rahul , Goilav Beatrice , Xia Yumin , Zuang Haoyang , Reeves Westley , Putterman Chaim
Published: January 2023

NGAL (lipocalin-2), a member of the lipocalin superfamily, is expressed by neutrophils, hepatocytes, and renal resident cells following ischemia or inflammatory conditions & is involved in innate immune responses during infection. However, its role in adaptive immunity has not been described. Previously, we had found that NGAL is instrumental in the pathogenesis of antibody mediated nephritis. To investigate a possible role for NGAL in the generation of autoreactive antibody responses, we examined its effect in an experimental lupus model. We injected pristane (0.5 ml per mouse i.p.) into wild type B6 (n=10) and NGAL-deficient (NGAL-/-) (n=10) mice and followed the course of disease. Analyzing the levels of serum autoantibodies at 3 months post injection, NGAL-/- mice had significant increases in IgG2a (p=0.007) and IgG2b (p=0.019) anti-double stranded DNA antibodies, and IgG2a (p=0.002) & IgG2b (p=0.010) anti-single stranded DNA antibodies, and IgG anti-histone antibodies (p=0.025) as compared to pristane challenged, NGAL sufficient mice. Immunoprecipitation studies showed elevated serum anti-Sm/RNP antibodies in NGAL-/- mice compared to the WT mice at 3 months post injection, with an increase in titer and epitope diversity of this response at 6 months post injection. In conclusion, NGAL delays the onset of anti-nuclear antibodies in pristane induced lupus. The mechanisms by which NGAL plays a protective role in humoral autoimmunity are under investigation.
IRF7-dependent IFNβ production in response to RANK facilitates medullary thymic epithelial cell development. (60.3)
Authors: Otero Dennis , Baker Darren , David Michael
Published: January 2023

The signal transducer and activator of transcription 1 (STAT1) is an essential part of signaling cascades induced by type I and II interferons. Deficiency of STAT1 results in heightened susceptibility to infections and autoimmunity in mouse and humans. While the role of IFNγ in T cell development is well known, the role of type I interferons in thymic development has largely remained unexplored. Here we show that STAT1 and IFNAR-deficient mice harbor a defect in medullary compartment characterized by a significant reduction in self-antigen presenting, AIRE expressing medullary thymic epithelial cells. Using a novel reporter mouse we demonstrate that constitutive IFNAR signaling is taking place in the thymic medulla in the absence of any infection or inflammatory signals. In addition, we found that RANK stimulation in thymic stromal cells results in the upregulation of IFNβ which in turn is inhibits RANK signaling thereby facilitating AIRE expression. Finally, we show that IRF7 is required for the induction of IFNβ by RANKL, and that IRF7-, but not IRF3-deficient mice display a defect in thymic architecture and medullary thymic epithelial cell differentiation similar to that observed in IFNAR- or STAT1-deficient animals. These results illustrate that a spatially and temporally coordinated crosstalk between the RANKL/RANK signaling pathway and the IRF7/IFNβ/IFNAR/STAT1 axis is essential for the differentiation of AIRE-expressing medullary thymic epithelial cells.
p21 selectively regulates the activation of autoimmune of lpr memory T cells and attenuates autoimmunity (60.5)
Authors: Weber Kathrin , Vázquez-Mateo Cristina , Daszkiewicz Lidia , Balomenos Dimitrios
Published: January 2023

Fas drives activated T cell apoptosis in vitro, but lymphadenopathy and lupus in Fas-deficient (lpr) mice cannot be explained only on the basis of the lpr T cell apoptotic defect. Unexplained also remains the hyperproliferation of T cells in lpr mice, including CD4+, CD8+ and CD4-CD8-TCR+ cells. We investigate these questions by generating T cell-specific p21-transgenic and p21-/- BL/6 lpr mice. p21, a cell cycle inhibitor and a lupus-like autoimmunity suppressor, regulates memory T cell but not naïve T cell expansion. p21 overexpression in lpr T cells did not affect primary T cell responses, but profoundly diminished lpr T cell expansion upon repeated activation, without affecting the apoptosis defect of lpr T cells. Importantly, p21 overexpression restrains lpr T cell memory expansion and IFN-gamma and IL-17 production, resulting in reduced autoantibody production, glomerulonephritis and lymphadenopathy in lpr mice. In p21-/- lpr mice, lack of p21 enhanced the activation and accumulation of memory T cells, which produced higher levels of IFN-gamma and IL-17. Lack of p21 led to severe glomerulonephritis and death in BL/6 lpr mice, which normally develop non-lethal mild autoimmunity. Overall, we conclude that independently of their lpr apoptotic defect, T cell hyperactivation and autoimmunity development can be controlled by p21. Thus, analysis of the role of p21 in T cell immunity may be of therapeutic value for both Lupus and the Autoimmune Lymphoproliferative Syndrome.
Stressor-enhanced colitis is abrogated in mice deficient in CCL2 (61.6)
Authors: Mackos Amy , Galley Jeffrey , Parry Nicola , Eubank Tim , Bailey Michael
Published: January 2023

Exposing mice to a social stressor called social disruption (SDR) has been reported to increase CD11b+Ly-6ChiCCR2+ inflammatory monocytes in the circulation and spleen. This study was designed to test the hypothesis that stressor-induced exacerbation of pathogen-induced colitis is dependent upon CCL2-mediated recruitment of inflammatory monocytes. Mice deficient in CCL2 (i.e., CCL2-/-), a chemokine involved in the recruitment of CCR2+ monocytes, and wild type C57BL/6 mice were challenged with the colonic pathogen Citrobacter rodentium during exposure to SDR. Exposing mice to SDR, which consists of intermale aggression for 2 hrs a night on 6 consecutive nights, during challenge with C.rodentium significantly increased pathogen-induced colitis. This increase was marked by an enhanced accumulation of F4/80+ colonic monocytes/macrophages, increased pathogen burden and pathogen-induced colonic histopathology, and increased inflammatory cytokine and chemokine gene expression in comparison to C. rodentium-challenged non-stressed control. These stressor effects were dependent upon CCL2, since exposing CCL2-/- mice to SDR did not lead to an increased accumulation of colonic monocytes/macrophages in the colon and did not significantly increase gene expression or pathogen-induced histopathology. Together, these data illustrate the deleterious effects of stressor-enhanced recruitment of inflammatory monocytes during bacterial challenge.
Modulation of TNFα and TGFβ1 in botox treated rats with burn wounds (62.2)
Authors: Jurjus Abdo , Abdallah Hajj Hussein Inaya , El-Khatib Arij , Dali-Balta Nour , Shaalan Tareq , Watfa William , Atiyeh Bechara , Jurjus Rosalyn
Published: January 2023

Abstract Many attempts have been made to improve wound healing using growth factors and a large array of medications. This study reexamined the effect of Botox on second degree burn wound in rats. Histological alterations and temporal evolution of two cytokines TGFβ1, and TNF-α were assessed in presence and absence of Botox. Fifty adult Sprague Dawley rats were used; one group was injected 10 units of Botox into wound. Daily observation and transepidermal water loss were assessed. Punch biopsies were taken on days 0, 3, 8, 14 and 28 for RNA extraction and PCR, and for routine light microscopy. Botox treated rats showed a delayed inflammation, necrosis, and earlier crust detachment compared to controls. On day 14, a better and smoother epithelialization was noted in the Botox groups. Finally, by day 28, the wound surface area was smaller and the quality of the skin was better with more fibroblasts in the regenerated dermis of Botox group, which expressed 2-4 times more TGFβ1. It peaked on days 3 and 8 and then decreased to almost half the control group by day 28. On the other hand, the TNF-α peaked immediately after burn to almost 20 times the control, then decreased between 2.72 to 5.5 times during the rest of the experiment very much like TGFβ1, or lower. Botox could possibly play an alleviating role and have a positive effect on burn wound healing.
Distinct regulatory networks control B-lymphoid specification and lineage commitment. (63.1)
Authors: Sigvardsson Mikael , Zandi Sasan , Åhsberg Josefine , Qian Hong , Stjenrberg Jenny
Published: January 2023

The development of B-lymphoid cells from hematopoetic stem cells in the bone marrow involves an orchestrated interplay between transcription factor networks and external signals. In order to better understand B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early B-lineage cells from Pax-5 and Notch-1 deficient animals carrying a B-lineage restricted reporter gene, allowing us to identify B-lineage specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of B-cell progenitors marked by expression of a λ5 (Igll1) promoter controlled reporter gene. Gene expression analysis of ex vivo isolated cells revealed that Pax-5 deficient cells expressed several B-cell restricted genes, however, they displayed a high degree of plasticity for development into other lymphoid lineages at a single cell level. Even though the Pax-5 deficient progenitors expressed higher levels of Notch-1 and respond to delta-signaling in vitro, these cells expressed lower levels of the Notch-1 target gene Deltex-1, arguing for a minor role of Notch signaling in B-cell commitment in the BM. By comparison of gene expression patterns in ex vivo isolated Pax-5 and Ebf-1 deficient progenitors, it was possible to identify a set of B-cell restricted genes dependent of Ebf-1 but not Pax-5, revealing that B-cell specification and commitment is controlled by distinct regulatory networks.
Loss of MyD88 promotes the persistence of skin lesions in filaggrin deficient mice (64.1)
Authors: Leisten Sabine , Oyoshi Michiko , Bartnikas Lisa , Geha Raif
Published: January 2023

Flaky tail (ft/ft) mice have a skin barrier defect due to a loss-of-function mutation in the gene for filaggrin. They spontaneously develop eczematous skin lesions and are a model for human atopic dermatitis. We tested the hypothesis that the disrupted skin barrier in these mice facilitates the entry of environmental antigens, including microbial antigens, which are recognized by Toll-like receptors (TLRs) and may trigger the development of skin lesions. To examine the role of TLR signaling in the development of eczematous skin lesions in ft/ft mice, we created MyD88KO-ft/ft mice. Unexpectedly, the MyD88KO-ft/ft mice developed persistent skin lesions over the course of 32 weeks, while the ft/ft mice developed relapsing lesions throughout the study period. Lesional skin of both strains was characterized by epidermal thickening, infiltrating eosinophils and neutrophils as well as high mRNA levels of IL-17, IL-23 and CXCL2. These results indicate that MyD88 may be a negative regulator of skin inflammation in a mouse model of filaggrin deficiency.
Peripheral Foxp3+ regulatory T cell development in response to self-peptides (65.2)
Authors: Weissler Katherine , Bedoya Felipe , Kropf Elizabeth , Garcia Victoria , Caton Andrew
Published: January 2023

It is generally accepted that developing thymocytes can become Foxp3+ regulatory T cells (Tregs) through recognition of self-antigens during thymic selection. Tregs can also develop from conventional CD4+ T cells responding to exogenously administered peptides (including food antigens) in vivo, and via TGF-β signaling during activation in vitro, but the processes by which Treg induction may occur in response to self-peptides in the periphery remain unclear. We are examining this question by transferring conventional CD4+CD25-Foxp3- T cells specific for influenza virus hemagglutinin (HA) into mice that express varying amounts of the HA molecule as a self-antigen (HA Tg mice). We find that conventional CD4+ T cells can convert into Foxp3+ Tregs upon recognition of a self-antigen in the lymph nodes and spleens, and that accumulation of HA-specific Tregs is greater in mice expressing HA at lower levels than it is in mice expressing relatively higher levels of HA. Conversion occurs most efficiently at sites draining mucosal surfaces, in agreement with published studies demonstrating the existence of specialized subsets of dendritic cells at these locations that promote Treg induction. We also find that the extent of Treg induction in response to a self-antigen is essentially unaffected by acute viral infection. These studies are defining parameters that determine the ability of self-peptides to induce the formation of Foxp3+ Tregs in the periphery.
Novel Role for Btk in the Induction of Classically Activated M1 Macrophages (65.9)
Authors: Ni Gabhann Joan , Wynne Claire , Smith Siobhan , Jefferies Caroline
Published: January 2023

This study defines a critical role for Btk in regulating TLR-mediated APC activation and differentiation. Using Btk-deficient mice we examined the effects of LPS in regulating macrophage activity and function. Reduced levels of pro-inflammatory cytokines IL-12, IL-18 and IFN-γ were observed in Btk-deficient mice and ex vivo generated macrophages and dendritic cells (DCs) following acute LPS administration, whilst enhanced production of the anti-inflammatory cytokine IL-10 was observed. In response to diverse stimuli specific populations of macrophages are induced such that macrophage activation can be either pro-inflammatory or anti-inflammatory. Exposure to bacteria and IFN-γ results in the generation of classically activated (M1) macrophages which are microbicidal and secrete pro-inflammatory cytokines, whereas IL-10 is a key cytokine produced by alternatively activated immunomodulatory (M2) macrophages. Given the immunosuppressive environment observed in the absence of Btk we investigated macrophage population induction following treatment of APCs with either polarizing cocktails or with TLR4. Comparison of gene induction profiles for WT or Btk-deficient APCs revealed an inability of Btk deficient macrophages to become M1 effector macrophages. The predominance of M2 macrophages when Btk is absent is of crucial importance when considering therapeutic inhibition of Btk for the treatment of cancer or autoimmunity given the role of M2 macrophages in driving these pathologies.
Low phosphorylation of STAT-3 on interleukin-21 pathway in common variable immunodeficiency (66.2)
Authors: Alcántara Julio , Berrón Laura , Mogica Dolores , Espinosa Sara , Espinosa Francisco , Santos Leopoldo
Published: January 2023

Introduction: Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by recurrent infections and defective immunoglobulin production. Interleukin-21 (IL-21) is expressed by activated CD4+ T cells. Its receptor (IL-21R) is expressed on T, B, dendritic cells, macrophages and NK cells. The role of IL-21 in B cell differentiation make this an attractive candidate as a potential cause of CVID. Objective: To evaluate IL-21, IL-21R and its pathway in patients with CVID in naïve and memory B cells populations. Methods: Six patients with established diagnosis of CVID, according to European Society for Immunodeficiencies criteria, were included. CVID patients were previously analyzed to discard known genetics causes of CVID (ICOS, CD40L, TACI, CD19). To evaluate the pathway of IL-21R, PBMCs were stimulated with rhIL-21 for 15 minutes and phosphorylation of STAT-3 was evaluated by flow cytometry. Results: The patients show consistent low phosphorylation of STAT-3 after the stimulation with rhIL-21 for 15 minutes. IL-21 was equally expressed by PBMC from patients and healthy controls. Patients showed over expression of IL-21R on T and B cells. Conclusion: An unexpected discover in IL-21R signaling was the low phosphorylation of STAT-3 in CVID patients. This suggested that the production and regulation of STAT-3 may have an important role in B cell activation. So far, we have found no evidence of defects in IL-21, IL21R.
TLR4 and alveolar macrophages mediate lung ischemia reperfusion (IR) sterile inflammatory injury in a mouse model of ventilated lung ischemia (67.6)
Authors: Prakash Arun , Mesa Kailin , Hellman Judith
Published: January 2023

Background: Ischemia reperfusion (IR) injury is sterile inflammatory process associated with diverse clinical situations, for example, hemorrhage followed by resuscitation, transient embolic events, and organ transplantation. IR injury can result in lung dysfunction whether the IR occurs in the lung itself or in a remote organ. Increasingly it appears that receptors and pathways of the innate immune system are involved in recognizing sterile inflammation and overlap considerably with those involved in recognition and response to pathogens and pathogen-derived products. Methods: We used a mouse surgical model of transient unilateral pulmonary artery occlusion to the left lung without bronchial involvement to create IR injury and tracked injury in vivo and ex vivo. Additionally, we mimicked IR injury in vitro by transiently depriving cells of nutrients. Results: In the mouse ventilated lung IR model we demonstrate an upregulation of inflammatory markers and recruitment of neutrophils that is characteristic of IR injury. We show that the early inflammatory response to IR requires TLR4. Furthermore, we demonstrate in vitro that cooperativity and cross-talk exists between macrophages and endothelial cells resulting in an augmented inflammatory response to nutritional deprivation and that in vivo macrophages are indeed required for early sensing of IR injury. Remarkably, we find that selective depletion of alveolar macrophages rendered mice resistant to lung IR injury.
The local immune response in a mouse model of acute Clostridium difficile infection (67.8)
Authors: Sadighi Akha Amir , Theriot Casey , McDermott Andrew , Reeves Angela , Young Vincent , Huffnagle Gary
Published: January 2023

The current report presents our initial findings on the immune response to C. difficile infection (CDI) in an acute mouse model. 5-8 week old male C57BL/6 mice were either left untreated or received a cocktail of 5 antibiotics in drinking water for 3 days followed by a single intra-peritoneal dose of clindamycin. The antibiotic-treated mice were then infected with 105 CFUs of the C. difficile strain VPI10463 and sacrificed 42 hours later. Flow-cytometric study of the spleens, mesenteric lymph nodes (MLN), colons and ceca of the infected mice showed a significant increase in the numbers of B cells, CD4 and CD8 T cells, FOXP3+ CD4 cells, NK cells and neutrophils in the colons and ceca, as well as cells of the monocyte/macrophage lineage and dendritic cells in the ceca of the infected mice. The spleens and MLN of the infected mice did not display such an increase. A substantial fraction of the recruited neutrophils in the colons and ceca of the infected mice displayed upregulated levels of CD11b, whereas a similar fraction of CD4 and CD8 cells in the colons and ceca of uninfected and infected mice showed an upregulated level of CD69. Multiplex qPCR analysis of colonic and cecal tissue from the infected mice showed a significant increase in the mRNA levels of numerous neutrophil-attracting chemokines and a number of cytokines produced in an innate immune response, but no indication of T cell polarization, thereby collectively underscoring the innate, local nature of the response.
Cytokines production by PBMC from pregnant and non pregnant women after addition lives tachyzoites of Toxoplasma gondii (67.9)
Authors: de Oliveira Karine , Alves Silva Jéssica , Peghini Bethânea , Silva Marcos Vinicius , Gómez-Hernández César , Silva Neide , Mineo José Roberto , Rodrigues Junior Virmondes
Published: January 2023

Introduction: During pregnancy, mother’s immune system modulates immune response in order to allow the maintenance of fetal tolerance. Besides, this tolerogenic environment the adaptative immune response exist and are effective against microbes such as T. gondii, causative agent of toxoplasmosis. Objective: Evaluate the production of cytokines by PBMC from parturient and non-pregnant women exposed to live tachyzoites ME49 strain of T. gondii. Methods: PBMC from 31 parturient, which 21 were T. gondii IgG+ and 22 non-pregnant women, where 8 were T. gondii IgG+ were cultivated (105cells/well) in presence of anti CD3 or live tachyzoites (2 parasites/cell) of T. gondii ME49 strain. The cells were incubated for 72h and cytokine titration by CBA in supernatants. Results: The cytokine’s levels produced by parturient’s cells and non pregnant women showed existence of correlation between the production of Th2 and Th1 cytokines on live tachyzoites culture, independent of group or serology: IL4/TNF-α (r=0.51, p<0.05), IL5/TNF-α (r=0.68, p<0.05), IL5/IFN-γ (r=0.28, p<0.05), IL10/TNF-α (r=0.89, p<0.05) and IL10/IFN-γ (r=0.50, p<0.05). The group with previous exposure to parasite (IgG+) presented correlation between the cytokines: IL4/TNF-α (r=0.70, p<0.05), IL5/TNF-α (r = 0.81, p<0.05) and IL10/TNF-α (r=0.90, p<0.05). Conclusion: T. gondii induces pro or anti-inflammatory cytokines production independent of patient’s pregnancy condition or previous exposition to parasite.
Loss of the ITAM receptor CD200R1 attenuates HSV-1 infection and encephalitis. (68.15)
Authors: Kurt-Jones Evelyn , Soberman Roy , Vaine Christine , Mackay Christopher , Ryan Glennice , Knipe David
Published: January 2023

We have discovered a novel role for the Immunoreceptor tyrosine-based activation motif (ITAM) containing receptor CD200R1 in virus infection. The host response to viral pathogens is driven by the activation of innate immune receptors that are triggered by pathogen-associated molecular pattern (PAMPs). These receptors include the transmembrane Toll-like receptors as well as the intracellular nucleic acid sensors RIG-I, Mda5, AIM2 and IFI16. ITAM receptors are known to both attenuate and activate signaling by innate receptors like TLRs but little is known about the potential impact of ITAMs on the outcome of viral infection. We generated CD200R1 knockout (KO) mice and infected with herpes simplex virus (HSV)-1. CD200R1 KO mice were protected from lethal HSV-1 encephalitis with >75% of the CD200R1 KOs surviving compared to 25% of the wild type (WT) mice, i.e., loss of CD200R1 conferred a survival advantage. This was not due to any obvious effects on the inflammatory response to infection. CD200R1 KO and WT mice had equivalent cytokine responses and leukocyte infiltration during the initial infection. Remarkably, HSV-1 virus replication was attenuated in CD200R1 KOs. Thus 50% of the CD200R1 KOs had cleared the virus from their brain by day 3-4 post-infection. In contrast, HSV-1 continued to replicate in the brains of 100% of the WT mice. Infection of cultured cells confirmed that CD200R1 expressing WT cells were more permissive for HSV-1 infection than CD200R1 KO cells.