bluuebunny/crossref_metadata_2025_split huggingface dataset:
CXCL4, a chemokine upregulated in systemic sclerosis patients, abrogates TLR9 signaling and central tolerance in B cells
Authors: Ah Kioon Marie Dominique , Cakan Elif , Garcia-Carmona Yolanda , Cunningham-Rundles Charlotte , Meffre Eric , Barrat Franck J 
Published: August 2023

Abstract

Background
Systemic sclerosis (SSc) is an autoimmune connective tissue disease, characterized by defective central B cell tolerance. Deficiency of MYD88, which mediates the function of TLRs, results in a failure to silence autoreactive B cells. CXCL4, a chemokine found elevated in the sera of SSc patients, potentiates IFN-α secretion from TLR9-induced plasmacytoid dendritic cells. The objective is to investigate whether CXCL4 regulates TLR9 signaling and central tolerance in B cells.

Methods
B cells from SSc patients or healthy donors were cultured with TLR9 ligand (TLR9-L) alone or with CXCL4. Cytokines gene expression and secretion were analyzed. Delivery of TLR9-L was analyzed by Amnis and confocal microscopy. NOD-scid-common gamma chain (γc) knockout immunodeficient mice were engrafted with CD34+ human fetal hematopoietic stem cells transduced with lentivirus expressing human CXCL4. New emigrant/transitional B cells were sorted. Immunoglobulins chains of these cells were cloned and transfected in fibroblasts. Polyreactivity of the antibodies released by the fibroblasts were assessed.

Results
Defective TLR9 signaling was observed in B cells of SSc patients. CXCL4 abrogated TLR9 response in human B cells. TLR9-L when given to B cells alone, was localized in the lysosomes and LAMP-2+ compartments while CXCL4 prevented the delivery of TLR9-L to these vesicles. CXCL4 in vivo expression led to defective TLR9 responses and increased number of polyreactive B cells.

Conclusions
Our data provide evidence for a novel mechanism by which CXCL4 impairs central B cell tolerance by altering the intracellular trafficking of TLR9 ligands, hence inhibiting TLR9 response which is required for the removal of developing autoreactive B cells.
17β-estradiol and B-cell intrinsic type I interferon amplify toll-like receptor 7 signaling loop in B cells of female lupus prone BXD2 mice
Authors: Sullivan Kathryn , Wang Yong , Chatham Winn W , Mountz John D , Hsu Hui-Chen 
Published: August 2023

Abstract
While systemic lupus erythematosus (SLE) disproportionally affects women versus men, elevation in 17β-estradiol (E2) alone is not sufficient to promote the development of autoantibody producing B cells. The objective of the present study is to determine if B-cell intrinsic mechanisms contribute to the increased TLR7 response to E2 stimulation. We identified that there were elevated circulating levels of E2 in young African American (AA) SLE patients (24–41 yr-old), compared to older AA (42–56 yr-old) and European American SLE patients (24–66 yr-old). Circulating E2 levels positively correlated with the levels of anti-Smith (Sm) and the expression of interferon-beta (IFNβ) in naïve B cells of SLE patients (n=39). Mouse studies were used to determine if E2 stimulates the expression of IFNβ in B cells and if sex plays a role to influence B cell responses to E2. Serum levels of Sm/RNP and RNP autoantibodies positively correlated with the levels of E2 in female lupus prone BXD2 mice post-puberty (12-wk-old) but not pre-puberty (4–6-wk-old). At the post-puberty stage (&gt;12 wk-old), there were significantly elevated levels of anti-DNA and anti-Sm in female BXD2 mice, compared to male mice. This was associated with an increased TLR7-induced expression of IFNβ and CD69 in transitional stage 1 (T1: CD23− IgM+ CD93+) B cells in female BXD2 mice, compared to male mice. Interestingly, E2 stimulation promoted intracellular levels of IFNβ and TLR7 in T1 B cells from female but not male BXD2 mice. Together, our results suggest that elevation of E2 in combination with an increased B-cell susceptibility to E2 induction of IFNβ may play a role in the increased B cell responses to TLR7 stimulation in individuals predisposed to the development of SLE.
This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01-AI-071110, R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311
OC-STAMP-reactive IgG auto-antibody may upregulate osteoclastogenesis in association with elevated anti-P. gingivalis antibody
Authors: Nakamura Shin , Shindo Satoru , Leon Elizabeth , Kumagai Tomoki , Pastore Maria Rita , Ouhara Kazuhisa , Kawai Toshihisa 
Published: August 2023

Abstract

Objectives
Periodontal disease (PD) is a polymicrobial inflammatory lesion in which pathogenic bone loss is caused by osteoclasts (OC) differentiated from MCSF/RANKL-primed monocytes. Elevated IgG antibody to a keystone opportunistic pathogen, Porphyromonas gingivalis (Pg), is a hallmark humoral response in PD that is correlated with pathogenic bone loss. Whereas, a cell membrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), is engaged in cell-fusion event required for OC-genesis. This study examined the humoral immune responses to OC-STAMP expressed by human monocytes in relation to anti-Pg antibody.

Methods
Blood serum (N=10) collected from systemically healthy human subjects (Precision for Medicine) were measured for IgG antibody titer to Pg or OC-STAMP peptides. Human peripheral blood mononuclear cells were stimulated with MCSF/RANKL with or without anti-OC-STAMP rabbit polyclonal antibody or control rabbit IgG. Affinity purified human anti-OC-STAMP antibody was also tested for its activity to alter the OC-genesis. OC-genesis induced in vitro was evaluated by TRAP staining and pit formation assay.

Results
IgG antibody titer to Pg positively correlated with that to human OC-STAMP peptides (r=0.579, p &lt;0.05). Anti-OC-STAMP auto-antibody purified from human serum with high IgG antibody titer to Pg, as well as anti-OC-STAMP rabbit antibody significantly promoted in vitro OC-genesis (p &lt;0.05).

Conclusions
The results indicated that Pg infection may be associated with development of anti-OC-STAMP auto-antibody which can promote OC-genesis, suggesting a novel immunological intervention in the pathogenesis of PD.

Supported by grants from NIH NIDCR grants, DE-027851, DE-028715 and DE-331851
Development of a seven-color immunofluorescent panel to identify a rare B cell population (IgM+ IgDlo) and its effect on T regulatory cell (Treg) proliferation
Authors: Beltrame Angela , Dittel Bonnie 
Published: August 2023

Abstract
Multiple sclerosis (MS) is caused by a T cell orchestrated immune response to myelin sheath antigens of the central nervous system. Autoimmune responses can be suppressed by CD4+ Foxp3+ T regulatory cells (Treg); therefore, a therapeutic strategy is to increase endogenous Treg numbers. Our studies using the mouse model of MS, experimental autoimmune encephalomyelitis, have identified a unique subset of IgM+ IgDlow/− B cells (BDL) that induce Treg proliferation in a glucocorticoid-Induced TNFR-related (GITR:GITRL)-dependent manner. However, where BDLlocalize with Treg in the spleen is unknown and imaging is challenging due to rarity of both cell populations. Because Treg remain within the T cell zone, we hypothesize BDL-Treg interactions take place within the T-B boundary. To visualize and localize BDL-Treg-interactions within the spleen, a seven-color microscopy panel was developed to identify splenic architecture using the Nikon Inverted Eclipse Ti2 microscope and SPECTRA Light Engine filter cube. Fluorescent conjugated antibodies to identify the marginal zone (MAdCAM-1), BDL(IgM, IgD), Treg (CD4, Foxp3), proliferation (Ki-67), and chemokine receptors (used for lymphocyte localization in the spleen) were selected. To select fluorescent antibodies with low background and spectral overlap thereby ensuring detection of real signal, cytospin slides of sorted CD19+ B cells or CD4+ T cells and single-stained controls were utilized. This method can be applied for a variety of imaging experiments and will be beneficial for other applications. By completing this project, we hope to better understand BDL-Treg interactions to develop a BDL-based therapeutic to increase Treg numbers and reduce autoimmunity.
MCW Center for Immunology GRIT Award
B cell apoptosis and inflammation conspire to promote lupus in mice
Authors: Khan Wasif N , Bazile Cassandra , Clark Emily S , Shaikh Kashif , López-Rodríguez Darlah M , LaPorte Kathryn M , Wright jacqueline A , Carlesso Gianluca , Riggs Jeff , Altman Norman , Greidinger Eric L , Malek Thomas R , Harhaj Edward W 
Published: August 2023

Abstract
The pathogenesis of Systemic lupus erythematosus (SLE) is driven by autoreactive B and T cells and autoinflammation. Impaired apoptosis in T and B cells has been shown to result in autoimmune disease, and we recently demonstrated that reducing apoptosis in B cells alone, by B cell-specific deletion of Bim (B.Bimf/f) could lead to SLE with prominent features of Sjogren’s Syndrome in C57BL/6 mice. Consistently, B cell targeting therapies are successful in reducing SLE pathogenesis in mouse models, however, their clinical success requires a better understanding of the contributions of B cells and the mechanisms that drive inflammation to autoimmunity. To address the role of inflammation in the development and progression of autoimmunity we generated a novel mouse model that lacks ZFAND6 (Zfand6−/−) and crossed with the B.Bimf/f mice. ZFAND6 is an A-20 like ZF domain-containing protein with a potential role in the negative regulation of NF-kB activation, however, its physiological function is unknown. The compound mutant mice displayed accelerated and exacerbated overlapping lupus and Sjogren’s autoimmune symptoms with a higher incidence of splenomegaly and kidney pathology than either single mutant alone including the early appearance of anti-SSA, anti-SSB, and anti-RNP autoantibodies. The compound mutant mice displayed increased spontaneous and TLR-induced TNFa and IL-6 and B cells from these mice produced much greater levels of these cytokines in response to TLR7 and TLR9. These results suggest that inappropriate B cell survival and persistent inflammation cooperate in the initiation and severity of lupus pathogenesis.
Sylvester Comprehensive Cancer Center, University of Miami, Miami FL
Distinct roles of CD4+ and CD8+ Tregs in regulating human autoreactive T cells, B cells, and antibodies in genetically engineered tonsil organoid system
Authors: Chen Xin , Mallajosyula Venkata , Ghanizada Mustafa , Sola Elsa , Chen Lei , Kamalyan Lilit , Li Jing , Davis Mark M 
Published: August 2023

Abstract
CD4+ and CD8+ regulatory T cells (Treg) are critical for maintaining immune tolerance, and alternations in number and function are associated with detrimental outcomes in infectious and autoimmune diseases. Despite their importance, their distinct roles and mechanisms in regulating self-tolerance need to be better characterized in humans. Here we analyzed the relative contribution of CD4+ and CD8+ Tregs to control autoreactive T cells, B cells, and antibodies in an entirely human tonsil organoid system in vitro. We disrupted their suppressive functions by knocking out FOXP3 and GZMB genes using CRISPR/Cas9 technology. Normally tonsil organoids make robust antibody and cellular responses to live attenuated flu vaccine, but not to autoantigens. In contrast, FOXP3 KO tonsil organoids produced autoantibodies when stimulated with a panel of classical autoantigens, including double-stranded DNA and others. We also detected increased CD8+ T cells specific for autoantigens, including SMCY antigen. With the GZMB KO, we found a marked increase in follicular helper T cells and autoreactive CD4+ and CD8+ T cells. Interestingly the GZMB KO tonsils showed an expansion of plasmablasts, but only a low level of autoantibodies and autoreactive B cells was detected. This indicates that CD8+ Tregs control the early T and B cell activation but not autoantibody production. Moreover, knocking out FOXP3 generated high-affinity HA-specific antibodies in tonsil organoids compared to control, indicating that CD4+ Tregs are a key checkpoint for high-affinity antibodies and the production of autoantibodies. We conclude that CD8+ and CD4+ Tregs have distinct and complementary roles in regulating cellular and humoral responses and preventing autoimmunity.
Eos is a critical transcription factor for T regulatory cell (Treg) function
Authors: Xie Xuan , Tanwar Shalini , Thornton Angela M , Korty Pat , Schwartz Owen M , Smelkinson Margery , Shevach Ethan M 
Published: August 2023

Abstract
Eos is a member of the Ikaros transcription factor family and is highly expressed in Tregs. Mice with a selective deficiency of Eos in Treg develop systemic or organ-specific autoimmune disease at at a young age. We have generated a specific anti-Eos monoclonal antibody. The percentage of Tregs expressing Eos rapidly increases with aging from almost 0% at birth to 30~50% of thymic Tregs and 35~60% of splenic Tregs at 3 weeks of life and then remains stable. Eos is expressed at very low levels on induced Tregs, conventional CD4+ T cells and CD8+ T cells in vivo or in vitro. In the thymus, Eos is only expressed on CD73+ CCR6+ CXCR4+ Tregs, which suggests that Eos+ thymic Tregs represent peripheral Tregs that have recirculated to the thymus. In thymus, Eos expression on Tregs is also highly correlated with CXCR4 and CCR6. Using these markers, we sorted Eos+ and Eos− Tregs and in vitro expanded them using anti-CD3/CD28 beads and IL-2; Eos expression remained stable. Using immunofluorescence microscopy, we have found that Eos is highly co-localized with Helios, another Ikaros family member and HDAC1, but not other Ikaros family members including Ikaros or Aiolos, suggesting that Eos and Helios may form heterodimers and together with HDAC1 collaborate in Treg function. Nevertheless, expression of Eos is independent of Helios and vice versa, as Treg from Helios deficient mice express normal or elevated levels of Eos, and Treg from Eos deficient mice express normal levels of Helios. Taken together, these strongly suggest that Eos plays a related, but distinct role, from Helios in Treg function.
This work was supported by Division of Intramural Research (DIR), NIAID, NIH.
Regulatory T cells limit opportunistic colonization of Staphylococcus aureus to promote lesion resolution in cutaneous leishmaniasis
Authors: Singh Tej Pratap , Amorim Camila Farias , Lovins Victoria , Bradley Charles W , Carvalho Lucas P , Carvalho Edgar M , Grice Elizabeth A , Scott Phillip 
Published: August 2023

Abstract
The severity and responsiveness to treatment in L. braziliensis patients is influenced by a variety of factors, including microbiota and cytolytic T cells. Recently, we studied the skin microbiome in patients and demonstrated that high levels of S. aureus are associated with delayed healing in patients. To understand how S. aureus might influence cutaneous leishmaniasis, we studied the immune responses in mice following colonization with a S. aureus isolate from a L. braziliensis patient. Colonization of the skin with S. aureus alone induced a dominant type-17 response as well as an accumulation of Foxp3 T regulatory cells, and subsequent infection of mice with L. braziliensis led to increased IL-17 dependent pathology. Unexpectedly, transient depletion of total Tregs or RORγtin Foxp3 Tregs in S. aureus colonized mice increased IFN-γ and decreased the IL-17A response in skin. These mice exhibited increased pathology, which was accompanied by epidermal cell death and loss of barrier integrity. Additionally, the mice had substantially more S. aureus in the skin, and in the lymph nodes. In mice colonized with S. aureus and infected with L. braziliensis partial reduction led to a substantial increase in disease and a higher burden of S. aureus. Consistent with these results, in a transcriptional analysis of lesions from L. braziliensis patients, we found that low Foxp3 expression in patients had increased expression of cytolytic genes and IFN-γ, increased S. aureus in the lesions and had a delay in lesion resolution compared to patients with high Foxp3. Taken, together, these results suggest that in L. braziliensis patients Tregs are critical to regulate type-1 responses that in the context of S. aureus can promote more severe disease.
Oxidized phospholipid oxPAPC induces a dysfunctional Th1-like phenotype in regulatory T cells
Authors: Appleton Brenna , Palmer Sydney , Smith Harrison , Stephens Lilly , Major Amy 
Published: August 2023

Abstract
Regulatory T cells (Tregs) are protective in atherosclerosis but numbers are reduced during disease progression due to cell death and loss of stability. Oxidized lipids are abundant in atherosclerotic lesions and can activate innate immune cells, but there is limited information regarding their impact on T cells. Given the Treg loss observed during atherosclerosis progression and increase in oxidized lipid levels in the microenvironment, we sought to determine whether an oxidized phospholipid associated with atherosclerosis, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), alters Treg differentiation and function. In vitro skews of naïve T cells showed that oxPAPC-treated Tregs were less viable with a greater proportion expressing the Th1 markers T-bet, CXCR3, and IFN-γ. Th1 and Th17 skewed cultures were unaltered by oxPAPC suggesting a Treg specific effect of oxPAPC. OxPAPC-treated Tregs were less suppressive in vitro and were insufficient to inhibit atherosclerosis progression in an adoptive transfer in Ldlr−/− mice fed Western diet for up to 16 weeks. A role for IFN-γ signaling in Treg dysregulation was examined using Treg specific IFNγR1 deficient mice. IFNγR1 deficient CD4+ T cells skewed in the presence of oxPAPC did not adopt the Th1-like Treg phenotype compared to control oxPAPC-treated Tregs, indicating that IFNγR signaling was important for oxPAPC-mediated Treg dysregulation. Bone marrow transplant of Ldlr−/− showed that Treg IFNγR1 signaling was atherogenic in males but protective in females. Overall, our findings demonstrate that oxidized phospholipids directly impact Treg differentiation and decrease their atheroprotective potential.
Effect of malaria parasites’ infection on CD4 cells, haemopoietic cells and haemopoietic factors
Authors: Amadi Emmanuel Chike 
Published: August 2023

Abstract
Malariology Immunity is essential for understanding the pathology, treatment & vaccine production. Changes in CD4, haemopoietic cells & factors (RBC/HB/PCV/MCV/MCH & MCHC) in malaria parasites-infected patients were studied at a teaching hospital in Enugu, Nigeria. 45 patients on doctor’s provisional diagnosis of malaria were examined for Plasmodium infections & the degree of parasitaemia (0/+/++/& +++). Positive (+) & negative (0) samples were thereafter counted for CD4/haemopoietic cells & factors using Flow Cytometry/Automated Blood Cell Counter, respectively. Plasmodium-negative specimens were within reference ranges for CD4 cell count (464–1308); mean value: 835. Fifteen (+) parasitaemia showed lower ranges of CD4 count (502–1282); mean: 678; with immune falls in one (427). Nine from twelve (++) parasitaemia showed crash in CD4 cells counts, range: 301–415; mean: 399. Seven (+++) parasitaemia showed crashes in CD4 cell counts, range: 160–357; mean: 225. Significant rise with increase in parasitaemia occurs in neutrophils and Total WBC. Opposite occurred in the three other primary immune cells & platelets. There were variances in the basic immune cells’ percent & numeric. The haemoglobin factors didn’t exhibited significant change, except platelet that showed marked steady decrease with increase in parasitaemia. Plasmodium infection causes immunosuppression in patients, vivid changes in haemopoietic cells populations/percentages, & platelets. Malaria infection in the immunodeficients patients should accelerate the complications, including death, unlike prevailing reports. Haemolytic anaemia in malaria is likely complication resulting only from chronic cases since there was no vivid anaemia in this study
None
Identifying spatial immuno-oncological parameters of mouse tumors using Imaging Mass Cytometry
Authors: Pfister Thomas D , Raza Qanber , Lim Liang , Loh Christina 
Published: August 2023

Abstract
Identification of immuno-oncological (IO) processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates in preclinical models. Imaging Mass Cytometry™ (IMC™) is a proven high-plex imaging technology that enables deep characterization of the complexity of tumor tissue without disrupting spatial context while eliminating artifacts caused by tissue autofluorescence. Here, we showcase the Maxpar® OnDemand™ Mouse Immuno-Oncology IMC Panel Kit for application on mouse tumor tissues.
We compiled a 33-plex antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray containing a large variety of mouse tumors. We digitized high-plex data from mouse tissues using the Hyperion™ Imaging System and generated images demonstrating the detailed layout of the tumor microenvironment (TME). We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME.
The Mouse IO IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the TME tissue architecture. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells.
Our quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumor cells and activity of immune cell infiltrates.
For Research Use Only. Not for use in diagnostic procedures.
Decoding the structural and cellular composition of the tumor microenvironment in multiple human cancers using Imaging Mass Cytometry
Authors: Pfister Thomas D , Lim Liang , Ouladan Shaida , Zabinyakov Nick , Raza Qanber , Loh Christina 
Published: August 2023

Abstract
High-plex imaging techniques such as Imaging Mass Cytometry™ (IMC™) have become key tools in understanding and decoding the spatial complexity of the tumor microenvironment (TME). The TME contains tumor-infiltrating lymphocytes that have been associated with positive therapeutic outcomes. IMC enables detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide without spectral overlap or background autofluorescence.
We customized the Maxpar® Human Immuno-Oncology IMC Panel Kit using antibodies from the Standard BioTools™ catalog to create panels for tissue-based immuno-oncology research. Data acquisition was performed using a Hyperion™ Imaging System. Cell segmentation was facilitated using an IMC Cell Segmentation Kit. A pixel classification approach and CellProfiler™ were applied for single-cell segmentation. HistoCAT™ was used for single-cell analysis to visualize protein expression in various cancer types via PhenoGraph clustering and t-SNE maps.
Our custom panels were applied to normal and cancer human tissue microarrays to phenotype and analyze cell populations in these tissues. We classified the activation state of immune cells, epithelial-to-mesenchymal transition (EMT) progression, and composition of the extracellular matrix. In-depth single-cell analysis quantitatively evaluated the cellular makeup and immune cell component in the TME of cancer tissues. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers, including quantitative and spatial identification of multiple immune parameters in the TME, in tumor microarrays of cancer subject samples.