bluuebunny/crossref_metadata_2025_split huggingface dataset:
A simple and fast method for the isolation of untouched mouse panDCs from spleen (100.43)
Authors: Tabatabaei-Zavareh Nooshin , Luong Wendy , Thomas Terry , Fairhurst Maureen 
Published: January 2023

Abstract
Dendritic cells (DCs) control immune responses through their robust antigen presenting activity. Steady-state mouse spleen contains three major DC subsets with distinct functional and phenotypic properties including CD8+ and CD8− conventional DCs (cDCs), and plasmocytoid DCs (pDCs). These subsets are present at a very low frequency (&lt; 4%). Typically, elaborate purification protocols such as FACS-based cell sorting or expansion in culture are needed to obtain enough DCs for subsequent studies. Here, we describe a negative selection method to isolate all DC subsets (panDC) from mouse spleen. This method uses immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, non-DCs are labeled for depletion with biotinylated antibodies and cross-linked to magnetic particles using bispecific antibody complexes. The unwanted cells are then removed using an EasySepTM magnet. The selection steps can be fully automated using RoboSepTM . The panDCs are assessed by flow cytometry and defined as Lin−CD11c+ (cDCs) or Lin−CD11cloPDCA-1+ (pDCs). PanDC purities of 80 ± 7% (n=8) are achieved. The rare pDCs are enriched 36-fold with purity of 11.4 ± 1.4% as compared to 0.3 ± 0.1% in the start spleen. Both CD8+ and CD8− cDC subsets are represented in the cDC fraction. The freshly isolated DCs are not activated but upregulate maturation markers upon stimulation with LPS. This method enables fast, easy isolation of panDCs required for immune regulation studies.
Characterization of HLA-DM susceptibility of MHC II-peptide complex in antigen presentation and epitope selection (100.54)
Authors: Yin Liusong , Calvo-Calle Mauricio , Stern Lawrence 
Published: January 2023

Abstract
HLA-DM (DM) is a non-classic major histocompatibility complex II (MHC II), which mediates the exchange of peptides loading to MHC II during antigen presentation. However, the role of DM-mediated peptide exchange in epitope selection is still unclear. In this study, we addressed this question systematically using overlapping peptides from vaccinia virus protein A10L. We measured the binding affinity, intrinsic half life, DM-mediated half life and immunogenecity of 126 A10L peptides bound to HLA-DR1. Correlation coefficient analysis shows that immunogenecity of peptides is very weakly correlated with either binding affinity or intrinsic half life, while strongly correlating with half life in the presence of DM. Moreover, receiver operating characteristics analysis (ROC) indicates that DM-mediated half life is a very strong predictor for epitopes, while binding affinity and intrinsic half life can only mildly predicts epitopes. Taken together, our present quantitative and qualitive analysises demonstrate that DM-susceptibility is a strong and independent factor governing peptide’s immunogenicity.
TIM-3- and TIM-4-mediated phagocytosis of apoptotic cells induces cytokine production in peritoneal macrophages (100.55)
Authors: Bu Xia , DeKruyff Rosemarie , Umetsu Dale , Freeman Gordon 
Published: January 2023

Abstract
TIM-3 and TIM-4 mediate phagocytosis of apoptotic cells by recognizing phosphatidylserine on the surface of the apoptotic cell but are expressed by different cell populations, suggesting each has distinct functions in regulating immune responses. TIM-4 is exclusively expressed on APCs while TIM-3 is expressed on some T cell and APC subsets. We found that TIM-4 was constitutively expressed on mouse resident peritoneal macrophages (PMϕ) and played a predominant role in phagocytosis of apoptotic cells. Following phagocytosis of apoptotic cells, PMϕ secreted CCL2 (MCP-1), CCL3 (MIP-1 α), CCL4 (MIP-1β), CCL7 (MCP-3), CCL9 (MIP-1γ), CXCL-2 (MIP-2), CCL19 (MIP-3), CCL22 (MDC), IL-10 and TGF-β. mRNA levels of these cytokines increased very early (peaking at 0.5-4 hours), but the peak of protein level occurred 48-72 hours after phagocytosis. When TIM-4 on PMϕ was blocked by TIM-4 antibody, phagocytic activity was dramatically decreased and cytokine expression reduced. Following in vivo thioglycollate treatment, expression of TIM-4 by PMϕ was gradually lost, whereas expression of TIM-3 increased. TIM-3-mediated phagocytosis by thioglycollate-induced PMϕ resulted in a generally similar cytokine production pattern compared with TIM-4 mediated phagocytosis by PMϕ, but also resulted in the production of IL-1β and TNF-α. In summary, both TIM-4 and TIM-3 mediated phagocytosis of apoptotic cells and promoted macrophage production of cytokines that further regulates the immune response.
Infection with murine gamma herpesvirus 68 modifies experimental autoimmune encephalomyelitis (101.1)
Authors: Casiraghi Costanza , Horwitz Marc 
Published: January 2023

Abstract
Recent findings have identified Epstein-Barr virus (EBV) as a putative environmental trigger of multiple sclerosis (MS) but the mechanisms by which this virus causes MS remains elusive. We used murine gamma herpesvirus 68 (MγHV), the murine homolog to EBV, to examine how MγHV infection could aid in the enhancement of an autoimmune reaction against the CNS. C57Bl/6 mice were infected with MγHV and five weeks later, when the virus is cleared and has established latency, EAE was induced. Mice previously infected with MγHV developed a more severe EAE course showing both signs of paralysis and other neurological symptoms such as ataxia. MγHV EAE mice had higher levels of IFN-γ and TNF-α in the serum when compared to EAE mice and showed pronounced CD4 and CD8 T cell infiltrations both in the spinal cord and the brain parenchyma. On the other hand, CD4 T cells were less prominent in the brain parenchyma of uninfected EAE mice and CD8 T cells were absent. CD4 and CD8 T cells isolated from the CNS of MγHV EAE mice produced higher levels of IFN-γ accompanied by IL-17 suppression, whereas CD4 T cells isolated from the CNS of EAE mice produced high levels of IL-17 and lower IFN-γ levels. The absence of IL-17 production in infected mice suggests that the disease might be triggered by different T cell subsets than typical EAE. In conclusion, the ability of MγHV to elicit a more pathogenic T cell response could represent a new mechanism through which EBV triggers autoimmunity.
The role of Epstein-Barr Virus Latent Membrane Protein 2A in Experimental Autoimmune Encephalomyelitis (101.12)
Authors: Chang Rhoda , Miller Stephen , Longnecker Richard 
Published: January 2023

Abstract
Multiple Sclerosis (MS) is an inflammatory, autoimmune disease of the central nervous system. The cause of MS is still unknown but epidemiological and immunological studies have implicated Epstein-Barr Virus (EBV), which infects B cells, as a possible etiological agent. Of particular interest is EBV Latent Membrane Protein 2A (LMP2A). Previous work in our lab has demonstrated that LMP2A enhances the expansion and differentiation of B cells upon antigen stimulation, revealing a potential contribution of LMP2A in autoimmunity. As B cells may be a key player in MS, we wanted to examine the role of LMP2A in the animal model Experimental Autoimmune Encephalomyelitis (EAE). Recombinant MOG protein was immunized into transgenic mice in which B cells express LMP2A. LMP2A mice show increased severity and incidence of disease, and faster onset of disease. The explanation of this difference is not due to lymphocyte recruitment into the CNS or differences in serum antibody levels. Further in vitro analysis will address the role of LMP2A in lymphocyte activation and antigen presentation.
Establishment of a sustained delayed type hypersensitivity model with mono-arthritic manifestations in C57Bl/6J mice (101.17)
Authors: Nansen Anneline , Atkinson Sara , Usher Pernille , Markholst Helle , Haase Claus 
Published: January 2023

Abstract
RA is a progressive, inflammatory and destructive autoimmune disease, characterized by chronic synovial joint inflammation. Various RA models of both mono-and polyarthritis have been developed in different inbred strains of mice. However, establishment of sustained arthritis models in the C57Bl/6J mice has been difficult. As most genetically modified mice are bred on the C57Bl/6J background, there is a need to explore new approaches of obtaining robust models in this mouse strain. We have established a sustained DTH model with monoarthritic manifestations in C57Bl/6J mice. The model builds on a classical transient DTH model, where a DTH reaction is elicited by antigen injection into the right hind footpad at 7 day post Antigen sensitisation. Interestingly, we find that administration of a single low dose of a monoclonal antibody cocktail of anti-collagen type II antibodies before elicitation of the DTH reaction results in a sustained footpad swelling that is maintained for at least two weeks. Histopathological examination revealed inflammation and bone destruction with characteristics of arthritis with only the Antigen challenged footpad being affected (monoarthritis). This was not observed in mice receiving an isotype antibody cocktail. Moreover, no mice receiving anti-CII antibodies alone developed polyarthritis. Similar results were obtained using Balb/C mice thus the presence of anti-CII antibodies appears crucial for development of sustained joint tissue inflammation.
PPARγ is required for CD4+ T cell proliferation, survival, and disease-mediation in lymphopenia-associated autoimmunity. (101.21)
Authors: Housley William , Adams Catherine , Clark Robert 
Published: January 2023

Abstract
Ligands for the nuclear hormone receptor PPARγ ameliorate many autoimmune conditions in both mice and humans. Recent reports have also suggested that PPARγ is important in regulatory T cell function and in preventing the development of pathogenic Th17 cells. In contrast to this role of PPARγ in suppressing autoimmunity, we now find that CD4+ T cell PPARγ expression is required for development of autoimmunity under lymphopenic conditions. PPARγ-deficient (PPARγ-/-) CD4+ T cells are unable to mediate disease in both murine graft-versus-host disease and an adoptive transfer model of colitis. In the colitis model, PPARγ is required for the accumulation and cytokine production of CD4+ T cells in the spleen, mesenteric lymph nodes, and colonic lamina propria. This defect is specific for CD4+ T cells as PPARγ-/- CD8+ T cells accumulate equally to wild type CD8+ T cells. Mechanistically, PPARγ-/- CD4+ T cells show diminished in vivo proliferation as assessed via Ki67 staining, but no difference in activation marker expression demonstrating that PPARγ is required for proliferation and cytokine production but not for activation. Endogenous PPARγ-/- CD4+ T cells recover normally after mice are sub-lethally irradiated demonstrating that loss of proliferative potential requires antigen-induced activation as well as lymphopenia. Our results identify a novel function for PPARγ in the proliferation and survival of CD4+ T cells in lymphopenia-associated autoimmunity.
MHC Dextramers enhance the detection sensitivity of antigen-specific autoreactive CD4 T cells (101.22)
Authors: Upadhyaya Bijaya , Massilamany Chandirasegaran , Gangaplara Arunakumar , Reddy Jay 
Published: January 2023

Abstract
Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 cells, detection of autoreactive cells by major histocompatibility complex (MHC) class II tetramers is challenging because of their low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers designated ‘MHC Dextramers’ that contain a large number of peptide-MHC complexes per dextramer molecule. We prepared MHC Dextramers for proteolipid protein (PLP) 139-151 using soluble IAs molecules by coupling them to an activated dextran backbone (kindly provided by Immudex, Denmark). We used PLP-reactive T cells generated from immunized SJL mice to validate the reagents by flow cytometry. Using Theiler’s murine encephalomyelitis virus 70-86 Dextramers as controls, we showed that the MHC Dextramers detect PLP-reactive T cells with specificity, and the detection sensitivity was enhanced by 6-fold when compared with uncoupled tetramers. The MHC Dextramers also allowed us to enumerate the frequencies of antigen-specific cells from immunized mice ex vivo . Similar results were obtained when the antigen-sensitized cells were enumerated in A/J mice immunized with cardiac myosin heavy chain (Myhc)-α 334-352 using IAk MHC Dextramers. Taken together, the data suggest that MHC Dextramers can be efficiently used to track the generation of self-reactive CD4 cells in various experimental systems.
Memory cells specific for myelin oligodendrocyte glycoprotein govern the transfer of experimental autoimmune encephalomyelitis (101.26)
Authors: Williams Jessica , Kithcart Aaron , Smith Kristen , Shawler Todd , Cox Gina , Whitacre Caroline 
Published: January 2023

Abstract
Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system thought to be initiated by CD4+ T cells specific for myelin antigens. Experimental autoimmune encephalomyelitis (EAE), a model of MS, is induced in susceptible mouse strains by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). MOG35-55-specific T cell receptor transgenic (2D2) mice are susceptible to EAE following immunization with MOG peptide. To better understand the role of T cells in EAE initiation, we explored several approaches for effecting adoptive transfer of EAE using 2D2 T cells. Surprisingly, despite robust proliferation and production of IFN-γ and IL-17 following a variety of culture conditions, EAE was not transferred. Only when 2D2 whole splenocyte preparations contained CD44+ T cells and were Th1-differentiated using MOG peptide and IL-12 did they reliably transfer EAE. Further, we found that CD44hiCD62Llo effector/memory CD4+ T cells are likely responsible for disease transfer due to up-regulation of the adhesion molecule CD44 following differentiation. We also observed a sex dimorphism in 2D2 mice with males transferring more severe EAE, associated with a larger population of effector/memory CD4+ T cells in males compared to females. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.
Tissue-expressed coinhibitory B7 molecules can control both adaptive and innate immunity (101.28)
Authors: Zang Xingxing 
Published: January 2023

Abstract
Some new members of the B7/CD28 families were discovered over the last several years and their functions in vivo need to be elucidated. We show that B7x (B7-H4 or B7S1) was expressed in pancreatic b cells but not in immune cells. Using a CD8 T cell-mediated diabetes model, we demonstrate that mice lacking B7x developed more severe diabetes than the control, conversely mice overexpressing B7x in b cells were diabetes free. Pathogenically effector CD8 T cells were capable of migrating to the pancreas but failed to kill target cells and destroy tissue when encountering local B7x. In response to the local B7x these CD8 T cells achieved an overlap yet distinct gene-expression profile from anergic or exhausted CD8 T cells. Our findings reveal that B7x in the periphery abrogates CD8 T cell-mediated tissue destruction. B7-H1 (PD-L1) is widely expressed in immune cells and is important in the coinhibition of T cell function via binding its receptor PD-1. We show that both human and mice intestinal epithelium expressed B7-H1 and mice deficient for B7-H1 were highly susceptible to gut injury and associated mortality and morbidity. Defective B7-H1 led to loss of epithelial integrity and systemic dispersion of commensal bacteria during intestinal inflammation. B7-H1 expressed on parenchymal cells but not on hematopoietic cells controlled intestinal inflammation, which was not dependent on PD-1, B7-1(CD80), and the adaptive immunity.
The role of Th40 cells in EAE mice. (101.29)
Authors: Carter Jessica , Vaitaitis Gisela , Wagner David 
Published: January 2023

Abstract
Auto-aggressive T cells are responsible for the onset and perpetuation of autoimmune diseases, including Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). A subset of CD4 T cells, CD4+CD40+ (Th40) cells, have an expansive role in the Non-Obese Diabetic (NOD) mouse model of T1D. Not only do Th40 cells increase in number with the onset of disease, but they are necessary and sufficient in transferring T1D from NOD to NOD.scid mice. Because Th40 cells have an imperative role in the development of disease in T1D, we considered that Th40 cells might also be involved in Experimental Autoimmune Encephalomyelitis (EAE), a neurological disease which mimics human MS. When given myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin (PT), C57Bl/6 mice develop neurological symptoms and impaired motor functions similar to those in human MS patients. Data shows an increase, not only in CD4 T cells, but also in Th40 cells in mice receiving induction of EAE. This does not occur in their control counterparts. Further, purified splenic Th40 cells from asymptomatic disease induced C57Bl/6 mice rapidly transfer EAE to C57Bl/6.scid recipients with only 1.2x106 cells, demonstrating that Th40 cells alone can induce EAE. Also, we show cerebral infiltrates of immune cells in localized regions of the brain of the Th40 recipient mice, suggesting that these lesions are similar to the lesions found in human MS patients.
Loss of T cell CD98hc specifically ablates T cell clonal expansion and protects from autoimmunity. (102.16)
Authors: Cantor Joseph , Slepak Marina , Ege Nil , Chang John , Ginsberg Mark 
Published: January 2023

Abstract
CD98hc (CD98 heavy chain, 4F2 antigen, Slc3a2) is a lymphocyte activation antigen with unclear function in T cells. Deletion of CD98hc in B cells leads to complete failure of B cell proliferation, plasma cell formation, and antibody secretion. Here we examined the role of T cell CD98 in protective and auto- immune responses by specifically deleting it in mouse T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with reduced T cell clonal expansion. However, initial T cell homing to pancreatic islets was unimpaired. In contrast to B cells, CD98-null T cells showed only moderately impaired antigen-driven proliferation in vitro and nearly normal homeostatic proliferation. Furthermore, these cells were activated by antigen and produced cytokines (CD4) and efficiently killed targets (CD8). The integrin binding domain of CD98hc was necessary and sufficient for full clonal expansion, highlighting the role of adhesive signaling in T cell proliferation and autoimmune disease. When CD98-null T cells were pre-expanded in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. The integrin binding domain of CD98hc is thus required for antigen-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.
L. amazonensis infection differentially affects migration of phagocyte populations from inflammatory site to the draining lymph node (102.27)
Authors: Hermida Micely , Malta Rafaela , Doria Priscila , Taguchi Angela , Utsch Lara , Maciel Maira , Mengel José , dos Santos Washington 
Published: January 2023

Abstract
Leishmania infection interferes with integrin function. In this work we use a model of chronic peritonitis to study the effect of L. amazonensis infection upon the ability of phagocytes populations to migrate from an inflammatory site to the draining lymph node (dLN). First we showed that macrophage recruitment into the peritoneum and migration to the dLN were both maximal 4 days after thioglycollate injection into the peritoneum. At this time point, cell tracking assays using cell transfer between MHC-mismatched mice showed that 1% of the leukocytes injected into the peritoneum migrated to the dLN after 24h. In the mice injected with cells cultivated with medium alone adoptive cells represented 14% of the peritoneal cells. Among these leukocytes 57% were macrophages, 2% were neutrophils and 35% were myeloid dendritic cells. In the LN, 22% of the migrating cells were macrophages, 4% were neutrophils and 25% were myeloid dendritic cells. In the mice injected with leukocytes co-cultivated with Leishmania adoptive cells represented 12% of the peritoneal cells. Among these cells 44% were macrophages, 1% were neutrophils and 46% were dendritic cells. In the LN 19% of the migrating cells were macrophages, 2% were neutrophils and 12% were myeloid dendritic cells. Our data show that: (1) A variety of phagocytes are able to migrate from the inflammatory site to the dLN. (2) Populations of Leishmania-infected dendritic cells appear to be retained in the peritoneum.
Latent CMV and EBV infections and the mobilization of viral-specific and senescent T-cells with exercise (102.6)
Authors: Simpson Richard , Spielmann Guillaume , Bigley Austin , Rector Jerrald , Morrison Mark , LaVoy Emily , Lowder Thomas 
Published: January 2023

Abstract
Exercise mobilizes senescent T-cells into the blood but the impact of latent CMV and EBV infections on this response is unknown. This study aimed to determine if: (1) CMV or EBV specific T-cells are mobilised in response to exercise; and (2) the impact of CMV and EBV on the exercise-responsiveness of senescent T-cells. 24 healthy males (age 28.5 ± 5) completed a 30-min cycling protocol at 85% of maximum power. PBMCs isolated before, immediately after and 1hour after exercise were labelled with MHC class I Pentamers specific to CMV (pp65) or EBV (LMP-2) and assayed for Naïve (KLRG1-/CD28+/CD57-), effector-memory (KLRG1+/CD28+/CD57-) and senescent (KLRG1+/CD28-/CD57+) surface markers. Exercise increased CMV and EBV-specific T-cell numbers by 158% and 200% respectively, and altered the proportions of senescent (+44%) and naïve (-20%) CD8+ T-cells. CMV+ had a greater (+136%) proportion of senescent and a lower (-33%) proportion of naïve CD8+ T-cells compared to CMV-, and demonstrated a greater egress of senescent T-cells in response to exercise. Senescent markers on both CMV and EBV-specific T-cells increased with exercise (+53% and +76% respectively). To conclude, CMV and EBV specific CD8+ T-cells are mobilized into blood with acute exercise and those infected with CMV demonstrate a greater relative egress of senescent T-cells during exercise recovery. Future work will determine the potential impact of exercise in altering senescent T-cell frequency in those infected with CMV