bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying the role of antigen-specific resident memory CD4 T cells in maintaining disease during Experimental Autoimmune Encephalomyelitis a murine model of Multiple Sclerosis.
Authors: Yuzefpolskiy Yevgeniy , Oukka Mohamed , Bettelli Estelle
Published: November 2024

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) driven by antigen-specific CD4+ T cell mediated destruction of the myelin sheath. Most T cells circulate between lymphoid organs and peripheral tissues, but a fraction can establish tissue residence and do not recirculate. Growing evidence suggests resident T cells exist in the CNS of MS patients, however, their contribution to disease progression and whether they can cause relapses independently of peripheral T cells is unclear. Current disease modifying therapies for MS patients are not curative and none target CNS resident T cells. Using a murine model of MS, Experimental autoimmune encephalomyelitis (EAE), we investigated whether resident T cells form in the CNS of diseased mice and their contribution to disease pathogenesis. Our data show that antigen-specific CNS resident T cells can be detected during EAE by intravital antibody staining and CD69 expression. Following depletion of circulating CD4+ T cells, CNS resident CD69+ CD4+ T cells maintained their numbers, while the number of CD69- T cells in the CNS was significantly depleted. These CNS resident T cells can drive severe disease in the absence of circulating CD4+ T cells and showed memory-like properties including high proliferative capacity and increased cytokine polyfunctionality compared to early effector T cells. Therefore, disruption of resident T cell function in MS patients could represent a novel target for MS therapies.
IL-10 promotes mast cell homeostasis and differentiation towards a mucosal mast cell phenotype
Authors: Ranjitkar Saurav , Maldonado Perez Nicole , Tedeschi Caitlin , Jordan Nathan , Mathias Clinton
Published: November 2024

We recently demonstrated an unexpected role for IL-10 in driving mast cell (MC) activation and proliferation. IL-10 stimulation significantly enhanced IgE-mediated MC activation, upregulated FceRI expression, and promoted MC-mediated food allergy. Surprisingly, IL-10 also amplified cytokine production in un-activated MCs and enhanced their proliferation and survival, suggesting a role for IL-10 in MC homeostasis. Herein, we demonstrate that IL-10 can significantly expand MC proliferation and activity during homeostatic conditions. Treatment with IL-10 enhanced the proliferation and survival of both IL-3 and IL-3+SCF cultured bone marrow-derived MCs (BMMCs). This corresponded with a dramatic increase in cytokine production in IL-10-treated cells, along with upregulation of FceRI expression. While SCF was dispensable for the increased FceRI, it significantly enhanced cytokine production compared to cells cultured in IL-3 alone. Further analysis revealed a distinctive mucosal MC phenotype in IL-10-treated cells, characterized by beta7-integrin expression and enhanced secretion of murine MC protease-1. Lastly, autocrine IL-10 also supported MC expansion and function, as blocking IL-10 signaling attenuated MC expansion, inducing apoptosis, and decreasing cytokine production. Taken together, these data suggest an important role for IL-10 in maintaining MC responses during physiological conditions.
Profiling autoreactive IgG, IgM, IgA and IgE antibodies in system lupus erythematosus patients using antigen microarrays
Authors: Li Quanzhen , Wei Junxia , Zhu Chengsong , Luo Hui
Published: November 2024

OmicsArray™ antigen microarrays bearing 120 antigens were used for profiling IgG, IgM, IgA and IgE autoantibodies in 240 well defined SLE patients and matched healthy controls (HCs). Our analysis show that the average number of positive IgG autoantibodies in SLE is 28.5 with over 95% of SLE patients harbor 5 or more autoantibodies, comparing with 3 positive IgG Autoantibodies in HCs with 25% have 5 or more autoantibodies (p<0.05). A group of 19 IgG autoantibodies targeting to various nuclear antigens (dsDNA, chromatin, histone, Sm/RNP, PCNA, CENP-B, etc.) were significantly enriched in SLE by clustering analysis. The presence of IgG autoantibodies against DNA-associated antigens were most significantly associated with disease activity (SLEDAI) and lupus nephritis (r=0.56, p<0.001) in SLE. The IgG autoantibodies which are positive in HCs were mostly those targeting non-nuclear antigens, such as collagens, alpha-actinin, heparin and cardiolipin. However, some ANA positive HCs exhibited higher IgM specificities targeting to both non-nuclear and nuclear antigens whereas in SLE the IgM autoAb reaction were relatively lower. IgA autoantibodies were detected in over 50% of SLE patients, preferentially targeting to DNA- and RNA-associated antigens indicating the high prevalence of IgA autoantibodies may play a role in SLE. IgE autoantibodies were not detectable for most autoantigens except about 15% of SLE patients showed positive IgE autoantibodies to a small subset of DNA antigens.
Klebsiella michiganasis as a tissue repairing commensal in gut inflammation
Authors: White Zachary , Sano Teruyuki , Kapustka Isabel
Published: November 2024

Commensal bacteria are recognized as contributors for both tissue inflammation and tissue homeostasis. Therefore, we tested their potential for gut inflammation and tissue repairing using Germ-Free (GF) mice using stools from healthy human donors. Although, DSS-treated WT GF mice all die within 10 days due to severe colonic epithelial damage, gnotobiotic WT mice colonized with a healthy microbiome survived and carried more IL-22 producing innate and adaptive immune cells. This suggests that healthy stools contain wound healing commensals, however, IL10 KO mice with the same stools developed colitis, indicating a colitogenic potential. To eliminate colitogenic from wound healing commensals we utilized various antibiotics in human stool inoculated mice. These treated mice showed significant reductions in their microbiomes. Importantly, IFNg/IL-17A double producing pathogenic CD4 T cells were decreased leading to no colitis in gavaged IL10 KO mice. Additionally, gnotobiotic WT mice colonized with cecum contents from treated human stool gavaged IL10 KO survived lethal DSS treatment. We isolated Klebsiella michiganasis (K. michiganasis) from their cecum and confirmed that monocolonization of K. michiganasis in GF WT mice reproduced the wound healing ability via both the IL-23 and IL1b pathways stimulating type 3 innate lymphoid cells to strongly produce IL-22. Our results suggest that non-pathogenic K. michiganasis is a promising commensal for combating gut inflammation.
Mast cell-intrinsic TGF-b signaling modulates mast cell effector function and allergic responses
Authors: Haque Tamara , Leak Lashawna , Youngblood Emily , Laky Karen , Frischmeyer-Guerrerio Pamela
Published: November 2024

Mast cell (MC) activation via the high affinity IgE receptor, FcεRI, causes the release of mediators that are directly involved in the allergy diathesis. TGFβ has been shown to regulate MC development and function in vitro however it’s role in allergic diseases is unclear. Patients with Loeys Dietz Syndrome (LDS), a disorder caused by loss-of-function variants in genes encoding the TGFβ receptors are highly predisposed to develop atopy, thus provide an opportunity to study the role of MC TGFβ signaling in allergic diseases. Unexpectedly, IgE mediated degranulation of LDS MC were reduced compared to healthy controls in vitro. We examined active and passive IgE responses in a mouse model of LDS and mice with conditional deletion of Tgfbr1 in MCs. Both models exhibited less IgE mediated anaphylaxis and degranulation. This could not be attributed to alterations in IgE or stem cell factor receptor expression on MCs, or to the distribution of MCs in tissues. In contrast, LDS MCs exhibited enhanced responses to the type 2 alarmin, IL-33, in vitro and in vivo. Mechanistically, altered responses to IgE, IL-33, and TGFβ were linked, as the reduction of anaphylaxis in LDS mice was partially restored in IL-33RKO LDS mice. Moreover, this was tied to both transcriptional and proximal signaling mechanisms. Taken together, TGFβ signaling upregulates MC function by disrupting an IL-33/ST2 mediated regulatory pathway, which likely plays a major role in controlling IgE mediated MC functions.
Borrelia Burgdorferi mediated mitochondrial DNA release into the cytosol induces host inflammatory response via the activation of cGAS-STING signaling pathway
Authors: Priya Raj , Raghunandanan Sajith , YANG FRANK
Published: November 2024

Background: Mitochondrial DNA (mtDNA) is a potent ligand of inflammation when it leaks from stressed mt into the host cytosol during bacterial infections. In this study, we uncover mtDNA as one of the ligands involved in inflammatory response against B. burgdorferi (Bb) infection via the activation of cGAS-STING signaling pathway in Raw264.7 cells. Methods: Raw264.7 cells were infected with Bb & the mt stress was studied by measuring mt reactive oxygen species (mtROS) using MitoSOX dye & mt membrane potential (MMP) by JC-1 dye staining. The release of mtDNA was assessed by Immunofluorescence assay & q-RT-PCR. The co-localization of mtDNA & cGAS was determined by IP assay. To explore the requirement of mtDNA for Bb-induced inflammation, both WT & cGAS-KO cells were treated with Ethidium bromide (EtBr) to deplete the mtDNA & ELISA was done to detect the level of inflammatory cytokines. Results:Bb infection to Raw264.7 cells caused mt stress, that results in decrease of MMP, enhanced mtROS & promoted mtDNA leakage into the cytosol. Leaked mtDNA activates cGAS-STING signaling pathway & results in inflammation. Both stable knockdown of cGAS & depletion of mtDNA reduced Bb induced inflammation. Conclusions: We uncover a novel mechanism of Bb induced inflammation, which involved mtDNA leakage from stressed mt into the cytosol stimulating the cGAS-STING signaling pathway. The mtDNA-cGAS-STING pathway could be a therapeutic target to prevent Bb induced inflammatory disease.
Suppression of the Immune System in Borrelia burgdorferi Infection Shilpa Sachan, Heather Kulaga, and Nicole Baumgarth
Authors: Sachan Shilpa
Published: November 2024

Lyme Disease, caused by tick-transmitted infection with Borrelia burgdorferi (Bb), is an emerging vector-borne disease. Bb establishes persistence in their natural reservoir hosts and in laboratory mice. Our previous work established Bb-infection induced immune suppression, manifested in lymph tissue remodelling, rapid collapse of germinal centres, and inability to generate B cell memory. The objective of this study was to further explore the T-dependent humoral responses to Bb infection and to identify mechanisms of immune dysregulation. For this, we infected or sham-infected C57BL/6 mice with Bb, bled mice over time and measured total and influenza-specific serum IgG serum antibody levels following subsequent immunization with influenza A/PR8 virus in alum. Serum analysis revealed strong and continued hypergammaglobulinemia, contrasting the significantly reduced influenza-specific serum IgG responses in Bb-infected mice compared to sham-infected controls. Reductions in influenza-IgG levels was explained by reduced bone marrow IgG ELISPOT and were seen only when mice were infected with Bb. Flow cytometric analysis of CD4 T cells revealed the presence of CD4 Tfh cells, as well as effector CD4 T cells that showed an exhausted phenotype, even at early timepoints after in Bb infection. Together, our data reveal a profound defect in effective T and B cell responses to Bb, explaining the inability of the immune system to clear this infection.
Label-free imaging of microbial-infected cells and tissues using 2p-FLIM and MALDI-MSI identify altered NADH metabolism.
Authors: Snyder Greg , Omomo Ayotunde , White Erica , Snyder Michelle , Yang Hyojik , Scott Alison , Ray Krishanu
Published: November 2024

We have developed label-free imaging methods to identify and characterize microbial infection of live cells and tissues using a novel application of Fluorescence lifetime (FLT) imaging with 2-photon excitation (2p-FLIM) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). 2p-FLIM is often used in cancer biology to image metabolically altered cells of affected tissues and tumors. Whereas MALDI-MSI is one of the growing number of spatial ‘omic methods, that is also label-free and allows mapping of individual metabolites at cellular and tissue scales. We have previously reported the application of 2p-FLIM to characterize HIV-1-infected cells and tissues. We now report the novel application and integration of 2p-FLIM with MALDI-MSI for characterizing pathogenic bacteria patient isolates and infected tissue. We observe increases in NAD(P)H intensity and unique distributions of fluorescence lifetimes (FLTs) for select pathogenic bacteria patient isolates, which may reflect differences in OxPhos metabolism. We have further characterized Pseudomonas-infected mouse lung tissues which exhibit unique NAD(P)H fluorescence, average FLTs and spatial mapping of individual NAD+-associated metabolites. A highly integrative, cross-platform, label-free, multi-omics approach involving 2p-FLIM and MALDI-MSI may provide a better understanding of microbial infection for developing and assessing novel antimicrobial and immune-modulatory interventions.
Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis
Authors: Choi Ji Hyun , Jeong Gi Uk , Hwang Insu , Lee Wooseong , Yoon Gun Young , Kim Hae Soo , Yang Jeong-Sun , Kim Kyung-Chang , Lee Joo-Yeon , Kim Seong-Jun , Kwon Young-Chan , Kim Kyun-Do
Published: November 2024

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Notably, double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.
SARS-Cov-2 antigen exposure history shapes post-infection antibody epitope and immune pressure distribution
Authors: Yisimayi Ayijiang , Jian Fanchong , Wang Jing , Song Weiliang , Cao Yunlong
Published: November 2024

The COVID-19 pandemic has led to a complex immunological landscape shaped by various vaccination regimens and SARS-CoV-2 variant infections. The effect of diverse exposure history on antibody response and virus evolution is a critical, unsolved issue.

Using high-throughput single-cell V(D)J sequencing, high-throughput deep mutational scanning, and neutralization assays, we characterized epitope distributions and immune pressure of more than 6000 Receptor Binding Domain (RBD)-antibodies from various SARS-CoV-2 exposure histories, including ancestral strain vaccinated or unvaccinated individuals with single or dual infections with different variants.

We found that single variant breakthrough infections post-vaccination induce a high proportion of non-neutralizing antibodies due to immune imprinting. In contrast, dual breakthrough infections exhibit fewer non-neutralizing antibodies, akin to those in unvaccinated individuals. Furthermore, the initial infection variant influences the antibody repertoire to second infections, indicating that variant infections also induce immune imprinting. Antibody profiles shaped by diverse exposure histories exert distinct pressures on RBD, promoting the virus to evolve at convergent immune pressure sites. This is evidenced by frequent RBD mutations at positions 420, 455, 456, and 475 across different variants.

These findings highlight the potential of diversifying the antibody pressure across populations to reduce the pace of viral evolution.
Generation of a novel agonist of the human CD137 costimulatory pathway for cancer immunotherapy
Authors: Jackson Christa , Nur Arguc Feyza , Ulker Vahap , Powell Noah , Yolcu Esma , Shirwan Haval
Published: November 2024

The use of immune checkpoint stimulators is an increasingly attractive therapeutic approach against cancer, and costimulatory pathway agonists are being developed for clinical use. Our laboratory has focused on the 4-1BB (CD137) pathway because of its critical role in CD8+ T cell expansion, survival, acquisition of effector function, and establishment of long-term memory. We previously reported the generation of a recombinant oligomeric form of the mouse ligand, muSA-4-1BBL, which, unlike the natural 4-1BB ligand, demonstrated robust costimulatory activity in soluble form. muSA-4-1BBL showed therapeutic efficacy as the immune adjuvant component of subunit vaccines in various mouse transplantable tumor models. In this study, we generated a human version of this ligand, huSA-4-1BBL, and characterized its structure and function. The molecule binds to 4-1BB on LPS-activated human THP-1 monocytes, but not ConA-stimulated mouse T cells expressing the 4-1BB receptor. Conversely, muSA-4-1BBL binds to ConA-stimulated mouse T cells, but not LPS-activated human THP-1 cells, demonstrating species-specific binding for both molecules. To assess the cancer immunotherapeutic efficacy of huSA-4-1BBL, we generated a transgenic mouse model where mouse 4-1BB was replaced with the human ortholog. The assessment of huSA-4-1BBL anti-tumor efficacy in this model will serve as a prelude for further development and translation of this novel agonist to the clinic for cancer immunotherapy.
Characterizing functions of endogenous bile acid pools altered by Asbt-deficiency in the ileal transcriptional regulation.
Authors: Sudo Koichi , Dionne Henry , Sundrud Mark
Published: November 2024

Bile acids (BAs) are a diverse and abundant class of metabolites synthesized in the liver, absorbed in the ileum, and metabolized by the colonic microbiota. Besides the classical functions of BAs for lipid and vitamin absorptions in the small intestine (SI), BAs have emerged as potent immunoregulatory metabolites via interactions with G protein-coupled and nuclear receptors (NRs; e.g., FXR/NR1H4). Alterations of BA metabolism have been reported in human diseases, e.g., inflammatory bowel diseases. However, it remains unclear whether or how altered BA pools may influence the intestinal homeostasis and pathology. As in vivo studies cannot uncouple the regulatory functions of BAs from their impact on microbiota, I have developed a novel intestinal explant culture system, in which the ileal fragments can be treated with the endogenous BA pools from normal or diseased mice. The ileal, not colonic, fragments induced mRNA expressions of BA-targeted genes in the presence of (t)CDCA which is a representative and FXR-agonistic BA. Then, the fragments were cultured with the endogenous SI BA pools from healthy or Asbt-/- mice – which have altered BA pools. RNA-seq showed that some genes were similarly induced, but others were differentially regulated by each BA pools. In NR reporter assays, the BA pools from Asbt-/- mice potently activated RXRa, PXR, and ERb. Together, this study suggests abnormal BA pools differentially modulate the ileal environment via distinct NR pathways.
Antioxidant and anti-Inflammatory effects of maltol on inflammasome activation in macrophages and mice
Authors: Ahn Huijeong , Lee Geun-Shik
Published: November 2024

Maltol (3-hydroxy-2-methyl-4-pyrone) is used widely as a food and cosmetic supplement, and it has antioxidant and anti-inflammatory activities. Inflammasome causes the maturation and secretion of interleukin (IL)-1β and -18 through activation of caspase-1 (Casp1), which contributes to various inflammatory diseases. This study examined the effects of maltol on inflammasome activation in macrophages and mice. Lipopolysaccharide (LPS)-primed macrophages were treated with a trigger of NLRP3, NLRC4, AIM2, or non-canonical (NC) inflammasomes in the presence of maltol. The secretion of IL-1β and IL-18, and cleavage of Casp1 were analyzed as indices of inflammasome activation. Mice were injected with LPS and an NLRP3 trigger with or without maltol, and the peritoneal IL-1β secretions were observed. The effects of maltol on reactive oxygen species (ROS) production and Casp1 activity were analyzed to determine the mechanism. Maltol inhibited the activation of NLRP3 and NC inflammasomes, but it did not alter the other inflammasomes. Maltol also attenuated IL-1β secretion resulting from the inflammasome activation in mice. The anti-inflammatory mechanism of maltol was revealed by the inhibition of ROS production and Casp1 activity. Maltol is suggested as a candidate of anti-inflammasome molecules.
Tpl2-/- macrophages exhibit impaired mitochondrial respiration due to type I interferon signaling in response to LPS stimulation.
Authors: Fahey Denise , Heo Junwon , Call Jarrod , Watford Wendy
Published: November 2024

Macrophages are essential to the innate immune system and their dysfunction has been implicated in many diseases, including autoimmunity, obesity, and cancer. Activated macrophages can alter their oxidative phosphorylation, metabolite production, and reactive oxygen species generation to elicit their diverse functions. Tumor progression locus 2 (Tpl2, MAP3K8, or COT) is a serine-threonine kinase involved in macrophage activation and function. In TLR-stimulated macrophages, TPL2 ablation induces high type I interferon (IFN) production. Previous research implicates type I IFNs in metabolic regulation, but their exact mechanism remains unclear. We hypothesize that TPL2 promotes oxidative phosphorylation in LPS-stimulated macrophages by suppressing type I IFN signaling. LPS-stimulated Tpl2-/- bone marrow-derived macrophages had decreased oxygen consumption, ATP production, and maximal respiration compared to LPS-stimulated wild type and Tpl2-/-IFNAR-/- macrophages. LPS-stimulated Tpl2-/- macrophages had reduced antioxidant gene expression compared to wild type macrophages, including SOD2, HMOX-1, and PRDX5. Disrupted IFN signaling in IFNAR-/- and Tpl2-/-IFNAR-/- macrophages significantly increased antioxidant gene expression, suggesting the absence of IFN signaling mitigates macrophage oxidative stress. These data indicate that Tpl2-/- macrophages are metabolically defective and type I IFNs contribute to the underlying dysfunction.
Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome
Authors: Pandey Abhimanu , Shen Cheng , Man Si Ming , Ngo Chinh
Published: November 2024

Pattern-recognition receptors are critical in the host defence against infectious diseases and cancer. DNA is the genetic code of life but also represents a form of microbial or damage-associated signal which can induce an immune response. Central to immune surveillance are the cytoplasmic DNA sensors that respond to microbial or endogenous DNA liberated from microbes or damaged and/or dying host cells. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. The effect of Ku70 on attenuating intestinal cancer was independent of the role of Ku70 in DNA repair, and the production of inflammatory markers and interferons. Instead, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex that docks at Rab5+Rab7+ early- late endosomes. We identified that the ⍺/β domain and the C-terminal domain of Ku70 interact with the GTPase domain of Ras, whereas the C-terminal domain of Ku70 interacts with the conserved region 2 of Raf. The Ku70-Ras-Raf signalosome activates the MEK-ERK signalling pathway, leading to impaired activation of cell cycle proteins, Chk2, Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. These findings suggest that therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
You too Brutus! Faculty fighting death-of-teaching in the classroom.
Authors: Fazal Nadeem , Graefen Bahar
Published: November 2024

In this study, we explored the hypothesis that active learning approaches to immunology improve comprehension and retention by increasing student engagement in class and their subsequent mastery of complex topics. What is “active learning”? Literature suggests that students participating in active learning “must do more than just listen: They must read, write, discuss, or be engaged in solving problems. The larger fear is that if this battle is lost it will change the battlefield forever. If faculty, classrooms, physical space of university, administration were to disappear physically if not virtually one can forecast a life-long learner, be of any age, be a student or teacher is positioned to stay amid machines. Pedagogical theory has taught us that students learn best by engaging with complicated questions and by thinking metacognitively about how to approach solutions. Building this skill set in today's students, who now hail from a broad demographic and who are accustomed to acquiring their knowledge from a variety of different media, requires a new set of teaching tools. A college-wide survey was sent to all the instructors and data was collected as per the requirements of IRB. To surmise faculty has lost motivation and enthusiasm for sustainability of classroom teaching methods and are testing unchartered water for teaching current sets of incoming professional students.
A novel method for inferring B cell clonal lineages from single-cell RNA sequencing data
Authors: Weber Leah , Reiman Derek , Roddur Mrinmoy , Qi Yuanyuan , El-Kebir Mohammed , Khan Aly
Published: November 2024

Understanding the adaptive and evolutionary mechanisms of B cells in response to specific stimuli remains a significant challenge in the field of immunology. We introduce a new method, TRIBAL, which aims to infer the evolutionary history of clonally related B cells from single-cell RNA equencing data (scRNA-seq). The key insight of TRIBAL is that inclusion of isotype data into the B cell lineage inference problem is valuable for reducing phylogenetic uncertainty that arises when only considering the receptor sequences. Consequently, TRIBAL inferred B cell lineage trees jointly capture the somatic mutations introduced to the B cell receptor during affinity maturation and isotype transitions during class switch recombination. In addition, TRIBAL infers isotype transition probabilities that are valuable for gaining insight into the dynamics of class switching.

We validated our method on simulated data, demonstrating more accurate reconstruction than existing methods. Using experimental scRNA-seq datasets, we show that TRIBAL recapitulates expected biological trends in a model affinity maturation system. Furthermore, the B cell lineage trees inferred by TRIBAL were equally plausible for the BCR sequences as those inferred by competing methods but yielded lower entropic partitions for the isotypes of the sequenced B cell. TRIBAL holds the potential to advance our understanding of vaccine responses, disease progression, and the identification of therapeutic antibodies.
The Igh distal enhancers modulate locus topology and repertoire diversity in pro-B cells
Authors: Drake Ellen , Priyadarshi Saurabh , Naiyer Sarah , Kenter Amy
Published: November 2024

The Igh locus undergoes large-scale locus contraction prior to V(D)J recombination in pro B cells. Our Hi-C analyses have identified chromatin loops anchored by a series of distal VH enhancers, EVH1-4 and Pax5-activated intergenic repeat 4 (PAIR4) implying that these interactions contribute to Igh locus contraction. Indeed, EVH1 modulates Igh locus spatial organization and VH gene usage in VDJ recombination linking Igh locus conformation with function (Nat Comm. 14:1225). Loop extrusion is a process that forms chromatin loops by extruding chromatin through cohesin’s ring structure and is a critical regulator of distal V(D)J recombination. However, the relationship between chromatin looping and loop extrusion remains incompletely understood. We posit that EVHs and PAIR4 provide structure independent of loop extrusion which then interacts with loop extrusion to shape higher order locus architecture. Wings apart-like protein (Wapl) is a cohesin release factor. Acute WAPL-depletion increases loop extrusion, enabling us to directly test the relationship between Igh regulatory elements and loop extrusion in Abelson transformed (Abl-t) pro-B cell lines. We show that EVH4 and PAIR4 deletion reduce distal Igh locus interactions despite increased loop extrusion. Our studies support a model in which cis regulatory elements modulate loop extrusion to shape Igh locus architecture and consequently direct VH gene usage.
Uterine Tissue-Resident Natural Killer Cells Derived from Conventional Natural Killer Cells are Regulated by Progesterone
Authors: Sojka Dorothy , Tatematsu Bruna
Published: November 2024

Innate lymphoid cells (ILCs) respond to pathogen-induced tissue distress and maintain tissue homeostasis. The uterus contains three subsets of ILCs: ILC1, conventional natural killer (cNK) cells, and tissue-resident NK (trNK) cells. ILC1s and cNK cells seed the uterus before puberty. In contrast, trNK cells emerge at the onset of puberty and persist with varied frequency throughout the estrous cycle. Here, we considered the origin of uterine trNK cells and the potential influence of hormones on their differentiation in the uterus. We intravascularly labeled immune cells to distinguish cells in the vasculature by flow cytometry of ovariectomized (OVX) mice supplemented with hormones, intact and parabiosed mice. We found that mice OVX at three weeks of age and analyzed as adults did not have trNK cells in the uterine tissue unless progesterone (P4) was administered. In parabiosed mice, we identified cNK-derived trNK cells in estrous cycling but not in the noncycling parabionts. Delivery of P4 to OVX or intact mice induced the expansion of cNK-derived trNK cells which was abrogated if RU486 was administered. Uterine ILCs from Nfil3-/- mice, that lack cNK cells, did not expand in response to P4. Finally, sorted cNK cells transferred into OVX mice supplemented with P4 differentiated into trNK cells in the uterus, but not the spleen. Taken together, we identified a novel differentiation pathway for uterine trNK cells tightly regulated by P4 in the uterine tissue.
Stabilization of β-Catenin directs HEB to limit Thymic Selection
Authors: Tousinas Georgios , Emmanuel Akinola Olumide , Tracy Melissa , Arnovitz Stephen , Friedman David , Khazaie Khashayarsha , Gounari Fotini
Published: November 2024

Activation of β-catenin in CD4+CD8+ (DP) thymocytes stalls progression through thymic selection and predisposes to transformation. Leukemogenesis but not the developmental block depends on Tcf-1, β-catenin’s DNA binding partner. Here we show that β-catenin deregulation directs the DNA-binding protein HEB to stall thymocyte development at the thymic selection stage. Conditional loss of HEB along with β-catenin activation restores the frequency of TCRβ+/CCR7+ DP thymocytes, with reduced CXCR4 expression indicating their potential to migrate to the thymic medulla. It also restores the emergence of TCRβ+/CD69+ DPs and enhances the overall CD69+DP expression profile that were diminished by activating β-catenin, indicating a functional response to TCR stimulation. β-Catenin stabilization redirects the binding of HEB to ~11,000 novel DNA sites throughout the genome. A major fraction of these sites occupy promoter regions of genes associated with cell cycle regulation and mitotic processes. HEB binding to those regions corelates with downregulation of the associated genes, and HEB inactivation restores their expression to physiologic levels. These findings highlight the complex molecular interplay between HEB and β-catenin in impairing thymic development.
The role of TCR in regulating T cell function in acute and chronic infections and cancer
Authors: Krause Jenny , Gavil Noah , Rollins Meagan , Wijeyesinghe Sathi , Matson Courtney , Künzli Marco , O'Flanagan Stephen , Smith Olivia , Quarnstrom Clare , Weyu Eyob , Yu Yun , Webber Beau , Vezys Vaiva , Masopust David
Published: November 2024

In contrast to naive T cells, memory T cells do not require TCR stimulation to survive and are maintained by cytokines to undergo homeostatic proliferation. However, permanent TCR stimulation of T cells by persistent antigen in chronic infections and cancer causes a dysfunctional state, termed T cell exhaustion (TEX). Yet, how TCR signaling influences the location, maintenance, and function of T cell subsets remains incompletely understood.

Using an inducible TCR knockout model (UBC-CreERT2 x Trac flox/flox), longevity of tissue resident memory T cells (TRM) and persistent CD69 expression after TCR deletion was shown. However, this model was limited due to the polyclonal memory T cell population used.

To study the role of TCR in the regulation of TRM and TEX in infections and cancer, we designed a rAAV6 vector construct that encodes the floxed P14 TCRa and TCRb to the endogenous TRAC locus using CRISPR/Cas9. We validated the construct in vitro by transfecting UBC-CreERT2 cells, adoptively transferred into CD45.1 mice and infected with LCMV Armstrong before TCR deletion after tamoxifen treatment. We next generated UBC-CreERT2 x P14a flox mice using the same approach, and confirmed the expression by PCR and H-2Db/gp33 MHC I tetramer stain by flow cytometry.

We will use this model to understand how TCR signaling influences T cell differentiation, residence and exhaustion by inducing TCR ablation at the effector, contraction, memory and exhaustion state.
Exportin 1 (XPO1) blockade regulates immunosuppressive function of tumor myeloid derived suppressor cells
Authors: Daneshmandi Saeed , Yan Qi , Choi Jee Eun , Katsuta Eriko , MacDonald Cameron , Goruganthu Mounika , Roberts Nathan , Repasky Elizabeth , Singh Prashant , Attwood Kristopher , Wang Jianmin , Landesman Yosef , McCarthy Philip , Mohammadpour Hemn
Published: November 2024

Myeloid-derived suppressor cells (MDSCs) are key regulators of immune responses in the tumor microenvironment (TME). Here we report the pivotal role played by Exportin 1 (XPO1) in shaping the phenotype and suppressive function of tumor associated MDSCs, thereby influencing tumor progression. Using the EL-4 murine tumor model, we demonstrated that tumor MDSCs exhibit elevated levels of XPO1, that drive their immunosuppressive activity. We showed that the XPO1 inhibition can effectively remodel MDSCs phenotype and functions, resulting in enhanced anti-tumor responses and the suppression of tumor growth. We showed that depletion of MDSC by anti-Ly6C antibody, restore tumor growth post XPO1 blockade. Unbiased proteomic analysis revealed that nuclear export of ERK1/2 by XPO1 is pivotal for IL-6 mediated MAPK signaling, which underpins MDSC immunosuppressive functions. Importantly, our data confirm that blockade of XPO1 function by Selinexor (an XPO1 inhibitor), sequesters ERK1/2 in the MDSC nucleus. This results in the suppression of IL-6 mediated MAPK signaling, a shift in transcriptional profiles toward the induction of neutrophil-like immunostimulatory cells. Our study demonstrates that XPO1 inhibition transforms tumor MDSCs into immune-stimulating neutrophil-like cells, reducing tumor growth and prolonging survival in pre-clinical tumor models. These results reveal novel targets for MDSC reprogramming to enhance cancer treatment outcomes in clinical practice.
Differential proteomic signatures of host responses following COVID-19 vaccination in individuals with or without prior SARS-CoV-2 infection
Authors: Echeverri Tribin Felipe , Williams Erin , Carreno Juan , Krammer Florian , Hoffer Michael , Pallikkuth Suresh , Pahwa Savita
Published: November 2024

The goal of this study is to characterize how proteomic signatures differ following COVID-19 vaccination in individuals naïve to SARS-CoV-2 infection (CoV-N) and those with a prior history of infection (CoV-P) to better understand the influence of pre-infection on host responses to vaccination. A subset of individuals (n = 63) enrolled in our IRB-approved, longitudinal cohort (COVID-19 Immunity Study [CITY]) were included in this study. Serum samples collected at baseline and ≤ 14 days following two doses of primary vaccination (PV) were analyzed with the proteomic aptamer-based SomaScan assay across 1500 targets. Significant (α ≤ 0.05) differentially expressed proteins (DEPs) were those with ≥ 2.5 log2-adjusted fold changes following PV among both CoV-P and CoV-N. Proteins identified in both groups were utilized in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to elucidate biological regulation following PV. Twenty DEPs were identified in the CoV-P group and 91 DEPs in the CoV-N group. CoV-P displayed downregulation of fibrinogen, the active compound in fibrin clot synthesis. CoV-N displayed increased activation of C3, the essential activator of the alternative complement pathway, and downregulation of alternate pathway inhibitors CR1 and complement decay acceleration factor. The significant activation of complement pathways in CoV-N indicates an enhanced innate immunoprotected effect following vaccination not observed in CoV-P individuals.
Keap1-Nrf2 regulate nutrient metabolism to direct CD4+ T-cell activation and inflammatory functions.
Authors: Pyaram Kalyani , Tripathi Aprajita , Dasgupta Debolina , Pant Anil , Bugbee Ashlyn
Published: November 2024

CD4+T-cells are critical orchestrators of antigen-specific adaptive immunity. Here we elucidate the role of nuclear factor erythroid 2–related factor2 (Nrf2), negatively regulated by Kelch-like ECH-associated protein (Keap1), beyond the traditional oxidative stress regulation, in modulating the effector responses of CD4+ T-cells by influencing their nutrient metabolism. We noted that in CD4+ T-cells, Nrf2 protein levels increase during early activation but decrease during their expansion. With the objective of identifying the effect of T-cell intrinsic Nrf2, we used T-cell specific knockout (KO) mice of Keap1 or Nrf2. In vitro CD4+ T-cell activation and Th subset differentiation assays were performed, and the findings were validated in vivo. Constitutively high Nrf2 in activated CD4+ T-cells lead to increased proliferation as well as apoptosis, increased expression of early activation markers and augmented TCR signaling in addition to dampened Th1 differentiation yet elevated Foxp3+Treg generation. Further, Nrf2-high Keap1-KO CD4+ T-cells caused a milder disease in mouse models of inflammatory bowel disease than WT or Nrf2-KO cells. Mechanistically, Nrf2 drives glutaminolysis to support increased proliferative capacity but inhibits glycolysis in CD4+ T-cells. Overall, our data uncover a novel role of Nrf2 as a metabolic modulator of activation driven expansion and inflammatory function of CD4+ T-cells, thus providing a framework for improving Nrf2-targeting drugs.
Dynamic recruitment of inhibitory complexes by CD25 controls B-cell development and selection
Authors: Sun Ruifeng , Lee Jaewoong , Robinson Mark , Kume Kohei , Ma Ning , Cosgun Kadriye Nehir , Chan Lai , Antoshkina Irina , Khanduja Dhruv , Leveille Etienne , Katz Samuel , Vaidehi Nagarajan , Müschen Markus
Published: November 2024

Initiation of BCR signaling, and subsequent antigen-encounter in germinal centers are both marked by sharp increases of CD25 surface-expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B cells was unclear.

Our genetic studies revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCd, ITIM-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling. Conditional CD25-deletion resulted in clonal expansion and autoimmunity.

Mechanistic studies revealed that BCR engagement induced PKCd-mediated phosphorylation of CD25, which anchored CD25 on cell surface in proximity to the BCR complex. Moreover, CD25 recruited inhibitory receptors CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g., SHP1). The positively charged tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations, Co-IP and TIRF microscopy results revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain interacts with the CD79B-ITAM to terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25.
Determining the natural time course of immune response
Authors: Zeng Wei-ping , Zhou Baohua
Published: November 2024

Given the dynamic nature of immune response, it is important to study and medically interfere the molecular events in lymphocytes in accordance with the kinetics of the immune response. However, unlike in laboratory settings, in clinical settings the time of antigen exposure is usually uncertain. We found that the natural time course of immune response can be tracked by the dynamic changes of the intracellular pH of the lymphocytes. Therefore, the stage of immune response at a given time can be determined without the knowledge of the time of initial antigen exposure.
The Transcription Factor ELF4 Restrains the Inflammatory Potential of CD4+ T Cells
Authors: Bender Mackenzie , Olyha Sam , Bucklin Molly , Siddapureddy Sindhura , Attanasio John , Pan Jie , Muise Aleixo , Lucas Carrie
Published: November 2024

ELF4 is a member of the ETS family of transcription factors, known to be involved in the regulation and development of immune cells. Yet, the primary function of ELF4 was unknown prior to our discovery of patients with loss-of-function mutations in ELF4 that cause the monogenic autoinflammatory disease Deficiency in ELF4, X-linked (DEX), driven by aberrant inflammation. While it has been demonstrated by our lab that global loss of ELF4 results in hyper-cytokine release in macrophages and restrains Th17 differentiation in mice, the breadth and mechanisms of CD4+ T cell regulation by ELF4 remain enigmatic. Here, we show that loss of T cell-intrinsic ELF4 leads to enhanced interferon gamma and IL-17A production upon activation of naïve CD4+ T cells and demonstrate modulation of CD4+ T cell cytokine production by ELF4 is independent of its presence during T cell development as well as extrinsic signaling in vivo. Further, absence of T cell-intrinsic ELF4 leads to decreased expression of ATF4, a master regulator of the integrated stress response demonstrated to be crucial in restraining cytokine release upon initial activation of naïve T cells. Notably, we found ELF4 binds to the promoter region of ATF4, and expression of ATF4-induced genes is significantly reduced in CD4+ T cells in the absence of ELF4 post-activation. Thus, we elucidate an essential mechanism for restraining CD4+ T cell inflammatory potential dependent on regulation of the integrated stress response by ELF4.
Vasculopathy Leading to Stroke in Adult Onset Still’s Disorder
Authors: Qiu Esther , Kaddouh Firas
Published: November 2024

Background

Rheumatological disorders may predispose patients to hypercoagulability and/or vasculopathy increasing stroke risk. Adult-Onset Still’s Disease (AOSD) is not usually associated with either, with only two reported cases in the literature to have had strokes. We present a case of AOSD with acute stroke manifesting as lateral medullary syndrome. We hypothesize that vasculopathy and thrombocytosis may have contributed to stroke occurrence. We further explore the possible underlying pathomechanism in this patient population.

Case Presentation

A 35-year-old woman with AOSD on methotrexate, tocilizumab and daily prednisone presented with vertigo and numbness involving her right face and left arm. She was found to have an ischemic stroke in the lateral medulla with vertebral artery thrombotic changes. No recent trauma. Investigation of other underlying stroke etiologies was unyielding with negative lupus anticoagulants, ANCAs and normal ESR and CRP.

Discussion & Conclusion

Our case marks the third report of stroke occurrence in AOSD in the literature. While our patient had no elevated inflammatory markers, she still developed stroke due to ongoing endothelial inflammation and thrombotic changes in the vertebral artery. The presence of vascular involvement leading to stroke may provide initial insight into the nature of this event. Further research should thus be done to investigate the exact mechanism of stroke and vasculopathy in AOSD patients.
Metabolic pathways in T cells are modulated by CCR7 and IL-7R to facilitate T cell motility.
Authors: Morrissey Kimberly , Gridley Rebekah , Byrum Janie , Cannon Judy
Published: November 2024

T cells play an essential role in eliminating infections by migrating through tissues and interacting with antigen-presenting dendritic cells within lymph nodes, leading to activation. A continuous supply of energy is required for T cell movement to enable scanning of dendritic cells. Many studies have established that signaling through chemokine receptor CCR7 facilitates rapid movement, however, how CCR7 signaling induces T cell metabolism remains poorly understood. IL-7 signaling regulates glycolysis to promote T cell survival, but how signaling through the IL-7R affects T cell movement is unexplored. This study investigates the roles of CCR7 and IL-7R in metabolism to drive naïve T cell movement. Utilizing Seahorse analysis, we observe that CCL21 signaling to CCR7 has no effect on glycolysis, yet decreases cellular respiration. Conversely, IL-7 has no effect on respiration but promotes glycolysis. We then determined whether oxidative phosphorylation and glycolysis play a role in CCL21 and IL-7 driven T cell movement. We employ inhibitors of oxidative phosphorylation such as rotenone to examine the influence of different metabolic pathways on T cell mobility. Our findings provide new insights into the intersection of metabolism and motility, suggesting potential differential mechanisms of ATP production that drive interactions.
Flow cytometry-based FRET to monitor β2 integrins activation
Authors: Wang Yan
Published: November 2024

β2 integrins is very important for leukocyte arrest, rolling and transmigration. Neutrophils mainly express two β2 integrins, αLβ2 and αMβ2. Neutrophil arrest is triggered by rapid conformational change of β2 integrins from bent and low affinity to open and high affinity. Recent studies have reported a new conformation of β2 integrins bent high affinity (PMID: 27578049, PMID: 30605669). This conformation is very difficult to monitor. By dynamic molecular simulation, we found that bent high affinity conformation of integrin is close enough to membrane, less than 10nm. Förster resonance energy transfer (FRET) is a powerful technology to interrogate close association between two fluorescent molecules. Importantly, FRET requires a distance of 10nm or less. Using real-time flow cytometry-based FRET, we can not only detect bent high affinity conformation but also the dynamic conformational change of β2 integrins along time. Interestingly, the FRET signal was detected between bent high affinity β2 integrins and cell membrane using conformational antibody mAb24 and membrane dye octadecyl rhodamine B (ORB). We developed an easy and efficient way to monitor the bent high affinity conformation of β2 integrins.