bluuebunny/crossref_metadata_2025_split huggingface dataset:
VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation (IRM10P.744)
Authors: Wang Li , LeMercier Isabelle , Suriawinata Arief , Chen Wenna , Li Jiannan , Noelle Randolph 
Published: January 2023

Abstract
V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed.
Regulation of E3 ubiquitin ligase Cbl-b expression by protein tyrosine phosphatase SHP-1 (IRM10P.753)
Authors: Xiao Yun , Qiao Guilin , Zhang Jian 
Published: January 2023

Abstract
Casitas B cell lymphoma protein-b (Cbl-b) is a RING finger family E3 ubiquitin ligase. It has been well documented that Cbl-b plays a crucial role in T cell activation and tolerance induction. Our recent study also indicates that Cbl-b regulates proallergic T cell development and allergic airway inflammation. Therefore, the regulation of Cbl-b expression in T cells is important for its biological functions. Our previous studies demonstrate that CD28 costimulation potentiates Cbl-b sel-ubiquitination, CTLA-4-B7 interaction induces Cbl-b expression. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b tyrosine phosphorylation in primary mouse T cells requires CD28 costimulation. The failure to detect Cbl-b tyrosine phosphorylation upon CD3 stimulation is not due to defective PTK activation, but rather interestingly SHP-1 is recruited to Cbl-b upon CD3 stimulation, while CD28 costimulation abrogates the interaction between Cbl-b and SHP-1. Furthermore, we identified Cbl-b N-terminal tyrosine residues within TKB domain and linker region (Y106, Y133, and Y363) are required for its E3 ubiquitin ligase activity. The aberrant Th2 phenotype observed in T cell-specific SHP-1-/- (tSHP-1-/-) mice is reminiscent of our recent observation that Th2 responses are heightened in Cbl-b-/- mice. In support of this notion, Cbl-b expression is down-regulated in naïve T cells of tSHP-1-/- mice.
MALT1 has both protease activity-dependent and -independent roles in innate and adaptive immunity (IRM10P.739)
Authors: Foley Kevin , Yu Jong , Hoffman Sandy , Finger Joshua , Kasparcova Viera , Rickard David , Dare Lauren , Dykon Angela , Ramanjulu Joshi , Emery John , Gough Peter , Bertin John 
Published: January 2023

Abstract
Hetero-trimeric CARMA/BCL10/MALT1 CBM complexes play important roles in B and T cell antigen receptor signaling and other pathways. Previous studies have suggested that MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a cysteine protease to cleave and inactivate downstream signaling molecules such as A20 and CYLD. However, little is known about the relative importance of these two distinct activities in orchestrating immune responses. Utilizing mice homozygous for either null or protease-dead C461A mutations in MALT1, we found that some but not all MALT1 functions were dependent upon its protease activity. Although protease-dead mice had normal follicular B cell and T cell populations in the spleen, defects were observed in the generation of splenic marginal zone and peritoneal B1 B cells. Ex vivo, B cell receptor-stimulated proliferation was normal, but CD4+ and CD8+ T cells displayed decreased activation-induced proliferation and IL-2 production. C-type lectin receptor stimulation of cytokine production by dendritic cells was also found to be partially dependent upon protease activity. In vivo, protease-dead mice displayed severely reduced basal immunoglobulin levels, and defective T-dependent and T-independent immune responses. Our findings suggest that pharmacologic inhibition of MALT1 protease activity may be a useful therapeutic approach for diseases linked to uncontrolled immune cell activation.
Molecular mechanism of transcription and epigenetic alteration at immunoglobulin variable gene heavy chain locus induced by GANP (IRM11P.761)
Authors: Singh Shailendra , Maeda Kazuhiko , Eid Mohammed , Almofty Sarah , Shimoda Mayuko , Kawabata Teruya , Sakaguchi Nobuo 
Published: January 2023

Abstract
Epigenetic modification at IgV-region gene is the key base to generate somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID). However, the details how AID-targeting to the rearranged IgV-region locus have remained unclear. As a key player, we demonstrate that the histone-acetyl transferase activity of the germinal center-associated protein GANP is necessary for efficient AID-accession. GANP is upregulated in germinal center B-cells upon T-cell dependent antigen responses. GANP forms a complex with AID and accompanies into the nucleus. GANP co-precipitates with RNA polymerase II (Pol-II) and its stalling factor Spt5 that also interacts with AID, indicating that the recruitment of GANP to IgV-region locus undergoes transcription-coupled manner. GANP augments the transcription elongation toward the downstream of the IgV-region gene with active RNA Pol-II. The histone acetyltransferase (HAT) domain of GANP is involved in the chromatin modification selectively at the rearranged IgV-region segment with the interference of nucleosome occupancy, controlling the accessibility of AID positioning at IgV-region gene. This suggests a specific role for the GANP associated complex in antigen specific high affinity B-cells.
CMV and inflammation contribute to decreased B cell vaccine response in the elderly (LYM6P.777)
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Landin Ana Marie , Frasca Daniela 
Published: January 2023

Abstract
We previously showed intrinsic age-related defects in the in vivo and in vitro B cell responses to the seasonal influenza vaccine. Serum antibody responses were associated with the in vitro B cell response to the vaccine, measured by AID (activation-induced cytidine deaminase). Both responses decrease with age and are significantly correlated. In order to develop predictive markers for a beneficial humoral immune response, we measured markers of systemic and B cell-intrinsic inflammation. We hypothesized that the increased pro-inflammatory status of the elderly (including CMV) would directly impact B cell function, thus impairing the capacity of the individual to respond to vaccination. Our results show that aged B cells themselves make TNF-α before vaccine challenge, which positively correlates with serum markers of inflammation, but negatively correlates with B cell function, measured by AID expression after B cell stimulation. From the last 2 years (2011/2012 and 2012/2013) we show that CMV seropositivity is associated with lower in vivo and in vitro vaccine responses in both young and elderly. Recent studies will also be presented on the CD4 and CD8 TEMRA shown to be associated with lower antibody response. Our results reveal new molecular mechanisms which contribute to reduced antibody responses in aging and suggest that these will have an impact for crucial development of effective vaccines to protect the elderly from infectious and other debilitating diseases.
Distinct roles for TLR7 and TLR9 in regulation of B cell signal strength and differentiation (LYM6P.766)
Authors: Nundel Kerstin , Mande Purvi , Shaffer Art , Sindhava Vishal , Cancro Michael , Marshak-Rothstein Ann 
Published: January 2023

Abstract
BCR signal strength plays a key role in the fate decision of B cells. Strong BCR signals promote plasma cell differentiation and weaker BCR signals drive germinal center B cell fate. Signals through toll-like receptors (TLRs) also influence B cell differentiation, but it is unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and AM14 B cell activation depends on TLR7 and/or TLR9. ICs formed by the anti-nucleosome antibody PL2-3 can activate AM14 B cells through either TLR7 or TLR9, allowing us to dissect signaling pathways unique to each TLR. We find that activation of AM14 TLR7-deficient B cells leads to sustained activation of the NF-κB pathway and p38 phosphorylation, subsequently leading to cell death. In contrast, TLR9-deficient AM14 B cells activated with ICs are unable to sustain the initial NF-κB and p38 signaling response. Intriguingly, these TLR9-deficient AM14 B cells differentiate into short-lived plasmablasts as characterized by increased levels of plasma cell associated proteins such as IRF-4, Blimp-1 and CD138. Plasma cell development is not observed in IC activated TLR7-deficient AM14 B cells. Our results suggest divergent roles for TLR7 and TLR9 in B cell activation and plasma cell differentiation.
ENPP1-positive B cells and activation in human peripheral blood (LYM6P.772)
Authors: Yoon Jeong Heon , Wang Hongsheng , Morse Herbert 
Published: January 2023

Abstract
ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), also known as PC1 (plasma cell alloantigen 1), is a homodimeric type II transmembrane glycoprotein that mediates nucleotide recycling by breaking down ATP to AMP. Previous studies of mouse B lineage cells showed that levels of PC1 expression could distinguish two distinct subsets of peritoneal B -1a cells. In addition, PC1 expression increases progressively on germinal center B cells and splenic plasma cells with highest levels on bone marrow plasma cells. To determine if PC1 might also help define human B1-like cells, we have used a mouse mAb to human PC1 to characterize its expression on human cells. The frequency of PC1-positive cells from cord blood (mean 6%, n=10) was higher than that from adult peripheral blood (1.34%, n=15). These PC1-positive cells are mostly derived from CD27- naïve B cells expressing CD38, IgM and IgD, and were detected in plasmablast/plasma cells in peripheral blood. In contrast to B-1a cells in mice, human PC1-positive cells did not secrete natural Abs or produce IL10. Expression of PC1 was greatly increased on purified peripheral blood B cells stimulated with anti-CD40, IL4 and IL21. These activated PC1-positive cells expressed low levels of CD5, HLA-DR, surface IgM, and high levels of B cell activation markers and CD138. Our results indicated that PC1-positive B cell populations in humans are different from either CD20+CD27+CD43+ B1 cells in human, or CD5+ B-1a cells in mice.
Recognition of the cyst life cycle stage induces a robust immune response to the opportunistic fungal pathogen Pneumocystis murina (MPF3P.814)
Authors: Evans Heather , Garvy Beth 
Published: January 2023

Abstract
Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of a vegetative trophic stage and an ascus-like cystic stage. We demonstrate that infection of wild-type adult or neonatal mice with a mixture of trophic and cystic P. murina life forms elicits a more robust CD4+ and CD8+ T cell response than infection with the trophic form alone. β-1,3-glucan is a predominant structural component in the cyst wall, but is not produced by the trophic stage. Depletion of cysts with the β-1,3-glucan synthase inhibitor anidulafungin prevents expansion of the remaining trophic organisms in immunocompromised adult mice. Depletion of cysts in immunocompetent adult mice results in fewer infiltrating CD11b+ cells in the bronchoalveolar lavage fluid. In addition, bone marrow-derived dendritic cells pulsed with mixed P. murina express more IL-1β and induce greater in vitro CD4+ T cell interferon-γ production than cells pulsed with the trophic form alone. Dendritic cells have been previously reported to activate CD4+ T cells in response to Dectin-1 stimulation by purified Pneumocystis β-1,3-glucan. We propose that dendritic cells stimulated by β-1,3-glucan on cystic Pneumocystis direct a robust pro-inflammatory response, while stimulation with the trophic form induces a less exuberant immune response that is sufficient for clearance in immunocompetent individuals.
Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its localization in the cell wall envelope and affects phagolysosomal fusion (MPF3P.818)
Authors: Shukla Supriya , Richardson Edward , Athman Jaffre , Shi Libin , Wearsch Pamela , McDonald David , Banaei Niaz , Boom W. , Jackson Mary , Harding Clifford 
Published: January 2023

Abstract
Genetic deletion of the lipoprotein LprG reduces the virulence of Mycobacterium tuberculosis (Mtb), but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, e.g. lipoarabinomannan (LAM). Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (MtbΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining Mtb cell envelope structure. Furthermore, this mutant allowed accelerated Mtb phagosome-lysosome fusion, consistent with the role for LAM in blocking phagosome maturation. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages
Bacterial membrane vesicles mediate the secretion of immunomodulatory factors during Mycobacterium tuberculosis infection (MPF3P.804)
Authors: Athman Jaffre , Wang Ying , McDonald David , Harding Clifford , Wearsch Pamela 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages and releases microbial factors to promote immune evasion and survival. These factors include the lipoglycan LAM which blocks phagosome maturation and Mtb lipoproteins which modulate MHC-II antigen presentation and cytokine production via TLR2 signaling. It is unknown how lipoproteins and lipoglycans are shed from the Mtb cell wall, but they are dispersed throughout Mtb-infected macrophages and released within extracellular vesicles. It was initially presumed that Mtb lipoproteins and LAM trafficked into exosomes, small vesicles (50-150 nm) of endocytic origin, but studies have shown that bacteria can secrete membrane vesicles of similar size (50-250 nm). Using imaging and biochemical approaches, we show that Mtb produces bacterial vesicles within infected macrophages that are subsequently released into the extracellular environment. These Mtb vesicles are enriched for cell wall components (LAM, TLR2 agonists) and are distinct from exosomes labeled with host cell markers (CD9, CD63). Our data suggest that bacterial vesicles are the mechanism employed by Mtb to transfer cell wall components and impair effector functions within the infected macrophage. Furthermore, we propose that released Mtb vesicles, not exosomes, circulate bacterial components beyond infected cells to further modulate immune responses. These mechanisms may promote the survival of Mtb and pathogenesis of infection.
Microbiota enhances intestinal immunity through JNK/ROS pathways (MUC4P.838)
Authors: Chen Lee-Wei , Wu Ying-Ying , Chung Pei-Hsuan , Hsu Ching-Mei 
Published: January 2023

Abstract
Microbiota maintains normal mucosal immunity through complex mechanisms. To examine the role of JNK signaling pathway in microbiota-related gut immunity, we assessed the microflora in ileal lumen and mucosa, the antibacterial protein expression, and the RORγt cells of lamina propria in WT and JNK-/- mice. The bacterial counts in ileal lumen and mucosa show a significant decrease of Lacobacilli in JNK-/- mice. Moreover, JNK-/- mice demonstrated a significant decrease of RORγt cells in lamina propria and an increased translocation of Klebsiella pneumoniae to the portal vein as well as blood. The generation of reactive oxygen species (ROS) in intestinal mucosa was also significantly decreased in JNK-/- mice as detected by using 2',7'-dichlorodihydrofluorescein diacetate. Oral supplementation of fructooligosaccharides (FOS) or L-NAME but not D-NAME increased the production of ROS, IL-1β, IL-23 as well as RORγt cells in intestine, and decreased the translocation of K. pneumoniae in JNK-/- mice compared to WT mice. N-Acetylcysteine, an antioxidant, treatment inhibited ROS production and reversed the effect of FOS on intestinal immunity in JNK-/- mice. Collectively, JNK pathway plays an important role in microbiota-induced intestinal immunity through the induction of ROS generation, and FOS or iNOS inhibitor increase the numbers of RORγt cells and the expression of IL-1β as well as IL-23 in JNK-/- mice through enhanced production of ROS.
Mucosal antimicrobial protection toward enteroaggregative Escherichia coli infection is modulated by dietary tryptophan through an IL17-dependent mechanism (MUC4P.846)
Authors: Philipson Casandra , Bassaganya-Riera Josep , Viladomiu Monica , Carbo Adria , Eden Kristin , Hontecillas Raquel 
Published: January 2023

Abstract
Enteroaggregative E. coli (EAEC) is a predominant extracellular pathogen that causes inflammatory diarrhea in individuals worldwide, especially in persons suffering from malnutrition. We previously reported that protein-energy malnutrition abrogates protective Th17-dependent antimicrobial immunity in a murine model of EAEC infection. However, mechanisms whereby malnutrition enhances host-susceptibility to EAEC colonization remain unreported. This study presents evidence that tryptophan is crucial for antibacterial protection against EAEC infection. Mice fed tryptophan-free diet had reduced antimicrobial peptide production coinciding with significantly high pathogen levels. Likewise, bacterial burden occurred in a dose dependent manner when mice were supplemented with glycly-L-tryptophan dipeptide. Low serum tryptophan directly correlated with hampered gene expression levels of cytokines regulating effector IL-17A responses (e.g. IL-23 and IL-6). Mechanistically, orally administered tryptophan enhanced IL-17A and IL-17C production at the colonic and ileal mucosa facilitating antimicrobial defense and protection against EAEC colonization. IL17a-/- and IL17ra-/- mice receiving nourished diets or tryptophan supplements were unable to recover from disease due to impaired intestinal epithelial barrier function. Thus, our data suggests that tryptophan modulates IL17-dependent antimicrobial responses and is required for preventing EAEC colonization and pathogenic disease.
Microbial metabolites modulate GI mucosal damage from graft versus host disease (GHVD). (MUC4P.850)
Authors: Mathewson Nathan , Mathew Anna , Oravecz-Wilson Katherine , Wu Julia , Toubai Tomomi , Cummings Emily , Pennathur Subramaniam , Reddy Pavan 
Published: January 2023

Abstract
The microbiome of the GI tract is known to affect GI mucosal responses and regulate disease processes. GI tract damage is the major cause for graft versus host disease (GVHD) mortality. Studies have shown the microbiome affects the course of allogeneic bone marrow transplant and modulates GVHD. However, little is known about the microbial metabolites crucial for achieving the effects of the microbiome on immune responses, specifically GVHD. We examined the metabolome of naïve mice and murine recipients of syngeneic (B6→B6) and allogeneic (B6→BALB/c) BMT, in an unbiased way. We made a surprising observation that short chain fatty acids (SCFA), including butyrate, were altered in the intestinal epithelium but not in the luminal contents of allo-BMT recipients. Butyrate, a known metabolite of the microbiome was previously shown to have HDAC inhibitor (HDACi) and salutary effects on the intestinal epithelium. Additionally, recent data showed systemic chemical inhibition of HDAC protects GI epithelium from GVHD severity. The decrease of butyrate in the epithelial cells of the small intestine (P&lt;0.03) suggested poor uptake of butyrate following allo-BMT. Consistent with this notion, we found that treatment of epithelial cells with butyrate confers protection from irradiation and decreases allo-induced cell death. Our results suggest that the microbiome regulates the damage from allogeneic GVHD due to the reduction in the tissue level of the SCFA-metabolite and HDACi, butyrate.
Understanding the effect of early-life pulsed antibiotic treatment on mucosal T-lymphocyte populations (MUC4P.827)
Authors: Ruiz Victoria , Gottwick Cornelia , Livanos Alexandra , Li Huilin , Blaser Martin 
Published: January 2023

Abstract
Broad-spectrum antibiotics are frequently prescribed for children. The gut microbiota have functional roles in the development and differentiation of the host immune system. Early-life antibiotic use may have dynamic effects on the host microbiota, promoting long-term immunologic dysregulation and subsequent allergic and autoimmune pathology. Using a model of early-life antibiotic use, we hypothesize that early-life pulsed antibiotic treatment (PAT) -induced perturbation of the intestinal microbiota leads to alterations in tissue-specific and systemic T-cell populations. To test this hypothesis, we characterized splenic and ileal T-cell populations in control and PAT-treated C57BL/6 mice. Flow cytometric analysis demonstrated that early-life PAT significantly (p&lt;0.05) decreased the frequency of splenic CD4+ and CD8+ T-cells and ileal CD4+ IL-17A+ Th17 populations. In contrast, ileal CD4+ Tbet+ (Th1) T cells and CD4+ Foxp3+ regulatory T-cell populations were significantly increased in PAT-treated mice. Alterations in immune cell populations were associated with microbial compositional changes, including significantly higher relative abundance of Enterobacteriaceae and Akkermansia muciniphila in PAT-treated mice. These results provide evidence that early-life perturbation of the intestinal microbiota by PAT modulates systemic and mucosal T-cell populations, phenotypes that may persist after microbial recovery, promoting life-long consequences to host immunity.