bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenic role for chemerin receptor CMKLR1 in experimental pulmonary fibrosis (P5138)
Authors: Monnier Justin , Lewen Suzanna , Carlson Jeffrey , Zabel Brian
Published: January 2023

Pulmonary fibrosis (PF) is a debilitating interstitial lung disease with few treatment options. The pathogenesis of idiopathic PF likely involves recurring microinjuries to the alveolar epithelium, leukocyte infiltration and inflammation, and an aberrant pro-fibrotic wound-healing response. CMKLR1 is a chemoattractant receptor for chemerin that is expressed on NK cells and other immune cells implicated in PF. Our goal was to determine the role of CMKLR1 in the pathogenesis of bleomycin-induced pulmonary inflammation and fibrosis. During the inflammatory phase of the model, neutrophilic infiltration of the airways was significantly increased in CMKLR1-deficient mice compared to wild-type (WT). During the fibrotic phase, CMKLR1-deficient mice developed more severe pulmonary fibrosis than WT as measured by lung collagen content and histopathology. The levels of key pro-inflammatory and pro-fibrotic cytokines (TNFα, IFNγ, IL-10, IL-17, and TGFβ) were significantly elevated in interstitial lung tissue from bleomycin-treated CMKRL1 KO mice. There was also a significant reduction in the recruitment of NK cells into the airways in CMKLR1 KO mice during the fibrotic phase. We hypothesize that chemerin and CMKLR1 play a protective role in lung fibrosis by dampening the initial inflammatory response and by recruiting anti-fibrotic leukocytes. Augmentation of the CMKLR1/chemerin system may therefore provide a novel approach to engage anti-fibrotic leukocytes for PF therapy.
Orai1 function is essential for T cell homing to lymph nodes (P5102)
Authors: Greenberg Milton , Yu Ying , Leverrier Sabrina , Parker Ian , Cahalan Michael
Published: January 2023

In T lymphocytes, Ca2+ release-activated Ca2+ (CRAC) channels composed of Orai1 subunits trigger antigen-induced gene expression through the NFAT pathway. Lymphocyte homing into lymph nodes may also require CRAC channel function. We evaluated the requirements for human lymphocyte homing, using expression of a dominant-negative Orai1-E106A mutant to suppress Ca2+ signaling. To investigate homing and motility of human lymphocytes we transferred human T cells into immunocompromised mouse hosts following stable reconstitution. Human lymphocyte homing was assessed by flow cytometry, and cells were tracked within lymph nodes by two-photon microscopy. Our results demonstrate that human T lymphocytes home into and migrate within the lymph nodes of immunocompromised NOD.SCID.B2 mice, similar to murine lymphocytes. Human T cells migrated in a random walk at velocities of 12.8 ± 0.4 µm/min. Expression of Orai1-E106A inhibited CRAC channel function in human T cells and prevented homing from high endothelial venules into murine lymph nodes. Ca2+ signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel-mediated Ca2+ influx for T cell homing to lymph nodes mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.
The role of pTα/pre-TCR in susceptibility to autoimmunity (P1464)
Authors: Mayans Sofia , Lambolez Florence , Roh Sujin , Larange Alexandre , Becart Stephane , Cheroutre Hilde
Published: January 2023

Autoimmune diseases result from a breakdown in self-tolerance, with a major contribution of pathogenic self-reactive T cells. Individuals with a particular autoimmune disease are often at risk of developing additional autoimmunity directed towards other target tissues. These observations suggest that there might be a mechanism leading to a general defect in self-tolerance, resulting in increased susceptibility to autoimmune disease. Tolerance towards self is initially established in the thymus during the process of thymic selection. Developing thymocytes are first immature CD4 and CD8 double negative thymocytes. Cells with a productive TCRβ gene rearrangement will express a pre-TCR/CD3 complex on the cell surface by combining a TCRβ chain with the pTα chain. Signaling through the pre-TCR, β-selection, plays a crucial role in the generation αβT cells, by enabling further maturation to the CD4 and CD8 double positive stage. The crucial role of pTα at β-selection together with the genetic localization of the ptcra gene and the fact that variations in pTα will affect all developing T cells, suggests that pTα could be a common factor leading to a general defect in self-tolerance. We have discovered differences in the isoform expression pattern and amino acid sequence of pTa between NOD and B6 mice. These findings are consistent with the hypothesis that alterations in the pre-TCR might affect selection, giving a modified TCR repertoire and increased susceptibility to autoimmunity.
Matricellular protein CYR61 expands thymic stroma (P1423)
Authors: Emre Yalin , Irla Magali , Dunand-Sauthier Isabelle , Ballet Romain , Meguenani Mehdi , Jemelin Stephane , Vesin Christian , Reith Walter , Imhof Beat
Published: January 2023

T-cell development is dependent on microenvironments generated by stromal cells such as thymic epithelial cells (TEC). TEC population, which is phenotypically and functionally heterogeneous, controls the migration, proliferation and selection of differentiating thymocytes. CYR61 (cysteine-rich protein 61) is a matricellular protein reported to promote proliferation and migration of cancer cells. Here we investigated the impact of CYR61 treatment on T-cell development. We found that matricellular protein CYR61 was strongly expressed in TEC and favored TEC/thymocyte interaction by binding to LFA1 and ICAM1. Treatment of thymic lobes with recombinant CYR61 expanded the thymic stromal compartment. Thymocyte-independent TEC proliferation occurred through CYR61 binding to integrin alpha6/beta1 and the activation of Akt signaling pathway. Engraftment of thymic lobes mildly overexpressing CYR61 into athymic nude mice drastically boosted thymic output through the expansion of TEC. Actually, CYR61-expanded stroma offered more space for the recruitment of circulating hematopoietic progenitors and for the development of T-cells. Our results reveal CYR61 as a potent target for the development of thymic stroma and T-cells.
Comparison of neonatal and adult T cells’ responses to TCR, CD43 and TLR5 signals (P1427)
Authors: Santana M. Angelica , Galindo-Albarrán Ariel , Rodríguez-Jorge Otoniel , Labastida-Conde Rosario , Ramírez-Pliego Oscar
Published: January 2023

Characterisation of neonatal T cell response is largely incomplete, despite infants under 6 months old are highly susceptible to infections. It has been reported that neonatal cells require strong signals for differentiation into the pro-inflammatory Th1 phenotype, which is needed to fight viral and intracellular bacteria infections. We decided to evaluate the response of human neonatal and adult naïve T cells to two accessory molecules that have been reported to induce a Th1 profile. CD43 is a transmembrane Type One mucin-like molecule that has been proposed as one of the first molecules to make contact with APCs, regulating cell adhesion and function. We found that toll like receptor 5 (TLR5), which recognizes the flagellin monomer, is highly expressed in T-lymphocytes. It was reported that human neonatal CD8 T cells respond to flagellin with a strong expression of IFNγ. We compared the response of ex-vivo human neonatal CD4 and CD8 T cells to TCR and either CD43 or TLR5 signals alone or in combination. We evaluated the expression of cytokines of the major differentiation profiles (Th1, Th2, Th17 and Treg) and found that neonatal cells produce lower levels of all cytokines, with the exception of TGFβ, and have a higher requirement for TCR signals as compared to the adult cells.
Bifidobacterium longum subsp. longum KACC 91563 from Korean neonates alleviate the food allergy by decrease of mast cells in the gut. (P3172)
Authors: Kim Jung-Hwan , Jeun Eun Ji , Ham Jun-Sang , Jang Min Seung , Hong Chun Pyo , Lee Eun-Jung , Yang Bo Gie , Jang Myoung Ho
Published: January 2023

Gut microbiota which establish the habitat in our gut, have formidable effects on human body via complex mechanisms, including metabolism, development, behavior, immune defense and diseases. Probioitcs are live microorganism that confers a benefit on the host when administrated adequate amount. Food allergy is a potentially serious disease caused by abnormal immune response to food or food additives and hospital visits for food allergy have been dramatically increased in the last several decades. Here we investigated the effects of new probiotics that isolated from the feces of healthy Korean infants on food allergy. Among them, we found that new subspecies of Bifidobacterium Longum suppress the diarrhea in allergen-induced food allergy mouse model. When we administrated B. longum subsp. longum KACC 91563, the accumulation of mast cells was significantly decreased in small intestinal lamina propria and mast cell proteases were down regulated in serum. However Th1, Th2, Th17 and regulatory T cells were not involved in the alleviation of allergen-induced food allergy in small intestinal lamina propria. These findings suggest that oral administration of newly isolated B. longum, ameliorates the severity of allergen-induced food allergy by decrease of mast cells.
Regenerating islet-derived III-gamma regulates pulmonary Th17 immunity by altering the gut microbiome (P3164)
Authors: McAleer Jeremy , Nguyen Nikki , Chen Kong , Ricks David , Binnie Matthew , Sheppard Dean , Ouyang Wenjun , Kolls Jay
Published: January 2023

Commensal microbes are critical for the development of intestinal lymphoid tissues, but little is known about how they shape systemic immunity. Lung infection with Aspergillus fumigatus induces a potent IL-17 response from T cells. Vancomycin drinking water diminished lung il17a expression during infection, demonstrating a role for gram-positive commensals in amplifying pulmonary immunity. Since RegIIIγ is an intestinal anti-microbial factor that targets gram-positive bacteria, we hypothesized that it would decrease lung Th17 cytokine expression, similar to the effects of oral vancomycin. To test this, RegIIIγ-/- mice and C57BL/6 (WT) mice were infected with A. fumigatus, and lung cytokines were analyzed two days later. RegIIIγ-/- mice had elevated expression of il17a, il17f and il22, and this was positively correlated with intestinal colonization by segmented filamentous bacteria. Further, reconstitution of il22-/- mice with recombinant RegIIIγ by oral gavage decreased steady-state IL-17+ γδ T cells in the lung. To test if RegIIIγ regulates lung Th17 priming by interacting with intestinal microbiota, mice were treated with vancomycin water and then immunized with OVA plus cholera toxin. Notably, antibiotic treatment reduced lung Th17 numbers in RegIIIγ-/- mice to the level observed in WT mice. These data demonstrate that modulating the expression of an intestinal anti-bacterial lectin can have profound effects on pulmonary immunity by altering the gut microbiome.
The variation of CD4+ memory T cells in the development of asthma in mice (P6233)
Authors: Li Yanchun , Hua Shucheng , He Xiaoyan , Li Yun , Sun Ye
Published: January 2023

Objective To understand the role of CD4+ memory T cells in pathogenesis of asthma in mice Method Female BALB/c mice were sensitized and challenged with OVA for continuous 7 days. After one month, another challenge was given for one day. We detected the ratio changes of CD4+ memory T cells in CD4+ T cells during the treatment course, the membrane surface markers of CD3+, CD4+, CD44High, CD62L- were examined by using a flow cytometry. We further compared the different challenge time durations effect on CD4+ memory T cell numbers. Results In control mice, the ratios of CD4+ memory T cells in lung and spleen were 11.32% and 20.48%, respectively; After challenge, the ratio of CD4+ memory T cells increased gradually and reached the peak value ( 53% in lung and 45% in spleen) at one week after the last challenge. After one month these ratios returned to the levels of before the challenge. However, these memory T cells raised up rapidly on the next day during the second challenge, especially in the lung and kept relatively stable levels (about 35% in lung and 30% in spleen) within one week. We observed that the different challenge time durations had similar effect on the ratio of CD4+ memory T cells in lung, but it differ in spleen the peak value of CD4+ memory T cells at 30min challenge was 10% lower than that with 1 hour challenge and dropped earlier. Conclusion The CD4+ memory T cells play important roles in asthma pathogenesis, the detailed mechanisms warrant further exploration.
Allergenicity of the major birch pollen allergen Bet v 1 might arise from molecular mimicry of homologous bacterial proteins (P6210)
Authors: Smole Ursula , Ackerbauer Daniela , Lengger Nina , Radauer Christian , Wallner Michael , Svoboda Martin , Wills-Karp Marsha , Mechtcheriakova Diana , Breiteneder Heimo
Published: January 2023

Certain families of allergens contain domains that bear structural and functional similarity to homologs from pathogens enabling them to sabotage host immunity by hijacking crucial innate immune pathways. We focused on members of the Bet v 1-like superfamily that share a remarkably conserved architecture, the so called Bet v 1-fold, with the aim to identify structural mimicry as one possible cause of allergenicity. Here we show that Bet v 1, the major birch pollen (BP) allergen, has functional homology with a bacterial homolog, namely TTHA0849 from Thermus thermophilus. TTHA0849 and Bet v 1 competed for the same uptake mechanism by monocyte-derived DCs (MoDCs) of BP allergic and normal donors as visualized by fluorescence microscopy. Using a pre-designed antibacterial response PCR array, we could reveal striking similarities in gene expression patterns induced by Bet v 1 in MoDCs of both donor groups to those induced by TTHA0849. Importantly, Bet v 1 and its bacterial homolog also triggered the transcription of the Th2 signature cytokines IL-4 and IL-13, as well as EGR-3, a marker of early cell activation, in MoDCs from BP allergic but not normal donors. These findings propose that conserved structural features are present on both Bet v 1 and TTHA0849 targeting the same pathways common to allergic and normal individuals. Altered signal processing and translation in predisposed allergic individuals might be a mechanism underlying the phenomenon of allergic sensitization.
Induction of immunological tolerance by pulmonary macrophages during chronic exposure to house dust mite antigen (P6212)
Authors: Bracken Sonali , Guernsey Linda , Szczepanek Steven , Natarajan Prabitha , Adami Alexander , Beck Jeremy , Ehsan Mohsin , Thrall Roger
Published: January 2023

Allergic asthma is a leading cause of morbidity in industrialized nations. While the emphasis of most studies pertaining to the attenuation of asthma after chronic antigen exposure has focused on the local generation of Tregs, little attention has been paid to the potentially crucial role of pulmonary macrophages (MΦ) in disease resolution. To determine whether these MΦ are involved in tolerance generation, C57BL/6 mice were challenged intranasally with house dust mite (HDM) extract both acutely (2 and 5 weeks) and chronically (11 weeks) to induce an asthma-like phenotype (n=3-6 per group). Compared to acutely-challenged mice, MΦ frequency increased over two-fold in the airways of chronic HDM-exposed mice (P<0.001). This finding correlated with increases in local Foxp3+ Tregs as well as resolution of pulmonary eosinophilia (P<0.01) and airway hyper-reactivity (P<0.01). Flow cytometric analysis of naïve IL-10 GFP mice confirmed that pulmonary MΦ produce high amounts of IL-10. Following chronic HDM exposure, these MΦ may expand in the lungs and influence tolerance development directly through IL-10 or indirectly through effects on Tregs. We also observed 3- to 7-fold increases in serum MCP-1, MIP-1α, and MIP-1β following chronic but not acute HDM exposure, which suggests that these MΦ are derived from monocytes recruited to the site of inflammation. Collectively, these data suggest a novel role for pulmonary MΦ in the generation of tolerance in an experimental model of asthma.
Complement regulatory protein properdin promotes complement activation on platelets and increases platelet-leukocyte aggregate formation (P1311)
Authors: Ferreira Viviana , Saggu Gurpanna , Nauli Surya , Cortes Claudio
Published: January 2023

An increased number of activated platelets and platelet-leukocyte aggregates are found in patients with chronic inflammatory diseases. Activated platelets can activate the complement system. We have recently determined that properdin, a positive regulator of the alternative pathway that is produced mainly by stimulated leukocytes, promotes complement activation on activated, but not resting, platelets. Unfractionated properdin (that has non-physiological aggregates) induces formation of platelet-leukocyte aggregates. However, the roles that physiological dimers, trimers, and tetramers of properdin play, as well as the molecular mechanisms by which the alternative pathway is involved in this phenomena remain unknown. Here, we have determined that (a) activated platelets, as well as neutrophils, bind physiological forms of properdin and neutrophil-derived properdin; (b) properdin mediates a novel mechanism for alternative pathway activation on activated platelets; and (c) physiological properdin leads to increased platelet-leukocyte aggregate formation in thrombin receptor-stimulated whole blood. The effects of shear stress in properdin-induced aggregate formation and differences between leukocyte subpopulations in platelet-leukocyte aggregate formation have also been assessed. Our data support a role for properdin in cellular microenvironments, contributing to complement activation and aggregate formation, with potential consequences in inflammation pathophysiology.
Pro-apoptotic chemotherapeutic drugs induce non-canonical processing and release of IL-1β via caspase-8 activation in dendritic cells (P1353)
Authors: Antonopoulos Christina , Kaiser William , Mocarski Edward , El Sanadi Caroline , Dubyak George
Published: January 2023

The emergence of non-canonical (caspase-1 independent) IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We compared the relative contributions of canonical versus non-canonical IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) stimulated with pro-apoptotic chemotherapeutic agents such as doxorubicin (Dox). Dox induced a release of mature (17 kDa) IL-1β from LPS-primed BMDC that was temporally correlated with caspase-8 activation and greatly suppressed in BMDC from Casp8-/-Rip3-/- mice. The magnitude of Dox-stimulated IL-1β release from Casp1-/-Casp11-/- BMDC or wild-type (WT) BMDC treated with YVAD caspase-1 inhibitor was similar to that in non-YVAD-treated WT BMDC. Neither ASC nor NLRP3 were required for Dox-stimulated IL-1β release. In contrast, Dox-induced IL-1β release was suppressed in LPS-primed TRIF-/- BMDC. This indicated interaction with a TLR4-TRIF pathway for assembly of RIP1/FADD/procaspase-8 ripoptosome complexes. Current studies focus on characterizing the ability of Dox to induce down-regulation of cIAPs and/or cFLIP which act to restrain the stability/proteolytic activity of caspase-8 containing ripoptosomes. These findings show that Dox promotes caspase-8-dependent release of IL-1β, implicating direct effects of such genotoxic chemotherapeutic drugs on a non-canonical inflammatory cascade that may modulate immune responses in tumor microenvironments.
IRF-5 and NF-κB p50 co-regulate IFN-β and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells (P1363)
Authors: McFarland Adelle , Steinhagen Folkert , Rodriguez Luis , Tewary Poonam , Jarret Abigail , Savan Ram , Klinman Dennis
Published: January 2023

Robust production of type I interferon (IFN) by plasmacytoid dendritic cells (pDCs) occurs in response to Toll-like receptor 9 (TLR9) activation by unmethylated CpG dinucleotides present in bacterial and viral DNA. Previous work using different CpG oligodeoxynucleotides (ODN) revealed that CpG-A ODN but not CpG-B ODN robustly induce IFN-α production in pDCs. Although CpG-B ODN do not efficiently induce IFN-α, they are capable of highly inducing IFN-β. However, it is currently unclear what factors mediate CpG-B ODN induction of IFN-β in human pDCs. Using nuclear translocation, siRNA and proximity ligation assays, we demonstrate a requirement for both IRF-5 and NF-κB p50 in the induction of IFN-β and IL-6 by CpG-B ODN in human pDCs. The finding that CpG-B ODN activation of IRF-5 leads to IFN-β production in human pDCs may have clinical importance as IRF-5 has recently been identified as a major risk locus in systemic lupus erythematosus (SLE), a disease largely driven by aberrant type I IFN production by pDCs. Overall, this study provides new insights into the transcriptional regulation of IFN-β and IL-6 induced by CpG-B ODN in human pDCs and identifies a clinically important link between IRF-5 and IFN-β.
Towards understanding the influence of lipid metabolism on CD8 T cell biology (P1136)
Authors: Kidani Yoko , Elsaesser Heidi , Hock M. , Vergnes Laurent , Williams Kevin , Argus Joseph , Marbois Beth , Komisopoulou Evangelia , Wilson Elizabeth , Osborne Timothy , Graeber Thomas , Reue Karen , Brooks David , Bensinger Steven
Published: January 2023

A hallmark of the adaptive immune system is the ability of CD8 T cells to undergo rapid clonal expansion in response to antigenic stimulation. To meet the biosynthetic requirements of rapid growth and proliferation, effector T cells must reprogram their metabolism to provide essential macromolecules. We have previously shown that mitogen signaling induces a gene expression program that drives de novo cholesterol and fatty acid biosynthesis, however the molecular events linking antigen receptor signaling with lipid anabolism remain poorly understood. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8 T cells are unable to grow and acquire the effector cell metabolism program, resulting in attenuated clonal expansion during viral infection. In contrast, SREBPs are dispensable for homeostatic proliferation, demonstrating a context-specific requirement for SREBP signaling during effector T cell blastogenesis. Mechanistic studies indicate that SREBP activity is required to maintain a level of cellular cholesterol sufficient to permit rapid ER and cellular growth. Taken together, these studies provide novel insights into the molecular signals underlying the metabolic reprogramming of CD8 T cell during the transition from quiescence to activation, and demonstrate the importance of SREBPs in regulating a cholesterol metabolic checkpoint during blastogenesis.
New insights into TRAF-mediated regulation of BAFF receptor signals (P1110)
Authors: Lin Wai , Hildebrand Joanne , Bishop Gail
Published: January 2023

BAFF receptor (BAFFR), a member of the TNFR superfamily, binds to the pro-survival cytokine BAFF to promote B cell development and survival, and deficiencies of either BAFFR or BAFF result in inhibition of B cell development. BAFF-R engagement recruits TRAFs 2, 3 and 6 and activates the non-canonical NF-κB2 pathway. Current models propose that TRAF3 mediates degradation of the NF-κB activating kinase NIK, concluding that BAFFR-mediated TRAF3 degradation is necessary and sufficient for NF-κB2 activation. However, our recent studies of a mutant BAFFR indicate that this model is inadequate to fully explain how BAFFR activates the NF-κB2 pathway. The A/WySnJ mouse harbors a spontaneous mutation of BAFFR in which 8 residues of the cytoplasmic tail are replaced by 22 amino acids from a viral genome. Engagement of this mutant BAFF-R fails to activate the NF-κB2 pathway, and A/WySnJ mice have a substantial B cell deficiency. However, the mutant BAFF-R shows no decrease in recruitment or receptor-mediated degradation of TRAF3, demonstrating that TRAF3 degradation is not sufficient to allow NF-κB2 activation. Our recent data instead suggest that recruitment of TRAFs 2 and 6 and the signaling protein Act1 may play important roles in BAFFR-mediated NF-κB2 activation.
H3K4 methylation profiling reveals distinct epigenetic signatures in CD4 T cell activation and memory formation. (P1146)
Authors: LaMere Sarah , Thompson Ryan , Komori H. , Mark Adam , Salomon Daniel
Published: January 2023

The roles of histone modifications in T cell activation and memory formation are poorly understood. Thus, we used RNAseq and ChIPseq in tandem to assess the impact of promoter H3K4 di- and trimethylation in naive vs memory human CD4 T cells at rest and 5 days after activation. Changes in H3K4me2 and me3 correlated strongly with changes in RNA expression when comparing naive and memory cells; e.g. increased H3K4 methylation with increased gene expression. These changes define unique signatures comprised of >200 genes at rest and >400 post-activation. Dramatically, activation of naïve CD4 resulted in differential H3K4 methylation of >4000 genes while only ~800 changed with activation of memory. In contrast, activation-induced changes in gene expression and methylation correlated nearly perfectly in memory but poorly for naïve CD4 T cells. These data suggest a higher level of H3K4 methylation-based commitment in the memory subset 5 days after activation. We are presently doing the same studies for days 1 and 14 after activation to understand how quickly activation-induced H3K4 methylation changes occur and how well H3K4 methylation signatures are reflected 14 days after activation of naive cells. In sum, naïve and memory CD4 T cells demonstrate specific H3K4 methylation signatures, T cell activation is clearly an epigenetically dynamic process, and profiling of H3K4 methylation provides important insight into the epigenetic mechanisms driving the commitment to memory.
Polyclonal anergic CD4 T cells give rise to pathogenic TH1 and suppressive regulatory T cells during lymphopenia. (P1024)
Authors: Kalekar Lokeshchandra , Jenkins Marc , Mueller Daniel
Published: January 2023

Anergy is defined as a state of hyporesponsiveness during which T cells fail to respond to their cognate antigen. Studying anergy using non-transgenic animals has been difficult due to lack of distinguishing markers for anergic cells. We identified CD73 and Folate Receptor 4 (FR4) to be highly expressed in anergic cells from transgenic animals following gene array analysis. Here, we show that CD44hi polyclonal CD4 T cells from non-transgenic animals expressing CD73 and FR4 are hyporesponsive following in vitro stimulation. Anergic T cells with specificity for a known self-antigen also express these markers. Adoptive transfer of these cells into lymphopenic animals leads to reversal of their anergic phenotype and differentiation to Tbet+ TH1 T cells and Foxp3+ regulatory T cells. Selective ablation of Treg cells, which arise following adoptive transfer, leads to accelerated weight-loss and lymphoproliferative disease in mice receiving these anergic cells. Our data suggest that anergy is not a stable state and anergic cells can reverse to diverse functional phenotypes. This finding has important implications for cancers, where anergy reversal and helper T cell differentiation in tumor infiltrating lymphocytes would be a favorable outcome, as well as for autoimmunity, where anergy reversal associated with Treg development could be beneficial.
In vivo treatment with MK801, an ionotropic NMDAR antagonist, reduces experimental autoimmune encephalomyelitis. (P1042)
Authors: Schatzmann Peron Jean , Brandão Wesley , Fickinger Andira , Rossato Cristiano , Costa Matheus , Oliveira Daniel , Saraiva Câmara Niels Olsen
Published: January 2023

Glutamate is the most abundant and perhaps the most important neurotransmitter. It acts through metabotropic and ionotropic receptors. Reports demonstrated the expression of glutamate receptors on immune cells, as macrophages and T cells. Its role however is a matter of debate. Thus, we used MK801, a NMDAR antagonist to evaluate its role during an immune response. To evaluate the pathways involved, DO11.10 T cell clones were activated in vitro with or without MK801 or N-methyl D-Aspartate. Total and phospho ERK 1-2 were evaluated by western blot. Our data demonstrated that NMDAR activation increases ERK phosphorylation, reversed by MK801. Further, we performed in vitro Treg differentiation and the presence of MK801 increased the percentage of CD4+Foxp3+ cells. For in vivo experiments, C57BL/6 mice were immunized with MOG35-55 and treated or not with MK801 on days 0,1,2 and 3. At the peak of disease animals were sacrificed and Th1, Th17 and Treg cells were analyzed by flow cytometry. MK801 treated animals had milder disease when compared to controls. Interestingly, animals showed an increased frequency of Th1, Th17 and CD4+p-STAT-3+ infiiltrating brain and spinal cord. Our results demonstrated that glutamate signaling boosts the commitment of Th1 and Th17 cells. However, less-encephalitogenic cells are being generated. These findings contribute to the understanding of NMDAR on immune cells and also for a new point of view concerning its role in neuroinflammatory diseases.
T cell responsiveness to retinoic acid is required for immune mediated liver injury (P1046)
Authors: Deng Jie , Celaj Stela , Noelle Randolph , Gorham James
Published: January 2023

Inflammatory liver disease (ILD) contributes to significant morbidity and mortality. T cell mediated injury is a central event in the pathogenesis of ILD of a variety of etiologies; however, the specific driving mechanisms are not fully elucidated. Retinoic acid (RA), the active metabolite of vitamin A, plays fundamental roles in shaping the immune response. Although liver is the major site of retinoid storage in the body, the specific role of RA during liver inflammation has not been evaluated. Using the Concanavalin A (Con A) mouse model of ILD, we tested the hypothesis that RA is required for T cell mediated liver injury. Con A induced a significant increase in RA concentration in liver via heightened expression of RALDH, the rate-limiting enzyme in RA synthesis. Surprisingly, RALDH expression was found in liver CD11b+CD11c- macrophages, but not in liver CD11c+ dendritic cells. Using bioluminescence imaging of whole liver, we observed that RA signaling increases 25-fold after Con A. We evaluated the requirement for RA response in T cells using transgenic mice in which T cells specifically express a dominant negative RA receptor alpha construct. Mice with disrupted RA signaling in T cells developed significantly less liver damage than did mice with intact RA signaling. Thus, our findings illustrate that RA function is critical for immune-mediated injury in ILD, and suggest that liver macrophages have a specific role in provision of RA for liver T cells.
Identification of a novel cytokine amplification loop during influenza infection (P6142)
Authors: Teijaro John , Walsh Kevin , Rosen Hugh , Oldstone Michael
Published: January 2023

Pathogenic influenza viruses cause significant morbidity and mortality in humans. Influenza virus causes lung tissue damage by direct pulmonary epithelial injury and by its initiation of an aggressive innate inflammatory response. The sphingosine 1-phosphate (S1P) signaling system is well known for its role in modulation of immune cell trafficking. Using genetic and chemical tools to probe functions of the S1P1 receptor (S1P1-rec), we recently published that pulmonary endothelial cells (ECs) are central modulators of innate immune cell recruitment and cytokine responses early following influenza virus infection. Use of S1P1-rec agonist results in reduced influenza morbidity and mortality. While S1P1 receptor is found on ECs and lymphocytes within lung tissue, an S1P1-selective agonist suppressed cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, clearly identifying pulmonary ECs as central regulators of cytokine storm. Now, we report a novel cytokine amplification loop requiring cellular cross-talk between pulmonary epithelial cells, ECs and plasmacytoid dendritic cells (pDCs). Cytokine amplification through this loop requires MyD88 and IRF7 signaling. Importantly, S1P1 signaling on ECs elicits a negative signal to pDCs leading to global suppression of cytokine amplification. These findings identify therapeutic control points to blunt pathology associated with influenza virus infection and further illuminate influenza pathogenesis.
Late cognate antigen interactions are required for CD4+ T cell survival during influenza infection. (P6146)
Authors: Bautista Bianca , McKinstry Kai , Strutt Tara , Swain Susan
Published: January 2023

At the termination of an influenza infection, the majority of antigen-specific effector CD4 T cells undergo Bim-driven apoptosis leaving a small, heterogeneous population of memory cells capable of protecting against subsequent infection. The kinetics of the CD4 response is known to be influenced by the levels of inflammation and antigen, but the contribution of these to contraction and memory formation is unclear. To analyze these we isolated OT-II transgenic CD4 T cells at various days following PR8-OVAII infection and transferred them to either uninfected, PR8-infected (inflammation), or PR8-OVAII-infected (inflammation plus antigen) hosts. In hosts lacking cognate antigen, effectors underwent minimal cytokine-driven proliferation and began to contract as early as two days post transfer, while those in PR8-OVAII-infected hosts proliferated 3-fold and underwent apoptosis after a delay of 2-3 days, leading to greater recovery. To dissect how CD4 effectors nearing the time of contraction interpret external signals, we isolated donor OT-II CD4 effectors on days 6-8 following PR8-OVAII infection and cultured them with or without TCR stimulation. TCR signaling resulted in increased cell recovery, enhanced proliferation, and reduced ratios of Bim:Bcl2 promoting effector survival. Blocking IL-2 diminished the recovery of TCR-stimulated effectors, suggesting that the beneficial impact of late TCR signaling is dependent on autocrine IL-2 signaling.
β-catenin modulates inflammatory cytokine production and disease development in NOD mice (P5231)
Authors: Zirnheld Arin , Fan Chunlan , Kosiewicz Michele , Alard Pascale
Published: January 2023

Because of their various immune defects including the excess production of proinflammatory cytokines by dendritic cells, non-obese diabetic (NOD) mice provide a powerful model to understand Type I diabetes. We have found increased levels of nuclear and cytosolic β-catenin in BMDC from NOD mice. We have assessed its association with inflammatory cytokine production and disease development, and the reason for its accumulation. First, we transfected NOD or B6g7 BMDC with β-catenin siRNA or β-catenin plasmid and measured IL-12 production. We found that modulating β-catenin expression influences IL-12 production. We next examined potential defects in β-catenin phosphorylation in NOD BMDC as a mechanism of β-catenin accumulation. We found high levels of phosphorylation at two residues, S675 and S552, mediated by PKA and Akt respectively. Because these types of phosphorylation have been associated with increased stabilization, these data suggest that β-catenin accumulation may result from defective PKA or Akt activity. Finally, we inhibited β-catenin in vivo in NOD mice and measure the incidence of type I diabetes. We intraperitoneally administered quercetin, a β-catenin inhibitor, or intravenously injected BMDC treated with β-catenin siRNA into NOD mice. We found that both treatments reduced disease incidence. Overall, these data point to the importance of β-catenin as a therapeutic target for reducing the inflammation that leads to the development of Type I diabetes.
Foxp3-independent suppression of autoimmunity by anti-CD4-induced recessive tolerization of autoreactive T cells (P5222)
Authors: Sparwasser Tim , Mayer Christian
Published: January 2023

Non-depleting anti-CD4 monoclonal antibodies potently induce dominant infectious tolerance and Foxp3+ regulatory T cells (Tregs) have recently been implicated as a crucial underlying mechanism. However, it has not been explored whether anti-CD4 treatment has residual activity in the complete absence of Foxp3+ Tregs. Utilizing DEREG mice which allow for the inducible depletion of Foxp3+ Tregs by diphtheria toxin (DT), we found that the anti-CD4 clone YTS177.9 is able to fully inhibit CD4+ T cell activation in the absence of Foxp3+ Tregs in vitro. Moreover, anti-CD4 treatment of neonatal scurfy (sf) mice genetically devoid of functional Foxp3+ Tregs resulted in a rescue of the autoimmune phenotype. We thus discovered a novel Foxp3-independent mechanism of anti-CD4-induced tolerance. Foxp3-independent tolerance was recessive as opposed to classical dominant infectious tolerance requiring Foxp3+ Tregs. Given that anti-human CD4 antibodies are currently developed as therapeutics, our results provide valuable insights into anti-CD4-mediated tolerance and unexpectedly highlight a potential synergy of anti-CD4 antibodies with Foxp3+ Treg-modulating regimens for the treatment of human inflammatory diseases. Beyond, anti-CD4 antibodies might represent a novel therapeutic option for the human IPEX syndrome which is caused by Foxp3 mutations similar to the autoimmune disease of sf mice.
T cell miRNA in multiple sclerosis pathogenesis (P5209)
Authors: Guerau-de-Arellano Mireia , Lovett-Racke Amy , Racke Michael
Published: January 2023

Multiple sclerosis (MS) is an immune-mediated disease that results in destruction of the myelin sheath and axonal degeneration. Interestingly, while myelin-specific T cells are present in peripheral blood mononuclear cells (PBMC) of both healthy controls and MS patients, they only become pathogenic in MS patients. This suggests that some regulatory checkpoint is bypassed in MS T cells, unleashing their autoimmune potential. By regulating protein-coding mRNAs, microRNAs (miRNAs) control important cellular processes. Specifically, we have previously shown that dysregulated miRNAs in MS patient’s naïve T cells promote pro-inflammatory Th1 immune responses. In this study, we explored whether differences in miRNA expression in memory T cells of MS patients further contribute to the pathogenesis of MS. Memory CD4 T cells were purified by magnetic bead sorting to 95% purity and subjected to a comprehensive Nanostring technology miRNA profiling. In a study on healthy (n=17) and seven MS patients (n=21), a number of miRs were found to be significantly different in MS compared to healthy controls. Among the identified miRs, many target genes known to be involved in T cell proliferation and apoptosis, activation, adhesion and regulation. These results identify miRNAs in memory T cells as MS biomarkers of biological significance and potential clinical value, and reveal potential new therapeutic targets in MS.
HMGB1 preconditioning protects against kidney ischemia reperfusion injury (P2126)
Authors: Wu Huiling , Steenstra Renske , C.S. de Boer Elianne , Zhao Cathy , Ma Jin , Chadban Steven
Published: January 2023

High-mobility group box1 (HMGB1) is an endogenous ligand for TLR4. We have reported that HMGB1 mediates kidney ischemia reperfusion injury (IRI) via TLR4. We aimed to determine: 1) the potential of preconditioning with rHMGB1 to block kidney IRI; 2) whether this effect is dependent upon TLR4; 3) whether this involves down-regulation of TLR signaling. Methods: Wild-type (WT) and TLR4-/- mice were receive rHMGB1 (20 ug/animal) or saline by i.p. injection 2 hours before 21 minute kidney ischemia. Results: WT mice pre-treated with rHMGB1 were protected against kidney IRI, with lower serum creatinine, less tubular damage, less tubulo-interstitial neutrophil, macrophage and CD4+ cell infiltration and less tubular epithelial cell apoptosis versus controls at day 1 and/or 5 post IRI. mRNA expression of cytokines IL6 & TNFa and chemokines CXCL2 & CCL2 in the kidney were significantly reduced by rHMGB1 pre-treatment at day 5. TLR4-/- mice were protected against kidney IRI, with no additional protection afforded by pre-administration of rHMGB1. The protective effect of rHMGB1 preconditioning on WT kidney involved Siglec-10, a negative regulator of HMGB1-TLR4 activation, which is induced through NF-kB signalling (p<0.05 versus control). Conclusion: HMGB1 preconditioning affords significant protection from kidney IRI by negatively regulating TLR4 signaling through Siglec-10 and NF-kB signalling, indicating the therapeutic potential.
MyD88-dependent expansion of an immature GR-1+CD11b+ population from donor bone marrow reduces early mortality after allogeneic hematopoietic stem cell transplantation (P2166)
Authors: Lim Ji young , Youn Hyewon , Choi Eun Young , Min Chang-Ki
Published: January 2023

Myeloid-derived suppressor cells coexpressing both CD11b and Gr-1 antigens are a heterogeneous population of immature myeloid cells. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs. We investigated how the MyD88-dependent expansion of MDSCs from donor BM contributes to protection of GVHD. Lethally irradiated B6D2F1 mice were transplanted with C57BL/6 WT or MyD88 KO mice TCD-BM together with WT spleen T cells. Morbidity and mortality of GVHD were significantly worse in recipients of KO TCD-BM. The expression of MDSCs in blood, MLN and liver was reduced in the recipients of KO TCD-BM, whereas the percentage of donor T cells infiltrating gut and liver was markedly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of KO TCD-BM. Moreover, in vivo administration of isolated MDSCs to recipients of KO TCD-BM attenuated the severity of GVHD. Administration of LPS to donor mice expanded MDSCs in BM with enhanced expression of MyD88 mRNAs. Recipients of TCD-BM from WT mice injected LPS also showed attenuated GVHD severity. In summary, MyD88-dependent expansion of MDSCs population from donor non T-cell component appears to be critical for attenuating GVHD severity. Incomplete expansion of MDSCs in target organs correlates with decreased apoptosis and increased infiltration of donor T cells.
In vivo reconstitution of human natural T regulatory cells in NOD/SCID IL2rγcnull mice by expression of human IL-2 (P2174)
Authors: Abraham Sojan , Pahwa Rajendra , Ye Chunting , Pahwa Savita , Jaggaiahgari Shashidhar , Manjunath N. , Shankar Premlata
Published: January 2023

Regulatory T cells (TRegs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment graft versus host disease (GVHD). The potential clinical translation of targeted therapy with in vitro expanded human nTRegs is being tested to prevent GVHD, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model is a major impediment for investigating the behavior of adoptively transferred nTRegs in vivo. The aim of this study is to develop TRegs reconstituted humanized mice as a preclinical model for understanding their therapeutic usefulness in the transplantation setting. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nTRegs in NOD-SCID IL2rγcnull mice. Furthermore, the reconstituted TRegs trafficked to different organs of the body and retained their characteristic suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted TRegs are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel hu-TReg mouse model offers a pre-clinical platform to study the in vivo function and stability of human nTRegs and their ability to modulate autoimmune diseases and GVHD.
The role of caspase-1 involved in autophagy activation in graft-versus-host disease model (P2178)
Authors: Lee Kyung Ho , Lee Hye Jin , Park Minhwa , Ryu Kyung-Ha , Woo So-Youn
Published: January 2023

Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic cell transplantation. Although donor-derived T cells recognize host alloantigens and induce detrimental acute GVHD, the mechanism of the onset and progression of acute GVHD remain uncertain. In order to reveal the involvement of autophagy activation in acute GVHD pathogenesis, we used reduced intensity conditioning model (e.g., BU-CY conditioned mice) and compared the tissue damage and expression of autophagy-related genes. Recipient C57BL/6 and caspase-1 KO mice received busulfan (BU, 80 mg/kg) daily for 4 days, followed by cyclophosphamide (CY, 200 mg/kg) daily 2 days via intraperitoneal injection. Two days after, bone marrow cells and spleen cells were prepared from Balb/c mice and injected via tail vein. For three weeks, the clinical and histological GVHD score and Kaplan-Meier survival rate was observed. We found that the GVHD score showed the difference between C57BL/6 and caspase-1 KO recipient mice. In addition, the expression level of LC3B and Beclin-1 was decreased in caspase-1 KO mice. It implies that caspase-1 activation is involved in GVHD pathogenesis via autophagy pathway.
IVIg-induced anti-inflammatory monocytes (CD14 low, HLA-DR high) with a low CD80 and high PDL1 expression inhibits T cell activation in allogeneic mixed lymphocytes reactions (P2194)
Authors: Padet Lauriane , Bazin Renée
Published: January 2023

Intravenous immunoglobulin (IVIg) is a therapeutic preparation of human IgG isolated from plasma donations and has been proposed as a therapy to improve the rate of graft survival in patients with a high risk for antibody-mediated-rejection. Blocking of anti-HLA antibodies by anti-idiotypic IgG present in IVIg was proposed to explain the rapid effect of IVIg. However, a long-term reduction of anti-HLA alloreactivity in IVIg-treated patients suggests that IVIg modulates the functions of immune cells. In the present study, we showed using the allogeneic mixed lymphocyte reaction (MLR) as an in vitro model of allograft rejection and GvHD that IVIg strongly inhibits IL-2 secretion (T cell activation) and modulates the level of other pro- and anti-inflammatory cytokines secretion. To determine the mechanisms underlying the inhibition of T cell activation in MLR, we studied the effect of IVIg on the phenotype of the cells involved in MLR. Our results revealed that MLR inhibition by IVIg correlates with the induction of anti-inflammatory monocytes (CD14 low, HLA-DR high) with a low CD80 and high PDL1 expression. To evaluate the importance of PDL1 on the MLR inhibition, anti-PDL1 was added during the MLR. Blocking of PDL1 restored the MLR, as evaluated by IL-2 secretion. We thus propose that PDL1 plays a central role in the inhibition of MLR. Our results help to better understand how IVIg induces long-term peripheral tolerance and improves graft survival in transplanted patients.