bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory T cells use LAG-3 as the “brake” in autoimmune diabetes but the “accelerator” in tumor environment (IRC4P.487)
Authors: Zhang Qianxia , Beres Amy , Vignali Dario 
Published: January 2023

Abstract
Lymphocyte Activation Gene-3 (LAG-3, CD223) is an inhibitory receptor that is expressed upon TCR activation, is highly up-regulated on exhausted conventional T cells, and regulates T cell expansion, homeostasis and function via both cell intrinsic and extrinsic mechanisms. LAG-3 is also required for maximal regulatory T cell (Treg) function. Our lab has previously shown that LAG-3 modulates tolerance mechanisms, but the specific function of LAG-3 expressed by Tregs remains unknown. To elucidate the role of LAG-3 in Treg-mediated immune suppression, we generated mice in which active, membrane-expressed LAG-3 is specifically deleted in Foxp3+ Tregs. We used both the non-obese diabetes (NOD) and B6 mouse backgrounds to study the role of LAG-3-deficient Tregs in autoimmune diabetes and B16 melanoma, respectively. Surprisingly, the deletion of LAG-3 in Tregs both protected NOD mice from autoimmune diabetes and resulted in decreased B16 melanoma growth. In an autoimmune environment, the number of Tregs infiltrating the islets was limited by LAG-3, which facilitated the breakdown of self-tolerance. However, in the tumor microenvironment, LAG-3 activity was required for Treg-mediated immune suppression. These data suggest opposing roles for LAG-3 on Tregs in an inflammatory versus an immunosuppressive microenvironment. Therefore, specifically targeting LAG-3 on Tregs may provide a strategy for limiting tumor growth without subsequent development of autoimmunity.
Reduced T-bet induction in proliferation- interferon-γ decoupled CD4+ T cells as a mechanism for propagated T cell tolerance (IRC4P.491)
Authors: Dutta Avijit , Drake Charles , Powell Jonathan , Pardoll Drew , Huang Ching-Tai 
Published: January 2023

Abstract
Infectious tolerance of immunity has been proposed as a mechanism of tolerance propagation. Limited effort of tolerance induction may be exploited for maintaining stable long-term tolerance. In our hemagglutinin (HA) antigen-specific transgenic mouse model system, adoptively transferred naïve HA-specific CD4+ T cells became tolerized in HA bearing mice and developed into regulatory T cells (Treg) with LAG-3 expression. These Treg suppressed the activation of secondly transferred naïve HA-specific CD4+ T cells. The suppressed HA-specific CD4+ T cells of second transfer were impaired for their IFN-γ capacity with reduced T-bet induction and STAT1 phosphorylation. However, they still substantially proliferated and acquired LAG-3 expression. They themselves became regulatory T cells as well. They further suppressed HA specific naïve CD4+ T cells both in vitro and in vivo. Compromised T-bet induction and IFN-γ capacity with T cell activation under Treg suppression ensure the transition into Treg without significant effector activity and ultimate propagated tolerance.
Structure-functional analysis of Foxp3 (IRC4P.478)
Authors: Zeng Wei-ping 
Published: January 2023

Abstract
Foxp3 is responsible for the major immunological features of Treg cells, including hypoproliferation in vitro, immune suppression of conventional T cells and resistance to Th2 cell differentiation. In addition to the Forkhead domain, the Foxp3 protein contains the N-terminal, zinc finger and leucine zipper domains. To understand how these domains contribute to Foxp3 functions, we systematically compared the roles of these domains in determining the 3 major immunological features of Treg cells. We designed a bridge-mediated mutagenesis method to generate Foxp3 mutants with complete deletion of each of the domains. CD4 T cells expressing the Foxp3 mutant with deletion of the N-terminal, leucine zipper or the forkhead domain showed robust TCR dependent proliferation in vitro, differentiated into Th2 cells, and lost immune suppressive activities in vitro and in vivo, demonstrating a complete loss of all 3 functions of Foxp3. In contrast, deletion of the zinc finger domain only partially impaired these functions of Foxp3. This result suggests that mutations in the zinc finger domain could lead to nonlethal autoimmune and allergic diseases, in which reduction rather than complete loss of Foxp3 functions is expected. In any case, deletion of a particular domain showed similar effects on all 3 functions of Foxp3. Therefore defining each of the immunological features of Treg cells requires intact Foxp3 proteins.
Differential requirement for IRF4 in CD8+ T cell responses to acute versus chronic LCMV infection (IRM4P.498)
Authors: Nayar Ribhu , Schutten Elizabeth , Brehm Michael , Welsh Raymond , Berg Leslie 
Published: January 2023

Abstract
The magnitude of the CD8+ T cell response to acute infection is regulated by the strength of TCR signaling. A key component to this is the level of expression of the transcription factor IRF4, which controls CD8+ T cell expansion in a dose-dependent manner. During an acute response to LCMV-Armstrong, a modest reduction in IRF4 levels, such as that achieved by a haplo-deficiency at the irf4 locus, leads to a 2-4 fold reduction in the number of LCMV specific CD8+ T cells without impacting viral clearance (RN manuscript submitted). These data indicate that acute LCMV infections are associated with an expansion of cognate CD8+ T cells that is in excess of that needed to clear the virus. In contrast, during LCMV-clone 13 infection, the modestly-reduced CD8+ T cell response of irf4+/- T cells fails to control the infection, resulting in long-term viral persistence. In this system, loss of one allele of irf4 results in reduced CD8+ T cell function, increased exhaustion due to loss of PD1 low Tbet high precursors, and less weight loss than in LCMV-clone 13-infected WT mice. These data indicate differential requirements for high levels of IRF4 in generating functional CD8+ T cell responses capable of clearing acute versus chronic virus infections.
A new genetic model for separating and analyzing Treg cells in unchallenged mice based on their proliferative history (LYM3P.738)
Authors: Zhang Baojun , Dai Meifang , Zhuang Yuan , Li Qijing 
Published: January 2023

Abstract
Apart from other cell types, antigen-primed conventional CD4 T cells integrate their functional differentiation into the process of proliferation. However, it is unknown whether the functional maturation of Tregs is also associated with their proliferative history, especially in unchallenged mice. To aid dissection of Treg populations, we developed a reporter system for marking and isolating Tregs undergone proliferative expansion during development or tolerance response towards endogenous antigens. We designed a tandem loxP cassette, named Tlox, to prevent intramolecular recombination. Tlox also acts as a genetic switch placed in front of a fluorescent marker. Cell division provides an opportunity for inter-molecular pairing between the duplicated Tlox on sister chromatids. Upon pairing, Cre recombinase can convert Tlox to a single loxP and consequently express the fluorescent marker permanently. We showed that, in vitro and in vivo, reporter activation is indeed dependent on both active cell cycles and Cre expression. By combining Ox40-Cre with the Tlox reporter, we separated Tregs based on their proliferative history in naïve animals. Functional and transcriptomic analysis indicted that, within the Treg pool, proliferated Tregs form a distinct population and are highly enriched with effector phenotypes. Thus, the newly established lineage tracking system provides a novel tool for assessing dynamitic changes among Treg cells during their tolerance response in live animals.
The RNA-binding protein HuR is a posttranscriptional on switch for CD4+ Th2 and Th17 differentiation (LYM3P.730)
Authors: Atasoy Ulus , Chen Jing , Techasintana Patsharaporn , Gubin Matthew , Magee Joseph , Cascio Jason , Calaluce Robert , Yu Shiguang 
Published: January 2023

Abstract
Posttranscriptional control by RNA binding proteins (RBPs) in CD4+ T cells is poorly understood. The RBP, HuR, regulates IL-4, IL-13 and GATA-3, by binding to AU-rich (ARE) regions in their 3’ UTRs. We hypothesized HuR is regulating CD4+ Th2 differentiation by controlling mRNA stability. We previously showed that HuR over-expression results in increased Th2 differentiation and Th2 cytokines. We generated a new model to study Th2 differentiation, distal Lck-cre ROSA HuRfl/fl, in which HuR is ablated prior to T cell activation. This resulted in profound suppression of Th2 but not Th1 cytokines, under in vitro conditions. We used the ova challenge model of airway inflammation to study antigen presentation and in vivo cytokine regulation. HuR KO mice had completely abolished lung cellular inflammation to levels similar to non-immunized mice. This further resulted in significant reductions in IL-4, IL-13 secretion, and serum IgE levels. We also investigated effects of HuR ablation in Th17 differentiation. We show HuR regulates IL-17 expression by controlling transcript stability. HuR KO T cells have greatly reduced Th17 differentiation (66% reduction) and diminished onset and severity of inflammation in an EAE model. These data indicate that HuR is required for efficient CD4+ Th2 and Th17 differentiation both in vitro and in vivo but not Th1. These findings reveal nuanced control by HuR upon different CD4+ T cell subsets.
Resistance to invasive pneumococcal infection in mice with acute allergic lung inflammation (MPF1P.766)
Authors: Sanfilippo Alan , Furuya Yoichi , Metzger Dennis 
Published: January 2023

Abstract
A recent publication by Talbot et al (N Engl J Med 2005) first identified asthma as a risk factor for invasive pneumococcal disease (IPD). However, more recent reports suggest that mortality is unaffected following IPD in patients with asthma and that COPD, an obstructive condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the immune response to a pneumococcal infection in the lung, we used an acute murine model of ovalbumin (OVA)-induced allergic lung inflammation (ALI) and hypothesized that mice with acute ALI would show increased susceptibility to IPD. Surprisingly, mice with acute ALI demonstrated enhanced resistance to infection. Resistance in mice with ALI correlated with a greater capacity for bacterial clearance from the lungs within eight hours post infection. This early clearance resulted in decreased bacterial invasiveness into the lung tissue as well as a blunted cytokine response to infection. Despite an increase in alveolar macrophage levels following OVA challenge, the increased bacterial clearance appeared to be mediated by MHCII- SiglecF+ eosinophils. These findings suggest that while eosinophils are pathogenic in asthma they could be protective against IPD, highlighting the need for a clearer understanding of the risk of IPD in patients with different asthma phenotypes.
Leukotriene B4 controls methicillin-resistant Staphylococcus aureus skin infection (MPF2P.815)
Authors: Brandt Stephanie , Wang Sue , Delafield Nathan , Serezani Henrique 
Published: January 2023

Abstract
Methicillin resistant Staphylococcus aureus (MRSA) was traditionally a nosocomial pathogen but community-associated MRSA clones have emerged as a cause of severe necrotizing cutaneous infection. Leukotriene B4 (LTB4) is a potent lipid mediator produced by 5-lipoxygenase (5LO) that is important in host defense against many pathogens including Streptococcus pneumoniae lung infections and Leishmania amazonensis skin infections. We hypothesize that 5LO-/- and mice lacking LTB4 receptor (BLT1-/-) are more susceptible to MRSA skin infection than wild type (WT) mice. MRSA was inoculated subcutaneously in shaved backs of 5LO-/- or BLT1-/- and WT mice. We observed delayed skin lesion healing in 5LO-/- or BLT1-/- compared to WT. Both genotypes had higher bacterial burden in the site of infection than WT. H&E staining showed more leukocyte recruitment and epidermis thickening in both genotypes compared to the WT. We did not observe any change in phagocytosis of MRSA in macrophages from WT and BLT1-/-, which suggests a defect in killing. However, macrophages from 5LO-/- mice produced less nitric oxide, a potent microbicide, than WT macrophages. Topical treatment of 5LO-/- mice with an ointment containing LTB4 at the site of infection reduced lesion size when compared to untreated mice. These findings suggest that LTB4 is an important agent in skin host defense mechanisms. LTB4 is a likely candidate to be used as an immunotherapeutic agent to control MRSA infections.
Antibody and complement-mediated enhancement of red cell invasion by Plasmodium falciparum. (MPF2P.806)
Authors: Biryukov Sergei , Stoute José 
Published: January 2023

Abstract
Malaria is an infectious disease caused by an intracellular protist of the genus Plasmodium that invades red blood cells (RBCs). Experimental vaccines that target the blood stage of the parasite (merozoites) induce a potent humoral immune response but there is no protection despite inhibition of invasion in vitro in the presence of heat-inactivated (HI) serum. The reason for this discrepancy is unknown. We hypothesize that complement activation and opsonization of merozoites enhances complement receptor 1 (CR1)-mediated invasion of RBCs. We tested the effect of anti-merozoite surface protein 1 (MSP-1) antibody 5.2 (mAb5.2) in the presence of fresh or HI serum on the in vitro invasion of RBCs by P. falciparum. We observed inhibition of RBCs invasion with inactivation of serum for 3 min, 5 min, or 30 min at 56°C. The inhibitory effect of 3 min HI serum was rescued upon the addition of C2 and Factor B, but only in the presence of mAb5.2. Compstatin, a C3 inhibitor, negated the enhancing effect of mAb5.2 in fresh serum. The invasion enhancing effect of mAb5.2 was absent in 3 min HI serum. Similar results were seen in fresh serum when sCR1 was used as a competitor. Purified antibodies from MSP-1 vaccinees showed inhibitory activity in 3 min HI serum, but inhibition was reduced in fresh serum. Our results suggest that anti-merozoite antibodies in the presence of complement can enhance RBCs invasion, constituting a novel mechanism of immune evasion by P. falciparum.
CCL21 defect impairs dendritic cell trafficking in SAMP1/YitFc mice (MUC2P.817)
Authors: Mikulski Zbigniew , Johnson Rebecca , Shaked Iftach , Kim Gisen , Nowyhed Heba , Chodaczek Grzegorz , Cominelli Fabio , Ley Klaus 
Published: January 2023

Abstract
SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to Crohn’s disease. The disease mechanism is unknown but thought to involve adaptive and innate immune responses. Here, we discovered that the chemokine CCL21, a ligand for CCR7, is almost completely absent in mesenteric lymphatics and other tissues of SAMP mice. Lymphatic CCL21 is known to be required for dendritic cell (DC) trafficking. Absence of CCL21 results in a severe defect of CD11b+CD103+ DC migration from the ileal lamina propria to the mesenteric lymph nodes (MLN), similar to the defect seen in CCR7-deficient mice. The ability of DCs to produce retinoic acid (RA) supporting regulatory T cells (Tregs) is also drastically reduced in SAMP mice. In young mice, the defects in CCL21 expression and DC trafficking preceded the clinical manifestation of ileitis. As a therapeutic intervention, we mobilized DCs by oral treatment with the TLR7 ligand R848, which increased Tregs in MLN and dramatically improved disease scores. Thus, absence of CCL21 expression represents a major pathogenic defect contributing to ileitis in SAMP mice. Our data suggest that therapies aimed at improved DC trafficking might be useful in patients with Crohn’s disease.
Poor expression and activation of the NLRP3 inflammasome in resident but not inflammatory colonic monocytes/macrophages (MUC2P.831)
Authors: Filardy Alessandra , He JianPing , Silveira Danielle , Kelsall Brian 
Published: January 2023

Abstract
Colonic macrophages (cMPs) play an important role in maintaining intestinal homeostasis since they are able to tolerate commensals and kill pathogenic microorganisms. One critical inflammatory pathway tightly controlled in cMPs is the assembly of the NLRP3 inflammasome that drives the maturation and secretion of IL-1β and IL-18 in response to multiple stimuli, including ATP and bacterial toxins. Here we found poor secretion of IL-1β by F4/80+CD64+ cMP after NLRP3 inflammasome activation. Steady-state cMPs expressed low constitutive levels of pro-IL-1β and NLRP3 protein. Following LPS/P3C4 stimulation, both cMPs and bone marrow-derived MP (BMDMs) increased expression of pro-IL-1β and NLRP3 mRNA. Surprisingly, however, pro-IL-1β and NLRP3 protein expression was significantly impaired in cMPs compared with BMDMs. Using the adoptive transfer model of colitis, recently recruited F480+CD64+ inflammatory monocytes/cMPs expressed similar levels of pro-IL-1β mRNA to cMPs under resting conditions; however in contrast, inflammatory monocytes/cMPs secreted high levels of mature IL-1β after NLRP3 inflammasome activation. Collectively, these results show that cMPs, but neither BMDMs nor newly recruited inflammatory monocytes/cMPs, have a functional impairment of NLRP3 inflammasome activation; and suggest that the poor NLRP3 activity observed in resident cMPs is largely due to the selective inhibition of the translational of NLRP3 and pro-IL-1β mRNA to protein.
A structural approach to the study of viral immune evasion mechanisms (TECH1P.850)
Authors: Sgourakis Nikolaos , Natarajan Kannan , Margulies David , Bax Ad 
Published: January 2023

Abstract
Mouse cytomegalovirus encodes immunoevasin proteins that interfere with MHC-I antigen presentation to CD8+ T cells and natural killer (NK) cells. Members of the m02-m16 family contribute to viral survival and infectivity. One member of this family, m04/gp34, binds MHC-I molecules in the endoplasmic reticulum and accompanies them to the cell surface, while the related m06 binds and directs MHC-I to endosomes. Biochemical evidence suggests that m04 counters MHC-retaining functions of other viral proteins. Despite many genetic and functional studies, the structural basis of immunoevasin specificities and functions is poorly understood, due largely to difficulty in cocrystallization of these proteins with their ligands. There is no identifiable amino acid homology of any member of this family to any protein structure available. To bypass this structural bottleneck, we have developed a new technology that combines sparse datasets recorded from Nuclear Magnetic Resonance spectroscopy with sophisticated Rosetta macromolecular modeling methods. We apply this approach to determine the structure of the m04 core domain and its interactions with the H2-Dd MHC-I molecule. m04 has a novel Ig-like fold, with long loops providing a flexible scaffold for MHC-I binding. This study provides insight into a key site on MHC-I “discovered” by the virus and helps us to understand the function of m04 with respect to T and NK cell MHC recognition.
Analysis of mass cytometry data with viSNE reveals phenotypic heterogeneity within human Tregs (TECH1P.854)
Authors: Konrad Mark , Simonds Erin , Bendall Sean , George Thaddeus , Delucchi Pam 
Published: January 2023

Abstract
Regulatory T (Treg) cells play a fundamental role in the development and maintenance of immunological tolerance. Tregs are characterized by constitutive expression of the transcription factor Foxp3, in addition to a surface phenotype of CD4+CD25hiCD127lo. While this pattern of expression has proven highly useful for studying Tregs, the use of additional markers should provide greater resolution of minor subsets within the Treg compartment. Moreover, identification of a concise surface marker profile will facilitate isolation of viable Tregs by FACS. Mass cytometry is a new platform that couples flow cytometry with mass spectrometry, and enables single-cell analysis of at least 30 parameters simultaneously without the requirement for compensation. To profile Treg phenotypes in human peripheral blood, we employed a panel of metal-conjugated antibodies, against both surface and intracellular targets. High-dimensional analysis with the viSNE algorithm, a cytometry analysis tool based on the t-distributed stochastic neighbor embedding algorithm, revealed heterogeneity within the Treg compartment with respect to expression of the markers CD39, CTLA-4 and HLA-DR. Furthermore, we found these markers to be co-expressed primarily in Tregs with the highest level of Foxp3 expression. We propose a gating strategy for identification of Tregs by mass cytometry that can be used to further phenotype human regulatory T cells.
Defining accuracy and sensitivity of T-cell receptor repertoire profiling for monitoring recurrent/persistent disease in patients with mature T-cell neoplasms (TECH1P.857)
Authors: Sherwood Anna , Robins Harlan , Fromm Jonathan , Greisman Harvey , Sabath Daniel , Chung Moon-Wook , Bertucci Caterina , Wilcox Cara , Emerson Ryan , Rieder Mark , Wood Brent , Wu David 
Published: January 2023

Abstract
Accurate and sensitive identification of recurrent or persistent disease in T-cell neoplasms is important for proper patient care. We developed a method that amplifies rearranged TCR CDR3 sequences and uses high-throughput sequencing (HTS) to sequence tens of thousands of chains simultaneously. Because the technology utilizes gDNA the frequency of sequenced CDR3 chains is representative of the relative frequency of each CDR3 sequence in the sample population of T cells. Thus these assays can describe both the breadth of the T-cell receptor repertoire and quantify individual clones; enabling tracking the presence and frequency of neoplastic clones. To demonstrate the potential of this technology, we first test the assay’s sensitivity and accuracy (1 lymphocyte in 1M total cells). Then, to show utility, we apply the technology to monitor persistent/recurrent disease in a set of mature T-cell lymphoma samples. To identify the neoplasm’s CDR3 chain, we sequence the TRB and TRG repertoire of 35 index samples. Then, to diagnose persistent disease, we sequence the TCR repertoire of follow-up samples and search for the neoplastic sequence. We then define persistent disease based on the sensitivity and accuracy of the assay. Concurrently, as part of standard clinical practice, a number of other methods were used to identify persistent disease. We compare these two methods to demonstrate that HTS technology is a viable alternative to traditional methods to detect and monitor lymphoma.