bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dnmt3a mediated de novo DNA methylation programming enforces T cell exhaustion (IRM14P.451)
Authors: Youngblood Ben , Hale Scott , Ahn Eunseon , Im Sejin , Ghoneim Hazem , Abdelsamed Hossam , Ahmed Rafi 
Published: January 2023

Abstract
Virus-specific CD8 T cells play a critical role in controlling chronic infections such as HIV and HCV, but progressively lose their antiviral functions during prolonged antigen exposure. Repression of CD8 T cell effector functions, commonly referred to as T cell exhaustion, limits the ability of the immune system to purge viral reservoirs from the host. Therapies that transiently rejuvenate exhausted virus-specific CD8 T cells have been highly successful at recovering antiviral functions, but a current challenge for the field is to identify mechanisms for stably reprogramming exhausted CD8 T cells so that recovered antiviral properties can be long-lived. We have recently demonstrated that epigenetic modifications acquired in pathogen-specific CD8 T cells during prolonged antigen exposure (chronic viral infections) reinforces T cell exhaustion. Using a mouse model system of chronic viral infection we investigated the role of Dnmt3a mediated de novo DNA methylation in regulating CD8 T cell exhaustion. Strikingly, conditional deletion of Dnmt3a in activated CD8 T cells blocked the cells from becoming exhausted. Moreover, in the absence of acquiring a de novo exhaustion program, pathogen-specific CD8 T cells are able to control what normally is a chronic viral infection. These results have significant implications for therapeutic strategies that utilize reactivation of host pathogen-specific CD8 T cells to control chronic viral infections or cancer.
Aptamer-targeted siRNA inhibition of raptor promotes the persistence of vaccine-induced CD8+ T cell responses and enhances protective immunity against influenza virus (IRM14P.452)
Authors: Berezhnoy Alexey , Termini James , Andreansky Samita , Stone Geoffrey , Levay Agata , Gilboa Eli 
Published: January 2023

Abstract
Inhibition of mediators of effector differentiation redirects activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR with rapamycin led to the enhenced differentiation of antigen activated CD8+ T cells into memory cells. Nevertheless, pharmacological agents are not available for many “undrugable” targets and often exhibit undesirable effects reflecting the broad distribution of their targets. We are developing a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi is used to downregulate intracellular mediators of effector differentiation that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice downregulated mTORC1, but not mTORC2, activity in vaccine-activated CD8+ T cells. In OT-I model, CD8+ memory cells generated with 4-1BB-raptor exhibited normal cytotoxic effector functions while rapamycin generated CTL were defective in their cytotoxic effector functions. Prophylactic immunization with DNA-based vaccine in combination with 4-1BB raptor increased persistence of NP-specific memory CD8+ T cells and protected animals from influenza challenge. Overall, these studies demonstrate the feasibility and efficiency of aptamer-targeted delivery of siRNAs to immune cells.
Erk2-deficiency impairs the effector function of mature CD4+ T cells (IRM15P.455)
Authors: Tudyk Julie , Zboril Emily , Forsthuber Thomas 
Published: January 2023

Abstract
Extracellular signal-regulated kinase 2 (Erk2) is required for CD4+T cell development and positive selection in the thymus, however the role of Erk2 in mature CD4+T cells is unknown. Erk2 is a key player of the mitogen activated protein kinase (MAPK) pathway which, in T cells, transduces crucial signals such as TCR engagement, CD28 co-stimulation, and cytokine binding. Upon activation, these signals are carefully coordinated to initiate gene expression programs, thus directing the expansion and differentiation of Ag-specific cells. The exquisite control of these signaling mechanisms is needed to maintain tolerance; dysregulation can result in inappropriate immune responses. In this study we used a mouse model with T cell-specific deletion of Erk2 to determine the role of Erk2 for CD4+T cell function. Our results show that increased numbers of Erk2∆CD4+T cells from naïve mice express markers for activation (CD25, CD44, and CD69). Further, Erk2∆CD4+T cells are able to survive and expand in vivo and proliferate upon IL-2, IL-7, and IL-15 cytokine stimulation. Importantly however, although the frequency and magnitude of Ag-specific Erk2∆CD4+T cell responses was comparable with controls upon antigen recall, secretion of IFN-g and IL-17 was significantly impaired. As CD4+T cells orchestrate immune responses primarily via cytokine production, our results suggest that Erk2 is required for CD4+T cell effector function.
Transcriptional regulation of IL-4 expression in T follicular helper cells (IRM15P.456)
Authors: Nurieva Roza , Sahoo Anupama , Alekseev Andrei 
Published: January 2023

Abstract
Apart from Th2 cells T follicular helper (Tfh) cells are a major class of IL-4 producing T cells, required for regulation of type 2 humoral immune responses. Although the transcriptional control of IL-4 in Th2 cells has been extensively investigated, the precise regulation mechanism(s) of IL-4 production in Tfh cells remains mainly unknown. In the current study, we found that the transcription factor Batf, a member of the AP-1/Jun family is essential for IL-4 expression in Tfh cells. Remarkably, Batf deficiency leads to decreased IL-4 production selectively in Tfh cells but not in conventional Th2 cells and subsequently to reduction in their pro-allergic pathogenicity. Batf in cooperation with Interferon regulatory factor (IRF)4 controls IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region of the IL-4 locus. The transcription factors Stat3 and Stat6 also aid in Batf/IRF4 binding to the CNS2 locus thus playing a crucial role in Batf-mediated regulation of IL-4 in Tfh cells. We also found that enhanced expression of Batf in Tfh cells is largely dependent on IL-4/Stat6 signaling, indicating towards a positive feedback loop in controlling Batf-IL-4 expression in Tfh cells. Our results thus identify a novel and critical role of Batf in promoting the generation of pro-allergic IL-4 producing Tfh cells.
Phospholipid biosynthesis and antigen-specific B cell fate and function (LYM7P.621)
Authors: Barrington Robert , Solodushko Viktoriya , aragon Ileana , Brewer Joseph 
Published: January 2023

Abstract
In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. Previous studies revealed that CD19Cre-mediated deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, consistent with defective germinal center reactions. To more specifically assess the influence of PtdCho on B cell fate during germinal center and long-term humoral responses, we examined immune responses in Cg1Cre CCTaflox mice. Serum IgG1 was undetectable in naïve Cg1Cre CCTaflox mice, while levels of IgM and other IgG subclasses were similar in naïve Cg1Cre CCTaflox and control mice. Following immunization with T cell-dependent antigen NP-KLH, control mice predictively generated high titer IgG anti-NP. In contrast, IgG anti-NP titers were markedly reduced in immunized Cg1Cre CCTaflox mice. Interestingly, though antigen-specific IgM responses were comparable between Cg1Cre CCTaflox and control mice, the IgM anti-NP response in Cg1Cre CCTaflox mice failed to increase in affinity. Lastly, the frequency of antigen-experienced NP-specific B cells and antibody-secreting cells (ASC) were both reduced in Cg1Cre CCTaflox mice compared with controls. These data indicate that PtdCho plays a pivotal role in the generation and persistence of both germinal center B cells and ASC.
Atg7 is critical for the functional maturation of germinal center B cells (LYM7P.625)
Authors: Zheng Biao , Fang Lei , Zhang Qi , Jiang Wen , Han Shuhua , Qin Jun 
Published: January 2023

Abstract
The role of autophagy, especially individual components of the autophagy machinery, in antibody response has not been well established. Atg7 is an integral part in autophagesome formation and elongation. Using lineage- and stage-specific conditional knockout (CKO) mice, we have found that, in GC B cell-specific Atg7 CKO mice, although GC formation and structure were not affected by Atg7 deficiency, both primary and memory antibody responses were severely impaired in Atg7 CKO mice. Class-switch recombination (CSR) and Ig somatic hypermutation (SHM) were defective in Atg7-/- GC B-cells, leading to a severely inhibited production of class-switched high-affinity antibodies. Analysis of GC B-cell clones showed that Atg7-deficiency in GC B-cells causes a dramatic change in Ig gene composition in the responding B-cell repertoire, which is dominated by non-canonical Ig genes encoding lower affinity antibodies. Thus, our study demonstrated for the first time that a single gene mutation affects GC function without inhibiting GC formation. The underlying molecular mechanism for impaired GC function in Atg7 mutant mice is that Atg7 is critical in regulating activation-induced deaminase (AID). Atg7-deficiency in GC B cells led to delayed onset and reduced disease severity in collagen-induced arthritis (CIA), demonstrating that Atg7 is critical in the development and pathogenesis of autoimmune disease.
Involvement of the serine/threonine kinase DRAK2 in the modulation of T cell metabolism (LYM7P.611)
Authors: Fracchia Kelley , Walsh Craig 
Published: January 2023

Abstract
Clonal expansion of T cells is often essential for effective immune responses, but in order to avoid lymphoproliferative disease and autoimmunity, negative regulation of T cell activation is crucial. The serine/threonine kinase DRAK2 is a negative regulator of T cell receptor (TCR) signaling. A loss of this kinase leads to an altered threshold for activation, allowing T cell proliferation under suboptimal stimulation conditions. Despite enhanced T cell activation, DRAK2-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE). However, the basis for this autoimmune resistance is currently unknown. Emerging evidence highlights an intimate correspondence between cellular metabolism and immunity. Of note, T cell activation via TCR stimulation results in extensive metabolic reprogramming, a process required to meet the biosynthetic demands of rapid clonal expansion. The bioenergetic profile adopted by specific T cell subsets depends on a variety of factors including the type of activation and the substrate availability within the environment. Here we show that, in the absence of DRAK2 signaling, T cells exhibit selective alterations in metabolic profile, leading to differential transcriptional and translational responses. These findings demonstrate that DRAK2 is selectively important for T cell metabolism and highlight the potential that DRAK2 modulation of metabolic activity may serve as an attractive therapeutic target for the treatment of autoimmune diseases.
T cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome (LYM7P.612)
Authors: Peters Bjorn , Bresciani Anne , Schommer Nina , Sette Alessandro , Nielsen Morten 
Published: January 2023

Abstract
Several mechanisms exist to avoid or suppress inflammatory T cell immune responses that could prove harmful to the host by targeting self-antigens or commensal microbes. We hypothesized that these mechanisms could be evident when comparing the immunogenicity of a peptide with the conservation of its sequence in the human proteome or the healthy human microbiome. We utilized the Immune Epitope Database (IEDB) to retrieve experimentally characterized T cell epitopes derived from bacteria that are targets of inflammatory human immune responses. Indeed, we found that epitopes that are homologous with self-antigens above a certain threshold showed lower immunogenicity, presumably as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. Interestingly, we found evidence that the microbiota of different body sites play different roles in the tolerization of T cells. For example, peptides conserved among bacteria of the gastrointestinal tract had lower immunogenicity than those conserved in bacteria of the lung microbiome. Overall, these findings indicate that the existence (and potentially the polarization) of T cell responses to a given epitope is influenced and to some extend predictable based on its homology to antigens shaping the immune response.
The Tec family kinase Itk is required for Th9 cell differentiation (LYM8P.642)
Authors: Gomez-Rodriguez Julio , Meylan Francoise , Handon Robin , Hayes Erika , Siegel Richard , Schwartzberg Pamela 
Published: January 2023

Abstract
One of the main functions of the adaptive immune system is to mount specific responses to pathogens. These effector CD4 T helper cells, include Th1, Th2, Th9 and Th17 cells, produce distinct cytokines important in the eradication of different infectious pathogens--however excessive activation of these cells can be harmful to the host, contributing to immunopathology, including autoimmunity and asthma. ITK-deficient mice exhibit reduced responses in models of allergic asthma, a disease attributed to increased Th2 responses. Polymorphisms in the Itk promoter in humans have also been linked to increased asthma incidence. Th9 cells are a recently appreciated T helper cell population that produce IL-9, a cytokine implicated in allergic asthma, responses to microbial infections, inflammatory gut diseases and autoimmunity. However, the factors contributing to IL-9 regulation are still poorly understood. We have found that ITK-deficient CD4 cells completely fail to produce IL-9 in culture. This defect is one of the most profound phenotypes we have observed for Itk-/- cells to date, and is associate with reduced levels of IRF4 during Th9 differentiation. Furthermore, in preliminary experiments, we found decreased IL-9 and IL-13 expression in vivo using a papain hypersensitivity model. Our studies demonstrate important roles for Itk in Th9 differentiation, suggesting Itk as a therapeutic candidate for treatment of diseases involving Th9-mediated inflammation.
Immunological study of recombinant protein fragments from Clonorchis sinensis thioredoxin peroxidase on inhibitory effect of macrophage activation (MPF6P.656)
Authors: Bae Young Mee , Wi Hae Joo , Son Hyeon Seok 
Published: January 2023

Abstract
Clonorchis sinensis (C. sinensis) estabilishes long term parasitism within bile ducts without notable symptoms. General down-modulated host immune responses in helminth infestation are maintained by excretory-secretory products (ESP), produced by the live parasites and considered as an important immunomodulator in this context. Reference based study found out that Thioredoxin peroxidase (TPX) of helminth parasites had an immune-modulating property toward Th2 type through induction of regulatory phenotype of macrophages. Hence, full length of TPX and a number of recombinant TPX fragments of C. sinensis were produced by using bacteria system. The CS-TPX was able to inhibit LPS-induced surface expression of TLR4 in mouse macrophage cell line, RAW264.7, and LPS-induced secretion of pro-inflammatory mediators NO, TNF-α and IL-6. Moreover, CS-TPX induces IL-10, but not TGF-β. Pull-down assay results revealed that the direct interaction between CS-TPX and TLR4 protein. Furthermore, we found that the smallest recombinant fragment of CS-TPX, which is T44, has comparable anti-inflammatory property as compared with full length CS-TPX. In conclusion, present study describes the macrophage regulatory function of C. sinensis ESP and the smallest fragment of CS-TPX, T44, could function of the negative regulator in LPS treated macrophage cell line RAW264.7 cells. Defined CS-TPX T44 will be one of the best candidates for therapeutic immunosuppressant in allergies and autoimmune diseases.
Passive immunization with CotH3 antibodies protects neutropenic mice from lethal mucormycosis through enhancing macrophage killing (MPF6P.660)
Authors: Lin Lin , Gebremariam Teclegiorgis , Ibrahim Ashraf S. 
Published: January 2023

Abstract
Mucormycosis is a fatal fungal infection caused commonly by Rhizopus. We recently identified R. oryzae CotH3 as a surface protein which mediates invasion of host cells. CotH3 is present in other fungi that cause mucormycosis. Previously, we found polyclonal anti-CotH3 antibodies (CotH3-Abs) to block host cell invasion by R. oryzae and protect diabetic ketoacidotic mice from mucormycosis. Because neutropenic patients are also predisposed to mucormycosis, we used a cyclophosphamide/cortisone acetate injected mice to detect if CotH3-Abs protect against mucormycosis. Mice were infected intratracheally with R. oryzae, Mucor, Lichtheimia, Apophysomyces, Rhizomucor, or Cunninghamella prior to treating with CotH3-Abs (0.3mg, 0.1mg or 0.03mg) at different times post infection (4, 16, or 48h). All treatment doses showed enhanced survival of mice infected with R. oryzae vs. placebo-treated mice when used 16h post infection (p&lt;0.005). The 0.03mg dose given 16h post infection demonstrated better survival outcome vs. higher doses. Protection was time dependent with higher survival of mice receiving CotH3-Abs at earlier time points post infection. Further, CotH-Abs prolonged survival of mice infected with each of the other tested organisms. Finally, CotH3-Abs reduced tissue fungal burden in target organs and dramatically enhanced mouse macrophage killing via enhancing lysosomal synthesis. Our studies warrant further development of CotH3-Abs as a novel therapy for mucormycosis.
The bacterial second messenger cyclic-di-GMP regulates Brucella melitensis phenotype and host immune responses (MPF6P.645)
Authors: Khan Mike , Harms Jerome , Armon Leah , Eskra Linda , Hall Cherisse , Banai Menachem , Smith Judith , Splitter Gary 
Published: January 2023

Abstract
With greater than 500,000 new infections per year, brucellosis is the most prevalent zoonosis worldwide. Brucella melitensis are intracellular bacteria that invade and replicate within macrophages and dendritic cells. The bacterial second messenger cyclic-di-GMP plays an important role in a variety of cellular processes such as differentiation and cell cycle, and regulates B. melitensis virulence in IRF1-/- mice. Deletion of bpdA or bpdB phosphodiesterase genes resulted in attenuated virulence whereas deletion of the cgsB guanylate cyclase gene produced a hypervirulent strain. In this study we examined host and mutant bacterial responses to explain the altered virulence. Proteomics analysis of ΔbpdA and ΔcgsB revealed differences in expression profiles of amino acid and carbohydrate transport/metabolism, cell wall biogenesis, and defense mechanisms. Scanning electron microscopy revealed smaller bacterium size and abnormal shape of the attenuated ΔbpdA strain. BALB/c mice infected with ΔbpdA had significantly less splenic colony forming units as well as 16S rRNA compared to wild-type. Bone marrow-derived macrophages infected with ΔbpdA induced the greatest increase in genes involved in the Type 1 interferon pathway compared to ΔbpdB, ΔcgsB, and wild-type. These data suggest a critical role of cyclic-di-GMP in promoting the infectious success of B. melitensis by altering bacterial phenotype and host immune responses.
Chimeric mouse/human Fab antibodies that selectively binds to the surface of Mycobacterium tuberculosis (MPF6P.649)
Authors: Batista-Gonzalez Ana , Prados-Rosales Rafael , Casadevall Arturo 
Published: January 2023

Abstract
The current diagnostic tools for Tuberculosis (TB) are time consuming and not suitable for point of care devices. Antibody-based diagnostic approaches could solve this issue. We have used antibody phage-display to isolate chimeric mouse/human Fab antibodies that binds to the surface of live Mycobacterium tuberculosis (Mtb). For that purpose, mice were immunized with live or heat killed (HK) Mtb and splenic mRNA was used to generate 2 phage-display Fab libraries. Several rounds of bio panning were performed against whole cell live and HK Mtb and a substraction round was performed against non-tuberculous Mycobacteria. Of the 24 clones analyzed we found 6 Fab-expressing phages that specifically bind to live Mtb over HK or non-tuberculous Mycobacteria. Further characterization of these Fab antibodies consisted of recombinant expression and purification in E coli and selective binding to Mtb-specific subcellular fractions. This is the first study on Mtb-specific Fab antibodies that specifically bind a live and virulent Mtb strain. These molecules could represent a good platform to set up highly specific TB biomarkers.
Oral colonization with Neisseria highlights differences in wild derived mouse immune responses (MPF6P.650)
Authors: Powell Daniel , Ma Man , Weyand Nate , So Magdalene , Frelinger Jeffrey 
Published: January 2023

Abstract
Neisseria species have the ability to colonize the host as either a commensal or pathogen depending on the strain and anatomical location. Human Neisseria species carriage estimates can reach as high as 50% of the population. Investigations of the immunological changes induced by Neisseria carriage, to date, have been inhibited by the lack of a small animal model. Here we set out to develop a mouse model of commensal Neisseria colonization. Three wild derived inbred mouse strains (PWK, WSB and CAST) along with C57BL/6 were tested. These mice were inoculated orally with a Neisseria strain isolated from wild caught mice in Tucson, Arizona. Mice were then sampled by oral swab weekly to determine Neisseria burdens in the oral cavity. Only the CAST/EiJ mice were completely susceptible to colonization (6/6) while C57BL/6 mice were weakly colonized (2/7). PWK and WSB mice were completely resistant to colonization. To determine if the immune response was responsible for the resistance of C57BL/6 mice both RAG-1-/- and MyD88-/- were inoculated. Interestingly, MyD88-/- mice were completely susceptible to colonization (5/5) while the RAG-1-/- mice were completely resistant (0/8), indicating the innate response in C57BL/6 mice was responsible for the resistance to colonization. Current work is aimed at dissecting the innate immune responses to different stimuli between C57BL/6 and CAST mice.