bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of T cell responses by a novel tissue-resident-macrophage derived immunomodulatory molecule-CRIg (IRC11P.434)
Authors: Yuan Xiaomei , Dong Yi , Fu Wenxian
Published: January 2023

Costimulatory and coinhibitory molecules of B7 family and TNF superfamily play critical roles in almost all aspects of T cell biology. Recently, a novel immuno-modulatory molecule -complement receptor of the immunoglobulin superfamily (CRIg), has been identified. CRIg is exclusively expressed on CD11b+ F4/80hi tissue-resident macrophages. In addition to act as a receptor for complement component C3 fragments, CRIg has also been implicated as a B7 family member to suppress T cell proliferation. Our previous studies suggest that CRIg has a regulatory role in dampening the autoimmunity in type 1 diabetes. Here we attempted to study how CRIg regulates T cell activities. Using in vitro T cell culture system, we found that suppression of conventional T (Tconv) cells by CRIg, in a form of Fc fusion protein, was elicited at the early activating, rather than the late dividing phase of T cells. These data suggest that CRIg may preferentially influence T cell activation signaling cascades. More strikingly, we found that CRIg can promote the conversion of inducible regulatory T (iTreg) cells, by synergizing with TGFb signaling. Together, these data define novel mechanisms of regulating adaptive immunity by tissue-resident macrophages.
Neuronal circuits modulate antigen flow through lymph nodes (IRC11P.420)
Authors: Hanes William , Olofsson Peder , Tsaava Tea , Levine Yaakov , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Initial exposure to an antigen, by infection or vaccination, is a critical step in developing protection against subsequent infection. Antigen injected into the dorsum of the mouse hind foot flows to the popliteal lymph node, then to the sciatic lymph node, through the lymphatics and eventually reaching the blood stream. In mice immunized to Keyhole-Limpet Hemocyanin (KLH), flow of IrDye-labeled KLH was restricted through the popliteal and sciatic lymph nodes. Imaging one hour after antigen administration revealed a significant decrease in sciatic and popliteal KLH levels in immunized mice compared to naïve animals (antigen signal in naïve 0.75 ± 0.15 vs sensitized 0.23 ± 0.11, p<0.05). Flow cytometric analysis of lymph node cellular populations revealed that antigen was associated mainly with a CD21+CD23+ mature subset of B-cells (51.9 ± 1.6% of total antigen positive cells)). Blocking neuronal activity with bupivacaine at the lymph nodes of immunized animals resulted in restoration of antigen flow, with an increase in antigen signal (control 0.27 ± 0.016 vs bupivacaine 0.38 ± 0.037, p<0.05). Conversely, direct activation of neuronal signals by noninvasive magnetic stimulation resulted in a significant decrease of antigen trafficking compared to sham controls (sham 1.39 ± 0.18 vs stimulated 0.47 ± 0.11, p<0.001). Taken together, these studies reveal a neuronal circuit that modulates antigen trafficking through a pathway involving a mature subset of B-cells.
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

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

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

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

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

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

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

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

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

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

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

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<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

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

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

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.
Interferon gamma is involved in Borrelia-induced arthritis in mice lacking interleukin-17 (MPF7P.702)
Authors: Kuo Joseph , Nardelli Dean , Schell Ronald
Published: January 2023

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis. Neutralization of IL-17 with antibodies inhibits the progression of arthritis in immunocompetent mice. Our study is the first to examine if Borrelia-induced arthritis occurs with IL-17-deficiency. We hypothesized that mice lacking IL-17 would have low to no Borrelia-induced arthritic symptoms. IL-17 knockout C57BL/6 mice were vaccinated with heat-inactivated Borrelia and/or infected with live spirochetes. Hind paw swelling, histopathology of ankle joints, serum cytokine levels, and T cell populations in tissues were measured. We found insignificant hind paw swelling and pathological changes in mice that were only vaccinated or infected when compared to control naïve mice, due to the Borrelia-resistant C57BL/6 background. However, mice that were both vaccinated and infected had more severe swelling and pathological changes, eliciting a robust immune response disregarding resistance. Also, elevated serum interferon gamma (IFNγ) levels and increasing frequencies of IFNγ-expressing lymphocytes with CD4 or CD8 were also observed in Borrelia-vaccinated and -infected mice. Therefore in these mice, IFNγ is directly involved in Borrelia-induced arthritis without IL-17. Future studies include identifying the role of IFNγ and other understudied IFNγ-expressing immune cells that induce Lyme arthritis in the absence of IL-17.
EphA2 promotes the development of experimental cerebral malaria. (MPF7P.707)
Authors: King Thayer , Mimche Patrice , Bray Christian , Brady Lauren , Aneke Janessa , Meyer Esmeralda , Galinski Mary , Lamb Tracey
Published: January 2023

Cerebral malaria is a severe complication of infection with the Plasmodium falciparum parasite, yet the etiology of the disease is not fully understood. In the well-established Plasmodium berghei ANKA (PbANKA) mouse model of experimental cerebral malaria (ECM), sequestration of parasitized red blood cells (pRBCs) and CD8+ T cells in the brain microvasculature is required for ECM development. However, our understanding of the receptors involved in CD8+ T cell adherence to the brain endothelium during ECM is currently incomplete. We hypothesize that EphA2, a member of the Eph family of receptor tyrosine kinases, is involved in the sequestration of CD8+ T cells in the brain during ECM through its interactions with membrane-bound ephrin-A ligands on CD8+ T cells. Using the PbANKA model of ECM, we show that EphA2 is upregulated in the brains of mice six days post-infection at the onset of cerebral symptoms in a lymphotoxin-α dependent manner. Ephrin-A ligands are also upregulated on splenic T cells of mice infected with PbANKA three days post-infection prior to potential trafficking to the brain. Importantly, EphA2-/- mice show significant resistance to death from ECM compared to EphA2+/+ mice. Similar numbers of pRBCs accumulate in the brains of EphA2-/- and EphA2+/+ mice, but EphA2-/- mice have less CD8+ T cells in the brain six days post-infection correlating with improved survival. Our data demonstrates a novel potential role for the EphA2 receptor in the pathogenesis of ECM.
Host vitamin D status is dependent on the microbiota. (MUC9P.747)
Authors: Bora Stephanie , Smith Philip , Patterson Andrew , Cantorna Margherita
Published: January 2023

Vitamin D deficiency is linked to susceptibility to inflammatory bowel disease (IBD). Mice deficient in vitamin D are more susceptible to experimental colitis than supplemented mice. Vitamin D alters the microbiota suggesting some of the effects of vitamin D on IBD are due to changes in the microbiota. Here we explored how the microbiota affect vitamin D metabolism using germ-free (GF) mice. Vitamin D is converted to 25D3. 25D3 is converted primarily in the kidney to 1,25D3, by 1α-OHase (Cyp27B1), or to 24,25D3 for degradation. Expression of Cyp27B1 was significantly higher in conventional (CV) versus GF kidneys. GF mice were given vehicle or 5μg vitamin D3 for 14 days and then conventionalized while maintaining their respective treatments for another 14 days. Liquid chromatography mass spectrometry was used to measure plasma vitamin D3, 25D3, 24,25D3, and 1,25D3 from GF mice before and after conventionalization. Vitamin D supplementation was less effective in GF mice. Conventionalized mice absorbed more vitamin D and had higher 25D3, 1,25D3, and 24,25D3. Furthermore, after correcting for differences in absorption between GF and CV mice, 1,25D3 was still significantly decreased in GF mice. This indicates GF mice neither absorb nor metabolize vitamin D normally. Differences in vitamin D metabolism between GF and CV mice suggest the microbiota play a role in regulating host vitamin D status, which could affect host immune responses and susceptibility to diseases like IBD.
Aryl hydrocarbon receptor activation enhances total but not antigen-specific IgA secretion in Leishmania major-infected mice (MUC9P.748)
Authors: Metten Maddie
Published: January 2023

Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some mucosal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to transiently decrease total fecal IgA levels but increase antigen-specific IgA levels after mucosal antigen challenge. The goal of this study was to determine if TCDD exposure would alter secretion of antigen-specific IgA levels after systemic pathogen challenge. Female C57Bl/6 mice were administered TCDD at 0 or 40 μg/Kg (po) one day prior to infection with Leishmania major promastigotes. Three weeks later, IgA levels were measured by ELISA. L. major-specific IgA levels in serum were readily detected but unchanged by TCDD treatment. L. major-specific lgA was undetected in the feces of any animal. In contrast, total fecal IgA levels (OD) were significantly increased by 34% in TCDD treated animals (p < 0.05). Total spleen and bone marrow cell numbers were reduced significantly in TCDD-treated mice. The expression of IgM heavy chain, IgJ chain, or kappa light chain in these cells (measured by RT-RT-PCR) was unchanged by TCDD treatment. In contrast, the expression of IgA heavy chain was significantly reduced by approximately 50% in TCDD-treated animals. These results suggest that AhR activation enhances bulk IgA secretion but does not enhance secretion of antigen-specific IgA following systemic pathogen challenge.
Dynamic vaginal microbiota in pigtail macaques is associated with menstrual cycle and inflammation (MUC9P.753)
Authors: Klatt Nichole , Srinivasan Sujatha , Gile Jillian , Hensley-McBain Tiffany , Fiedler Tina , Fredricks David
Published: January 2023

A challenge in prevention of mucosal HIV transmission is incomplete understanding of correlates of vaginal transmission, including mucosal inflammation. Vaginal SIV/SHIV transmission in pigtail macaques (Macaca nemestrina) is an excellent model for HIV transmission, however little is known about how genital microbes may influence inflammation and transmission. Broad-range 16S rRNA gene PCR and pyrosequencing was performed on vaginal swabs to assess vaginal microbiome in healthy pigtail macaques. Bacteria were identified to the species or genus level. Luminex was used to measure cytokine and chemokine levels in vaginal swabs. Pigtail macaques had diverse bacterial communities at all phases of the menstrual cycle, with a range of dominant bacteria. Several species were identified that are commonly found in bacterial vaginosis (BV) in humans, and three animals exhibitied Lactobacillus-dominance. Longitudinal sampling demonstrated that the vaginal microbiome is dynamic and possibly influenced by the menstrual cycle. Animals with Lactobacillus-dominant vaginal microbiota had decreased IL-8 (p=0.009) and IL-1Ra (p=0.044). In conclusion, the macaque vaginal microbiota is diverse, dynamic, and can resemble the human vaginal microbiota, including BV-associated bacteria as well as Lactobacillus spp, which is associated with decreased innate inflammation. These data provide a foundation for understanding how the vaginal microbiome may impact risk of HIV/SIV transmission.
Gut commensal dependence of autoreactivity and Th17 cells in systemic autoimmunity (MUC9P.740)
Authors: Manfredo Vieira SILVIO , Ruff William , Hiltensperger Michael , Yu Andrew , Goodman Andrew , Kriegel Martin
Published: January 2023

Infectious triggers have been implicated in transient antiphospholipid antibody production but the cause of chronic antiphospholipid syndrome (APS) is unknown. We disrupted the gut microbiota composition in the (NZWxBXSB)F1 model of APS using antibiotics after maturation of the immune system. Targeting the microbiota with broad-spectrum antibiotics (ABX), vancomycin or ampicillin, but not neomycin or metronidazole, lowered pathogenic autoantibodies and mortality (p=0.014). On histology, manifestations of the autoimmune clotting disorder, i.e. myocardial infarctions, pulmonary emboli and strokes, were prevented. 16S rDNA sequencing on the MiSeq platform revealed preliminarily depletion of similar gram-positive anaerobic communities with vancomycin and ampicillin as were enriched in longitudinally collected human APS samples compared to controls. Systemic Th17 cells were detectable in our cohort in the absence of Th17-inducing segmented filamentous bacteria (SFB) but were reduced with ABX. IL-17 and Th17-differentiating factors IL-1b and IL-21 were diminished in serum whereas regulatory IL-10 was increased. Autoantigen- but not global anti-CD3-induced splenocyte proliferation was blunted after ABX (p=0.0012) supporting antigen-specific effects of the gut microbiota in systemic autoimmunity. In summary, gram-positive anaerobic gut microbiota are fundamentally involved in the pathogenesis of murine APS and systemic Th17 responses with broad implications for systemic autoimmunity.
Commensal flagellated A4 bacteria inhibit intestinal Th2 responses through induction of innate cell TGFβ production (MUC9P.742)
Authors: Wu Wei , Liu Houpu , Cao Anthony , Yao Suxia , Chen Feidi , Liu Han , Evans-Marin Heather , Duck Wayne , Elson Charles , Cong Yingzi
Published: January 2023

Interaction between host and microbiota maintains intestinal immune homeostasis. While commensal bacteria promotes Th1, Th17, and Tregs cells in lamina propria (LP) in the steady condition, it suppresses mucosal Th2 cells. Some specific commensal organisms have defined effects on mucosal immune function, e.g., SFB preferably promotes intestinal Th17 while certain Clostridium spp induce Tregs. However, it is still unclear whether there are specific commensal organisms down-regulating Th2 responses, and the mechanism involved. In this report, we showed that there were more LP Th2 cells in germ-free mice compared to the mice under SPF conditions. A4 bacteria, a commensal isolated from mouse cecum and produces immunodominant microbiota antigen, CBir1 flagellin, presented in intestinal lumen. In CBir1 TCR Tg mice, Th1, Th17 and Treg but not Th2 cells were present in the intestinal LP. When stimulated with CBir1 flagellin, naïve CBir1 Tg T cells differentiated into Th1, Th17 and Tregs but resisted Th2 differentiation. Aaddition of A4 bacteria, as well as pretreatment of APC with A4 bacteria, inhibited Th2 differentiation of OTII T cells stimulated with OVA and of wild-type T cells stimulated with anti-CD3 under Th2 polarizing conditions. A4 bacteria stimulated DC production of TGFβ, blockade of TGFβ abrogated A4 inhibition of Th2 cell development. Collectively, our data reveal that A4 bacteria inhibit intestinal Th2 responses through induction of innate cell production of TGFβ.
Chronic ingestion of low doses of cadmium alters the gut microbiome and immune homeostasis to enhance allergic sensitization (MUC9P.743)
Authors: Kim Eunsoo , Xu Xiaohua , Steiner Haley , Ahmer Brian , Cormet-Boyaka Estelle , Boyaka Prosper
Published: January 2023

Low doses of cadmium can be ingested due to its presence in contaminated water and its accumulation in leafy vegetables, fish and grains. Pollutants are believe to contribute to the increased incidence of allergy diseases. We addressed whether chronic ingestion of low doses of cadmium could impact allergic sensitization and thus, favor the development of allergic diseases. Conventional C57BL/6 mice given low doses of cadmium in the drinking water for 28 days exhibited a significant reduction of bacterial diversity, and an alteration of the Firmicutes to Bacteriodetes ratio. This treatment also activated both the canonical and the non-canonical NF-κB pathway and promoted proinflammatory cytokine and antimicrobial responses in the gut. The effects of cadmium were at least partially independent of the gut microbiome since germ-free C57BL/6 mice subjected to the same treatment developed the same profile of responses although at a lower degree. Finally, conventional mice chronically treated with low doses of cadmium developed higher antigen-specific IgE responses upon oral sensitization with OVA and cholera toxin as adjuvant. Furthermore, upon nasal antigen, cadmium-treated mice developed higher airway allergic response, which were characterized by the increased levels of IL-17 and Th1 responses control mice. In summary, the environmental pollutant cadmium can be a major regulator of gut immune homeostasis and a cause for increased allergic responses at distant mucosal sites.
Immuno-spin trapping to detect immune-derived free radicals in type 1 diabetes (TECH2P.903)
Authors: Tse Hubert , Anderson Brian , Ganini Douglas , Mason Ron
Published: January 2023

In autoimmune Type 1 diabetes (T1D), islet-infiltrating leukocytes synthesize noxious free radicals to collectively mediate b-cell lysis and enhance diabetogenic T cell responses as a pro-inflammatory third signal. We recently demonstrated a crucial role for NADPH oxidase (NOX)-dependent superoxide in T1D, as NOX-deficient Non-obese diabetic (NOD.Ncf1m1J) mice were highly resistant to autoimmune diabetes due to an enhanced alternatively-activated M2 macrophage phenotype, diminished pro-inflammatory cytokine and Type I interferon synthesis upon Toll-like receptor stimulation, dampened Th1 cytokine responses, and blunted CD8 T cell cytotoxicity. To further define the synergy between free radicals and T1D, we employed a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) immuno-spin trapping technique to identify the generation of free radicals upon T cell activation and infection with Coxsackie B4 (CB4) virus, a putative diabetogenic enterovirus and hypothesized that NOD.Ncf1m1J immune cells would exhibit severe reductions in DMPO adducts. In the absence of superoxide, NOD.Ncf1m1J macrophages and CD4 T cells exhibited a significant decrease in DMPO adducts upon CB4 infection and autoantigen stimulation, respectively, in contrast to wild type cells. Future studies will utilize this technique to visualize the interface between free radicals and immune cells during spontaneous T1D progression and determine if immuno-spin trapping can be used as a biomarker during spontaneous T1D progression.
Ribosome profiling to investigate in vivo molecular mechanisms of immune function (TECH2P.906)
Authors: Jin Hyun Yong , Xiao Changchun
Published: January 2023

Genomic analyses of gene expression of immune cells have so far focused on their transcriptional program by quantifying mRNA abundance, measured by microarray or more recently by RNA-seq. The mRNA abundance is often presumed as a proxy of the gene expression, but the correlation between mRNA abundance and protein level, which is the true end point of gene expression, frequently shows lack of sufficient correlation due to the apparent regulation of gene expression at the layer of translation. Here, we present the applications of an emerging technique, Ribosome profiling (Ribo-seq), on primary immune cells derived from various genetic mutant mice developing immune diseases. This directly captured genome-wide maps of protein synthesis (translatome) by allowing quantification of ribosome footprint of each mRNA with high resolution and depth. Side-by-side comparison of transcriptome and translatome in the disease models revealed the signaling pathways that are predominantly regulated in translation levels are prevalent, highlighting the critical roles of translational control in immune function.
Novel single wall carbon nanotube electrode system capable of quantitative detection of CD4+ T cells (TECH2P.909)
Authors: Park Gayoung , Kim Joon Hyub , Min Nam Ki , Lee Kyung-Mi
Published: January 2023

We developed a fully-integrated SWCNT-based immunosensor capable of selective capture and linear quantification of CD4+ T cells by employing the two-step O2 plasma treatment processes. These processes overcome the current hurdle in the quantification of a specific subset of lymphocytes by removing the byproduct of spray-coated SWCNTs that hinders charge transfer and by creating large number of anchoring sites for anti-CD4 antibodies for stable cell binding. As a result, a linear range of electrochemical signal is generated by SWCNT electrode in proportion to the bound CD4+ T cells. Our CD4+ T cell-immunosensor provides a simple and low-cost platform which can fulfill the requirement for the development of point of care diagnostic technologies for HIV patients in the resource-limited countries.
Intranasal immunization with a chimeric influenza antigen delivered by detoxified anthrax toxin induces broad spectrum immunity against influenza viruses (TECH2P.756)
Authors: Diaz-Arevalo Diana , Arévalo Maria T. , Li Junwei , Navarro Ashley , Chen Yanping , Zeng Mingtao
Published: January 2023

Influenza remains to be a significant public health problem worldwide, despite the heavy campaign for flu vaccination in many countries. The conventional influenza vaccines are based on stimulating immune responses against hemagglutinin by virus attenuation or inactivation. However, they must be reformulated annually because of antigenic shift and drift of circulating influenza viral strains. These seasonal vaccines do not always match the circulating strains, and there is the ever-present threat that avian influenza viruses may adapt to be transmitted in humans. Development of a broadly protective or universal influenza vaccine would have a significant impact on public health. We constructed a new chimeric influenza antigen, which is composed of the relatively conserved matrix protein 2 (M2) and stem region of hemagglutinin (HA2) of influenza A viruses in combination with a detoxified anthrax edema toxin delivery system. This vaccine candidate has been shown to elicit T cell and humoral responses against influenza antigens and cross-strain protective immunity against influenza viruses, when it is intranasally delivered to mice. In addition, the vaccination of CD4+ and CD8+ T cell knock out mice do not protect against influenza virus infection. Thus, this novel vaccine strategy induces the CD4+ and CD8+ memory T cell responses which play an important role in cross-strain protection against influenza viruses.
A model of somatic hypermutation targeting in mice based on high-throughput immunoglobulin sequencing data (TECH2P.910)
Authors: Cui Ang , Diniro Roberto , Briggs Adrian , Adams Kris , Vander Heiden Jason , O'Connor Kevin , Vigneault Francois , Shlomchik Mark , Kleinstein Steven
Published: January 2023

Analyses of somatic hypermutation (SHM) patterns in B-cell immunoglobulin (Ig) genes have important basic science and clinical applications, but are often confounded by the intrinsic biases of SHM targeting on DNA motifs (i.e., hot/cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized heavy-chain transgenic mice with nitrophenyl (NP) to generate NP-specific λ+ B cells, and used next-generation sequencing to examine the unproductive κ light chains. Most of these sequences had out-of-frame junctions and were presumed uninfluenced by selection. Despite being non-functionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We identified 29,005 mutations and used them to construct a quantitative SHM targeting model, which was consistent with classic hot/cold-spots, but revealed additional highly mutable motifs. We observed comparable targeting for functional and non-functional sequences, suggesting similar biological processes operate at both loci. In contrast, the level of C to T transitions in mice was higher than in human κ chains, suggesting lower levels of DNA repair activities. In summary, our model of SHM targeting for mice provides insights into the SHM process and supports future analyses of mutation patterns.
Quantitative proteomics reveals a role for epigenetic reprogramming during human monocyte differentiation (TECH2P.757)
Authors: Zhang Kangling , Nicholas Dequina , Tang Hui , Zhang Qiongyi , Xu Feng , Langridge William
Published: January 2023

Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. While highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.