bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel scaffolding proteins and mechanisms mediating RIG-I signaling complex assembly (INM2P.351)
Authors: Krishnan Manoj , Pulloor Niyas , bist pradeep 
Published: January 2023

Abstract
The cytosolic innate-immune receptor RIG-I is very important for the detection and host response against several RNA-viruses. During viral challenge, RIG-I activates mitochondria-associated adaptor MAVS to recruit several downstream cytosolic signaling proteins, resulting in transcription factor IRF3 activation and Type-I interferons transcription (TI-IFN). However, the mechanisms coupling activation of organelle-anchored MAVS with key downstream cytosolic components is incompletely understood. Through a human genome-wide RNAI screen, we identified several novel regulators of RIG-I signaling. Furthermore, we identified a key novel scaffolding protein (named RIG-I assembly mediator protein, RAMP) that links MAVS with downstream TBK1 activation, IRF3 phosphorylation and TI-IFN transcription during virus infection. RAMP functioned as a scaffolding platform that interacted and assembled a complex containing MAVS, TBK1, NEMO and IRF3. Lack of RAMP expression led to the failure of MAVS to interact with downstream NEMO, TBK1, and IRF3. Recombinant RAMP supported IRF3 activation in a cell free assay. RAMP was essential for antiviral cellular resistance, and infection induced subcellular re-localization of RAMP. Furthermore, our data demonstrated that RAMP and TRAF proteins act in parallel to transduce signals from MAVS to IRF3. This study provided important novel insights into the regulation and assembly of the RIG-I signalosome and TI-IFN response.
Ubiquitination-mediated regulation of RIP2 in NOD2 signaling and MDP tolerance induction (INM2P.354)
Authors: Sukumaran Bindu , bist pradeep , Ng Aylwin , Kim Bae Hoon , Kudukkil Pulloor Niyas , Khameneh Hanif , Hedl Matija , Dikshit Neha , Shenoy Avinash , Balamuralidhar Vanniarajan , Hong Michelle , Neutzner Albert , Chin Keh-Chuang , Kobayashi Koichi , Bertoletti Antonio , Mortellaro Alessandra , Abraham Clara , MacMicking John , Xavier Ramnik 
Published: January 2023

Abstract
The cytoplasmic innate immune receptor NOD2 is required for antibacterial immune response. Optimal regulation of NOD2 is essential for controlling bacterial infections and inflammatory disorders. Recently, ubiquitination has emerged as an important posttranslational modification required to control NOD2-signaling. Yet, the specific ubiquitination mechanisms that negatively regulate NOD2-signalling during both acute and chronic (e.g., MDP-tolerance) stimulations are incompletely understood. Through a human genome-wide-RNA-interference screen, we identified an E3-ubiquitin ligase {termed as NOD2 Signaling Regulator (NSR)} as a novel negative-regulator of NOD2-mediated NF-KB, cytokine, and antibacterial responses. Mechanistically, NSR induced K48-linked ubiquitination of RIP2 (NOD2-adaptor), and promoted RIP2 degradation through the endoplasmic-reticulum-associated-degradation (ERAD) pathway. We identified the endoplasmic reticulum as a new regulatory site for NOD2-signaling, where RIP2 was localized and interacted with NSR and ERAD. In vivo NSR knock-down in mice resulted in increased NOD2-mediated NF-KB activation. NSR-mediated RIP2 degradation was critically required for MDP-tolerance induction both in vivo and in vitro. Thus, this study unravels a key molecular mechanism by which NOD2 pathway is attenuated, and provide new directions to understand the role of NOD2 pathway in both infectious and inflammatory diseases.
NLRP12-deficiency results in TNFα-mediated bone marrow apoptosis during emergency hematopoiesis (INM2P.355)
Authors: Linz Brandon , Neely Crystal , Kartchner Laurel , Malik Julia , Brickey Willie , Ting Jenny , Cairns Bruce , Maile Robert 
Published: January 2023

Abstract
The hematopoietic system can rapidly expand in response to tissue damage. NLRP12, expressed in leukocytes and bone marrow cells, has been implicated in regulation of immune responses but remains controversial if it acts as an inflammasome or a regulator of NF-κB signaling. We tested the hypothesis that NLRP12 regulates immune responses and reconstitution as a direct regulator of NF-κB signaling using an emergency hematopoiesis model. To test this, C57BL/6 wild type and Nlrp12-/- female mice underwent a radiation-thermal combined injury (RCI) consisting of 20% total body surface area burn and 5Gy γ-radiation. In wild type mice, we observed elevated NLRP12 expression in target tissues after RCI compared to controls. Following RCI, Nlrp12-/- mice failed to reconstitute peripheral innate neutrophil and monocyte populations, had increased levels of serum TNFα, and TNFα-mediated apoptosis of bone marrow progenitor cells. These data suggest that NLRP12 is essential for immune reconstitution after combined radiation and burn injuries. We have uncovered a central role for NLRP12 in regulating granulopoiesis following immune ablation and NFκB cytokine- and receptor-expression. Accordingly, we propose that NLRP12 acts as an important regulator of NF-κB signaling during events that trigger hematopoeisis, suggesting a novel role of NLRP12 in immune reconstitution following injury and immune depletion.
Streptolysin O hijacks the membrane repair system to blind natural killer cell (INM3P.405)
Authors: Bhattacharjee Pushpak , Romero Matthew , Keyel Peter 
Published: January 2023

Abstract
The pore-forming toxin Streptolysin O (SLO) secreted by Group A Streptococcus binds to cholesterol and oligomerizes on the membrane to form a lytic pore. Immune cells survive pore-formation through a Ca2+ dependent process that sequesters and sheds pores on vesicles. These vesicles are enriched in cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins, raising the possibility that immune receptors are shed during the repair process. NKG2D, IL-18 receptor and IL-12 receptor promote Interferon-γ (IFNγ) production in natural killer (NK) cells and all associate with cholesterol-rich microdomains or GPI-anchored proteins. Here we found SLO challenge impaired IFNγ secretion from NK cells when stimulated with K562 cells or IL-12 and IL-18 without affecting NK cell viability. We found that the IL-12 Receptor β1 was shed from NK cells on microvesicles along with SLO in a Ca2+ dependent fashion. These data suggest that shedding of activation receptors during membrane repair impairs NK cell function. Taken together, these data describe one way in which bacterial toxins can hijack membrane repair to blind cells to the presence of pathogens.
USP18 as a novel regulator of PD-1 and IFNAR1-mediated immune dysfunction (IRM11P.629)
Authors: Wood Laurence , Mavinkurve Vikram , Muthukumaran Geetha , cabasal marian , dauphinee shauna , Malo Danielle , Paterson Yvonne 
Published: January 2023

Abstract
Immune dysfunction induced by cancer and chronic viral infection contributes to the pathology and progression of each of these diseases. Recent studies have identified a novel player associated with poorly functioning immune cells during chronic viral infection, the ubiquitin-specific protease Usp18. Usp18 is a negative regulator of innate anti-viral responses through direct inhibition of Type I IFN receptor signaling. However, the role of Usp18 in maintaining functional immunity is still unclear. To investigate the role of Usp18 in immunity, we have utilized Usp18-deficient mice and assessed immune function during infection with the Th1 pathogen Listeria monocytogenes (Lm). We find that Usp18 deficiency results in severe susceptibility to acute Lm infection and an inability to develop functional adaptive immunity to Lm. Severe susceptibility to Lm infection in the Usp18-/- mouse is associated with poor inflammatory cytokine responses and blunted splenic T cell function. The immune tolerant state in Usp18-/- mice is partially regulated by the PD-1 inhibitory receptor as blockade partially restores innate responses to Lm. However, blockade of IFNAR1 signaling resulted in significant reversal of this immunosuppressive state and a restoration of T cell function. This study identifies Usp18 and IFNAR1 signaling as critical mediators of immune dysfunction, a finding that may have broad implications in the treatment of cancer and chronic viral infection.
Molecular screening of Mycobacterium vaccae for identification of novel anti-inflammatory compounds (IRM12P.651)
Authors: Smith David , Cantley Alexandra , Clardy Jon , Lowry Christpher 
Published: January 2023

Abstract
Mycobacterium vaccae is a saprophytic, non-pathogenic bacterium. Administration of M. vaccae in activates regulatory T cells (Treg) and attenuates allergic responses. M. vaccae-induced activation of Treg may be initiated through interactions with sentinel immune cells, such as macrophages. We are interested in identifying the particular immunoregulatory molecules produced by M. vaccae that suppress inflammatory signaling in macrophages. Consequently, we developed a reiterative cell-based screen as an unbiased approach to search the M. vaccae chemical landscape for potential anti-inflammatory small molecules. M. vaccae was grown in 7H9 broth as a mixture of both smooth and rough variants.We prepared hexane, ethyl acetate, and aqueous extracts of live M. vaccae cultures; cell-based assays were used to guide successive steps of purification. For cell-based assays, 100 µg/ml of the extracts were incubated with freshly isolated murine peritoneal macrophages, and, after one hour, the cultures were stimulated with 1 µg/ml of lipopolysaccharide (LPS). Interleukin 6 (IL-6) concentrations in the supernatant 12h after stimulation were measured via ELISA as a determinant of inflammatory response. After 5 iterations of screening and evaluation of 80 fractions, we identified 3 fractions from the ethyl acetate extract with potent anti-inflammatory properties. From mass spectrometry, we expect the molecules to be lipid conjugates, and we are in preparation for a structural analysis.
SLAM family receptors are key drivers of CD8 T cell immunity towards B cells (IRM12P.657)
Authors: Priatel John , Huang Yu-Hsuan , Tsai Kevin , Harder Kenneth , Tan Rusung 
Published: January 2023

Abstract
Epstein-Barr virus (EBV) poses serious health consequences for immunocompromised individuals, such as patients undergoing transplant or chemotherapeutic regimens, and is strongly associated with an expanding list of human cancers. To gain insight into immunity against EBV, a rare congenital immunodeficiency defined by exquisite sensitivity to EBV but not other pathogens called X-linked lymphoproliferative disease, a syndrome caused by mutations in the SH2D1A gene that encodes SLAM-associated protein (SAP), has been the subject of intense investigation. The unique B cell tropism of EBV led us to speculate that SAP and SLAM family receptors are especially critical for optimal CD8 T cell immunity against antigen-expressing B cells and B lymphoma cells. Consistent with this hypothesis, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells or B lymphoma cells in vitro. By contrast, both wild type and Sh2d1a-/- CD8 T cells responded robustly when stimulated with either antigen-coated B cell-depleted splenocytes or antigen-presenting tumor cells lacking SLAM family receptor expression. Collectively, these observations suggest that the recognition of SLAM family receptors on the surface of antigen presenting-B cells and -B lymphoma cells are essential for antigen priming and immunosurveillance by CD8 T cells.
Recombinant human IL7 enhances naïve and T stem cell memory reconstitution and enhances repertoire diversity following cytotoxic chemotherapy (LYM5P.705)
Authors: Zhang Hua , Robins Harlan , Mackall Crystal 
Published: January 2023

Abstract
T cell immune reconstitution in humans is limited by poor thymic reserve. Hence, dose intensive cytotoxic chemotherapy often induces profound, prolonged lymphopenia. Lymphopenia can increase the effectively of adoptive cell therapy but also has detrimental consequences related to diminished repertoire diversity, which limits the hosts’ capacity to respond to new antigenic challenges. In this study, we analyzed circulating T cell subsets in a cohort of 50 children and young adults rendered lymphopenic via cytotoxic chemotherapy, and compared immune reconstitution ± rhIL-7. Chemotherapy induced profound T cell depletion with decreased frequencies of CD4+ and CD8+ naïve cells, but increased frequencies of CD4+ and CD8+ T stem cell memory cells. RIL-7 therapy (20 mcg/kg SQ every 2 wk) beginning 6 wk after completion of chemotherapy, increased total numbers of CD4+ and CD8+ T cells as well as CD4+ and CD8+ naïve, central memory, stem cell memory and CD4+ effector memory cells. Repertoire diversity of specific subsets was measured sequentially in three subjects from each treatment group using next generation sequencing. Chemotherapy induced substantial reductions in the number of unique TCR sequences/mcl of blood, which was dramatically reversed in patients treated with rhIL7 compared to those who were not. Thus, rhIL-7 therapy can rapidly restore T cell repertoire diversity in patients following cytotoxic chemotherapy.
Efficient culture of human naïve and memory B cells (LYM5P.711)
Authors: Su Kuei-Ying , Kuraoka Masayuki , Watanabe Akiko , Kelsoe Garnett 
Published: January 2023

Abstract
The ability to culture and expand B cell numbers in vitro has become a useful tool for studying human immunity. A limitation of current methods in human B cell cultures is the capacity to support continued proliferation by mature B cells. We have adapted the culture methods established by Luo et al. (Blood, 2009) to efficiently (60% cloning efficiency) activate and expand both naïve and memory human B cells. Briefly, naïve or memory B cells recovered from blood are cultured with cytokines on CD154+ feeder cells; this culture supports extensive B cell proliferation, with approximately 103 fold increases in a week; by splitting cultures onto new feeder layers, culture-derived (CD) B cells continuously proliferate, achieving 106 fold increases by day 16. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells upregulate AID expression, undergo isotype switching without V(D)J hypermutation, and terminally differentiate into plasmacytes. CD B cells are readily cryopreserved and retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. We have examined the APC function of CD B cells and find that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.
Levels of the pro-apoptotic molecule Bim are determined early in the response and positively correlated with memory T cell fate (LYM5P.702)
Authors: Li Kun-Po , Sholl Allyson , Hildeman David 
Published: January 2023

Abstract
We and others have shown that the pro-apoptotic molecule Bim plays a critical role in controlling the contraction of T cell responses. Paradoxically, T cells destined to become memory (pre-memory) cells have higher levels of Bim than their effector counterparts. Similarly, after memory development, central memory (TCM) cells have higher levels of Bim relative to their effector memory (TEM) counterparts. However, due to the intracellular location of Bim, sorting and subsequent tracking of T cells based on Bim expression has been challenging. To circumvent this issue and determine the relationship between Bim expression and cell fate, we generated Bim-mCherry reporter mice, inserting an IRES-mCherry cassette into the 3’-UTR of the Bim gene. Similar to intracellular staining for Bim, mCherry levels were higher in pre-memory cells and TCM cells than their effector or TEM counterparts. In addition, the expression and function of Bim in T cells is not altered in Bim-mCherry reporter mice. The transfer of Bimhi or Bimlo T cells on the basis of mCherry expression favored their development into TCM or TEM cells, in recipient animals, respectively. Further, the levels of mCherry (and Bim) remained stable in the transferred mCherryhi or mCherrylo T cells across contraction of the response. These data suggest that the levels of Bim in CD8 T cells are determined at an early stage of infection and are paradoxically positively correlated with T cells having a memory fate.
Virtual memory cells: a consequence of self-affinity-linked naïve CD8 T cell heterogeneity (LYM5P.703)
Authors: White Jason , Walton Kendra , Danhorn Thomas , O'Connor Brian , Kedl Ross 
Published: January 2023

Abstract
Memory phenotype (MP), CD44hi CD49dlo CD8 T cells are found in nearly all mice—lymphoreplete, pathogen naïve, and even TCR transgenics. The presence of MP was long thought to be due to environmental pathogen exposure (or, in the case of transgenics, heterologous immunity to same), but studies establishing the presence of these cells in germ-free mice suggested that this was not the case. These cells, dubbed Virtual Memory (VM), are of special relevance due to their increased effector functioning, and, in contrast to other MP CD8 T cells, develop in the periphery with a dependence on IL-15. Our recent studies have determined that VM cells develop in a manner reminiscent of lymphopenic homeostatic expansion, whereby the highest self-affinity T cells (as measured by CD5) interact with available IL-15 to turn into VM cells. This indicates that the naïve CD8 T cell pool is not as homogeneous as usually assumed, and RNA sequencing has further confirmed the heterogeneity of the CD8 naïve T cell pool (separated on CD5). The sequencing has further implicated VM cells in mediating CD8 bystander protection, and ongoing studies suggest that this is indeed the case, with the VM bystander effects being IL-15 dependent.
Generation of central memory T cells by vaccination that mimics natural infection (LYM5P.704)
Authors: Dalai Sarat , Bhurani Vishakha , Mathur Aditi , Patel Hardik 
Published: January 2023

Abstract
The goal of vaccination is to generate long-lived antigen specific memory T & B cells. Often we notice that the immune responses generated by the vaccines fail to achieve the desired goal, particularly the T cell responses. In many infection models it is well established that long-lived memory T cells could be generated in hosts exposed to pathogens through natural infection. Here we are adopting the strategies that would mimic natural infection with the hope to induce immune responses in a manner similar to that triggered during natural infection. In our study we have mimicked the natural infection to vaccinate the host by (i) providing the danger signals to the immune system before or during encounter of Ag to mimic the course of infection, (ii) combining non-target Ag(s) with target Ag, while priming the host in the presence of danger signals.We have used OVA as the model antigen to immunize C57BL/6 mice, and Poly I:C/R848 as danger signals to represent viral infection. For non-target antigens, we have used immunogenic (HEL) as well as non-immunogenic (MBP) antigens. We found that vaccination through mimicking course of infection favors generation of Central MemoryT cells whereas conventional vaccination favors effector memory T cells. When we mimicked natural infection to vaccinate the host by including poorly immunogenic non-target antigen with the vaccine target we found it to favor generation of central memory T cells compared to that of immunogenic non- target.
In situ hematopoiesis-derived mast cells promote protective immunity to Trichinella spiralis (MPF4P.738)
Authors: Siracusa Mark , Henry Everett , Sy Chandler 
Published: January 2023

Abstract
In situ hematopoiesis (ISH) refers to the differentiation of hematopoietic stem/progenitor cells (HSPCs) into effector cells that occurs at active sites of inflammation. Previous studies have demonstrated that ISH contributes to the population expansion of basophil and mast cell populations in the context of multiple helminth infections. However, whether ISH-derived effector cell populations are critical regulators of TH2 cytokine-mediated inflammation and protective immunity to helminth parasites remain unknown. Here, we demonstrate a previously unrecognized role for ISH in promoting the population expansion of intestinal mast cell populations, TH2 cytokine responses and protective immunity to Trichinella spiralis. Further, discovery based-studies suggest that helminth-induced HSPCs exhibit distinct transcriptional profiles compared to their bone marrow-resident counterparts. These data demonstrate that ISH contributes to the development of protective immunity to helminth parasites and that helminth-induced HSPCs are phenotypically distinct cells that may represent viable therapeutic targets.
Activation of inflammasome by rickettsiae in human and murine macrophages (MPF4P.727)
Authors: Smalley Claire , Bechelli Jeremy , Zhao Xuemei , Walker David , Fang Rong 
Published: January 2023

Abstract
Rickettsiae are Gram-negative, obligately intracellular bacteria that infect macrophages. However, the molecular mechanisms involved in interaction of rickettsiae with macrophages remain poorly understood. In this study, we investigated the inflammasome activation by rickettsiae in human and mouse macrophages. R. australis activated the inflammasome in mouse bone-marrow derived macrophages (BMDM), marked by caspase (casp)-1/11-dependent secretion of IL-1β and IL-18, and induction of pyroptosis. Kinetic studies revealed that low dose (MOI 2) R. australis activated the inflammasome in mouse macrophages at 12 h post infection (p.i.), while high dose (MOI 6) activated at 8 h p.i., with no significant difference in activation between doses at 24 h p.i.. In contrast, R. australis activated the inflammasome in human THP-1 derived macrophages at as early as 3 h p.i., resulting in production of IL-1β, IL-18, induction of pyroptosis, and activation of casp-1. Studies using siRNA and inhibitors specific for casp-4 suggest that rickettsiae activated non-canonical inflammasome pathway in human macrophages. Taken together, our data, for the first time, illustrated the activation of inflammasome by rickettsiae in both mouse and human macrophages, although with different kinetic mechanisms. This study provides invaluable information for developing novel therapeutic interventions for rickettsioses by employing current knowledge derived from mouse infection models.