bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rescuing cancer immunity through formins and the actin cytoskeleton (VAC3P.1063)
Authors: Howard Andrew , Alberts Arthur 
Published: January 2023

Abstract
Immunotherapy strives to replace radiation, chemo and surgery in the fight against cancer by reversing cancer’s suppression of the immune system. CTLA-4 and PDL1 checkpoint blockade inhibitors are emerging therapies that release anti-tumor T cells to kill the tumor. However, cancer also alters myeloid activation to systemically suppress the immune system, which may explain the range of responses to checkpoint blockade inhibitors. We strive to better understand myeloid activation in cancer and develop therapies to modulate these altered responses. Through in vitro myeloid activation assays, we investigated the mechanism of action for two established immunomodulatory compounds, lenalidomide and tipifarnib, as well as tested novel small molecule Intramimics, developed by our lab, for immunomodulatory function. We found that lenalidomide and tipifarnib modulate immune responses through their activation of Rho GTPases and their downstream effectors to promote F-actin assembly. Interestingly, Intramimics prove to be better at modulating immune responses as they directly activate the diaphanous related formin effectors of Rho GTPases. We propose that modulation occurs mechanistically through the MADS box transcription factor SRF, which senses actin dynamics within the cell. We intend to confirm the dependency of SRF in immunomodulation and test these therapeutics in combination with checkpoint blockade inhibitors in preclinical cancer models in the future.
Genetically modified live attenuated L. donovani parasite induces classical activation of macrophages (M1 phenotype) leading to generation of Th1 response in BALB/c mice (VAC4P.1102)
Authors: Bhattacharya Parna , Dey Ranadhir , Dagur Pradeep , kruhlak Michael , Debrabant Alain , Okeeffe Nevien Ismail , McCoy John , Nakhasi Hira 
Published: January 2023

Abstract
Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. We have tested live attenuated L. donovani parasites (Ldp27-/-and LdCen-/-) (LAPs) as vaccine candidates and demonstrated the protection through induction of an adaptive immune response. However the immunological basis of LAP mediated protection in terms of host innate response is poorly understood. In this study we showed, LAPs induced classical activation in bone marrow derived macrophages (BMDMs) as observed by significantly higher production of proinflammatory cytokines and nitric oxide and attenuated arginase-1 expression compared to LdWT parasites. Unlike LdWT infection, LAP infection did not alter the membrane fluidity of BMDMs, showed no impairment of antigen presentation and induced robust CD4+ T cell activation in vitro. Similarly, parasitized splenic macrophages from LAP infected mice showed up regulation of genes encoding classical activation markers such as IL-1β, TNF- α , IL-12 and iNOS2 and down regulation of genes associated with alternative activation like IL-10, YM1, Arg-1 and MRC-1 compared to LdWT infection. Furthermore, BMDM from LAP infected mice induced more IFN-γ and less IL-10 production by Leishmania antigen specific T cells than WT infected mice, thereby leading to proliferation of Th1 cells suggesting that LAP infection promotes a state of classical activation in macrophages in vitro and in vivo, leading to the generation of host protective Th1 response.
MicroRNA-155 plays a critical role in the establishment of latency in murine γ-herpesvirus infection (VIR1P.1148)
Authors: Crepeau Rebecca , Usherwood Edward 
Published: January 2023

Abstract
Gammaherpesvirus infection in immunosuppressed populations is associated with severe disease. MicroRNA-155 (miR155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). We sought to determine the relative contribution of miR-155 in the T and B cell compartments to latent murine gammaherpesvirus infection. Mice deficient in miR-155 had a 100-fold decrease in latent viral load, measured by qPCR, and exhibited decreases in both follicular helper cells and germinal center B cells. However, infection of mixed bone marrow chimeric mice showed a similar latent viral burden in both WT and miR-155 deficient B cells. Therefore the role of miR-155 may not be B cell intrinsic, but is likely mediated in part due to the regulation that miR155 exerts on the T follicular helper cell population. Studies using a YFP marker virus also address roles for miR-155 in virus reactivation and maintenance of latency. Further elucidating the relative role of miR155 on these cell populations will provide key insights into the development of latency in this model system.
Regulation of EBI2 expression by the EBV BRRF1 gene in B lymphocytes (VIR1P.1152)
Authors: Poole Brian , Cornaby Caleb , Birrell Cameron 
Published: January 2023

Abstract
Background: Epstein Barr Virus Induced gene 2 (EBI2), also known as GPR183, plays a role in migration of B cells. Expression of EBI2 is activated by Epstein Barr Virus (EBV). We investigated the mechanism of EBV activation of EBI2. By regulating EBI2 expression EBV may be able to manipulate B cell migration and interactions between B and T cells. Materials and Methods: EBI2 and EBV gene expression was measured using quantitative-PCR. The effects of the EBV gene BRRF1 were investigated using an EBV strain deficient for the Na protein and lentiviral transfection of the Na gene (BRRF1). Results: B cell lines and ex vivo B cells up-regulate EBI2 after EBV infection (p &lt; .005). Treatment with inactivated virus did not affect EBI2 expression. EBI2 expression is higher in primary B cells than cell lines containing EBV (LCL and Ramos cells; LCL p =0.012, Ramos p=0.027, N=3). EBV BRRF1 expression correlated with EBI2, and BRRF1 deficient EBV did not affect EBI2 expression. Overexpression of BRRF1 using a lentiviral vector resulted in elevated expression of EBI2. Conclusions: EBI2 expression is directly influenced by EBV infection, rather than general cellular activation. BRRF1 is the viral gene responsible for EBI2 upregulation during infection of B cells with EBV. Since BRRF1 is expressed early in infection and during lytic infection, there may be an advantage to EBV in disrupting B cell migration at these times.
TCR-independent suppression of IFNγ production in vivo (VIR1P.1156)
Authors: Hosking Martin , Flynn Claudia , Whitton J Lindsay 
Published: January 2023

Abstract
After encountering cognate antigen in vivo, CD8+ rapidly secrete the pleiotropic anti-viral cytokine IFNγ. Interestingly, after producing a transient burst of IFNγ immediately following secondary viral infection, memory CD8+ T cells fail to sustain IFNγ production in vivo, even though ample viral antigen remains (Hosking et al. JI 2013). We did not know whether CD8+ T cells were (i) refractory to further TCR stimulation or (ii) actively suppressed and thus incapable of IFNγ production. We have, therefore, attempted to model discrete encounters with cognate antigen in vivo by injecting LCMV-immune mice with a cocktail of peptides corresponding to a subset of immunodominant CD8+ T cell epitopes. Virus-specific CD8+ memory T cells receiving TCR stimulation transiently produced a burst of IFNγ in vivo and phenotypically resembled antigen-specific CD8+ T cells responding to a secondary viral infection. On the other hand, bystander virus-specific CD8+ T cells that did not receive peptide stimulation did not produce IFNγ. To determine whether prior TCR stimulation limits subsequent cytokine production, peptide stimulated LCMV-immune mice were immediately rechallenged with LCMV, and CD8+ T cell cytokine production was assessed in vivo. Both prior-peptide stimulated and bystander virus-specific CD8+ memory T cells failed to produce IFNγ following infection. TCR-independent suppression of cytokine production was also observed within CD4+ T cells and appears to be mediated by IFNγ.
Intra-spike crosslinking overcomes antibody evasion by HIV-1 (VIR1P.1131)
Authors: Galimidi Rachel , Klein Joshua , Politzer Maria , Bai Shiyu , Seaman Michael , Nussenzweig Michel , West Anthony , Bjorkman Pamela 
Published: January 2023

Abstract
Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV’s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a “molecular ruler” to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders-of-magnitude increased potency (&gt;100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection.
Study of binding characteristics of human monoclonal antibodies to differentially matured dengue virus particles (VIR2P.1172)
Authors: Tsai Wen-Yang , Lloyd Yukie , Wu Yi-Chieh , Lin Hong-En , Wang Wei-Kung 
Published: January 2023

Abstract
Dengue virus (DENV) is the leading cause of arboviral disease. The ectodomain of envelope protein, containing three domains, DI, DII and DIII, is the major target of DENV vaccine. Studies on monoclonal antibodies (mAbs) showed that DIII mAbs are more potent neutralizing (NT) than those mAbs binding DII fusion-loop (FL). Due to the inefficiency of furin protease activity, produced DENV is a mixture of mature, immature and partially immature virions. It remains unknown regarding the epitope accessibility, avidity and NT potency of different mAbs on different maturation status of DENV. Immature and mature virions were generated from DENV1 infected BHK cells with treatment of NH4Cl and transfected with furin, respectively. Capture ELISA were performed to examine human anti-FL and anti-DIII mAbs (kindly provided by Dr. J. Mongkolsapaya and Dr. F. Sallusto). Anti-FL mAbs had higher maximum binding (Bmax) to immature than mature virions (P=0.0015) and no difference in dissociation constant (Kd) to both virions, suggesting that they have more accessible epitopes on immature virions and bind both with similar avidity. Anti-DIII mAbs showed no difference in Bmax or Kd to both virions but had lower Kd to mature virions than Anti-FL mAbs (P=0.04). Considering the greater infectivity of mature than immature virions, our findings of higher avidity of Anti-DIII mAbs to mature virions compared with Anti-FL mAbs provide a mechanistically explanation for stronger NT potency of Anti-DIII mAbs.
Increased susceptibility to influenza A in a mouse model of MeCP2 duplication syndrome (VIR2P.1163)
Authors: Cronk James , Kim Taeg , Moser Emily , Sharma Ashish , Braciale Thomas , Kipnis Jonathan 
Published: January 2023

Abstract
MeCP2 duplication syndrome is a devastating neurodevelopmental disorder caused by duplication of the Xq28 region of the X chromosome, primarily affecting males. Methyl-CpG-binding protein 2 (MeCP2) ubiquitously binds methylated CpG sites in the genome, and regulates gene expression by recruitment of transcriptional mediators. MeCP2 is expressed highly in both neurons and immune cells, thus mutations or over-expression of MeCP2 are associated with neurologic pathology (including seizures, motor dysfunction, and cognitive impairment), and immunologic pathology. Many patients die by the age of 25, and recurrent respiratory infections are a common cause of death. We are studying the pathogenesis of influenza A virus in a mouse model of MeCP2 overexpression, the MeCP2Tg3 mouse, which expresses MeCP2 at ~3 fold normal levels. MeCP2Tg3 mice display significant mortality approximately one week post infection, with T and B cell deficiencies in both number and activation, further characterized by high viral titer and low virus-specific antibody levels in the infected lung. Preliminary evidence suggests that vascular and/or lymphatic impairment may underlie both immunologic defects and the increased mortality observed in MeCP2Tg3 mice during influenza A virus infection. In sum, these findings uncover MeCP2 as an important regulator of host response to respiratory virus infection and may inform treatment of human patients with MeCP2 duplication syndrome.
Location, abundance, and phenotype of follicular SIV-specific CTL during chronic infection (VIR2P.1169)
Authors: Skinner Pamela 
Published: January 2023

Abstract
The inability of HIV- and SIV-specific CTL to fully suppress virus replication may be due to a deficiency of CTL in B cell follicles. HIV-1 and SIV replication is concentrated within B cell follicles during chronic disease; whereas HIV and SIV-specific CTL fail to accumulate in large numbers in follicles. It is not known whether virus-specific CTL migrate throughout the entire follicular area including germinal centers, nor whether virus specific-CTL within B cell follicles are functional. We evaluated these questions using in situ tetramer staining and IHC in tissues from chronically SIV infected macaques. We found that SIV-specific CTL migrated into germinal centers at similar levels detected in other follicular areas. In addition, subsets of follicular SIV-specific CTL expressed the activation and proliferation marker Ki67, and subsets expressed the effector molecules perforin and granzyme B. However, we also found some in contact with Foxp3+ Tregs. Thus, the inability of SIV-specific CTL to clear follicular SIV producing cells is not due to a complete inability of these cells to migrate into germinal centers. In addition, although subsets of follicular SIV-specific CTL express Ki67 and effector molecules, their relative low abundance and contact with Tregs likely inhibits their ability to clear follicular SIV+ cells. These findings suggested that a functional cure for HIV might include strategies that augment functional virus-specific CTL within B cell follicles.
Heterologous protective immunity can lead to impaired activation, memory differentiation and inflation of new naive responses (VIR2P.1170)
Authors: Agarwal Priti , Kraft Anke , Watkin Levi , Kenney Laurie , Daniels Keith , Chen alex , Brehm Michael , Selin Liisa 
Published: January 2023

Abstract
Activation of crossreactive memory CD8 T cells leads to heterologous protective immunity. Here we question whether early activation of crossreactive memory CD8 T cells alters the activation of new naïve CD8 responses to the second virus. Pichinde virus (PICV) and lymphocytic choriomeningitis virus (LCMV) contain a crossreactive usually subdominant epitope, NP205, which differs in 6 of 8 amino acids. During PICV infection of LCMV-immune mice the NP205 memory response become immunodominant and mediates protective immunity. PICV is cleared early and does not persist. The usually dominant non-crossreactive new naïve PICV NP38 CD8 T cell response appears to be subdominant day 7 post infection. However, surprisingly over the next 100 days post infection the NP38 response continues to gradually expand and then stabilizes. These NP38-specific CD8 T cells appear to arrest in an intermediate effector T cell phenotype (KLRG1low, CD62Llow IL-7Rhi), with altered functionality (decreased TNFα production). They appear to have altered expression of apoptotic genes on gene array. This NP38 inflationary response did not occur when mice were immunized with an LCMV mutant that did not present the crossreactive NP205 epitope. Our data shows that the strong crossreactive response kills DC’s expressing NP205 early (day 2-3) in PICV infection. This may result in an early termination of antigen-presentation leading to inadequate activation of the naïve NP38 response and inflation.
Engineering MHC class I fine structure for activation of self-tolerant T cells
Authors: Parks Christopher A , Hansen Michael J , Ju Josephine M , Reed Brendan K , Kranz David M , Bordner Andrew J , Pease Larry R 
Published: January 2023

Abstract
Central tolerance imposed during thymic development and mechanisms of peripheral tolerance result in a mature T cell repertoire capable of cross-reacting weakly on self, but usually incapable of activation. We have observed mutations in class I heavy chains capable of activating normally tolerant T cells which can then cross-react effectively on self. We hypothesized that mutation of the class I heavy chain, based on our understanding of MHC fine structure, could be used to generate engineered MHC molecules capable of activating T cells normally incapable of activation due to self-tolerance that could then cross-react effectively on self, but without altering the presentation of bound peptides. Known ternary complex (pMHC:TCR) crystal structures were modeled to predict amino acid substitutions within the class I heavy chain resulting in increased stable contacts between pMHC and TCR without altering the structure of bound peptides. Four substitutions within the class I heavy chain meeting these criteria were predicted by the structure based modeling. Cell lines were established which demonstrated stable surface expression of the engineered class I molecules. In a direct test of binding, a single chain TCR was detected with 20 fold greater intensity on the surface of cells expressing engineered MHC molecules compared with wild-type MHC, while competitive inhibition of binding a labeled peptide demonstrated unaltered peptide presentation. Stimulation of T cells with a weak antigen presented by the engineered MHC resulted in enhanced activation and proliferation compared with wild-type MHC. In an in vivo transplant model, protective immunity was induced against a native tumor after challenge with tumor expressing engineered MHC.
An internalizing antibody induces the production of pro-inflammatory cytokines via Fc-dependent pathway in THP-1 cell.
Authors: Park Hyun Joon , Kwon Myung-Hee 
Published: January 2023

Abstract
Conventionally, the extracellular effector functions of antibodies are mediated by the heavy chain constant regions of Ig molecules, but recently, intracellular effector functions of antibodies have been reported. After antibodies binds to the virus and the antibody-virus complex enters the cytosol, the complex can be detected by TRIM21 that is an intracellular Fc receptor, leading to the degradation of the virus and activation of transcription factors related to immune response. In this process, the minimum requirement is the presence of antibody in the cytosol for interaction with TRIM21. We previously have shown that 3D8 IgG originated from an autoimmune-prone MRL-lpr/lpr mouse is an internalizing antibody that can spontaneously enter the cytosol. Here, we investigated whether 3D8 IgG regulates the immune response in diverse cells without viral infection. Treatment of 3D8 IgG wild type induces pro-inflammatory cytokines (IL-8 and TNF-α) in human monocyte cell line THP-1. In contrast, treatment of 3D8 IgG mutant which does not bind to TRIM21 up-regulates production of the cytokines, compared to 3D8 IgG wild type. Moreover, 3D8 scFv (single chain fragment variable) which is devoid of Fc region did not induce the production of cytokines. Our results demonstrate that the production pathway(s) of cytokines are TRIM21-independent and Fc-dependent manner. We suppose that Fc-interacting unknown molecules would induce the secretion of pro-inflammatory cytokines. Our study would provide a new insight to the pathogenesis of autoimmune disease.
Characterizing surface costimulatory molecule expression on plasmacytoid dendritic cells in male and female B6.Nba2 lupus-prone mice.
Authors: Gubbels Bupp Melanie R , Thoner Timothy , Jorgensen Trine N 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disorder that primarily affects women and is characterized by the production of autoreactive antinuclear autoantibodies and elevated levels of serum interferon-alpha (IFN-α). Female B6 mice congenic for the Nba2 lupus susceptibility interval develop a lupus-like disease including antinuclear autoantibodies, glomerulonephritis, and elevated serum IFN-α. Plasmacytoid dendritic cells (pDC) are capable of secreting high amounts of IFN-α and also present antigen. Ablation of Siglec H+ plasmacytoid dendritic cells in female B6.Nba2 mice is sufficient to reduce serum IFN-α and prevent the production of antinuclear autoantibodies as well as the development of glomerulonephritis. We hypothesized that genetic and/or sex-specific differences in pDC activation states may contribute to differential susceptibility to lupus. Therefore, we compared the expression levels of specific costimulatory molecules on pDCs in male and female control (B6) and lupus-prone (B6.Nba2) mice. Plasmacytoid DCs were categorized into 3 subsets: Siglec H+ CD19−, CD19+ Siglec H−, or double negative. Using flow cytometry, the expression of CD80, CD86, CD40, CD40L, ICOS, B7RP1, and MHC Class II were quantified in each subset. This study may provide insights into the genetic and sex-specific mechanisms by which pDCs contribute to SLE pathogenesis.
NADPH-derived superoxide influences CD4 T cell activation by oxidizing membrane thiols
Authors: Morgan Nadine N , Tse Hubert Ming 
Published: January 2023

Abstract
Reactive oxygen species play a key role in mediating T cell autoreactivity in the development of autoimmune diseases including Type 1 diabetes (T1D). It was recently shown that redox regulation of cell surface thiol levels can influence T cell activation, and the development of autoimmunity. Enhanced susceptibility in developing autoimmune arthritis has been attributed to an increase in the number of reduced cell surface thiols on arthritogenic CD4 T cells. We hypothesize that NAPDH (NOX)-derived superoxide can modulate the oxidation status of cell surface thiols on diabetogenic CD4 T cells, and thereby, influence T cell effector responses. We utilized the NOD.BDC-6.9.Ncf1m1J TCR transgenic mouse that possesses superoxide-deficient CD4 T cells that recognize the putative autoantigen, islet amyloid polypeptide. BDC-6.9.Ncf1m1J CD4 T cells stimulated with their cognate BDC-6.9 hybrid peptide displayed a 3.5- and 1.5-fold increase in IFN-γ and TNF-alpha production, respectively, in comparison to NOX-sufficient BDC-6.9 CD4 T cells. Hybrid peptide-stimulated BDC-6.9.Ncf1m1J CD4 T cells also displayed a 1.8-fold increase in the percentage of CD25 and a 1.5-fold increase in the percentage of CD69 in comparison to BDC-6.9 T cells. We examined the number of reduced cell surface thiols on CD4 T cells using Alexa Fluor 647 coupled with maleimide. Interestingly, hybrid peptide-stimulated BDC-6.9.Ncf1m1J CD4 T cells exhibited a 2.3-fold increase in the percentage of reduced cell surface thiols in comparison to stimulated BDC-6.9 CD4 T cells. Our data indicates that NOX-derived superoxide mediates the oxidation status of T cell surface thiols to influence diabetogenic CD4 T cell effector responses.