bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Wiskott-Aldrich Syndrome protein (WASp) controls iNKT cell effector function. (50.39)
Authors: Locci Michela , Catucci Marco , Draghici Elena , Bosticardo Marita , Roncarolo Maria Grazia , Villa Anna 
Published: January 2023

Abstract
The Wiskott-Aldrich Syndrome protein (WASp) is a regulator of actin remodeling in hematopoietic cells. In T cells, WASp is required for a long-lived Immunological Synapse (IS) and TCR-mediated cytokine production. We recently published that WASp is fundamental in the biology of iNKT cells, a peculiar T cell subset activated by glycosphingolipid antigens presented by APC. We showed that murine was-/- iNKT cells were defective in IL-4 and IFN-γ production upon in vivo activation. It remains an open issue whether this functional impairment is caused by a cell autonomous functional defect of was-/- iNKT cells or by an impaired crosstalk between iNKT cells and APC. To address this issue we analyzed cytokine production upon in vitro activation of wt and was-/- iNKT cells by wt and was-/- DC. The data obtained indicate the defective IL-4 production due to a was-/- iNKT cell autonomous defect and the lower IFN-γ production caused by an inefficient crosstalk between was-/- iNKT cells and was-/- DC. However was-/- iNKT cells produced sustained levels of cytokines upon in vitro stimulation with TPA and Ionomycin, suggesting a TCR-mediated functional impairment of these cells. Supporting this hypothesis was-/- iNKT cells presented a decreased TCR avidity. Altogether these results demonstrate the relevance of WASp in TCR-mediated effector function of iNKT cells. Ongoing studies are aimed at evaluating the ability to properly form the IS by was-/- iNKT cell and APC.
IL-2/ IL-2 mAb treatment generates prolonged expansion and increased cytokine production by NKT cells (50.42)
Authors: Webster Kylie , Kyparissoudis Konstantinos , Godfrey Dale , Sprent Jonathan 
Published: January 2023

Abstract
Regulatory T cells can be dramatically boosted in vivo by stimulation with a complex of IL-2 and an IL-2 mAb (JES6-1). The swift 15-fold expansion creates a tolerogenic environment that significantly perturbs autoimmunity and allograft rejection. We recently observed that IL-2/mAb treatment also expands Natural Killer T (NKT) cells, a glycolipid-specific, CD1d-restricted T cell lineage implicated in both regulatory and immunogenic responses. Interestingly, the phenotype of the IL-2/mAb-expanded NKT cells differed from those expanded with the prototypic NKT ligand, alpha-galactosylceramide (α−GalCer). The IL-2/mAb-expanded NKT cells were relatively stable and contracted slowly. They were hyper-functional, with the capacity to rapidly produce cytokines for several days after stimulation. In comparison, α−GalCer stimulated NKT cells characteristically produce a quick burst of Th1 and Th2 cytokines, an ability that then dramatically declines along with their number in the following days. Interestingly, the range of cytokines produced by IL-2/mAb-NKT cells was broad and included not only IFNγ and IL-4, but also significantly high amounts of IL-10 and IL-13. IL-17, however, was notably reduced. These variations in cytokine production appeared to alter the phenotype of dendritic cells in their vicinity. These data suggest an alternate approach to the expansion of NKT cells, specifically within the context of a regulatory milieu that may be appropriate for tolerance induction.
Distinct sites in ADAP control IKKα/β phosphorylation, IKKγ ubiquitination and cell cycle progression in T cells. (50.43)
Authors: Srivastava Rupa , Burbach Brandon , Shimizu Yoji 
Published: January 2023

Abstract
NF-κB activation following TCR engagement involves PKCθ-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IκB-kinase (IKK). We previously identified a novel role for the adhesion and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. The ADAP interacts with another adaptor protein SKAP55 which is critical for for T cell integrin activation and T:APC conjugate formation. In this study, we identify a novel site in ADAP that is critical for association with the TGFβ activated kinase (TAK1). ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to PKCθ. While both the TAK1 and CARMA1 binding sites in ADAP are essential for IκB phosphorylation and degradation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKγ. Loss of ADAP in ADAP-/- primary T cells led to a block in G1-S transition. Expression of ADAP mutants lacking TAK1 and CARMA1 binding sites do not rescue the block in cell cycle progression while ADAP mutant lacking the SKAP55 binding site rescued cell cycle progression. Thus, distinct sites within ADAP control two key activation responses that are required for NF-κB activation and provide unique signals for cell cycle cycle progression and T cell poliferation.
RIAM and RapL regulate distinct signaling events and functional outcomes upon TCR-mediated activation (50.8)
Authors: Patsoukis Nikolaos , Lafuente Esther , Boussiotis Vassiliki 
Published: January 2023

Abstract
Rap1, a small GTPase of the Ras superfamily, regulates inside-out activation of integrins and cell adhesion. Two new Rap1 effectors, RapL and RIAM, have been implicated in this function. Significant differences in their structure indicate that these molecules may mediate distinct signaling events. RIAM has a coiled-coil region, RA and pleckstrin homology (PH) domains, and proline-rich regions with specific motifs capable of interacting with the EVH1 domains of Ena/VASP proteins, with profilin and with SH3 domain containing proteins. RapL has an RA domain and a C-terminal coiled-coil region. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. Using shRNA we determined that activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by elimination of RIAM, whereas activation of these kinases was unaffected by elimination of RapL. Activation of p38 was unaltered in RIAM-KD but was abrogated in RapL-KD cells. Moreover, knockdown of RIAM but not RapL resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. Strikingly, RIAM-KD resulted in impaired IL-2 production, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon TCR/CD3-plus-CD28 crosslinking. Thus, RIAM and RapL have distinct roles in regulating signaling and functional outcomes of T cell responses.
Regulation of IL-13 expression in lymphocytes by the Let-7 miRNA family (51.11)
Authors: Gamper Christopher , Lee Youl-Nam , Brandal Stephanie , Mendell Joshua , Takemoto Clifford , McDevitt Michael , Powell Jonathan 
Published: January 2023

Abstract
IL-13 is a Th2 cytokine that plays a critical role in antibody production, parasite immunity, allergic disorders, and as a negative regulator of anti-tumor immunity. Curiously, microarray screens of activated Th1 clones demonstrated high levels of IL-13 RNA in spite of essentially no IL-13 protein production. Such findings were subsequently confirmed in primary cells cultured in Th1 conditions. We hypothesized that these observations might be due to regulation by miRNAs. Indeed, a highly conserved Let-7 site is predicted in the 3’ UTR of IL-13. Furthermore, several Let-7 family members are more highly expressed in activated Th1 cells when compared with activated Th2 cells. To test our hypothesis, we generated luciferase reporter constructs containing the native 3’ UTR of IL-13 (WT) or a 3’ UTR with the Let-7 site mutated (Mut). Overexpression of Let-7 mimetics inhibited the IL-13 WT but not the IL-13 Mut construct. In addition, the transfection of the mimetics or infection with Let-7g expressing retrovirus led to the inhibition of native IL-13 protein production. Alternatively, inhibitors of Let-7 family members or infection with a retrovirus encoding the Let-7 antagonist LIN28B led to enhanced IL-13 production. Overall our data suggest Let-7 miRNA plays a critical role in the regulation of IL-13 and that such molecules might be exploited to inhibit IL-13 in allergic diseases and to enhance anti-tumor immunity.
The Crohn’s Disease protective SNP rs11209026 mediates alternative splicing in human IL23R. (51.14)
Authors: Gallagher Grant , Brazaitis Jonothan , Yu Raymond 
Published: January 2023

Abstract
SNP rs11209026 in the human IL23R gene has been associated with susceptibility in human autoimmune diseases. Notably, the rarer “A” allele confers protection against Inflammatory bowel diseases. IL-23 and IL-23R signalling are key to the development of the pro-inflammatory T-cell subset, Th17 cells, where they stabilise the functionally-mature Th17 phenotype. Despite causing an Arg&gt;Gln change at residue 381, inside the cell membrane, no differences in IL-23 signalling resulting from this change have been reported. Multiple splice-forms of the human IL23R transcript exist and one of these, lacking exon-9 (“Δ9”), encodes a potent IL-23 inhibitor comprising a truncated, soluble HuIL23Rα chain. Here, we show that the disease-protective “A” allele destroys the affinity of this site for the splice-factor Sf2 and so favours transcription of HuIL23RΔ9. Using both BAC and mini-gene approaches, we used anti-sense oligonucleotides to target the Sf2 binding site and cause dose-responsive, specific induction of Δ9. We applied this strategy to primary human CD4+ lymphocytes, again inducing Δ9 mRNA. In experiments designed to differentiate human Th17 cells in vitro this caused a reduction in wild-type IL-23R expression on the cells’ surface and reduced the expression and secretion of the signature Th17 cytokines, IL-17A and IL-17F. Thus, the presence of allele "A" predicts the elevated expression of Δ9 and a consequent lower ability to respond to IL-23 or to maintain Th17 activity.
Regulation of IL-27 Production by Antigen Presenting Cells in Mice (51.15)
Authors: Qin Fenghua , Li Sky , Dong Lingpu , Lin Xiao , Yang Xifeng 
Published: January 2023

Abstract
Interleukin-27 (IL-27) is a novel IL-6/IL-12 cytokine family member that consists of EBV-induced gene-3 (EBI3) and p28 subunits. IL-27 triggers expansion of antigen specific naïve CD4+ T cells, promotes polarization of Th1 cells, and inhibits the development of Th2 and Th17 cells. It is known that IL-27 is an early product of activated antigen-presenting cells. But the regulation of IL-27 production has not been well documented. Here, we studied the effects of IFN-γ and LPS on IL-27 production by mouse macrophages. Thioglycollate-elicited peritoneal cells from Balb/c mice were stimulated with IFN-γ and LPS with various combinations and different time periods. IL-27 expression was examined by flow cytometric analyses of intracellular staining with fluorochrome-conjugated anti-mouse IL-27 mAb (MM27.7B1) and bead-based analyses of the cultured supernatants. The data showed that LPS alone-stimulated macrophages yielded less than 10% of IL-27 positive cells on 24 hrs after stimulation. However, if the cells were primed with IFN-γ for 2 hours and then stimulated with LPS, higher frequency of IL-27 producing cells was observed (25% to 45%). Kinetic studies demonstrated that a significant level of IL-27 secretion was detected as early as 2 hours and the peak production was observed around 22 hours post stimulation.
Retinoic acid modulates IL-4 receptor alpha chain expression and signaling pathways in intestinal epithelial cells and macrophages (51.3)
Authors: Dawson Harry , Chen Celine , Fendlay Devin , Chitko-Mckown Carol , Wagner Bettina , Boyd Patricia , Lunney Joan , Urban Jr. Joseph 
Published: January 2023

Abstract
We have shown that all-trans retinoic acid (ATRA) increased mRNA expression of several IL-4-induced chemokines in explanted porcine alveolar macrophages. We now describe that ATRA modulated IL-4-induced signaling pathways in primary macrophages, and several macrophage and epithelial cell lines. ATRA increased epithelial cell mRNA and protein for the IL-4 receptor alpha chain (IL4RA), and IL-4 induced phosphorylation of STAT6 and expression of CLCA1 mRNA in vitro. In primary macrophages and several macrophage cell lines, ATRA-induced IL4RA mRNA and enhanced IL-4-induced expression of DHSR3, MRC1, PLAT, PTPN1, TGM2, and WARS mRNA and facilitated IL-4-inhibited expression of TLR2 and CYBB mRNA. However, ATRA blocked IL-4-induced expression of DHSR9, MAF, SEPP1 and STAT5A mRNA and counteracted IL-4 inhibited expression of BCL2L1, CCL2, NCF4, and PDGFB mRNA. These activities were largely shared by the RAR-a selective retinoid, Am580. ATRA variably affected the expression of a large number of other IL-4-induced mRNAs. These data indicated that ATRA and related retinoids can modulate Th2-related responses through mechanisms that utilize IL-4 and IL-4 receptor signaling pathways.
A naturally antagonist of human IL-23 inhibits the development and function of human Th17 cells (51.4)
Authors: Gallagher Grant , Yu Raymond 
Published: January 2023

Abstract
The Th17 subset of CD4 cells is critical to inflammation. Secretion of IL-17 by Th17 cells drives inflammation in human diseases and their mouse models, including IBD, psoriasis and arthritis. The differentiation of mature Th17 cells depends upon stimulation in the presence of IL-6, TGF-β and IL-21 and the induction of RORγt, but their phenotype is stabilised by IL-23, which is essential to Th17 stability and function. Initial induction of Th17 cells can be antagonised by IL-4 or IFN-γ, but mechanisms through which terminal differentiation can be inhibited have not been identified. We found that HuIL23Rα chain mRNA transcripts exist that lack exon-9 (“Δ9”); these are translated to a truncated receptor form containing the entire external domain of the wild-type protein. Here, we show that this soluble variant of the HuIL23Rα chain antagonises IL-23 signalling and Th17 maturation. The variant is secreted and present at low levels in the blood. This soluble form represents 10% of HuIL23Rα chain mRNA. It binds IL-23 in solution and inhibits the phosphorylation of STAT3 caused by IL-23. In experiments designed to differentiate human Th17 cells in vitro, the Δ9 form inhibits the production of Th17-associated cytokines, including IL-17A and IL-17F. Δ9 does not bind IL-12; thus it is a specific inhibitor of IL-23 and a modulator of Th17 cells. Our results indicate that this soluble form of HuIL23Rα likely functions to regulate Th17 activity.
Effects of cell-type specific hIL-10 expression on disease susceptibility (51.5)
Authors: Ranatunga Dilini , Hedrich Christian , Wang Fengying , McVicar Daniel , Norwak Nathan , Joshi Trupti , Feigenbaum Lionel , Grant Lindsay , Stager Simona , Bream Jay 
Published: January 2023

Abstract
IL-10 is an immunoregulatory cytokine produced by many cell types. Studies in mice suggest the cellular source of IL-10 plays a critical role in determining disease outcome. Yet the effects of tissue specific human IL-10 (hIL-10) expression are not well understood. To address this issue, we generated a hIL-10 transgenic mouse using a bacterial artificial chromosome (hIL10BAC). The hIL10BAC contains most if not all the regulatory sequences that govern hIL-10 production thus allowing tissue specific expression. As hIL-10 is biologically active in the mouse, reconstituting Il10-/- mice with the hIL10BAC allowed us to evaluate the effects of hIL-10 on known IL-10 dependant phenotypes. Expression of IL-10 from myeloid sources in WT mice protects against lethal endotoxic shock. Faithful expression of the hIL10BAC in myeloid cells rescued Il10-/- mice from this fate. In contrast, T-cell-derived IL-10 leads to high parasite burdens in the livers and spleens of WT mice infected with Leishmania Donovani. However both Il10-/- and Il10-/-/hIL10BAC mice cleared the parasites. The failure to reproduce the susceptibility phenotype is attributed to weak hIL-10 expression in CD4+ T cells. Similarly, inflammatory bowel disease (IBD) is also IL-10-dependant and resistance is thought to involve T-cell-derived IL-10. Surprisingly, we found that the hIL10BAC rescues Il10-/- mice from IBD, suggesting differential regulation of hIL-10 in CD4+ T cells found in the gut versus the spleen.
Generation of fully human monoclonal antibodies following anthrax vaccine absorbed (AVA) vaccination that provide protection from lethal toxin challenge. (52.15)
Authors: Crowe Sherry , Vineyard Amanda , Smith Kenneth , Carla Guthridge , Morris Jennifer , Wilson Patrick , James Judith 
Published: January 2023

Abstract
The release of Bacillus anthracis spores can cause significant morbidity and mortality and effective vaccination strategies and immunotherapeutics are needed. This study seeks to identify and characterize the major humoral targets that provide protection. To this end, we isolated antibody secreting cells from AVA vaccinated individuals and generated a panel of fully human monoclonal antibodies. Ten of these antibodies were highly specific for the protective antigen (PA), with affinities ranging from 3.7x10-7 to 1.4x10-10. Antibodies p6C01 and p6F01 were able to neutralize lethal toxin activity in vitro even at low concentrations (EC50 = 0.1µg/ml). Passive transfer of either human monoclonal antibody to A/J mice prior to lethal toxin challenge conferred 80-90% protection. Using a combination of western blotting and subunit ELISAs, we have shown that the p6C01 antibody binds to domain 3 of PA, which is critical for heptamer formation. Experiments are ongoing to further characterize the mechanism(s) of protection by these antibodies. These fully human antibodies can be used as a tool to identify protective humoral epitopes, as well as provide directed immunotherapeutics.
Immunization with Adenoviral-expressed salivary gland proteins (SALPs) decreases spirochete load in a murine model of Lyme borreliosis (52.2)
Authors: Ullmann Amy , Dolan Marc , Fikrig Erol , Piesman Joseph , Zeidner Nordin 
Published: January 2023

Abstract
It is known that tick saliva contains a vast array of proteins that function as anti-haemostatic, anti-inflammatory, and immunomodulatory factors when delivered to the vertebrate host. Previous studies have also demonstrated that prior exposure to tick saliva induced specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. In our current studies we explored a vaccination technique to modulate tick feeding, using an adenovirus (Ad) expression system to deliver four salivary proteins (previously shown to modulate host immunity) derived from Ixodes scapularis; Salp15, Salp25A, Salp25D, and ISAC. C3H/HeJ mice were immunized individually with Ad-Salp15 and Ad-ISAC constructs, as well as all four constructs simultaneously. Immunized mice were then challenged with B. burgdorferi-infected I. scapularis ticks. Utilizing quantitative PCR, spirochete burden in target organs like heart and bladder was reduced by 55% compared to animals immunized with an empty Ad-cassette. The generation of anti-salivary protein antibodies in a murine model was also confirmed. Our results indicate that adenovirus expressed tick salivary proteins can stimulate dendritic cell expression of tick salivary proteins, as well as tick-specific humoral and cellular immune responses in mice, affording at least partial protection against subsequent tick-transmitted B. burgdorferi infection.
Multiple antigenic peptide vaccine targeting the loop neutralizing determinant of protective antigen from Bacillus anthracis completely protects rabbits from aerosol spore challenge (52.24)
Authors: Oscherwitz Jon , Yu Fen , Cease Kemp 
Published: January 2023

Abstract
The loop neutralizing determinant (LND) from domain 2 of Bacillus anthracis protective antigen (PA) has been shown to represent a potentially important target for an epitope-specific vaccine for anthrax. This region in the 2β2-2β3 loop of PA is immunologically inert, since rabbits immunized with PA or otherwise immune to PA do not contain antibody specific for this region. Herein we evaluated the immunogenicity in rabbits of a MAP peptide comprising the T* helper T cell epitope from Plasmodium falciparum colinearly synthesized at either the N- or C-terminus with a.a. 304-319 from PA. In initial studies, the MAP peptides formulated in Freund’s adjuvant were found to be highly immunogenic in rabbits, rapidly eliciting high levels of neutralizing antibody. To more fully evaluate this peptide as an anthrax vaccine, we immunized separate groups of rabbits (n=7) five times at two-week intervals with either of the two LND MAP peptides or with soluble PA in Freund’s adjuvant and then subjected all rabbits to a 200 LD50 aerosol spore challenge with B. anthracis Ames strain. 100% of rabbits immunized with the LND MAP in the B-T orientation and with soluble PA, and 89% of rabbits immunized with the LND MAP in the T-B orientation, survived the spore challenge, while all naïve rabbits died. Analysis of post-challenge serum suggested little or no spore germination in challenged rabbits. We conclude that the LND MAP peptide is a promising epitope-specific prototype vaccine for anthrax.
Recombinant human antibodies to IFN-α from the immune repertoires of MG/thymoma donors (83.13)
Authors: Hammond Philip , Boozer Christy , Branum Mark , Fox Raymond , Lilly Rebecca , Mosley Bruce , Ward Courtney , Whitman Darbie , Frey Steve , Cicirelli Mike , Olsen Ole , Moyle Matthew 
Published: January 2023

Abstract
A serum titer of autoantibodies to IFN-α has been reported in several autoimmune conditions and in many cases has been shown to neutralize IFN-α. The IFN-α antibody repertoire from 10 patients with autoimmune myasthenia gravis and associated thymoma (MG/T) was characterized. Surface IgG positive memory B-cells from peripheral blood mononuclear cells (PBMC) were cultured in 384-well plates to produce assayable quantities of antibody. Sensitive antibody microarray and AlphaScreen™ binding assays were used to identify antibodies reactive with IFN-α(2a). A range of IFN-α subtype specificity profiles was observed in competition assays for binding to the 12 subtypes of IFN-α. A subset of these antibodies was expressed recombinantly after recovery of heavy and light chain variable regions by RT-PCR and these recombinant antibodies recapitulated the activities demonstrated in the B-cell culture supernatants. These antibodies fell into 2 groups, blocking binding of IFN-α(2a) to either the IFNAR2 or IFNAR1 subunit of the Type 1 interferon receptor. The ability of these antibodies to neutralize the activity of IFN-α(2a) in a Daudi cell proliferation assay was confirmed. The antibody repertoires of MG/T patients include high affinity antibodies with both broad and narrow specificities for the different subtypes of IFN-α. These function-blocking human antibodies may have therapeutic utility in IFN-α dependent diseases such as SLE.