bluuebunny/crossref_metadata_2025 huggingface dataset:
Two subtypes of microvesicles can be isolated from tissue culture (174.17)
Authors: Inal Jameel , Stratton Dan , Antwi-Baffour Samuel , Grant Ryan
Published: January 2023

Microvesicles or MVs released from the plasma membrane of eukaryotic cells, during early apoptosis, are 100-1000 nm in diameter, rich in phosphatidylserine and important mediators of intercellular communication. Analysis of MVs revealed two distinct subtypes that can be collected from cells in culture: cMVs are constitutively released and sMVs that are stimulated for release. There are marked differences in size and composition between cMVs and sMVs, the former being smaller, denser, ‘well made’ and more slowly released from cells whereas sMVs are larger, lighter, less robust and released rapidly in response to a stress stimuli. Furthermore these differences allude to different physiological roles, indeed, analysis of calcium homeostasis having revealed MVs to contain high calcium levels, that are deliverable to cells. The implications of this research highlights that detailed characterisations of MVs should be performed to further understand the underlying mechanisms in MV biology, as the subtypes are generated through different processes and undoubtedly will perform different roles.
IL-4/IL-13 signaling responses are dictated by the extracellular/transmembrane domain of the receptor complex, not the cytoplasmic domain (174.3)
Authors: Heller Nicola , Qi Xiulan , Gesbert Franck , Keegan Achsah
Published: January 2023

Previously, we demonstrated that the γC subunit of type I IL-4 receptor was required for robust tyrosine phosphorylation of the downstream adapter protein, IRS-2, correlating with expression of genes (ArgI, Retnla, Chi3l3) characteristic of alternatively-activated macrophages. We hypothesized that the γC cytoplasmic domain possesses a putative I4R motif, not found in IL-13Rα1, which enhanced IRS-2 recruitment. A mutant of the key I4R motif tyrosine (Y325F) and different γC truncation mutants (γ285, γ308, γ318, γ323, γFULL LENGTH) were co-expressed in L- or CHO cells with wild-type (WT) IL-4Rα and signaling responses to IL-4 were examined. IRS-2, STAT6 and JAK3 phosphorylation was restored in CHO expressing γ323 and γFL but not in γ318 and γ285 mutants. IRS phosphorylation was not diminished in Y325F L-cell mutants. Our data suggest that Y325 is not required for IRS activation and a smaller (4 amino acid) interval than previously determined is necessary for IL-4-induced JAK3 activation/γC-mediated signaling. We predicted that cytoplasmic domain swaps between γC and IL-13Rα1 would endow signaling responses corresponding to the nature of the cytoplasmic domain. Surprisingly, IRS-2/STAT6 signaling responses were dictated solely by the nature of the extracellular/transmembrane domains in our chimeric receptor-expressing CHO or M12.4.1 cell models. A more complete understanding of receptor activation/signaling will lead to improved rational drug design for allergic disease.
Mechanism of walnut induced anaphylaxis-like shock reaction in mice (175.1)
Authors: Gangur Venugopal , Gonipeta Babu , Kim Eunjung , Parvataneni Sitaram
Published: January 2023

Walnut is a leading cause of life-threatening anaphylaxis in humans. Such reactions are largely thought to be mediated by the Type-I hypersensitivity mechanism. We have found that English walnut extract (EWE) upon oral or i.p., administration in naive mice without pre-sensitization results in anaphylaxis-like shock response as quantified by hypothermia. We have been studying the mechanism underlying this unusual reaction. The Fcg receptor knockout (KO) mice, IgE non-responder SJL mice and TLR4 mutant C3H/HeJ mice, all exhibited hypothermia response indicating that the reaction was antibody and LPS independent respectively. We then examined the role of complement system. The C3a receptor (C3aR) KO mice were protected from shock responses. Among the C5 deficient (DBA/1J) and C5 sufficient (DBA/2J) mice, both exhibited hypothermia responses with DBA1 being a slightly lower responder. Since the shock reaction was complement dependent, we reasoned and tested whether EWE causes hemagglutination (HA) of RBC. Using a standard HA assay protocol we evaluated the HA activity of EWE. We found that the EWE had a significant HA activity for RBC obtained from BALB/c, DBA/1J and DBA/2J mice (HA titre: 1/32; translates to ~35,555 HA units per gram of EWE protein). These data are consistent with the hypothesis that EWE causes HA in vivo leading to complement system activation with consequent C3aR dependent anaphylaxis-like shock reaction.
Lyn but not Fyn kinase controls IgG-Mediated Systemic Anaphylaxis (175.17)
Authors: Falanga Yves
Published: January 2023

Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcϵRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage and neutrophil secretion of platelet activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38 and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (KO) cells, but decreased in Fyn KO cells, compared to wild type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features while no change was observed for Fyn KO mice, compared to wild type littermates. Intriguingly, we establish that mast cells account for the total amount of the serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.
Plasma cell gene expression depends on ELL2 (176.15)
Authors: Milcarek Christine , Arumemi Fortuna , Park Kyung
Published: January 2023

From studies with splenic B cells and cell lines ELL2, a transcription elongation factor, was found to play a vital role in the differentiation of B cells into plasma cells. It is responsible for Igh mRNA being both alternatively processed to the secretory-specific form and increased in abundance, leading to high levels of secreted Ig protein and a robust immune response. To investigate the molecular mechanisms that occur as a result of ELL2 action, we studied histone H3 methylation at K4 and K79 on the Igh in plasma cells by chromatin IP. We found that knocking out ELL2 with siRNA decreases the H3K4 di- and tri-methylation and K79 tri-methylation patterns as well as the association of CstF-64 and pTEFb, a part of the super elongation complex. Recent chromatin IP studies show that ELL2 is associated with RNA polymerase II on essential regulatory plasma cell genes including IRF-4 and blimp-1. Using luciferase transcription assays we show that ELL2 is able to increase transcription from its own and the IRF-4 promoter. Elongation is thus linked to increased mRNA production and the use of a proximal poly(A) site in the IRF-4 gene. ELL2 and ELL1 do not have identical patterns of action. Taken together, these studies define a fundamental role for ELL2 in the transcription elongation machinery and RNA processing in plasma cell differentiation. Studies on the in vivo role of ELL2 in Ig secretion are in progress.
IgG1 protects against IgG3 immune complex mediated renal disease. (176.21)
Authors: Strait Richard , Mahler Ashley , Barasa Nathaniel , Stringer Keith , Witte David , Herr Andrew , Finkelman Fred
Published: January 2023

Immunoglobulins (Ig) have been reported to inhibit or activate effector mechanisms with varying potency. In the mouse, IgG1 has less ability to activate complement (C), interact with stimulatory Fc receptors (FcRs) or form large immune complexes (IC) than other Ig isotypes. Therefore, we hypothesized that IgG1 might suppress IC immunopathology mediated by other isotypes. Consistent with this hypothesis, IgG1-deficient, but not IgG1-sufficient, mice developed renal failure at the time of the peak antibody response when immunized with a potent antigen. Renal disease was both C- and stimulatory FcR-independent, resulted from IgG3 IC cryoglobulin obstruction of glomerular capillaries and usually caused death within 2 weeks. Disease was prevented by administration of antigen-specific IgG1. A passive model of this obstructive glomerulopathy was induced by administration of a TNP-protein conjugate plus IgG3 anti-TNP mAb but not IgG1, IgG2a or IgG2b anti-TNP mAb. IgG3-dependent renal disease could be prevented by co-administration of IgG1, but not IgG2a or IgG2b anti-TNP mAb. These observations demonstrate the adaptive significance of Ig isotypes that poorly activate effector mechanisms and reveal an IC-dependent, complement- and stimulatory FcR-independent mechanism that can rapidly destroy renal structure and function. Our results suggest the possibility of using Ig isotypes that poorly activate effector mechanisms to inhibit IC-induced immunopathology.
Peripheral tolerance induction by PD-L1+ lymphatic endothelial cell is due to deficient costimulation that leads to rapid, high-level expression of PD-1 on CD8 T cells (176.24)
Authors: Tewalt Eric , Cohen Jarish , Rouhani Sherin , Guidi Cynthia , Qiao Hui , Fahl Shawn , Bender Timothy , Tung Kenneth , Vella Anthony , Adler Adam , Chen Lieping , Engelhard Victor
Published: January 2023

It has been shown that lymph node (LN) stromal cells directly present peripheral tissue antigens (PTA) leading to systemic peripheral tolerance. In particular, lymphatic endothelial cells (LEC) directly present an epitope from tyrosinase to CD8 T cells (TCD8). Here, we determined the roles of costimulatory and inhibitory pathways in the induction of LEC-mediated tolerance. LEC do not express costimulatory ligands but do express ligands for the PD-1, CD80, CD160, BTLA, and LAG-3 inhibitory pathways. Nonetheless, LEC-mediated deletion of tyrosinase-specific TCD8 is solely dependent on the PD-1:PD-L1 pathway. Based on radioresistance and the fact that LEC express the highest levels of any LN resident cell, we conclude that PD-L1 expressed by LEC is necessary and sufficient for tyrosinase-specific TCD8 deletion. Presentation of tyrosinase by LEC leads to deletion by inducing more rapid, high-level PD-1 expression on TCD8 than that induced by professional APC. This is due to lack of costimulation by LEC, and is correctable with agonistic anti-4-1BB and OX-40. Apoptosis of tyrosinase-specific TCD8 induced by PD-1 is a consequence of failure to upregulate the IL-2 receptor. Rescue of tyrosinase-specific TCD8 from LEC-mediated deletion by interference with PD-1:PD-L1 or provision of costimulatory signals leads to the induction of autoimmune vitiligo. As LEC express numerous PTA, this mechanism may be of general importance in the development of autoimmune diseases.
PGE2 promotes apoptosis induced by cytokine deprivation through EP3 receptor on mast cells (177.1)
Authors: Kovarova Martina , Koller Beverly
Published: January 2023

Increased mast cells numbers are observed at sites of allergic inflammation. Although a critical factor in the resolution of these inflammatory responses is the restoration of normal mast cell numbers. Early studies showed that cytokines protect mast cells from programmed cells death, suggesting a simple model in which diminished cytokines levels during resolution leads to cell death. The report that prostaglandins (PGs) can contribute to the resolution of inflammation together with the demonstration that mast cells express all four PGE2 receptors raises the possibility that PGE2 act in part by augmenting mast cell apoptosis. We report here that, while exposure of mast cells to PGE2 alone does not lead to apoptosis, PGE2 through the EP3 receptor promotes cell death initiated by cytokine withdrawal. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨm), increased caspase-3 activation, and chromatin condensation with low molecular weight cleavage. PGE2 augmented cell death is dependent on increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE2 contributes to the resolution of inflammatory response in part by augmenting the removal of inflammatory cells particularly mast cells.
Modulation of mast cell function by TIM-1 signaling (177.4)
Authors: Phong Binh , Kane Lawrence
Published: January 2023

Transmembrane, immunoglobulin and mucin domain (TIM)-1 belongs to a family of transmembrane proteins with emerging roles in immune modulation. As a putative atopy susceptibility gene, polymorphisms in human and mouse TIM-1 are associated with atopic diseases, including asthma and atopic dermatitis. Antibodies targeting different extracellular domains of Tim-1 either attenuated or exacerbated airway inflammation in a mouse model of asthma. Recent studies found that Tim-1 is expressed on mast cells. Tim-1 crosslinking by its ligand Tim-4 enhanced secretion of pro-inflammatory cytokines in IgE and Ag-activated bone marrow-derived mast cells (BMMCs). However, the mechanism(s) by which Tim-1 modulates mast cell function and how Tim-1 activity may be altered by antibody treatment is unknown. We hypothesize that Tim-1 engagement provides a co-stimulatory signal to enhance mast cell degranulation and mediator release in allergic inflammation. We found that Tim-1 requires a tyrosine phosphorylation motif in its cytoplasmic tail to co-stimulate NF-AT/ AP1, NF-κB, and IL-6 promoter transcriptional activation as well as IL-6 cytokine secretion. Furthermore, IgE-Ag and Tim-1 costimulation of BMMCs using a panel of Tim-1 antibodies and its ligand Tim-4 demonstrate that IL-6 and TNF-alpha secretion can be modulated by activity of agonistic and blocking Tim-1 antibodies. These studies suggest that Tim-1 may serve as an important modulator of mast cell effector functions in atopic diseases.
Exploring airway smooth muscle-mediated mast cell function at the single cell level using carbon-fiber microelectrode amperometry (177.6)
Authors: Manning Benjamin , Haynes Christy
Published: January 2023

An important distinguishing feature of asthma pathology is the migration of mast cells (MCs) into the airway smooth muscle (ASM). MCs are known to influence the immune response in a variety of capacities, including the release of many stored immunoactive mediators from secretory vesicles via exocytosis. Conversely, ASM is known to produce several mediators that potentially influence MC activity including the chemoattractant CXCL10. However, the specific consequences of interaction between MCs and ASM on asthma pathophysiology have yet to be fully explored. Carbon-fiber microelectrode amperometry (CFMA) is a unique analytical tool that allows the direct and label free detection of secreted serotonin as it is released from a single MC. Individual packets of serotonin corresponding to single vesicle fusion events are manifested as discrete current spikes in the collected amperometric trace. Individual spike analysis of each trace provides information such as vesicular content and delivery capacity, vesicle fusion efficiency and other kinetic information pertaining to exocytosis regulation. To explore the relationship between MCs and ASM in asthma, CFMA was used to monitor the effects of ASM and asthma-related factors, such as the cytokines CXCL10 and RANTES, on MC function in vitro. Analysis of CFMA data has shown significant modulation of MC secretory function in response to these factors, demonstrating the value of CFMA for the study of MC-related inflammatory processes.
Long-term oral delivery of encapsulated rapamycin is not detrimental to immunity in old mice (178.11)
Authors: Benavides Adriana , Fernandez Elizabeth , Sharp Z. , Strong Randy , Stacy Sue , Infante Anthony , Javors Martin , Richardson Arlan , Kraig Ellen
Published: January 2023

Recently, encapsulated rapamycin (e-rapa) was shown to extend lifespan in mice when given at an old age (Harrison Nature 2009), so it has been suggested that this mTOR inhibitor may be efficacious in slowing the aging process. However, use of rapa in the elderly might further compromise immune competence. Therefore, we sought to test the effects of e-rapa on immunity in older individuals by looking at T cell and B cell responses. Because rapa has been shown to be protective against some bacteria and viruses, effects on humoral immunity to a viable microbe were tested. Adult (4 months) and old (20 months) mice on e-rapa or control diets were challenged with the periodontal bacteria Aggregatibacter actinomycetemcomitans; e-rapa did not negatively impact the antibody titers seen in either age group. E-rapa effects on responses to a protein antigen, TAChRα, were also tested. No significant e-rapa effect was seen on antibody titers following primary or boost immunizations. Moreover, pilot data show that T cell proliferation to p146-162, the immunodominant epitope of TAChRα, was not significantly affected by the encapsulated drug. Since TAChR is the autoantigen in Myasthenia Gravis, we also immunized TAChRα transgenic mice (JI 169:6570) and showed that tolerance was maintained and the levels of TREGS were unaffected by e-rapa treatment. To-date, our data suggest that encapsulated rapamycin will not negatively impact immune regulation in old individuals.
Interaction of TNF and TNFR2 stabilizes the phenotype and function of CD4+FoxP3+ regulatory T cells in the inflammatory environment (179.16)
Authors: Chen Xin , Wu Xueqiang , Howard O.M.Zack , Oppenheim Joost
Published: January 2023

Our previous results showed that TNF-TNFR2 signaling promotes activation and expansion of Tregs. Thus we hypothesize that TNF-TNFR2 signaling may play a critical role in maintaining phenotypic and functional stability of Tregs during an inflammatory response. We were able to confirm that, in colitis model induced by transfer of naïve CD4 cells into Rag KO mice, the disease could be inhibited by co-transfer of WT Tregs, but not by co-transfer of TNFR2-deficient Tregs. Furthermore, in the colon lamina propria of the colitis model, the majority of WT Tregs maintained FoxP3 expression. In contrast, increased number of TNFR2-deficient Tregs lost FoxP3 expression. In vitro, FoxP3 expression by highly purified Tregs was reduced to almost undetectable levels after potent TCR-stimulation, which was partially abrogated by TNF. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. In addition, TNF was not able to increase FoxP3 expression by TNFR2-deficient Tregs undergoing TCR-stimulation. A full mouse genome array study revealed that, as compared with TNFR2- Tregs, TNFR2+ Tregs from normal mice expressed 4-fold lower levels of a gene encoding SATB1, in line with a recent study show that the repression of this genome organizer play a crucial role in maintenance of Treg phenotype and function. Thus, our data clearly show that the TNF-TNFR2 signaling is critical for the phenotypic and functional stability of Treg in the inflammatory environment.
Two histone/protein acetyltransferases (HATs), p300 and CBP, promote Foxp3+ T regulatory (Treg) cell production and function (179.18)
Authors: Liu Yujie , Wang Liqing , Han Rongxiang , Beier Ulf , Brindle Paul , Hancock Wayne
Published: January 2023

The master Treg transcription factor, Foxp3, is pivotal to the development and function of Treg cells. Foxp3 stability and function are regulated by acetylation, and we now identify p300 and CBP as controlling Treg production and function in vivo and in vitro. We found both p300 and CBP bind and acetylate Foxp3, and increase Foxp3 expression, DNA binding, and transactivation. We investigated their roles in Tregs by conditional deletion of p300, CBP, or both. While p300flflFoxp3cre and CBPflfl/Foxp3cre mice were born at the expected Mendelian ratios, p300flfl/Foxp3cre mice developed autoimmunity by several weeks of age, whereas CBPflfl/Foxp3cre mice showed long-term survival. However, p300flfl/CBPflfl/Foxp3cre mice developed severe autoimmunity and survived for only 3-4 weeks. Compared to littermate controls, both p300flflFoxp3cre and CBPflfl/Foxp3cre mice had decreased Foxp3 expression and impaired Treg suppressive function, whereas p300flfl/CBPflfl/Foxp3cre mice had even lower Foxp3 level and their Tregs essentially lacked suppression function. Additional studies showed p300 and CBP affect Foxp3 expression by regulating the Foxp3 promoter or CNS2 region, respectively. Our findings provide genetic evidence for central roles of these HATs in Treg biology, and provide a rationale for development of pharmacologic inhibitors to modulate Treg function in the context of oncology and other settings where it may be desirable to dampen but not eliminate Treg suppressive functions.
Constitutive nuclear localization of NFAT in FoxP3+ regulatory T cells independent of calcineurin activity (179.4)
Authors: Li Qiuxia , Shakya Arvind , Guo Xiaohua , Zhang Hongbo , Tantin Dean , Jesen Peter , Chen Xinjian
Published: January 2023

FoxP3 plays an essential role in conferring suppressive functionality to CD4+/FoxP3+ regulatory T (Treg) cells. While studies have shown that FoxP3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Treg cells for FoxP3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm and its nuclear translocation depends on calcineurin activation. We report that a fraction NFAT protein constitutively localizes in the nucleus of primary Treg cells, where it selectively binds to FoxP3 target genes. Treating Treg cells with calcineurin (CN) inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity. Consistently, Treg cells are resistant to CN-inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 stimulation, while proliferation of non-Treg cells is abrogated by CN-inhibitors. In addition, PMA, which activates other transcription factors required for T cell activation but not NFAT, selectively induces Treg cell proliferation in the absence of ionomycin. TCR-interaction with self-MHC class II is not required for PMA-induced Treg cell proliferation. Treg cells expanded by PMA or in the presence of CN-inhibitors maintain Treg cell phenotype and functionality. These findings shed light on Treg cell biology, paving the way for strategies to selectively activate Treg cells.
Sphingosine analog AAL-R enhances TLR7-mediated dendritic cell activation via p38 MAPK and type I IFN signaling pathways (180.12)
Authors: Seo Young-Jin , Pritzl Curtis , Vijayan Madhuvanthi , McClain Mariah , Hahm Bumsuk
Published: January 2023

Sphingosine analogs display immunosuppressive activities and are therapeutically used for the treatment of autoimmune diseases. Here, we investigated the effects of the sphingosine analog AAL-R (FTY720 derivative) on dendritic cell (DC) response to toll-like receptor (TLR) stimulation. AAL-R inhibited DC maturation in response to TLR3 or TLR4 activation, representing its previously known immunosuppressive action. In contrast, AAL-R increased TLR7-mediated DC responses by increasing the expression of MHC-I and co-stimulatory molecules and type I interferon (IFN), and by enhancing the capacity of DCs to induce CD8+ T cell proliferation. Importantly, the stimulatory activity of AAL-R was dependent on type I IFN signaling, since type I IFN receptor-deficient DCs failed to respond to AAL-R. Further, AAL-R activated p38 MAPK signaling to increase type I IFN synthesis and TLR7-mediated DC maturation. Thus, our results indicate that AAL-R’s regulatory action is strongly affected by the form of pathogenic molecular patterns and is stimulatory when DCs are treated with a TLR7 agonist. These findings enhance our understanding of sphingosine regulation of host immune responses particularly in pathogenic infections.
Roles of TRAF5 in TLR signaling (180.4)
Authors: Buchta Claire , Bishop Gail
Published: January 2023

Dysregulation in Toll-like receptor (TLR) signaling has been implicated in many disease states, including chronic inflammatory conditions such as atherosclerosis. The cytoplasmic adaptor proteins TNF receptor associated factor (TRAF)3 and TRAF6 are known to mediate TLR signaling. Recent data from our lab demonstrate for the first time that TRAF5 also plays a significant role in immune cell TLR functions. B lymphocytes, macrophages, and dendritic cells from TRAF5-/- mice show increased cytokine production and enhanced early signaling pathways after stimulation through various TLRs. Interestingly, B cell TLR responses are the most greatly affected by TRAF5 deficiency. Our data show that TRAF5 is a negative regulator of TLR signaling and restrains proinflammatory cytokine production. It has been reported that TRAF5 deficiency accelerates the development of atherosclerosis in mice, while human patients with coronary heart disease express lower levels of TRAF5 mRNA in blood compared with healthy controls. Elucidating the role of TRAF5 in TLR signaling may provide a new target for therapies against a variety of inflammatory conditions, including those predisposing to atherogenesis. Our current studies focus on identifying the key molecular interactions by which TRAF5 mediates its effects on TLR pathways.
TLR2 molecular recognition of PorB from commensal Neisseria lactamica (180.6)
Authors: Massari Paola , Toussi Deana , Liu Xiuping , Carraway Margareta
Published: January 2023

Toll-like receptors recognize diverse conserved pathogen-associated molecular patterns (PAMPs). TLR2 has the broadest ligands repertoire, including bacterial lipidated and non-lipidated proteins such as porins. TLR2/porins interaction is possibly based on electrostatic attraction. PorB porin, expressed by all Neisseriae, has significant strain-specific sequence variability within the putative TLR2-binding regions. PorB from commensal N.lactamica (NL) and pathogenic N.meningitidis (NM) bind to TLR2 in vitro with different specificity and induce inflammatory responses of different magnitude. Whole NM is also more potent than NL in inducing cell activation. A TLR2-binding signature sequence in the PorB surface exposed loops might influence specific interaction with TLR2 and activation of signaling pathways. Exchange of NM PorB for NL PorB in NM induces attenuated inflammatory responses. To identify PorB/TLR2 interactive domains (and possibly TLR1) and to provide a mechanistic explanation for their different activity, we have expressed PorB mutants with altered loops sequences and show a gradient of cell activation. However, hybrid PorB mutants bind to TLR2 in vitro. This possibly suggests that the reduced activity of PorB mutant could be due to non-functional TLR2 heterodimerization with TLR1.
Differential roles of DNA-PKcs in the innate response to CpG-DNA (180.9)
Authors: Chu Wen-Ming , Ma Chi , Muranyi Marianna , Chu Catherine
Published: January 2023

CpG-DNA and its analog CpG-ODN powerfully activate dendritic cells (DCs) to produce pro-inflammatory cytokines, which are critical for vaccine strategies for allergy, infectious disease and cancer. There are two major types of CpG-DNAs, CpG-A and CpG-B. CpG-A activates the IFNa/b pathway, which requires IRF1 and 7, while CpG-B triggers the pro-inflammatory pathway, which requires AP-1 and NF-kB. Primarily, CpG-A triggers the IFNa/b response in pDCs in a complete TLR9- dependent manner. Yet, CpG-A together with DOTAP clearly activates DCs to produce IFNa/b in both TLR9 dependent and independent manners. Also, CpG-B induces IFNb production in DCs. To further understand how CpG-DNA activates innate immunity, we investigated a role of DNA-PKcs in this activation. Our results showed that loss of DNA-PKcs reduced pro-inflammatory cytokine response to CpG-B in DCs in a time and dose dependent manner. Interestingly, the IFNa/b response to CpG-A was defective in DNA-PKcs-deficient DCs, whereas the IFNb response to CpG-B was enhanced in the same cells. Also, Stat1 phosphorylation was diminished in either DNA-PKcs or TLR9 deficient DCs, and was further reduced in DNA-PKcs/TLR9 double deficient DCs. Additionally, our data showed that DNA-PKcs associated with TRAF6, which is critical for the IFNa/b and the pro-inflammatory cytokine responses to CpG-DNA. Together, our results suggest DNA-PKcs plays a differential role in regulating the innate immune response to CpG-DNA.
Rapid evolution of the binding specificities and expression patterns of CD33-related Siglecs in primates (181.4)
Authors: Padler-Karavani Vered , Hurtado-Ziola Nancy , Verhagen Andrea , Sonnenburg Justin , Chen Xi , Varki Ajit , Angata Takashi
Published: January 2023

Siglecs (Sialic acid-binding Immunoglobulin-like Lectins) are vertebrate sialic acid (Sia) binding proteins mostly expressed in hematopoietic and immune cells. They are type-I proteins with an extracellular N-terminal Sia-binding Ig-like V-set domain, one or more C2-set Ig-like domains, a transmembrane domain and a C-terminal cytoplasmic tail. CD33-related Siglecs (CD33rSiglecs) are a genetically clustered subgroup. Prior comparative genomic analysis indicated accelerated evolution of CD33rSiglecs selectively occurring in the Sia-binding V-set domain, suggesting that this domain is under the greatest selection pressure. To gain further insight into this rapid evolution of CD33rSiglecs, we prepared recombinant form of the N-terminal domains of CD33rSiglecs from human, chimpanzee, and baboon, and compared their sialoglycan binding-specificities. Using ELISA on a panel of gangliosides or analysis on a sialoglycan microarray we show that binding patterns are widely divergent between the three species. Some show preference to Neu5Gc over Neu5Ac, especially in chimpanzees and baboons, indicating an evolutionary adjustment to the loss of Neu5Gc expression in humans. Expression patterns of CD33rSiglecs on circulating blood cells of humans and related great apes also show evidence for rapid evolution, and overall expression is greater in apes compared to human. The selection mechanisms driving this rapid evolution will be discussed.
The complex relationship between TRAF3 and B cell function (42.2)
Authors: Lin Wai , Hildebrand Joanne , Bishop Gail
Published: January 2023

TRAF3 is a negative regulator in B lymphocytes of CD40 and BAFF-R mediated signaling events, as well as basal B cell survival and homeostasis. Loss of function mutants of TRAF3 are associated with multiple myeloma (MM) and B-cell lymphoma, and TRAF3-/- B cells in mice show greatly enhanced survival, as well as constitutive activation of the alternative NF-κB2 pathway. Current dogma is that TRAF3 promotes degradation of the kinase NIK to control activation of the NF-κB2 pathway, and stimulation through CD40 or BAFF-R induces TRAF3 degradation, allowing NIK to activate NF-κB2, which promotes survival. However, our recent findings indicate a more complex regulatory mechanism. The A/WySnJ mouse expresses a mutant BAFF-R in which the C terminal 8 amino acids are replaced by a proviral sequence. BAFF ligation of this mutant receptor does not activate NF-κB2, but induces TRAF3 degradation comparable to Wt BAFF-R. Additionally, we analyzed a human MM-derived TRAF3 mutant and found that its expression enhanced NF-κB2 activation in B cells, but it is not degraded. Finally, we found that T cells or dendritic cells lacking TRAF3 also have constitutive NF-κB activation, but show no enhanced survival. Taken together, these data suggest that TRAF3 degradation or loss is not always necessary or sufficient to promote the NF-κB2 pathway, that activation of this pathway is insufficient to promote cell survival, and that TRAF3 plays cell-type-specific unique roles in B cell biology.
Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes (42.5)
Authors: Lucas Christopher , Cordero-Nieves Hector , Campbell Kerry , Bhatia Sumeena , Hodes Richard , Sanders Virginia
Published: January 2023

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that increase Oct-2 and IgG1 mRNA and protein expression, independent of class switch recombination. One of the most proximal intermediates identified is PLCγ2. Since the cytoplasmic domain of CD86 lacks tyrosine residues reported to bind PLCγ2, we used a proteomics-based identification approach to show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and prohibitin-2 (Phb2), bind to CD86. Although basal expression of Phb1/2 and association with CD86 was low in resting B cells, expression and association increased after CD40 engagement. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by shRNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine PKC-phosphorylation sites, but did not affect Phb1/2 binding to CD86. Furthermore, Phb1/2 reduction and CD86 truncation revealed that the CD86-induced phosphorylation of PLCγ2 required Phb1/2 alone; whereas, IκBα and NF-κB (p65) phosphorylation required both Phb1/2 and the CD86 cytoplasmic domain. Together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell to regulate IgG1 production and is the first evidence to define the molecular basis of membrane-proximal CD86 signal transduction in B cells.
The transcription factor T-bet regulates parasitemia and promotes pathogenesis during Plasmodium berghei ANKA murine malaria (43.16)
Authors: Oakley Miranda , Solanki Nehal , Lotspeich Cole Leda , Majam Victoria , Sahu Bikash , Derrick Steven , Morris Sheldon , Kumar Sanjai
Published: January 2023

CD4+ T cells are required for the pathogenesis of experimental cerebral malaria (ECM) during the induction phase of disease in mice. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the C57BL/6 background, we demonstrate that while Th1 CD4+ T cells play a role in the regulation of parasite burden, they also promote the pathogenesis of ECM possibly by invoking a robust proinflammatory response. We find that while 100% (10/10) of wildtype mice developed ECM by day 9 post-infection, only 30% (3/10) of T-bet deficient mice succumbed to disease during the cerebral phase of infection (p=0.000058). In addition, T-bet deficient mice had higher parasitemia than wildtype controls during the ECM phase of disease (17.7 +/- 3.1% verses 10.9 +/- 1.5%). These results indicate that T-bet dependent regulation of parasite burden may promote pathogenesis in this murine model of ECM. We are also investigating the effect of T-bet on protective immunity in the P. yoelii NL murine model and the P. chabaudi AS model during the acute and clearance phases and in recrudescence. In depth analysis of immune cells and cytokines is currently being performed to better understand how Th1 CD4+ T cells mediate their protective as well as pathogenic functions during malaria infection.
T-cells contribute to the pathogenesis of Staphylococcus aureus pneumonia (43.19)
Authors: Parker Dane , Prince Alice
Published: January 2023

Staphylococcus aureus is a leading cause of pneumonia and increasing evidence indicates the host response contributes to disease. We investigated the role of T-cells in S. aureus pneumonia. We initially utilized Rag2 null mice and infected them with S. aureus MRSA strain USA300 (the strain currently epidemic in the USA) for 20h. Rag2-/- mice showed reduced pulmonary pathology and improved bacterial clearance in bronchoalveolar lavage fluid (BALF) and lung tissue (85% reduction, P<0.05) compared to wild-type mice. Examination of immune cells in the BALF and lung showed no differences between infected WT and Rag2 null mice. Levels of TNF, KC, IL-6 and IL-17 were significantly reduced (50%, 85%, 52%, 100% respectively, P<0.01). We then focused on the role of CD4 cells. CD4 null mice infected with USA300 also exhibited reduced pulmonary pathology compared to wild-type mice as well as significantly fewer bacterial numbers in the BALF and lung parynchyma (reduced by 80% (P<0.05) and 75% (P<0.05) respectively). Although Cd4-/- mice also had no changes in lung cell populations they did have significantly reduced levels of TNF (68%, P<0.01), KC (86%, P<0.01), IL-6 (61%, P<0.05) and IL-17 (48%, P<0.05). Our data from these mouse models suggest that T-cells in response to S. aureus contribute significantly to inflammation and interfere with bacterial clearance in the lung. The modulation of T-cells may have a role in modifying the immune response to S. aureus and improving outcomes.
IgM memory B cells generated during bacterial infection are required for secondary IgG responses to antigenic challenge (43.2)
Authors: Yates Jennifer , Racine Rachael , McBride Kevin , Jones Maura , Winslow Gary
Published: January 2023

Immunological memory was long considered to be harbored in B cells that express high affinity class-switched IgG. More recently, IgM memory B cells have been identified following immunization with nominal antigens and adjuvants. Based on the expression of CD11c, we have identified a large population of such cells using a natural model of infection by the bacterium Ehrlichia muris. These CD11c+ IgM memory cells exhibit phenotypic characteristics of memory B cells, including expression of CD73, CD11b, and PD-L2. In addition, these CD11c+ IgM memory cells lack expression of CD138, are largely quiescent, and have accumulated somatic mutations. Although these cells did not proliferate or secrete antibody ex vivo, they produced antigen-specific IgM upon in vitro stimulation with mitogens. Furthermore, in vivo depletion abrogated the IgG recall response to specific antigenic challenge. Our findings are consistent with previous data demonstrating that IgM memory cells undergo class switch recombination and affinity maturation following re-encounter with cognate antigen. We propose that these CD11c+ IgM memory B cells are responsible for the IgG produced following secondary challenge in ehrlichial infection, and likely function to provide greater flexibility to variant antigenic challenges.
The Role of B Cells in the Immune Response to Mycobacterium tuberculosis. (43.3)
Authors: Kozakiewicz Lee , Maglione Paul , Xu Jiayong , Casadevall Arturo , Chan John
Published: January 2023

The role of humoral immunity in the immune response to Mycobacterium tuberculosis (Mtb) is poorly defined. B cells form aggregates with features of germinal centers and are prominent components of the lung granuloma in Mtb-infected humans, mice and macaques. Using muMT B cell-deficient mice, we showed that B cells were required for optimal granulomatous response and protection upon aerosol infection. muMT mice were more susceptible to Mtb with increased mortality and elevated lung bacterial burden. B cell-deficiency also resulted in exacerbated immunopathology with increased neutrophil influx and IL-10 production independent of tissue bacterial load. Transfer of B cells reversed the phenotypes of the muMT mice. This occurred in the presence of immunoglobulins (Igs) in the serum but without the presence of lung B cells. We showed that engagement of distinct Fcγ receptors (FcγR) divergently affected susceptibility to Mtb. Mice lacking activating FcγR showed increased mortality and bacterial load with enhanced lung neutrophil influx and IL-10 expression compared to wildtype, whereas mice lacking inhibitory FcγRIIB exhibited an augmented lung Th1 response. Thus, B cells via their ability to produce Igs may regulate T cell responses by immune complex engagement of FcγR on antigen presenting cells. The mechanisms by which B cells modulate the immune response to Mtb are being characterized by testing the immune response of transgenic mice deficient in B cells or Igs to infection.
Recombinant proteins encoded by genes present in Mycobacterium tuberculosis-specific regions of difference induce delayed-type hypersensitivity skin responses (43.31)
Authors: Mustafa Abu
Published: January 2023

The world-wide control of tuberculosis (TB) requires affordable and easy to apply test(s), which could differentiate TB from BCG vaccination and exposure to environmental mycobacteria. In this study, the proteins encoded by Mycobacterium tuberculosis-specific regions of difference (RD) genes, which are absent in BCG and most environmental mycobacteria, were studied for delayed-type hypersensitivity (DTH) skin responses in guinea pigs. The genes of RD1 (CFP10 and ESAT-6), RD7 (Rv2346 and Rv2347) and RD9 (Rv3619 and Rv3620) proteins were cloned in plasmid vectors and expressed in Escherichia coli. The expressed recombinant proteins were purified to homogeneity using affinity columns. Guinea pigs were injected with 1x1010 heat-killed M. tuberculosis, and phosphate buffered saline (PBS), as a placebo control. After four weeks of injection, all the animals were tested for DTH skin responses by intradermal injection with PBS (negative control), purified protein derivative (PPD) and sonicates of M. tuberculosis (positive control), and purified recombinant proteins. The results showed that PBS failed to induced DTH responses in any group, whereas PPD, M. tuberculosis sonicates and the recombinant proteins induced positive DTH responses in animals injected with heat-killed M. tuberculosis, but not in PBS-injected animals. These results suggest that purified RD proteins are active in inducing DTH responses in vivo, and may be useful in specific diagnosis of TB.
Acute alcohol exposure induces tolerance to endotoxin and reduces pro-inflammatory cytokine production: role for hsp70 and hsp90 (44.21)
Authors: Ambade Aditya , Deshpande Janhavee , Catalano Donna , Mandrekar Pranoti
Published: January 2023

Alcohol, a commonly abused drug in US, is linked to impaired host defense, increased susceptibility to infections. Acute alcohol exposure in humans and experimental animals attenuates endotoxin/LPS induced pro-inflammatory cell activation. Molecular mechanisms involved in these processes are unclear. Earlier, we showed that acute alcohol induces hsp70, reported to exert anti-inflammatory effects by inhibiting NFκB activity. Here we hypothesize that acute alcohol induces tolerance to a subsequent LPS challenge by regulation of hsp70 and hsp90. To test our hypothesis, RAW 264.7 mouse macrophages were exposed overnight to alcohol (25mM, 50mM) followed by LPS (100ng/ml) challenge. Pro-inflammatory cytokine, NFκB activity and heat shock transcription factor (HSF) 1, hsp70 and hsp90 expression was analyzed. Results show that overnight acute alcohol exposure decreased inflammatory cytokine, TNFα, production to a subsequent LPS challenge. Concomitantly acute alcohol induced HSF1 binding activity and hsp70 but reduced hsp90 levels in RAW macrophages. Hsp70 immunoprecipitation revealed that acute alcohol induces hsp70-NFκB-p50 complexes likely preventing NFκB DNA binding. Treatment of macrophages with hsp70 inhibitor, quercetin, partially blocked acute alcohol mediated down regulation of LPS-induced pro-inflammatory cytokine TNFα. Summarizing, our results indicate that acute alcohol induces endotoxin tolerance and reduced pro-inflammatory cytokines via HSF1 and hsp70 induction.
Adaptive immunity mediated by Natural Killer cells. (45.20)
Authors: Paust Silke , Flynn Michael , Wang Baozhong , Dougan Stephanie , Ploegh Hidde , Telenti Amalio , Compans Richard , von Andrian Ulrich
Published: January 2023

Adaptive immunity is thought to be a unique feature of T and B lymphocytes. However, there is mounting evidence that adaptive immunity can be mediated by a discrete subset of liver-resident natural killer (NK) cells. To date, Rag-independent NK memory has been documented for four distinct haptens and six viral antigens (Ag). We have developed a sorting strategy to identify and isolate Ag-specific NK cells from livers of Rag-/- mice sensitized with the hapten DNFB. By transplanting nuclei from sorted Rag-/- NK cells into enucleated oocytes, embryonic stem (ES) cell lines were generated that were used to clone mice. NK cells from the cloned animals instantly responded to DNFB without requiring prior sensitization, but they could not be sensitized to other Ags. Thus, the nuclear 'information' for Ag specificity of memory NK cells persists even in animals generated from a donor nucleus whose epigenetic state was reset by nuclear reprogramming. This apparent genetic fixation would be expected if Ag receptor specificity were encoded at the level of genomic DNA, rather than by epigenetic regulation of conventional 'hardwired' NK receptors, and suggest an entirely novel mechanism to generate Ag receptor diversity. Thus, current views of the immune system as a duality of adaptive (T and B cells) and innate (everything else) arms, need to be re-imagined as a trinity that includes memory NK cells as a third branch.
Obligate role of CXCR3 chemokine receptor for trafficking of effector CD8 T cells in the tumor microenvironment (46.14)
Authors: Mikucki Maryann , Skitzki Joseph , Fisher Daniel , Luster Andrew , Evans Sharon
Published: January 2023

Trafficking of tumor-reactive T cells across vascular checkpoints is a critical juncture in the effector phase of cancer immunity although this step is largely unexplored. We recently showed that poor trafficking of blood-borne CD8 effector T cells across tumor microvascular checkpoints is a significant obstacle to antitumor immunity. This bottleneck can be overcome by preconditioning regimens using systemic thermal therapy (STT, 39.5 ± 0.5°C, 6 h) that promotes E/P-selectin and ICAM-1-dependent trafficking of adoptively transferred CD8 T cells and subsequent lysis of tumor targets. Here we investigated the chemokine receptor requirements for trafficking of tumor-specific OT-I CD8 T cells across microvascular barriers in B16-OVA murine melanoma. We identified a non-redundant role for CXCR3, but not CXCR4, in T cell trafficking to tumors under both homeostatic and STT conditions using CXCR3-deficient OT-I T cells. Parallel results were observed by intravital microscopy whereby CXCR3 was necessary for T cells to undergo chemokine-dependent transition from rolling to firm arrest in tumor vessels of STT-pretreated mice. Taken together, these studies provide the first evidence that CXCR3 is obligatory for T cell trafficking across tumor vascular gateways and support strategic use of therapies to augment chemokine-dependent delivery of immune cells for the treatment of cancer. Supported by the NIH (CA79765, CA085183), and the Jennifer Linscott Tietgen Family Foundation.