bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide binding characteristics of NOD2 proteins (INM1P.442)
Authors: Davis Beckley , Talento Suzanna , Tocker Aaron , Duncan Joseph
Published: January 2023

Nucleotide binding and oligomerization domain containing protein 2 (NOD2) is an intracellular protein that is involved in the recognition of bacterial cell wall derived muramyl-dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders including Crohn’s disease and Blau syndrome. NOD2 is a member of the Nucelotide-binding domain and leucine-rich repeat containing protein gene family (NLR). Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by pathogenic NOD2 proteins remains unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl-dipeptide in cellular assay. We demonstrate that nucleotide binding is necessary for wild type and pathogenic NOD2 protein activation of the NF-κB signaling pathway. Mutations of the Walker A motif inhibit binding to RIP2k in wild type NOD2, Crohn’s disease-associated NOD2 and Blau syndrome-associated NOD2. Loss of RIP2k binding correlates with decreased NF-κB-dependent luciferase activity. These data suggest that nucleotide binding is necessary for NOD2 function.
Neutrophil extracellular traps activated by damage associated molecular patterns exacerbates sterile inflammatory liver injury (INM1P.427)
Authors: Huang Hai , Tohme Samer , Al-khafaji Ahmed , Tsung Allan
Published: January 2023

The initiation of liver ischemia/reperfusion (I/R) injury results in the release of damage associated molecular patterns (DAMPs) such as HMGB1 and histones, which trigger innate immune and inflammatory cascade via Toll-like receptor 4 (TLR4) or TLR9. Infiltrated neutrophils contribute to the organ damage, innate immune and inflammatory responses after liver I/R. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo, associated with increased serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 (NET markers) compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I conferred significant protection after liver I/R evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. DAMPs, such as HMGB1 and histones stimulate NET formation through TLR4 and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. Conclusion: DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.
Rapid activation of lymph node lymphangiogenesis (IRC4P.458)
Authors: Ruddell Alanna , Farr Andrew , Jordan-Williams Kimberly
Published: January 2023

The lymphatic system delivers fluid and molecules from tissues to the lymph node (LN) to mediate immune system surveillance of self and foreign antigens. LN lymphatic endothelial cells (LECs) function in orchestration of the immune response, and in reinforcement of peripheral tolerance. Extensive LN lymphatic sinus growth (lymphangiogenesis) is involved in multiple diseases including cancer, arthritis, and inflammation. Lymphocytes and leukocytes regulate this lymphangiogenesis; however, the mechanism is not well defined. We identified a monoclonal antibody (10.1.1) that recognizes the LEC protein mCLCA1. Our studies identified mCLCA1 as an interacting partner for LFA-1 and Mac-1 that mediates lymphocyte and leukocyte adhesion. The 10.1.1 Ab blocks adhesion of lymphocytes to LECs in vitro to a greater extent than ICAM-1 neutralizing Ab, suggesting that while ICAM-1 interactions predominate in vascular endothelium, mCLCA1 interaction is more important for adhesion to LECs. In addition to mediating adhesion, the 10.1.1 Ab (but not control Ab) induces rapid LN lymphangiogenesis within 23 hours after injection into mice, which involves proliferation of LN LECs. This lymphangiogenesis is accompanied by LN remodeling involving multiple stromal cell types, resembling the LN response to inflammation. These findings suggest that 10.1.1 Ab mimics lymphocyte or leukocyte B2-integrin binding to LEC mCLCA1, to drive LN remodeling and lymphangiogenesis during immune responses.
Recovery of S100A8 in the absence of S100A9 exacerbates TNFα-mediated psoriatic-like arthritis (IRC4P.462)
Authors: Thurainayagam Sumita , Wixler Viktor , Roth Johannes , Vogl Thomas
Published: January 2023

Psoriasis is a chronic autoimmune disorder and frequently associated with arthritis (PsA). Alarmins S100A8 and S100A9, expressed in granulocytes, monocytes and activated keratinocytes, are highly up-regulated in psoriatic skin and synovium. Although S100a8 mRNA remains unchanged in S100A9-deficient (S100A9-/-) mice, S100A8 protein expression is abrogated, thus generating a functionally double knockout mouse model. In general, S100A9-/- mice show reduced inflammatory activities in several mouse models of infection and inflammation. Recently, we generated ihTNFtgxS100A9-/- mice by crossing doxycycline-inducible human TNFα-transgenic mice (ihTNFtg) with S100A9-/- mice. Unexpectedly, we observed an enhanced disease progression in these mice as evidenced by increased loss of body weight, paw swelling and cartilage destruction and decreased grip strength compared to ihTNFtg mice upon TNFα induction. Interestingly, the enhanced disease progression was associated with re-expression of S100A8 protein upon TNFα induction, observed in bone marrow, blood, skin and nail keratinocytes by IHC staining and optical imaging. We hypothesize that in the absence of its binding partner S100A9, constitutively active S100A8 homodimers aggravate TNFα-mediated psoriatic-like arthritis. Our data indicate a complex S100A8/S100A9 regulatory mechanism of an alarmin-driven inflammation.
AN2728, a new boron-based topical anti-inflammatory agent, inhibits phosphodiesterase 4 (IRC4P.611)
Authors: Freund Yvonne , Dong Chen , Virtucio-Frates Charlotte , Rock Fernando , Mak Yvonne , Zhou Yasheen , Zane Lee , Jarnagin Kurt
Published: January 2023

The novel boron-containing compound, AN2728, is being investigated in phase 3 trials for atopic dermatitis treatment. It exerts its anti-inflammatory effect by inhibiting phosphodiesterase-4 (PDE4), which catalyzes the breakdown of cAMP to AMP. AN2728 competes with cAMP to inhibit the PDE4B1-catalytic domain with Ki of 173±26 nM; thus AN2728 interacts at the enzyme-active site. The X-ray structure of PDE4B-catalytic domain with AN2728 and its structural relative AN2898 reveals that the boron atom interacts with the bimetal center and occupies a position in the catalytic site similar to that of the cAMP phosphate. AN2728 has good affinity across PDE4 gene products A-D. Its selective affinity for PDE4 is 4-10-fold greater than its affinity for PDE1, 2, 3A, 6, or 7B. It is inactive on PDE3B, 5, 7A1, and 8-11. AN2728 activity increases intracellular cAMP and activates PKA, followed by phosphorylation and negative regulation of various cytokine transcription factors. AN2728 inhibits production of specific cytokines with a pattern similar to that of other established PDE4 inhibitors and distinct from those of a glucocorticoid and calcineurin inhibitor. To investigate reactivity against other targets, AN2728 was tested against a set of 50 receptors and ligand-gated ion channels for inhibition at 10 µM. Inhibition was <25% for all receptors; thus, AN2728 is specific for PDE4 and appears to exert an anti-inflammatory effect through inhibition of inflammatory cytokine production.
Genetic control of isotype specific antibody responses to flagellin in mice (IRC4P.450)
Authors: LU CHUNCHI , Lopez-Yglesias Americo , CHEN HSINHUI , Zhao Xiaodan , VandenBos Tim , Strong Roland , Smith Kelly
Published: January 2023

Adjuvants can improve the efficacy of vaccines by inducing class-switched immune responses. Host factors controlling the magnitude of class-switched antibody responses are poorly understood. We have observed flagellin-immunized mouse strains differ in the magnitude of isotype specific antibody responses. Some strains are high responders (A/J, Swiss Webster) and other strains are low responders (C57BL6, Balb/c). All strains had comparable primary anti-flagellin IgM responses, suggesting common genetic variations influence isotype class switching. We hypothesized different genetic differences between A/J and C57BL/6 mice determine magnitude of responder phenotype. We bred C57BL/6 and A/J mice and tested F1 and F2 offspring for isotype specific antibody responses. Mice were immunized with a FliC protein mutated in the Naip5/6 recognition domain to overcome known differences in Naip5-dependent inflammasome activation between these mouse strains. We demonstrate anti-flagellin IgG1, IgG2a/c and IgA responses are inherited as a quantitative trait. Using consomic mice, we show the high responder phenotype is not controlled by the IgH locus on A/J chromosome 12. Using mutant flagellin proteins, we demonstrate the high responder phenotype is primarily controlled by the FliC D2/D3 domain. Understanding the host factors contributing to FliC D2/D3 induced antibody responses aid in the rationale design of vaccines, and help establish principles for future adjuvant design and development.
Effects of short chain fatty acids and GPR43 stimulation on human Treg function (IRC5P.631)
Authors: Mamontov Polina , Neiman Eric , Cao Tinghua , Perrigoue Jacqueline , Friedman Joshua , Das Anuk , Mora J.
Published: January 2023

SCFA (acetate, propionate, butyrate) are produced when dietary fiber is metabolized by commensal microbiota. In the mouse, SCFA increase colonic Foxp3+ regulatory T cell (Treg) function, promote immune tolerance, and ameliorate intestinal inflammation through the G protein-coupled receptor GPR43 (FFAR2). However, despite the compelling literature in the mouse, it has not been reported whether SCFA can modulate human Treg function. Moreover, whether GPR43 stimulation is sufficient to mediate any effects of SCFA on human Treg remains unknown. Here we show that acetate or butyrate did not increase Foxp3 expression in ex vivo-differentiated human Treg. Nonetheless, consistent with a proposed effect of SCFA on Treg tolerogenic function, butyrate increased IL-10 production by ex vivo-differentiated Treg. Of note, we show that synthetic GPR43-agonists increased the in vitro suppressive capacity of human peripheral blood CD4+/CD25+ Treg in a T cell/DC co-culture system, an effect that correlated with a decreased production of pro-inflammatory cytokines. Thus, pharmacological GPR43-agonists may partially emulate SCFA effects on Treg. Our results show for the first time that SCFA can modulate human Treg function, and that pharmacological GPR43-agonists are sufficient to increase Treg suppressive capacity, with potential therapeutic implications for inflammatory/autoimmune diseases.
Inhibition of phosphorylation of 4E-BP1 by resveratrol suppresses monosodium urate crystal-induced production of interleukin-1β in human monocytes (IRM5P.646)
Authors: Chung Yeon-Ho , Chung Doo Hyun , Lee Won-Woo
Published: January 2023

Monosodium urate (MSU) crystal-induced production of interleukin-1β(IL-1β) from innate immune cells has a critical role for the pathogenesis of gouty inflammation. Recently, it has been reported that resveratrol has a protective effect on gouty arthritis in mouse model. However, the underlying mechanism remains unknown. In this study, we investigated the immunoregulatory role of resveratrol affecting MSU crystal-induced IL-1β production in human CD14+ monocytes. Our data showed that resveratrol inhibited the release of IL-1β by monocyte in response to MSU crystal stimulation and it was closely associated with a decreased level of production of intracellular pro-IL-1β. To investigate the mechanism involved with the negative effect of resveratrol on pro-IL-1β synthesis, we evaluated the phosphorylation status of cap-dependent translation inhibitor 4E-BP1 in MSU crystal-treated monocytes. Resveratrol markedly diminished the phosphorylation of 4E-BP1 in MSU crystal-treated monocytes. Moreover, the autoproteolytic activation of caspase-1 induced by MSU crystal-treatment was also greatly prevented by resveratrol. These results reveal the mechanism underlying how resveratrol prevents MSU crystal-induced production of IL-1β in monocytes and suggest its novel function in inhibition of gouty inflammation.
Rhbdd3 controls innate inflammatory diseases via negatively regulating TLR signaling (IRM5P.647)
Authors: Liu Juan
Published: January 2023

Rhomboid domain-containing protein 3 (Rhbdd3), which belongs to a family of proteins with rhomboid domain, is widely expressed in immune cells; however, the roles of Rhbdd3 in immunity remain unknown. Rhbdd3-deficient mice spontaneously developed autoimmune diseases due to increased IL-6 production, and developed more severe inducible autoinflammatory colitis and acute hepatic injury. Using Rhbdd3-deficient mice, we reveal that Rhbdd3 negatively regulates Toll like receptor ( TLR)-mediated NK cell and DC activation. Rhbdd3 respectively interacts with DAP12 and NEMO to inhibit MAPK and NF-kB signaling activation in TLR-triggered NK cells and DC. Furthermore, Rhbdd3 directly binds to K27-linked polyubiquitin chains on NEMO via its UBA domain, and recruits the deubiquitinase A20 to induce K63-linked deubiquitination of NEMO and thus inhibits activation of the transcription factor NF-kB in DCs. Our study outlines a mechanism for the negative regulation of TLR signaling and autoimmune diseases.
Calcium-dependent PKC is required for chromatin decondensation during the activation of peripheral T cells (IRM6P.651)
Authors: Rawlings Jason , Eikenbusch Sarah , Shields Molly , Bingham Kellie , Lee Megan , Meredith Jenna , Mitchell Taylor
Published: January 2023

Activation of the T cell receptor (TCR) through antigen binding results in chromatin decondensation required for the clonotypic proliferation observed during a proper immune response. Activation of the TCR results in the cleavage of phospholipase biphosphate (PIP2) into diacylglyercol (DAG) and inositol trisphosphate (IP3) by phospholipase C-γ (PLC-γ). Previous studies have shown that both the DAG and IP3 pathways can decondense chromatin, and that intracellular calcium is required for proper TCR-mediated chromatin decondensation. In this study, we used flow cytometry to analyze histone accessibility of chromatin in T cells and found that PKC activation is also required for proper activation-induced chromatin decondensation. Furthermore, we show that calcium is required for proper PKC-mediated chromatin decondensation. Our studies suggest that the PKC isoform(s) required for decondensation may be calcium-dependent. Finally, we show that activation of PKC and not IP3 is required for T cells to gain competence to respond to cytokine stimulation. Taken together, our data suggest that IP3 and PKC signaling work cooperatively to decondense chromatin and regulate clonotypic expansion of T cells during an immune response.
H3K9 trimethylation silences Fas expression to confer colon carcinoma immune escape and chemoresistance (IRM6P.654)
Authors: Paschall Amy , Yang Dafeng , Li Xia , Choi Jeong-Hyeon , Liu Feiyan , Figueroa Mario , Oberlies Nicholas , Pearce Cedric , Bollag Wendy , Nayak-Kapoor Asha , Liu Kebin
Published: January 2023

The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colorectal carcinoma (CRC) often uses silencing of Fas expression as a mechanism of immune evasion. The molecular mechanism of FAS transcriptional silencing in human CRC is unknown. We performed genome-wide ChIP-Sequencing analysis and determined that the level of H3K9me3 in the FAS promoter region is significantly higher in metastatic than in primary human CRC cells, and is inversely correlated with the level of Fas expression. We discovered that verticillin A is a selective inhibitor of histone methyltransferases and inhibits H3K9 trimethylation in human CRC cells. Fas expression is abolished in metastatic human CRC cells and verticillin A treatment effectively restored Fas expression in these metastatic CRC cells. Furthermore, verticillin A exhibited greater efficacy than Decitabine and Vorinostat in overcoming metastatic human CRC resistance to FasL-induced apoptosis. Interestingly, verticillin A also effectively overcomes metastatic human CRC resistance to 5-Fluorouracil in vitro and in vivo. Using an orthotopic CRC mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL+ and FasL-mediated cancer immune surveillance is essential for CRC inhibition in vivo. Our findings determine that H3K9 trimethylation of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant CRC immune evasion and metastasis.
Nucleosomal remodeling defects at gene promoters underlie cross-phenotype effects in X-linked thrombocytopenia/Wiskott-Aldrich syndrome (IRM6P.655)
Authors: Sarkar Koustav , Sadhukhan Sanjoy , Han Seong-Su , Vyas Yatin
Published: January 2023

Loss-of-function mutations in Wiskott-Aldrich-Syndrome (WAS) gene associate with either X-linked thrombocytopenia (XLT) or XLT that progresses to immunodeficiency, yet the biological basis for gene-pleiotropy remains unknown. WASp, the protein deficient in WAS, generates F-actin in the cytosol via VCA-domain and supports transcription in the nucleus, the latter by an unknown mechanism. We report that nuclear-WASp, independently of its VCA-domain, is required for the recruitment of BAF47, BAF170, and EP400 to IFNG and TBX21 promoters during TH1-differentiation, but not to IL4 or GATA3 during TH2-differentiation. XLT-causing Thr45Met mutation that progresses to immunodeficiency, impairs recruitment of the above chromatin-remodelers, NF-κB(p65), and STAT1 to TH1-gene promoters, which show paucity of H2A.Z containing nucleosomes, and are therefore sub-optimally transcribed. Dissimilarly, Ala236Gly and Arg477Lys mutations that manifest in XLT without progressing to immunodeficiency do not impair TH1-transcriptional reprogramming. We identify a functional link between nuclear-WASp and SWI/SNF-complexes in T lymphocytes, perturbation of which provides a mechanism for the development of clinical crossphenotypes in human XLT/WAS.
Regulation and function of absent in melanoma 2 in human CD4 T cells (IRM7P.710)
Authors: Komori Heather
Published: January 2023

Recognition of dsDNA by absent in melanoma 2 (AIM2) leads to inflammasome formation, activation and secretion of IL-1β and IL-18 in innate cells. While AIM2 inflammasome function has been well characterized in macrophages, it has not been explored in adaptive cells. Recently, we have shown that human CD4 T cells robustly upregulate expression of AIM2 following T cell activation. Interestingly, memory CD4 T cells have a higher resting level of expression of AIM2 and more rapid upregulation of AIM2 mRNA following activation. High-throughput targeted bisulfite sequencing and ChIPseq show that AIM2 expression in CD4 T cells is epigenetically regulated by DNA methylation and histone modifications. The AIM2 promoter exhibits both hypomethylated CpGs and increased H3K4me3 and H3K4me2 in memory CD4 T cells compared to naïve. Such epigenetic marks allow for both a higher basal level of AIM2 expression and stronger induction of AIM2 expression following T cell activation. Robust protein expression of AIM2 is also found following stimulation of these cells. While AIM2 is known to form inflammasomes and drive IL-1β and IL-18 expression in macrophages, CD4 T cells do not express either of these cytokines, suggesting a non-canonical role for AIM2 function in T cells. Our current studies are focused on determining the function of AIM2 in CD4 T cells, and preliminary work suggests that AIM2 plays a role in the regulation of apoptosis following T cell receptor crosslinking
SLAM-associated protein deficiency in T cells from patients with systemic lupus erythematosus contributes to early signaling abnormalities and reduced IL-2 production (IRM7P.704)
Authors: Karampetsou Maria , Kis-Toth Katalin , Comte Denis , Tsokos George
Published: January 2023

T cells are key players in the pathogenesis of systemic lupus erythematosus (SLE), a chronic autoimmune disease. Following TCR stimulation, SLE T cells display increased levels of tyrosine phosphorylated proteins (pTyr) and augmented calcium (Ca++) responses compared to healthy donors. Impaired IL-2 and excessive IFNγ production also characterize SLE T cells. X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency caused by loss-of-function mutations of the SLAM-associated protein (SAP), a SH2-domain-containing cytoplasmic adaptor molecule. XLP T cells display TCR-mediated early signaling aberrations and a cytokine profile similar to SLE. We determined SAP protein and mRNA levels in SLE T cells and found them significantly decreased. Silencing of SAP in normal T cells resulted in increased pTyr levels and Ca++ flux following aCD3 stimulation. Interestingly, silencing of SAP resulted in decreased IL-2 production. Forced expression of SAP in SLE T cells restored pTyr levels and Ca++ responses to normal levels while the production of IL-2 was corrected to normal levels. In conclusion, SLE T cells display reduced levels of the protein adaptor SAP and that restoration of SAP protein levels corrects the early signaling transduction defects and restores IL-2 production. Approaches to restore SAP levels in SLE T cells may prove of therapeutic value.
TRAF3 regulates CSK and PTPN22 to enhance T cell receptor signaling (IRM7P.705)
Authors: Wallis Alicia , Yi Zuoan , Bishop Gail
Published: January 2023

TCR signaling is critical for T cell activation, so further elucidation of this process is necessary to understanding T cell biology, and to manipulate T cell activation in clinical settings. Extensive research on TCR signaling has revealed many details, but additional information on the molecular mechanisms continues to be revealed. Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 plays an important positive role in TCR signaling and T cell functions. After TCR engagement, TRAF3 associates with the TCR complex, and TRAF3-/- cells display a marked reduction in the early phosphorylation of Lck and CD3-ζ activation sites. This impact of TRAF3 on proximal TCR signaling events led us to hypothesize that TRAF3 regulates CSK, a T cell membrane-localized protein that binds to and inhibits Src family kinases, such as Lck. Through immunoprecipitation, TRAF3 was shown to interact with CSK upon TCR stimulation. The interaction between TRAF3 and CSK occurs in both the membrane and cytoplasmic fractions. In the absence of TRAF3, elevated amounts of CSK remain in the plasma membrane of stimulated T cells. Another TCR inhibitor, the protein tyrosine phosphatase PTPN22, also interacts with the TRAF3:CSK complex. Similar to its impact on CSK, loss of TRAF3 results in elevated levels of PTPN22 in the plasma membrane. These data identify a new role for TRAF3 in regulating the localization of the TCR signaling inhibitors CSK and PTPN22.
A single amino acid residue of CDR3β alters the antigenicity and autoreactivity of human invariant natural killer T cell receptors (IRM7P.707)
Authors: Guo Tingxi , Chamoto Kenji , Hirano Naoto
Published: January 2023

Detection of human invariant natural killer T (iNKT) cells relies on staining for their T cell receptor (TCR), invariant Vα24 TCRα chain paired with semi-variant Vβ11 TCRβ chain, or α-GalCer-CD1d tetramer. It has been assumed that all Vα24+ iNKT cells are stained with similar intensity by anti-Vα24 mAbs, clones C15 and 6B11, regardless of the hypervariable Vβ11 CDR3β sequences. By analyzing a large panel of autoreactive human iNKT TCRs, we found that TCRs encoding CASR at the start of CDR3β were stained with significantly lower intensity by C15 than those encoding CASS. Substitution of S to R at the fourth CDR3β residue was sufficient to diminish staining intensity by anti-Vα24 mAbs. Interestingly, the autoreactivity was also reduced in the TCRs encoding CASR. Importantly, α-GalCer tetramer positive population of human primary T cells presented low and high intensity subsets when stained with anti-Vα24 mAbs. Vβ11 TCRβ chains were cloned from each sorted population, and the Vα24 low population contained a significantly higher frequency of Vβ11 TCRs encoding CASR compared to the Vα24 high population. Therefore, human iNKT TCRs encoding CASR may falsely be negative when stained with anti-Vα24 mAbs, which underestimate the frequency of bona fide Vα24+ iNKT cells. Our data demonstrate that the fourth CDR3β amino acid residue affects iNKT TCR autoreactivity and binding of anti-Vα24 mAbs, suggesting that the CDR3β sequence alters the structural conformation of the Vα24 TCRα chain.
Dendritic cells coordinate the development and homeostasis of organ-specific regulatory T cells (LYM2P.730)
Authors: Leventhal Daniel , Nishi Saki , Gilmore Dana , Malchow Sven , Kline Douglas , Kline Justin , Savage Peter
Published: January 2023

Foxp3+ regulatory T cells (Tregs) are critical for the maintenance of tolerance to self. However, the antigen presenting cells (APCs) controlling the thymic development and peripheral homeostasis of these cells have yet to be clearly defined. Previously, we identified a monoclonal population of prostate-specific Tregs, termed MJ23 Tregs, that require Autoimmune Regulator (Aire) for their thymic development. Using a variety of mixed bone marrow chimera experiments, we sought to determine the contributions of various thymic APC subsets towards MJ23 Treg development. We demonstrate that the thymic development of MJ23 Tregs requires both MHC-II and B7.1/.2 on bone-marrow derived cells. Moreover, development is abolished by the depletion of CD11c-expressing cells. Together, these data indicate that the antigen recognized by MJ23 Tregs in the thymus must be transferred from Aire-expressing medullary thymic epithelial cells to CD11c-expressing dendritic cells to coordinate MJ23 Treg development. In the periphery, we demonstrate that the depletion of CD11c-expressing cells eliminates the enrichment of MJ23 Tregs in the prostate-draining lymph nodes, demonstrating that dendritic cells also coordinate the anatomical distribution of organ-specific Tregs in the secondary lymphoid organs. In all, our results reveal a critical role for dendritic cells in orchestrating the development and homeostasis of Aire-dependent, organ-specific Treg cells.
Heterochronic parabiosis: allowing the dissection of the aged immune system (LYM2P.723)
Authors: Davies John , Pulko Vesna , Thompson Heather , Nikolich-Zugich Janko
Published: January 2023

Parabiosis is the surgical union of two organisms resulting in the development of a single, shared circulatory system. When animals of different ages are conjoined (i.e. heterochronic parabiosis), blood-borne factors from the younger animal can often beneficially affect the older animal and recapitulate the youthful phenotype & function in target tissues. However, the effects of heterochronic parabiosis on the aged immune system remain unexplored. An important question to be answered is whether the cellular defects involved in the aged immune system are due to intrinsic deficiencies or if they can be rescued by extrinsic factors. Also, T cell ontogenesis is known to diminish with age due to thymic involution as well as defects in lymphopoiesis that affect pre-T cells. The heterochronic parabiosis model allows testing of contributions of the aged thymus, pre-T cells and of systemic environment in immune aging. The preliminary data demonstrate differential migration patterns of T cells, B cells & dendritic cells as a function of age. Of interest, pre-T cells from either animal adequately seed the opposing thymus and undergo thymopoiesis and other data suggest that there are no intrinsic defects in old thymocytes, but rather of the old thymic environment. That latter defect may not be corrected by heterochronic parabiosis.
Homeostasis and function of Tregs at the site of chronic inflammation (LYM2P.728)
Authors: Spence Allyson , Kim Youmin , Tang Qizhi
Published: January 2023

This study is aimed at characterizing Treg dynamics at the site of chronic inflammation in the islets of NOD mice. Tregs expressing CD103 were present primarily in the inflamed islets and low in the pancreatic LN and spleen. Both CD103+ and CD103- Tregs from inflamed NOD islets could prevent diabetes when transferred to Treg-deficient CD28KO mice. CD103 was selectively induced and maintained in inflamed islets after transfer. However, fewer CD103+ Tregs were recovered after transfer compared with CD103- Tregs, suggesting that CD103+ Tregs have a survival defect. Indeed, CD103+ Tregs had lower Bcl2 expression despite higher CD25 expression and higher responsiveness to IL-2. CD103+ islet Tregs have reduced TCRb repertoire diversity and higher BrdU incorporation, indicating they clonally expand in the islets. Lastly, in NOD mice expressing a TCR signaling reporter, the Nur77-GFP transgene, CD103+ islet Tregs were found to be uniformly Nur77hi suggesting they were more recently activated by antigens. TCRb sequences used by high frequency clones of CD103+ Tregs could be found in Nur77hiCD103- but not Nur77loCD103- Tregs, consistent with sequential TCR activation and CD103 expression. Together, our results demonstrate that chronically inflamed islets in NOD mice contain functional Tregs, but fail to maintain the highly antigen-specific activated subset. Maintenance of this subset may have important implications for the treatment of chronic inflammatory disease.
Thioredoxin peroxidase secreted by Echinococcus granulosus induces the alternative activation of macrophages in mice (MPF2P.755)
Authors: Wang Hui , Li Jun , Zhang Wenbao , Wen Hao
Published: January 2023

Alternatively activated macrophages (AAM) are key to promoting Th2 responses and have been associated with a variety of roles during helminth infection. We have shown that E. granulosus infection induces a polarized Th2 response in its intermediate hosts. The role of AAM in the regulation of the immune response is unclear. Here we describe the effects of a thioredoxin peroxidase (TPx) derived from E. granulosus, an enzymatic scavenger of H2O2, on murine macrophage phenotype in vivo and in vitro. The activation status of macrophages was analyzed by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMs were recruited to the peritoneum of mice of E. granulosus infection. Administration of hydatid cyst fluid (HCF) and recombinant EgTPx or its inactive recombinant variant (rvEgTPx) also induced the recruitment of AAM to the peritoneum indicated that the immunomodulatory property of EgTPx is not due to its antioxidant activity. In vitro studies showed that HCF and EgTPx or rvEgTPx can directly converts PECs from normal mice to an alternatively activated phenotype characterized by the production of high levels of Arg1. Our data suggest that the Th2 responses induced by the E. granulosus infection are mediated through the secretion of molecules, one of which is EgTPx, that induce the recruitment and alternative activation of macrophages.
Memory Tfh cells promote rapid antibody production after secondary Plasmodium yoelii infection (MPF2P.756)
Authors: Wikenheiser Daniel
Published: January 2023

The antibody (Ab)-mediated immune response contributes to protection against a secondary blood-stage Plasmodium yoelii infection. Follicular helper T (Tfh) cells promote B cell survival and facilitate somatic hypermutation and affinity maturation within germinal centers, resulting in the production of high-affinity Ab in a primary infection. The presence of memory T cells is important for the rapid induction of an effector response in secondary infections; however, it is unclear what role memory T cells play in supporting Ab production in a secondary response. Here, we report the identification of a splenic-resident population of CXCR5+ T cells that possess a central memory phenotype (CXCR5+ Tcm) that is maintained greater than 90 days post-P. yoelii infection. Co-transfer of these CXCR5+ Tcm and isotype-switched memory B cells into naive congenic or CD28-/- mice resulted in early plasma cell and germinal center B cell differentiation, as well as class-switched parasite-specific Ab production relative to control mice that did not receive transferred cells. Ultimately, mice receiving CXCR5+ Tcm cells controlled parasitemia more quickly than mice receiving memory B cells alone or no transferred cells. Collectively, these data suggest Plasmodium-specific CXCR5+ Tcm can differentiate into functional Tfh cells upon re-exposure to antigen, and participate in secondary humoral responses leading to rapid production of protective, isotype-switched Abs.
Protective and non-protective CD8 T cell responses against Plasmodium infection (MPF2P.760)
Authors: Doll Katherine , Pewe Lecia , Harty John
Published: January 2023

Despite decades of research, malaria remains a global health crisis. Currently, subunit vaccine approaches do not provide long-term, sterilizing immunity against Plasmodium infection. Conversely, immunization using whole parasites can confer protection in rodents through eliciting protective CD8 T cell responses, likely against a broad spectrum of Plasmodium antigens. However, it is unknown whether all of these CD8 T cell specificities contribute to protection. Recently, four novel H-2b-restricted Plasmodium-specific CD8 T cell epitopes were described from the following antigens: GAP50, PbT, S20, and TRAP. Here, we show that priming with peptide-loaded mature dendritic cells followed by boosting with recombinant L. monocytogenes expressing these epitopes results in memory antigen-specific CD8 T cell responses constituting 5-40% of the CD8 T cell compartment. To date, only TRAP-specific CD8 T cells generated through this vaccination approach can provide sterilizing immunity following a sporozoite challenge. This result occurs despite similar numbers, function and phenotype for each of the memory CD8 T cell populations. These data suggest that timing of antigen presentation during liver-stage infection coupled with a requirement for direct MHC presentation predicts the protective capacity of a CD8 T cell specificity. These data may aid in identifying novel target antigens to elicit sterilizing protection against Plasmodium infection.
Flagellar hook protein FlgE initiates a proinflammatory response via mixed pathways (MPF2P.762)
Authors: Wang Yiqiang , Shen Ying , Yuan Qing , Lin Dandan , Wang Meixiang , Liang Zhongjie , Li Weihua , Tang Hua , Zhang Xuemin , Song Wengang , Zhao Ge
Published: January 2023

Among components of bacterial flagellum, filament flagellin C mediates immunostimulation via TLR5 pathway. Here we showed that hook protein FlgE also participates in host-pathogen interactions and stimulates innate immunity. Recombinant P. aeruginosa FlgE was obtained via routine prokaryotic expression system. After treatment of immortalized human corneal epithelial cells (HCEC) with FlgE, microarray profiling revealed that up-regulated genes were highly enriched in inflammation/immunity-related pathways. Inclusion of anti-TLR5 antibodies or 130mM KCl in culture each partially decreased the effects of FlgE, suggesting a mixed signaling pathways involvement. Pull-down assay performed on FlgE with HCEC lysates and following Mass Spectrum assay of bound proteins suggested that caveolin, membrane type ATP Synthase, Rab5 subunits and prohibitin might couple with FlgE. In an intranasal delivery model in mice as well in an organoid culture of murine lung slices, FlgE induced upregulation of IL1b, IL6 and CXCL1 at both mRNA and protein levels as detected by real-time PCR or ELISA. When administrated with ovalbumin in mice, FlgE enhanced OVA-specific CD4 T cells expansion. 3D modeling of FlgE disclosed two segments that would participate in FlgE polymerization during hook formation, and altering these two sites decreased immunostimulatory potency of FlgE. In conclusion, FlgE bears immunostimulation activity when confronting host cells, and might be used as an immunomodulator/adjuvant.
CD4 signaling and Pneumocystis clearance: a critical role of IL-21R and STAT3 for effector function (MPF2P.745)
Authors: Elsegeiny Waleed , Zheng Mingquan , Eddens Taylor , Alcorn John , Kolls Jay
Published: January 2023

Pneumocystis jirovecii is an opportunist infection that is a significant cause of disease in immunocompromised patients. Patients whose blood CD4 counts drop below 200 cell/μl due to HIV infection or immunosuppressive therapies are at high risk of developing Pneumocystis jirovecii pneumonia (PJP). However, the exact role of CD4 cells and the specific effector subtype that is required for clearance has yet to be determined. We investigated this question with several cytokine and STAT knock out mice using both in vivo infection as well as the assessment of effector CD4+ T-cell function by adoptive transfer into Pneumocystis infected Rag1-/- mice. We observe that classical Th1, Th2, Th17, and Tfh cell effector functions are dispensable for organism clearance. However we found an essential role for both IL21 receptor signaling on CD4 cells as well as CD4+ T-cell intrinsic STAT3 activation are required for clearance. These data are consistent with the recent identification of human IL21R deficiency and susceptibility to PJP. We are currently examining potential novel downstream mechanisms that may mediate immunity against Pneumocystis pneumonia in this context.
Independent of bacterial clearance, interleukin-17A acts to exacerbate pulmonary inflammation in BALB/c mice infected with Mycoplasma pulmonis (MUC1P.901)
Authors: Mize Maximillion , Simecka Jerry
Published: January 2023

As a leading cause of pneumonia in man and animals worldwide, mycoplasmas cause persistent infections that induce debilitating chronic lung inflammation. An incomplete understanding on the pathogenesis of disease has contributed to the absence of effective vaccines against these organisms. Using a naturally occurring murine model mirroring human and animal infections, Interleukin-17A (IL-17A) was found to increase within the lungs of susceptible BALB/c and resistant C57BL/6 mice inoculated with M. pulmonis. In a previous study, C57BL/6 mice deficient in IL-17 receptor expression were shown to have impaired clearance of mycoplasma. We hypothesized that IL-17A may have a different role in susceptible BALB/c mice. Consistent with this idea, we report that injection of neutralizing antibodies against IL-17A reduced respiratory inflammation in BALB/c infected with M. pulmonis. This was seen through abrogated lymphoid and neutrophil recruitment when compared to littermate controls. In addition, both gross lung and alveolar lesions were significantly reduced upon IL-17A neutralization. Surprisingly, blocking IL-17A did not alter bacterial burden, thus solely influencing the inflammatory process during infection in BALB/c mice. Interestingly, anti-IL-17A antibody administration did not have an effect on mycoplasma infection in resistant C57BL/6 mice, suggesting that the IL-17A pathways differ between susceptible and resistant strains of mice.
A novel subset of lung tissue-resident memory CD4 T cells generated by influenza virus infection (MUC1P.903)
Authors: McKinstry Karl , Strutt Tara , Swain Susan
Published: January 2023

We recently identified a late IL-2-dependent ‘checkpoint’ during the effector phase of the T cell response against influenza A virus (IAV) that is required for the generation of most long-lived CD4 T cell memory. Here, we describe a novel subset of IL-2- and IL-7-independent IAV-specific memory CD4 T cells that survive only in the lung (the site of IAV infection). These cells (i) are shielded from labeling by fluorescent anti-CD4 antibodies administered intravenously, (ii) express a CD69high, CD127low surface phenotype, and (iii) are highly functional upon re-stimulation both in terms of cytokine production and the ability to rapidly activate dendritic cells. These characteristics are consistent with recently described ‘tissue-resident’ memory T cells (Trm). In contrast to most described CD8+ Trm populations, the IAV-specific CD4+ Trm are not CD103high, and do not require TGF-β signaling for their generation. We show that this novel CD4+ Trm subset survives poorly upon adoptive transfer to unmanipulated hosts, suggesting that this Trm subset is adapted to survive in a unique non-lymphoid niche. Transcriptional analysis further supports this hypothesis as broad differences distinguish Trm from conventional memory CD4 T cells present in the lung. In summary, our studies identify a population of IAV-specific Trm CD4 T cells that develops independently of signals required for the generation of virtually all other subsets of CD4+ memory T cells.
Nasal plasmid Flt3 ligand and CpG ODN alone enhances immediate, influenza virus-specific recall secretory IgA Ab responses in aging (MUC1P.904)
Authors: Ohori Junichiro , Asanuma Hideki , Aso Kazuyoshi , Ikeda Yorihiko , Sugita Gen , Fujihashi Keiko , McGhee Jerry , Fujihashi Kohtaro
Published: January 2023

The elderly have already experienced various pathogens and thus possess pathogen-specific Abs, whose titers are too low to combat reinfection. Thus, it would be of great benefit to the aged population if one could use an innate adjuvant system alone, without vaccine, in order to enhance mucosal immunity against past respiratory infections. We tested whether a combined plasmid Flt3 ligand (pFL) and CpG ODN adjuvant would enhance influenza virus-specific recall Ab responses without a need for additional vaccination. Young adult mice were nasally immunized with A/Puerto Rico/8/34 (PR8) hemagglutinin (HA) plus cholera toxin as mucosal adjuvant three times at weekly intervals. Seven months later, these mice were given nasal pFL and CpG ODN only or combined adjuvant together with PR8 HA. Saliva and plasma samples were collected 7 days later and PR8 HA-specific IgA Ab responses were evaluated. Both groups of mice showed significantly increased and comparable levels of anti-PR8 HA secretory IgA Ab responses in saliva when compared with those responses seen without innate adjuvant. The increase in total IgG Ab responses in mice given pFL and CpG ODN were limited when compared with those seen after primary nasal immunization. Total plasma IgE Ab titers in mice given nasal pFL and CpG ODN only were the same as IgE levels seen in naïve mice. These results suggest that nasal pFL and CpG ODN alone can enhance pre-existing Ag-specific Ab responses without adverse effects in the elderly.
MAVS plays an essential role in host immunity against human metapneumovirus (MUC1P.907)
Authors: Chen Yu , Liu Guangliang , Ren Junping , Ivanciuc Teodora , Casola Antonella , Garofalo Roberto , Bao Xiaoyong
Published: January 2023

Human metapneumovirus (hMPV) is a common cause of respiratory tract infection in the pediatrics population. Recently, we and others have shown RIG-I-like receptors are essential for hMPV-induced cellular antiviral signaling. However, the contribution of those receptors in host immunity against pulmonary hMPV infection is largely unexplored. In this study, mice deficient in MAVS, an adaptor of RIG-I-like receptors, was used to investigate the role(s) of these receptors in pulmonary responses to hMPV infection. MAVS deletion significantly impaired the induction of IFN-I and other proinflammatory cytokines by hMPV. Compared to wild type mice, mice lacking MAVS harbored a higher viral load and demonstrated impaired abilities to activate pulmonary dendritic cells (DCs), subsequently leading to abnormal memory T cell responses to hMPV infection. In addition, we found that MAVS is required for the recruitment of DCs and other immune cells to the bronchoalveolar lavage fluid (BALF). Taken together, our data indicate that MAVS-mediated pathways are essential for the pulmonary immune responses, both innate and adaptive, to hMPV infection.
Border patrol gone awry: lung NKT cell activation by F. tularensis exacerbates tularemia-like disease (MUC1P.908)
Authors: Hill Timothy , Gilchuk Pavlo , Cicek Basak , Boyd Kelli , Khanna Kamal , Joyce Sebastian
Published: January 2023

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning for border patrol, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia—because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain results in rapid recruitment of NKT cells to the lung interstitium. Interstitial NKT cell activation resulted in protective interferon-γ production, and affected both local and systemic proinflammatory responses. Hence, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet survived the infection whilst their wild type counterparts did not. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F tularensis infection hampers iBALT formation, which in conjunction with NKT cell-dependent proinflammatory response causes severe pulmonary tularemia-like disease in mice.
Selective programming of regulatory CCR10+ innate lymphoid cells in skin-draining lymph nodes for skin immune homeostatic regulation (MUC2P.918)
Authors: Yang Jie , Hu Shaomin , Xiong Na
Published: January 2023

Innate lymphoid cells (ILCs) are preferentially localized in barrier tissues where they function in homeostatic regulation but also contribute to inflammatory diseases. Mechanisms regulating establishment of ILCs in barrier tissues with diverse functions in homeostasis and inflammation are poorly understood. We found that microenvironments of lymph nodes (LNs) program ILCs with homing and functional potentials for their tissue-specific functions. In skin- but not mucosal tissue-draining LNs, ILCs were continuously activated to acquire regulatory properties with high expression of CCR10 for their localization into the skin. Upon analyzing Rag1-/-, Foxn1-/-, Foxp3-/- and skin-inflammation-induced mice, we found that the generation of CCR10+ ILCs in skin-draining LNs and their establishment in the skin required T cell-regulated homeostatic environments and were suppressed during inflammation. Reciprocally, CCR10+ ILCs also promote homeostasis of resident T cells. When transferred into IL2Rγ-/-Rag2-/- mice that lack ILCs, CD4+ T cells could not establish homeostasis in the skin, with notably reduced regulatory T cells and increased effector T cells expressing high levels of inflammatory cytokines, demonstrating cross-regulation of CCR10+ ILCs and CD4+ T cells to promote immune homeostasis in the skin. Our studies reveal a process programming tissue-specific ILCs in LNs for their localization and function in local tissues.