bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-27 priming of T cells controls IL-17-production in trans via induction of PD-L1 (59.1)
Authors: Hirahara Kiyoshi , Ghoreschi Kamran , Yang Xiang-Ping , Takahashi Hayato , Laurence Arian , Vahedi Golnaz , Sciumè Giuseppe , Hall Aisling , Dupont Christopher , Francisco Loise , Chen Qian , Tanaka Masao , Kanno Yuka , Sun Hong-Wei , Sharpe Arlene , Hunter Christopher , O’Shea John
Published: January 2023

Interleukin (IL)-27 is a key immunosuppressive cytokine that counters Th17-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in naïve CD4+ T cells rapidly induced by IL-27 stimulation for 3 hours. We discovered that IL-27 priming of naïve T cells upregulated expression of the ligand of PD-1 (PD-L1) in a STAT1-dependent manner. To assess the relevance of IL-27 in controlling PD-L1 expression in vivo, we employed a T. gondii infection model in which IL-27 is important for controlling immunopathology. We found that expression of PD-L1 on CD4+ T cells was significantly lower in EBI3-/- mice. When added to naïve CD4+ T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans in vitro. In vivo, co-administration of IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Moreover this inhibitory effect of IL-27 in vivo was largely reversed by elimination of PD-L1 on T cells. Thus, these data identify a novel suppressive activity of IL-27, by which CD4+ T cells can restrict differentiation of Th17 cells in trans. It is conceivable that the strategy of IL-27-pretreatment of naïve T cells ex vivo with subsequent adoptive transfer might be used therapeutically to attenuate autoimmune disease.
A conserved AU-rich element in IL-17A mRNA’s 3’-UTR confers posttranscriptional control (59.4)
Authors: Ramgolam Vinod , Huntenburg Sarah , Yarovinsky Timur , Bender Jeffrey
Published: January 2023

Modulated decay of labile mRNAs encoding inflammatory cytokines provides dynamic posttranscriptional control of their expression. AU-rich elements (AREs) in 3’-UTRs are cis-regulatory elements that determine mRNA half-life, modulated by association with mRNA binding proteins (RBPs) or miRNAs. We hypothesized that human and mouse IL-17A 3’-UTR AREs confer instability to the IL-17A transcript. In mRNA reporter assays, introduction of destabilizing AREs shortens the half-life of the intrinsically stable rabbit β-globin (RBG) transcript. We inserted full-length (FL), fragmented or mutated human and mouse IL-17A 3’-UTRs into the serum-inducible RBG-encoding plasmid pBBB. pBBB-transfected NIH3T3 cells were serum-stimulated and RBG mRNA levels analyzed by qRT-PCR at 5 hr. RBG mRNA levels were dramatically reduced (>50%) in cells transfected with pBBB containing either the mouse-FL or human-FL IL-17A 3’-UTRs, whereas the fragment with a deletion in the ARE lost its RBG-destabilizing effect, maintaining levels identical to those in the wild-type pBBB-expressing cells. Destabilizing activity was also lost with single, double and triple point mutations in IL-17A’s conserved 30 bp ARE, demonstrating its importance. Model analysis predicts that these mutations convert IL-17A mRNA’s secondary folding structure, likely affecting RBP and miRNA binding sites, leading to our future focus on the regulation of IL-17A-mediated disease by IL-17A 3’-UTR-interacting RBPs and miRNAs.
CD4+ T cell affinity can affect gender bias, disease severity and B cell requirement in inflammatory arthritis. (60.6)
Authors: Perng Olivia , Aitken Malinda , Garcia Victoria , Kropf Elizabeth , Caton Andrew
Published: January 2023

CD4+ T cells make a crucial contribution to the development of inflammatory arthritis both in humans and in mouse models. However, how the affinity with which T cells recognize target antigens might shape disease development and influence treatment modalities is poorly understood. We have examined these phenomena in mouse models of autoimmune arthritis: TS1xHACII and TS1(SW)xHACII mice express influenza hemagglutinin (HA) as a neo-self peptide and co-express transgenic TCRs that have either high affinity (TS1xHACII) or low affinity (TS1(SW)xHACII) for the HA-derived MHCII determinant, S1. Despite extensive deletion of autoreactive TCRs, arthritis spontaneously develops in both strains and in each case arthritis can be prevented by IL-17 blockade. Notably, the low affinity model displays less severe extra-articular disease manifestations and a prominent female bias emerges. In addition, B cells are required for arthritis development in the low affinity setting; by contrast, B cells are not required in the high affinity setting where the disease is accompanied by higher levels of pro-inflammatory cytokines. These studies demonstrate that the overall affinity of the CD4+ T cell response to an autoantigen can play a prominent role in guiding the pathways to inflammatory arthritis development and presentation. These studies may also explain why treatment modalities targeting particular pathways (cytokines vs B cells) can exhibit different efficacies in arthritis patients.
Interaction of CR1 with Par3/Par6 polarity complex mediates anti-inflammatory reprogramming of neutrophils during phagocytosis. (61.1)
Authors: Ghiran Ionita , Glodek Alecsandra , Chang Robert , Nicholson-Weller Anne , Lawler Matthew
Published: January 2023

Partitioning defective protein 6 (Par6) belongs to a family of proteins first discovered to mediate partitioning in C. elegans. In mammals, the Par family of proteins is involved in several critical functions such as cell polarity, vesicle trafficking, cell migration, and signal transduction. Presence of Par proteins in T cells, suggests that spatial polarity could play important roles in mediating and/or regulating immune responses. Our data demonstrate that Par proteins, particularly as Par3/Par6/aPKC polarity complex, regulate neutrophil responses during phagocytosis. Our results show that during complement-mediated phagocytosis, engagement complement receptor 1 (CR1) by complement-opsonized particles, promotes the interaction between Par6/Par3 complex and CR1. The binding between CR1 and the Par complex is mediated by the interaction of the PDZ motifs found in the cytoplasmic domain CR1 with the PDZ domains of Par6/Par3. During IgG- and complement-mediated phagocytosis, ligation of CR1 by complement fragments, inhibits FcgammaR-induced ROS production as well as the FcgammaR-mediated release of pro-inflammatory cytokines . In conclusion, our data suggest that during phagocytosis, engagement of CR1 by complement fragments, down regulates the IgG-mediated pro-inflammatory response by disrupting the temporal/spatial assembly of the inflammatory complex at the sites of phagocytosis.
Anti-Obesity Effect of Ethyl Acetate Extracts from Agrimonia pilosa Ledeb. in 3T3-L1 preadipocytes (62.3)
Authors: Lee Jung A , Ahn Eun-Kyung , Hong Seong Su , Oh Joa Sub
Published: January 2023

To evaluate the anti-obesity effect of Agrimoniae pilosa L., this study investigated that ethyl acetate extract from A. pilosa L. (EAAP) suppresses lipid accumulation and inhibits expression of adipogenic marker genes, such as peroxisome proliferator activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and adiponectin in 3T3-L1 preadipocytes. We demonstrated that EAAP inhibited adipocyte differentiation and expression of PPARγ and C/EBPα mRNA levels in a dose-dependent manner. In addition, EAAP reduced the PPARγ transcriptional activity stimulated by rosiglitazone in HEK 293T cells and decreased the expression of GLUT4 and adiponectin in 3T3-L1 cells. These results suggest that EAAP inhibits preadipocyte differentiation and adipogenesis by blocking of PPARγ and C/EBPα gene expression in 3T3-L1 cells.
Identification of SNARE Proteins in Dendritic Cell Secretion (62.4)
Authors: Collins Laura , Canavan Mary , Loscher Christine
Published: January 2023

Dendritic cells (DC) act as link between innate and adaptive immunity with the ability to drive a range of T helper cell subsets. The DCs responsible for this differ primarily in the cytokines they secrete. Soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor (SNARE) family members are proteins that drive membrane fusion and have been shown to play a key role in cytokine release. Little is known about the specific mechanisms involved in cytokine secretion from DCs, Therefore we examined the expression of SNAREs in DCs as they represent potential therapeutic targets. In this study we used JAWS II; a C57BL/6 murine DC line, to examine the expression of SNAREs following stimulation with a range of Toll-Like Receptor (TLR) ligands. We have shown that activation of TLR 4 with lipopolysaccharide (LPS) in these cells results in an increased transcription of the SNAREs Syntaxin 3 and Vti1b mRNA which was concomitant with increased protein expression of these SNAREs. This pattern of expression differed using other TLR ligands. The presented data demonstrates the differential expression of SNARE proteins in DCs following activation of TLRs. Given the different cytokine profiles elicited by activation of different TLRs, this data may provide some information on the SNAREs necessary for secretion of key inflammatory cytokines.
Deletion of NF-κB/p50 influences the outcome of Notch3-dependent T cell leukemia (63.4)
Authors: Campese Antonio , Grazioli Paola , Scafetta Gaia , Noce Claudia , Verkhovskaia Sofia , Mari Chiara , Felli Mari Pia , Gulino Alberto , Screpanti Isabella
Published: January 2023

The deregulation of Notch3 signaling inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic lymphoma (T-ALL), sustained by a constitutive activation of the NF-κB canonical pathway, mainly represented by the p50/p65 heterodimer. To clarify the Notch/NF-κB relationships in the development of T-ALL, we generated double mutant mice, deleted of the NF-κB/p50 subunit in a Notch3 transgenic background (N3-tg/p50-/-). The follow-up of N3-tg/p50-/- versus N3-tg mice revealed that p50 deletion strongly inhibits the development of Notch3-dependent T-ALL. Surprisingly, double mutant succumb earlier than N3-tg mice, displaying the trait of a myeloproliferative disease, with an aberrant accumulation of Mac1+Gr1+ myeloid cells in both spleen and peripheral blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Our preliminary results suggest that Notch3 overexpression in T-cell compartment is able to influence, possibly in trans, the equilibrium of the myeloid compartment and that the ablation of NF-κB canonical pathway may impact on the outcomes of a T-cell specific deregulation of Notch signaling. We provide a useful experimental model to extend our understanding of Notch/NF-κB interplay in hemopoietic system and to unravel novel relationships between lymphoid and myeloid differentiation.
Haploinsufficiency for the hematopoietic transcription factor IKAROS causes common variable immunodeficiency (63.6)
Authors: Kumanovics Attila , Voelkerding Karl , South Sarah , Rowe Leslie , Crim Jeremy , Stephens Jack , Whitney Tiffany , Martins Thomas , Augustine Nancy , Hill Harry
Published: January 2023

Common variable immunodeficiency (CVID) is characterized by defective immunoglobulin production that leads to recurrent infections and increased risk of autoimmune and malignant diseases. CVID is the most common primary immunodeficiency disease with an estimated 1:25,000 to 1:50,000 population frequency world-wide. Substantial progress has been made in determining the genetic basis of CVID. Mutations in ICOS, TACI, BAFF-R, CD19, CD81, CD21, and CD20, all encoding cell-surface receptors required for B-cell activation, have been found in a few individuals and families and are recognized to cause CVID. Only mutations in TACI are found frequently (both in sporadic cases and familial cases); representing 8-12 % of CVID cases. The cause of CVID is unknown in most patients (>85%). Here, we describe a large family with autosomal dominant hypogammaglobulinemia and B cell deficiency in which all affected members have a 4.7 Mb hemizygous deletion (7p12.3-p12.1) that includes IKZF1, encoding IKAROS, a transcription factor required for B-cell development. This study demonstrates that constitutional hemizygous deletion of IKZF1 leads to IKAROS haploinsufficiency, and patients with hemizygous IKZF1 deletion present with late-onset humoral immunodeficiency and low B-cell counts, complicated in one family member with pre-B-cell acute lymphoblastic leukemia requiring bone marrow transplantation.
Different CD4+ Treg subsets, different suppressive abilities: A comparison of CD27+CD127- and CD39+ Treg cells (65.3)
Authors: Magid-Bernstein Jessica , Rohowsky-Kochan Christine
Published: January 2023

T regulatory (Treg) cells are important in the development of autoimmunity. They are usually identified by their expression of CD25 and Foxp3, although functionally and phenotypically diverse subsets exist. To identify human Treg subsets we analyzed expression of CD27, CD127, CD39, and CTLA4 on CD4 T cells. A CD27+CD127- phenotype correlates with high CD25 and Foxp3 expression. CD39 expression is higher on CD25hi than on CD25med/lo T cells. We co-cultured sorted Treg subsets with T effector (Teff) cells to study suppression of proliferation and cytokine production. CD4+CD25hiCD27+CD127- Treg cells were suppressive of both Teff cell proliferation and cytokine production when activated with anti-CD3 and anti-CD28. CD39+ Treg cells did not inhibit Teff cell proliferation under these culture conditions, but did suppress proliferation and cytokine production when activated with anti-CD3 and antigen-presenting cells (APCs). The varied suppressive abilities of Treg cells in these different culture conditions may be a result of the ability of APCs, but not anti-CD28, to signal through the negative costimulatory molecule CTLA4. We are currently investigating the function of CTLA4 in these Treg populations. We also examined whether these Treg subsets vary in their cytokine profile. Elucidating Treg mechanisms and subsets will enable us to clarify how Treg cells suppress, how they are altered in autoimmunity, and ultimately how this defect can be overcome to treat autoimmunity.
FVIII-Fixed Splenocytes Induce Tolerance in Hemophilic Mice (65.4)
Authors: Su Yan , Miller Stephen , Scott David
Published: January 2023

Monogenic diseases, such as hemophilia A, can be treated with protein therapeutics, such as recombinant factor VIII. A problem with this therapy is the patient’s immune response to the human protein to which they are not tolerant. This is manifested by antibody formation in up to 30% of patients. Called “inhibitors”, such antibodies block the efficacy of therapeutic FVIII. Administration of human FVIII to hemophilia A mice (Exon 16 deleted FVIII-/- [E16]) is a small animal model for the immune response and tolerance to FVIII. These mice manifest a robust, T-cell dependent, antibody response to exogenous FVIII treatment even when given via tolerogenic routes. Tolerance to FVIII requires multiple high doses of FVIII and is costly. Hence, therapeutics for inducing tolerance to these inhibitory antibodies are desired. Ethylene carbodiimide (ECDI) cross-linking of antigens to splenocytes has been utilized to generate tolerance in multiple models. We show that ECDI-fixed splenocytes specifically suppress anti-FVIII responses in the E16 mouse hemophilia model. We coupled a FVIII immunodominant peptide (C22310-2330) and transferred coupled splenocytes into FVIII deficient Balb/c E16 mice. Upon challenge with C22310-2330 and OVA323-339 peptides, these recipients exhibited specifically reduced T-cell responses to FVIII. This strategy has potential as a novel therapeutic for treating hemophilia patients with inhibitors
Drak2 contributes to type 1 diabetes by mediating effects on effector and regulatory T cells (65.7)
Authors: Creasy Blaine , McGargill Maureen
Published: January 2023

Drak2 is a serine/threonine kinase that is primarily expressed in B and T lymphocytes. Previously, we showed Drak2 deficient mice are resistant to autoimmune disease in models of type 1 diabetes and multiple sclerosis. Our studies suggest the resistance to disease is due to a defect in the survival of autoreactive T cells. However, Mao et al. demonstrated expression of Drak2 in pancreatic β-islet cells and suggested that Drak2 is involved in cytokine induced apoptosis of β-islet cells. Thus, we investigated whether the resistance to diabetes is T cell intrinsic or due to the role of Drak2 in β-islet cell apoptosis. Adoptive transfer experiments revealed the resistance to diabetes is T cell intrinsic. Upon further investigation we found that not only do Drak2-/- T effector cells have an inherent survival defect, but mice lacking Drak2 have an increased proportion of T regulatory cells. In addition, Drak2-/- T regulatory cells were more effective at suppressing the proliferation of WT or Drak2-/- cells in vitro and in vivo. We are currently investigating the possible suppressive mechanisms responsible for these results. Most interestingly, Drak2-/- Treg cells were able to prevent WT pathogenic T cells from inducing diabetes in adoptive transfer models. These data reveal the resistance to diabetes in Drak2-/- mice is T cell intrinsic and occurs due to a slight shift in the ratio of pathogenic T cells to Tregs along with enhanced Treg suppressive activity in the absence of Drak2.
Calpastatin is a crucial regulator of macrophage activation that limits in vivo inflammation (66.1)
Authors: Hoffmann Peter , Rose Aaron , Hoffmann FuKun , Norton Robert , Huang Zhi
Published: January 2023

Calpain enzymes regulate cellular function through the proteolytic modulation of structural and signaling proteins. Calpain activity is positively regulated by the binding of calcium and negatively regulated by the endogenous inhibitor, calpastatin (CAST). We have revealed a novel role for CAST in attenuating macrophage activation. While calpain is constitutively expressed in macrophages, CAST levels increase upon activation of macrophages and decreases cleavage of specific calpain targets. Interestingly, we found that CAST increased when macrophages were stimulated with LPS, IFNγ, and TNFα, but not with IL-4, -10, or -17A. We utilized siRNA to inhibit the increase in CAST protein levels that arises from stimulation with TNFα, and decreased CAST expression led to increased NF-κB nuclear translocation and over-production of IL-6 upon stimulation with CpG DNA. CAST knockout mice were examined for markers of inflammation, and results showed that CAST-deficiency led to increased inflammation in the colons of these mice. In addition, serum levels of the pro-inflammatory chemokine, MCP-1, was increased in CAST knockout mice compared to wild-type mice. Overall, CAST plays a key role in limiting macrophage activation and defects in CAST expression increase susceptibility to the development of inflammation in vivo.
Identification of novel subsets of CD5+ B-1a cells reveals layered populations with distinct innate-like functions (67.2)
Authors: Wang Hongsheng , Shin Dong-Mi , Abbasi Sadia , Kovalchuk Alexander , Jain Shweta , Morse Herbert
Published: January 2023

B-1a cells are found to contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. These various functional attributes of B-1a are often presented as reflecting the repertoire of all B-1a cells, while understanding how these functions are fulfilled at the clonal level is rather limited. Here we show that B-1a cells can be subdivided into two distinct stable subsets based on differing expression of the plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), an enzyme involved primarily in hydrolysis of ATP at the cell surface. One subset that we term PC1lo, expresses ENPP1 at low levels, whereas the second subset, designated PC1hi, expresses ENPP1 at high levels. These subsets are distinguishable by gene expression profiles, VH gene usage and time of development. Adoptively transferred PC1lo cells secret significantly more circulating natural IgM and intestinal IgA than PC1hi cells. In contrast, PC1hi cells produce more IL-10 than PC-1lo cells when stimulated with LPS and PMA. Furthermore, PC1lo cells generate antigen-specific IgM responses to pneumococcal polysaccharide antigens whereas PC1hi cells do not. We conclude that the classic B-1a functions segregate quite cleanly between these two subsets and the identification of PC1lo and PC1hi cells extends the concept of a layered immune system.
Bacterial lipopeptides and heat-killed Staph aureus modulate lung vascular endothelial permeability via Toll-like receptor 2 (TLR2) (67.3)
Authors: Hellman Judith , Wilhelmsen Kevin , Mesa Kailin , Xu Fengyun
Published: January 2023

Background: Endothelial cell (EC) TLR2 activation modulates endothelial permeability. We hypothesized that heat-killed Staph aureus (HKSA) induces lung vascular permeability via TLR2, and mapped out the role of NF-κB and MAP kinases in TLR2 agonist modulation of EC permeability. Methods: Electric Cell-substrate Impedance Sensing was used to quantify permeability of lung microvascular EC (LMVEC) monolayers treated with bacterial lipopeptides or HKSA. Signaling pathways were probed by inhibiting MAP kinases and NF-κB. Lung permeability was assessed after IT challenge with HKSA. Results: Bacterial lipopeptides had a biphasic effect on permeability of human LMVEC, with an initial decrease at 1-2 hours that was JNK-dependent, followed by a sustained increase that was NF-κB-dependent. HKSA also induced LMVEC permeability. Anti-TLR2 IgG reduced the HKSA-induced LMVEC permeability. The permeability effects of HKSA were also reduced in LMVEC from TLR2 KO versus WT mice. IT challenge with bacterial lipopeptide and HKSA induced lung vascular leak in mice via TLR2. Conclusions: Bacterial lipopeptides and HKSA modulate endothelial and lung vascular permeability via TLR2. We speculate that the early TLR2-induced reduction in permeability may serve to limit spread of bacteria out of the vascular space, thereby reducing the development of tissue foci of infection, whereas the later increased permeability facilitates WBC trafficking and later reparative processes during active infection.
CD163/WC1 functions as pattern recognition receptors and co-receptors for the gamma delta TCR (68.17)
Authors: Chen Chuang , Herzig Carolyn , Hsu Haoting , Telfer Janice , Baldwin Cynthia
Published: January 2023

γδ T cells are deemed critical to immune surveillance and protection since they are found as resident cells in many organs and tissues including in humans, cattle and mice. It is known that γδ T cells contribute to cellular immunity and protection against important pathogens, organizing granulomas in response to Mycobacteria and producing IFNγ following vaccination to Leptospira. However, it is not clear how best to engage γδ T cells and few efforts have directly addressed the contribution of co-receptor stimulation for doing so. We are filling this gap by addressing the hypothesis that the CD163/WC1 co-receptors on γδ T cells, as an example of PRRs on nonconventional T cells, can be exploited to fully activate these cells when combined with TCR activation. In this study, the complexity of the WC1 multi-gene family in cattle has been resolved. The WC1 family is composed of 13 genes with three structural forms whose sequences are conserved among individual cattle and breeds. These three structural forms have different intracytoplasmic tail sequences which require phosphorylation of distinct tyrosines for transmission of signaling through the TCR and lead to different functional outcomes. In addition, the specific activation of subpopulations of γδ T cells can be defined by the WC1 gene products expressed. The work will inform how PRR families might be exploited for efficacious vaccine design to activate nonconventional T cells that bridge innate and adaptive immune responses.
Leucine rich repeats and calponin homology containing protein 4 (Lrch4) is a novel regulator of Toll-like receptor signaling (68.8)
Authors: Aloor Jim , Gowdy Kymberly , Azzam Kathleen , Madenspacher Jennifer , Draper David , Guardiola-Bright John , Merrick Alex , Dhungana Suraj , Fessler Michael
Published: January 2023

Toll-like receptor (TLR)4 signaling involves assembly of multi-protein complexes in lipid rafts. We identified Leucine rich repeats and calponin homology containing protein (Lrch)4 in a proteomic screen as a protein recruited to rafts of lipopolysaccharide (LPS)-exposed macrophages. Lrch4 had no published function, but structure predictions, including a transmembrane domain and leucine rich repeats, were suggestive of a receptor. Subcellular fractionation studies verified Lrch4 localization to the plasma membrane. qRT-PCR of 14 mouse tissues revealed that Lrch4 is ubiquitous. Lrch4 silencing in RAW 264.7 cells using lentiviral shRNAs and siRNAs attenuated LPS induction of TNFα and activation of mitogen-activated protein kinases and NF-κB. Lrch4 knockdown also reduced TNFα induction by ligands to several other TLRs. Lrch4 knockdown in TLR2 or TLR4 stable MD2-CD14-293 cells revealed that Lrch4 regulates human TLR but not TNFα -receptor signaling. Lrch4 and MD2 co-precipitated with biotin-LPS, and Lrch4 knockdown reduced cell binding of biotin-LPS without altering surface display of TLR4, together suggesting that Lrch4 interacts either directly or indirectly with LPS as part of a receptor complex. Lrch4 moreover co-precipitated with both MyD88 and TLR2. Lentiviral shRNA-mediated knockdown of Lrch4 in the murine airway reduced LPS-induced neutrophil influx into the airspaces in vivo. Taken together, we hypothesize that Lrch4 is a novel regulator of the innate immune response.
Enhanced T cell activation in lymphocytes from transgenic mice expressing LAT molecules resistant to ubiquitination (69.5)
Authors: Rodriguez-Pena Ana B. , Balagopalan Lakshmi , Gomez-Rodriguez Julio , Kortum Robert L. , Feigenbaum Lionel , Sommers Connie L. , Samelson Lawrence E.
Published: January 2023

Linker for activation of T cells (LAT) is critical for the propagation of T cell signals from the plasma membrane to the cytoplasm upon T cell activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KRLAT) resulted in a decrease in LAT ubiquitination and elevated T cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KRLAT. Upon TCR stimulation, proximal T cell signaling was elevated in lymphocytes expressing 2KRLAT versus WT cells as evidenced by higher levels of phospho(p)-tyrosine proteins, p-LAT, p-phospholipase C-gamma and cytosolic calcium flux. Evaluation of various cell surface markers also revealed an enhanced memory T cell profile in 2KR lymphocytes. Moreover, upon TCR stimulation, a higher percentage of T cells expressing greater levels of effector molecules such as intracellular interferon-gamma, tumor necrosis factor-alpha and interleukin-2 was found in 2KR versus WTLAT mice. We have thus identified enhanced proximal T cell signaling, an enhanced memory T cell profile and higher expression of cytokine effector molecules in lymphocytes from 2KRLAT transgenic mice. These results suggest that 2KR mice may respond more efficiently to pathogenic challenges, an hypothesis that will be tested in various experimental models of mouse infection.
Antigen specific changes of CDR3 fragment distribution of Vδ2 chain of human γδ T cells stimulated by Mycobacterium tuberculosis heat resistant antigen (70.11)
Authors: Li Baiqing , Ma Fei , Guo Shujun , Lü Hezhuo , Xue Zhuping
Published: January 2023

Recent studies show that phosphate antigen activated γδ T cells fail to, but Mycobacterium tuberculosis (Mtb) activated γδ T cells potentially inhibit intracellular Mtb growth, and BCG-stimulated γδ T cells display more restricted TCR diversity than phosphate activated γδ T cells. Mtb heat resistant antigen (Mtb-HAg), peptide antigen, has been shown to preferentially stimulate Vγ9Vδ2 T cells. However, the characteristics of diversity of TCR Vδ2 region of Mtb-HAg stimulated Vγ9Vδ2 T cells has not been investigated. In this study, by using size-spectratyping analysis of length of complementarity-determining region (CDR) 3 fragment of Vδ2 gene, the length of the predominant peaks of CDR3 fragment of Vδ2 gene in γδ T cells that stimulated by Mtb-HAg was significantly larger than that in γδ T cells cultured with IL-2 only and that in fresh PBMC groups. However, there were no significant different changes in the variations and length of CDR3 fragment of all other subfamilies of V gene of γδ T cells stimulated with Mtb-HAg. By sequencing Vδ2-CDR3 transfected clones, Vδ2-CDR3 regions showed two conserved “CACD” and “FGXG” at the N-terminus and the C-terminus, respectively. In conclusion, our results indicate that biased distribution of CDR3 fragment of Vγ9Vδ2+ T cells stimulated by Mtb-HAg is antigen specific changed and the clonally restricted. There might be important implications for the development of prophylactic vaccines related to protective and pathogen-specific γδ T cells.
Campylobacter jejuni modulates mucosal immune responses in the murine small intestine (70.13)
Authors: Maue Alexander , Mohawk Krystle , Hill Christina , Poly Frederic , Ferderber Jason , Patterini Dawn , Guerry Patricia
Published: January 2023

Campylobacter jejuni is among the most common causes of diarrheal disease worldwide. Evidence of immunity to C. jejuni following natural infection has been documented; however, relatively little is known regarding the mucosal immune response and correlates of protection against C. jejuni. To determine the role that the capsular polysaccharide (CPS) of C. jejuni plays in colonization and its effect on cytokine production by intestinal T cells, we utilized a mouse intestinal colonization model following oral infection with either CPS-deficient or wild type (CPS-replete) strains of C. jejuni. Initially, mouse colonization levels were similar between strains. Around 7 days post-infection, a significant reduction in bacterial shedding was seen in mice infected with the CPS-deficient strain. The production of IFN-γ and IL-17 was analyzed in lymphocytes from the epithelium, lamina propria and Peyer’s patches at various timepoints post-infection. IL-17 expression was significantly higher in lamina propria and Peyer’s patch CD4+ cells from mice infected with CPS-deficient bacteria compared to mice infected with wild type C. jejuni. Additionally, the percentage of CD8+ intraepithelial lymphocytes expressing IFN-γ was increased in mice infected with CPS-deficient bacteria. These data suggest that the production of IFN-γ and IL-17 is associated with clearance of unencapsulated C. jejuni from the mouse intestine and that CPS modulates cytokine production from intestinal T cells.
Qa-1b deficiency results in increased inflammatory response in aerogenic Mycobacterium tuberculosis infection (70.14)
Authors: Bian Yao , Shang Shaobin , Shanmuganad Sharmila , Wang Chyung-Ru
Published: January 2023

The murine MHC Ib molecule Qa-1b is known to play roles in both antigen presentation during infection by intracellular pathogens, as well as an immunoregulatory role in autoimmune and viral infection disease models. It has been shown that Qa-1b is able to suppress NK cell and CD8+ T cell activity through the interaction of Qa-1b /self-peptide complexes with inhibitory CD94/NKG2A molecules on activated effector cells. We find that Qa-1b expression is upregulated on a variety of cell types in a mouse model of low-dose, aerogenic infection with Mycobacterium tuberculosis (Mtb). Further, Mtb-infected, Qa-1b -deficient mice demonstrate increased production of INF-γ and IL-17A as compared to wild-type mice, suggesting that Qa-1b primarily plays an immunoregulatory role in Mtb infection. Qa-1b -deficient mice also display increased expression of inhibitory CD94/NKG2A complexes on CD8+ T cells during infection, indicating a potential mechanism by which Qa-1b is able to control immune responses during Mtb infection.
Deficiency of prolactin-inducible protein leads to a lower Th1 immune response in vitro and in vivo (70.2)
Authors: Li Jintao , Liu Dong , Blanch Anne , Jia Ping , Myal Yvonne , Uzonna Jude
Published: January 2023

The prolactin-inducible protein (PIP) is a secretory protein strategically located at several ports of pathogen entry into the body and hence might play a role in the first line of immune defense against invading pathogens. To date, there are no studies that address the contributions of PIP against infection. Our previous studies show that PIP knockout (KO) mice had enlarged submandibular lymph nodes and thymic medulla. Here, we assessed the phenotype and responsiveness of immune cells from PIP KO mice to polyclonal T cell stimulators and model antigens in vitro and in vivo. We found that PIP KO mice have comparable numbers or frequency of immune cells, including T, B, natural killer and dendritic cells. In depth phenotypic analysis revealed that the PIP KO mice had slightly but significantly lower numbers of CD4+ T cells in their spleens and lymph nodes. Furthermore, CD4+ T cells from PIP KO mice showed significantly decreased proliferation, IL-2 production and impaired differentiation into Th1 subsets in vitro. The impaired in vitro Th1 response was confirmed in vivo where CD4+ T cells from OVA-immunized PIP KO mice showed significantly impaired proliferation and IFN-γ production following restimulation with OVA. Collectively, our findings implicate PIP as an important CD4+ modulatory protein that enhances Th1 cell response, and suggest that this protein might play critical roles in cell-mediated immunity and resistance to intracellular pathogens.
Goblet cell-associated antigen passages and luminal antigen delivery to intestinal dendritic cells (71.6)
Authors: Miller Mark , McDole Jeremiah , Wheeler Leroy , McDonald Keely , Knoop Kathryn , Newberry Rodney
Published: January 2023

Understanding how pathogen-specific immunity is elicited in the gut against the overwhelming background of foreign antigens from food and normal flora is essential for treating intestinal infections and inflammatory diseases. Oral tolerance is mediated in part by intestinal dendritic cells (DCs) that promote the development of regulatory T cells. Beneath the villous epithelium, the lamina propria (LP) contains a large population of DCs (CD11c+ CD11b+ MHCII+ cells), CD103+ CX3CR1- DCs, which can promote IgA production, imprint gut homing on lymphocytes, and induce the development of regulatory T cells, and CD103- CX3CR1+ DCs, which can contribute to TNFα production, colitis, and the development of Th17 T cells. How luminal antigens are captured by LP-DCs in the steady-state is controversial. Using a combination of in vivo two-photon imaging and ex vivo analyses we show that goblet cells (GCs) within the small intestine function as passages to deliver low-molecular weight antigens from the intestinal lumen to underlying CD103+ LP-DCs, a phenomenon we have termed goblet-cell associated antigen passages (GAPs). The preferential delivery of antigens to CD103+ LP-DCs has important implications for peripheral tolerance induction at the mucosa. The mechanisms that regulate GAP function during homeostasis and pathogenic infection are the focus of ongoing work.
Higher numbers of Gr1hiCD11b+ cells suppress B cell differentiation and autoantibody production in male BWF1 mice (72.7)
Authors: Trigunaite Abhishek , Khan Ayesha , Der Evan , Jorgensen Trine
Published: January 2023

(NZBxNZW) F1 (BWF1) mice spontaneously develop a lupus-like disease characterized by elevated levels of anti-nuclear autoantibodies (ANA) and systemic inflammation and with a similar female bias as observed in humans. Bone marrow (BM) chimera studies have shown intrinsic disease-driving properties of female BM cells and subsequent flow-based analyses identified significantly less Gr1hiCd11b+ myeloid cells in BWF1 female mice as compared to males. As Gr1hiCD11b+ cells have been suggested to be immunosuppressive, we hypothesized that these cells suppress B cell activation and differentiation and protect against lupus-like disease in male BWF1 mice. We analyzed the effect of male and female Gr1hiCD11b + cells on 1) cytokine-driven B cell differentiation in vitro, 2) antibody responses to immunization, and 3) spontaneous autoantibody production. We found that Gr1hiCD11b+ cells from both male and female 4 wk old BWF1 mice significantly suppressed cytokine-driven plasma cell differentiation in vitro in a dose dependent manner. Likewise, treatment of male BWF1 mice with depleting anti-Gr1 antibody resulted in elevated antibody response to Np-CGG immunization. And finally, spontaneous production of ANA were significantly increased in male BWF1 mice during and after anti-Gr1 antibody treatment, with ANA levels comparable to those of unmanipulated BWF1 females. These data suggest a new and important role for immunosuppressive Gr1hiCD11b+ cells during antibody-mediated autoimmunity.
Paradoxical requirement for T cell expressed MyD88 for long term allograft survival (73.2)
Authors: Borges Christopher , Kim Beom , Turka Laurence
Published: January 2023

The TLR-MyD88 signaling pathway enables the innate immune system to sense inflammation and promote adaptive immune responses. Previously, we have shown that T cell intrinsic MyD88 signaling promotes T cell survival and is required for optimal responses to pathogens such as T. gondii and LCMV. Here, we utilized MyD88flox/flox x CD4-Cre (MyD88ΔT) mice to test how T cell expressed MyD88 affected the alloimmune response. MyD88ΔT mice (B6 background) rejected bm12 allogeneic skin with the same kinetics as C57BL/6 wild type mice, but, were resistant to the induction of long-term graft survival with αCD154, rapamycin, CTLA4Ig and donor splenocytes. Furthermore, in the bm12→B6 cardiac allograft model, where wild-type recipients spontaneously accept their grafts in a Treg dependent manner, we found that MyD88ΔT recipients acutely rejected their transplants (MST - ~30d). Consistent with this, although MyD88ΔT mice had normal numbers of Foxp3+ cells in lymphoid organs, infusion of wild-type Tregs into MyD88ΔT mice prior to heart transplantation reconstituted long-term graft survival, while depletion of CD25+ cells in MyD88ΔT recipients enabled long-term graft survival. Our data show that MyD88ΔT mice can mount an effective response against alloimmune antigen despite having abnormal responses to infection. Intriguingly, MyD88ΔT mice appear to have a defective regulatory compartment, suggesting that MyD88 signaling may be required for Treg function and/or stability.
AGO-CLIP-Chips reveal a distinct miR-mRNA interaction maps in alloantigen activated T cells (73.3)
Authors: Sun Yaping , Toubai Tomomi , Nieves Evelyn , Tawara Isao , Reddy Pavan
Published: January 2023

Following allo BMT, donor T cells that respond to allo-antigens cause GVHD and those that respond to non-alloantigens are critical for reconstitution, the molecular landscape and whether they are distinct is unknown. miRs regulate gene expression, but target prediction is a challenge. AGO is the central component in AGO-miR-mRNA complex. CLIP followed by microarray allows to look into the miRs’ direct target mRNAs. 45 miRs that differentially enriched in T cells, activated either by Allo-antigen or CD3/CD28 were divided into 4 groups and only 19 miRs were enriched in allo-T cells when compared to Ab-T cells. Six of the 19 were selected in further analysis. The enrichments of transcripts for mRNAs were screened with Affy. microarray. After CLIP, 1426 transcripts were detected in Allo-T cells and 1514 transcripts were in Ab-T cells. Amongst these, 200 transcripts were differentially enriched in allo-T cells and 288 in Ab-T cells. We predicted the miR targets by multi-algorithms based on CLIP-chip results and hypothesized that reduced miR enrichments should be companied with reduced mRNA transcript enrichments. Cyb5r3, LPP, Wapal, Synji1, Med17 and Fchsd2 were targets of miR-142-3p, -669D,-142-5p and -16. These miRs and above targets were validated with pool transcripts, PCR, WB and in vivo following BMT. We demonstrated for the first time, the distinct miR-mRNA interaction map based on CLIP-Chip and identified the specific targets in allo-activated T cells.
The role of NKT cells in immune evasion of liver metastases arising from intraocular tumors (74.12)
Authors: Sadegh Leila , Niederkorn Jerry , Chen Peter
Published: January 2023

Uveal melanoma is the most common intraocular tumor in adults within the United States. Half of the patients develop liver metastases, which is almost universally fatal. We have previously demonstrated that liver natural killer (NK) cell-antitumor immunity was suppressed in mice bearing ocular tumors, and natural killer T (NKT) cells were responsible, in part, for liver NK cell suppression. This study examines the role NKT cells in promoting liver metastases arising from intraocular tumors. We showed that when wild type and NKT-deficient C57BL/6 mice were injected with B16LS9 melanoma cells, NKT-deficient mice developed less liver metastasis than wild type mice. The cytotoxicity of liver NK cells was also assessed. Results indicated that NK cells from NKT-deficient mice were more cytotoxic compared to NK cells from wild type mice. However, in-vitro assays suggested that NKT cells were unable to directly suppress NK cell-mediated cytotoxicity. To investigate the potential role of a third-party cell, myeloid derived suppressor cells (MDSCs) were depleted by anti-GR1 antibody. Surprisingly, tumor bearing MDSC-depleted mice developed more liver metastases than isotype-treated mice. Our results imply that liver NKT cells suppress NK cell cytotoxicity through a GR1+ third-party cell population other than MDSCs. We are currently investigating whether liver NKT cells impart their immunosuppressive function through GR1+ neutrophils.
Treatment of pancreatic cancer with bifunctional siRNA silencing TGF-b and activating RIG-I (74.13)
Authors: Düwell Peter , Ellermeier Jonathan , Wei Jiwu , Hoves Sabine , Endres Stefan , Schnurr Max
Published: January 2023

A hallmark of pancreatic cancer is deregulated transforming growth factor-b (TGF-b) signaling which promotes tumor growth and entertains a potent immunosuppressive network correlating with poor prognosis. To brake immune suppression, we designed siRNA molecules silencing TGF-b1 and introduced a 5’ppp-modification (ppp-TGF-b) to additionally activate the cytosolic helicase retinoic acid-inducible gene I (RIG-I). RIG-I recognizes ppp-RNA motives in viral RNA and triggers type-I IFN production. We found that human pancreatic cancer cells express functional RIG-I. Treatment with ppp-RNA resulted in IRF-3 phosphorylation, type-I IFN and CXCL10 secretion, and tumor cell apoptosis. Treatment of mice with syngeneic orthotopic pancreatic tumors with ppp-TGF-b (i.v.) resulted in reduced tumor-associated TGF-b levels, systemic immune activation and high IFN levels in serum and tumor. Furthermore, ppp-TGF-b induced caspase-9-mediated tumor cell apoptosis in vivo and significantly prolonged survival of tumor-bearing mice. The development of bifunctional ppp-siRNAs that combine TGF-b silencing and RIG-I activation in one molecule represents an innovative strategy for the treatment of pancreatic carcinoma.
A small molecule inhibitor of the TGF β type I Receptor suppresses UVB-Induced mouse Skin Carcinogenesis (74.3)
Authors: Ravindran Anand , Mohammed Javed , Gunderson Andrew , Glick Adam
Published: January 2023

Ultraviolet radiation is a key environmental mutagen and chronic exposure to UVB is the leading cause of Skin Cancer. Transforming Growth Factor (TGF-β) represents an important immunoregulatory cytokine in the skin microenvironment with context dependent role in modulating tumor formation and progression. The immune system plays a critical role in containing the toxic damage caused by UVB; TGF-β has been known to play a key role in the activation and survival of immune cells. However, the specific role of TGF-β signaling in UVB induced skin cancers is not clear. To study this interplay between UVB, TGF-β and the immune system, we performed a UVB carcinogenesis study in the presence or absence of SB431542, an inhibitor of TGF-β1 type I receptor. We observed a significant decrease in the number of papillomas with TGF-β1 pathway inhibition and a concomitant decrease in the tumor infiltration and activation status (IFNγ secretion) of CD4 and CD8 lymphocytes. In order to understand the mechanism of suppression of T cell response by SB431542, we analyzed antigen presentation by dermal dendritic cells and langerhans cells. Preliminary data suggests that inhibition of TGF-β1 pathway suppresses the migration of skin DC subsets to the lymph nodes in response to UV, in turn suppressing the T lymphocyte response. In summary, the TGF-β1 pathway is important for the activation of the inflammatory response to UVB irradiation of the skin, which maybe mediated through Dendritic cells.
Adjuvant-carrying targeted Synthetic Vaccine Particles (tSVP™) augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release (75.3)
Authors: Ilyinskii Petr , Roy Chris , O’Neil Conlin , Browning Erica , Pittet Lynnelle , Altreuter David , Kishimoto Kei
Published: January 2023

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and also by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions resulting in safety concerns. Here we show that co-delivery of an antigen encapsulated into synthetic polymer nanoparticles with TLR7/8 or TLR9 agonists results in a strong elevation of immune responses which does not involve systemic production of inflammatory cytokines. In contrast, antigen admixed with free TLR7/8 agonist leads to lower immunogenicity and to rapid induction of serum cytokines associated with toxicity and immune suppression (e.g., TNF-α and IL-6 levels are 50-200 times higher upon injection of free than of encapsulated TLR7/8 agonist). Conversely, local immune stimulation as seen by cellular infiltration of draining lymph nodes and by intranodal cytokine production was much more pronounced and persisted longer if particle-encapsulated TLR agonists were used. Moreover, particle encapsulation permitted the efficient utilization of immunostimulatory CpG sequences with an enzyme-labile phosphodiester backbone which are known to rapidly degrade in vivo. The strong local immune activation in lymph nodes which resulted in a marked augmentation of immunogenicity was achieved using a modular self-assembling polymer nanoparticle platform and was equally efficient whether antigen and adjuvant were encapsulated in a single particle or in two separate particles.