bluuebunny/crossref_metadata_2025 huggingface dataset:
Human invariant chain Iip35 isoform expressed in Ii-deficient mice fully restores thymic selection (130.16)
Authors: Genève Laetitia , Labrecque Nathalie , Thibodeau Jacques
Published: January 2023

During biosynthesis, invariant chain (Ii) associates with MHC II molecules, prevents peptide binding in the endoplasmic reticulum (ER) and targets the complexes to the endocytic pathway. Iip35, a human-specific isoform, displays a cytoplasmic ER retention motif that is masked upon MHC II binding. Little is known on the role of this ER retention motif and we asked if Iip35 alone was capable of supporting the functions of Ii. We generated a Tg mouse expressing the human Iip35 isoform (Tgp35). Breeding with murine Ii deficient mice produced mice expressing only Iip35 (Tgp35/mIiKO). In these mice, Iip35 is highly expressed by about 30 % of B cells, 50% of splenic DC and 74% of IFNγ-induced macrophages. Interestingly, B cells expressing Iip35 were lost during the maturation process and not found in the T2 stage. We have demonstrated that Iip35 interacts with both murine Ii and MHC II. Also, we observed MHC II compacts forms typical of tight peptide binding. Iip35 partially rescued MHC II cell surface expression in mIiKO mice and was sufficient to fully restore thymic selection as judged by normal CD4+ T cell numbers in thymus and periphery. In contrast, as observed in mIiKO mice, no antigen-specific antibody production was observed in Tgp35/mIiKO following injection of Ova. Altogether, our results suggest that the Iip35 isoform is able to restore thymic selection but may not be sufficient to perform all Ii functions.
B cell receptors micro-cluster by antigen cross-linking and macro-cluster in a cap by biased transport (130.2)
Authors: Alla Srinivas , Raychaudhuri Subhadip
Published: January 2023

It is known from experiments that, in the presence of soluble antigens B-cell receptors (BCRs) assemble into small micro clusters and then structure into a cap macro cluster. However the underlying mechanisms of the antigen interaction with the BCRs and their cluster formation remain unclear. Such mutual attraction between two adjacent BCR molecules could arise, among other possibilities, due to cross-linking by bivalent soluble antigens. Recently, we have developed and studied a Monte Carlo model of B cell receptor clustering caused by binding and cross-linking of soluble antigens. The results of our study demonstrate the formation of small micro-clusters of BCR molecules (typically of size 2-10 molecules). But antigen cross-linking only is not adequate enough for the formation of large macro-clusters. A biased diffusion may drive the BCRs to form large macro-cluster. A simple model of biased diffusion where BCR molecules experience a biased directed motion towards the (a) largest cluster or (b) towards a random BCR micro-cluster is then applied, which results in a single macro cluster (cap) of receptor molecules. The mechanisms for both types of biased transport are compared using suitable network based metrics. We also develop and use appropriate network measures to analyze the effect of BCR and antigen concentrations on the receptor clustering, the stability of the formed clusters over the time, and size of BCR-antigen cross-linked chains.
Drug Controlled Antigen Degradation Defines Sources of MHC Class I Peptides and Enables Pathway Discovery (130.29)
Authors: Dolan Brian , Bennink Jack , Yewdell Jonathan
Published: January 2023

Defective Ribosomal Products (DRiPs) are an important source of MHC class I peptide ligands. How and where DRiPs are generated is an unanswered question with important implications for rationale design of T cell vaccines. To better understand DRiP antigen processing, we developed a novel antigenic protein, based upon the ProteoTuner technology, whose metabolic stability is controlled by a small molecule termed Shield-1. We show robust MHC class I antigen presentation derived from this shield controlled recombinant antigenic protein (SCRAP) regardless of the overall stability conveyed by shield. We use Shield-1 to generate a large pool of SCRAP, that is rapidly degraded upon Shield-1 removal, and show that these “retirees” are a much less efficient source of peptides than DRiPs. These findings demonstrate that true DRiPs are the source for a large proportion of peptide-MHC class I complexes. We also exploit the unique properties of this system to screen for chemicals and siRNAs that specifically inhibit DRiP antigen presentation, and have generated results demonstrating a role for deubiquitinating enzymes in DRiP antigen presentation.
Mechanisms of antigen presentation in recombinant adeno-associated virus (rAAV) vector mediated vaccination and gene therapy (130.36)
Authors: Xu Dan , Velazquez Victoria , Schaffer David , Kaspar Brian , Walker Christopher
Published: January 2023

Recombinant adeno-associated virus (rAAV) vectors are being considered for vaccination against infectious diseases and gene replacement therapy. Direct transduction of dendritic cells (DC) is thought to be required for initiation of CD8+ T cell immunity, but a role for antigen cross-presentation has not been excluded. Here we evaluated the requirement for DC transduction in T cell priming important to the outcome of vaccination and gene therapy strategies. Persistent eGFP expression was established in murine skeletal muscle after rAAV vector transduction. CD8+ T cells were primed but failed to clear transduced myocytes. Remarkably eGFP-positive DC were detected in draining lymph nodes (DLN) after vector transduction. They expressed co-simulatory and inhibitory molecules that regulate T cell priming. To determine if the DC were transduced by rAAV, F1 (H2bXd) mice were lethally irradiated several weeks after vector transduction of muscle and reconstituted with parental (H2b or H2d) bone marrow. eGFP-positive DC expressing only parental (H-2b or H-2d) class I molecules were visualized in the DLN after bm reconstitution. This result indicates that DC were not transduced by the vector, but instead captured antigen in the muscle before migrating to the DLN. Moreover T cells primed in these bm-reconstituted animals were restricted only by parental class I molecules, indicating a critical role for parental DC but not transduced myocytes in antigen presentation.
Humoral responses to haptenated and native proteins: The role of MyD88 (130.39)
Authors: Snowden Pilar
Published: January 2023

Recently, Palm and Medzhitov claimed that antibody responses to native proteins require MyD88 whereas responses to haptenated protein do not. Haptens are small antigenic molecules that are capable of inducing humoral responses only when covalently coupled to larger, “carrier” molecules; most carrier molecules are proteins. Antibodies elicited by carrier-hapten can be hapten- or carrier specific, or can recognize the hapten-carrier junction. Often, the majority of antibody induced by hapten-carrier is hapten-specific, but haptenation was not thought to alter fundamentally the nature of the carrier antigen. To compare the capacity of native and haptenated protein to elicit antibody responses in MyD88 deficient and congenic control mice, we immunized cohorts of animals with chicken γ-globulin (CGG) or CGG haptenated with (4-hydroxy-3-nitrophenyl)acetyl (NP9-CGG) in alum. Subsequently, we compared the humoral responses of control and MyD88-/- mice by quantifying serum antibody and germinal center (GC) responses. Under these experimental conditions, control and MyD88-/- mice mounted comparable IgG and GC responses to CGG and NP9-CGG. In all cases, NP9-CGG elicited higher amounts of NP-specific antibody compared to that against CGG; native CGG elicited higher levels of carrier antibody but less than the anti-NP response. By FACS, GC responses were comparable in both strains also. Our findings suggest that immune responses to native CGG and NP9-CGG are MyD88 independent.
Carbohydrate Tumor Antigen Vaccines using Unique Strategies (131.27)
Authors: Rittenhouse-Olson Kate , Abdulla Julia , Heimburg-Molinaro Jamie , Sahoo Padmini , Almogren Adel , Roy Rene , Chang Richard , Morey Susan , Barchi Joseph
Published: January 2023

The Thomsen-Friedenreich antigen (TF-Ag), a carbohydrate tumor associated antigen, is differentially expressed on carcinomas including those of the breast, colon, and prostate. It is involved in adhesion and metastasis and even low levels of naturally formed antibody to TF-Ag is related to better prognosis. We hypothesize that vaccination to create antibody to TF-Ag may create a survival advantage for patients with TF-Ag+ tumors. The innovation of this work is that both peptide mimics and other novel constructs have been utilized as immunogens. Peptide mimics of TF-Ag, with and without C3d N- terminal peptide constructs and synthetic TF-Ag-Muc 4 constructs with the C3d N- terminal peptide, were prepared to target T cells, and CD21+ splenic B cells and follicular dendritic cells for an increased response and memory B cell production. CD21 is the C3d receptor and MUC-4 is a tumor associated peptide. The immune response was measured using TF-Ag -BSA in enzyme immunoassays. The significance of antibody production to TF-Ag can be seen in the fact that passive immunotherapy with a monoclonal Ab to this Ag decreases lung metastasis and extends survival time of mice bearing breast cancer. The improved survival time is of particular interest because the McAb was not cytotoxic, and the enhanced survival was due to blocking metastasis. A successful vaccine would replicate this inhibition of metastasis and in addition provide cytotoxic effects to improve prognosis.
Tumor immunotherapy using chimeric receptor redirected allogeneic T cells across MHC barriers (131.39)
Authors: Marcus Assaf , Waks Tova , Eshhar Zelig
Published: January 2023

We present a strategy for adoptive immunotherapy using fully mismatched allogeneic chimeric receptor redirected T cells (allo-'T-bodies') in the absence of Graft vs. Host disease (GvHD). The strategy hinges on creating a therapeutic time-window using mild preconditioning which on one hand allows the allogeneic T-bodies enough time to effectively attack the tumor but on the other hand does not ablate the Host vs. Graft response (HvG) ensuring the eventual rejection of the allogeneic T cells thus preventing GvHD. We found that transfer of fully mismatched allogeneic T cells expressing a HER2/neu specific chimeric receptor following mild preconditioning roughly tripled the median survival of mice bearing pulmonary Renca-HER2 metastases with around 50% of mice surviving long term (>200 days) in the absence of GvHD mortality. We show that allo-T-bodies provide comparable benefit to syngeneic T-bodies, and are far superior to non-specific allogeneic T cells, demonstrating that the response is indeed tumor-specific and not merely allo-specific. Taken together these data provide proof of principle for safe and effective allogeneic adoptive therapy allowing for the use of 'universal effector cells' as a standardized treatment for cancer.
Resveratrol enhances CD8+ T cell mediated effector responses by inducing type 3 deacetylase dependent mTOR kinase activity to promote T-bet expression (132.3)
Authors: Rao Rajesh , Li Qingsheng , Shrikant Protul
Published: January 2023

Caloric restriction (CR) augments cellular immunity and extends lifespan in mammals; however the mechanisms underpinning these observations remain unclear. The CR mimic; resveratrol, activates SIRT-1; a NAD+ dependent type III deacetylase, for promoting life-span. Herein, we show that resveratrol present during antigen stimulation of naïve CD8+ T (OT-I) cells enhances T-bet dependent type I effector maturation. Resveratrol mediated enhanced T-bet expression required Sirt-1-type III deacetylase activity dependent increases in mTOR kinase activity. In agreement, resveratrol administration enhanced viral immunization induced CD8+ T cell effector responses and restored the poor CD8+ responses observed in diet-induced obese mice. These results demonstrate that the CR-mimic resveratrol promotes effective CD8+ T cell responses in metabolically imbalanced hosts, which may lead to life-span extension. By delineating the role of Sirt-1; NAD+ dependent type III deacetylase activity for T-bet expression, we have identified a new pathway to regulate CD8+ T cell mediated host immunity.
A novel pro-survival and proliferation-inducing role for CCR4:TARC interaction in chronic lymphocytic leukemia (CLL) B cells (133.4)
Authors: Damle Rajendra , Temburni Sonal , Malavasi Fabio , Kaufman Matthew , Kolitz Jonathan , Allen Steven , Rai Kanti , Chiorazzi Nicholas
Published: January 2023

CLL is a lymphoproliferative disorder characterized by an accumulation of CD5+ B cells. Somatic mutation of Ig V genes and cell surface CD38 expression by CLL cells stratifies cases into clinically distinct subgroups. CLL cells are likely sustained by the stimulation via BCR, TLR, and cytokine/chemokine:ligand interactions. Here we report on CCR4 expression and function in CLL. Cases with high numbers of CD38+ cells (≥30%) showed significantly higher percentages of CCR4-expressing cells (52.8 ± 5.2; p< 0.01) than counterparts with low numbers of CD38+ cells (<30%) (33.7 ± 5.3). Among CD38+ clones, those with mutated Ig V genes exhibited more CCR4-expressing cells than those with unmutated Ig V genes (p< 0.01). CCR4 molecules were functional as defined by proximal events (significant increases in phosphorylation of Btk and STAT5 in response to varying doses of TARC, specific decreases in surface CCR4 expression, and, migratory responses to TARC). Among its downstream effects, TARC alone rescued 25-60% of CCR4+ CLL cells from apoptosis. Simultaneously TARC induced proliferation of CLL cells in a dose-dependent manner. BCR crosslinking, TLR-9 agonists, T cell help, and CD38:CD31 interactions increased density of CCR4, likely facilitating ligand-mediated effects. Taken together, these findings suggest an important and novel role for CCR4:TARC interaction in promoting CLL cell survival and proliferation downstream of several triggering molecules.
IL-27 promotes Tc1 polarization of human CD8 T lymphocytes and enhances their proliferation and effector functions (134.2)
Authors: Arbour Nathalie , Schneider Raphael , Yaneva Teodora , Beauseigle Diane
Published: January 2023

IL-27 has been shown to exhibit both pro- and anti-inflammatory properties. This cytokine favors naïve CD4 T cell differentiation into Th1 cells to the detriment of Th17 and Th2 skewing. Moreover, IL-27 promotes proliferation and cytotoxic functions of mouse CD8 T cells, but no data is available for human cells. We investigated the impact of IL-27 on human CD8 T cells. Surface IL-27 Receptor (IL-27R), composed of TCCR (also known as WSX-1) and gp130, is detectable on subsets of peripheral blood T cells. More CD8 than CD4 T cells are IL-27R positive and these proportions increase upon polyclonal activation. IL-27 activates rapid STAT1 and STAT3 signaling and transcription of SOCS1 and SOCS3 mRNA in CD8 T cells. Addition of IL-27 to anti-CD3 activated CD8 T cells leads to a significant augmentation of proliferation, and IFN-gamma and granzyme B production. Furthermore, purified naïve (CD45RA) but not memory (CD45RO) CD8 T cells are susceptible to those IL-27-enhancing effects. Moreover, anti-CD3 activated CD8 T cells express higher levels of T-bet upon IL-27 addition. Thus, our results support the capacity of IL-27 to drive naïve CD8 T cell polarization toward a Tc1 phenotype via STAT phosphorylation and induction of T-bet expression. Finally, the presence of IL-27 in the environmental milieu of naïve human CD8 T cells being activate through their TCR enhances the proliferation, IFN-gamma and granzyme B production thus contributes to generate more efficient effector cells.
Enhanced liver regeneration in IL10 deficient mice after partial hepatectomy via activating hepatocyte STAT3 and stimulating inflammatory response (134.6)
Authors: Yin Shi , Wang Hua , Park Ogyi , Wei Wei , Shen Jilong , Gao Bin
Published: January 2023

A series of evidence show that pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), play a critical role in the initiation and progress of liver regeneration. Relatively little is known regarding the role of anti-inflammatory cytokine IL-10 in liver regeneration after partial hepatectomy (PHx). Here, we examined the role of IL-10 in liver regeneration using a model of PHx in several strains of genetically modified mice. IL-10 -/- mice, and IL10-/-STAT3hep-/- double knockout mice in which IL-10 gene was globally disrupted and STAT3 gene was deleted specifically in hepatocytes; and their littermate controls underwent 70% PHx. Liver regeneration was evaluated by BrdU incorporation. After PHx, expression of IL-10 mRNA was significantly elevated after PHx. Compared to wild type mice, IL-10-/- mice had enhanced liver regeneration as determined by measuring hepatocyte proliferation and mitosis with the peak of hepatocyte proliferation occurring 48 hours post PHx. This increased liver regeneration in IL-10-/- mice was associated with elevated serum levels of TNF-alpha and IL-6, and enhanced activation of the IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes reduced liver regeneration in IL-10-/- mice. These findings suggest that IL-10 may negatively regulate liver regeneration via limiting inflammatory cytokine responses and subsequently tempering hepatic STAT3 activation.
Increased autoimmune disease activity in MRL-lpr mice following influenza infection. (135.21)
Authors: Crowe Sherry , James Judith , Edwards Erica , Anderson Jourdan
Published: January 2023

Influenza virus infection induces IFNα and TNFγ, which have been linked to increased severity of select autoimmune diseases. Influenza also stimulates TRL3 and TLR7, which are associated with increased kidney damage in mouse models of lupus. Therefore it seems likely that influenza infection of individuals with systemic lupus erythematosus, or lupus, would accelerate autoimmune disease progression. We used the MRL-lpr mouse model of spontaneous lupus-like disease to examine the impact of influenza infection on end stage organ damage. Influenza infection of MRL-lpr mice resulted in significant peri-vascular cellular infiltrates in the kidneys, composed primarily of CD4+ T cells. The infected mice also had accelerated proteinuria and lymphadenopathy of the cervical and mediastinal lymph nodes. Infection also resulted in a transient but significant increase in the concentration of anti-cardiolipin antibodies, but no changes in other autoimmune specificities. Experiments are underway to determine if this end-stage organ damage is dependent upon virus replication or if exposure to viral proteins, such as following vaccination, would result in increased kidney damage. This model will provide a unique system to dissect the immune-pathogen interaction in an autoimmune disease setting. These data will also provide valuable information on how infection impacts autoimmune disease development and progression.
CCR6-dependent recruitment of dendritic cells to the central nervous system during autoimmune encephalomyelitis (135.26)
Authors: Wu Gregory , Archambault Angela , Debes Gudrun , Laufer Terri
Published: January 2023

The antigen presentation requirements during experimental autoimmune encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS), are incompletely known. Dendritic cells (DCs) are specialized antigen presenting cells that critically participate in neuro-inflammation. Previously, we found that DCs alone are sufficient as antigen presenting cells for the induction of active EAE. We hypothesized that DCs arising from the periphery are specifically recruited to the central nervous system (CNS) during EAE. We utilized radiation bone marrow chimeras to address the role of peripheral DCs during EAE. We found that bone marrow-derived DCs accumulate within the CNS during disease. Further, limiting expression of MHCII to radiation-sensitive DCs was sufficient to support wild-type levels of disease. Using mixtures of CCR6-deficient and wild-type bone marrow as donors for radiation chimeras, we determined the proportion of each that arose in the CNS during EAE. These mixed bone marrow chimeras were equally susceptible to EAE compared to chimeric mice with entirely wild-type bone marrow. However, the fraction of DCs lacking CCR6 was reduced compared to CCR6-expressing DCs. Thus, in a competitive environment, CCR6-deficient DCs are inefficiently recruited to the CNS during EAE. These results suggest a potential mechanism for the entry of DCs during the development of neuro-inflammation.
Anti-neutrophil autoantibodies in tissue damage in systemic lupus erythematosus (135.31)
Authors: Yu Yangsheng , Fan Run , Zhang Yan , Yue Yinshi , Gould Karen , Thiele Geoffrey , Klassen Lynell , Fassina Giorgio , Kimberly Robert , Zhang Zhixin , Su Kaihong
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which abnormal immune responses attack multiple organs, leading to life-threatening complications. Although the pathogenic role of autoantibodies in SLE has been well established, the most disease-relevant reactivity of autoantibodies and their use as disease progression biomarkers are not fully understood. Using a single-cell PCR approach, we generated a large panel of recombinant antibodies from B cells of SLE patients. We found that a high frequency of SLE-derived antibodies (~ 20%) bind to and activate neutrophils as assayed by degranulation, oxidative burst, calcium influx, and tyrosine phosphorylation. One of the antibodies, S4Ab4, also induces neutrophil extracellular trap (NET) formation and increases cell death to human vein endothelial cells. Using mass spectrometry, we found that S4Ab4 reacts with two neutrophil proteins: lactoferrin and nucleotide-binding oligomerization domains 27 (NOD27). While lactoferrin is a known neutrophil autoantigen, NOD27, a family member of NOD-like pattern recognition receptors, might be a novel autoantigen in SLE. Furthermore, S4Ab4 antibody can cause proteinuria and kidney and lung manifestations when administered into NZB/W F1 lupus-prone mice. Our results show that SLE-derived autoantibodies can directly bind to and activate neutrophils. These anti-neutrophil autoantibodies may play important roles in the disease progression and tissue damage in SLE.
Clonally expanded α-chain TCR transcripts are present in skin biopsies of patients with Systemic Sclerosis (135.33)
Authors: Zacharakis Nikolaos , Oleszak Emilia , Klaiber Nicholas , Myers Allen , Platsoucas Chris
Published: January 2023

Systemic Sclerosis (SSc) is a chronic autoimmune disease characterized by excessive deposition of collagen in the skin and many internal organs, production of autoantibodies and microvascular alterations. Previous studies in our laboratory have demonstrated the presence of high proportions of identical β-chains TCR transcripts in skin biopsies from patients with SSc of recent onset, suggestive of clonal expansions of T cells. To identify the antigen(s) recognized by clonally expanded T cells in SSc biopsies, we examine the clonality of the α-chain TCR transcripts in patients with SSc. Sequence analysis of α-chain TCR transcripts following non-palindromic PCR/Vα specific PCR and cloning revealed the presence of clonally expanded α-chain TCR transcripts in skin biopsies from four patients with SSc. Substantial proportion of identical α-chain TCR transcripts were found in these skin biopsies accounting from 16% to 32% of the transcripts sequenced. These clonal expansions were confirmed with Vα-specific PCR followed by cloning and sequencing. Identical α-chain TCR transcripts, ranging from 68% to 100%, were identified in individual Vα families. In one of these patients an identical T cell clone was clonally expanded in both peripheral blood and skin. These T cells have undergone clonal expansion in response to, as yet unidentified, specific antigen(s).
IP-10-dependent terminal differentiation of human plasma cells: an implication for autoimmunity (135.5)
Authors: Xu Wei , Dullaers Melissa , Pascual Virginia , Oh Sangkon , Banchereau Jacques
Published: January 2023

AAntibodies secreted by terminally differentiated plasma cells are important in immune defense, but also contribute to the pathogenesis of autoimmune diseases. How do naïve and memory B cells efficiently and promptly undergo terminal differentiation after encountering an antigen remains to be fully understood. We found that in the human tonsil most CD138+ plasma cells are surrounded by CD163+ resident macrophages. This prompted us to explore the role of macrophages in B cell activation. Sorted tonsilar macrophages were co-cultured with autologous IgD+CD27- naïve B or IgD-CD27+ memory B cells that were activated by anti-Ig, CpG and IL-2. Both naïve and memory B cells co-cultured with macrophages underwent significant proliferation and differentiation towards CD138+CD38+ plasma cells, whereas B cells alone failed to do so. A similar finding was obtained by using in vitro-generated macrophages. Such a process was tightly regulated by the dominant production of IP-10 by macrophages and the receptor CXCR3 on B cells. We found that patients with systemic lupus erythematosus (SLE) with active disease display an expansion of CXCR3+ B cells (range 20-90%) compared to those with inactive disease (range 5-15%). Accordingly, serum levels of IP-10 were elevated in active SLE patients. Thus, our data support a previously unrecognized mechanism by which macrophages induce terminal differentiation of plasma cells through secretion of IP-10, which may have potential implications autoimmunity.
CpG-B oligodeoxynucleotides inhibit Toll-like receptor-dependent and -independent induction of type I IFN in dendritic cells (136.10)
Authors: Liu Yi , Gray Reginald , Hardy Gareth , Kuchtey John , Abbott Derek , Emancipator Steven , Harding Clifford
Published: January 2023

CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) in dendritic cells. CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFNαβ. Because IFNαβ may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFNαβ. In our studies, CpG-B ODN inhibited induction of IFNαβ by CpG-A ODN, while induction of TNFα and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFNαβ was observed in Flt3L-induced murine DCs, purified murine mDCs and pDCs, and human PBMCs. CpG-B ODN inhibited induction of IFNαβ by agonists of multiple receptors, including MyD88-dependent TLRs and MyD88-independent receptors. CpG-B ODN did not inhibit the IFNαβ positive feedback loop “second wave” IFNαβ, since IFNαβ-induced expression of IFNαβ mRNA was unaffected, and CpG-B inhibition of IFNαβ was manifested in IFNαβR-/- DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early “first-wave” IFNα4 and IFNβ. Chromatin immunoprecipitation revealed that association of IRF1 with the IFNα4 and IFNβ promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A-induced association of IRF1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFNαβ that selectively inhibits induction of IFNαβ downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFNαβ expression in vivo.
Inflammasome Activation of Dermal Fibroblasts by an Endogenous Peptide Activate Tissue Resident Immune Cells (136.18)
Authors: Artlett Carol , Sassi-Gaha Sihem , Thacker James
Published: January 2023

Activation of innate immunity is an important strategy for host defense as it is an early response to a pathogen derived danger signal. Recently, we discovered a lipopeptide 1-peptidyl-2,3-diacylglyceride that acts like a danger signaling molecule to induce an innate immune response. The peptide moiety alone (acALY-18) activates the inflammasome inducing the secretion of IL-1β, and subsequently IL6, IL8, MIP1α, and MCP1 in fibroblasts and keratinocytes. Subcutaneous administration of acALY-18 protected mice from a lethal dose of S. typhimurium (survival: 80% treated vs 0% untreated, P< 0.0001). We extended our initial observations with acALY-18 to further determine its effect in fibroblasts and subsequent effects on immune cells. Arrays confirmed the upregulation of IL1β, MIP1α, MCP1, and IL6; C8A, IL1α, IL1F8, IL1R2, IL1RN were also increased. THP1 cells responded to acALY-18 fibroblast conditioned media with the upregulation of CASP1 (2-fold), IL18 (2.6 fold), IL33 (7.5 fold), (IL1β 3600 fold), and IL6 (2.6 fold). The upregulation of IL1β and IL33 was greater than that observed in acALY-18 treated THP1 cells (2.5 fold and 1.6 fold respectively), suggesting that the fibroblast response to acALY-18 can mediate immune cell activation. Additional studies are further investigating the activation of the immune response by acALY-18. acALY-18 could directly translate to a novel therapeutic to enhance non-specific innate immune signaling against pathogens resistant to antibiotics.
Filaggrin knockdown induced the production of IL-6 and IL-8 from HaCaT cells via TLR3 and TLR1 (136.27)
Authors: Lee Kyung Ho , Cho Kyung-Ah , Kim Ji-Yoon , Woo So-Youn , Kim Jin-Woo
Published: January 2023

The skin acts as an indispensible barrier to protect various invaders from the outside. The disturbed skin barrier allows entering innocuous antigens into the skin followed by triggering immune responses and initiate inflammation. Filaggrin (filament-aggregating protein) aggregates the keratin filaments into tight bundles leading to collapse of the coneocytes. Filaggrin deficiency or aberrant expression result the phenotype of ichthyosis vulagaris and atopic dermatitis, characterized by a disturbed skin barrier and dry skin. Filaggrin-deficient mice showed the predisposition of skin inflammation and sensitization of irritants and haptens through epidermis. As keratinocytes are first barrier against exogenous antigens and they possess TLRs as pattern recognition molecules to activate antimicrobial innate immune response. To determine whether the level of filaggrin expression affected the TLR-mediated responses of keratinocytes, we transfected filaggrin siRNA into human keratinocyte cell line, HaCaT, and measured the IL-1β, IL-6 and IL-8 secretion. We found that TLR stimulation did not enhance the IL-1β but IL-6 secretion was increased by poly(I:C). In addition, IL-8 secretion was increased under Pam3CSK4 stimulus in filaggrin knockdown cells. Therefore, these results suggest that the decreased level of filaggrin may trigger innate immune response via TLR stimuli and contribute to pathogenesis of inflammatory skin disease.
Rapid differentiation of MSC into adipocytes and osteocytes is facilitated by Toll-Like receptor engagement (136.43)
Authors: Tas Ibrahim , Tokcaer-Keskin Zeynep , Ayhan Fatma , Gursel Mayda , Akcali Kamil , Gursel Ihsan
Published: January 2023

Mesenchymal stem cells are multipotent progenitor cells capable of differentiating into several lineages including adipocytes, and osteocytes. While they are regarded as non-immunogenic, accumulating evidence suggests that MSC expresses several TLRs. We checked the contribution of TLR ligands to MSC differentiation. Rat bone marrow derived MSC surface marker and TLR transcript levels were checked by FACS and RT-PCR respectively. MSCs were CD29hi, CD71dim, CD90hi, and negative for CD45 & CD34. They expressed TLR1,2,3,4,6, and low levels of 7 and 9 (no TLR5 was detected) at passage zero. Cells were incubated in adipogenic and osteogenic differentiation media either alone or supplemented with, TLR2L:PGN, TLR3L:pI:C, TLR7L:R848, and TLR9L:CpGODN as well as control ODN and followed for three weeks. Oil Red O staining (to assess adipogenesis) suggested that, induction in adipogenic media (by one week) gave no adipose positive cells whereas treatment with R848 or PGN induced significantly high adipose positive cells (1-2% vs 30-35%, respectively). The degree of adipogenic differentiation at wk1, was; TLR7≥TLR2>TLR3>TLR9>Control group≥medium. During osteogenesis (checked by alizarin red staining) by one week the highest differentiation was seen with CpG ODN treatment (TLR9≥TLR7>TLR2>TLR3>Control≥medium). These results strongly support the view that TLR ligands differentially contribute to transition of MSCs into either adipogenic or osteogenic differentiation.
Role of shingosine-1-phosphate receptor 1 in effector CD8 T cell migration (137.15)
Authors: Khanna Kamal , Xu Daqi , Rudenga Benjamin
Published: January 2023

Our previous studies have helped illuminate the landscape in which the endogenous CD8 T cell immune response occurs. However, the factors that regulate the anatomical program followed by antigen-specific CD8 T after infection are still unknown. Recent studies have revealed an important role of sphingosine 1-phosphate receptor 1 (S1P1) in mediating the migration of naive T cells. However, these studies have primarily focused on understanding the role of S1P-S1P1 signaling in T cell trafficking in steady state. The role of the S1P receptors in regulating endogenous CD8 T cell migration in the spleen or lymph nodes (LN) during infection has not been investigated. Here we investigated the role of S1P receptors in the migration and localization of CD8 T cells in lymphoid tissues after infection. We used genetic mouse models, as well as temporal blocking of S1P receptors to reveal a central role of S1P1 in controlling effector CD8 T cell migration from draining LN following viral infections. In the absence of S1P1 receptor signaling, antigen-specific CD8 T cells failed to utilize the lymphatic cortical sinuses to egress from the draining LN. Moreover, our data show that S1P receptors mediate the sequestration of effector CD8 T cells early after bacterial infection in specific compartments of the spleen. Therefore, our studies demonstrate that S1P1 receptor signaling is the dominant factor that regulates endogenous effector CD8 T cell migration in secondary lymphoid organs.
Respiratory syncytial virus hijacks the tumor-suppressor p53 to enhance its replication in lung epithelial cells (137.3)
Authors: Gibbs John , Bian Tao , Menendez Daniel , Resnick Michael , Imani Farhad
Published: January 2023

Respiratory syncytial virus (RSV) infection is the major cause of childhood lower respiratory illness and has been associated with the induction and exacerbation of asthma. Previously, we reported that RSV infection induced TGF-β expression that caused cell cycle arrest and subsequently enhanced RSV replication. In the current study, we demonstrate that a downstream target of TGF-β signaling; P53, is a pivotal transcription factor for RSV replication. Over-expression of p53 protein by an inducible system (Tet-off) enhanced RSV replication by 9 folds, whereas, knockdown of p53 using siRNA significantly reduced RSV replication in H1299 and A549 lung epithelial cell lines. Stabilization of p53 with nutlin-3 epithelial (PHBE) cells. We hypothesized that a plausible mechanism of enhanced RSV replication by p53 was through direct binding of p53 to RSV RNA. A search of RSV genome revealed several putative p53-responsive elements. To confirm interaction of p53, EMSA analysis was performed which showed a specific shift with one of the p53 response elements. Taken together, we have identified a novel role for p53 in positive regulation of RSV replication. These findings suggest that enhanced p53 expression through lung injury, smoking or air pollution could facilitate a significant increase in RSV replication leading to more severe respiratory pathologies.
Histone methylation by NUE1, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis (137.9)
Authors: Pennini Meghan , Perrinet Stephanie , Dautry-Varsat Alice , Subtil Agathe
Published: January 2023

Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism and detected its translocation to the host cell nucleus during infection. We therefore named the protein nuclear effector 1 (NUE1). Expression of NUE1 in mammalian cells by transfection reconstituted nuclear targeting, and the protein associated with chromatin. In vitro methylation assays confirmed NUE1 is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Treatment of infected cells with the methyltransferase inhibitor 5’-deoxy-5’-(methylthio)adenosine significantly impaired chlamydial development. Thus, NUE1 is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.
The role of TRAF1 in stabilizing TRAF2 from proteasome mediated degradation downstream of 4-1BB signaling (138.1)
Authors: McPherson Ann , Watts Tania
Published: January 2023

TNF receptor family members play important roles in the innate and adaptive immune response, inducing signals for cell survival or apoptosis. This signaling in most cases relies on TNF receptor associated factors (TRAFs) to relay the signal from the TNFR family member to activation of NF-κB or MAPK pathways. Results from our lab have shown that TRAF1 is critical in the downregulation of the proapoptotic molecule Bim and the survival of activated and memory CD8 T cells, acting downstream of the prosurvival TNF receptor family member 4-1BB. We show here that in the absence of TRAF1, signaling downstream of 4-1BB results in the rapid proteasome dependent degradation of TRAF2. To identify interacting binding partners of TRAF1 that may mediate its stabilizing role on TRAF2 we performed a yeast two hybrid screen using a cDNA library from activated mouse lymph nodes. Among the interacting proteins identified were PSMC3, a subunit of the 19S regulatory particle of the 26S proteasome. We have confirmed the interaction between TRAF1 and PSMC3 in T cells and are currently investigating its functional role in the signaling downstream of 4-1BB. The results suggest that TRAF1 may have an important role in controlling the duration of the signal.
Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells. (138.20)
Authors: Barbi Joseph , Pan Fan , Yu Hong , Dang Eric , Yang Huang , Pan Xiaoyu , Jinasena Dilini , Pardoll Drew
Published: January 2023

CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. Here we identify Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. By qRT-PCR analysis we found that Eos is highly expressed in the CD4+CD25+ and CD4+Foxp3+ population of T cells. Additionally, Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress naïve T cell proliferation in vitro. Furthermore, Tregs with Eos knockdown fail to protect recipient Rag2-/- mice from severe colitis when co-transferred with naïve (CD4+CD25- CD62Lhigh ) T cells. Eos silencing in Tregs also endows these cells with partial effector function. These results demonstrate the critical role that Eos plays in Treg programming. They also raise the possibility that the interaction between Foxp3 and Eos may be a therapeutic target for controlling pathological immune responses in which excessive Treg activity plays an important role such as cancer and chronic infections.
Dendritic cell paracrine IL-2 regulates phenotype and function of CD4+CD25+ Treg cells in vitro. (138.5)
Authors: Field Elizabeth , Nasr Mohamed , Kulhankova Katarina , Rouse Todd
Published: January 2023

The development, expansion and function of mouse CD4+CD25+ regulatory T cells (Treg) requires intact IL-2 receptor and paracrine IL-2, but the identity of the paracrine supplier remains obscure. Herein we show in mice that Treg suppressive function in vitro depends on IL-2 from dendritic cells (DC). Anti-CD25 blocked not only Treg inhibition of DO11.10 CD4+CD25- effector cells, but also Treg inhibition of DC stimulatory function, suggesting a DC source of IL-2. We showed by IL-2 ELISPOT, intracytoplasmic flow cytometry and RT-PCR, and we confirmed by transfection studies using IL-2-promoter-driven reporters, that splenic and bone marrow-derived DCs constitutively produced IL-2, and that CpG-B increased IL-2 production. Though secreted IL-2 protein was readily detected in DC cultures by ELISPOT, little or no IL-2 protein was detected in DC cultures that contained Treg cells. The addition of anti-CD25 preserved the level of IL-2 protein detected in co-cultures of DC and Treg cells. Tregs failed to maintain levels of CD25 and foxp3 or to suppress CD4+CD25- effector cells proliferation and IL-2 production in cultures where DCs from IL-2 knockout mice were substituted for wild-type DCs. These results show that dendritic cells supply IL-2 to Tregs and that Treg phenotype and inhibitory function critically depends on paracrine IL-2 from dendritic cells.
Oltipraz inhibits TGF-{beta}-mediated Foxp3+ regulatory T cell differentiation (139.15)
Authors: Kim Il-Kyu , Park Young-Jun , Kim Byung-Seok , Kang Chang-Yuil
Published: January 2023

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] is a synthetic dithiolethione that has been widely studied for the prevention of cancer formation as well as treatment of liver cirrhosis. Although the precise mechanism by which oltipraz inhibits cancer and liver diseases remains unknown, it may be attributed to its ability to induce the expression of numerous Phase 2 detoxification enzymes and to reduce hepatic triglyceride accumulation by S6K1-dependent pathway, respectively. However, whether oltipraz also has an effect on immune cells, especially on T helper cell differentiation has to be elucidated. Here, we found that addition of oltipraz significantly inhibited Th2 differentiation under neutral and Th2-polarizing conditions while there was little, if any, effect on Th1 differentiation. In addition, TGF-{beta}-mediated Foxp3 induction in naïve CD4+ T cells was considerably reduced in the presence of oltipraz. Furthermore, Foxp3 induction was inhibited by oltipraz even in IFN-{gamma}-deficient naïve CD4+ T cells, indicating that oltipraz can inhibit TGF-{beta}-mediated Foxp3+ regulatory T cell differentiation through an IFN-{gamma}-independent manner. Therefore, these results suggest that oltipraz can act as an immune-modulating agent.
IL-1β/p38 signaling controls IL-17 production by T regulatory cells (139.3)
Authors: Li Lequn , Kim Jin Sub , Boussiotis Vassiliki
Published: January 2023

T regulatory (Treg) cells expressing Foxp3 represent a distinct lineage of T lymphocytes committed to suppressive functions. Recent studies have shown that Treg cells can become IL-17 producers in the context of inflammatory cytokine milieu similarly to conventional CD4+ T cells (Tconv). In the present study, we studied the differentiation of IL-17 producing cells in the absence of exogenous cytokines and insults. We showed that under neutral culture conditions, simultaneous activation of naïve Tconv cells and Treg cells in the presence of antigen presenting cells (APC) promoted conversion of Treg but not Tconv into IL-17 producing cells. Using both an in vitro culture system and an in vivo model, we determined that endogenous IL-1β but not IL-6 played an essential role in the conversion process. Mechanistic analysis revealed that IL-1β induced strong activation of p38 MAPK in Treg but not in Tconv cells. Addition of the p38 inhibitor SB203580 in the co-culture abrogated the production of IL-17A by Treg cells, indicating that p38 downstream pathways might be responsible for regulating production of IL-17 by Treg. Our findings indicate that Treg cells are subjected to regulation that is mediated through APC and activated Tconv and reveal a central role of IL-1β and p38 MAPK in this process. These observations provide a new dimension to our understanding of Treg functions and may have important implications in therapeutic strategies using Treg cells.
Differential expression of IL-17A and IL-17F is coupled to TCR signaling via Itk-mediated regulation of NFATc1 (139.4)
Authors: Gomez-Rodriguez Julio , Sahu Nisebita , Handon Robin , Sacta Maria , Anderson Stacie , Kirby Martha , August Avery , Schwartzberg Pamela
Published: January 2023

Th17 CD4+ effector T cells play important roles in autoimmunity and responses to bacterial infections. The roles of cytokines in regulating the transcription factors STAT3 and RORγT in Th17 differentiation have been extensively studied. However, the role of T cell receptor (TCR) signaling in Th17 differentiation is less appreciated. To examine this issue, we evaluated IL17 production from T cells lacking Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ and downstream pathways. We find that Itk-/- CD4+T cells exhibit specific defects in the expression of IL-17A despite relatively normal expression of RORγT, RORα and the other Th17 cytokines, including the closely related IL-17F. Defects in IL-17A expression and differential regulation of IL-17A and IL-17F were also observed in vivo in Itk-/- mice challenged with an allergic asthma model. Although Itk-deficient cells have slightly depressed phosphorylation of STAT-3 in response to IL-6, expression of constitutively activated STAT-3 failed to rescue their IL-17A production. In contrast, expression of IL-17A could be rescued by pharmacologically-induced Ca2+ influx or by a constitutively active NFATc1. The effects of Itk on transcriptional complexes and chromatin at the IL-17 locus are under further investigation. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1 and that TCR signaling pathways differentially influence Th17 cytokine production.
The LFA-1/ICAM-1 bond: A Spatio-Temporal Cue in PMN Recruitment in Acute Inflammation (140.3)
Authors: Dixit Neha , Simon Scott
Published: January 2023

Polymorphonuclear leukocyte (PMN) adhesion to the inflamed endothelium is in part mediated by LFA-1(CD11a/CD18) which recognizes its ligand ICAM-1. It has long been known that ICAM-1 rapidly forms homodimers and this conformation supports stable bonds specifically with high affinity LFA-1 on the PMN in the presence of shear flow. We hypothesize that shear stress on these dimeric pairs of bonds between high affinity LFA-1 and ICAM-1 result in intracellular signaling that serves as a gatekeeper in the transition from PMN arrest to a migratory phenotype. Imaging PMN rolling on an inflammatory mimetic substrate expressing allosteric activating and blocking antibodies to CD18 under shear flow revealed that calcium flux as well as Talin recruitment was mediated primarily through shear stress acting on high affinity CD18 bonds. Immunofluorescence revealed colocalization of cytoplasmic F-actin, Talin, Rap-1, and phospho-Src within regions correlating with high affinity clusters of LFA-1. In summary, these data sets reveal that an integrin mediated outside-in signal is dependent on shear and the affinity of LFA-1 for its ligand ICAM-1 and that a minimum dimerization of high affinity LFA-1/ICAM-1 bonds elicits the assembly of a macromolecular complex that provides a key navigational step in the transition from PMN arrest to a migratory phenotype.