bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel characterization of lethenteron japonicum complement C3 involved in immune response (INM2P.429)
Authors: Li Qingwei , Pang Yue , Zan Qi , Ba Wei , Pei Guangying
Published: January 2023

As an important model organism for the research of adaptive immune system, lampreys have been recognized as the key of the discovery of immune factors. In this paper, we discovered a novel immune factor named LjC3-like which participated in immune deposition and clearance. Datas indicated that the transcript and protein expression of LjC3-like was distributed in immune associated tissues, such as marrow, gill, kidney, intestines. A part of LjC3-like cDNA from lamprey liver, was successfully constructed and expressed in E.coli Rosatte and obtained anti-LjC3 polyclonal antibody. Flow cytometry analysis and laser scanning confocal microscopy revealed the existence of LjC3 in leukocytes and myelocyteson, especially in cytoplasm. The functional interaction between VLRB and LjC3 were demonstrated by coimmunoprecipitation in parallel with western blot assays. Furthermore lamprey serum destroyed target cells while the deposition of LjC3-like on their surface was detected. These results show novel characterization of LjC3-like, which lay a foundation to further research the vertebrate immunology , bring to the rethink of immune system in the lowest vertebrates.
Antibacterial and pro-resolving mechanisms in lung diseases: role of C-type lectin receptors (INM2P.430)
Authors: Sharma Jyotika , Sharma Atul , Steichen Anthony , Jondle Christopher , Binstock Brandilyn , Mishra Bibhuti
Published: January 2023

Lower respiratory tract infection with bacteria can lead to sepsis development, a complex immune disorder with systemic hyperinflammation. There are currently no effective therapies for sepsis. To understand the function of specific pulmonary components in regulation of immune responses in this immune disorder, we utilize an acute pulmonary bacterial infection model of Klebsiella pneumoniae (KPn). Nosocomial infections with this opportunistic pathogen account for 5-20% of Gram-negative sepsis cases. C-type lectin receptors (CLRs), expressed mainly by myeloid cells, can shape immune responses in diverse pathological conditions. However, their role in development of pneumonic sepsis is unknown. Our preliminary analysis of a panel of CLRs showed an upregulated expression of two CLRs, Clec4d and Clec4e in the lungs of KPn infected mice. While the wild-type mice were able to resolve a sublethal pneumonic KPn infection, the Clec4d-/- and Clec4e-/- mice displayed increased susceptibility with a progressive increase in bacterial burden, hyperinflammatory response and massive accumulation of neutrophils in lungs. Importantly, we show that coordinated function of Clec4d and Clec4e in neutrophils during bacterial pneumonia, and in a chronic lung disease, leads to control of bacterial growth and hyperinflammation by regulating phagocytosis and efferocytosis (clearance of dead cells). Our studies provide novel insights into regulation of inflammatory derangements in immune disorders.
Actin confinement of activating receptors does not dictate natural killer cell licensing (INM2P.431)
Authors: Thomas Louis , Long Eric
Published: January 2023

The responsiveness of an individual Natural Killer (NK) cell is calibrated by licensing (aka education), a process that depends on inhibitory receptor binding to major histocompatibility complex (MHC) class I. Previous data suggested that unlicensed NK cells have decreased responsiveness because the mobility of their activating receptors is hindered by actin. To test this hypothesis, we compared activating receptor-induced calcium mobilization in licensed and unlicensed human NK cells before and after actin depolymerization by Latrunculin. Licensed NK cells mobilized more calcium following stimulation by single or multiple activating receptors, as compared to unlicensed cells. Notably, the amplitude but not the induction of calcium mobilization was different. Actin depolymerization elevated the amplitude of calcium mobilization and accelerated calcium induction in both licensed and unlicensed NK cells. Therefore, licensed NK cells mobilized more calcium than unlicensed cells even after actin depolymerization. Furthermore, actin depolymerization resulted in faster lateral mobility of activating receptors in both licensed and unlicensed NK cells. Ongoing studies are evaluating the movement of activating receptors by single particle tracking. In conclusion, the results of our study show that actin-dependent confinement of activating receptors is not the only determinant of reduced responsiveness by unlicensed NK cells.
Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (INM2P.434)
Authors: Sauer Karsten , Park Eugene , Siegemund Sabine , French Anthony , Wahle Joseph , Sternberg Luise , Rigaud Stephanie , Jonsson Helena , Yokoyama Wayne , Huang Yina
Published: January 2023

NK cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their limited efficacy requires a better understanding of the molecular mechanisms dampening their effector functions. NK cells recognize and kill pathogen-infected or tumor cells through invariant NK cell receptors (NKR). Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble IP4. We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product PIP3 and control PIP3-mediated protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.
Covalently coupled immunostimulant heterodimers (INM2P.419)
Authors: Mancini Rock , Tom Janine , Esser-Kahn Aaron
Published: January 2023

We report increased stimulation of dendritic cells via heterodimers of immunostimulants formed at discrete molecular distances. Many vaccines present spatially organized agonists to immune cell receptors. These receptors cluster suggesting that signaling is increased by spatial organization and receptor proximity, but this has not been directly tested for multiple, unique receptors. In this study we probe the spatial aspect of immune cell activation using heterodimers of covalently attached immunostimulants including pyrimido[5,4-b]indoles, imidazoquinolines, palmitylated peptides, lipoteichoic acids, and CpG oligonucleotides.
Lung MDSC-derived IL-10 in resolution of inflammation during bacterial pneumonia (INM2P.420)
Authors: Chakraborty Krishnendu , Khare Anupriya , Raundhal Mahesh , Poe Stephanie , Morse Christina , Huff Rachael , Ray Anuradha , Ray Prabir
Published: January 2023

Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight against pathogenic microorganisms, persistent inflammation leads to collateral tissue damage and precipitates acute lung injury. Recently we have shown that interleukin (IL)-10 is essential for resolution of lung inflammation with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia. Although IL-10-/- mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b+Gr1(Ly6G)intF4/80+ cells resembling myeloid-derived suppressor cells (MDSCs). These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. However, our ongoing studies show that IL-10 production from lung MDSCs can be negatively regulated during pneumonia. Previous studies have documented increased levels of the lipid cardiolipin, which is generated from the mitochondria of dead cells, in the lungs of patients with severe pneumonia. We have observed that cardiolipin blunts pulmonary IL-10 production thereby compromising the efficiency of the MDSC-IL-10 system to dampen inflammation. Considering the important role of IL-10 in limiting inflammation and tissue damage, our study identifies new targets for maintaining IL-10 expression during resolution of inflammation in pneumonia.
The actin-bundling protein L-plastin regulates alveolar macrophage pro-inflammatory cascade (INM2P.426)
Authors: Morley Sharon Celeste , Deady Lauren , Todd Elizabeth , Zhou Julie
Published: January 2023

Regulation of the actin cytoskeleton is essential to multiple immune cell functions, including cell motility, adhesion, and activation. In lymphocytes, the actin-bundling protein L-plastin (LPL) plays critical roles in sustaining cell polarization and formation of the immunological synapse. We now show defects in the innate immune system that lead to profound susceptibility to pulmonary pneumococcal infection. LPL is required for generation of alveolar macrophages: there are fewer alveolar macrophages in the bronchoalveolar lavage fluid of LPL-/- mice, and regeneration of alveolar macrophages following sublethal irradiation is delayed when bone marrow is donated by LPL-/- mice. Activation of LPL-/- alveolar macrophages is also defective, with reduced upregulation of IL-1-beta and IL-6 following LPS stimulation. Reduced numbers and activation of alveolar macrophages in LPL-/- mice correlate with disrupted podosome formation. Reduced numbers and activation of alveolar macrophages would be expected to result in an inability to clear pulmonary pathogens. When challenged with pulmonary infection with pneumococcus, LPL-/- mice show a clearance defect within 3 hours, with a ten-fold increase in recoverable pneumococci compared to wild-type mice. LPL-/- mice thus offer a model system in which to detail how actin cytoskeletal integrity regulates macrophage development and activation.
Carma1 and MALT1 are required for stimulation-dependent mTORC1 activation in T cells (IRC2P.455)
Authors: Hamilton Kristia , Shiva Sruti , Kane Lawrence
Published: January 2023

Stimulation of T cells through the T cell receptor (TCR) and co-stimulatory molecules induces a diverse set of signaling events leading to T cell activation. Some characteristics of T cell activation are cellular proliferation, increase in cell size and changes in metabolic pathways. Our work has primarily focused on understanding the roles that Carma1, MALT1 and Bcl10 (the CBM complex) play in T cell activation, specifically, elucidating their requirement in distinct T cell signaling pathways. Here, we show a requirement for Carma1 and MALT1 in activation of mTORC1. In the absence of Carma1 or MALT1, we noted impaired TCR/CD28-induced stimulation of ribosomal protein S6, p70S6K and 4E-BP1 phosphorylation. Further investigation into the specific molecular mechanism revealed constitutive association of MALT1 and p70S6K. Surprisingly, Bcl10 was not required for S6 or S6K phosphorylation in T cells. Pharmacological inhibition of this pathway with a MALT1 protease inhibitor (z-VRPR-fmk) impaired T cell proliferation upon TCR and co-stimulatory molecule stimulation, though this did not affect T cell “blasting” or early activation. Additionally, inhibition of MALT1 activity impaired T cell activation-induced glycolysis, as seen by a decrease in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Thus, these findings present a novel mechanism for mTORC1 activation in T cells, through Carma1 and MALT1, leading to cellular proliferation and increased glycolysis.
Negative signalling in T cells with aging (IRC2P.446)
Authors: Fulop Tamas , Dupuis Gilles , Le Page Aurélie , Larbi Anis
Published: January 2023

Immune responses are generally impaired in aged humans. There is impaired production of IL-2 and T lymphocyte proliferation in response to antigenic stimulaton. These decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery of T cell activation. We have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms. We used tyrosine-specific mAbs and Western blot analysis to show that a dysregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Tyr505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels not significantly different than those of young donors. Our data suggest that the role of SHP-1 in T cell activation extends to its increased effect in negative feedback in aging. Modulation of SHP-1 activity could be a target to restore altered T cell functions in aging.
Nedd4-family interacting proteins regulate T cell homeostatic processes via activation of catalytic E3 ubiquitin ligases (IRC2P.447)
Authors: O'Leary Claire , Riling Christopher , Oliver Paula
Published: January 2023

Ubiquitylation tunes signaling pathways in stimulated T cells to regulate activation and function. Catalytic E3 ubiquitin ligases, like Itch, have known roles in these processes. We have found that Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 promote ubiquitin charging of Itch, a requisite step in protein ubiquitylation. In vitro, Ndfip1 and Ndfip2 have overlapping function. In vivo, Ndfip1 negatively regulates T cell activation and Th2 polarization. The in vivo role of Ndfip2 is unknown. To investigate this, we generated Ndfip2-/- mice. We found that, unlike Ndfip1, Ndfip2 is not a prominent negative regulator of T cell activation or Th2 polarization. However, loss of Ndfip2 exacerbates the inflammatory Ndfip1-/- phenotype, suggesting that, like Ndfip1, Ndfip2 dampens inflammatory processes. Our data indicate a T cell intrinsic role for Ndfips in limiting T cell activation and function, as Ndfip1/Ndfip2 DKO CD4+ T cells in mixed fetal liver chimeras are more activated and produce more cytokine than WT CD4+ T cells in the same host. In vitro coculture of naïve WT and DKO CD4+ T cells supports that Ndfip deficient T cells have increased viability and proliferative capacity. These results suggest that Ndfip1 and Ndfip2 together limit T cell survival and proliferation downstream of TCR engagement. We are taking a quantitative proteomic approach to identify ubiquitylation pathways engaged during TCR stimulation that are nucleated by Ndfip-E3 ligase interactions.
Activation of the aryl hydrocarbon receptor during development leads to an altered CD4+ T cell response to influenza A virus infection (IRC2P.448)
Authors: Boule Lisbeth , Lawrence B. Paige
Published: January 2023

Epidemiological evidence suggests developmental exposures to certain pollutants correlate with lower antibody responses to childhood immunizations, but the mechanism by which this occurs is unknown. The transcription factor aryl hydrocarbon receptor (AhR) is activated by a variety of chemicals, expressed by immune cells, and has been shown to influence immune function, such as CD4+ T cell subset specification and function. The effect of maternal exposure to AhR ligands on the function of CD4+ T cells has never been directly examined. We report here the consequences of developmental triggering of the AhR on the CD4+ T cell response to influenza virus infection. Mice developmentally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical AhR ligand, have fewer activated, virus-specific CD4+ T cells, Th1, and T follicular helper cells, yet an increase in regulatory T cells in their lung draining lymph nodes. Consistent with this, developmentally exposed mice have a reduction in their class-switched, virus-specific antibody response. By using reciprocal adoptive transfers, we demonstrate that the effects of developmental activation of the AhR on CD4+ T cells occur through both direct and indirect pathways. These findings suggest changes in the functional capacity of CD4+ T cells may be a key contributor to the decreased antibody response to immunizations observed in children of mothers exposed to AhR-binding chemicals.
Pivotal role of STAT3 signaling in germinal center B cell and T follicular helper cell differentiation (IRC3P.458)
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

Germinal center (GC) B cells and T follicular helper (Tfh) cells are critical for long-lived plasma cell differentiation and high-affinity antibody production in response to T cell-dependent antigens. Previous studies demonstrated that GC formation was intact in the absence of STAT3 in B cells at day 12. However, emerging evidence suggests that IL-21/STAT3 signaling is a potent regulator for B cell differentiation. To better understand the role of STAT3 in the kinetics of GC formation and GC B cell response, we assessed GC B cells at consecutive time points following immunization with antigen OVA. In B cell conditional STAT3-dificient mice, fewer GC and GC B cells were generated upon immunization in the late phase (day 21) but not on day 14 compared with that of control mice. Increased cell death and decreased cell proliferation were found in the STAT3 deficient GC B cells. Consequently, plasma cell differentiation and antibody production were impaired in B cell STAT3-deficient mice. In addition, the Tfh cell response was correlated with the magnitude of the GC B cell response and significantly decreased in B cell STAT3-deficient mice. Furthermore, GC B cells and autoantibody production were also significantly decreased in autoreactive B cell STAT3-deficient mice after apoptotic cell immunization. These data provide new insights into the GC and Tfh differentiation and the function of STAT3 in humoral immune responses.
TIM-4 expression by B cells identifies an inflammatory B effector 1 subset that promotes allograft rejection and inhibits tumor metastases (IRC3P.471)
Authors: Mohib Kanishka , Ding Qing , Camirand Geoffrey , Rothstein David
Published: January 2023

B cells, like T cells, give rise to polarized subsets, termed Be1, Be2 and Breg that produce IFNγ, IL-4 and IL-10, respectively. We previously reported that TIM-1 is an inclusive marker for Be2 cells and Breg that can transfer allograft tolerance. We now show that TIM-4, a TIM-1 ligand, is expressed by a distinct subpopulation (~6-10%) of B cells. TIM-4 B cells are enriched for IFNγ (2.5%) and express relatively less IL-4 (1%) and IL-10 (4%) while TIM-1 B cells are enriched for IL-4 (6.5%) and IL-10 (14%) and express less IFNγ (1%). Accordingly, transfer of TIM-4 B cells to B cell deficient (JhD) islet allograft recipients accelerated rejection (MST 11d) vs. controls (no B cells; MST 15d) or TIM-1 B cell transfer (MST 70d), p<0.05. TIM-4 B cell-mediated rejection required IFNγ as transfer of IFNγ-/- TIM-4 B cells modestly prolonged MST (15d to 25d, p<0.05). In this model, transfer of TIM-4 B cells enhanced IFN-γ+ T cells (1.5X) while decreasing IL-4+, IL-10+ and Foxp3+ T cells (1.2-1.8X) whereas TIM-1 B cells increased IL-4+, IL-10+ and Foxp3+ T cells (1.2-1.7X) while decreasing IFNγ+ T cells (1.4X) vs. no B cell controls (p<0.05 for each). Moreover, transfer of TIM-4 B cells into μMT mice decreased B16 melanoma lung metastases ~ 2-fold, while TIM-1 B cells increased lung metastases ~2-fold vs. controls (p < 0.0001). Thus, we provide strong evidence that TIM-4 expression by B cells identifies a distinct Be1 inflammatory subset that can directly impact T cells responses.
Aging of the B-1a cell pool (IRC3P.473)
Authors: Holodick Nichol , Vizconde Teresa , Rothstein Thomas
Published: January 2023

B-1a cells arise predominately during fetal development, and their persistence throughout adult life is maintained through self-renewal. These cells are essential for immediate protection from S. pneumoniae infection. The unique ability of fetal derived B-1a cells to provide protection against S. pneumoniae is attributed to their spontaneously secreted natural IgM, which is germline-like due to minimal insertion of N-region additions and little somatic hypermutation. The B-1a cell pool is also maintained by B-1 cell progenitors found in the adult bone marrow. We reported these bone marrow derived (BMD) B-1a cells are functionally and phenotypically similar to native B-1a cells; however, BMD B-1a cells contain an increased number of N-region additions in their natural IgM as compared to native B-1a cells. Natural IgM derived from native B-1a cells in aged mice also contains more N-region additions. We show an increase in N-region addition in mice 6 months to 24 months of age. This suggests the repertoire of B-1a derived natural IgM changes with age, and the adult B-1a cell pool is not a closed system. We hypothesized the level of natural IgM protection changes with age as a result of repertoire alterations to the B-1a cell pool over time. We found the natural IgM from aged mice is less effective at protection from S. pneumoniae infection. Data presented here suggest age related changes of natural IgM can lead to less effective initial protection against infection.
B cells promote obesity-associated periodontitis and oral pathogen-associated inflammation (IRC3P.461)
Authors: Zhu Min , DeFuria Jason , Carr Jordan , Belkina Anna , Van Dyke Thomas , Gyurko Robert , Nikolajczyk Barbara
Published: January 2023

Chronic inflammation likely underlies the predisposition of type 2 diabetes (T2D) patients to periodontitis (PD), and the exacerbation of each disease when both are present. Several lines of evidence indicate that pro-inflammatory B cells promote T2D and PD individually, but the possibility that B cells are a key link between T2D to PD is untested. We compared outcomes from oral P. gingivalis challenge of lean WT or B cell-null mice to outcomes from mice that were obese and insulin resistance prior to oral challenge. Obese WT mice responded to oral P. gingivalis challenge with significant periodontal bone loss, while obese B cell-null mice were completely protected from PD. By contrast, lean WT and B cell-null mice suffer similar periodontal bone loss in response to oral pathogen. B cells from obese/insulin resistant hosts also support oral osteoclastogenesis, and both oral and systemic production of inflammatory cytokines, including the PD-associated cytokines TNF-α and MIP-2. B cells furthermore impact adipose tissue inflammation in obese P. gingivalis-infected hosts. Taken together, these data show that fundamentally different mechanisms regulate PD in lean and obese hosts, with B cells promoting PD only if the hosts are “primed” by obesity. These results justify more intense analysis of obesity-associated changes in B cells that predispose PD in human T2D, and may indicate the use of B cell depletion to alleviate PD and T2D simultaneously.
Tissue-specific tolerance of endogenous self antigen-specific CD4+ T cells (IRC4P.484)
Authors: Moon James , Lim Jong-Baeck , Legoux Francois
Published: January 2023

CD4+ T cells specific for self antigens are involved in autoimmunity, but little is known about the extent to which these potentially harmful T cells develop within a normal T cell repertoire or how they are maintained in a tolerant state. We modeled Cre recombinase as a neo-self antigen that is expressed by various different tissue-specific promoters in several strains of Cre transgenic mice. We mapped a CD4+ T cell epitope within the Cre protein and generated a corresponding peptide:MHCII tetramer, which we used in a magnetic enrichment strategy to directly detect rare populations of endogenous Cre-specific CD4+ T cells that naturally develop in the unmanipulated immune systems of these mice. We found that deletion of Cre-specific T cells was efficient in mice ubiquitously expressing Cre, but almost nonexistent in mice expressing Cre in tissue-restricted patterns. In mice expressing Cre exclusively in the pancreas, immunization with Cre antigen induced strong Cre-specific T cell expansion and cytokine production, suggesting immune ignorance to the pancreatic self antigen. In contrast, tolerance to Cre self antigen expressed in mucosal tissues such as the gut and lung relied on elevated steady state frequencies of Cre-specific regulatory T cells. While these Tregs efficiently controlled primary autoimmune responses, their ability to maintain tolerance was lost over successive antigenic challenges, indicating limits of peripheral tolerance to tissue-restricted self antigens.
An endogenous aryl hydrocarbon receptor (AhR) ligand, ITE induces regulatory T cells (Tregs) and ameliorates experimental colitis (IRC4P.490)
Authors: Singh Udai , Abron Jessicca , Singh Narendra , Guan Honbing , Hegde Venkatesh , Mishra Manoj , Scissum-Gunn Karyn , Price Robert , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that affects millions of people with high morbidity and health-care cost. Recent studies have shown that activation of the aryl hydrocarbon receptor (AhR) with the nontoxic ligand 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces regulatory T cells (Tregs) and suppresses autoimmune diseases. However, the effects of ITE on experimental colitis have not been investigated. We hypothesized that ITE induces in-vitro and in-vivo Tregs through activation of AhR and suppresses experimental colitis. Our data showed that ITE increases the frequency of Tregs in spleen, MLN and LPL as compared to vehicle. This induction of Tregs was reversed by AhR antagonist treatment in-vitro. Next, in-vivo colitis pathogenesis including a decrease in body weight was significantly reversed and systemic inflammatory cytokines were reduced in mice that received ITE treatments as compared to vehicle. ITE treatment also increased CD11c+, decreased activated T and CD11b+ cells from the spleen, MLN and LP as compared to vehicle alone. These findings clearly suggest that ITE diminishes colitis pathology through induction of Tregs, reduces inflammatory cytokines, decreases the inflammation score, and induces dendritic cells resulting in amelioration of colitis. Thus, our work demonstrates that the nontoxic endogenous AhR ligand, ITE, may serve as unique treatment modality against IBD.
TNFR2-deficiency contributes to sex-biased spontaneous experimental autoimmune encephalomyelitis (IRC4P.481)
Authors: McKarns Susan , Miller Patrick
Published: January 2023

Tumor necrosis factor-α (TNF) antagonism has improved the treatment of autoimmune disorders including rheumatoid arthritis and inflammatory bowel disease but is contraindicated in multiple sclerosis (MS) due to disease exacerbation. The mechanisms underlying these contrasting responses remain elusive. The objective of this study was to investigate the effect of selective TNFR2 ablation on the autoreactivity of myelin oligodendrocyte glycoprotein (MOG)-specific T cells. We report that 92% of female C57BL/6 TNFR2-/- MOG-specific (2D2) TCR transgenic mice spontaneously develop experimental autoimmune encephalomyelitis (EAE); whereas, only 8% of male TNFR2-/- 2D2 littermates developed an autoimmune phenotype. Demyelination accompanied B cell and CD4 T cell infiltration in the spinal cords and optic nerves of TNFR2-/- 2D2 mice with EAE. CNS inflammation in these mice correlated with increased production of IFN-γ and IL-17 and elevated sera titers of MOG35-55-specific IgG2b. Ex vivo co-culture studies demonstrated autonomous TNFR2-/- 2D2 CD4 FoxP3+ regulatory T cell (Treg) dysfunction as well as increased resilience by FoxP3-/- 2D2 CD4 T cells to respond to Treg suppression. These results suggest that TNFR2 plays a key role in the functional tolerance of MOG-specific CD4 T and B cells in a sex-related manner. This novel spontaneous autoimmune model provides a unique paradigm to study T and B cell interactions as well as sex-related factors in multiple sclerosis susceptibility.
Different roles for Smad4-dependent and independent TGFβ signaling pathways during the differentiation of virus-specific effector CD8 T cells (IRM4P.494)
Authors: Hu Yinghong , Cauley Linda
Published: January 2023

Transforming growth factor-β (TGFβ) is a regulatory cytokine that modulates the activities of other cells via multiple signaling pathways. The predominant pathways in hematopoietic cells are mediated by Sma and Mad-related family members (SMAD) and depend on a ubiquitous activating protein SMAD4. Other Smad4-independent pathways can also be activated by TGFβ however their role in immune regulation is less clearly defined. We examined the role of Smad4-dependent and -independent TGFβ signaling during the development of the CD8 T cell response to influenza virus infection. Our studies show that a complete blockade of all TGFβ-signaling accelerates the development of virus-specific CD8+ effector T cells (Teff) which express KLRG-1 and produce large quantities of cytokines in the lungs. In contrast, selective inhibition of only Smad4-dependent signaling prevents the development of a robust Teff response. These data point to a previously unexamined role of Smad4-independent TGFβ signaling in the regulation of virus-specific Teff cells. We are using this model to determine whether TGFβ regulation plays an major role in protective immunity in the lungs.
Simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells generates expanded population of exhaustion-resistant memory T-cells (IRM4P.495)
Authors: Berezhnoy Alexey , Levay Agata , Puplampu-Dove Yvonne , Rajagopal Anugraha , Brenneman Randall , Gilboa Eli
Published: January 2023

It is possible to redirect recently activated T-cells toward memory differentiation by altering intracellular signaling of the mTORC1 pathway. As previously reported, treatment with rapamycin increased formation of memory cells; however, animals treated with the drug exhibited signs of immune suppression and the rapamycin-generated memory T-cells failed to protect animals against a tumor challenge. We introduced highly efficient and specific method to downmodulate mTORC1 in CD8 T cells, whereby siRNA against raptor, was targeted in vivo to activated CD8 T-cells by conjugating to a 4-1BB-binding RNA aptamer, leading to the generation of an enhanced memory response and protective antitumor immunity. Memory T-cells are resistant to immune suppression; yet, in vivo the population is experiencing attrition mediated in part via PD-1 signalling. We developed the conjugate with two siRNAs against raptor and PD-1 linked to a 4-1BB aptamer. In vitro, both siRNAs efficiently dissociated from the conjugate inside target cells and downregulated their respective mRNAs. In vivo, we demonstrated that treatment with 4-1BB-raptor-PD1 conjugates increased formation of memory CD8+ T-cells in a rate similar to the 4-1BB-raptor conjugate. Notably, CD8+ memory T-cells expanded with 4-1BB-raptor-PD1 conjugates expressed significantly less PD-1 on cell surface. Thus, we introduced a new method of cell-targeted delivery of two siRNA to generate CD8 memory cells with enhanced functional potential.
The role of genes with bivalent chromatin in memory CD8 T cells (IRM4P.496)
Authors: Kim Jiewan , Subedi Kalpana , Wersto Robert , Zhao Keji , Peng Weiqun , Weng Nan-ping
Published: January 2023

Genes containing both histone methylation H3K4me3 and H3K27me3 markers at the promoter are so called bivalent genes that have been implicated in stem cell differentiation and function. Our previous study identified several dozen bivalent genes in memory CD8 T cells (central memory, TCM and effector memory, TEM). But the role of bivalent genes in CD8 T cells is not understood. Our current focus is to determine the characteristics and functions of these bivalent genes during CD8 memory T cell activation. We used freshly isolated TCM and TEM from human blood that were stimulated by anti-CD3 plus anti-CD28 in vitro, and analyzed histone methylation and mRNA levels of these bivalent genes in resting and activated CD8 memory T cells. Our preliminary results showed that the bivalent status of most genes was stable but corresponding mRNA change was more detectable. We also identified a few bivalent genes whose status changed to become “monovalent”; either H3K4me3 or H3K27me3 after activation. We plan to determine the functional role of these bivalent genes that are altered after activation, and whether the bivalent histone methylation is present on a single gene allele or on two different alleles by sequential ChIP. Our study will shed new insight into the details of histone methylation on gene loci and the function of these bivalent genes in memory CD8 T cells. “This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging “
FoxO1 directly regulates the expression of PD-1 and sustains viral specific CD8+ T cell responses during chronic infection (IRM4P.501)
Authors: Staron Matthew , Gray Simon , Kaech Susan
Published: January 2023

Understanding the signaling and transcriptional pathways that regulate the function, survival and homeostasis of CD8+ cytotoxic T lymphocytes (CTLs) during chronic infection is fundamental to the development of therapies to treat chronic infections such as HIV, HSV, and HCV. Of these, the PI3K/AKT/mTOR signaling pathway is of significant interest because it controls T cell differentiation, proliferation, metabolism and survival. We show that during murine chronic lymphocytic choriomeningitis virus (LCMV) infection, chronic antigen stimulation greatly impairs the ability of virus-specific CD8 T cells to activate PI3K/AKT/mTOR signaling in response to TCR and cytokine stimulation, resulting in enhanced nuclear retention of the transcription factor, FoxO1. Therapeutic blockade of the inhibitory PD-1 receptor, which restores antiviral CTL responses, was dependent on increased PI3K/AKT/mTOR activity. FoxO1 expression was critical to sustain virus-specific CTL responses and control viral infection. Lastly, we demonstrate that FoxO1 was both necessary and sufficient to induce the expression of the inhibitory receptor PD-1 on virus-specific CD8 T cells, and therefore represents a critical regulatory factor in balancing effector and exhausted CTL states during chronic infection. These findings highlight a novel role for AKT-FoxO1 pathway in the regulation of exhaustion CTL responses during chronic infection.
Divergent signaling pathways downstream of TLR7 regulate TNFα production by bone marrow polymorphonuclear leukocytes and monocytes in experimental lupus (IRM5P.700)
Authors: Han Shuhong , Zhuang Haoyang , Xu Yuan , Li Yi , Yang Lijun , Reeves Westley
Published: January 2023

Pristane-treated mice develop TLR7/IFNα dependent lupus autoantibodies and nephritis. In contrast, anemia results from TLR7-driven TNFα production by bone marrow (BM) polymorphonuclear leukocytes (PMN). Little TNFα is produced by BM monocytes. We examined the regulation of TNFα in BM of mice with pristane-lupus, emphasizing the role of IL-10, whose over-productionis implicated in SLE pathogenesis. Intracellular TNFα and IL-10, surface IL-10Rα, and p38MAPK activation were assessed by flow cytometry. In response to TLR7 ligand R848, BM PMN and monocytes both produced TNFα and IL-10. Inhibiting p38MAPK suppressed both PMN and monocyte TNFα production. In contrast, blocking NFκB had opposite effects on TNFα production in PMN (inhibition) vs. monocytes (enhancement). The differential effects were a consequence of NFκB mediated IL-10 production and the higher IL-10 responsiveness of monocytes vs. PMN. IL-10 suppressed TNFα in monocytes to a far greater degree than in PMN, consistent with levels of IL-10Rα expression by the different cell types.In contrast, PMN were more sensitive than monocytes to dexamethasone (which acts in part via NFκB) suppression of TLR7-driven TNFα production. Cell-type-specific regulation of NFκB and p38MAPK downstream of TLR7 leads to production of a complex array of cytokines (IFNα, TNFα, IL-10).Patients with disease mediated by IFNα and monocytes may respond differently to certain therapies than those with PMN-mediated clinical manifestations.
Regulation of TLR4-promoted inflammatory signaling by β-arrestin 2 (IRM5P.706)
Authors: Yin Deling , Denney James , Hu Dan , Yan Hui , Caudle Yi , Li Hui
Published: January 2023

The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that β-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 MAPK-dependent mechanism. β-arrestin 2 forms complexes with p38 mitogen-activated protein kinase (MAPK) and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of β-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking β-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which β-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.
ATF4 is induced by ROS and regulates Th1 and Th17 responses (IRM6P.718)
Authors: Yang Xi , Gao Xin , Yang Qianting , Zhu Yibei , Li Gang , Zhang Xueguang , Xiao Guozhi , Cao Huiling , Chen Xinchun , Lu Binfeng
Published: January 2023

Reactive oxygen species (ROS) play a major role in the pathogenesis of chronic inflammatory and autoimmune diseases. Little is known about underlying molecular mechanisms regulating T cell immune responses to ROS. Here, we demonstrated that ROS induced ATF4 in activated T cells. We found, in the high ROS condition, ATF4-/- T cells failed to proliferate and produced much less IFN-γ when cultured in the Th1 polarization condition. Inhibition of ROS by addition of antioxidants significantly recovered proliferation and IFN-γ production by ATF4-/- CD4 T cells in the Th1 condition, suggesting a crucial role of ATF4 in preventing oxidative stress during T cell activation and Th1 differentiation. In addition, we demonstrated that ATF4-/- mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), which is associated with elevated levels of IL17 but reduced IFN-gamma production. The in-depth molecular mechanisms are further studied and the result will be presented. Our study establishes that the ROS/ATF4 axis promotes Th1 and suppresses Th17 immune responses and likely plays an important role in autoimmunity and cancer immunology.
Identification of candidate genes for type 1 diabetes by microarray expression analysis of islet-specific CD4+ T cells (IRM6P.726)
Authors: Waldner Hanspeter , Berry Gregory , Frielle Christine , Salzberg Anna
Published: January 2023

Type 1 diabetes (T1D) is a complex genetic disease, resulting from the interplay of multiple insulin dependent diabetes loci (Idd) in both humans and nonobese diabetic (NOD) mice. NOD.B10 Idd9 congenic mice contain the Idd9 from T1D-resistant C57BL/10 mice, mediating significant T1D protection compared to NOD mice. However, the genes and the pathways by which Idd9 controls T1D development remain to be fully elucidated. We have developed novel NOD.B10 Idd9 congenic mice that harbor predominantly islet-specific CD4+ T cells expressing the BDC2.5 T cell receptor (BDC-Idd9.905 mice). To establish functional links between Idd9 genotype and its phenotype, we have performed microarray gene expression analyses of purified CD4+ T cells from these mice and BDC2.5 NOD control mice. We found that among the limited number of differentially expressed genes, those located within the Idd9 region were greatly enriched. Bioinformatics analyses identified Eno1, Rbbp4 and Mtor as differentially expressed Idd9 genes that were part of significant gene networks involved in T cell growth and development. Accordingly, antigen-specific proliferation and Th1 and Th17 responses between BDC-Idd9.905 and BDC2.5 splenocytes differed significantly and were impaired in BDC-Idd9.905 splenocytes. Our findings suggest that these Idd9 genes differentially control proliferation and differentiation of islet-specific CD4+ T cells in the two strains, which may contribute to their differential T1D development.
The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation (IRM6P.710)
Authors: Lappas Courtney
Published: January 2023

Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A2A receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A2AR playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A2AR-dependent mechanism. Specifically, zeatin riboside inhibits the production by CD3+CD4+ T cells of IFN-γ, IL-2, TNF-α, IL-4 and IL-13, and the production by CD3+CD8+ T cells of IFN-γ, IL-2, and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A2AR antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.
The antigen sensitivity of effector but not memory CD4 T cells is regulated by the pathogen-induced inflammatory milieu. (IRM6P.712)
Authors: Richer Martin , Kim Marie , Segura Stephanie , Harty John
Published: January 2023

CD4 T cells play an important role in orchestrating immune responses following recognition of antigens through their T cell receptor (TCR). As such, their sensitivity for cognate antigen is likely to be tightly regulated. Previously, we demonstrated that the antigen sensitivity of CD8 T cells was tuned by inflammatory cytokines. Whether inflammatory cytokines also tune the antigen sensitivity of CD4 T cells remains unknown. Herein, we demonstrate that the antigen sensitivity of effector CD4 T cells is tuned by the inflammatory milieu. Mechanistically, we demonstrate that, in contrast to CD8 T cells, increased antigen sensitivity is not linked to enhanced TCR signal transduction capacity. In addition, tuning of the antigen sensitivity of CD4 T cells by inflammatory cytokines is independent of their role in the polarization of these cells towards a Th1 phenotype. Further, we demonstrate that, unlike CD8 T cells, inflammatory cytokines do not regulate the antigen sensitivity of memory CD4 T cells. Taken together, these data demonstrate that while inflammatory cytokines tune the antigen sensitivity of both CD4 and CD8 T cells, they do so through distinct mechanisms. Understanding the regulation of the antigen sensitivity of T cells can yield important insight leading to potential therapeutic avenues aimed at manipulating the antigen sensitivity of T cells. These approaches may be useful to prevent chronic viral infections, allow for clearance of tumors and prevent autoimmunity.
T-bet and Runx transcription factors are required for the ontogeny of pathogenic IFNγ-producing Th17 cells (LYM3P.729)
Authors: Lazarevic Vanja , Wang Yan , Godec Jernej , Ben-Aissa Khadija , Cui Kairong , Zhao Keji , Pucsek Alexandra , Weaver Casey , Yagi Ryoji
Published: January 2023

Functional plasticity of CD4+ Th cells can be advantageous for the host as it creates diversity that is required to establish a productive immune response against a variety of pathogenic microbes. However, the acquisition of a gene expression profile associated with the opposing CD4+ Th lineages can lead to increased susceptibility to autoimmune diseases. For example, Th17 cells can give rise to IL-17A/IFNγ-double producing cells, which have been implicated in the development of autoimmune diseases. However, our understanding of the molecular mechanisms that govern the generation of IFNγ-producing Th17 cells during an inflammatory response remains incomplete. Here we report that, contrary to current concepts, co-expression of Th1- and Th17-cell master transcription factors, T-bet and RORγt, respectively, did not generate Th cells with strong dual Th1 and Th17 features. Instead, the development of Th1-like Th17 cells required T-bet and Runx1 or Runx3. Indeed, interleukin (IL)-12 stimulated Th17 cells upregulated Runx1, which bound to the Ifng locus in a T-bet dependent manner. Conversely, T-bet or Runx1 deficiency, or inhibition of Runx transcriptional activity, impaired the development of Th1-like Th17 cells during experimental autoimmune encephalomyelitis in vivo. Thus, our studies identify a critical role for T-bet and Runx transcription factors in the generation of pathogenic IFNγ-producing Th17 cells.
Analysis of the formation of antigen-specific memory CD4+ T cell populations derived from single naïve precursors (LYM3P.731)
Authors: Tubo Noah , Fife Brian , Jenkins Marc
Published: January 2023

Following infection, a naive antigen-specific CD4+ T cell population proliferates and differentiates into to effector cell progeny that are either Th1 cells, which activate the microbicidal functions of macrophages, or T follicular helper cells, which help B cells make antibodies. After the resolution of infection, most antigen-specific cells die, while a small number of cells survive to form long-lived memory cells. The origins of these memory cells remain unclear. In this study, we tracked the differentiation clonal populations of CD4+ T cells, from single naïve cells into effector cells, and subsequently memory cells. We find that not all naïve CD4+ T cells that produce effector cell populations give rise to memory populations. However, when memory populations are formed, they are phenotypically similar to the effector populations in terms of Th1 and T follicular helper cell differentiation, suggesting that effector cell phenotype is maintained into memory, and that memory cells are derived from effector cells. Ongoing work is focused on determining the contributions of TCR signal strength and effector population burst size has in memory cell differentiation.