bluuebunny/crossref_metadata_2025 huggingface dataset:
Time-lapse imaging of contact dynamics between natural killer cells and cancer cells (TUM10P.1035)
Authors: Kim Jisung , Kim Yong Guk , Park Eun Jae , Kim Bo Yeong , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Natural killer (NK) cell -mediated killing of cancer cells is essentially dependent on the direct cell-cell interaction. Using time-lapse imaging, we examine how NK cells contact melanoma cells at the single cell level. First, we analyzed contact dynamics of NK cells. Among 1,134 NK cells appeared in 36 movies in time-lapse imaging for 6 h, 121 NK cells (named competent NK, cNK) showed lytic contact with melanoma cells and 158 NK cells (named incompetent NK, iNK) showed non-lytic contact. Although contact numbers of both cell types was similar each other, the duration of contact appeared to be different. cNK cells contacted melanoma cells for 25 min without movement, but iNK cells contacted target cells for 13~14 min with moving around cancer cells. Both cell types moved at average speed of 5~6 um/min before contact and 1.6~2.1 um/min during contact. Next, we analyzed lytic process of melanoma cells, which could be described by two phases: rounding and membrane damage. Immediately after contacting cNK cells, all melanoma cells rounded up and it took 35 min (6-120) having small deviation. Then, melanoma cells lost membrane integrity and it took 103 (6-298) showing big deviation. Together, our data demonstrate that a small fraction of NK cells are cytotoxic to melanoma cells.
OVA-Texo/4-1BBL helped restore exhausted OVA-specific CD8+ T cell function during Adenovirus induced chronic infection. (VAC12P.1115)
Authors: Wang Rong , Xiang Jim
Published: January 2023

CD8+ T cell functional exhaustion has been adequately studied during established persistent viral infections such as LCMV in mice models as well as HIV and hepatitis C virus (HCV) in human chronic infections. Recombinant Adenovirus infection has also been reported to cause CD8+ T cell dysfunction and deletion. In this study, we established an Adenovirus induced chronic infection in C57BL/6 mice, characteristic by a long persistence of high percentage of antigen-specific CD8+ T cells, with increased inhibitory markers expression (PD-L1, PD-1, LAG-3, CD43) and compromised effector functions. Exosome-targeted CD8+ T cell vaccine is constructed through ConA stimulated non-specific CD8+T cells uptaking OVA pulsed DC-derived exosome (EXO), acting as CD8+Tc antigen presenting cells (Th-APCs), and capable of stimulating DC- and CD4+T cell-independent OVA-specific CTL responses leading to long-term immunity. We demonstrated that OVA-Texo/4-1BBL vaccine is able to provoke a successful immune response in chronic infected mice, increasing OVA-specific CD8+ cell level massively by approximately 3-fold, the primed CTLs could differentiate into functional CD8+ memory T cells. OVA-Texo/4-1BBL also helped restore exhausted CD8+ T cell function in the absence of CD4+ T cells. A sufficient increase of IFN-r production CD8+ T cell was measured after OVA-Texo/4-1BBL immunization compared with mice without treatment.
Virus reactivation is driven by inflammatory monocytes that are controlled by T cells (VIR9P.1163)
Authors: Chandran Ramakrishna , Cantin Edourad
Published: January 2023

The current dogma is that episodes of transient immunodeficiency triggered by various stressors reactivate herpesviruses that are ubiquitous in the human population. We established a novel model of Herpes simplex virus 1 (HSV) latency in immunodeficient B6.Rag mice inoculated with either a low (LD) or high dose (HD) inoculum by using IVIG and acyclovir to suppress virus replication and promote latency. Remarkably, heat stress induced in vivo reactivation (IVR) of latent HD-Rag mice resulted in uncontrolled HSV replication and fatal encephalitis (HSE) in all mice compared to transient reactivation in LD-Rag mice that all survived. The major difference between the two groups was the massive invasion of inflammatory Ly6Chigh monocytes (IM) into the brainstem (BS) of latent HD-Rag but not LD-Rag mice. T cell transfer prevented IVR induced fatal HSE in HD-Rag recipients by reducing inflammation, especially IM invasion of the BS. Surprisingly, IVR of lethally irradiated latent HD-Rag and wildtype B6 mice induced only transient reactivation resulting in 100% survival; notably, IM were absent in the BS of these mice. Thus, counter intuitively, our data show that IM resident at sites of latency promote rather than suppress HSV reactivation and by modulating IM invasion into the BS, T cells control virus reactivation. This is a novel finding with important clinical implications, especially for treatment of neonates and immunosuppressed cancer patients undergoing transplant therapy.
CD4+ FoxP3+ regulatory T cells control effector T cell responses to murine cytomegalovirus (VIR10P.1169)
Authors: almanan maha , Raynor Jana , wang Mei , Chougnet Claire , cardin Rhonda , Hildeman David
Published: January 2023

Human cytomegalovirus causes a persistent, lifelong infection. The virus persists in a latent state and undergoes intermittent subclinical viral reactivation. Such reactivation drives the accumulation of “inflationary” T cells that suppress viral replication. While T cells are critical to maintain control of infection, the factors that prevent T cells from driving complete viral elimination remain unclear. Here, we investigated the role of regulatory T cells (Treg) in a mouse model of persistent CMV infection using FoxP3-diphtheria toxin receptor (DTR) mice. FoxP3-DTR mice were infected with murine CMV (MCMV) and then Treg were depleted via administration of DT several months later. Treg depletion resulted in a dramatic increase in the numbers of MCMV-specific CD4 and CD8 T cells in the spleen which functionally, produced significantly higher levels of IFN-γ and IL-2. Strikingly, Treg depletion led to a significant decrease in splenic viral load. We are initiating experiments using a spleen viral reactivation assay, to determine the effect of Treg depletion on latent virus. Combined, these data suggest that Treg promote persistent MCMV infection, in part, by suppressing T cell activation.
Localization of iNKT subsets determines lipid response (APP2P.101)
Authors: Wang Haiguang , Lee You Jeong , Hogquist Kristin
Published: January 2023

iNKT cells can be rapidly activated by α-galactosylceramide (α-GalCer), and α-GalCer or analogues of it are being tested for clinical use against certain cancers and autoimmune diseases. Recent studies in our lab showed that iNKT cells are comprised of NKT1, NKT2 and NKT17 effector subsets. In the current study, we asked whether these different iNKT subsets respond equivalently to α-GalCer using Nur77GFP transgenic mice to quantify the antigen specific response. We observed a robust response by NKT1, but not NKT2, in the spleen, despite in vitro stimulation showing they could be equivalently activated. Using an in vivo antibody-labeling technique, we found that NKT1 possess significantly higher accessibility to blood than NKT2, suggesting that NKT1 cells are preferentially localized to red pulp. This was confirmed by tetramer-based immunofluorescence studies. As expected, i.v. labeled iNKT cells showed a greater response than unlabeled cells. Thus, iNKT cells with accessibility to blood are the predominant cells to respond to lipid. Consistent with this, we observed a robust response of liver iNKT cells, and no response by lymph node iNKT cells, regardless of the subset. Because iNKT subsets produce distinct cytokines, their differential response to i.v. lipids is a critical consideration in therapeutic applications.
Increased PKC signaling in lupus La reactive B cells promotes development of the transitional T3 population and memory B cells in autoimmune BXD2 mice (BA3P.108)
Authors: Hamilton Jennie , Li Jun , Wu Qi , Yang PingAr , Luo Bao , Li Hao , Bradley John , Taylor Justin , Randall Troy , Mountz John , Hsu Hui-Chen
Published: January 2023

We recently identified that lupus prone BXD2 mice exhibited defective clearance of apoptotic debris. In vivo administration of excessive autologous apoptotic cells lead to elevation of IgG anti-La13-27 responses in BXD2 mice but not normal B6 mice, suggesting chronic self-antigen stimulation plus B-cell tolerance defects may be related to the IgG anti-La13-27 responses. To interrogate if there was abnormal maturation of anti-La13-27 B cells in naïve BXD2 mice, a recently developed La13-27 tetramer was used with CD80 and PD-L2 co-staining to reveal that there was a significantly higher frequency of memory CD80+PD-L2+ La13-27+ B cells in BXD2 (17%), compared to that in B6 (3%) mouse spleens. Tetramer analysis of CD93+ transitional B cells showed that there was an abnormal skewing of the La13-27+ T3 population, but not T1 or T2 B cells, in BXD2 (40%) compared to that in B6 (18%) mouse spleens. Survival of anti-La13-27 producing B cells in BXD2 but not B6 mice was associated with increased protein kinase C (PKC) activation, as stimulation of B cells with PMA+ionomycin which promotes survival of BCR stimulated transitional B cells resulted in an increase in IgG. As T3 B cells are considered anergic B cells maintaining self-tolerance through rapid turnover in vivo, the abnormal expansion of these B cells coupled with increased BCR signaling response in BXD2 mice may present a risk for B-cell tolerance loss to La13-27, leading to maturation into IgG secreting cells.
Lupus risk-variant increases pSTAT1 binding and decreases ETS1 expression (BA4P.137)
Authors: Lu Xiaoming , Zoller Erin , Weirauch Matthew , Wu Zhiguo , Namjou-Khales Bahram , Shen Nan , Greis Kenneth , Kaufman Kenneth , SLEGEN The , Harley John , Kottyan Leah
Published: January 2023

Genetic variants near gene ETS1 have been associated with Systemic Lupus Erythematosus (SLE) in independent cohorts of Asian ancestry. ETS1 is a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects using multi-ancestral cohorts, starting with genotyped variants and imputing to all common variants spanning the ETS1 locus. By constructing genetic models using frequentist and Bayesian association methods, we identified a set of 16 variants that are statistically most likely to be causal. We functionally assessed each of these variants based on their biological function predictions. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found increased binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 compared to the non-risk allele. Western blot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. eQTL analysis indicates that the risk allele of rs6590330 is only associated with decreased ETS1 expression in Han Chinese. We propose a model in which the risk allele of rs6590330 increases SLE risk by enhancing the binding of pSTAT1, resulting in repression of the ETS1 expression.
Alleviation of joint inflammation by lymphatic pump treatment of rats with adjuvant-induced arthritis (CAM1P.163)
Authors: Volin Michael , Zanotti Brian , Larimer Kaitlin , Gustafson Nicholas , Shah Pratik
Published: January 2023

Rheumatoid arthritis (RA) is characterized by synovial inflammation, cytokine production and lymph node collapse. Lymphatic pump treatment (LPT) is an osteopathic manipulative treatment used to reduce edema through increasing lymphatic circulation. This study examined the therapeutic potential of LPT on rats with adjuvant-induced arthritis (AIA). We hypothesize that LPT would lessen joint inflammation through increasing lymphatic drainage resulting in the reduction of cytokines in synovial space. AIA was induced in female Lewis rats and randomly placed into a LPT groups or sham groups. Experiments were repeated using different LPT protocols varying the frequency and number of LPT treatments. However in each experiment LPT consisted of rhythmically pressing below the rib cage once a second for 30 seconds, while the sham group was held for 30 seconds. Joint inflammation was determined by measuring ankle circumferences and by articular index scoring. Ankles were collected and homogenized to determine changes in synovial cytokine levels by magnetic immunoassays and ELISA. Clustering LPT treatments three-times a day at the peak of joint inflammation had the greatest impact on arthritis. Animals treated with LPT in this manner exhibited significantly reduced ankle edema and inflammation. Additionally, synovial levels of inflammatory cytokines (GRO, IL17, and IL-6) were reduced with LPT. These results indicate that LPT can reduce joint edema and inflammation in a rat model of RA.
A computational model for mechanistic investigation of negative feedback in interleukin-17 receptor signaling (CCR3P.201)
Authors: Sheehan Robert , Garg Abhishek , Gaffen Sarah , Faeder James
Published: January 2023

IL-17 is a pro-inflammatory cytokine that promotes autoimmunity and protects against certain pathogens. Here, we present a computational model of signaling downstream of the IL-17 receptor. To manage the complexity of the system we use a rule-based modeling approach, in which signaling proteins are modeled as structured objects and rules describe their biochemical interactions, allowing us to consider all possible complexes and phosphoforms generated from a set of basic molecules. This model encompasses major signaling components downstream of IL-17R, which activate NF-κB and in turn promote the production of pro-inflammatory cytokines. NF-κB also promotes production of A20, a deubiquitinating enzyme that inhibits NF-κB activation in multiple settings. We recently showed that A20 acts as a negative regulator of the IL-17 signaling pathway. By modeling A20 inhibition of TRAF6 and IKK, we are able to recapitulate experimentally-observed oscillations in NF-κB activity and A20 expression. Novel experimental data identifying the dynamics of signaling intermediates has further constrained model behavior and informed model development. Modeling the depletion of IκB, followed by its rebound above baseline levels, required the addition of a stochastic model of transcription factor binding, paired with a deterministic model of signaling events. We are currently using the hybrid model to identify which of several potential mechanisms A20 uses to attenuate IL-17 signaling.
The introduction of RNA-DNA differences underlies inter-individual variation in the human IL12RB1 mRNA repertoire (CCR3P.204)
Authors: Robinson Richard
Published: January 2023

IL12 and IL23 promote numerous aspects of human immunity, including autoimmunity and mycobacterial disease resistance. IL12RB1 encodes human IL12Rβ1, a surface receptor that signals in complex with IL12Rβ2/IL23R to relay the pro-inflammatory signals of IL12/IL23. Here, we present data demonstrating that in primary human T-lymphocytes, sequence diversity reminiscent of TCRA/TCRB diversity is introduced into IL12RB1 mRNAs via RNA DNA differences (RDDs). RDDs are ribonucleotides present in an mRNA that are not genome encoded; as such they are distinct from genomic single nucleotide polymorphisms. First observed in protozoa, RDD-introduction is now known to be a mechanism employed by many species to suppress protein function. In the case of IL12RB1, we show that in T-cells of healthy individuals the majority of IL12RB1 mRNAs expressed contain RDDs that concentrate in exons encoding IL12Rβ1’s cytokine binding region. IL12RB1 RDDs comprise multiple mutation types, are detectable by both Ion Torrent sequencing and classic Sanger sequencing, and decrease in frequency following T-cell activation. As a result of RDD-introduction, IL12Rβ1 proteins have an altered amino acid sequence that could not be predicted based on genome sequencing alone. While the underlying mechanisms of sequence diversification differ for TCRA/TCRB and IL12RB1, the consequence of this diversification is the same - the expression of proteins of variable amino acid sequence and variable immunological function.
Chromodomain helicase DNA-binding 4 is required for proliferation, distal VH rearrangements and developmental progression of B cell progenitors (HEM1P.221)
Authors: Hagman James , Dege Carissa , Straign Desiree , Jia Haiqun , Walton Kendra , Lukin Kara , Lei Hong , Danhorn Thomas , Feeney Ann
Published: January 2023

Chromodomain Helicase DNA-binding protein 4 (CHD4, or Mi-2β) is a catalytic core subunit of Nucleosome Remodeling and Deacetylase (NuRD) complexes, which regulate chromatin structure and transcription in lymphocytes. CHD4 activities include ATP-dependent mobilization of nucleosomes, DNA binding and binding to histone tails. Here, we investigated requirements for CHD4 in B cell development in a mouse model system. We utilized Chd4flox/flox:Cd79a-Cre (Chd4 cko) mice, which inactivate Chd4 genes selectively in early B cell progenitors. These mice confirmed that CHD4 is essential for B lymphopoiesis. Following the loss of CHD4 expression, B cell development is arrested at the pro-B cell stage. Peripheral B220+ cells were nearly absent. To address the basis of the observed developmental arrest, we measured effects of the lack of CHD4 on proliferation and Igh gene rearrangements. CHD4-deficient pro-B cells fail to proliferate in response to IL-7. Furthermore, pro-B cells lacking CHD4 complete proximal VH to DJH rearrangements, but rearrange distal VH segments only rarely. Overall, our data demonstrate that CHD4 and NuRD complexes are essential for multiple aspects of early B cell development, including V(D)J recombination, proliferation and survival of pro-B cells.
The T cell receptor alpha/delta locus adopts distinct contracted conformations during primary and secondary T cell receptor α rearrangement (HEM2P.242)
Authors: Carico Zachary , Krangel Michael
Published: January 2023

In DP thymocytes, primary TCRα rearrangements are restricted to proximal Vα segments at the 3’ end of the Tcra/d locus. We have shown that this restricted Vα usage is supported by a 3’ contracted and 5’ decontracted conformation of Tcra/d. Secondary rearrangement, however, generates a far more diverse pool of Vα-Jα pairings. To investigate the structure the 5’ end of the locus might adopt after primary rearrangement, we performed 3D-FISH on Tcra/d alleles engineered to mimic a natural TRAV17-TRAJ57 rearrangement. We found that the artificially rearranged Tcra/d alleles were markedly contracted at the 5’ end relative to unrearranged Tcra/d. Notably, we observed a similar 5’ end contraction of the locus in CTCF-deficient DP thymocytes with unrearranged Tcra/d. This led us to hypothesize that loss of a critical CTCF binding site as a result of primary rearrangement might promote the 5’ end contraction. Knockout of the T early alpha promoter (TEA), but not other CTCF-bound elements in the region deleted by primary rearrangement, resulted in 5’ contraction of Tcra/d. These data suggest that diversity in secondary TCRα rearrangements is supported by contraction of the whole length of Tcra/d, and that CTCF binding by TEA plays a critical role in enforcing a 5’ decontracted conformation for primary rearrangement. Using 4C, we are now working to understand specific changes in long-range DNA contacts between pre- and post-rearrangement alleles.
IL10 mediates the protective effect of voluntary exercise on stroke-induced injury (HUM1P.255)
Authors: Selvaraj Uma Maheswari , Poinsatte Katherine , Ortega Sterling , kong xiangmei , Plautz Erik , Noorbhai Ibrahim , Stowe Ann
Published: January 2023

Previous data show that voluntary exercise prior to stroke reduces neuronal injury in wild type (WT) mice. Our lab found that exercise specifically modulates B cells to upregulate IL10, which could reduce post-stroke inflammation. To determine the influence of IL10 on exercise-mediated neuroprotection, IL-10-null mice (male, 12-16 weeks old) were randomly separated into sedentary and voluntary exercise (3 weeks on monitored running wheels) groups. Both groups were subjected to a 60 minute transient middle cerebral artery occlusion, with infarct volumes and splenic immune populations assessed 24 hours after stroke by a blinded observer. Voluntary exercise (n=11) did not reduce infarct volume compared to sedentary mice (n=8) in the absence of IL-10. Exercise increased B2 cell (CD1d-CD5-; p<0.05) and B regulatory (Breg) cell (CD1d+CD5+; p=0.08) populations in the spleen. Mice with the highest exercise intensity exhibited both the lowest splenic Breg populations (p<0.05) and the lowest infarct volumes (p=0.09). This would suggest an IL10-independent mobilization of Bregs by exercise prior to stroke, though future studies should determine if this neuroprotective Breg population moves into the ischemic brain to reduce neuronal injury.
Alarmin HNPs 1-3 predicts theonset of lung injury after cardiac surgery in children (HUM1P.259)
Authors: Liu XiWang , Chen QiXing , Lai Dengming , Qin ChaoJin , Fang XiangMing , Shu Qiang
Published: January 2023

Introduction:Human neutrophil peptides (HNPs) 1-3 are endogenous mediators to be characterized as alarmins. Methods:Children younger than 3 years after cardiac surgery were prospectively enrolled. Plasma concentrations of HNPs 1-3 and inflammatory cytokines were measured by ELISA during and after operation. Results: Thirty patients were enrolled which included 18 (60%) infants. Plasma HNPs 1-3 and the pro-inflammatory cytokine IL-6 levels significantly increased immediately after CPB (P < 0.001), while IL-8 increased 1 hour after operation (P = 0.002). The anti-inflammatory cytokine IL-10 levels immediately after CPB were also significantly elevated compared with baseline (P < 0.001). The multiple linear regression analysis showed that the plasma HNPs 1-3 levels immediately after CPB was independent correlated with declined lung function, as reflect by the PaO2/FiO2 ratio on the first 2 days after operation (for the first day: OR, -1.067, 95% CI, -0.548 to -1.574; P < 0.001; for the second day: OR, -0.667, 95% CI, -0.183 to -1.148; P = 0.009) and prolonged mechanical ventilation time (OR, -0.039, 95% CI, -0.005 to -0.056; P = 0.022). Plasma levels of HNPs 1-3, IL-8 and IL-10 returned to normal values, while IL-6 levels remained significantly higher than baseline 24 hours after operation (P < 0.001). Conclusions: Elevated HNPs 1-3 levels correlate with impaired lung function, and HNPs 1-3 is a useful early alarmin biomarker for lung injury inchildren after cardiac surgery.
Inhibition of fatty acid oxidation blocks asthma in mice (HYP2P.340)
Authors: Al-Khami Amir , Ghonim Mohamed , Pyakurel Kusma , Ibba Salome , Trillo-Tinoco Jimena , Hossain Fokhrul , Del Valle Luis , Rodriguez Paulo , Boulares Hamid , Ochoa Augusto
Published: January 2023

Background: There has been a growing appreciation for the role of energy metabolic pathways in the differentiation and function of immune cells. Unpublished findings from our laboratory suggest that inhibiting fatty acid oxidation (FAO) modulates the activation and function of tumor-infiltrating myeloid cells, indicating a potentially important role for FAO in inflammation and thus inflammatory diseases such as asthma. Objective: We sought to test the hypothesis that FAO plays an important role in asthma manifestation. Methods: Etomoxir, an inhibitor of the FAO rate-limiting enzyme carnithine palmitoyltransferase-1, was used to test our hypothesis in an ovalbumin-based murine model of asthma. Results: Etomoxir treatment thirty minutes after ovalbumin challenge resulted in a significant reduction in the recruitment of eosinophils and macrophages into the lungs without a prominent effect on the total number of lymphocytes. Etomoxir treatment also prevented ovalbumin-induced hyperresponsiveness. The protective effects of etomoxir were associated with a decrease in Th2 cytokines and ovalbumin-specific IgE production. Additionally, the frequency of splenic macrophages was lower in etomoxir-treated mice, compared with control. Conclusions: Our preliminary data suggest the inhibition of FAO as a likely strategy to block asthma traits. As such, we are currently uncovering the mechanisms by which FAO inhibition reduces the pathogenesis of asthma.
ICOS: ICOS-ligand interaction is essential for ILC2 function and survival (HYP2P.322)
Authors: Maazi Hadi , Patel Nisheel , Sankaranarayanan Ishwarya , Riagas Diamanda , Soroosh Pejman , Freeman Gordon , Sharpe Arlene , Akbari Omid
Published: January 2023

Type 2 Innate lymphoid cells (ILC2s) are a newly identified subset of immune cells that play important roles in the pathogenesis of allergic asthma by producing large amounts of IL-5 and IL-13. ILC2 lack lineage markers but express CD45, IL-2Rα, IL-33R and IL-7Rα. All murine ILC2s and a significant portion of human ILC2s express Inducible T-cell COStimulator (ICOS) that is essential for T cell activation and function, however, the role of ICOS in ILC2s remains unknown. We investigated the role of ICOS in the function and survival of murine and human ILC2s using ICOS-/- and RAG2-/- and humanized mice in different experimental setups. We found that:1) ICOS-/- mice show lower IL-33-induced airway hyperreactivity (AHR) and eosinophilia than wild type (WT) mice. 2) Survival and the number of ILC2s is substantially lower in ICOS-/- than WT mice. 3) Blocking anti-ICOS antibody reduces survival and the number of ILC2s in WT mice. 4) Ex vivo stimulated ICOS-/- ILC2s show impaired IL-5 and IL-13 production compared to WT ILC2s. 5) Human peripheral ILC2s express ICOS and blocking ICOS:ICOS-Ligand interaction impairs their IL-5 and IL-13 production in vitro and IL-33 induced AHR and eosinophilia in humanized mice. 6) Human and murine ILC2s express ICOS-Ligand. 7) ICOS signaling modulates STAT5 pathway. Our results indicate that ICOS is required for the optimal function and survival of ILC2s and can be a novel therapeutic target in patients with ILC2-mediated asthma and lung inflammation.
Cell membrane stabilization prevents the natural IgM-mediated complement response to hypoxia in human coronary artery endothelial cell (INC1P.359)
Authors: Zhang Ming , Lee Haekyung
Published: January 2023

Hypoxia occurs in many clinical conditions, including ischemic diseases (e.g. heart attack, cerebral stroke and pulmonary hypertension), surgery (e.g. organ transplantation) and trauma. The goals of this study were to determine whether natural IgM-mediated complement activation occurs during hypoxia of human tissue prior to reoxygenation and whether pre-incubation with the sugar trehalose, a membrane stabilizer, can prevent hypoxia-related injury. Primary human coronary artery endothelial cells (hCAECs) were used in two in vitro hypoxia models. Subjection of hCAECs to hypoxia, without subsequent reoxygenation, was found to produce IgM deposition and C3 activation and deposition. Trehalose blocked these effects, indicating that cell membrane instability due to hypoxia is upstream of natural IgM-mediated complement activation. The results indicate that trehalose has potential as a therapeutic intervention in hypoxia-related tissue injury.
Hepatitis C virus-infected Huh7.5 cell reduces NK cell functions via HCV’s serine protease NS3/4A (INC2P.408)
Authors: Yang Chang Mo , Park Jeon Han , Lee Jae Myun
Published: January 2023

Hepatitis C virus (HCV) infection is characterized by high frequency of chronic cases and impairment of innate and adaptive immune responses are the causes. Innate immune response impairment may involve modulating NK cell functions. However, NK cell functions’ impairment mechanism in the early phase of HCV infection and viral proteins’ roles are controversial. Therefore, we investigated which protein of HCV modulates NK cell functions. Human hepatoma Huh7.5 cells were infected with cell culture-generated HCV virions and cocultured with ex vivo NK cell. Cell-to-cell contact between NK cell and infected cells significantly reduced NK cell’s natural cytotoxicity and IFN-γ production. We also assessed effects on NK cell functions using HCV’s replicon cells. These replicon cells affected NK cell functions’ reduction. Whereas, we observed the restoration of NK cell functions when contacted with infected Huh7.5 cells, which were treated with HCV NS3/4A serine protease inhibitor, BILN-2061. The capacity of NK cell to degranulation against K562 and IFN-γ production level was increased significantly compare to infected Huh7.5 cells. These restored functions of the NK cell, correlated with some activating receptors expressed on NK cell surface, especially NKp46 expression level. Our observations suggest that HCV’s serine protease role in NS3/4A protein responsible for abrogation of the NK cell functions in the early phase of infection through downregulation of NKp46 receptor on NK cell.
Involvement of IL-1β on cognitive impairment after experimental pneumococcal meningitis. (INM1P.432)
Authors: Barichello Tatiana , Generoso Jaqueline , Simoes Lutiana , Ceretta Renan , Dominguini Diogo , Carvalho Vilela Márcia , Comim Clarissa , Lucio Teixeira Antonio , Quevedo Joao
Published: January 2023

Pneumococcal meningitis is associated with high rates of mortality and long-term cognitive impairment. In the current study, we evaluated the effects of an interleukin (IL)-1β receptor antagonist (IL-1Ra) on memory, cytokine, and BDNF levels in experimental pneumococcal meningitis. Adult Wistar rats were divided into four groups: control/saline, control treated with IL-1Ra, meningitis/saline and meningitis treated with IL-1Ra. The animals were killed at 24 h post-infection. In the meningitis/saline group IL-1β and CINC-1 levels increased at 24 h post-infection; however, treatment with IL-1Ra reversed these increases in the hippocampus. In a second experiment, the rats received antibiotic and were subjected to behavioural tasks (open field, step-down inhibitory avoidance and object recognition), cytokine and BDNF levels ten days after meningitis induction. In the open-field task, the meningitis group did not exhibit differences in motor and exploratory activity, indicating habituation memory impairment. The same impairment occurred in the novel object recognition memory and step-down inhibitory avoidance task (aversive memory) in the meningitis group; however, adjuvant treatment with IL-1Ra prevented memory impairment. The IL-4, IL-6, IL-10 and BDNF levels were increased in the meningitis/saline and meningitis/IL-1Ra groups. These findings illustrate a neuroprotective role of IL-1Ra inhibitor and the involvement of IL-1β on cognitive impairment after pneumococcal meningitis.
In vivo plasma LPS levels in HIV negative subjects alter T cell proliferative responses, stimulate cytokine release and induce TLR cross tolerance in monocytes ex vivo (IRC4P.604)
Authors: Palmer Christine , Romero-Tejeda Marisol , Sirignano Michael , Sharma Siddhartha , Allen Todd , Altfeld Marcus , Jost Stephanie
Published: January 2023

Multiple studies have shown correlates of immune activation with microbial translocation and plasma lipopolysaccharide (LPS) levels during HIV-1 infection. It is unclear whether this activation is solely due to LPS, residual viral replication, or both. LPS and other pathogen-associated molecular patterns (PAMPs) are recognized by Toll-like receptors (TLRs). Binding of PAMPs to TLRs can alter subsequent responses by inducing self/cross-tolerance or priming. These effects of can alter immune functions in the context of chronic LPS exposure. Few studies have addressed the effects of chronic in vivo levels of LPS on specific immune functions in humans in the absence of chronic viral infection. We previously reported on a cohort of HIV negative high-risk men who have sex with men with elevated plasma LPS levels. This cohort allowed us to assess cellular immune functions in the context of plasma LPS levels ex vivo without confounding viral effects. Using 2 time points with different plasma LPS levels, we now show that in vivo plasma LPS levels altered ex vivo cytokine expression by monocytes, without stimulation and in response to several TLR ligands. We further showed that high levels of plasma LPS in vivo delayed early T cell proliferative responses in vitro, whereas intermediate plasma LPS levels enhanced T cell proliferation. These data suggest that increased levels of plasma endotoxemia can have profound effects on immune function.
IL-10 inducing bacteria are preferentially depleted in the gut of HIV infected subjects (IRC4P.607)
Authors: Neff Charles , Rhodes Matthew , Donnelly Jody , Schneider Jennifer , Mazmanian Sarkis , Lozupone Catherine , Palmer Brent
Published: January 2023

Chronic HIV infection is associated with a dramatic change in gut microbiota composition, characterized by a drop in the Bacteroides genus and increase in Prevotella genus. Bacteroides fragilis, which is depleted during HIV infection, interacts with CD4+ T cells and induces IL-10 producing T regulatory cells (Tregs) using polysaccharide A (PSA). We hypothesize that massive depletion of CD4+ T cells during HIV infection results in the inability of B. fragilis to induced IL-10 producing T regulatory cells and persist in the gut. To characterize whether other bacteria depleted during HIV infection also produce PSA we screened genomes of bacteria for the presence of close homologues to essential genes in the PSA operon. Genome screening for PSA-like gene elements showed that many of the species of bacteria predicted to encode for PSA decreased with HIV infection. Stimulation of PBMC and Lamina Propria Mononuclear Cells (LPMC) with these bacteria demonstrated that compared to non-predicted PSA producers, PSA producing bacteria induced more Tregs and higher levels of IL-10. These data suggest that the compositional changes seen in HIV infection may be in part a result of the inability of PSA producing bacteria to stimulate IL-10 producing Tregs critical for damping innate immune responses and allowing colonization. The loss of these IL-10 inducing gut bacteria may also contribute to the generalized inflammatory state and chronic immune activation seen in HIV infected subjects.
Glutamate ionotropic receptor (NMDAR) modulates T cell survival (IRM7P.712)
Authors: Peron Jean Pierre , Brandão Wesley Nogueira , Rossato Cristiano , Camara Niels Olsen
Published: January 2023

Introduction: T cells play a crucial role in adaptive immune responses and neuroinflammatory diseases such as multiple sclerosis. Glutamate is one of the most abundant neurotransmitter in the central nervous system; it can acts through two different receptor families: metabotropic and ionotropic. It has been demonstrated that T cells can be the target of glutamate during antigen presentation and immunological synapses, however, little is known about its role on T cell survival. Methods and Results: To evaluate whether NMDAr ligands exert a role in T cell survival, we harvested spleen cells from C57BL/6 female mice and cultivated in the presence of anti-CD3 (1µg/mL) in vitro for 5 days. On day 5, cells were stained for CD4+/CD8+ and 7-AAD and data were acquired by flow cytometric analysis. NMDAr blockage by MK801 induced cell death while NMDA agonist did not affect T proliferation and T cell numbers. Conclusion: Our data demonstrate that NMDAr exert a crucial role in T cell survival. These findings not only contribute to the advantage of the neuroimmunology field but also suggest that T cells which infiltrate the central nervous system could be target of the neurotransmitter glutamate via NMDAr in neuroinflammatory diseases.
Novel interactions between IL17 and IFNγ signaling mediate native host defense to Staphylococcus (MPF2P.753)
Authors: Barin Jobert , Talor Monica , Gebremariam Elizabeth , Schaub Julie , Miller Lloyd , Rose Noel , Cihakova Daniela
Published: January 2023

In these studies, we characterize a novel spontaneous inflammatory disease that develops in the concurrent absence of the shared IL17 receptor subunit IL17RA, and the receptor for IFNγ. As part of previously published studies on convergent signaling between IFNγ and IL17, we generated double-knockout mice, deficient in both IL17RA and IFNγ. We observed large purulent abscesses developing in the IL17RA-/-IFNγRI-/- double-knockout strain, but neither of the parental single-knockout controls. Chronic mucocutaneous abscesses developed spontaneously in both sexes, in the buccal mucosa and the skin of the forelimb, and were found to contain uncontrolled Staphylococcal outgrowth. Histopathologic examination of the oral abscesses and flow cytometric enumeration of these tissues demonstrated extensive neutrophilic infiltration of the surrounding tissues. Interrogation of circulating immunoglobulins revealed elevated serum IgE in IL17RA-/-IFNγRI-/- double-knockout animals, positioning their disease as a novel model of features of Hyper IgE syndrome (Job’s syndrome).
The role of B cell-intrinsic gp96 /grp94 in sepsis (MPF2P.747)
Authors: Liu Bei , Fan Hongkuan
Published: January 2023

gp96, also known as grp94, is a master protein chaperone for Toll-like receptors (TLRs) and integrins in the endoplasmic reticulum (ER) and plays critical roles in innate immunity. However, the role of B cell-intrinsic gp96 in sepsis has not been studied. Sepsis is a clinical syndrome caused by a severe immune response to infection. B cell has been implicated in protecting against bacterial sepsis. We addressed this question using a unique B cell specific gp96 knockout mouse model. KO mice do develop mature and functional B cells, in spite of defects of gp96 null B cells in integrin-dependent migration and failure to proliferate in response to multiple TLR ligands such as Pam3 and LPS. We challenged WT and KO mice with a lethal dose of LPS, and we found that WT mice produced significantly more serum TNF-α, IL-6 and IL-12 than did KO mice. Consistent with the detrimental effect of these cytokines in sepsis, we found that the WT mice were more susceptible to LPS-induced sepsis than the KO mice. By day 2, 100% WT mice succumbed to endotoxemia. In contrast, 40% of KO mice survived to the same dose of LPS challenge. In addition, using a well-defined sepsis model of cecal ligation and puncture (CLP), we demonstrated that KO mice were unable to mount effective response against polymicrobial sepsis, underscoring the importance of B cells in innate immunity. This study indicates that B cell-intrinsic TLR signaling contributes to sepsis-induced B cell activation and outcome of sepsis.
Altered gastrointestinal environment and immune cellular plasticity during disease progression of type 1 diabetes (MUC2P.931)
Authors: Wallet Shannon , Hulme Maigan , Nelson Michael , Graves Christina , Amador Byron , Sorenson Heather , Brusko Todd
Published: January 2023

The gastrointestinal (GI) tract is the largest environmental interface which is a barrier to environmental insults and a site of immune activation and tolerance induction. The initiating events leading to the loss of self-tolerance which results in the autoimmune process of type1 diabetes (T1D) have yet to be fully defined. Leakiness at the GI barrier has been observed preceding symptomatic onset of T1D. Using the non-obese diabetic (NOD) mouse, we assessed measures of barrier integrity, global intestinal inflammation, as well as the frequency of gastrointestinal and pancreatic innate lymphoid (ILC), regulatory and inflammatory cell subsets at key checkpoints of disease to determine kinetics associated with the loss of tolerance. Early in the disease process, NOD mice exhibited minor losses in barrier function along with elevated levels of duodenal TNFα and IFNɣ. Concomitant with an increased frequency of inflammatory T cells and reduced frequency of regulatory T cells, NOD mice also presented with an increased frequency of type 3 ILCs and a decreased frequency of type 1 ILCs within the mesenteric lymph node (MLN) at all stages of disease and in the pancreatic lymph node (PLN) at 8 wks of age. Overall, these data support a model wherein early in disease, minor changes in barrier function can result in intestinal inflammation shifting the balance of immunoregulation to that of inflammation potentially resulting in the loss of self-tolerance responsible for T1D.
CD27 co-stimulation controls tissue-specific memory CD8 T cell formation. (MUC3P.949)
Authors: Sowell Ryan , Rogozinska Magdalena , Marzo Amanda
Published: January 2023

Memory CD8 T cells comprise niches of circulating cells and non-circulating tissue residents. The localization of memory cells defines phenotypic and functionally distinct populations. However, the mechanisms that direct tissue-specific T cell responses remain unclear. The co-stimulatory molecule CD27 is expressed on naïve cells and memory cells in secondary lymphoid and peripheral tissues. In response to virus infection, CD27 signals enhance the magnitude of effector CD8 T cells and their capacity to respond to subsequent pathogen challenge. Moreover, CD27 may actively maintain memory CD8 T cells in secondary lymphoid tissues. We previously reported that memory cells downregulate CD27 upon entry into peripheral tissues and acquire increased lytic potential. Thus our aim was to characterize the importance of CD27 signals during the formation of tissue-resident CD8 T cells. We determined that CD27 is critical for the generation of memory cells in secondary lymphoid tissues, however it is dispensable for tissue-resident memory cells. The defect was partially dependent on mTOR because administration of rapamycin during effector differentiation rescued memory formation in absence of CD27, despite no detectable effect on the magnitude of the effector response. Analysis of CD27low cells revealed functions distict from CD27hi memory cells. Downregulation of CD27 upon entry into the periphery may be an important event that enables CD8 T cells to take up tissue residence.
Contribution of MyD88-dependent PKD1 to the development of arthritis (THER2P.954)
Authors: Yi Ae-Kyung , Yoon Tae Won , Cho Hongsik , Brand David , Kim-Hoehamer Young-In , Park Jeoung-Eun , Hasty Karen , Stuart John
Published: January 2023

Rheumatoid arthritis (RA) may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of TLR signaling may provide an opportunity to halt the RA process in the earliest stages. We investigated whether protein kinase D1 (PKD1), which is essential for proinflammatory responses mediated by MyD88-dependent TLRs, plays a role in the development of experimental arthritis. The presence of TLR ligands in joint cavities induces development of reactive arthritis that is characterized by accumulation of activated inflammatory cells in the joints. Deletion of PKD1 in myeloid lineage cells prevented TLR-mediated accumulation of inflammatory cells in joints, thus protecting mice from developing reactive arthritis. Arthritis spontaneously occurring in IL-1R antagonist-deficient (IL-1rn-/-) mice was dependent on MyD88, and suppression of PKD1 expression resulted in inhibition of TLR-mediated cytokine production in IL-1rn-/- macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn-/- mice. In addition, a PKD inhibitor inhibited the synergistic effect of T cell receptor and TLR2 on IL-6 production in splenocytes, and significantly reduced the incidence and severity of collagen-induced arthritis that closely mimics the pathophysiologic process of human RA. This implies that PKD1 might be one of the key factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.
Desmoglein 3 chimeric autoantibody receptor T cells: a novel strategy for immunotherapy of pemphigus vulgaris (THER2P.958)
Authors: Ellebrecht C , Cho Michael Jeffrey , Mao Xuming , Bhoj Vijay , Tsai Ching-Yi , Nunez-Cruz Selene , Milone Michael , Payne Aimee
Published: January 2023

Pemphigus vulgaris (PV) is a blistering skin disease caused by autoantibodies (autoAbs) to desmoglein (Dsg) 3. PV therapy relies on general immunosuppression, risking severe infection with chronic use. Recently, chimeric antigen receptor (CAR) T cells have revolutionized immunotherapy. To evaluate this potent approach for PV, we engineered a series of Dsg3 chimeric autoAb receptors (CAARs), consisting of various Dsg3 extracellular (EC) domains fused to either T or NK receptor signaling domains. In vitro, Dsg3 CAAR T cells specifically kill target cells expressing surface anti-Dsg3 EC1, EC2, or EC3 autoAbs, with the most broad and potent cytolysis by the Dsg3 EC1-4 CAAR using the T cell CD137CD3ζ signaling domain (p=0.0001). Dsg3 CAAR T cells do not kill Fc-receptor+ cells coated with Dsg3 autoAbs or keratinocytes whose surface proteins might interact with the Dsg3 CAAR. TIRF microscopy reveals that autoAb binding by the Dsg3 CAAR forms supramolecular activation complexes, with segregation of the CAAR away from CD45, a regulator of T cell receptor activation. In a preclinical PV NSG mouse model, bioluminescent AK23 (anti-EC1 surface IgG+) hybridoma cells were efficiently killed by Dsg3 CAAR (p=0.0015), resulting in prolonged survival of CAAR-treated versus control mice (p=0.016) without skin toxicity. CAAR T cells represent a novel strategy for PV therapy that specifically targets autoantigen-specific B cells and that could readily be applied to other autoAb-mediated diseases.
CoenzymeQ10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice (THER3P.1000)
Authors: Seo Hyeon-Beom , Cho Mi-La , Jeong Jeong-Hee
Published: January 2023

Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA.