bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of granulysin in MDP-induced PGE2 production in human monocytes (68.2)
Authors: Endo Yukinori , Zaitseva Marina , Romantseva Tatiana , Golding Hana
Published: January 2023

Muramyl dipeptide (MDP) is a NOD2 agonist known to induce pyrogenic response in vivo in humans and in animal models. The mechanism of MDP-induced fever was studied in vitro in human primary cells by measuring PGE2, a final mediator of febrile response. Elutriated monocytes produced significant levels of PGE2 following stimulation with LPS but not MDP. However, addition of human CD3 bead-purified T cells significantly augmented COX2 mRNA expression and PGE2 protein production in MDP-activated monocytes. PI3 kinase inhibitor LY294002 and ROS inhibitor DPI all inhibited PGE2 production and COX2 mRNA up-regulation. Studies using trans-well system confirmed that PGE2 production in MDP-activated monocytes did not require direct cell to cell contact between monocytes and T cells. Anti-IFN gamma antibodies did not inhibit the effect of T cells suggesting that factor(s) other than IFN gamma play a role in MDP-induced PGE2 production in monocytes. Jurkat cells did not deliver the missing factor required for PGE2 production in MDP-activated monocytes. cDNA microarray analysis revealed high levels of Granulysin (GNLY) mRNA expression in bead-purified T cells but not in Jurkat cells. Recombinant GNLY induced high levels of PGE2 in monocytes activated with MDP and the effect of GNLY was sensitive to ROS and PI3K inhibitors. In conclusion, our data suggest that GNLY might be one of the molecules that are produced by T cells and contribute to PGE2 production by MDP-activated monocytes.
Molecular mechanisms of Innate DNA recognition by the AIM2 and IFI16 inflammasomes (68.4)
Authors: Jin Tengchuan , Perry Andrew , Jiang Jiansheng , Smith Patrick , Curry James , Unterholzner Leonie , Jiang Zhaozhao , Horvath Gabor , Rathinam Vijay , Hornung Veit , Johnstone Ricky , Latz Eicke , Bowie Andrew , Fitzgerald Katherine , Xiao T.
Published: January 2023

Structures of The HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of The AIM2 Inflammasome and IFI16
FLAGELLIN-DEPENDENT AND INDEPENDENT INNATE IMMUNE RESTRICTION OF LEGIONELLA PNEUMOPHILA INFECTION IN VITRO AND IN VIVO (68.5)
Authors: Lima Jonilson , Pereira Marcelo , Zamboni Dario
Published: January 2023

Recognition of L. pneumophila flagellin by Naip5-Nlrc4 inflammasome is required for innate immune restriction of the infection in restrictive mice background. Therefore, regardless to the mice background, flaA- mutant bacteria can be used to screen for important components of the immune system that account for growth restriction. flaA- bacteria were used to systematically infect mice and macrophages deficient for: 1) molecules important for effectors mechanisms of immune response (IFNγ, IL-6, 10, 12, 17, 18, 23, MIP-1α and TNFRp55); 2) cells of immune response (CD4KO-MHCII-/-, CD8KO-β2m-/-, BKO and neutrophils-anti-LY6G); 3) transmembrane innate immune receptors (CCR2, CCR4, Fas/Fas-L, ST2-, TLRs-); and 4) intracellular signaling molecules (PI3k, MyD88, Nod1, Nod2 and Rip2). Our data shows that mechanisms related with innate immune response, but not adaptive response are important for bacterial growth restriction in vivo and in vitro. Activation of these responses are critical for bacterial growth restriction and resolution of pulmonary infection in a murine model of Legionnaires’ disease.
The role of SIRT1 in the activation of innate immune responses during RSV infection (68.6)
Authors: Owczarczyk Anna , Petersen Bryan , Schaller Matthew , Reed Michelle , Demoor Tine , Mukherjee Sumanta , Ptaschinski Catherine , Morris Susan , Lombard David , Lukacs Nicholas
Published: January 2023

Respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract infection in infants and children worldwide. Dendritic cells (DC) prime the adaptive immune response by recognizing and delivering viral antigens to local lymph nodes, presenting antigen to T cells, displaying co-stimulatory markers, and releasing cytokines. Recent studies have linked macroautophagy to intracellular pathogen recognition and the activation of DCs leading to enhanced antigen-presenting cell (APC) function. SIRT1, a NAD+ dependent deacetylase, acts on many cellular substrates, including autophagy proteins (Atg), as shown with Sirt1-/- mice that accumulate damaged organelles similar to Atg5-/- mice. Our study investigates the role of SIRT1 in DC maturation, subsequent T cell activation, and RSV-induced pulmonary pathology. RSV infected bone marrow-derived DCs (BMDC) pre-treated with EX-527, a SIRT1 inhibitor, downregulate expression of key cytokines, MHCII, and CD86. EX527 treatment of RSV- infected wild-type mice resulted in increases in lung mucus production, viral load, and TH2 cytokine production. Likewise, EX527 significantly antagonizes TLR7-induced cytokine release in RSV-infected BMDCs, suggesting a link with the TLR/PAMP recognition response. These results suggest that SIRT1 may be a critical determinant in the innate immune response and resultant pulmonary pathology during RSV infection.
The role of nucleotide-binding oligomerization domain 2 (Nod2) in initiation of rheumatoid arthritis. (68.7)
Authors: Franca Rafael , Peres Raphael , Talbot Jhimmy , Cunha Thiago , Cunha Fernando
Published: January 2023

The etiology of several inflammatory diseases such as rheumatoid arthritis (RA) remains unknown. Some studies including data from our group, report that the receptor NOD2, a member of NLR family, is overexpressed in synovial fibroblasts of patients with RA. Since NOD2 activation might contribute to the initiation and perpetuation of chronic destructive inflammation in RA, we decided to investigate the role of this receptor in arthritis. Initially, WT and NOD2-/- mice were immunized with mBSA, and arthritis was induced by an i.a. injection of mBSA. Our data shows that knockout NOD2 mice did not develop arthritis, as evidenced by the low number of infiltrating cells into the articular cavity, and that transfer of wild type neutrophils into NOD2-/- mice did not restored the inflammatory status. Additionally, to assess if synovial fluid of RA patients could activate NOD2 in vitro, HEK293-Luciferase-NF-κB cells were transfected with a plasmid encoding the NOD2 receptor, 24 hours later these cells were stimulated with MDP or synovial fluid in different concentrations, osteoarthritis synovial fluid were used as control. Luciferase activity was measured 8 hours after stimulation. We observed that stimulation of NOD2 transfected cells with synovial fluid from RA patients leads to increased luciferase activity, suggesting the NF-κB activation dependent of NOD2 signaling in HEK293 cells. Together, these results suggest that NOD2 receptor has an important role in the pathogenesis of RA.
Characterization of the functional role of VAMP8 in granule exocytosis of T lymphocytes (69.12)
Authors: Marshall Misty , Pattu Varsha , Halimani Mahantappa , Maier-Peuschel Monika , Hoth Markus , Rettig Jens
Published: January 2023

One salient event in the adaptive immune response is the activation of T lymphocytes by foreign antigens. They patrol our body for providing a defense against intracellular pathogens, viruses and malignant cells. Upon recognition, T lymphocytes exert their cytotoxic activity through secretion of cytotoxic granules at a specialized cell interface, the immune synapse (IS), or engage in stimulus-coupled release of immune mediators in pre-formed granules. To induce target cell death, T lymphocytes rely on exocytosis of granules, which is the result of a cascade of events consisting of docking, priming, and finally Ca2+-dependent fusion involving soluble NSF attachment receptor (SNARE) proteins. VAMP8 is a SNARE protein that is a key component of a variety of secretion in other immune cell types and whose function in T cell killing is unknown. VAMP8 re-localizes to the immunological synapse following CTL activation suggesting it plays an important role in granule fusion. VAMP8 has also been shown to localize abundantly at the membranes of early and late endosomes. Additionally, VAMP8 plays a role in the exocytosis of organelles formed in the secretory pathway, such as exocrine granules and secretory lysosomes. Hence, this research is aimed at elucidating the involvement of VAMP8 in T lymphocytes of secretory granule release allowing us to better understand the contribution of VAMP8 to the killing process of T lymphocytes.
CD47low status on CD4 effectors is necessary for the contraction of the immune response in humans and mice (69.13)
Authors: Van Vu Quang , Baba Nobuyasu , Rubio Manuel , Wakahara Keiko , Panzini Benoit , Richard Carole , Susin Genevieve , Franchimont Denis , Fortin Genevieve , Martinez Torres Ana Carolina , Cabon Lauriane , Susin Santos , Sarfati Marika
Published: January 2023

To execute a memory program, CD4 effector T cells must escape cell death and elimination and survive beyond the contraction of the immune response (IR). The involvement of Fas/FasL in the crash of IR has been challenged and the elimination of CD4 effector T cells appeared to occur independently of caspase activation. We report here that in vitro TCR-activated human CD4 T and ex vivo isolated effector memory T cells displayed a CD47low status that was linked to thrombospondin-1/CD47-induced caspase-independent cell death. By contrast, a CD47highstatus that was observed on central memory T cells and on activated CD4 T cells cultured in the presence of IL-2 correlated with resistance to CD47-mediated killing. A CD47low status was required on murine antigen (Ag)-specific CD4 effectors T cells for the contraction of the IR in vivo, as lower numbers of Ag-specific CD47low CD4 T cells were recovered when compared to Ag-specific CD47-/- CD4 T cells. Finally, the maintenance of a CD47high status on CD4 effectors despite high level of TSP-1 production was observed in inflamed tissues of patients with Crohn’s disease when compared to non-IBD donors, suggesting that a CD47high status on tissue-destructive CD4 effectors prevents the resolution of inflammatory response. In conclusion, a transient change in the status of the self-marker CD47, i.e. from CD47high to CD47low, regulates the decision-making process that leads to cell death during the IR.
The antigen dose regulates T-cell fate by controlling the B-cell response (69.2)
Authors: Kalies Kathrin , Stamm Claudia , Westermann Juergen
Published: January 2023

The nature of the antigen determines the type of T-cell response and thereby the course of the resulting immune reaction. Especially, the binding affinity of the peptide-MHCII complex to the T-cell receptor and the presence of cytokines are crucial. In addition, it has been reported that the antigen dose used for immunization regulates T-cell fate. For example, intravenous priming with sheep red blood cells (SRBC) induces two functionally distinct T-cell populations. A high dose (10e9 SRBC) induces T cells that provide B cell help (Th2 cells) whereas a low dose (10e5) induces T helper cells that mediate delayed type hypersensitivity reaction in the skin (Th1 cells). The mechanism behind the induction of Th1 or Th2 cells is not well understood. In this study, we investigated the expression of Th1 and Th2 cytokines in the compartments of the spleen in vivo using laser microdissection and real-time RT-PCR to understand how these distinct T-cell populations are formed. Our results show that cytokine producing effector T cells appear in the spleen only after high dose priming and only in the presence of activated B cells. In contrast, T cells induced by a low dose do not start to produce cytokines in the spleen but after encounter SRBC in the skin. We conclude that the induction of cytokine producing effector T cells requires a second contact between T cells and SRBC, which is given by B cells in the spleen after high dose priming and in the skin after low dose priming.
POU2AF1(+) follicular helper T (Tfh) cells in obstructive sleep apnea syndrome (OSAS) (69.3)
Authors: Ichimiya Shingo , Matsumiya Hiroshi , Kubo Terufumi , Ara Shihoko , Ye Rui , Sato Akinori , Kikuchi Tomoki , Yamashita Keiji , Mitsuhashi Yukari , Seki Nobuhiko , Shirasaki Hideaki , Kimura Sachiko , Yokoyama Shigeaki , Sato Noriyuki , Himi Tetsuo
Published: January 2023

OSAS is attributable to periodic collapse of the upper airway due to adenotonsillar hypertrophy, featuring prominent germinal centers and wide interfollicular zones. However, the mechanism is still unclear. Here we present the possible involvement of Tfh cells in the pathogenesis of OSAS as indicated by comprehensive analysis of tonsillar lymphocytes of OSAS patients. The results revealed that tonsillar Tfh cells were plentiful in OSAS compared to recurrent tonsillitis (RT) without any discrepancy of T-cell components between the tonsils of OSAS and RT patients, indicating a ‘Tfh shift’ in OSAS tonsils. In addition, in vitro experiments delineated a unique function of the Tfh cells of OSAS, which could help expand the number of naive B cells and, more extensively, memory B cells, which allowed the Tfh cells to produce larger amounts of IL-4 and IFN-γ. Microarray studies newly identified transcripts characterizing the Tfh cells, including TIGIT and POU2AF1 regulating CXCR5 of B cells. Indeed, B-cell expansion was under the control of TIGIT, and large numbers of POU2AF1(+) Tfh cells were observed in and outside of germinal centers of OSAS tonsils. Overall these findings indicate that POU2AF1(+) Tfh cells cooperate with regional B-cell subsets to induce lymphoid hyperplasia.Focusing on the TIGIT-selective regulation of POU2AF1(+) Tfh cells, further investigations may provide a new modality for a noninvasive approach to treatment of this condition.
RANK-ligand activates stroma in postnatal lymph node growth (69.4)
Authors: Mueller Christopher , Hess Estelle , Golub Rachel , Choi Yongwon , Hideo Yagita
Published: January 2023

The lymph node (LN) is a well-organized immune organ, favoring immune cell interactions in a spacio-temporally-controlled manner. Studies of embryonic LN formation have identified TNFSF11 (RANKL), the lymphoid tissue inducer cells and the fibroblastic stromal cells as key players in this process. Yet the cellular and molecular mechanisms that underlie postnatal LN growth remain unclear. We have studied a mouse mutant overexpressing RANKL in the skin and found massive postnatal growth of skin-draining LNs. T cells and dendritic cells are not activated and respond well to immunization by foreign antigen and adjuvant. The overall organ architecture is preserved with the exception of an increase in the numbers of small B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines as well as MAdCAM-1 and VCAM-1 cell adhesion molecules. As for hematopoietic cells, they appear to receive anti-apoptotic signals as T cells from hyperplasic LNs survive better in lymphopenic hosts than T cells from control mice. These findings identify RANKL as a key molecule for controlling homeostasis of the immune system through stroma.
The role of CD11c in IL-17A and IL-22 production in innate A. fumigatus infection (70.10)
Authors: Gessner Melissa , Lilly Lauren , Dunaway Chad , Bullard Daniel , Steele Chad
Published: January 2023

We have previously reported that mice deficient in the beta-glucan receptor Dectin-1 displayed increased susceptibility to A. fumigatus lung infection in the presence of lower IL-17A and IL-22 production in the lungs. Our data suggests that IL-23 contributes to most, but not all, Il-17A and IL-22. As the presence of CD11b+ and CD11c+ cells are required for optimal IL-17A production by neutrophils (Werner, et al.), we examined the role of the CD11b and CD11c receptors in lung cell production of IL-17A and IL-22 during A. fumigatus infection. Here, we show that CD11c deficiency, and CD11b deficiency to a lesser extent, results in compromised IL-17A and IL-22 production by lung cells from A. fumigatus challenge mice. We further observed a contribution for CD11c in the production of IL-1a, IL-1b and TNF-a as well as neutrophil recruitment during A. fumigatus infection. Despite impaired levels of IL-17A and IL-22, CD11c deficiency did not result in impaired Il23 mRNA levels. Collectively, our results indicate that CD11c is required for optimal IL-17A and IL-22 production in early innate lung defense against A. fumigatus, although this defect is not a result of impaired IL-23 induction.
BAFF neutralization blocks T cell-independent IgM production (70.3)
Authors: Jones Derek , Jones Maura , DeIulio Greg , Racine Rachael , Winslow Gary
Published: January 2023

Infection of mice with the intracellular bacterium Ehrlichia muris induces a protective T cell-independent (TI) IgM response. The TI IgM produced during acute infection is secreted almost exclusively by a population of CD11c-expressing B cell plasmablasts in the spleen. We investigated the role of B cell activating factor of the TNF family (BAFF) on the generation of the IgM-secreting plasmablasts. To this end, we neutralized BAFF in vivo using a monoclonal antibody directed against murine BAFF. Blockade of BAFF in infected mice did not affect the generation of splenic plasmablasts; however, treatment nearly eliminated the number of antigen-specific IgM-secreting cells, serum IgM titers were reduced, and the plasmablasts failed to express cell-surface CD138. Analysis of mRNA expression in FACS-sorted plasmablasts from anti-BAFF and isotype control-treated mice revealed no differences in transcript levels of prdm1 and xbp1, genes encoding the transcription factors Blimp-1 and XBP-1, which are essential for plasma cell differentiation. Thus, our data indicate that BAFF signaling regulates a checkpoint in plasmablast differentiation, downstream of Blimp-1 and XBP-1 transcription, but prior to CD138 expression.
Intestinal mast cell progenitors function as innate lymphoid cells (P497) (71.3)
Authors: Lee Youngae , Kumagai Yutaro , Akira Shizuo , Jang Myoung Ho
Published: January 2023

Small intestinal innate lymphoid cells (mainly NKp46+ILCs) are known to have a protective role from infection of pathogenic bacteria through the production of IL-22. The proportions of ILCs localized intestinal lamina propria are higher than those in the others lymphoid tissues, such as mesenteric lymph node, spleen, liver, and bone marrow. In this study, we did a microarray experiment to study whether small intestinal ILCs (Lin-c-Kit+Sca-1- cells) highly express which genes as compared with non-ILCs (Lin-c-Kit-Sca-1- cells). We selected 251 up-regulated genes and 219 down-regulated genes in Lin-c-Kit+Sca-1- cells. Especially, Lin-c-Kit+Sca-1- cells highly expressed genes coding to Il22, Csf2rb2, mast cell proteases (Mcpts), and Rorc. To address whether which ILC populations are main source of IL-22, we more detailed divided Lin-c-Kit+Sca-1- cells into the two groups [Lin-c-Kit+NKp46+ (NKp46+ILCs) and Lin-c-Kit+NKp46-CD4-]. Only Lin-c-Kit+NKp46-CD4- cells expressed Csf2rb2 and Mcpts transcripts. These cells expressed higher level of Il22 mRNA, IL-22 protein, and IL-17F protein in physiological condition and in IL-23 stimulated condition compared with NKp46+ILCs. Moreover, these cells could differentiate into mast cells in the presence of mIL-3 and mSCF. Taken together, Lin-c-Kit+NKp46-CD4- cells as mast cell progenitors may regulate small intestinal homeostasis through the production of Mcpts, IL-17F, and IL-22 in physiological condition and in pathophysiological condition.
Endogenous S-Nitrosothiol Induced hypertrophic Astrogliosis has potential to maintain CNS homeostasis under pathological Conditions (72.8)
Authors: Paintlia Manjeet , Paintlia Ajaib , Singh Avtar , Singh Inderjit
Published: January 2023

Compelling evidence suggests that reactive astrogliosis exert both detrimental and beneficial effects in response to central nervous system (CNS) inflammatory insult. The exact mechanism behind such diversity in astrocytic functions to maintain CNS homeostasis is not clear. Recent studies explicated the biphasic functions of nitric oxide (NO) in EAE, where reactive astrogliosis is known to play critical role in its pathogenesis. Here, we investigated the effect of endogenous NO donor, S-nitrosoglutathione (GSNO) on astrocytic functions in vitro settings. Interestingly, GSNO induced hypertrophic astrogliosis with increased expression of ciliary neurotrophic factor (CNTF) in treated astrocytes. This effect of GSNO on astrocytes was ascribed to an induction of NO-soluble guanylate cyclase-cGMP activated PKG signaling and activation of p38MAPK and PPAR-g with RhoA inhibition. Also, we found that GSNO-mediated increased GFAP expression in astrocytes was ascribed to CNTF induced JAK2/STAT3 signaling in an autocrine manner. This GSNO-mediated hypertrophic astrogliosis had potential to maintain CNS homeostasis under pathological conditions. Together, these data showed the beneficial functions of GSNO and suggest new therapeutic interventions to protect CNS against Inflammatory insult similar to MS brain.
CD4+CD25+FOXP3+ regulatory T cells and association with survival in epithelial ovarian cancer (74.9)
Authors: Preston Claudia , Maurer Matthew , Oberg Ann , Hartmann Lynn , Kalli Kimberly , Goode Ellen , Knutson Keith
Published: January 2023

Ovarian cancer is an immune reactive tumor with a complex suppressive network that blunts immune eradication. One putative pathway of evasion may be recruitment of CD4 regulatory T cells (Treg) into the tumor microenvironment. This study investigated the association of tumor infiltrating Tregs in ovarian cancer patients with poor survival. Methods: Immunofluorescence of intraepithelial CD4+CD25+FOXP3+ Tregs, CD3+ and CD8+ cells was performed in tumors of 52 ovarian cancer patients. Average and maximum count of cells were associated with survival via Cox regression models and summarized with hazard ratios (HR) and 95% confidence intervals (CIs). Results: The maximum counts of Tregs in these patients suggests an association between higher number of Tregs and poor clinical outcome (HR=1.48, CI=0.97, 2.28), although not statistically significant (p=0.07). Moreover, presence of high CD8/Treg ratio (HR=0.7, CI=0.53, 0.91; p=0.008) as well as high CD8/CD4 ratio (HR=0.76, CI=0.57, 1.0; p=0.05) appear to be associated with good outcome. The CD3 lymphocyte count was not associated with survival (HR=0.9, CI=0.66, 1.24; p=0.52). Conclusion: Although the present pilot study was conducted with a limited number of patients, our data suggests that higher numbers of infiltrating Tregs might be associated with poor survival and low CD8/Treg ratio may be correlated with improved survival in patients with epithelial ovarian carcinoma. We plan to confirm these results in a larger set of patients.
Development and preclinical evaluation of SEL-068, a novel targeted Synthetic Vaccine Particle (tSVP™) for smoking cessation and relapse prevention that generates high titers of antibodies against nicotine (75.11)
Authors: Pittet Lynnelle , Altreuter David , Ilyinskii Petr , Fraser Christopher , Gao Yun , Baldwin Sam , Keegan Mark , Johnston Lloyd , Kishimoto Takashi
Published: January 2023

Novel vaccine-based therapies provide new avenues for the treatment of chronic conditions such as addiction. Here we describe SEL-068, an entirely synthetic, self-assembling nanoparticle vaccine against nicotine for smoking cessation. SEL-068 is a targeted Synthetic Vaccine Particle (tSVP™) comprised of four main components: a biodegradable and biocompatible polymer matrix, a synthetic TLR agonist, a novel universal T cell helper peptide, and nicotine covalently conjugated to the nanoparticle surface (B cell antigen). The nanoparticles are designed to flow freely to the lymph nodes, enabling direct and concomitant delivery of all required components to the responsive cells of the immune system. Encapsulation and controlled release of the TLR agonist in the nanoparticle minimizes the generation of systemic inflammatory cytokines, improving the overall safety profile and enabling the use of novel adjuvants. The novel universal T cell helper peptide used in SEL-068 elicited T cell recall responses in PBMC from a broad panel of human donors. Immunization of mice with SEL-068 led to the generation of high titers of high affinity anti-nicotine antibodies. Rhesus and cynomolgus monkeys immunized with increasing doses of SEL-068 showed a robust and dose dependent induction of high titers of anti-nicotine antibodies with strong affinity to nicotine. No dose-limiting systemic toxicities were detected. SEL-068 is currently being evaluated in humans in Phase 1 clinical trials.
Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425-A1g8 (75.12)
Authors: Yu Xiaocong , Duval Mark , Lewis Christopher , Cavacini Lisa
Published: January 2023

With the majority of HIV infections resulting from mucosal transmission of HIV, induction of an effective mucosal immune response would be pivotal in preventing transmission. HIV-specific IgA but not IgG has been detected in genital tract, seminal fluid, urethral swabs, urine and vaginal wash samples of HIV-negative sex workers and HIV-status discordant couples. Purified mucosal and plasma IgA from HEPS individuals neutralize infection and though present at low levels, cross-clade neutralization activity. We generated a CD4i human monoclonal antibody F425-A1g8 and characterized the impact of its isotype variants to HIV neutralizing activity. The result showed that in contrast to little neutralization by F425-A1g8 IgG1 in the absence of sCD4, the IgA1 variant of the antibody displayed significant neutralization activity against a range of HIV clade B isolates in the absence of sCD4. This research clearly suggests that IgA isotype utilizing its unique molecular structure plays an important role in HIV neutralization. The studies of the neutralizing function of IgA isotypes and the functional relationship between epitopes and IgA antibodies may also suggest strategies for intervention in virus transmission or spread within the mucosal of host, as well as serve to inform the design of vaccine strategies that may be more effective at preventing mucosal transmission.
Adjuvanting influenza vaccines for enhanced immunity to seasonal or pandemic influenza virus. (75.9)
Authors: Fairman Jeffery , Carroll Tim , Liu Feng , Hancock Kathy , Katz Jacqueline , Lu Xiuhua , Matzinger Shannon , Miller Chris
Published: January 2023

The efficacy of FDA approved influenza vaccines depends on eliciting neutralizing antibodies that provide protection against antigenically well-matched viruses. To determine if the JVRS-100 adjuvant could enhance vaccine-induced protection against matched and non-matched influenza virus challenge, mice or non-human primates (NHP) were immunized with vaccine +/- the JVRS-100 adjuvant. All animals that received JVRS-100 adjuvanted vaccine had significantly lower levels of virus replication following viral challenge. In matched and cross-clade challenge experiments JVRS-100 adjuvanted H5N1 vaccine elicited enhanced humoral responses, T-cell responses, and protection in mice. In heterosubtypic challenge experiments, mice and nhp immunized with adjuvanted H1N1 vaccine demonstrated enhanced antibody responses, T-cell responses, and protection against H3N2 challenge compared with groups that received nonadjuvanted vaccine. These data demonstrate that JVRS-100 enhances antibody and T-cell responses, resulting in matched, cross-clade, and heterosubtypic immunity for influenza vaccination.
Selective Depletion of CD19+CD24hiCD38hiIL-10high Regulatory B cells (Bregs) in HIV-infected Aviremic Individuals (76.2)
Authors: Siewe Basile , Stapleton Jack , Martinson Jeffrey , Keshavarzian Ali , Kazmi Nazia , Demarais Patricia , French Audrey , Alan Landay
Published: January 2023

HIV-infection associated upregulation of IL-10 contributes to impaired antiviral effector T-cell functions promoting viral persistence. Bregs (CD19+CD24hiCD38hi) IL-10-mediated modulation of T-cell function has been reported in autoimmune disease but yet undefined during HIV infection. In this study, we confirmed high expression of IL-10 by Bregs in healthy controls (HC, n=16) and determined Breg frequency, T-cell immune activation (CD8+HLA-DR+CD38+, CD4+HLA-DR+CD38) and T-cell exhaustion (CD8+PD-1+) in aviremic (HIVavir, n=10) and viremic (HIVvir, n=15) HIV-infected individuals. We found a significant positive correlation between Breg frequency and viral load (r2=0.5166, p=0.0097); HIVavir-individuals exhibited a significant Bregs loss compared to HC (p=0.0004) and HIVvir-individuals (p=0.0122), despite no significant differences in total B-cell frequency. A significant positive correlation was observed between Breg frequency and immune activation (CD8+HLA-DR+CD38+, r2=0.6262, p=0.0041; CD4+HLA-DR+CD38+, r2=0.6298, p=0.0039) as well as T-cell exhaustion (r2=0.8853, p=0.0333). Additionally, we found a significant difference in Breg CD95 (Fas) expression between HIVavir and HIVvir-individuals (p=0.0119) yet no such difference between in HIVavir-individuals and HC. We hypothesize that, Bregs loss during HIV-infection may be due to Fas mediated apoptosis and that activated IL-10-secreting Bregs contribute to T cell impairment hence viral persistence in HIVvir individuals.
A novel human CD1d knock-in mouse model demonstrates potent anti-tumor potential of human CD1d-restricted iNKT cells (P5001)
Authors: Yuan Weiming , Wen Xiangshu , Rao Ping , Cresswell Peter , Porcelli Steven , Kim Seil , Carreño Leandro , Lawrenczyk Agnieszka
Published: January 2023

Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of Natural Killer T (NKT) cell-based anti-tumor therapies in human clinical trials, which contrast strongly with the powerful anti-tumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated the first hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vβ8, the homolog of human Vβ11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d-KI mice exert potent anti-tumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted anti-tumor therapies.
Therapeutic inhibition of gut antigen trafficking through scavenger receptor A (P5027)
Authors: Raycroft Maury , Mamula Mark
Published: January 2023

We have demonstrated that B cells acquire specific antigen (Ag) through B cell receptor for transfer to both mouse and human macrophages (MOs) and dendritic cells (DCs). Our studies also determined that this process is dependent on the MO/DC surface scavenger receptor A (SR-A). Interestingly, SR-A deficient, lupus-prone mice exhibit reduced autoantibody production and pathology. Thus, this new pathway of Ag trafficking is important in human immunity and/or autoimmunity, leading to current investigations in targeting SR-A for therapeutic intervention. To identify novel modulators of SR-A, we screened bioactive small molecule inhibitor (SMI) libraries and identified candidates that alter Ag transfer from B cells to MO/DCs ex vivo in human cell lines and in vivo in mice. An SR-A-specific and dose-dependent SMI, sennoside B, was effective in reducing autoantibody production and lupus pathology in lupus-prone mice. Interestingly, we have observed drug-mediated reduction in antigen trafficking between APCs in gut Peyer's patches. The data suggest that SRA inhibition alters the gut immune responses, which has recently been linked to autoimmune diseases such as diabetes and arthritis. Finally, several studies indicate that SR-A has regulatory affects on TLR4. We are presently investigating TLR4 and SRA-mediated interactions in mediating antigen trafficking in Peyer's patches and downstream autoimmunity.
GILT expression in medullary thymic epithelial cells is required for presentation of tissue-specific self antigens and deletion of autoreactive T cells (P5037)
Authors: Rausch Matthew , Metzger Todd , Anderson Mark , Hastings Karen
Published: January 2023

Clonal deletion of thymocytes with high avidity for self-peptide:MHC complexes is critical for the maintenance of tolerance and prevention of autoimmunity. We have shown that gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for thymic deletion of T cells specific for a self and melanoma Ag, tyrosinase-related protein 1 (TRP1). To define GILT’s role in central tolerance, we show that GILT expression is enriched in thymic APC capable of mediating deletion, medullary thymic epithelial cells (mTEC) and dendritic cells. TRP1 expression is restricted to mTEC. GILT facilitates the MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APC (TEC and bone marrow (BM)-derived APC). Using BM chimeras, we show that TRP1-specific T cells are deleted in chimeras in which GILT is expressed in both TEC and BM-derived APC or GILT expression is restricted to TEC, indicating that GILT expression in TEC is sufficient for deletion of TRP1-specific thymocytes. In contrast, a large number of TRP1-specific T cells develop in chimeras which lack GILT expression in both TEC and BM-derived APC. Chimeras in which GILT expression is limited to BM-derived APC have intermediate levels of TRP1-specific T cells, demonstrating that GILT expression in TEC is required for efficient deletion of TRP1-specific thymocytes. These findings suggest that GILT operates in mTEC to facilitate the presentation of tissue-specific self Ag and promote deletion of autoreactive T cells.
Inflammation-induced splicing of Deaf1 in the pancreatic lymph nodes during the progression of Type 1 diabetes (P5061)
Authors: Yip Linda , Creusot Remi , Whiting Chan , Taylor Cariel , Akter Rahima , Matsumura Teppei , Fathman C. Garrison
Published: January 2023

Peripheral tolerance is partially controlled by ectopic peripheral tissue antigen (PTA) expression in lymph node stromal cells (LNSCs). We previously showed that the transcriptional regulator deformed epidermal autoregulatory factor 1 (Deaf1) can directly control the transcription and indirectly regulate the processing and/or presentation of PTA genes in LNSCs of the pancreatic lymph nodes (PLN). We showed that Deaf1 is alternatively spliced in the PLN of Non-Obese Diabetic (NOD) mice and Type 1 diabetes (T1D) patients, to form a dominant negative isoform, Deaf1-Var. We previously demonstrated that Deaf1-Var expression was highest in the PLN of NOD mice during the onset of disease, compared to the non-inflamed PLN of healthy age-matched NOD.B10 and NOD.SCID mice. In NOD mice, Deaf1-Var expression in the PLN was found to increase as disease progressed from peri-insulitis to destructive insulitis. In this study, we asked if inflammation induces Deaf1 splicing in the PLN. Deaf1 splicing could be induced in the PLN of NOD.B10 control mice and NOD.SCID mice by i.p. injection of activated splenocytes that home to the PLN. The supernatant of activated splenocytes could also to induce Deaf1 splicing in pancreatic islets, a tissue previously shown to splice >500 genes in response to inflammation. Together, these data suggest that the inflammation that occurs in the PLN during the progression of T1D leads to Deaf1 splicing, loss of Deaf1 function, and reduced PTA expression.
Functionally distinct ERAP1 haplotype combinations define individuals with ankylosing spondylitis (P5047)
Authors: James Edward , Reeves Emma , Colebatch Alexandra , Elliott Tim , Edwards Christopher
Published: January 2023

Ankylosing Spondylitis (AS) is a chronic inflammatory disease which commonly affects the spine occurring in approximately 0.5% of adults of European descent. In addition to HLA-B27, recent genetic linkage studies have identified polymorphisms within the aminopeptidase, ERAP1, associated with the disease. ERAP1 lies on the antigen processing pathway, generating peptide antigens for immune presentation by HLA. Here we show that ERAP1 from individuals are polymorphic and comprise haplotypes that are predominantly made up of multiple SNPs. ERAP1 haplotype combinations found in AS cases are distinct from those in controls. AS case ERAP1 haplotype combinations are unable to trim peptide precursors to generate the final epitope for presentation to T cells and are also unable to reconstitute MHC class I to normal levels as observed with control haplotype combinations. We therefore provide strong evidence that ERAP1 variation predisposes to chronic inflammatory disease via its influence on the antigen processing pathway.
Characterization of human monocyte subsets (P4161)
Authors: Boyette Lisa , Macedo Camila , Elinoff Beth , Metes Diana , Lakkis Fadi
Published: January 2023

Monocytes are an important source of antigen-presenting cells and macrophages in tissues affected by infection or inflammation and in organ transplantation. Monocytes have been grouped into classical (CD14+CD16-), non-classical (CD14dimCD16+), and intermediate (CD14+CD16+) subsets. The biologic significance of these subsets is not completely understood. We phenotyped monocytes in peripheral blood from 25 healthy subjects and performed functional studies on monocyte subsets sorted from leukapheresis products to better characterize these subsets in humans. Monocyte subset frequencies were found to be tightly controlled over time and across healthy individuals: 83.95% classical, 95% CI [81.12, 86.78], 6.65% intermediate [4.32, 8.99], and 9.28% non-classical [7.12, 11.44]. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to Toll-like receptor agonists, and cytokine secretion by classical and intermediate monocytes was suppressed when all three subsets were recombined in physiologic ratios. After incubation with IL-4 and GM-CSF, classical monocytes acquired dendritic cell markers and morphology; intermediate and non-classical monocytes did not. From these studies we conclude that classical monocytes appear to be the principal source of antigen-presenting dendritic cells, and non-classical monocytes suppress secretion of inflammatory cytokines by the other subsets in mixed cultures, perhaps indicating a regulatory role.
Multiple pathways are employed by TLR and NOD agonists to induce PGE2 production in primary human monocytes and macrophages (P4163)
Authors: Zaitseva Marina , Endo Yukinori , Romantseva Tatiana , Godling Hana
Published: January 2023

Innate immune activators that are potent inducers of fever also up-regulate plasma PGE2. To determine whether TLR and NOD agonists activate production of PGE2 directly or through IL-1β, human monocytes (Mo) and differentiated macrophages (MF) were cultured with TLR agonists with or without Caspase-1 inhibitor ZVAD, which inhibited LPS-induced IL-1β in both cell types. PGE2 production in MF was not affected by ZVAD, while in Mo, ZVAD reduced PGE2 and COX2 expression by 30-60%. Surprisingly, IL-1β alone did not up-regulate PGE2 in Mo. However, in Mo pretreated with low dose of LPS, PGE2 and COX2 mRNA were up-regulated by IL-1β. LPS (but not TLR2 agonist) induced phosphorylation of IRF3 (Ser 396) in Mo. Knock-down of IRF3 or of TRIF by siRNA abrogated IL-1β induced COX2 mRNA expression in LPS-pretreated U937-CD14 cells. MDP did not induce PGE2 in Mo alone. However, COX2 mRNA and PGE2 protein in MDP-activated Mo was significantly augmented by human CD3 bead-purified T cells or T cell supernatant. Treatment of Mo with inhibitors of autophagy including ATG5 or ATG7 siRNA, inhibited MDP-induced PGE2 production. T cells were also required for PGE2 produced in response to flagellin and IE-DAP but not Poly I:C. In summary, Mo employ multiple pathways to produce PGE2, depending on the type of innate receptors involved: IRF3/TRIF pathway is employed in response to TLR4, while autophagy/ROS and T cell-derived signals are involved in PGE2 production after triggering of NOD1/2 and IPAF.
E3 ubiquitin ligase NKLAM positively regulates nitric oxide production and the generation of reactive oxygen species in macrophages (P4167)
Authors: Lawrence Donald , Kornbluth Jacki
Published: January 2023

Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. There is mounting evidence that ubiquitination plays a role in modulating macrophage bactericidal functions. Recently, we demonstrated that the ubiquitin ligase NKLAM was a component of the macrophage phagosome. In this present study we investigated the effects of NKLAM on macrophage generation of nitric oxide (NO) and reactive oxygen species. Stimulation with LPS induced wild type (WT) macrophages to produce NO in a time and concentration-dependent manner. NO production was attenuated in NKLAM-knock out (KO) macrophages. We also found that inducible nitric oxide synthase (iNOS) protein expression was lower and that iNOS translocation to the maturing phagosome was defective in KO macrophages. In vivo, intraperitoneal injection of LPS induced a time-dependent increase in serum nitrite (NO metabolite) in both WT and KO mice. However, nitrite levels in KO mice were lower than in WT mice. The generation of reactive oxygen species (ROS) is a potent killing mechanism; however the role of ubiquitination in regulating ROS generation is unclear. To this end, we investigated ROS production in WT and KO macrophages and found that ROS production was defective in KO macrophages stimulated with either PMA or Escherichia coli. Collectively, our data implicate NKLAM as a potent modulator of macrophage oxidative killing functions.
Cannabinoid receptor modulation of the endothelial cell inflammatory response (P4191)
Authors: Wilhelmsen Kevin , Tran Alphonso , Hellman Judith
Published: January 2023

The cannabinoid receptor (CBR) family is composed of the G-protein coupled receptors, CB1R and CB2R, and potentially GPR18, GPR55 and GPR119, and the transient receptor potential ion channel, TRPV1. Recent reports suggest that several cannabinoids have the ability to modulate the activation of the endothelium in response to inflammatory mediators; however, it is not clear which receptors and what mechanisms are responsible. Our studies aim to determine: 1) the expression repertoire of CBRs in different human endothelial cell (EC) niches; and 2) determine which cannabinoid receptors are responsible for modulating EC activation by toll-like receptor agonists and TNFα. Through qPCR analysis, we have found that all ECs express the same subset of CBRs. Additionally, by utilizing a panel of synthetic, endogenous and plant-derived cannabinoids with differing specificities for each CBR, our data thus far suggest that CB1R and/or TRPV1 activation may be responsible for dampening the inflammatory activation of ECs by TLR2 agonists, as determined by a decrease in IL-6 and IL-8 secretion. Future studies aim to definitively identify the CBR(s) responsible, determine the effect of phytocannabinoids on EC activation in more detail, delineate the cellular mechanisms by which CBRs modulate the EC inflammatory response and more globally, identify the role of endocannabinoids and their receptors in modulation of the inflammatory response to infection.
α1-Acid glycoprotein as an inducer of M2b macrophages (P4139)
Authors: Nakamura Kiwamu , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

Alpha 1 acid glycoprotein (α1-AGP) is an acute-phase protein, and its plasma levels are increased in patients with severe burn injuries and various cancers. It has been reported that α1-AGP has an ability to polarize macrophages (MΦ) from resident MΦ to M2MΦ (IL-10+IL-12− MΦ, an immunosuppressive MΦ with reduced capacity to kill pathogens or cancer cells). So far, three subsets of M2MΦ (M2aMΦ, M2bMΦ and M2cMΦ) have been described. All 3 subsets of M2MΦ are equally immunosuppressive. In this study, resident monocytes (CD14+IL-10−IL-12− cells) cultured with α1-AGP were demonstrated to be specifically converted to M2bMΦ. Healthy donor monocytes cultured for 24 to 72 hrs with 0.0625 to 4.0 mg/ml of α1-AGP produced CCL1 (a biomarker of M2bMΦ) into their culture fluids. The maximum CCL1 production was demonstrated when resident monocytes were stimulated with 4 mg/ml of α1-AGP. Similar to RPMI 1640 medium supplemented with heat-inactivated FBS, CCL1 was produced by healthy donor monocytes stimulated with α1-AGP in serum-free medium cultures. However, CCL17 (a biomarker of M2a monocytes) and CXCL13 (a biomarker of M2c monocytes) were not detected in their culture fluids. Also, these monocytes did not express DC-SIGN (a biomarker of M2a monocytes) and showed weak bactericidal activities against P. aeruginosa and E. faecalis. These results indicate that the MΦ conversion from resident MΦ to M2bMΦ is specifically stimulated by α1-AGP .
Poly (I:C) induces human endothelial barrier dysfunction by disrupting tight junction expression of claudin-5 (P4197)
Authors: Huang Coty , D'Agnillo Felice , Golding Basil
Published: January 2023

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 ligand Poly (I:C), a synthetic analog of viral double-stranded RNA commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular cells. Poly (I:C) produced a dose- and time-dependent increase in endothelial monolayer permeability accompanied by a corresponding decrease in the expression of claudin-5, a transmembrane tight junction protein. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of internalized claudin-5 with lysosomal-associated membrane protein 1 (LAMP-1), a marker of lysosomes, suggesting enhanced lysosomal degradation of claudin-5. Poly (I:C) also induced a dose- and time-dependent cleavage of TLR3 and TRIF as well as activation of NF-kB and IRF3, but not IRF7 (MyD88-dependent pathway), consistent with the direct stimulation of the TLR3 signaling. Pronounced inflammatory response with the release of IL-6, IL-8, MCP-1 and RANTES and a hallmark TLR3-mediated type 1 IFN production (IFNbeta) was also observed. Together, these findings provide new insight on how virally-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.