bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the molecular mechanism of Syntaxin11 function in cytotoxic T lymphocytes (P3032)
Authors: Halimani Mahantappa , Pattu Varsha , Krause Elmar , Hoth Markus , Schwarz Eva , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTL) play a crucial role in eliminating virus infected and cancer cells through exocytosis of perforin and granzymeB containing lytic granules (LGs). Soluble NSF Attachment Protein Receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs and SNARE associated proteins in CTL function and LG exocytosis is evident in the form of Familial Hemophagocytic Lymphohistiocytosis (FHL). Mutations within Syntaxin11 (Stx11), a Qa-SNARE protein, result in FHL subtype-4, however the mechanism by which Stx11 malfunction leads to this severe phenotype remains unknown. Here, we investigate the molecular mechanism of Stx11 function in primary human CTLs. High resolution live cell imaging with CTLs before and after target cell contact showed sequential polarization of Stx11 followed by LGs to the immunological synapse. Using TIRF (total internal reflection fluorescence) microscopy we further showed an association of LGs with plasma membrane-bound Stx11 at the IS. Further, knockdown studies using Stx11 specific siRNA resulted in a strong reduction of both LG fusion and dwell time at the IS. We therefore propose that Stx11 is required for the final fusion of LG at the IS, explaining the severe FHL4 phenotype.
Cytokine regulation of BAFF expression in human myeloid cells (P3005)
Authors: Ragheb Samia , Li Yanfeng , Dore-Duffy Paula , Lisak Robert
Published: January 2023

B-cell activating factor (BAFF) has an essential role in B cell homeostasis. It is synthesized and released by myeloid cells (monocytes & macrophages). BAFF over-expression promotes the survival and maturation of autoreactive B cells. Increased BAFF levels are found in several human autoimmune diseases, suggesting a role for BAFF in disease pathogenesis and progression. However, the regulatory signals that affect BAFF production are largely unknown. Using the human monocytic cell line THP-1, we examined the capacity of several cytokines to modulate BAFF expression; these included tumor necrosis factor-α (TNF-α), IL-4, IL-10, IL-12, IL-17, and the type I & type II interferons (IFN-α, IFN-β, and IFN-γ). Replicate cultures were used to measure BAFF RNA transcripts and released BAFF protein. Quantitative real-time PCR (QRT-PCR) was used to measure BAFF RNA at 6 hrs; β-actin was used as the reference housekeeping gene. BAFF protein in the culture supernatants was measured by ELISA at 96 hrs. All interferons upregulated BAFF expression (RNA & protein); however, IFN-β was the most potent (IFN-β > IFN-α > IFN-γ). We also tested various combinations of cytokines to assess synergistic or antagonistic effects on BAFF expression. TNF-α inhibited BAFF expression by IFN-β−stimulated cells. Similar results were obtained with primary cultures of human peripheral blood monocytes.
Polysaccharides isolated from Alchornea cordifolia modulate monocyte/macrophage function (P3006)
Authors: Quinn Mark , Kouakou Koffi , Yapi Ahoua , Kirpotina Liliya , Jutila Mark , Schepetkin Igor
Published: January 2023

Extracts of leaves from different species of the genus Alchornea have been used for centuries to treat medical problems in tropical Africa; however, little is known about the active components conferring therapeutic properties to these extracts. We investigated the phagocyte immunomodulatory properties of water-soluble polysaccharides extracted from the leaves of Achornea cordifolia. Extracted polysaccharides were fractionated by DEAE-cellulose, Diaion HP-20 resin, and preparative Sepharose 6B size-exclusion chromatography and characterized. Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the parent fraction AP-AU and its high-molecular weight sub-fraction AP-AU1 induced nitric oxide and cytokine (IL-1β, IL-6, IL-10, TNF-α, and GM-CSF) production by human peripheral blood mononuclear cells and human and murine monocyte/macrophages cell lines in vitro. We also evaluated the ability of selected fractions to activate mitogen activated phagocyte protein kinase (MAPK) pathways using a phospho-MAPK array and found that treatment of phagocytes with AP-AU1 induced phosphorylation of Akt2, p38δ/p38γ, p70S6K1, RSK2, and mTOR, as well as stimulation of NF-κB transcriptional activity. Thus, these results provide a molecular basis to explain a portion of the beneficial therapeutic properties of water extracts from A. cordifolia leaves in traditional folk medicine of Africa.
Induction of autophagy by Type I interferon in human cancer cells (P6217)
Authors: Zoon Kathryn , Fey Samuel , Horowitz Julie , Fischer Elizabeth , Balinsky Corey , Miyake Kotaro , Bekisz Joseph , Snow Andrew , Schmeisser Hana
Published: January 2023

Type I IFNs (interferons) are cytokines that induce an antiviral state in cells and have antiproliferative, pro-apoptotic, and immunomodulatory activities. Autophagy is a basic cellular mechanism that involves degradation of cellular components via lysosomes. While induction of autophagy is well documented for Type II IFN, induction of autophagy by Type I IFN has not been previously reported. We determined that Type I IFN can induce autophagy in multiple human cancer cell lines. IFN-α2c and IFN-β induces autophagy in Daudi B cells, as indicated by an increase in autophagy markers LC3-II, Atg5-Atg12 complexes, and a decrease in p62. An increase in LC3-II was also detected post IFN-α2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines was confirmed by electron microscopy analysis. An increase in autophagy markers correlated with inhibition of mTORC1 activity and cell proliferation as well as changes in the cell cycle progression in Daudi cells. Treatment of Daudi and T98G cells with IFN-α2c and rapamycin (mTOR inhibitor), or LY294002 (PI3K inhibitor) increased the level of LC3-II, indicating that the PI3K/Akt/mTORC1 signaling pathway may affect IFN-induced autophagy in Daudi and T98G cells. The role of mTOR in Type I IFN-induced autophagy was confirmed by siRNA silencing experiments. Taken together, our findings demonstrate a novel function for Type I IFN as inducer of autophagy in multiple cancer cell lines.
Differential requirement of C3a receptor in APC-driven emphysema in response to cigarette smoke (P6225)
Authors: Yuan Xiaoyi , Shan Ming , You Ran , Wetsel Rick , Drouin Scott , Seryshev Alexander , Song Li-zhen , Corry David , Kheradmand Farrah
Published: January 2023

Pathogenic lung antigen presenting cells (APCs) are abundant in the lungs of smokers with emphysema and are capable of inducing T helper type 1 (Th1) and Th17 cell differentiation. Mice exposed to chronic cigarette smoke develop emphysema and harbor CD11b+CD11c+ APCs that can transfer the disease to naïve mice. Proteolytic products from activated innate immune cells cleave complement proteins but the contribution of their downstream signaling in APC-driven lung parenchyma destruction is less clear. We show here that relative to control smokers, complement 3 (C3) is increased in the plasma and its cleaved products are deposited on the lung tissue of smokers with advanced lung disease. Matrix metalloproteinase 12 and neutrophils elastase directly cleave human C3, and generate multiple activated signaling products. Compared to wild type, C3 deficient mice exposed to chronic smoke showed reduced CD11b+CD11c+ cells in the lungs and were protected against emphysema development. C3a and C5a, two of the major anaphylatoxin and chemoattractant mediators downstream of C3 activation, are present in the lungs of mice exposed to smoke, but only C3a signaling was critical in emphysema development because deficiency in C3a receptor (C3aR) but not C5aR phenocopied C3-/- mice in this model. These findings suggest a critical role for C3aR in the pathogenesis of smoke induced lung disease, and could be explored to develop specific new therapeutic targets for emphysema treatment.
Cholinergic neuronal signals attenuate inflammasome activation through inhibiting mitochondrial stress (P1264)
Authors: Lu Ben , Levine Yaakov , Yang Huan , Kwan Kevin , Olofsson Peder , Andersson Ulf , Chavan Sangeeta , Tracey Kevin
Published: January 2023

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Immunity is highly linked with mitochondrial stress, which importantly regulates the activation inflammasome, a key component of innate immunity. Here we show that cholinergic neuronal signals attenuate inflammasome activation by inhibiting mitochondrial stress. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibited the inflammasome activation and mitochondrial stress; whereas genetic deletion of alpha 7 nicotinic acetylcholine receptor (a7 nAchR) significantly enhanced the inflammasome activation and mitochondrial stress. Immunohistochemical analysis revealed that a7 is localized in the mitochondria. We also found that extracellular acetylcholine rapidly influxes into macrophage cytoplasm upon ATP stimulation in a P2X7 receptor-dependent manner. Importantly, acetylcholine significantly attenuated calcium or hydrogen oxide-induced mitochondrial damage and the subsequent mitochondrial DNA release. Together, these findings unravel a novel neurotransmitter-mediated signaling pathway, in which acetylcholine translocates into cytoplasm of immune cells during inflammation, and inhibits inflammasome activation by preserving mitochondrial membrane integrity.
Specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single cell gene expression analyses (P1448)
Authors: Arsenio Janilyn , Kakaradov Boyko , Yeo Gene , Chang John
Published: January 2023

Although it is well established that heterogeneity of lymphocyte fate is an essential feature of adaptive immune responses, how and when these divergent cellular fates are specified remains unknown. It has been previously shown that a T lymphocyte responding to a microbial infection can undergo asymmetric division to yield two daughter cells that are differentially fated from inception. Such a model suggests that the progeny arising from each of the two differentially fated daughter cells might exhibit distinct patterns of gene expression. Because strategies analyzing bulk cell populations cannot distinguish differences among individual cells, we applied high-throughput single cell gene expression profiling to interrogate single CD8+ T lymphocytes throughout the course of an immune response to a bacterial infection. We demonstrate that terminally differentiated effector cells and self-renewing memory cells exhibit unique gene expression profiles. Moreover, we are able to identify cells early during the immune response, beginning with the first cell division, that exhibit distinct gene expression patterns characteristic of effector or memory cells. Finally, we derive a network model that allows us to predict the sequential activation of genes that orchestrate each cellular fate, providing a comprehensive view of CD8+ T lymphocyte differentiation. Together, these results provide novel insights into the specification of lymphocyte fates during an immune response.
The role of immunological tolerance in humoral responses to the 2F5 epitope of HIV-1 (P6183)
Authors: Yang Guang , Holl T. , Nojima Takuya , Verkoczy Laurent , Moody M. , Haynes Barton , Kitamura Daisuke , Kelsoe Garnett
Published: January 2023

Many human antibodies (Ab), e.g., 2F5, that neutralize multiple clades of HIV-1 are self-reactive. We previously identified kynureninase (KYNU) as the primary self-antigen recognized by 2F5, suggesting that generation of Ab to the 2F5 epitope of HIV-1 may be proscribed by immune tolerance, a notion supported by impaired B-cell development in mice expressing the 2F5 VH and VL regions (2F5 KI). Here, we use Nojima-Kitamura single B-cell cultures to determine the pre- and post-tolerance Ab repertoire to HIV-1. All cultured B cells differentiate into IgG-secreting cells, including autoreactive cells normally subject to tolerization in vivo. The pre-tolerance compartment (small pre-B) from 2F5 KI mice represents cells that express the 2F5 V(D)J rearrangements, and maintain reactivity to HIV-1 gp41, KYNU, and cardiolipin. Mature, post-tolerance B cells from 2F5 KI mice, however, are purged of gp41- and KYNU-reactivity, but retain cardiolipin-binding, and sequence analysis reveals extensive light-chain editing. This loss of HIV-reactivity is followed in unmodified C57BL/6 mice, where B cells expressing 2F5-like BCR are lost during maturation in the bone marrow, as determined by binding to 2F5 epitope. Our results demonstrate that tolerance of the 2F5 epitope is driven by specific reactivity to KYNU, but not general cross-reactivity to cardiolipin and offer a mechanism for determining how immunological tolerance impairs humoral responses to HIV-1.
The death receptor Fas mediates a non-apoptotic signaling cascade in apoptosis resistant CD4+ T cells via the MAPK pathway. (P1304)
Authors: Ramaswamy Madhu , Cruz Anthony , Barden Mary , Siegel Richard
Published: January 2023

The Fas death receptor mediates peripheral tolerance by apoptotic deletion of autoreactive T cells through Restimulation Induced Cell Death (RICD). Previously, we have shown that within a pool of T cells, effector memory were more sensitive to Fas-induced apoptosis than activated naïve CD4 T cells due to efficient DISC formation. Recently, we found that FasL induces an early MAPK response in the form of pJNK activation, preferentially in activated naïve, but not memory T cells. In apoptosis resistant FADD or Caspase-8 deficient Jurkat T cells, the pJNK response is exaggerated indicating that in the absence of a functional Fas apoptotic response, an alternate non-apoptotic pathway is elicited. Further, in HH cells, a CTCL resistant to Fas apoptosis, pJNK activation was upregulated when compared to HUT78, a highly apoptosis sensitive cell line. In an attempt to identify the upstream activators of Fas-induced pJNK, we studied the role of the kinase RIP1, a known component of the Fas DISC. Inhibition of RIPK1 decreased pJNK activation in Jurkat cells and RIP deficient Jurkat cells were unable to activate pJNK upon FasL stimulation. Immunoprecipitation studies also indicated a preferential recruitment of RIPK1 to the Fas DISC in apoptosis resistant T cells. These preliminary results indicate a role for Fas mediated non-apoptotic MAPK signaling in early activated naïve T cells that precedes its traditional apoptotic role in the elimination of effector memory cells.
Innate imprinting of resident alveolar macrophages in RSV-induced allergic asthma exacerbations (P6003)
Authors: Naessens Thomas , Schepens Bert , Pollard Charlotte , Bogaert Pieter , De Koker Stefaan , Saelens Xavier , Grooten Johan
Published: January 2023

Acute RSV-infections are seen as instigators of exacerbations in allergic asthma patients. The question which cells and/or molecules that orchestrate viral-induced asthma exacerbations remains unanswered. We now show that the innate (antiviral) functionality of post-asthma resident alveolar macrophages (rAM), present after the clearance of the allergic bronchial inflammation, is imprinted by this preceding allergic inflammation. In contrast to naïve rAM, post-asthma rAM exhibited strong proinflammatory reactivity during RSV-infection which significantly contributed to the increased global pulmonary inflammation observed in RSV-infected post-asthma mice. The strong proinflammatory reactivity of post-asthma rAM was mainly due to increased NF-κB p65 activation in these cells upon RSV-infection. Furthermore, compared to naïve rAM, post-asthma rAM displayed no direct contribution to virus clearance. In fact, post-asthma rAM did not show efficient uptake of the virus. Finally, compared to naïve rAM, the basal activation state of post-asthma rAM was found to be strongly skewed towards an M2-phenotype. Consequently, the polarized M2-activation status of post-asthma rAM can serve as a possible explanation for the aberrant antiviral innate responses of these cells. Together, innate imprinting of rAM by a preceding allergic inflammation strongly impairs RSV-antiviral responses of the post-asthma lungs and can therefore contribute to the onset of asthma exacerbations.
Sex differences in macrophages in allergic inflammation (P6043)
Authors: Heller Nicola , Robinson Dionne , Klein Sabra , Fang Jessie
Published: January 2023

Patients hospitalized for asthma are up to 3 times more likely to be female than male and women comprised 64% of asthma-associated deaths in 2009. Hallmark features of allergic lung inflammation are worse in female mice compared to male mice. Both allergen-sensitized and challenged female mice and humans with asthma have greater numbers of alternatively activated macrophages (AAM) in the lung, compared to males. IL-4 or IL-13 triggers AAM differentiation, characterized by expression of the arginase I, Fizz1 and Ym1 genes in mice. To test the role of estrogen (E2) in AAM differentiation in allergic lung inflammation, we subjected female mice to ovariectomy (OVX) and implantation with E2 or placebo, followed by a typical ovalbumin-induced allergic lung inflammation protocol. There were more Ym1+ macrophages, giant cells and epithelial cells and more mucus-producing cells (PAS+) the OVX + E2 allergic group, compared to placebo. Female bone marrow-derived macrophages stimulated with IL-4 expressed double the amount of mRNA for Ym1 compared to male cells and E2 augmented expression of IL-4-induced Fizz1 mRNA and protein in both male and female macrophages. Finally, we measured distinct differences in characteristic AAM surface markers on human male and female monocytes stimulated with IL-4. Taken together, these data indicate there are intrinsic sex differences in AAM differentiation and that E2 enhances AAM differentiation and allergic lung inflammation.
STAT6 controls Tregs in vivo to enhance allergic lung inflammation (P6049)
Authors: Dorsey Nicolas , Chapoval Svetlana , Smith Elizabeth , Keegan Achsah
Published: January 2023

STAT6 plays a pivotal role in IL-4-mediated allergic responses. Several studies indicate that regulatory T cells (Treg) can be modulated by IL-4 in vitro. We previously showed that STAT6-/- mice are highly resistant to allergic lung inflammation even when wild type (WT) Th2 effectors were provided and have increased numbers of Tregs. However, the role of STAT6 in modulating Tregs in vivo during allergic lung inflammation has not been thoroughly investigated. To investigate Treg and STAT6 interaction during allergic inflammation, STAT6-/-, STAT6xRAG2-/- and RAG2-/- mice were subjected to Ova sensitization and challenge following adoptive transfer of ova-specific, WT Th2 effectors. Treg depletion was performed in a duplicate STAT6-/- mouse group using anti-CD25 (PC61). As expected, STAT6-/- mice were highly resistant to airway inflammation and remodeling. In contrast, allergic lung inflammation was partially restored in STAT6-/- mice treated with PC61 to levels observed in STAT6xRAG2-/- mice. When the susceptible STAT6xRAG2-/- mice were also given Tregs along with Th2 effectors, allergic inflammation and airway remodeling was suppressed to levels comparable to STAT6-/- mice. These results demonstrate that STAT6 acts to suppress the development of Tregs in vivo in order to promote allergic airway inflammation.
Regulation of CD4+ T cell responses: The impact of limiting CD28 costimulation (P1083)
Authors: Briggs Zoe , Sansom David
Published: January 2023

Controlling T cell activation is important to prevent autoimmunity. Costimulatory molecules CD28 and CTLA-4 bind the same ligands (CD80 and CD86) but cause opposite outcomes: CD28 signalling enhances TCR signals whereas CTLA-4 inhibits T cell activation. The mechanism of CTLA-4 action has been unclear however we have identified a novel mechanism whereby CTLA-4 removes its ligands from antigen presenting cells by trans-endocytosis. This reduces the availability of costimulatory ligands for CD28 engagement and thereby regulates T cell activation. This model predicts that there is a threshold number of costimulatory ligands required for T cell stimulation. We are using a dose-dependent inducible expression system to determine this threshold and compare the two ligands. We have also begun to examine the functional consequences of stimulating CD4+ T cells while restricting costimulation and observed that CD28 costimulation is not always required for T cell activation and proliferation, but without CD28 engagement T cells do not fully differentiate or upregulate a number of functional proteins. Interestingly in our studies, human T cells activated in the absence of CD28 signalling are not classically anergic. Establishing the characteristics of T cells activated without adequate costimulation may be a useful surrogate for CTLA-4 function and improve our understanding of immune responses in the context of autoimmunity, tumour biology or transplant rejection.
Galectin-1 as a novel effector molecule of cytotoxic cells (P1061)
Authors: Amarante-Mendes Gustavo , Machado Tiago , Vieira Narciso , Zorn Telma , Dias-Baruffi Marcelo , Rodrigues Maurício , Martin Seamus
Published: January 2023

The mechanism of cytotoxicity mediated by Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTL) is involved in the control of cancer progression, virus mediated diseases and many other intracellular related pathologies. These cells are particularly adapted to promote contact mediated cell death due to their ability to produce a special type of organelles, namely secretory lysosomes, aka cytotoxic granules. By proteomic analysis, we identified dozens of putative novel cytotoxic granule proteins in NK and CTL, as well as most of the already known granule constituents. Here we show that galectin-1, a protein previously described to have immunomodulatory activity, including regulation of apoptosis, is indeed present inside the cytotoxic granules. Moreover, galectin-1 deficient mice display an impaired in vivo cytotoxic activity against antigen-specific targets, suggesting that galectin-1 is a novel effector component of the cytotoxic cell machinery.
FcRn overexpression in transgenic animals results in robust immune response (P1069)
Authors: Kacskovics Imre , Schneider Zita , Vegh Attila , Ilias Attila , Baranyi Maria , Balogh Peter , Cervenak Judit , Kurucz Istvan
Published: January 2023

Our previous studies have shown that overexpression of the neonatal Fc receptor (FcRn) in transgenic animals (mice and rabbits) leads to (1) improved IgG protection; (2) larger numbers of antigen specific B cells; (3) generation of antibodies against weakly immunogenic antigens; (4) generation of antibodies against highly conserved antigens (breaking immune tolerance); (5) increased humoral diversity and (6) higher levels of circulating antibodies. FcRn transgenesis thus confers a number of practical benefits, including faster antibody production, higher antibody yields and improved generation of hybridomas for monoclonal antibody production. To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized our FcRn transgenic animals. Our analyses showed strong expression of the FcRn transgene in professional antigen presenting cells and we also found that overexpression of the FcRn significantly improves antigen presentation in case of antigen-IgG immune complexes. We have recently analyzed several key factors in the maturation of the immune response, including the structure of the spleen, number and structure of germinal centers, the level of T cell activation and the affinity of the generated antibodies. These results provide further insights of those mechanisms that are involved in the augmented humoral immune response in transgenic animals that overexpress FcRn.
Detection of phosphorylated signaling proteins using phospho-specific monoclonal antibodies (P1115)
Authors: Miletic Ana , Funatake Castle , Scott Bradley , Alvarez Miguel , Chen Helen , Li Shourong , Schrantz Nicolas , Just Peggy
Published: January 2023

Triggering of extracellular surface receptors results in the initiation of numerous intracellular signaling cascades that are propagated via phosphorylation of specific tyrosine, serine, and/or threonine residues on signaling proteins. Until recently, analysis of intracellular phospho-signaling relied solely upon western blotting. The development of phospho-specific antibodies for use in flow cytometry has become a powerful tool for the detection of signaling events and has numerous advantages over western blotting; it is faster, requires fewer cells, and can be performed in a heterogeneous population of cells in conjunction with surface marker staining to allow further phenotyping of each population present in the sample. To this end, we have developed monoclonal antibodies specific for phosphorylation of various signaling molecules such as ERK1/2 (T202/Y204), STAT1 (Y701), STAT4 (Y693), and STAT5 (Y695). Here, we show the detection of phosphorylation of different intracellular signaling by flow cytometry across species in a treatment- and cell type-specific manner. One issue complicating phospho-specific flow cytometry is the inability to detect certain surface markers because of either epitope or fluorochrome destruction using the current, standard staining protocol. We show options that allow multi-parametric staining with different phospho-specific antibodies.
A West Nile Virus NS4B P38G mutant strain induces adaptive immunity via TLR7-MyD88-dependent and independent signaling pathways. (P4317)
Authors: Xie Guorui , Welte Thomas , Wang Jia , Wang Ye , Whiteman Melissa , Wicker Jason , Saxena Vandana , Cong Yingzi , Barrett Alan , Wang Tian
Published: January 2023

West Nile virus (WNV)- induced neurological disease has become a public health concern. No vaccines have been approved for human use. We have previously shown that an attenuated WNV, the nonstructural (NS) 4B-P38G mutant, induced stronger innate cytokine and T cell responses than wild-type WNV. Mice immunized with this mutant were all protected from subsequent lethal wild-type WNV infection. Here, we found both Toll-like receptor (TLR7) and MyD88 expression are required for host protection from NS4B P38G mutant infection. Infected MyD88-/- and TLR7-/- mice had lower IL-12 expression, IgM production, and reduced effector T cell functions. MyD88-/- and TLR7-/- dendritic cells (DC)s exhibited impaired antigen-presenting functions compared to wild-type DCs. At the late stage of infection or during secondary challenge with wild-type WNV, MyD88-/- and IL-1R-/- mice displayed reduced memory T cell responses compared to wild-type mice; whereas TLR7-/- mice had normal T cell functions. Moreover, immunization of MyD88-/- and TLR7-/- mice with NS4B P38G mutant strain offer partial or full protection respectively from subsequent lethal wild-type WNV infection. Thus, TLR7-MyD88 signaling is required for initial T cell priming; whereas the TLR7-independent MyD88 signaling is involved in memory T cell development during NS4B P38G mutant infection. Results can be utilized as a paradigm to aid in the rational development of efficacious live attenuated flavivirus vaccines.
Prime-boost vaccination with a live attenuated influenza vaccine elicits T cell-mediated cross-strain protective immunity (P4287)
Authors: Zeng Mingtao , Li Junwei , Arévalo Maria , Chen Yanping
Published: January 2023

Antigen drift and shift of influenza viruses requires frequent reformulation of influenza vaccines. This stresses the need for a universal influenza vaccine. In this study, mice were vaccinated with the trivalent live attenuated (LAIV, nasal spray FluMist) and inactivated (TIV, flu shot FluZone) influenza vaccines by 1-3 dose prime-boost vaccination regimens. While vaccination with both LAIV and TIV elicited strong humoral responses, only LAIV induced significant protection against challenge with the non-vaccine strain H1N1 PR8 influenza virus. Furthermore, protective immunity against PR8 was dose-dependent. Of note, IL-2 and IFN-γ cytokine secretion in the lung alveolar fluid were significantly elevated in mice immunized with LAIV. Moreover, T cell depletion of LAIV immunized mice compromised protection, indicating that T cell-mediated immunities were required. In contrast, passive transfer of sera from mice immunized with LAIV into naïve mice failed to protect against PR8 challenge. To verify these results, we performed virus neutralization assays in vitro and confirmed that LAIV did not induce cross-strain neutralizing antibodies against PR8 virus. Finally, we showed that 3 doses of LAIV also provided protection against challenge with additional two heterologous viruses FM/47 (H1N1) and HK/68 (H3N2). These results support the potential use of the LAIV as a universal influenza vaccine under prime-boost vaccination regimen.
Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB (P3208)
Authors: Im Suhn-Young , Kim Kyoung-Jin , Kim Han-A , Lee Hern-Ku , Seo Kook Heon
Published: January 2023

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remain to be established. In this study, we investigated the ability of cholera toxin (CT) to induce the transcription factor NF-κB. Feeding of CT abrogated OVA-induced oral tolerance, as evaluated by OVA-specific serum Ab responses, footpad swelling reaction (FSR), and CD4+ T cell proliferation. CT feeding resulted in clear NF-κB activation in both mesenteric lymph node (MLN) and Peyer’s patch (PP) cells. Supershift analysis demonstrated that mainly one heterodimer type, p50-p65, i.e., canonical NF-κB, occurred in response to CT. In addition to NF-κB activation, CT induced the expression of mRNA in NF-κB-dependent proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α in vivo as well as in vitro. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs), but not in control siRNA. ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation in MLN and PP intestinal lymphoid cells. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.
TAM receptors and their role in the regulation of lung innate immunity (P3236)
Authors: Fujimori Toshifumi , Snelgrove Robert , Hussell Tracy
Published: January 2023

Background: The role of TAM receptors are well characterised in autoimmunity and cancer biology. Their role in infectious diseases is now being elucidated. The goal of this study is to characterise TAM receptor expression profiles of various innate immune components at homeostasis as well as in various infection models. The hypothesis of this study is that up-regulation of TAM receptors, AXL and MerTK are responsible for the de-sensitisation of airway macrophages to bacteria following influenza infection. Furthermore, I hypothesise that TAM receptors may be involved in limiting innate immune activation at homoeostasis. Methods: Using a murine model, we characterised TAM receptor expression on various innate populations in the lung following viral (influenza A) and bacterial (Streptcoccus pneumoniae) infections. We also investigated the role of AXL in influenza/bacteria single and co-infection using AXL-/- mice. Results: AXL and MerTK are differentially expressed by innate populations in the lung depending on their location. Monocytes and neutrophils which infiltrate into the alveolar space during influenza infection significantly up-regulate TAM receptor expressions. Lack of AXL significantly potentiates innate immunity by upregulating the release of inflammatory cytokines in vitro. Conclusion: Up-regulation of AXL following a viral infection may explain the exacerbation of secondary bacterial complications as its upregulation limits the innate inflammatory responses.
CXCR5-CXCL-13-pathway in protective immunity to tuberculosis (P3190)
Authors: Khader Shabaana , Slight Samantha , Gopal Radha
Published: January 2023

One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we show that organized ectopic lymphoid structures containing CXCR5+ T cells are found in Mtb-infected lungs. In addition, we show that in experimental Mtb infection models, the presence of CXCR5+ T cells inside ectopic lymphoid structures are associated with immune control. Furthermore, in a mouse model of Mtb infection, we show that activated CD4+ CXCR5+ T cells accumulate in Mtb-infected lungs, and produce proinflammatory cytokines. Absence of CXCR5 results in increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We show that CXCR5 expression on T cells mediates correct T cell localization within TB granulomas, efficient macrophage activation, promoting protection against Mtb infection and facilitating lymphoid follicle formation. We also show that inducing T helper 17 (Th17) memory responses using mucosal immunization strategies improves vaccine-induced immunity against TB highlighting the potential use of IL-17-CXCR5-CXCL-13 axis in future TB vaccine design and therapy.
Non-covalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact Gram-positive extracellular bacteria (P3083)
Authors: Colino Jesus , Duke Leah , Snapper Clifford
Published: January 2023

Intact Streptococcus pneumoniae, expressing type 14 capsular polysaccharide (PPS14) and type 3 Streptococcus agalactiae containing a PPS14 core capsule identical to PPS14, exhibit non-covalent associations of PPS14 and bacterial proteins, in contrast to covalent conjugates. Secondary immunization with conjugate and S. agalactiae, although not S. pneumoniae, results in a boosted response. However, in contrast to conjugate, PPS14-specific IgG responses to bacteria lack affinity maturation and use the 44.1-idiotype. To better understand the mechanism underlying this dichotomy we developed a minimal model of intact bacteria utilizing 1μm latex particles to which PPS14 and pneumococcal surface protein A (PspA) were stably attached, but not directly linked, in contrast to PPS14-PspA conjugate. PPS14+[PspA] beads, similar to conjugate, induced in mice boosted PPS14-specific IgG secondary responses, dependent on T cells and ICOS-dependent costimulation, and in which priming could be achieved with PspA alone. In contrast to conjugate, but similar to bacteria, the primary PPS14-specific IgG response to PPS14+[PspA] beads peaked rapidly, with the secondary response highly enriched in 44.1-idiotype and lacking affinity maturation. These results demonstrate that non-covalent association in a particle, of polysaccharide and protein, recapitulates essential immunologic characteristics of intact bacteria that are distinct from soluble covalent conjugates of these respective antigens.
IL-1α and IL-1β play non-redundant roles in limiting in vivo Aspergillus fumigatus pulmonary growth (P3092)
Authors: Obar Joshua , Lehmann Margret , Caffrey Alayna , Watschke Christopher , Shepardson Kelly , Barker Bridget , Cramer Robert
Published: January 2023

Aspergillus fumigatus is a mold that causes severe pulmonary infections in humans, such as invasive pulmonary Aspergillosis (IPA). Currently, our knowledge of how A. fumigatus growth is controlled in the respiratory tract is limited. Phagocytic alveolar macrophages constitute the first line of defense against inhaled A. fumigatus conidia; subsequently, neutrophils and macrophages are sequentially recruited to the respiratory tract to control fungal growth and germination . But how neutrophils and macrophages are recruited to the respiratory tract after A. fumiguatus infection remains ill defined. During sterile inflammation early neutrophil recruitment is dependent on IL-1α, while late macrophage recruitment is dependent on IL-1β. A. fumigatus infection induced the expression of both IL-1α and IL-1β. However, IL-1R1- and ASC-deficient mice displayed differential susceptibility to IPA. IL-1R1-deficient mice were highly susceptible to A. fumigatus infection as measured by increases in fungal growth, lung tissue damage, and fungal dissemination. In contrast, ASC-deficient mice were only mildly susceptible to A. fumigatus infection. IL-1R1- and ASC-deficient mice displayed a severe defect in neutrophil recruitment during the first 48h and a defect in macrophage recruitment after 48h, respectively. Taken together, our data reveal important non-redundant roles for the IL-1α and IL-1β in controlling A. fumigatus infection in the lung.
Highly immunogenic vaccine for prevention and therapy of malignant mesothelioma (P4437)
Authors: De Groot Anne , Bertino Pietro , Hoffman Peter , Carbone Michele , Siccardi Antonio , Terry Frances , Cousens Leslie , Moise Leonard , Martin William
Published: January 2023

Malignant mesothelioma (MM) is a deadly cancer with increasing incidence and no effective treatment options. We have previously constructed recombinant Fowlpox virus (FP) vectors encoding full-length survivin protein (FP-surv), which induced complete tumor regression in 40% of mice bearing subcutaneous MM. Macro- and microscopic evaluation of the tumor tissue showed that the tumors in vaccinated mice had large necrotic cores resulting from enhanced survivin-specific cytotoxic T lymphocyte (CTL). We aim to refine our novel FP vaccine by focusing on the most immunodominant survivin epitopes, thus enhancing its efficacy. We have identified additional high quality antigenic targets specifically upregulated in MM that will increase the breadth of T cell responses. We have used the EpiMatrix system to identify CD4+ and CD8+ T cell epitopes for synthesis and testing. Following T cell assays, highly immunogenic peptides will be selected that specifically stimulate CTLs, without activating T regulatory cells (Treg) that interfere with anti-cancer immune responses. We will include the selected peptides in the next-generation vaccine, FP-surv.2, which we expect to be more effective for inducing an immune response against MM cells than FP carrying the entire survivin gene (FP-surv). In addition, we anticipate that this response may include a higher number of long-lived memory T cells that can be exploited to prevent MM development in high-risk predisposed subjects.
The clinical implication of HAV viral load and HAV proteins-specific IFN-gamma production in patients with acute hepatitis A (P6120)
Authors: Lee Hyun Woong , Chang Dong-Yeop , Shin Eui-Cheol
Published: January 2023

The relationship between immune responses to hepatitis A virus (HAV) and the clinical manifestation of HAV infection have been obscured. We investigated the clinical manifestation of acute hepatitis A (AHA) and the relationship between HAV-specific, IFN-gamma-producing T cell response and clinical outcomes in patients with AHA. We analyzed 770 patients diagnosed with AHA for their clinical manifestation. Among them, 128 patients with a prothrombin time (PT) of less than 40% of normal were classified as “severe acute hepatitis (s-AH)”. Others were defined as having “mild acute hepatitis (m-AH)”. In 110 patients, direct ex vivo IFN-gamma ELISpot assays were performed with peripheral blood mononuclear cells using HAV proteins. Mean values of serum HAV RNA at admission were 4.79 log10 copies/ml in s-AH, and 3.30 log10 copies/ml in m-AH (P = 0.003). Viral load showed a significant correlation with ALT (r = 0.417, P = 0.0032) and PT levels (r = -0.453, P = 0.0012). In addition, the mean frequencies of IFN-gamma-producing T cells against VP1-2A (aa 669-782), and 2C(aa 1121-1234) proteins were significantly lower in patients with s-AH, compared to patients with m-AH (3.3 vs. 17.7, P < 0.001; 2.1 vs. 13.3, P < 0.001). In view of viral factors, the load of HAV is closely correlated with liver damage and disease severity in patients with AHA. As a host factor, HAV-specific, IFN-gamma-producing T cells might induce effective viral eradication and result in mild hepatitis
Gut bacteria modulates angiogenesis and corneal immunopathology after herpes simplex virus infection (P6136)
Authors: Richardson Raphael , Bhela Siddheshvar
Published: January 2023

The ocular disease herpetic stromal keratitis results in blindness due to an immunopathological attack mainly orchestrated by T cells and neutrophils. Recently, it has become apparent that lesion severity at local sites may be influenced by the balance of microbes in distal locations such as the gut, respiratory tract and even the skin. Reasons are still being assembled but appear to involve an influence on both innate and adaptive immunity. Since HSK is an uncommon outcome of ocular HSV infections in the infected individual, we sought to determine if the nature of enteric flora would influence the expression of HSK. A murine model of HSK was used to evaluate the concept. Mice were ocularly infected with HSV and separated into two groups. The controls received only sucrolose while the experimental group received sucrolose and a combination of antibiotics (Ampicillin, Gentamicin, Metronidazole, Neomycin, and Gentamicin) 3 weeks before infection to deplete gut flora and treatment continued until termination of the experiment at day 15. Significant decreases in corneal angiogenesis and HSK was evident in the antibiotic treated recipients. Analysis of inflammation cell numbers and molecules involved in angiogenesis also showed significant differences between treated and control groups. Further mechanistic studies to explain for the differences observed will I be reported and discussed.
Role of peripheral immune response in regulating chemokine and pro-inflammatory cytokine profiles during acute viral encephalitis (P6147)
Authors: Steel Christina , Brevis Kimberly , Tavakoli Susan , Kim Woong-Ki , Sanford Larry , Ciavarra Richard
Published: January 2023

Vesicular stomatitis virus (VSV) is a neurotropic virus that rapidly invades the central nervous system (CNS) following a single intranasal application and induces acute encephalitis essential for survival. Invasion of the CNS by inflammatory cells is thought to reflect virus-induced production of chemokines. To assess this possibility and to broadly characterize this response during the initial and acute phases of infection, brain chemokine and pro-inflammatory cytokine RNA profiles were determined in naïve and VSV-infected mice using qPCR arrays and flow cytometry. RNA transcript levels for several CC, CXC chemokines and cytokines were already elevated one day post infection with several ligand/receptor pairs co-induced which precedes peripheral leukocyte recruitment to the CNS. During acute VSV encephalitis (days 6-8), RNA levels became more robust with expression of additional chemokines and cytokines and a mixed leukocyte (previously reported). Chemokine responses associated with VSV encephalitis were blunted in mice acutely depleted of either peripheral dendritic cells or T cells. Several CC receptors for induced chemokines were highly expressed on microglia isolated from encephalitic brains. Infiltrating neutrophils and T cells failed to express these same receptors. These results suggest that different cytokines and/or chemokines may be responsible for recruitment of peripheral leukocyte subsets into the CNS, while others may drive the observed microgliosis.
Inflammasome regulates host immune responses to enteric pathogen (P4237)
Authors: Liu Zhiping , Kanneganti Thirumala-devi
Published: January 2023

Inflammasome is emerging as a central regulator of inflammatory immune responses. Nlrp3 inflammasome has been shown to be critical for intestinal homeostasis and inflammation using chemical dextran sodium sulfate -induced colitis model. However, the roles of inflammasome in other more natural models of colitis such as colitis induced by enteric bacteria (e.g., Citrobacter rodentium) are not well known. Our study identified that mice lacking the inflammasome components Nlrp3, Nlrc4 and Caspase-1 were highly susceptible to C. rodentium-induced colitis, demonstrated by more weight loss, higher bacterial burden and more severe histological lesions, in comparison to wild-type (WT) mice. Inflammasome-deficient mice displayed much higher infiltration of neutrophil and macrophage into spleen and colon. In addition, in comparison to WT mice, Caspase 1-deficient mice had higher percentage of active T cell that secret more IFN-γ, which might significantly contribute to the elevated inflammation in intestinal mucosa. We also identified that inflammasome-mediated production of IL-1β and IL-18 are critical for host defense against C. rodentium infection. Dissect the mechanisms how these inflammatory cytokines regulate host immune responses to enteric pathogens will be of great interest to the understanding of inflammatory bowel disease pathogenesis.
Genetic analysis of the role of mast cell- and basophil-derived TNF in severe sepsis (P4205)
Authors: Piliponsky Adrian , Lahiri Asha , Truong Phuong , Lilla Jennifer , Reber Laurent , Nedospasov Sergei , Galli Stephen
Published: January 2023

Mast cell-derived TNF can increase mortality in C57BL/6-KitW-sh/W-sh mice after severe cecal ligation and puncture (CLP). However, the mutation responsible for mast cell deficiency in these mice induces other abnormalities that may have influenced this finding. Therefore, we generated mice in which TNF production is specifically ablated in mast cells and basophils by crossing Cpa3-Cre mice with Tnffl/fl mice (Cpa3-Cre; Tnf fl/fl). Mast cells and basophils from Cpa3-Cre; Tnf fl/fl mice are impaired in their ability to produce TNF after stimulation by either IgE/antigen or LPS. Dermal leukocyte numbers in the ear pinnae of Cpa3-Cre; Tnf fl/fl mice were reduced at sites of IgE-dependent passive cutaneous anaphylaxis (PCA), suggesting that local release of mast cell- and/or basophil-derived TNF contributes to leukocyte recruitment after PCA. Serum TNF levels and intraperitoneal neutrophil numbers were significantly reduced after i.p. challenge with a sub-lethal dose of LPS, indicating that mast cells and/or basophils also contribute to systemic levels of TNF in this setting. Notably, Cpa3-Cre; Tnf fl/fl mice exhibited higher survival rates, reduced drops in body temperature, significantly lower numbers of intraperitoneal neutrophils, and decreased local and systemic TNF levels after severe CLP. Our findings in Cpa3-Cre; Tnf fl/fl mice provide strong evidence that mast cell- and/or basophil-derived TNF can contribute to increased mortality and inflammation in severe sepsis.