bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of the chronic inflammatory response in a murine model of chronic granulomatous disease (54.8)
Authors: Ryan Sean , Cobb Brian
Published: January 2023

Chronic granulomatous disease (CGD) is a primary innate immunodeficiency associated with a defective phagocyte NADPH oxidase complex and thus increased susceptibility to recurrent bacterial and fungal infections. Although antimicrobials have proven useful in resolving primary infection, CGD patients face complications thought to stem from an unresolved hyper-inflammatory state. Using a murine model of CGD we found that a sterile inflammatory insult induces a systemic and long-lived chronic inflammatory response not seen in wild type controls. This persistent inflammatory response is characterized by an increased and sustained expansion of myeloid cells, including Gr1+Ly6G+ cells actively producing the pro-inflammatory IFNγ cytokine and a Gr1+Ly6G- myeloid-derived suppressor cell (MDSC) population. Interestingly, although the expansion of a functional MDSC population would suggest a viable mechanism to control the persistent hyper-inflammatory response, we found that the Gr1+Ly6G+ subpopulation antagonizes MDSC function which significantly affects control of adaptive T cell responses, including responses to inhaled antigen in the lung, a frequently affected organ in CGD patients. Understanding CGD immune dysregulation beyond the primary innate immunodeficiency allows for the development of targeted therapies to potentially prevent inflammatory complications in CGD patients and possibly other patients at risk of complications due to chronic inflammation.
Acinetobacter baumannii virulence requires LPS activation of TLR4 (55.12)
Authors: Lin Lin , Tan Brandon , Baquir Beverlie , Ho Tiffany , Pantapalangkoor Paul , Spellberg Brad
Published: January 2023

Background: New treatments are needed for lethal infections caused by extreme-drug resistant (XDR)/pan-drug resistant (PDR) Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance during non-lethal A. baumannii pulmonary infection in mice. We sought to better to understand the role of LPS-TLR4 interactions in A. baumannii pathogenesis. Methods: LPS was harvested from nine clinical isolates of A. baumannii and then assayed for TLR4 activity using the HEK-Blue cells. Wild type and TLR4 mutant mice were infected via the tail-vein with A. baumannii. Results: After A. baumannii systemic infection, 100% of wild type vs. 0% of TLR4-mutant mice died. Wild type mice developed septic shock, typified by hypothermia, acidemia, and elevated inflammatory cytokine production, wherease TLR4 mutant mice did not, despite having similar bacterial burden in organs. Density of LPS in bacteria (ng/bacillus) and TLR4 activation by LPS extracted from the bacteria did not correlate with in vivo virulence. Amount of LPS shed during growth was the key correlate with in vivo virulence. Summary: LPS activation of TLR4 is a key pathogenetic factor of A. baumannii during systemic infection. Abrogation of TLR4 function protects mice from invasive A. baumannii infection by preventing induction of septic shock. These results suggest new areas of focus for developing novel therapeutics targeting these XDR/PDR infections.
Earlier protective response and milder clinical course of Mycobacterium bovis BCG infection in mice lacking an adapter molecule CARD9 (55.6)
Authors: Kawakami Kazuyoshi , Takahashi Yurie , Yamamoto Hideki , Nakamura Yuri , Ishii Keiko , Hara Hiromitsu
Published: January 2023

CARD9 plays a crucial role as an adaptor molecule in signal transduction triggered through C-type lectin receptors that are known to recognize polysaccharides from various fungal microorganisms. In this study, we examined the role of CARD9 in the host defense to Mycobacterium bovis BCG that has polysaccharide cell-wall components such as lipoarabinomannan. C57BL/6 (WT) and CARD9 knockout (KO) mice were infected intratracheally with M. bovis BCG (Tokyo strain) per mouse. The number of viable bacterium, cytokine synthesis and histopathological findings in lungs were evaluated. CARD9KO mice cleared more bacteria in lungs than WT mice on day 28 after infection. In a histopathological analysis, the lung tissues from CARD9KO mice showed poorer granuloma formation and fewer bacilli in lungs than WT mice on days 14 and 28 after infection. The levels of IFN-γ, a key cytokine for the host defense to this infection, in the bronchoalveolar lavage fluids and its expression in T, NK and γδ T cells on day 7 were increased in CARD9KO mice compared to those in WT mice, although the production of this cytokine was significantly reduced in Card9KO mice on day 28. By contrast, the expression of IL-17A in lungs was not much different between WT and CARD9KO mice. These results indicate that CARD9 is dispensable for elimination of M. bovis BCG and granulomatous response, which suggests a complicated mechanism involved in the role of this molecule in the earlier host response to M. bovis BCG.
Are YFAK-specific TCR retrogenic mice whose splenocytes secrete high levels of IL-10 protected from EAE? (56.9)
Authors: Ng Sze-Ling , Strominger Jack
Published: January 2023

Regulatory T cells (Tregs) are important mediators of a normal immune system response, and the misregulation of Tregs can result in susceptibility to various autoimmune diseases. The random amino acid copolymer YEAK (Copaxone) is widely used in the therapy of human multiple sclerosis (MS), as well as in the amelioration of experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. Our lab has identified a more effective copolymer, YFAK. Both YEAK and YFAK ameliorate EAE by inducing regulatory T cells that secrete the immunomodulatory cytokine IL-10, which mediates bystander immunosuppression. The precise mechanisms underlying the development, function, and maintenance of regulatory T cells are unknown. Therefore, we have generated a YFAK-induced regulatory T cell line and cloned its specific T cell receptors (TCRs) in order to investigate the role of the TCR in the epigenetic change that leads to selective IL-10 secretion. Retrogenic mice expressing YFAK-specific TCRs have been generated. Splenocytes from these mice secrete high levels of IL-10, indicating that the TCR encodes information for the secretion of this cytokine. In addition, experiments in progress asking whether these TCR retrogenic mice are better protected from EAE will be reported. These studies can inform the exploitation of regulatory T cells in the treatment of autoimmune diseases such as multiple sclerosis.
Mycoplasma pneumoniae CARDS toxin exacerbates ovalbumin induced asthma in BALB/c mice (57.9)
Authors: Medina Jorge , Brooks Edward , Le Saux Claude , Chaparro Adriana , Principe Molly , Kannan Thirumalai , Baseman Joel , Dube Peter
Published: January 2023

Mycoplasma pneumoniae is a common human respiratory pathogen that causes acute and chronic respiratory infections that can result in a variety of pulmonary and extra-pulmonary pathologies. M. pneumoniae is associated with exacerbations of human asthma and has significant impact on pathology through unclear mechanisms. We recently demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin induces an allergic asthma-like disease in naïve mice, as evidenced by a Th2 response, mucus metaplasia and airway hyperreactivity. In this study we examined CARDS toxin’s effects on preexisting asthma using an ovalbumin mouse model of asthma. We show that CARDS toxin can exacerbate pathologies associated with ovalbumin-induced asthma in BALB/c mice both histologically and physiologically. These results were highlighted by increased collagen deposition, shown by picrosirius red staining around medium airways and an increase in eosinophils in the alveolar spaces, shown by cellular differentials. Additionally, these findings are paralleled by heightened Th2 cytokine and chemokine responses as measured by qPCR or ELISA. Finally, lung function assessment via a Flexivent system revealed increases in airway hyperreactivity demonstrated by increased resistance and decreased compliance. The data demonstrate CARDS toxin as a causal factor in the exacerbation of experimental allergic asthma and implicates CARDS toxin as a mediator of morbidity in human asthma.
Mobilization of Notch ligand to a poly-HEMA scaffold to promote thymopoiesis and model T cell development (58.19)
Authors: Falcon Jessica , Lelkes Peter , Conway Daniel
Published: January 2023

Notch signaling is critical for thymopoiesis. Notch ligand’s expression on thymic epithelial cells is crucial to the development naïve T lymphocytes and therefore characterized as one of the primary signaling pathways. We are developing a biomaterial-based model of thymopoiesis. We hypothesize that by binding the Notch ligand onto a biomaterial based scaffold, we can maintain the Notch ligand in its active form, and differentiate mouse embryonic stem cells into T-lymphocytes. The common protocol for differentiating mouse embryonic stem cells into T lymphocytes is using the OP9 system. In this system, the stem cells are supported by a monolayer of stromal cells that express the Notch ligand on its surface. Our project aims to bypass the monolayer of living cells and replace them with a biocompatible inorganic matrix that provides the cells with the necessary environment to differentiate into T lymphocytes. We are currently using Poly(2-hydroxyethyl methacrylate) which is both biocompatible and has a similar Young’s modulus of the lymph nodes, which also expresses Notch. We have confirmed activation of the ligand through ELISA and are in the process of differentiating the embryonic stem cells. This project holds significant implications for the field on immunology. If successful, this early prototype can become the basis of the first artificial thymus.
Modulation of the Inflammatory and Metabolic Profile of Pancreatic β-cells Following TLR Ligation. (61.5)
Authors: Mc Evoy Fiona , Holthofer Harry , Loscher Christine
Published: January 2023

Type 2 Diabetes is a multifactorial metabolic disorder characterised by chronic hyperglycemia, insulin resistance and an insulin secretion deficit due to β cell exhaustion. Evidence from animal models and humans has indicated that systemic inflammation plays a role in the pathophysiological processes of diabetes and is facilitated by innate immune responses. TLRs (Toll-like receptors) are key innate immune receptors that recognise both exogenous (pathogen-associated molecular patterns) and endogenous (molecules generated after stress and free fatty acids) ligands. Their activation leads to a proinflammatory reaction that is fundamental for the initiation of an innate immune response and subsequent adaptive responses, but can also damage tissues. Although TLR expression is increased in many inflammatory disorders the effects of metabolic aberrations on TLRs and their role in diabetes is still emerging. Our objective was to elucidate the role of TLRs in the insulin secreting β cell line MIN6. We have shown that MIN6 cells express surface markers vital to the immune response including TLR2, TLR4, TLR5, MHCII, CD14 and CD40. Specific TLR stimulation alters β-cell insulin secretion in response to glucose. It also results in the secretion of various chemokines (Mip-1, Mip-2, MCP and RANTES), and alters the secretion of cytokines (IL-4 & IL-27). These results suggest that engagement of TLRs on β-cells has the ability to affect both the metabolic and inflammatory profile of the cell.
Fluorescent transgenic reporter mice can be used for in vivo Imaging of lymphatic vessels and high endothelial venules in a Sjögren’s model (61.7)
Authors: Ruddle Nancy , Truman Lucy , Bentley Kevin , Alonso-Gonzalez Noelia
Published: January 2023

Dramatic changes occur in the lymph node vasculature after immunization that affect antigen priming. There is a temporary defect in lymph flow and high endothelial venules (HEV) revert to an immature phenotype. To visualize these dynamic changes, we have made transgenic mice with red-fluorescent lymphatic vessels and green-fluorescent HEV that are suitable for live imaging. The lymphatic vessels of ProxTomxHec6st/eGFP reporter mice were successfully visualized in vivo using 2-photon laser scanning microscopy. We are interested in tertiary lymphoid organs (TLO) that develop in the autoimmune disease Sjögren’s-syndrome. Changes in lymphatic vessels and HEV may precede lymphocyte infiltration and autoantibody production. The presence of germinal centers in the minor salivary glands of Sjögren’s patients predicts the onset of extra-nodal lymphoma. We show lymphatic vessels, lymphocyte infiltration, HEV, T and B cell compartmentalization, and spontaneous lymphoma in BL/6 mice over 15 months old. We have made transgenic mice that express LTα and LTβ under the control of salivary amylase to model Sjögren’s syndrome. We are crossing the ProxTomxHec6st/eGFP reporter mice with mice that over-express lymphotoxin in their salivary glands, to understand the temporal sequence of the vascular changes that occur in Sjögren’s syndrome.
Hodgkin and Reed-Sternberg cells modulate endothelial cell function in classical Hodgkin lymphoma (61.9)
Authors: Lim Yaw-Chyn , Fhu Chee-Wai , Yap Celestial , Graham Anne , Chong Siew-Meng
Published: January 2023

Growth and survival of neoplastic Hodgkin and Reed-Sternberg cells (H-RS) cells in classical Hodgkin lymphoma (cHL) is dependent on cell-cell contact and soluble mediator signals derived from the inflammatory infiltrate within the lesion. However, the mechanisms underlying recruitment of the inflammatory infiltrate, particularly T cells, into lesional lymph nodes remain unclarified. In this study, we test the hypothesis that H-RS cells secrete soluble mediators to induce endothelial cell (EC) activation to modulate T cell recruitment in cHL. We examined in-vitro interactions, under static and flow conditions, between freshly isolated T cell subsets and EC treated with conditioned medium (CM) harvested from the H-RS cell line, KM-H2. Both CM-stimulated EC and TNFα-stimulated EC support CD4+ naive and memory T-cell interactions under flow conditions. Both CD4+ naive and memory T cell demonstrated enhanced transmigration across CM-stimulated EC monolayers in response to CXCL12 as compared to TNFα-stimulated or unstimulated EC. Our data show that the upregulation of inducible adhesion molecules on CM-stimulated EC is dependent on the NFκB pathway; but the dominant mediator involved is not TNFα. However, blockade of cyclooxygenase activity in KM-H2 cells partially attenuated the efficacy of the CM in activating EC. These findings suggest that H-RS cells secrete soluble mediators that can modulate endothelial cell function and influence recruitment of T cells into the cHL lesion.
A novel immunoassay strategy to measure total serum lymphotoxin-α levels in cynomolgus monkeys dosed with an anti-LTα antibody (62.5)
Authors: Young Judy , Nguyen Allen , Qiu Zhihua Julia , Ying Yong , Gao Xiaoying , Reed Chae , Chuntharapai Anan , Deng Rong , Luttman Jeff , Grogan Jane , Wong Wai Lee , DeForge Laura
Published: January 2023

Lymphotoxin α (LTα) is a TNF superfamily cytokine secreted from activated lymphocytes as a homotrimer (LTα3) or complexed on the cell-surface with LTβ as heterotrimers. Compelling evidence indicates that LTα is involved in the pathophysiology of autoimmune diseases. Given that blocking the activity of LTα holds promise as a treatment option, a neutralizing monoclonal antibody (mAb) is under clinical development as a potential therapeutic. In preclinical studies of mAb therapeutics, measuring the level of the target antigen is often of interest as a pharmacodynamic marker. We found that the presence of anti-LTα caused a dose-dependent decrease in ability to detect LTα by immunoassay due to the inability of detection antibodies to bind in the presence of bound therapeutic mAb. To circumvent this problem, an alternate assay strategy was devised to accurately measure total LTα3 (free LTα and LTα/anti-LTα complexes). Specifically, excess anti-LTα was added to fully saturate all binding sites on LTα3. Bound anti-LTα was detected using an anti-idiotype mAb. Using this approach, total LTα was measured in serum of cynomolgus monkeys dosed with anti-LTα. Circulating LTα concentrations showed dose related increases up to 8,000-fold that correlated with anti-LTα PK. This assay strategy may be useful in other situations where low molecular weight and/or multimeric structure of a target antigen makes accurate detection in the presence of therapeutic mAb problematic. KEY WORDS LTα mAb PD
Cytokine profiles of patients with Staphylococcus aureus skin infections (70.12)
Authors: Bartman Caroline , Chen Luqiu , Chong Anita , Daum Robert , Alegre Marisa
Published: January 2023

Methicillin-resistant S. aureus (MRSA) causes skin infections in epidemic proportions among otherwise healthy individuals. Several lines of evidence suggest that Th17 cells may be crucial to prevent localized S. aureus infections, as they occur in patients deficient in STAT3 or mice lacking IL-17A and F. In this study, we tested the hypothesis that patients presenting to the emergency room (ER) with skin infections have a reduction in Th17 differentiation, either globally, or specifically to S. aureus antigens because of inhibitory factors produced by MRSA strains. Blood samples from 71 patients presenting to the ER with skin infections and 142 controls were stimulated with MRSA lysates, or with the T cell mitogen OKT3, and supernatants were collected at 24h for detection of cytokines by ELISA. Upon stimulation with MRSA lysates, PBMCs of infected patients displayed increased production of IL-1β and IL-6, innate cytokines that can drive Th17 differentiation. In contrast, production of IFNγ and IL-17 in response to OKT3 was similar in patients and controls. These data suggest that unlike immunodeficient patients with recurrent S. aureus infections, our otherwise healthy ER patients with skin infections do not have a global intrinsic defect in Th17 differentiation. The next set of experiments is addressing the specific T cell recall responses to S. aureus antigens in patients and controls to determine whether patients can mount an anti-S.aureus T cell response in vivo.
Colitis in mice with WASP deficiency in innate immune cells is associated with impairment in IL-10 production (71.10)
Authors: Nguyen Deanna , Goettel Jeremy , Wurbel Marc-Andre , Marin Romela , Ahmed Osub , Horwitz Bruce , Snapper Scott
Published: January 2023

Colitis occurs in mice and some patients deficient in the Wiskott-Aldrich Syndrome protein (WASP), a hematopoietic-specific molecule that regulates the actin cytoskeleton. This colitis is lymphocyte-dependent, but severe disease is observed in chimeric mice with WT T cells and WASP KO innate immune cells, generated by transfer of WT CD4+ T cells into WASP/RAG DKO mice. Given the immunomodulatory function of IL-10, coupled with our findings that mixed bone marrow chimeras are free of colitis, we investigated whether alterations in IL-10 play a role in our colitis model. We found reduced IL-10 transcript levels in mesenteric lymph nodes of chimeric mice. To tease out the cellular source of decreased IL-10 production, WASP/RAG DKO DCs were stimulated in vitro and observed to produce less IL-10. We then transferred IL-10GFP CD4+ cells and found a reduction in the IL-10-expressing proportion of T cells in WASP/RAG DKO compared to RAG KO mice. Lastly, exogenous administration of IL-10-Ig completely abrogated disease in chimeric mice. In summary, besides impaired Treg homeostasis and function, severe colitis in our chimeric mice is associated with reduced IL-10 production in MLNs and in vitro stimulated DCs. Taken together with prevention of disease with IL-10 treatment, colitis in this model may result from decreased IL-10 production by WASP KO innate immune cells.
Imprinting CD103+ LP-DCs and the acquisition of luminal antigen is dependent upon CCR6 (71.12)
Authors: Wheeler Leroy , McDole Jeremiah , McDonald Keely , Miller Mark , Newberry Rodney
Published: January 2023

Dendritic cells (DCs) play a crucial role initiating and guiding immune responses. DCs are found within the gut associated lymphoid tissue, within the lamina propria (LP), and closely associated with the small intestinal epithelium. The epithelia associated DCs (EADC) are positioned to be imprinted by epithelial cells and to acquire luminal antigens, however the factors recruiting LPDCs to the epithelium are not known. We observed that CCL20 is expressed by small intestine villous enterocytes, and that the receptor for CCL20, CCR6, is expressed by small intestine EADCs and LPDCs. CCR6-/- mice had a diminished population of EADCs, but not LPDCs when compared with wildtype mice. Mixed bone marrow chimeric mice revealed that CCR6-/- LPDCs were less efficient at associating with the epithelium, demonstrating a requirement for CCR6 recruiting EADCs. Consistent with our observation that goblet cells (GC) act as passages delivering luminal antigens and GC antigens, LPDCs from CCR6-/- mice were inefficient at presenting luminal antigens to T-cells and rarely stained positive for the goblet cell protein cytokeratin 18. Moreover CD103+ LPDCs from CCR6-/- mice had decreased aldehyde dehydrogenase activity, consistent with the requirement for DC-epithelia associations to imprint DCs with a mucosal phenotype. These observations demonstrate an essential role for CCR6 in recruiting LPDCs to the epithelium where these DCs are imprinted and acquire luminal and GC antigens.
Isolation of neutralizing monoclonal antibodies against HIV-1 with predetermined conformational epitope specificity (76.4)
Authors: Sholukh Anton , Mukhtar Muhammad , Humbert Michael , Essono Sosthene , Watkins Jennifer , Shanmuganathan Vivekanandan , Hemashettar Girish , Hu Shiu-Lok , Montefiori David , Polonis Victoria , Schur Peter , Ruprecht Ruth
Published: January 2023

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. However, using the whole antigen for B-cell sorting implies a need for extensive screening of newly isolated mAbs, followed by epitope mapping. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. Using our strategy we isolated mAbs targeting the conformational V3 loop crown of HIV Env. The new mAbs showed strong dependence on epitope conformation and cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.
Elevated IL-17A levels in human lung of fatal cases of influenza A (76.9)
Authors: Rosas-Taraco Adrian , Rodriguez-Ramirez Heidi , Salinas-Carmona Mario , Melo Americo , Barboza-Quintana Oralia
Published: January 2023

Influenza A is an infectious disease and world health problem due to high morbi-mortality. Hypercytokinemia has been detected in sera or plasma of severe cases. However, little is known about the microenvironment in lung of patients in fatal cases. The aim of this study was to analyze the cytokines environment in human lung tissue from fatal cases of influenza A and its relation with viral load. A total of 6 biological samples from fatal cases of influenza A were included in this study. RNA was isolated from formalin fixed paraffin-embedded lung tissue of patients and RT-qPCR was performance to detect and quantify the viral load and T cells and macrophages related cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, IL-23, iNOS, Arg1, IFN-γ, TNF-α, TGF-β1 and RETNLB). Viral load was detected in all human lung samples (4-173 copies/mm3). Low levels of iNOS, IL-2, IL-6 and Arg1 gene expression were found in fatal cases of influenza A (p<0.05). On the other hand, high levels of IL-17A mRNA were found in the same samples (p <0.01). We also found a positive correlation between IL-6 expression and viral load. In conclusion, IL-17A could be associated with fatal outcome in influenza A infected patients.
The involvement of CD62L in the effect of IVIg on the cytotoxic activity of CD8 T cells. (P5031)
Authors: Chabot Dominique , Trépanier Patrick , Bazin Renée
Published: January 2023

Intravenous immunoglobulin (IVIg) is used for the treatment of an increasing number of autoimmune disorders. In the past decades, its uses increased constantly, creating a potential risk of shortage. In order to further understand the mechanisms by which IVIg exerts immunomodulatory effects, our group previously demonstrated that IVIg inhibits the in vitro CD8 T cell activation. The CD8 T cell response is known to contribute to the progression of several autoimmune diseases. Our results showed that antigen-mediated CD8 T cells activation was reduced by more than 50% (proliferation, CD69 expression) in the presence of IVIg (Trépanier et al., Blood 2012). In addition, recent results showed a decreased lytic activity of CD8 T cells in the presence of IVIg. In this work, we used an in vitro cross-presentation assay with bone marrow-derived dendritic cells (DC) from C57BL/6 mice and ovalbumin-specific CD8 T cells (OT-I) to study the mechanism by which IVIg affects cytotoxicity. Preliminary results suggest that IVIg prevents the shedding of CD62L/L-selectin from the surface of T cells. Shedding of CD62-L from the surface of cytotoxic T cells was recently shown to be linked to the acquisition of lytic activity. Therefore, the effect of IVIg observed on the expression of CD62L on the CD8 T cell surface may contribute to the decreased cytotoxicity of these cells. The elucidation of the mechanisms of immunomodulatory effects of IVIg could help to design a potent substitute.
Multiple defects in HLA class II-mediated immune recognition of Burkitt's lymphoma (P5055)
Authors: Haque Azizul , God Jason , Zhao Dan , Cameron Christine , Amria Shereen , Bethard Jennifer
Published: January 2023

While Burkitt Lymphoma (BL) has a well-known defect in HLA class I-mediated Ag presentation, the exact role of BL-associated HLA class II in generating a poor CD4+ T cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T cell recognition was not associated with low levels of costimulatory molecules on BL cells, as addition of external costimulation failed to elicit CD4+ T cell activation by BL. Further, the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II-peptide complexes were formed at acidic pH 5.5, which restored immune recognition. Acidic buffer (pH 5.5) eluate from BL cells contained molecules which impaired class II-mediated Ag presentation and CD4+ T cell recognition. Biochemical analysis showed that these molecules were greater than 30 kDa in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 kDa enolase-like molecule which enhances class II-mediated Ag presentation in B cells, macrophage and dendritic cells, but not in BL. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated Ag presentation and immune recognition, which may be exploited for future immunotherapies.
P2X4 receptor: A mediator of alcohol-induced effects on microglia cells. (P4185)
Authors: Gofman Larisa , Cenna John , Potula Raghava
Published: January 2023

Activation of purinergic receptors on glial cells is associated with functional responses under normal and pathophysiological conditions. Modulation of purinergic receptors in neurodegenerative and neuroprotective processes have become important therapeutic targets in inflammation and immune-related diseases. Chronic alcohol use causes structural and functional abnormalities of the brain and has a profound impact on the neuroimmune system. As immune effectors in the central nervous system (CNS), microglia are activated in pathological conditions and alcohol induces inflammatory processes in the brain leading to neurodegeneration. Among the P2XR subtypes, P2X4R is copious in the CNS and data suggests them to be important mediators of alcohol-induced effect. Our previous unpublished studies have shown that alcohol significantly increases microglia P2X4 mRNA and protein expression. In this study, we investigated the consequences of P2X4R expression on microglia immune function. Fractalkine induced microglia migration was decreased by 75% following 48hrs of alcohol treatment compared to control (p<0.001), which was attenuated 2.3 fold by pretreatment with 5-BDBD, a P2X4 antagonist. Similarly, 48hrs of alcohol treatment decreased phagocytosis by 15% compared to control (p<0.05), this was significantly reversed when pre-treated with 5-BDBD (p<0.001). Our findings suggest that P2X4 receptors play a role in mediating alcohol-induced effects on microglia immune effector functions.
Extracellular signal-regulated kinase 5, a central regulator of Toll-like receptor signaling in vivo and in vitro (P4193)
Authors: Wilhelmsen Kevin , Xu Fengyun , Tran Alphonso , Hellman Judith
Published: January 2023

Background: Sepsis is initiated by interactions between microbes and Toll-like receptors (TLRs), which leads to inflammation, coagulopathy and organ failure. We recently discovered that ERK5, a member of the mitogen activated protein kinase family, mediates TLR2-dependent activation of monocytes and endothelial cells, and promotes TLR2-dependent expression of the coagulation factor plasminogen activator inhibitor 1 (PAI-1). We tested the hypotheses that ERK5 also regulates TLR4-dependent endothelial activation, and that ERK5 mediates TLR2- and TLR4-dependent activation of inflammation and coagulopathy in vivo in mice. Methods: HUVEC were treated with FSL-1 or Pam3Cys (TLR2 agonists), or with LPS (TLR4 agonist), in the presence of the ERK5 inhibitor XMD8-92. Mice were pre-treated with ERK5 inhibitor (XMD-92) and then challenged IV with Pam3Cys or LPS. Cytokines were quantified in HUVEC supernatants and mouse plasmas. PAI-1 was quantified in mouse plasmas. Results: ERK5 inhibition resulted in the downregulation of TLR2- and TLR4-dependent cytokine production by HUVEC in vitro (p<0.05). Similarly treatment with ERK5 inhibitor reduced the induction of cytokines and PAI-1 in plasmas of mice treated with Pam3Cys or LPS (p<0.05). Conclusions: Our results highlight critical, previously overlooked functions for ERK5 in regulating TLR-dependent inflammatory signaling in sepsis.
Granzyme B contributes to impaired wound healing during chronic inflammation in apolipoprotein E knockout mice (P3036)
Authors: Hiebert Paul , Wu Dan , Granville David
Published: January 2023

Granzyme B (GzmB) is a serine protease expressed by immune cells during chronic inflammation and is capable of degrading extracellular matrix (ECM) proteins such as fibronectin (FN). Apolipoprotein E (ApoE) is a protein known to suppress inflammation and is expressed in skin. ApoE knockout (KO) mice develop an inflammatory skin phenotype when fed a high fat diet leading to impaired wound healing. We hypothesized that excessive GzmB activity contributes to impaired healing in ApoE KO mice through the degradation of ECM. Wild type (WT), ApoE KO and ApoE/GzmB double knockout (DKO) mice were fed a high fat diet for 30 weeks and given a 1 cm diameter skin wound on their mid dorsum. Wounds were allowed to heal for 16 days at which point the wounded tissue was harvested. All WT mouse wounds showed complete closure after 16 days while only 40% achieved closure in ApoE KO mice. Wound contraction was also slower in ApoE KO mice compared to WT controls (P<0.05). Histological analysis demonstrated persistent inflammation in ApoE KO mouse wounds at Day 16 featuring neutrophils, macrophages, T cells and GzmB. Wounded skin from ApoE KO mice also contained a FN fragment of similar size to that produced by GzmB in vitro. Compared to ApoE KO mice, DKO mice showed faster contraction (P<0.05) and better healing with 80% of wounds achieving closure by Day 16. These results suggest GzmB contributes to impaired wound healing in ApoE KO mouse wounds, possibly through excessive degradation of FN.
TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation (P3040)
Authors: Kim Brian , Siracusa Mark , Saenz Steven , Noti Mario , Monticelli Laurel , Sonnenberg Gregory , Hepworth Matthew , Van Voorhees Abby , Comeau Michael , Artis David
Published: January 2023

Innate lymphoid cells (ILCs) are a recently identified family of immune cells that regulate inflammation and tissue repair at barrier surfaces such as the intestine and lung. Previous studies have demonstrated that group 2 ILCs found in the intestine or lung respond to the predominantly epithelial cell-derived cytokines IL-33 and IL-25 and produce the type 2 cytokines IL-5 and IL-13. However, whether group 2 ILCs are found in the skin, whether they respond to epithelial cell-derived cytokines and whether they contribute to skin inflammation has not been characterized. Here, we identified a population of skin-resident group 2 ILCs present in healthy human skin and enriched in lesional human skin isolated from atopic dermatitis (AD) patients. Group 2 ILCs were also found in normal murine skin, accumulated in the skin and skin-draining lymph nodes in a murine model of AD-like inflammation and were critical for the elicitation of type 2 cytokine responses and AD-like inflammation. Remarkably, ILC responses and AD-like inflammation were independent of IL-33 and IL-25 but critically dependent on thymic stromal lymphopoietin (TSLP). Collectively, these results demonstrate an essential role for IL-33- and IL-25-independent group 2 ILCs in promoting skin inflammation.
An 8-color panel for detection of human blood dendritic cells by flow cytometry (P3014)
Authors: Miloud Tewfik , Dupas Nathalie , Montero-Julian Felix
Published: January 2023

Dendritic cells (DCs) are antigen presenting cells capable of presenting antigen and priming a T cell response. They form a heterogeneous group of cells based on phenotype, location and function. In human blood, DCs represent less than 1% of white blood cells, and can be separated into 2 main cell subsets, namely the myeloid DCs (mDCs) and the plasmacytoid DCs (pDCs). Among the mDCs, 3 distinct cell subsets are identified: CD1c+mDCs, CD141+mDCs and CD16+mDCs. In blood, the frequency of DCs is affected in certain pathological conditions such as HIV, diabetes, asthma, chronic viral hepatitis, and graft-versus-host-disease. Thus, the detection and enumeration of different blood DC subsets is important to understand immune regulation in pathological conditions and to guide specific patient treatments. Due to the lack of specific markers for DC definition, the combination of several markers is required to allow their identification. Based on current knowledge in human DC biology, we have evaluated the expression and association of several DC markers to design an optimized 8-color panel for flow cytometry which allows for the detection of all DCs subsets in whole blood samples or peripheral blood mononuclear cells (PBMCs). This panel (CD1c/HLADR/Lineage/CD11c/CD16/Clec9A/CD123/CD45) provides an easy and robust assay to study the role of DCs in healthy donors and patient samples.
Fli-1 transcription factor regulates inflammatory mediators in endothelial cells (P6240)
Authors: Karam Eva , Williams Sarah , Zhang John
Published: January 2023

Fli-1 transcription factor is implicated in the pathogenesis of human and murine lupus. A two-fold overexpression of Fli-1 protein in transgenic mice results in the development of a lupus-like disease. Expression of Fli-1 in lupus patients is higher compared to normal controls. Endothelial cells play a role in a number of biological processes ranging from inflammation to the regulation of blood flow. We have reported that Fli-1 directly regulates the expression of MCP-1 in endothelial cells. In this study we investigated if Fli-1 also regulates other inflammatory chemokines. We transfected an endothelial cell line with Fli-1 specific siRNA and negative control oligo siRNA. There was more than an 80% decrease in Fli-1 expression in cells transfected with Fli-1 siRNA when compared to those transfected with control siRNA. Next, we stimulated these cells with TLR4 ligand lipopolysaccharide and extracted mRNA. An mRNA array was used to screen the levels of 26 chemokines. A number of message levels were decreased, including MCP-1, in the cells transfected with Fli-1 specific siRNA when compared to the control. Expression of MIF was higher in the cells when Fli-1 was silenced. We further confirmed MIF mRNA expression levels by real-time PCR. Our data indicates that Fli-1 is likely involved in regulating the production of several chemokines in endothelial cells.
Isoliquiritigenin and glycyrrhizin inhibit the inflammasome activation in a different manner. (P1246)
Authors: Honda Hiroe , Nagai Yoshinori , Takatsu Kiyoshi
Published: January 2023

A soponin glycyrrhizin (GL) and a chalcone isoliquiritigenin(ILG) are representative components of Glycyrrhiza uralensis, which attenuate inflammatory responses mediated by TLR4. We have showed that GL and ILG suppressed different steps of the LPS sensor TLR4/MD-2 complex signaling at the receptor level. GL and ILG attenuated LPS binding to the TLR4/MD-2 complex and LPS-induced TLR4 homodimerization, respectively. In the present study, we examined effects of ILG and GL on IL-1β production in macrophage. When bone marrow-derived macrophages were stimulated with LPS to prime TLR4 signaling pathway followed by ATP to activate NLRP3 inflammasome, significant IL-1β production was observed. However, addition of ILG or GL together with ATP severely impaired the IL-1β production without changing the pro-IL-1β expression levels. We infer from these results that ILG and GL inhibit the NLRP3 inflammasome activation in both priming phase as well as activating phase. We also investigated effects of ILG and GL on the AIM2 inflammasome activation and found that GL but not ILG could suppress its activation, while both ILG and GL could suppress ATP-dependent inflammasome activation.We will discuss molecular mechanisms by which these compounds suppress inflammasome activation.
NFkB1 inhibits NOD2-induced cytokine secretion through ATF3-dependent and -independent mechanisms (P1252)
Authors: Zheng Shasha , Abraham Clara
Published: January 2023

NOD2, an intracellular sensor of bacterial peptidoglycan, is associated to Crohn’s disease (CD). In the intestine, NOD2 is continuously stimulated. Upon chronic NOD2 stimulation in human monocyte-derived macrophages (MDM), cytokine secretion is significantly attenuated upon restimulation of NOD2 or other pattern recognition receptors (PRR); cytokine downregulation is a prominent feature of intestinal tolerance. NFkB1 (p50/p105) is a critical transcription factor regulating PRR responses. However, the role of NFkB1 in NOD2-mediated tolerance reported is not known. Knocking down NFkB1 expression in primary human MDM significantly reversed NOD2-induced cytokine downregulation. MAPK and IkBa activation was dramatically reduced in NOD2-initiated tolerance; this reduced signaling was reversed upon NFkB1 knockdown. Furthermore, upon NOD2-initiated tolerance, NFkB1 knockdown significantly reversed the regulation of both activating (H3K4Me2, H4Ac) and inhibitory (H3K27Me3) histone modifications within cytokine gene promoters. Upon chronic NOD2 stimulation, the transcriptional repressor, ATF3, was induced and bound to the promoters of cytokine genes; both features were reversed upon NFkB1 knockdown. Restoring ATF3 expression under these NFkB1 knockdown conditions significantly restored NOD2-initiated tolerance. We therefore define a clear role for NFkB1 in the mechanisms through which the CD-associated protein NOD2 downregulates cytokines.
IL-12 priming and selective IL-2Rα engagement leads to enhanced CD8+ T cell persistence and anti-tumor immunity in the absence of lymphodepletion. (P1424)
Authors: Rubinstein Mark , Moore Caitlin , Su Ee , Songalia Neizel , Neitzke Daniel , Cloud Colleen , Johnson C. , Paulos Chrystal , Cole David
Published: January 2023

Effective T cell therapy against metastatic cancer requires high frequencies of functional tumor-reactive T cells. However, achieving sustained and elevated levels of tumor-reactive T cells has proven difficult and usually requires highly toxic preconditioning of patients with radiation or chemotherapy to destroy host lymphocytes and suppressive cells. Using the mouse CD8+ T cell pmel-1 TCR transgenic model, we found that the combination of 1) in vitro IL-12-priming of donor T cells during activation and 2) the administration of IL-2/anti-IL-2 mAb complexes after adoptive transfer promoted the dramatic and systemic increase in tumor-reactive T cells in the absence of host lymphodepletion. Importantly, this combinatorial therapy led to the cure of over half of mice with established subcutaneous B16 tumors. Interestingly, while IL-2/anti-IL-2 mAb complexes synergized with ex vivo IL-12-priming, IL-15/sIL-15Rα-Fc complexes failed to mediate comparable synergistic activity despite signaling through the shared IL-2Rβγ subunits. Given that IL-12 priming strongly promotes the upregulation of the high affinity IL-2 receptor subunit, IL-2Rα, our results suggest that selective IL-2Rα engagement uniquely promotes T cell persistence and anti-tumor efficacy in the absence of lymphodepletion. Our findings underscore the importance of T cell polarizing conditions in dictating the requirement of adoptively transferred T cells for exogenous cytokine.
Residual antigen presentation by CD40-licensed DCs instructs memory CD8+ T cells differentiation program to influenza (P1412)
Authors: Ballesteros-Tato André , León-Ruiz Beatriz , Lund Frances , Randall Troy
Published: January 2023

Here we show that residual antigen presentation by CD40-licensed dendritic cells (DCs) tunes memory CD8+ T cell responses to influenza. Primary and secondary CD8+ T cell responses to influenza nucleoprotein (NP) were reduced in Cd40-/- mice. When primed in Cd40-/- mice, NP-specific memory CD8+ T cells responded poorly to re-challenge, even in a CD40 sufficient environment. Defective programming in Cd40-/- mice was due to inefficient late cross-presentation of NP by CD70-expressing CD103-CD11bhi DCs. We demonstrated that residual antigen presentation by CD40 licensed CD70-expressing CD103-CD11bhi DCs prevented apoptosis of IL-2 responding CD8+ T cells and promoted memory CD8+ T cells differentiation by allowing combined TCR- CD25-CD27 signaling during the contraction phase of the primary response. We also show that CD40 signaling preferentially licensed CD70-expressing CD103-CD11bhi DCs to cross-present the more abundant influenza antigens. As a consequence, T cells of different specificities received different memory programing signals during the contraction phase depending on the antigen abundance, and the memory pool result enriched in T cell clones with a TCR repertoire selected for dominant epitopes. Taken together our results suggest that diferential residual antigen presentation by CD40 licensed CD70-expressing CD103-CD11bhi DCs represents a mechanism to enhance the fitness of the memory T cell responses
Polarized release and retroviral subversion of TCR-enriched microvesicles at the T cell immunological synapse (P6155)
Authors: Choudhuri Kaushik , Llodra Jaime , Tsai Jones , Roth Eric , Gordo Susana , Wucherpfennig Kai , Kam Lance , Stokes David , Dustin Michael
Published: January 2023

The recognition events that mediate adaptive cellular immunity depend on intercellular contacts between T cells and antigen presenting cells (APC). T cell signaling is initiated at these specialized junctions between T cells and APCs, known as the immunological synapse (IS), when surface-expressed antigen receptors (TCR) recognize peptide fragments of pathogens (pMHC) on APCs. Using high resolution optical and electron microscopy, optical-EM correlation, and electron-tomography, we show that centrally accumulated TCR at the IS is located on the surface of extracellular microvesicles that bud at the IS center and are recognized by APC bearing cognate antigen. An early endosomal-sorting complex required for transport (ESCRT) sorts TCR for inclusion in microvesicles, while terminal ESCRT components mediate scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process by displacing TCR from microvesicles, resulting in release of virus-like particles at the antigen-dependent IS. We conclude that post-signaling TCR accumulates at the IS center and is released in extracellular microvesicles by an ESCRT-dependent mechanism triggered by T cell activation. These microvesicles mediate intercellular communication in immune cell collaboration, and can be co-opted by HIV GAG for viral transmission across antigen-dependent synapses.
Loss and aberrant function of NKp44+ ILCs in the SIV-infected gut are associated with RORγt suppression (P6175)
Authors: Reeves R. Keith , Li Haiying
Published: January 2023

Recent studies have identified a novel NK cell-like population found in mucosal tissues and mucosae-draining lymph nodes distinguishable by high surface expression of NKp44 and referred to as innate lymphoid cells (ILCs). ILCs secrete IL-17/IL-22 which are critical for epithelial homeostasis and thus could play a role in gut integrity, a major factor in HIV/SIV pathogenesis. During chronic SIV infection, numbers of ILCs declined ~8-fold in gastrointestinal tissues, but remained comparable to naïve animals in distal sites such as mesenteric lymph nodes and lungs. Loss of ILCs was associated with up to 100-fold increases in apoptosis. Interestingly, gut ILCs expressed NFIL3, a classic NK cell-related transcription factor, as well as the IL-17 transcriptional regulator, RORγt, but during SIV infection RORγt was significantly downregulated associated with decreased IL-17 secretion. In these same animals increased IFN-γ secretion and acquired cytotoxicity were associated with an increase in ILC NFIL3. Loss and functional skewing of ILCs in vivo correlated with upregulated IDO-1 and IFN-α in the gut, and in vitro treatment of ILCs with IDO-1 catabolites and IFN-α also suppressed RORγt expression, ablated IL-17 production and induced apoptosis. SIV infection results in massive numerical and functional depletion of ILCs in the inflamed gut, but not distal sites. This phenomenon was due, at least in part, to IDO-1- and IFN-α-induced apoptosis and suppression of RORγt.