bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of human macrophages by the endogenous toll-like receptor 4 ligand high molecular weight hyaluronan (101.20)
Authors: Wallet Mark , Guiulfo Giorgio , Wallet Shannon , Sleasman John , Goodenow Maureen
Published: January 2023

Rheumatoid arthritis [RA] is caused in part by cytokines secreted by classically activated synovial macrophages. We reported that IFNγ, typically produced by CD4 T cells within synovitis, primes macrophages for activation by high molecular weight hyaluronan [HMW-HA], a major component of joint synovial fluid. HMW-HA is a ligand for both toll-like receptor 4 [TLR4] and CD44. The purpose of this study was to compare the molecular mechanism of HMW-HA (endogenous TLR ligand) versus LPS (exogenous TLR ligand) induced macrophage activation. Transcriptome bioprofiles were determined by cDNA arrays, and the roles of CD44 and TLR4 were determined using blocking antibodies and cytokine measurement by Milliplex®. Significant differences in gene expression profiles were revealed when comparing IFNγ priming versus mock priming under both LPS and HMW-HA activating conditions, although nearly identical gene expression profiles were observed when comparing HMW-HA to LPS stimulation under both mock and IFNγ priming conditions. Blockade of CD44 enhanced inflammatory cytokine secretion while blockade of TLR4 inhibited inflammatory cytokine secretion in response to either HMW-HA or LPS. HMW-HA provides the stimulus required for classical macrophage activation when cells are primed with IFNγ in a response that is essentially identical to the LPS response. Type 1 T cells in proximity to endogenous TLR ligands may provide sufficient signals to initiate classical macrophage activation in RA.
Pivotal role of dermal γδ T Cells in the pathogenesis of psoriasis (101.24)
Authors: Cai Yihua , Yan Jun
Published: January 2023

Interleukin (IL)-23 and CD4+ Th17 cells are thought to be critical in the development of psoriasis. Here, we report that IL-23 predominantly stimulates innate dermal γδ T cells to produce IL-17 that leads to disease progression. Dermal γδ T cells constitutively express high levels of IL-23R, transcriptional factor RORγt, and various chemokine receptors. However, dermal γδ T cells are CD27 negative and do not produce IFN-γ. IL-17 production from dermal γδ T cells is independent of αβ T cells. The epidermal hyperplasia and inflammation induced by IL-23 is significantly decreased in TCR δKO and IL-17RA KO mice but occurs normally in TCR αKO mice. The clinical manifestations including epidermal thickening and neutrophil infiltration in an Imiquimod (IMQ)-induced mouse model of psoriasis are also significantly decreased in TCR δKO mice. Perhaps further promoting disease progression, IL-23 preferentially stimulates dermal γδ T cell expansion in vitro. In human psoriasis patients, γδ T cells are also greatly increased in affected skin and similarly produce large amounts of IL-17. Thus, IL-23-responsive dermal γδ T cells are the major IL-17 producers in the skin and may represent a novel target for the treatment of psoriasis.
Eosinophils promote myocarditis and dilated cardiomyopathy by enhancing Th17 responses (101.27)
Authors: Baldeviano Geral , Barin Jobert , Wu Lei , Talor Monica , Ferrer Patricia , Chen Ping , Legault Jillian , Ong Sufey , Zheng Dongfeng , Bedja Djahida , Gabrielson Kathleen , Chatzidimitriou Dimitris , Rose Noel , Cihakova Daniela
Published: January 2023

Eosinophilic myocarditis is a rare but potentially fatal form of myocarditis that is occasionally associated with systemic eosinophilia. It is not known how eosinophils localize to the myocardium or which are the mechanisms underlying their involvement in inflammatory heart diseases. Here, we assessed the role of eosinophils using a murine model of experimental autoimmune myocarditis (EAM). We have previously observed that eosinophils are recruited to the inflamed heart. Eosinophil-deficient mice (ΔdblGATA1) had decreased EAM and did not develop dilated cardiomyopathy (DCM) compared to wild-type BALB/c control mice. In contrast, IL-5 transgenic mice (IL-5Cd3) developed peripheral eosinophilia, severe EAM, increased myocardial fibrosis and accelerated DCM. Transfer of bone marrow-derived eosinophils increased EAM severity in ΔdblGATA1 mice, suggesting a direct pathogenic role for eosinophils in myocarditis. The trafficking of eosinophils to the heart was independent of IL-5 and CCR3 but partially dependent on the cytosolic phospholipase-2. Flow cytometry analysis revealed that the frequency of IL-17A-secreting T cells tracked with the number of eosinophils; it was significantly increased in IL-5Cd3 and decreased in ΔdblGATA1 mice. This study provides the first direct evidence for a pathogenic role in eosinophils in myocarditis and DCM. Additionally, our findings support the hypothesis that eosinophils may promote Th17 responses during myocarditis.
Opposing roles for Fas ligand and IL-10 in regulating insulitis initiation (101.32)
Authors: Hamad Abdel Rahim , Uddin Sophia , Marshall Andrew , Nakata Chiaki
Published: January 2023

Type 1 diabetes (TID), leading autoimmune disease of childhood, is caused by destruction of beta-cells by autoreactive T cells. Despite daily insulin injections, patients often develop cardiovascular complications and there is a need for identifying therapeutic targets to prevent the disease without impinging on host defense. Fas ligand (FasL) can be such a potential target. Fas/FasL interactions primarily regulate T cell homeostasis but not activation. Nevertheless, mutation of FasL (gld) prevents autoimmune diabetes in NOD mice, the widely used model for T1D. Although homozygous gld mutations cause age-dependent lymphoproliferation, limiting the gld mutation to one allele (NOD-gld/+) or treating NOD-wt mice with FasL-neutralizing mAb inhibits insulitis and prevents disease development without causing lymphoproliferation or immunosuppression. Here we show that the gld mutation did not inhibit proliferation and expansion of diabetogenic T cells in pancreatic lymph nodes but inhibited their accumulation in the pancreata. A significant proportion of B cells found in pancreata of NOD-gld/+ mice surface-expressed CD5 and produced IL-10. Neutralization of IL-10 allowed CD4+ T cell accumulation in the pancreas and development of insulitis in NOD-gld/+ mice. The results provide novel mechanistic insights into the disease pathogenesis with potential implications for developing novel T1D therapy.
Investigating the role of interferon-alpha in Sjögren’s Disease (101.36)
Authors: Shen Long , Wu Jing , Suresh Lakshmanan , Ambrus, Jr. Julian
Published: January 2023

We have previously demonstrated that IL14a transgenic mice (IL14aTG) reproduce features of Sjögren’s disease (SD) seen in patients in the same relative time frame, including autoantibodies, early destruction of the salivary and lachrymal glands, infiltration of the salivary and lachrymal glands with lymphocytes, lymphocytic pneumonia, mild renal disease and eventually lymphoma (J.Immunol. 177: 5676, 2006 and Clin. Immunol. 130:304, 2009). We have more recently demonstrated that lymphotoxin is an important early mediator of salivary gland injury in IL14aTG mice and patients with SD (J.Immunol. 185: 6355, 2010). In our current studies we investigate the role of interferon-alpha (IFN-α) in SD in IL14aTG mice. We demonstrate that IL14aTG mice lacking the type I IFN receptor (IL14aTG.IFNa/bR-/-) have the same early injury to the submandibular glands as IL14aTG mice. However, IL14aTG.IFNa/bR-/- do not get the later parotid gland injury that is seen in IL14aTG mice. Both IL14aTG mice and IL14aTG.IFNa/bR-/- get lung injury, but IL14aTG mice get a lymphocytic pneumonia and IL14aTG.IFNa/bR-/- get a mild perivascular and peribronchial lymphocytic infiltration. Thus, IFN-α is not critical in early stages of SD but involved in disease manifestations that occur after original salivary gland injury. These data have important implications for the use of IFN-α inhibitors in the treatment of SD.
Analysis of Type I and III interferon levels in the sera of normal and autoimmune donors. (101.37)
Authors: Lavoie Thomas , Pandya Yognandan , Skawinski Michael , Crisafulli-Cabatu Sara , Moolchan Karlene , Stauffer Tara
Published: January 2023

We have begun examining serum levels of Type I and III interferons in normal and autoimmune donors by ELISA and a Type I reporter gene activity assay to determine if there are expression pattern signatures of these diseases. Samples were obtained from normal donors and patients with Systemic Lupus Erythematosus, Rheumatoid Arthritis or Multiple Sclerosis and assayed for IFN-α), IFN-β, IFN-ω, and IFN. Normal controls had little detectable IFN-α, IFN-ω, or Type I IFN activity. Low levels of IFN-β were detected in 15% of the normal controls. Approximately 35% of the normal controls had detectable IFN-λ. In the Lupus samples, 45% had detectable IFN-α. IFN-ω and IFN-β were detected in 20 and 40%, respectively, of the Lupus samples. Overall, greater than 80% of the Lupus samples had detectable Type-I IFN. Rheumatoid arthritis samples may have elevated IFN-β and IFN-ω relative to normal controls, but not IFN-α. Multiples sclerosis samples had a slight tendency towards elevated IFN-β. A low percentage of Lupus and multiple sclerosis samples had detectable Type-I IFN activity by the reporter gene assay. None of the samples displayed significant differences in Type III IFN expression. Multiplex ELISA was used to examine a subset of the samples for cytokine profiles and in Lupus samples IL-13 was decreased. In arthritis samples IL-8 and IL-23 were elevated, while in Multiple Sclerosis samples IL-2, IL-4 and IL-10 were elevated. The implications of these results will be discussed.
Galectins flip the CD40 survival-and-proliferation switch off and on in autoimmunity. (101.39)
Authors: Vaitaitis Gisela , Wagner David
Published: January 2023

CD40 is intimately associated with autoimmune disease. CD4+CD40+ T cells (Th40) are necessary and sufficient in transferring autoimmunity and CD40 engagement induces survival and proliferation of those cells. Several CD40 receptor constellations exist being composed of different glycoforms of CD40 and some are hybrid receptors with TNFR1 and 2. Autoimmune and non-autoimmune mouse Th40 cells differ in their CD40 glycosylation status. Here we reveal that in-vivo CD40 signals to non-autoimmune BALB/c mice access glycosylation processes and induce a reduction in glycan branching to levels seen in autoimmune strains. Such levels are known to activate T cells. CD40 signals induce a less glycosylated form of CD40 itself. We reveal an interaction of galectins 3 and 9 with CD40 in Th40 cells that differs between autoimmune and non-autoimmune conditions. Those galectins counteract each other in controlling CD40 receptor signals. Galectin-3 interaction is associated with CD40 signals leading to survival/proliferation of autoimmune NOD Th40 cells. However, the addition of galectin 9 counteracts that interaction to promote CD40 interaction with galectin-9 which thwarts the survival and proliferation signals. Therefore we suggest that CD40’s impact on glycosylation events may have far reaching consequences for immune effector molecules beyond CD40 itself and that galectins 3 and 9 are controlling a “switch” that determines whether CD40 signals lead to survival and proliferation.
FTY720 increases the ratio of Treg:CD4 T cells in vivo and inhibits the development of autoimmune arthritis. (101.9)
Authors: Rosloniec Edward , Whittington Karen , Brand David , Hasty Karen , Miller David
Published: January 2023

Treg cells play an integral role in preventing autoimmunity, and the enhancement of Treg function is an attractive approach for the treatment of autoimmunity. Toward this goal, we have investigated the efficacy and mechanism of action of FTY720 in preventing autoimmune arthritis elicited by immunization of humanized mice with type II collagen. Mice were treated on the day of immunization with 1 or 0.2 mg/kg ip of FTY720, 3 times/wk for 3 wks, and evaluated for incidence and severity of arthritis and the phenotype of their T cell responses. Both 1 and 0.2 mg/kg of FTY720 significantly delayed disease onset (day 35 vs 22) and reduced the incidence and severity in comparison to vehicle control groups. By day 18, the percentage of B cells in the lymph nodes of the FTY720 treated mice had increased and CD4 T cells had decreased. Foxp3+ cells were also significantly increased in the nodes of the FTY720 treated group, to >20%, more than 2 fold higher than the control group. This increase occurred in both the CD25hi and the CD25lo/neg Foxp3+ T cells. The increased number of Treg cells did not cause a reduction of autoimmune T cells as, based on tetramer analyses, the percent of CII-specific T cells in the lymph nodes were elevated 40% over the control group. These data indicate that the FTY720 did not interfere with the development of the autoimmune T cells response and that at least one mechanism of disease inhibition may be Treg cell inactivation of the autoimmune T cells.
Lung memory CD4 T cell subsets with different tissue retentive properties (102.1)
Authors: Turner Damian , Teijaro John , Yudanin Naomi
Published: January 2023

Memory T cells residing in the lung can mediate protective immunity to pathogens; however, the mechanism of lung cell recruitment and retention is unknown. We have shown that antigen-specific lung memory CD4 T cells can traffic to the lung and become irreversibly retained there, whereas lymphoid memory T cells distribute widely and are not retained in tissues. However, endogenous polyclonal lung memory CD4 cells migrate to the lung and additional tissue sites. We hypothesized that the lung memory CD4 T cell population is comprised of both tissue-resident and circulating subsets. To identify these subsets we labeled CD4 cells by intravenous administration of fluorescently labeled anti-CD4 antibody and found that 25% of CD4 cells in lungs were protected from labeling and were mostly CD11ahi CD69+. Inhibiting lymphocyte circulation with FTY720 resulted in a decrease in lung naïve cells but retention of the memory cells enriched for CD11ahi CD69+ cells. We have also found that tissue-specific recruitment of lung memory cells was inhibited by pertussis toxin, suggesting the involvement of chemokine receptors. Microarray and phenotypic analysis revealed higher expression of CCR6 and CD103 by lung memory CD4 cells. Transfer of lung memory CD4 cells resulted in CCR6+ and CD103+ donor cells being enriched in the lung-recruited population. Our data suggest that the lung memory CD4 T cell population consists of a circulatory subset and a tissue-retentive subset with distinct phenotypes.
Computer simulation of lymphocyte traffic through the homeostatic lymph node (102.14)
Authors: Billard Matthew , Kepler Thomas
Published: January 2023

Lymphocytes recognize their rare cognate antigens within the complex microenvironment of secondary lymphoid organs (SLO). Proper lymphocyte migration into, through, and out of SLO is essential to immune system function and is regulated via chemotactic signals transduced through a well-known set of receptors. Specifically, CCR7, CXCR5, and S1P1 are critical lymphocyte receptors with varied expression and function depending on the lymphocyte cell type, activation state, or differentiated subtype. Our group created a flexible, modular, 3D simulation environment to investigate chemokine control of lymphocyte traffic through SLO. Simulations using computational modeling compliment and drive experimental research by performing in silico experiments that can predict outcomes and inspire new hypotheses. In our system, lymphocyte behavior is principally controlled by two models, a Langevin motility model and a chemokine receptor dynamics model. Using this simulation software, we have asked questions pertaining to the basic cell biology of lymphocytes in order to better understand the mechanisms these cells use to initiate and coordinate inflammatory responses to pathogens. This robust software platform is designed for future modification and expansion, allowing the user to replicate published data, fit models, design in silico experiments, and test new hypotheses.
Podoplanin-rich stromal networks induce dendritic cell motility via activation of CLEC-2 (102.21)
Authors: Pinner Sophie , Turley Shannon
Published: January 2023

Cell motility is crucial for leukocytes to traffic between tissues and to interact with one another and their microenvironment. Stromal cells provide 3-dimensional scaffolds and molecular cues that coordinate leukocyte migration and compartmentalization within secondary lymphoid organs. Dendritic cells (DCs) are antigen presenting cells whose migration via stromal networks is critical for the induction of both immunogenic and tolerogenic T cell responses. DCs migrate in an amoeboid, integrin-independent manner, utilizing the chemokine receptor CCR7 to navigate their way to lymph nodes (LNs). However the molecules utilised by DCs and other leukocytes to interact with stroma are still largely unknown. Here we describe a novel motility mechanism driven by the glycoprotein podoplanin (gp38) that induces motility of DCs that express the C-type lectin-like receptor 2 (CLEC-2, CLEC-1b). Podoplanin-mediated activation of CLEC-2 promotes dynamic, actin-rich protrusions, and migration along stroma. The podoplanin-CLEC-2 interaction endows DCs with the capacity to crawl along stromal cell scaffolds of secondary lymphoid organs and to find and properly activate T cells. Our results illuminate a novel mechanism of leukocyte migration pertaining to multiple steps in the adaptive immune response which we propose functions in concert with chemokine gradients to ensure efficient, directional leukocyte trafficking.
Effects of aging on vaccine efficacy in nonhuman primate models (104.1)
Authors: Arteaga-Cortes Lourdes , Pasquali Amanda , Blevins Maria , Jorgensen Matthew , Leland Michelle , Kaplan Jay , Rice Karen , Dube Peter , Stacy Sue , High Kevin , Kraig Ellen
Published: January 2023

Vaccines are frequently less efficacious in the elderly and correlates of protection in older adults often differ from those seen in younger individuals. However, clinical testing of new vaccine formulations is typically limited and does not include elderly subjects. In order to identify a nonhuman primate (NHP) model that would recapitulate the effects of immunosenescence in humans, we assessed immune responses in three different NHPs: baboons (Papio), rhesus macaques (Macaca), and African green monkeys (Chlorocebus). The F1-antigen, a capsular protein encoded on the plague bacterium Y. pestis pFra plasmid, was chosen because it should elicit naive responses in all subjects. The animals were bled following primary and booster immunizations; B cell (antibody titer) and T cell parameters were assessed. As in humans, significant heterogeneity between individuals was seen. In rhesus and vervets, T cell responses to F1 were generally lower with increasing age. However, less of an age effect was observed in the baboons; this is consistent with the finding that antibody titers to F1 were not significantly diminished in older baboons and with our previous work showing immunization of old baboons with Y. pestis LcrV elicited high titer protective antibodies [JI 181:109]. Flow cytometric analyses of the T cell populations in old and young NHPs reveal differences across the species and provide additional insight into the immune parameters that correlate with healthy aging.
Elevated IL-7 availability does not account for T cell proliferation in moderate lymphopenia. (104.11)
Authors: Abraham Ninan , Patton Daniel , Seo Jung Hee , Osborne Lisa
Published: January 2023

Lymphopenia-induced proliferation (LIP) is a proliferative program initiated in response to T cell insufficiency caused by acute or chronic immunodepletion. Studies of lymphopenic mice have demonstrated that the cytokine IL-7 and TCR signaling are critical for LIP. We examined how these two factors impact T cell proliferation following transfer into moderately lymphopenic mice. We show here that moderate lymphopenia (~25% of WT lymphocytes) of IL-7Rα knock-in mutant (IL-7Rα449F) mice supports T cell proliferation, although with decreased frequency and kinetics compared to cells transferred to severely lymphopenic (5% of WT) IL-7Rα-/- hosts. Although previous studies have demonstrated that elevated IL-7 levels play an important role in LIP, IL-7 availability was not elevated in IL-7Rα449F mice. However, moderate lymphopenia increased access of transferred T cells to self-peptide presented on antigen presenting cells that can trigger TCR signaling and proliferation. Importantly, we did not detect significant changes in TCR Vβ usage of proliferated T cells recovered from either moderately or severely lymphopenic hosts. Our work demonstrates that polyclonal T cells retain a diverse TCR repertoire following proliferation mediated by either self-peptide/MHC interaction alone or in combination with IL-7, and that T cell reconstitution is most efficient in the presence of increased IL-7 availability.
Lasting alterations induced in murine skin T cell populations by chronic ethanol (EtOH) feeding (104.12)
Authors: Parlet Corey , Cook Robert , Coleman Ruth , Schlueter Annette
Published: January 2023

EtOH induced epithelial dysfunction is made evident by the diminished ability of alcoholics to fight skin infection and heal wounds; however the precise lesions responsible for these changes require clarification. Acute EtOH exposure in a rodent model induces alterations in inflammation and innate immunity detrimental to both host defense and wound healing responses in the skin. In addition, the skin is home to numerous immune cell populations including dendritic cells (DCs) and T cells. Using a murine model of chronic alcoholism, we previously reported that EtOH feeding imposes both numeric and functional defects upon skin DCs. There are currently no reports of how skin T cells are affected by chronic EtOH feeding. The present study used flow cytometry to evaluate the effect of chronic EtOH feeding on skin T cell numbers. EtOH feeding decreased the number of epidermal and dermal T cells expressing the γδ T cell receptor. These numeric alterations persisted five weeks after EtOH withdrawal. Given the established roles for γδ T cells in innate immunity and tissue repair, their deficiency in EtOH fed mice may help explain why alcoholism is associated with both impaired skin immunity and wound healing. Experiments are underway to investigate alterations in the number of skin regulatory T cells, further define the mechanism(s) for numeric changes, and potential functional alterations of skin T cells due to EtOH feeding.
BCL-2 allows effector and memory CD8+ T cells to tolerate higher expression of BIM (104.19)
Authors: Kurtulus Sema , Tripathi Pulak , Moreno-Fernandez Maria , Sholl Allyson , Katz Jonathan , Grimes Harry , Hildeman David
Published: January 2023

As acute infections resolve, most effector CD8+ T cells die, while some persist and become memory T cells. Recent work showed that subsets of effector CD8+ T cells, identified by reciprocal expression of KLRG1 and CD127, have different lifespans. Similar to previous reports, we found that effector CD8+ T cells reported to have a longer lifespan (i.e. KLRG1lo CD127hi) have increased levels of Bcl-2 compared to their shorter lived KLRG1hi CD127lo counterparts. Surprisingly, we found that these effector KLRG1lo CD127hi CD8+ T cells also had increased levels of Bim compared to KLRG1hi CD127lo cells. Similarly, central memory CD8+ T cells (TCM) expressed higher levels of Bim and Bcl-2 than did effector memory CD8+ T cells (TEM). Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8+ T cells required Bcl-2 to combat the pro-apoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8+ T cells. Finally, we found that Bim levels were significantly increased in effector CD8+ T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.
Changing access to STAT proteins in CD8 T cells responding to lymphocytic choriomeningitis virus infection (105.31)
Authors: Kallal Lara , Kim Kwang-Sin , Biron Christine
Published: January 2023

Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors that can be activated by particular cytokines regulating immune responses. Previous work in our laboratory has shown that STAT protein levels are dynamically regulated in cells, and this influences cytokine access to particular STATs with subsequent functional outcome. Herein, we examined STAT3 and STAT4 levels as well as cytokine activation of these STATs in CD8 T cells responding to infection with lymphocytic choriomeningitis virus (LCMV). The type I interferon (IFN), IFN-alpha, and IL-12 activated high phosphorylated (p)STAT4 levels in CD8 T cells only at late (day 8) timepoints following LCMV infection, whereas IL-6 and IL-21 activated high pSTAT3 levels only at early (day 2) and intermediate (day 5/6) timepoints after LCMV infection. Western blot analyses indicated that compared to day 0, STAT4 levels in CD8 T cells were highly increased at days 5 and 8, whereas STAT3 levels were decreasing at day 5 and very low at day 8. Assessment of T cell proliferation by BrdU incorporation showed that proportional to CD8 T cell numbers, the highest proliferation of cells occurred at day 5/6. Taken together, these studies suggest a key switch in STAT access from STAT3 to STAT4 in CD8 T cells responding to LCMV infection, which may correlate with the functional status of CD8 T cells.
Assessment of the role of CD4 and CD2 in plasmacytoid dendritic cells in response to HIV infection. (105.32)
Authors: Mwangi David , Fitzgerald-Bocarsly Patricia
Published: January 2023

pDC secrete type 1 interferons and pro-inflammatory cytokines upon viral stimulation through either TLR7 or TLR9. The numbers and function of pDC are impaired in HIV infection, but the mechanism of this deficiency is not fully understood. pDC express CD4, CXCR4 and CCR5, which serve as receptor and co-receptors for the HIV viral envelope protein, gp120. A subset of pDC also expresses CD2. The functional significance of these molecules on pDC in HIV infection is unknown. We hypothesize that CD4 is involved in DC:DC and/or TC:DC communication that leads to modulation of pDC function and that CD2 may augment the structural integrity of the virological or immunological synapse. Our data demonstrate that CD4 cross-linking inhibits activation of pDC as evidenced by a decrease in HSV-1-induced CD40 and HLA-DR and IFN-a production, and inhibits translocation of NF-kB to the nucleus. We compared TLR and NF-kB expression in the CD2+ and CD2- pDC subsets. Our preliminary data shows that CD2+ pDC expressed higher levels of TLR9 and TLR7 than that of CD2- pDC, while the CD2- subset shows higher levels of NF-kB. Neither IFN-a nor TNF-a production was different between CD2+ and CD2- subsets when stimulated with HIV-1. Interestingly, the CD2+ expressed higher levels of the HIV co-receptor, CXCR4, but not CCR5 than the CD2- subset. Future studies will seek to understand the significance of these findings with respect to activation of pDC and their potential interactions with HIV-1.
Exogenous and endogenous virus interactions in head and neck cancer immunity (105.34)
Authors: Sutkowski Natalie , Hoel Brian , Gibbs Kevin , Sun Wei , Halstead Lucinda , Huber Brigitte , Garrett-Mayer Elizabeth , Hill Elizabeth , Gillespie M. , Rubinchik Semyon
Published: January 2023

We report that two common tumor viruses etiologically associated with head and neck cancer, Epstein-Barr virus (EBV) with undifferentiated nasopharyngeal carcinoma, and human papillomavirus (HPV) with oropharyngeal squamous cell carcinoma, transactivate an endogenous retrovirus (HERV-K18), which encodes a superantigen that activates TCRBV13 T cells. EBV LMP-2 transactivated the HERV-K18 superantigen in nasopharyngeal epithelial cells, and EBV-associated nasopharyngeal carcinomas expressed the superantigen. HPV type-16 E6 and E7 proteins also transactivated the HERV-K18 superantigen in oropharyngeal epithelial cells, and superantigen expression was significantly increased in HPV16-associated carcinomas. Serum cytokine levels in head and neck carcinoma patients indicated that there were distinct inflammatory profiles associated with exogenous and endogenous viruses. HPV-associated patients had increased levels of serum IL-8, while HPV-negative patients had higher serum IL-6 and G-CSF levels. Interestingly, HERV-K18 superantigen levels correlated independently with serum levels of the Th2 cytokine IL-13. Among HPV+ tumors, gene expression array analyses indicated that B cell specific genes were significantly upregulated in the superantigen high group. Our results suggest that endogenous superantigen expression in HPV+ tumors was associated with a Th2 immune response, demonstrating that chronic and endogenous viruses comprising the virome affect both host and tumor immunity.
Liver-primed CD8+ T cells are dysfunctional but gain Treg activity on T effectors via the Lag-3 and Tim-3 inhibitory pathways (105.4)
Authors: Dolina Joseph , Krueger Peter , Hahn Young
Published: January 2023

The liver maintains a tolerogenic site as a result of constant exposure to bacterial constituents and food-derived antigens and facilitates the establishment of persistent infections by viruses such as hepatitis C virus (HCV). In these studies, we employ a murine model of intravenous (IV) adenovirus infection specifically targeting the virus to the liver. During IV infection, intrahepatic CD8+ T cells display an inability to produce proinflammatory cytokines and lyse targets compared to other sites of virus delivery (subcutaneous and intranasal). Notably, IV adenovirus infection results in local intrahepatic antigen presentation responsible for limiting the CD8+ T cell immune response. CD8+ T cells mounted by the IV infection demonstrate up-regulation of PD-1, Lag-3, and Tim-3 on their surface, correlating to IL-10 production. Tim-3 and Lag-3 blockade in vitro boosts IFN-γ and attenuates IL-10 production of αCD3-activated CD8+ T cells. Importantly, the liver-generated CD8+ T cells also appear to have a Treg function on the priming of antigen-specific T cells in vitro and in vivo. These results suggest that specific inhibitory pathways and IL-10 signaling are co-dependent. The presence of IL-10-producing CD8+ T cells in the livers of chronic HCV patients implicates that liver-primed CD8+ T cells play a pivotal role in dampening hepatic T cell responses in the local microenvironment.
Inhibition of p38 MAPK activity in mononuclear phagocytes abrogates HIV gp120-induced neurotoxicity while triggering expression of numerous anti-viral and cytoprotective factors. (105.42)
Authors: Kaul Marcus , Sejbuk Natalia , Maung Ricky , Medders Kathryn
Published: January 2023

Macrophages produce neurotoxins upon infection with HIV-1 or exposure to its envelope protein gp120. Both stimuli lead to activation of p38 mitogen-activated protein kinase (MAPK). In order to study the basis for the neurotoxic phenotype of monocytic cells that is initiated by HIV/gp120 and prevented by inhibition of p38 MAPK, we performed a genome-wide gene expression analysis. We treated monocytic THP-1 cells for 4 h or 24 hr with HIV gp120 (1 nM) in the presence or absence of the p38 MAPK inhibitor (SB203580; 10 μM) and subsequently isolated RNA. Microarray analysis indicated that HIVgp120 moderately affected gene expression in comparison to the p38 MAPK inhibitor. Blocking p38 MAPK suppressed some cytokines but, surprisingly, also significantly increased others, such as CCL3, CCL4 and CCL5, all of which have anti-HIV activity and protect against gp120 neurotoxicity. Using a multiplex protein array, we confirmed expression changes for thirteen cytokines. Thus, p38 MAPK signaling is critical for the neurotoxicity of HIVgp120-exposed monocytic cells but only moderately alters gene expression. In contrast, inhibition of p38 MAPK substantially changes gene and protein expression, including increases in potentially neuroprotective chemokines. Thus, inhibition of p38 MAPK may not only protect from HIVgp120-induced neurotoxicity of mononuclear phagocytes by interrupting toxic signaling pathways but also by inducing an array of potentially cytoprotective factors.
Effective control of influenza A virus infection by boosting γδ-T-cell immunity with pamidronate (106.13)
Authors: Tu Wenwei , Zheng Jian , Liu Yinping , Peiris Malik , Lau Yu-Lung
Published: January 2023

Influenza remains a global threat to public health. There are few antiviral drugs for treating influenza and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Pamidronate has been commonly used to treat osteoporosis by enhancing human Vγ9Vδ2-T-cell immunity. Using Rag2-/-γc-/- mice reconstituted with human peripheral mononuclear cells (huPBMCs) as a model, we demonstrate that the treatment of pamidronate decreases disease severity and/or mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. Importantly, pamidronate has no such effects in influenza virus-infected Rag2-/-γc-/- mice reconstituted with Vγ9Vδ2-T-cell-depleted huPBMCs. Our results demonstrate that pamidronate can control human and avian influenza A virus infections through a Vγ9Vδ2-T-cell dependent mechanism in vivo. This study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. This ‘new application of an old drug’ potentially offers a safe and readily available option for treating influenza.
TLR and ODN agonists used as adjuvants enhance the protection elicited in mice by a vaccine formulated with the Chlamydia trachomatis MoPn rMOMP (106.17)
Authors: Chang Chunmei , Jain Pooja , Pal Sukumar , Tifrea Delia , de la Maza Luis
Published: January 2023

Chlamydia trachomatis (CT) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant better enhances the immunogenicity of a vaccine, we formulated the CT recombinant major outer membrane protein (rMOMP) with several TLR and NOD agonists including: Pam2CSK4 (TLR2/6), Poly(I:C) (TLR3), Monophosphoryl lipid A (TLR4), Flagellin (TLR5), Imiquimod R837 (TLR7), Imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-DAP (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized i.m. with rMOMP and one of those adjuvants and were challenged i.n. with 104 CT MoPn inclusion forming units (IFU). Following the challenge the mice were weighed and, at 10 days post-infection (p.i.), they were euthanized, their lungs weighed and the number of IFU in the lungs determined. Based on the IgG2a/IgG1 ratio in sera collected the day before the challenge, mice immunized with rMOMP-Pam2CSK4 showed a strong Th2 bias. Furthermore, these mice develop a robust cellular immune response with high CT-specific T cell proliferation and levels of IFN-γ. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with rMOMP-Pam2CSK4, were significantly better protected than mice immunized with the other adjuvants. In summary, Pam2CSK should be considered as a good candidate adjuvant for a CT vaccine.
A live-attenuated HSV-2 ICP0- virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine (106.22)
Authors: Halford William , Gershburg Edward , Wilber Andrew , Rakowski Brandon
Published: January 2023

Many herpes simplex virus 2 (HSV-2) vaccines rely on combinations of HSV-2’s glycoprotein D (gD-2) and a potent adjuvant. The Simpilirix™ vaccine is a gD-2 subunit vaccine that recently failed in human clinical trials (Science, 2010, 330:304). We have proposed that immunization with an ICP0- mutant virus, HSV-2 0ΔNLS, would be more effective (PLoS ONE, 2010, 5:e12251). The current study was initiated to test this hypothesis. Mice immunized with a gD-2 subunit produced high titers of gD-2 antibody, but were poorly protected against HSV-2 challenge of the vagina or eyes (survival rate = 3 of 45). In contrast, 114 of 115 mice immunized with HSV-2 0ΔNLS survived the same HSV-2 challenges, and protective immunity endured for 6 months. We present several lines of evidence that immunization with HSV-2 0ΔNLS was 10- to 100-fold more protective against HSV-2 infection than a gD-2 subunit. We conclude that a gD-2 vaccine does not protect mice or humans from HSV-2 infections, and infer that live-attenuated HSV-2 would be 10 to 100 times more effective in preventing genital herpes.
Comparison of parenteral delivery routes of ACAM529, a replication-defective vaccine against genital herpes (106.24)
Authors: Hernandez Hector , Zhou Changhong , Mundle Sophia , Hamberger John , Catalan John , Pougatcheva Svetlana , Anderson Stephen , Londono-Hayes Patricia , Kleantous Harold , Delagrave Simon
Published: January 2023

Human herpes simplex viruses (HSV types 1 and 2) constitute the primary cause of genital ulcer disease. Genital herpes is the most prevalent sexually transmitted infection and is found throughout the world. Available treatments for HSV-infected patients are not 100% effective and cannot prevent acquisition of infection. ACAM529 is an HSV-2 virus made replication-defective by deletion of the genes UL5 and UL29. ACAM529 is propagated in a complementing cell line expressing the deleted genes (Da Costa et al., J. Virol.2000). Here we present the comparison of three routes of administration of ACAM529. We measured immunogenicity as well as protective efficacy in the mouse vaginal challenge model. ELISA and neutralizing antibody titers, as well as protection against morbidity, mortality and viral shedding of the challenge virus indicate that two doses of ACAM529 delivered intramuscularly are optimal.
Intranasal norovirus vaccination shows efficacy in preventing acute gastroenteritis following a live GI.1 NoV challenge (106.5)
Authors: Bargatze Robert , Bernstein David , Harro Clayton , Al-Ibrahim Mohamed , Chen Wilbur , Ferreira Jennifer , Estes Mary , Graham David , Richardson Charles , Mendelman Paul , Atmar Robert
Published: January 2023

NoVs are a common cause of both epidemic and endemic AGE. Currently, no vaccine is available to prevent illness. Vaccination with an IN GI.1 NoV virus-like particle (VLP) vaccine (adjuvanted with chitosan and monophosphoryl lipid A) was followed by a homologous viral challenge. The trial employed a randomized, double-blind, placebo-controlled design for assessing safety, immunogenicity and efficacy against AGE and infection. Two-doses of vaccine or placebo IN 3 weeks apart were administered to healthy adults 18-49 years of age. At least 3 weeks later ~10 HID50 of a GI.1 challenge virus was administered to subjects who were then monitored for illness and infection. Two doses of the NoV VLP vaccine were administered to 90 subjects with the vaccine being well tolerated. The per protocol (PP) population was 77 of the 84 persons who completed the challenge. Of the 84, 64% of vaccinees had a >4-fold serum IgA response, none receiving placebo. In the PP analysis the incidence of AGE decreased from 69% to 37% by vaccination and infection was reduced from 82% to 61%. Efficacy against illness was 47% (95% CI, 15-67%) and against infection was 26% (95% CI, 1-36%) relative to placebo and severity of illness was also reduced. Of note, protection from NoV illness and infection correlated with the level of 50% HBGA blocking activity prior to challenge. We report that a NoV vaccine candidate can prevent AGE and that an IN-delivered vaccine can prevent illness due to an enteric pathogen.
Assessment of T cell-mediated immunity by ELISPOT in preclinical studies of dengue in cynomolgus macaques (106.6)
Authors: Moldovan Ioana , Brewoo Joseph , Osorio Jorge , Tary-Lehmann Magdalena
Published: January 2023

We used ELISPOT assays to assess frequencies and cytokine signatures of dengue-specific T cells in preclinical studies. Cynomolgus macaques were either infected with one of the four wild type (wt) dengue viruses or immunized with a tetravalent, chimeric dengue vaccine (DENVax). Peripheral blood mononuclear cells (PBMC) were separated, cryopreserved at different timepoints of the studies and tested in the ELISPOT assay for recall responses to live dengue viruses (DENV). We show that dengue antigen-specific T cells can be consistently detected after the second infection or booster vaccination. In a third study three formulations of DENVax, based on the DENV-2 backbone, were tested. Upon stimulation with live DENV-2, PBMC from animals immunized with any of three DENVax formulations induced significant (p<0.05) higher levels of IL-2 compared to the controls. T-cell mediated IFN-γ responses were also significantly higher in DENVax-immunized macaques compared to the control group. IFN-γ responses in macaques receiving DENVax formulations 2 or 3 were significantly increased (p<0.05) after the second vaccination, whereas animals immunized with formulation 1 didn’t show any augmentation after the second immunization. Consistently, control animals failed to induce IL-2 or IFN-γ responses before wt virus challenge. These data show that DENVax had an efficacy in preclinical dengue studies and support the further evaluation of this vaccine in human clinical trials.
Kallikrein gene modified mesenchymal stem cell transplantation protected mice from anti-GBM induced and lupus nephritis (107.10)
Authors: Li Quanzhen , Li Yajuan , Shao Xinli , Ramin Indu , Yan Mei , Min So , Du Yong , Mohan Chandra
Published: January 2023

Mesenchymal stem cells (MSC) have been reported to be an attractive cell source for treatment of renal diseases. Recently, we reported that kallikreins (klk) play renoprotective role in anti-GBM-induced and lupus nephritis. Here, we investigated whether MSCs could be used as vehicle to deliver klks into injured kidneys and play synergetic protective role against experimental glomerulonephritis. Human KLK1 gene was incorporated into genome of mouse bone marrow-derived MSCs via retroviral vector pMSCV and a stable cell line, hKLK1-MSC, was established and proved to express high level hKLK1. NZW mice with anti-GBM induced nephritis were injected with 105 hKLK1-MSCs (i.v.) and hKLK1 expression was detected from blood and kidneys 2 weeks after injection. Comparing with MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria (0.188 mg/24 h vs. 0.766 mg/24 h, p<0.05), serum BUN (5.9 mg/dl vs. 12.5 mg/dl, p<0.05) and ameliorated renal pathology (p<0.05). hKLK1-MSC transplantation significantly reduced mesangial cell (MC) proliferation, monocyte/macrophage infiltration and mesangial matrix accumulation. Using the same strategy, we treated the B6.Sle1.Sle3 mice with hKLK1-MSCs and proved that hKLK1-MSCs reduced the renal injury caused by spontaneous lupus nephritis. In conclusion, our results indicate that Kallikrein modified mesenchymal stem cell transplantation is a potent therapeutic strategy for the treatment of immune-mediated and lupus nephritis.
Adeno-associated virus-mediated CCL11 shRNA reduced lung inflammatory responses in a mite allergen-sensitized mouse model (107.13)
Authors: Wu Chia-Jen , Huang Wen-Chung , Lin Chun-Yen , Kuo Ming-Ling
Published: January 2023

Asthma is considered as a chronic pulmonary inflammatory disease, characterized by eosinophilia. Lung epithelium cells secrete CCL11, the major chemokine for the recruitment of eosinophils. Hybrid adeno-associated virus serotype 2/9 might achieve long-term gene delivery specifically to lung epithelium cells. Thus, this study tested whether hybrid AAV2/9 virus carrying shRNA specific for CCL11, sh47 and sh137, could reduce eosinophilia in a mite allergen-sensitized mouse model. Both sh47 and sh137 viruses significantly reduced CCL11 level in CCL11-transformed NIH 3T3 cells, compared with others. Both viruses were delivered into Dp2 (group 2 allergen of Dermatophagoides pteronyssinus) sensitized mice before first challenge. The results showed that sh47 virus significantly reduced airway resistance, airway hyperresponsiveness, CCL11 levels and eosinophilia in the lungs of Dp2-sensitized mice. Th2 cytokines were also significantly reduced in the bronchoalveolar lavage fluid and Dp2-stimulated mediastinal lymphocytes of sh47 virus-treated mice. However, serum levels of Dp2-specific IgG1 and IgE as well as Th2 cytokine levels in Dp2-stimulated splenocytes culture supernatants were at the comparable levels to the untreated group. The results suggest that sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the hybrid AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma.
Disease-specific regulation of antibody-mediated immune diseases by the pathogenic antibody Fab fragments (107.15)
Authors: Yoshino Shin
Published: January 2023

Ag-specific immunotherapies for Ab-mediated diseases including asthma and rheumatoid arthritis have been unavailable. In the present study, we investigated whether Ab-mediated asthmatic responses as well as arthritis in mice were able to be Ag-specifically regulated by the pathogenic Fabs. Asthmatic responses were induced in BALB/c mice by i.p. injection of anti-OVA Abs followed by intratracheal (i.t.) challenge with OVA. Arthritis was induced in DBA/1J mice by i.v. injection of anti-OVA Abs and then by intraarticular injection of OVA. Fabs from anti-OVA Abs were prepared by digestion with immobilized papain and purified. Anti-OVA Fabs were i.t. administered at the same time or 30 min before OVA challenge to test their effect on asthmatic responses. To test the effect of anti-OVA Fabs on anti-OVA Ab-induced arthritis, the Fabs were i.v. injected immediately after administration of the intact Abs.The results showed that treatment with anti-OVA but not control Fabs was followed by a marked decrease in specific airway resistance (sRaw). The decrease in sRaw in anti-OVA Fabs-treated animals was associated with the suppression of infiltration of eosinophils in bronchoalveolar lavage fluids. Treatment of anti-OVA Ab-induced arthritis with anti-OVA Fabs was also followed by the significant inhibition of joint inflammation. Therefore, asthmatic responses and arthritis induced by the respective pathogenic Ab appear to be Ag-specifically downregulated by the Ab Fabs.