bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727 (LYM8P.641)
Authors: Lam Eric , Choi SungHee , Pareek Tej , Kim B , Letterio John
Published: January 2023

Cyclin-dependent kinase 5 (Cdk5) is a unique member of the family of cyclin-dependent serine/threonine kinases whose contextual and lineage-restricted activity is regulated by association with highly specific co-activators, p35 and p39. We previously demonstrated induction of both p35 expression and Cdk5 activity follows T cell receptor stimulation and is linked to post-translational modification of proteins that influence T cell activation and function. Here, we show IL-6-induced repression of Foxp3 gene expression in CD4+ T cells (Treg) requires Cdk5-mediated phosphorylation of the signal transducer and activator of transcription 3 (Stat3) specifically at Serine 727 (p-S727). Either lineage-restricted disruption of Cdk5 gene expression in T cells or pharmacological inhibition of Cdk5 activity abrogates the suppression of TGF-β-induced Foxp3 expression by IL-6. Cdk5-dependent phosphorylation of Stat3 does not alter either the abundance or nuclear localization of Stat3, nor phosphorylation of Stat3 at tyrosine 705, but it is required for proper Stat3 DNA binding to the Foxp3 gene on the enhancer II region and for the consequent disruption of Smad-dependent TGF-β-induced activation of Foxp3. These data define Cdk5 as molecular mediator of the repression of Foxp3 gene expression by inflammatory cytokines and suggest the potential to exploit specific inhibitors of Cdk5 to enhance the expansion of endogenous regulatory T cells.
Immunological characterization of human hosts to Ascaris lumbricoides and Trichuris trichiura infection in a population living in the rural municipality of Colomoncagua, Honduras (MPF6P.654)
Authors: Briggs Neima , Bottazzi Maria Elena , Beaumier Coreen , Sastry Jagannadha , Hotez Peter
Published: January 2023

Soil-transmitted helminth (STH) infections, specifically Ascaris, Trichuris, and Hookworm, compose three of the most prevalent Neglected Tropical Diseases, infecting over a billion people worldwide. Recurrent childhood STH infections have been shown to cause stunted physical growth, reduced physical fitness, and decreased school performance. Ascaris and Trichuris are of notable interest, given their abrupt decline from a peak prevalence and intensity in pre-adolescence to adulthood, with no known published works evaluating the possible underlying immunological mechanism. We used modern molecular techniques to (a) determine the burden of disease and (b) evaluate the immune profile of 30 adolescents and 51 adults between the ages 13-45, in rural Honduras, endemic for Ascaris and Trichuris. Using quantitative PCR of DNA extracted from stool we quantified the burden of disease of both Ascaris and Trichuris. Positive samples were stratified into groups based on the degree of infection, and controlled for with samples positive for 6 other common gastrointestinal parasites. Each group was immunologically characterized for serum Th1, Th2, and Th17 cytokines and associated antibodies. Results from this pilot study identify the basic serum immune profile associated with protection against Ascaris and Trichuris and serve as the foundation for a second, more comprehensive study that will guide vaccine antigen selection against each parasite by mimicking endemic host protective responses.
Timing effects on the inhibition of apoptosis by Toxoplasma gondii infection (MPF6P.655)
Authors: Peberdy Anna , Nash Paul
Published: January 2023

Toxoplasma gondii inhibits apoptosis in infected cells. This inhibition requires ongoing protein synthesis. Toxoplasma gondii is a protozoan that exists as an obligate intracellular parasite. Inhibiting apoptosis in infected cells may help preserve the parasite from damage by cytotoxic T lymphocytes during the immune response. Many inducers of apoptosis, such as cytokine withdrawal, have a long lag time and others, such as CTL killing, are cumbersome, so for this study, we focused on apoptosis induced by beauvericin and measured by nuclear morphology of cells observation in a UV microscope. Beauvericin is a toxin produced by certain fungi that is capable of inducing programmed cell death when cells are exposed. To determine the longevity of the inhibitory effect by Toxoplasma, we used cycloheximide to inhibit protein production after infection of the cells and monitored the loss of inhibition over time. We also evaluated how quickly after infection inhibition of apoptosis can be observed. We also share our observation of an in vitro artifact we inadvertently discovered: that mycoplasma infection interferes with infection of some cells by Toxoplasmam, resulting in a reduction in the inhibition of apoptosis.
The human protein resistin drives detrimental inflammatory monocyte responses in helminth infection (MPF7P.710)
Authors: Nair Meera , Jang Jessica , Barnes Mark , Le Gros Graham , Cooper Philip , Steel Cathy , Nutman Thomas , Lazar Mitchell
Published: January 2023

Resistin-like molecules (RELM) belong to a family of secreted proteins that are expressed in multiple helminth infections with important effects on the host immune response. However the importance of human RELM proteins in helminth infections is less well understood. To investigate this, we utilized transgenic mice that expressed the human resistin gene (Tg+). Following infection with Nippostrongylus brasiliensis, a hookworm that colonizes the lung and intestine, Tg+ mice exhibited significant lung inflammation, characterized by increased inflammatory monocytes and elevated parasite burdens compared to control Tg- mice. Genome-wide transcriptional profiling of the infected lung tissue from Tg+ and Tg- mice revealed that genes associated with cytokine and TLR signaling were significantly elevated in Tg+ mice. To delineate the downstream cellular mediators of resistin, we performed a resistin binding and chemotaxis assay and identified monocytes as the main cell-types that were responsive to resistin. Moreover, monocytes sorted from the infected lung tissue of Tg+ mice expressed significantly higher TNFα compared to Tg- mice. In human studies, increased serum resistin was associated with higher helminth burdens and was positively correlated with inflammatory cytokines. Together, these studies identify a detrimental role for human resistin in instigating a non-protective inflammatory response that may be mediated by monocytes.
Characterizing regulatory T cell differentiation to bacterial antigens in the gut (MUC8P.727)
Authors: Nutsch Katherine , Russler-Germain Emilie , Hsieh Chyi-Song
Published: January 2023

Regulatory T cells are an essential component of immune tolerance towards gut bacteria. Conversely, the microbiota shape the colonic Treg population. However, it is unclear if and how commensal bacteria direct naïve T cells to become peripheral Treg (pTreg) cells. Here, we define the process of pTreg development using transgenic T cells expressing commensal antigen reactive TCRs isolated from colonic Treg cells. We show that naïve TCR transgenic cells transferred into lymphoreplete mice are initially activated in a specific distal mesenteric lymph node. Treg cell differentiation is rapid, starting within 3 days, and efficient, with over 50% Foxp3+ cells arising 1 week after transfer. Contrary to prior observations, Foxp3+ cells are the most divided cells, suggesting that Treg cell differentiation is the primary cell fate upon commensal antigen encounter for these T cells. The most efficient period of Treg cell differentiation in TCR transgenic cells is around the age of weaning. Although T cells deficient in CNS1 showed a marked block in early Treg cell selection, dnTGFβRII cells were only partially deficient in Treg cell generation 1 week after transfer. By contrast, CNS1-deficient cells can upregulate Foxp3 at later time points. Thus, these data illustrate the kinetics, geography, and robustness of peripheral Treg cell selection to commensal bacteria, and suggests that CNS1 is an important region involved in early Foxp3 induction in a relatively TGFβ-independent fashion.
TLR-dependent anti-commensal antibodies regulate intestinal immune homeostasis (MUC9P.750)
Authors: Koch Meghan , Reiner Gabrielle , Lugo Kyler , Barton Gregory
Published: January 2023

Maternal antibodies have been suggested to be important for establishing intestinal immune homeostasis in developing offspring, yet the mechanisms by which maternal antibodies achieve this remain unclear. We analyzed the gut immune response in mice born to antibody-deficient dams and found that maternal antibodies regulate the activation of mucosal CD4+ T cells. Interestingly, maternal IgA, the dominant mucosal antibody isotype, was not necessary for this regulation. As such, we developed an unbiased flow cytometric based assay to assess the microbiota-specific antibody response, thereby allowing us to investigate if other antibody isotypes are important for enforcing intestinal immune homeostasis. Unexpectedly, we found that mice generate a robust anti-commensal IgG2b- and IgG3- response. This commensal-specific IgG response occurs through a pathway independent of T cells, yet dependent on signaling through TLR2 and TLR4. These TLR-dependent anti-commensal IgG antibodies are transmitted from mothers to their offspring. There, they function to regulate T follicular helper responses and subsequently dampen germinal center B cell responses. This work reveals a feedback loop whereby T cell-independent antibody responses function to regulate mucosal CD4+ T cell responses and thereby establish equilibrium between the host and its resident microbiota.
Impact of microbiota on ocular immunity (MUC9P.745)
Authors: Kugadas Abirami , Gadjeva Miheala
Published: January 2023

To evaluate the impact of microbiota on ocular immunity, the susceptibility of germ free mice (GFM) and conventional mice to ocular Pseudomonas aeruginosa infection was studied. We found that the GFM were significantly more susceptible to infection than the conventionally housed mice. This was exemplified by increased corneal bacterial presence and associated pathology. Quantitative LC-MS/MS analysis of the eyewashes showed that the elevated susceptibility of the GFM to infection correlated with alterations in the baseline levels of ocular immune effectors, suggesting that priming of ocular immunity with commensal microbiota strengthened the innate immune barrier function of the eye. Unlike the gut microbiota, not many bacteria are isolated from the conjunctiva, posing the question whether low levels of ocular commensals may be sufficient to prime local immunity. Colonization of GFM with ocular commensal Coagulase Negative Staphylococcus sp restored the resistance to infection. Consistently, similar corneal P. aeruginosa burden was detected in the challenged monocolonized GFM and conventional mice. Notably, the expression levels of innate immune effectors such as complement C3 was restored in the monocolonized mice. This increase in the lacrimal gland-derived immune effectors was IL-6 driven as neutralization of IL-6 increased the susceptibility to infection. In summary, these data show that tonic signals by local commensals are sufficient to promote innate immune responses.
Enhanced detection of eosinophils simultaneously with other leukocyte subsets using flow cytometry (TECH2P.902)
Authors: Guenther Garret , Kang Jia , Nunez Rene
Published: January 2023

Eosinophils differentiate in the bone marrow prior to migration to the vascular periphery and eventual residency in tissues. While they can either be a harmful or helpful component of the immune system, they help aid in the allergic response as well as modulate inflammation. Detection of eosinophils in blood samples was typically performed using H&E staining methods, however flow cytometric analysis of whole blood samples allows researchers to identify multiple cell types in a single sample that couldn’t be done before. Here, we were able to identify a distinct eosinophil population amongst clearly separated lymphocytes, monocytes, and granulocytes comparing FSC vs SSC using an ACEA NovoCyte flow cytometer. When compared to another flow cytometer, we were unable to clearly resolve other leukocyte subsets simultaneously with eosinophils; rather additional adjustments had to be made to resolve the eosinophil population at the expense of viewing the other leukocyte subsets. Eosinophils were also analyzed using their inherent auto-fluorescence in the FITC and PE channels, then back-gated to FSC vs SSC to confirm their identity. This shows that in order to identify multiple leukocyte subsets simultaneously using FSC vs SSC, a flow cytometer with a large dynamic range is essential for this type of analysis.
Enabling platforms for multiplex diagnostics (TECH2P.905)
Authors: Dufresne Claude
Published: January 2023

A number of technologies have evolved that are very well suited for the creation and manufacturing of multiplex diagnostics tests. This presentation will illustrate how biological content can be used to create high quality diagnostics devices. Examples covering different platforms will include R&D and production aspects of lateral flow strips, microarrays in microtiterplates, and of custom biochips. While Lateral Flow test strips have been around for a long time, their multiplex version is just now emerging in the next wave of low cost diagnostics. Printing several smaller lines or multiple dots, instead of single analyte lines, brings about multiplex benefits, such as providing more information per tested sample. In central diagnostic labs, the use of microarrays in microtiter plates allows for a high degree of multiplexing together with a very high sample throughput. When combined with the right content, microtiter plates can form the basis for robust IVD tests. We will illustrate this with an example for Human Papilloma Viruses, and sexually transmitted diseases. Finally, a number of companies have developed unique diagnostic format platforms. Often based on custom fabricated biosensors, electrodes or other CMOS structures need to be loaded with biological substances in a way that does not damage surface features. Combining advanced printing technology with a label-free photonic biosensor-based immunoassay platform, is one example that will be presented
A novel approach to generate LIGHT mutants with selectivity to binding partners (TECH2P.925)
Authors: Liu Weifeng , Nathenson Stanley , Almo Steven
Published: January 2023

LIGHT is a member of TNF superfamily, which participates in interactions with many TNF receptors, including membrane-anchored HVEM and LTβR, as well as solely soluble DcR3. Interaction of LIGHT with HVEM and LTβR directly triggers intracellular signaling, which regulates a wide range of immune response, such as T cell proliferation, thymocyte differentiation and lymphocyte trafficking and migration. LIGHT:DcR3 interaction tends to attenuate the immune response. Besides LIGHT, DcR3 also neutralizes two critical inflammatory molecules TL1A and FasL. The mechanisms that support the broad specificity of LIGHT need to be defined and the functions of LIGHT interacting with different receptors await to be dissected. We have solved the crystal structures of LIGHT:HVEM and LIGHT:DcR3 complexes, revealing the critical residues involved in the interactions of LIGHT with HVEM and DcR3. We have also developed a novel approach to generate LIGHT mutants with altered binding selectivities to different receptors, which allow the further dissection of the functions of LIGHT. This novel method creates LIGHT mutants by transplanting the loops responsible for receptor recognition from TL1A to the corresponding positions of LIGHT. The generated LIGHT chimeras still possesses the wild-type like binding abilities with DcR3 and LTβR, but dramatically compromise the binding affinities with HVEM. Applications of the LIGHT chimeras reveal cooperative signaling of LIGHT through HVEM and LTβR.
Immune regulatory effects of Fingolimod (FTY720) on T cells (THER7P.959)
Authors: Gandhi Roopali , Mazzola Maria
Published: January 2023

FTY720 is the functional analogue of sphingosine and is the first oral treatment for relapsing remitting MS. We investigated the immune regulatory effects of Fingolimod (FTY720) on T cells in Multiple Sclerosis (MS) and controls. FTY720 function by blocking immune cell trafficking and prevents relapses in Multiple Sclerosis. We found that T cells activated in the presence of FTY-720 were less inflammatory with decreased proliferation and cytokines expression. FTY720 treated T cells have decreased expression of granzyme B and induced less neuronal cytotoxicity. We showed, FTY720 treated T cells have unique anti-inflammatory immune gene profile. The anti-inflammatory effect of FTY720 on T cells is dependent upon increased expression of a regulatory transcription factor TCF1 (Transcription factor 1). TCF1 has been previously linked to T- cell differentiation and lineage specification in mice. We show that the TCF1 could regulate human T cell differentiation. Knock down of TCF1 gene expression in human T cells switched anti-inflammatory T cells to pro-inflammatory T cell phenotype. In summary, we showed that FTY720 could regulate human T cells differentiation and its immune regulatory function is dependent upon the expression of regulatory transcription factor TCF1.
IL-23 promotes the development of hepatocellular carcinoma through the induction of IL-17-producing ILC3 (TUM10P.1044)
Authors: Liu Haiyan , Liu Yonghao , Song Yuan , Hu Bo
Published: January 2023

IL-23 has been shown to play a pathogenic role in autoimmunity through the promotion of Th17 cells. However, its role in tumor development remains controversial. Some studies demonstrated that IL-23 could activate T cells and NK cells to suppress tumor metastasis and growth. However, it has also been shown to promote tumor growth in colon cancer, lung cancer, breast cancer and melanoma. In the current study, we established a murine hepatocellular carcinoma (HCC) model and found that IL-23 could promote HCC development. The in vitro study demonstrated that IL-23 could directly promote HCC cell proliferation but not affect its apoptosis. Furthermore, although IL-23 could significantly increase the percent of Tc17 (CD8+IL-17+), the majority of IL-17-producing cells induced by IL-23 in the liver was innate lymphoid cell type 3 (ILC3). IL-17 could further suppress anti-tumor immune responses through the recruitment and promotion of myeloid derived suppressor cells (MDSC). Our study, for the first time, demonstrated that IL-23 could enhance HCC development through direct promotion of tumor cell proliferation, as well as suppressing anti-tumor immune response by inducing IL-17-producng ILC3.
A role for IL-13 in the progression of prostate tumors (TUM10P.1046)
Authors: Gurusamy Devikala , Shoe Jennifer , Hocker James , Hurwitz Arthur
Published: January 2023

IL-13 is predominantly a Th2-derived cytokine that displays a variety of effects on inflammatory and stromal cells. Receptors for IL-13 are expressed by several cancers including prostate carcinoma. Although expression of IL-13 was previously positively correlated with human prostate cancer, there are no studies that validate the functional contribution of IL-13 during prostate cancer progression. In this study, we examined the importance of IL-13 during prostate tumor progression using Transgenic Adenocarcinoma of the Mouse Prostate [TRAMP] model of prostate cancer. Il13-deficient [Il13-/-] mice were bred onto the TRAMP background to generate TRAMP+Il13+/+,TRAMP+Il13+/-and TRAMP+Il13-/- genotypes. Mice were euthanized at 25, 30, 40 and 45 weeks of age and tumors were characterized for weight, leukocyte infiltration, and tumor histopathology. Preliminary data suggests IL-13 is important for prostate tumorigenesis, as tumors obtained from TRAMP+Il13-/- mice were smaller than those obtained from TRAMP+Il13+/- or TRAMP+Il13+/+mice. On-going studies continue to examine differences in histopathology, nature and function of immune infiltrates. Future studies are aimed at understanding distinct cellular sources of IL-13, as well as precise targets of IL-13 that contribute to tumor progression, focusing on both cells of hematopoietic lineage as well as epithelial and stromal cells. Findings from these studies may identify IL-13 as a novel target for treating prostate cancer.
IL-36γ promotes antitumor immune responses (TUM10P.1028)
Authors: zuo shuting , Wang Xuefeng , Lu Binfeng
Published: January 2023

Introduction of an appropriate inflammation to tumor should greatly increase tumor immunogenicity and thereby help breaking immune tolerance to tumor antigens and increase response rates of immunotherapy. Cytokines play a pivotal role in regulating tumor immunogenicity and antitumor immunity. IL-36γ is important for the IL-23/IL-17-dominated autoimmune diseases as well as anti-BCG Th1 immune responses. However, the impact of IL-36γ on tumor immunity is unknown. Here, we found IL-36γ stimulated IFN-γ production by CD8 T cells synergistically with TCR signaling and/or IL-12. Importantly, IL-36γ exerted profound antitumor effects in vivo and transformed the tumor microenvironment in favor of tumor eradication. Furthermore, IL-36γ strongly increased the efficacy of tumor vaccination. Our work also showed that IL-36γ expression in human cancers inversely correlated with cancer progression. Our study establishes a novel role of IL-36γ in promoting antitumor immune responses and suggests its potential clinical translation in cancer immunotherapy.
Combination therapy of radiation and interleukin 12 induced regression of large orthotopic liver cancers by modulation of tumor microenvironment (VAC12P.1111)
Authors: Tao Mi-Hua , Wu Chia-Jen , Tsai Yi-Ting , Wu Ping-Yi
Published: January 2023

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide with few therapeutic options. Radiation is one of the mainstay cancer therapies. We hypothesize that combination of radiation and local interleukin 12 (IL-12) immunotherapy may change the suppressive tumor microenvironment to a proinflammatory condition favorable in induction of antitumor immune responses. IL-12 has potent antitumor activities through activation of CD8+ T cells and natural killer cells, reprogramming several types of myeloid-derived suppressor cells to a phenotype that favors immune activation, and inhibiting new blood vessel formation within tumors. Our data showed that combination of radiation and intratumoral injection of an adenoviral vector encoding IL-12 (Ad/IL-12) led to tumor regression in the majority of animals bearing large orthotopic hepatocellular carcinoma and significantly prolonged their survival. Single treatment of radiation or Ad/IL-12 delayed tumor growth but did not result in tumor regression. Mechanistic studies revealed that combination therapy greatly increased the cell numbers and activities of tumor-infiltrating CD8+ T cells, while the cell numbers and suppressive functions of MDSC and tumor-associated macrophages (TAM) were significantly decreased. Our results strongly suggest that a combinatorial treatment of radiation and IL-12 may represent an alternative therapy for advanced HCC.
Rate of pESC plasmid loss in Saccharomyces cerevisiae in the absence of selective pressure (VAC13P.1126)
Authors: Jordan Jasmine , Nash Paul
Published: January 2023

Recombinant yeast has been proposed as a vaccine modality, especially for inducing CTL responses. The production of yeast expressing proteins of interest would be facilitated if they can be grown in less expensive non-selective medium. This would only be practical if the population of yeast retains high levels of plasmid over the duration of the growth. We tested the Saccharomyces cerevisiae multiple gene auxotroph with pESC-URA to determine the rate of plasmid loss in the population. The yeast maintain the plasmid much longer than anticipated, suggesting that growth in non-selective medium may be a viable method. Induction of protein expression increases the competitive advantage of plasmid loss. Growth rates of the yeast and of plasmid loss under these variable conditions was also determined.
A non-cytolytic role for NK cells during gp96-mediated immunotherapy (VAC13P.1129)
Authors: Sedlacek Abigail , Binder Robert
Published: January 2023

Immunogenic Heat Shock Proteins (HSPs) are capable of eliciting potent immune responses against their chaperoned antigens and are being investigated as a potential cancer immunotherapy. The current model of HSP mediated immune responses includes roles for Dendritic Cells (DCs) and T cells in priming and executing the HSP-mediated response. Additionally, NK cells are necessary to inhibit tumor growth following immunotherapy with HSPs. NK cells are innate lymphoid cells whose effector functions include cytokine production and target cell lysis. Despite their requirement, little is known about what role(s) NK cells play in HSP-mediated immune responses. In these studies, we aim to determine if NK cells are acting as helper cells or effector cells using the prototypical immunogenic HSP, gp96. Our results demonstrate that depletion of NK cells specifically during the effector phase (post-gp96 immunization) results in a loss of control of D122 tumor growth. Interestingly, NK cells isolated from mice immunized with gp96 do not exhibit increased killing ex vivo, suggesting NK cells may be acting as helper cells instead of directly killing tumor cells. Additionally, we determined that NK cells improve DC activation in response to gp96 in vitro, suggesting NK cells may also play a role in priming HSP-mediated T cell responses. Collectively, these data indicate that HSP-activated NK cells are capable of acting as helper cells, potentially during multiple stages of the immune response.
Trafficking of conventional dendritic cells into the central nervous system in response to SIV and Morphine creating viral reservoir in follicular DC within cervical lymph nodes of rhesus macaques (VIR9P.1146)
Authors: Jain Pooja , Nehra Artinder , Hollenbach Rebecca , Sagar Divya , Callen Shannon , Yao Honghong , Buch Shilpa , Khan Zafar , Byrareddy Siddappa
Published: January 2023

Neuroinvasion by HIV leads to neurocognitive diseases and alters the permeability of the BBB. Conventional dendritic cells (cDCs) infiltrating the CNS can potentially encounter HIV from infected perivascular macrophages lining the BBB. They can thereafter carry HIV to cervical lymph nodes (CxLNs), where HIV particles will be transmitted to CD4 T cells and eventually get trapped in CD35+ follicular DCs (fDCs) to create a viral reservoir within the germinal centers. HIV patients using drugs of abuse such as morphine can further compromise the BBB and enhance infectivity of the CNS. Research demonstrating the presence and role of DCs in the CNS and lymphoid organs during HIV infection has not well established. In this respect, we explored the presence of DCs in the brain parenchyma and CxLNs of rhesus macaques infected with SIV and administered with morphine. Cells positive for cDC markers were consistently found in the brain parenchyma of SIV-infected macaques and enhanced within infected macaques given morphine. Examination of CxLNs in SIV-infected macaques confirmed presence of SIV p27+ cDCs, and CD4+ T-cells entering CD20+ germinal centers further trapping of virus within CD35+ fDCs. Ongoing studies are assessing the effect of SIV chronic infection and Morphine on fDC viral entrapment. These results provide first evidence of DC trafficking and containment of SIV particles within fDCs of cervical lymph nodes in the context of NeuroAIDS vis-à-vis drugs of abuse.
Distinct impact of SIV infection in interstitial and alveolar lung macrophages on the pathogenesis of lung disease in rhesus macaques (VIR9P.1147)
Authors: Kuroda Marcelo , Cai Y , Sugimoto C. , Arainga M , Midkiff C. , Walker E , Haupt E , Alvarez X , Lackner A , Kim W. , Didier Elizabeth
Published: January 2023

Declining CD4+ T cells during HIV infection contributes to immunodeficiency, but we recently reported that increasing monocyte turnover better predicted onset of terminal disease progression to AIDS in SIV-infected rhesus macaques. Here we describe the kinetics of SIV infection on distinct lung macrophage subsets and on monocyte/macrophage turnover rates in relation to pulmonary disease progression in rhesus macaques. Monocyte and tissue macrophage turnover were monitored by in vivo BrdU injection and cell uptake. Cell-associated SIV DNA in lung tissue was quantified by qPCR and in situ hybridization. Massive SIV infection was associated with increasing death rate of shorter-lived lung interstitial macrophages (IM) with elevated monocyte turnover and progression to AIDS. Conversely, the turnover of alveolar macrophages (AM) and decreasing numbers of CD4+ T cells in lung tissue did not directly correlate with disease progression. SIV DNA levels within IM and AM of the lung increased as monocyte turnover increased, but did not change within lung CD4+ T cells regardless of the stage of disease. These data suggest that SIV infection and concurrent destruction of lung IM contribute to pulmonary pathogenesis during AIDS progression while the longer-lived AM that become infected may contribute to establishing a virus reservoir. Also, bronchoalveolar lavage (BAL) specimens contain AM but not IM, so may be insufficient for fully evaluating macrophage-mediated responses in the lung.
Lactobacilus johnsonii supplemented mice modulate RSV-induced disease and is associated with increased DHA metabolite that alters DC activation through PPAR- γ. (VIR9P.1148)
Authors: Fonseca Wendy , Jang Sihyug , Rasky Andrew , Morris Susan , Ptaschinski Catherine , Fuimura Kei , Lynch Susan , Lukacs Nicholas
Published: January 2023

The gut microbiome contributes to a variety of mammalian processes, including modulation of host immune responses. We have found that L. johnsonii-supplemented mice displayed altered gut microbiome profiles and reduced RSV-induced pulmonary immunopathology. Metabolomics analysis of plasma from L. johnsonii-supplemented mice identified docosahexaenoic acid (DHA) and 2-hydroxyisobutyrate (butyrate precursor) as increased metabolites. We hypothesized that DHA and butyrate played a role in the modulation of the immune response in lung. We found that bone marrow-derived dendritic cells (BMDC) treated with DHA or butyrate expressed and produced reduced levels of IL-12, IL-6, IL-1β, IFN-β and TNF-α when stimulated with LPS, CpG or RSV compared to untreated cells. DHA treatment of RSV-infected BMDC prior to their co-culture with T cells isolated from RSV-infected mice significantly reduced their production of Th2 cytokines and IFNΥ. Then, we determined whether DHA was activating the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) in BMDC, which has been described as an inflammatory response inhibitor. We observed that BMDC treated with DHA and PPAR- γ antagonist (GW9662), had altered expression of mRNA and production of cytokines compared to DHA treated cells. These results suggest that DHA is modulating the immune response through PPAR-γ in dendritic cells and may be one mechanism of attenuating RSV immunopathology in mice supplemented with L. johnsonii.
A novel CD141+ human blood plasmacytoid dendritic cell subtype produces high levels of IFN-α and acquires virus-associated cellular material from live tumor cells (VIR9P.1136)
Authors: Shih Tiffany , Dai Jihong , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Dendritic cells (DCs) are a heterogeneous population important in regulating both innate and adaptive immunity. Human blood DCs are broadly characterized as pDC(BDCA2+), mDC1(BDCA1+), and mDC2(BDCA3+(CD141)). While much work has focused on the functional role of CD141hi mDCs, we investigated the maturation potential and functional significance of a novel subtype of classically defined plasmacytoid DCs (BDCA2+) co-expressing intermediate levels of BDCA3. Using flow cytometry, we identified four human blood DC subsets: BDCA2+/3-, BDCA2+/3int, BDCA2-/3int, and BDCA2-/3hi. Upon stimulation with TLR9 agonists CpG-A or HSV-1, the BDCA2+/3int DCs had the highest frequency of IFN-α+ and TNF-α+ cells among these DC subtypes. The BDCA2+/3int compared to BDCA2+/3- DCs also become more activated and mature with relatively higher expression of co-stimulatory and migratory surface receptors in response to HSV-1. IRF7, the master regulator of IFN production in pDCs, was constitutively expressed by BDCA2+/3- and BDCA2+/3int ­DCs but not by BDCA2- DCs. Both BDCA2+/3int and to a lesser extent, BDCA2+/3- DC preferentially acquired cellular material from HSV-infected Raji cells. These data indicate that BDCA2+/3int DCs exhibit major functions associated with classical pDCs and are phenotypically and functionally distinct from BDCA2-/3int and BDCA2-/3hi DCs. Together these results describe a novel CD141+ pDC subset that is very potent and virus responsive as compared to the CD141hi mDCs.
Single clone expression cloning and protein microarray platforms for the identification of novel viral immunomodulatory proteins (VIR9P.1157)
Authors: Tom Irene , Martin Nadia , Ramani Sree , Gonzalez Lino
Published: January 2023

Viruses encode proteins that interact with surface receptors on immune cells to evade recognition and elimination by the host immune system. In this study, we focused on the E3/49K protein of human adenoviruses D (HAdV D) a highly diverse protein uniquely found in HAdVs D that was recently demonstrated to suppress leukocyte activation and effector functions through an interaction with receptor tyrosine phosphatase CD45 on immune cells. To advance the search for novel immunomodulatory receptors targeted by the E3/49K protein, we utilized two complementary high-throughput screening technologies consisting of single clone expression cloning and protein microarray platforms. Two highly divergent E3/49 proteins, encoded by HAdV-28 and HAdv-53 serotypes, were systematically probed against thousands of interactions either expressed on the cell-surface or displayed on a protein microarray. We found that both HAdV-28 and HAdV-53 E3/49K proteins interacted with CD45. Furthermore, we report the discovery of two additional receptors specifically targeted by HAdV-28, the leukocyte immunoglobulin-like receptor 1 (LILRB1), a well known immune receptor with inhibitory functions, and putative neuronal cell adhesion molecule (PUNC). Interestingly, we found that PUNC is expressed in immune cells, suggesting a previously undescribed function in immunity. Here we demonstrate the feasibility of this approach for identifying proteins with previously unknown involvement in the human immune response.
Targeting HSV-1 protein ICP6 by RIP1 and RIP3 initiates necroptosis to restrict HSV-1 propagation in mice (VIR9P.1159)
Authors: Huang Zhe , Wu Suqin , Liang Yaoji , Han Jiahuai
Published: January 2023

Necroptosis is a type of programmed necrosis that participates in host defense to eliminate certain pathogens. We show that receptor interacting protein 3 (RIP3 or RIPK3) restricts herpes simplex virus type 1 (HSV-1) propagation in mouse cells and in mice by mediating necroptosis of infected cells. ICP6 protein of HSV-1 triggers necroptosis by direct interaction with RIP1 and RIP3 through its RIP homotypic interaction motif (RHIM). ICP6 can be dimerized or oliogmerized by its C-terminal R1 homology domain. This leads to RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction and subsequent execution of necroptosis in HSV-1 infected cells. We show that both pro-necroptotic and anti-necroptotic viral RHIM exist since the RHIM in viral inhibitor of RIP activation (vIRA) of murine cytomegalovirus (MCMV) is known to inhibit necroptosis. Our data demonstrated that RIP1 and RIP3 can directly sense certain RHIM like sequences encoded by some viruses to initiate cellular responses for pathogen clearance.
HIV inhibits LPS-induced IL-27 production via HIV tat through the inhibition of TRAF-6, and consequent inhibition of PI3K and p38 and JNK MAPKs in human macrophages (VIR9P.1140)
Authors: Kumar Ashok , Gajnayaka Niranjala , Fernandez Jason , Angel Jonathan , Kozlowski Maya
Published: January 2023

Monocyte-derived macrophages (MDM) from HIV-infected patients and MDM infected in vitro with HIV manifest inhibition of cytokines including IL12. IL27, an IL12 family cytokine, was shown to inhibit HIV replication in macrophages. Whether HIV infection or HIV accessory protein(s) impact IL27 production in macrophages remains unknown. Herein, we show that in vitro HIV infection as well as intracellular HIV and HIV-tat peptides inhibited LPS-induced IL27 production in MDM suggesting that HIV-tat inhibits IL27 production by impairing TLR-4 signalling. To study the mechanism governing HIV-tat-mediated inhibition of LPS-induced IL27 production, we first established that p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), the phosphoinositide-3-kinase (PI3K), SRC homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1) and Src kinases regulated LPS-induced IL27 production in MDM. HIV-tat caused TNF receptor associated factor (TRAF)-6 inhibition and consequent decreased phosphorylation of downstream PI3K, and p38 and JNK MAPKs implicated in LPS-induced IL27 production. However, SHP-1 and Src kinases were not involved in HIV-Tat-mediated inhibition of LPS-induced IL27 production. In contrast to HIV-tat, in vitro HIV infection of MDM inhibited LPS-induced p38 and JNK activation. Overall, HIV inhibits LPS-induced IL27 production via HIV tat through the inhibition of TRAF-6, and consequent inhibition of PI3K and p38 and JNK MAPKs in macrophages.
Tetherin (BST-2) promotes control of a persistent infection by reducing viral spread and T cell exhaustion (VIR10P.1173)
Authors: Kenyon Elizabeth , Nayak Debasis , Moseman E , McGavern Dorian
Published: January 2023

Type I interferons play a critical role in host defense against most viral infections. This is achieved in part through induction of innate antiviral proteins like tetherin (BST-2). BST-2 was identified based on its ability to “tether” viral proteins to infected cells and decrease virion release in vitro. How this protein functions in the host defense against a persistent viral infection in vivo is not entirely understood. To gain novel insights into BST-2 function, we examined its role during the establishment of a persistent lymphocytic choriomeningitis virus (LCMV) infection. Transgenic overexpression of BST-2 in cell culture decreased release of LCMV from infected cells. We uncovered in vivo that BST-2 is significantly upregulated early after viral infection in plasmacytoid / myeloid dendritic cells, monocytes / macrophages, and brain-resident microglia. Deletion of BST-2 enabled a persistence-prone strain of LCMV to transition more efficiently from the splenic marginal zone to the white pulp following intravenous inoculation. This resulted in enhanced T cell exhaustion, which was evidenced by reduced cytokine production and altered motility. Interestingly, these early alterations in antiviral immunity translated into delayed control of LCMV in peripheral tissues and lifelong persistence in the brain. These data demonstrate that expression of single innate antiviral protein can influence adaptive immune cell function and dictate the fate of a viral infection.
Genetic risk alleles associated with serologic autoimmunity in normal individuals (BA3P.113)
Authors: Raj Prithvi , Li Quanzhen , Karp David , Olsen Nancy , Sivils K. , Dozmorov Igor , Song Ran , James J. , Kelly J , Lauwerys Bernard , Gregersen Peter , Wakeland Edward
Published: January 2023

We screened 2628 normal individuals for antibodies binding to the mix of antigens in a human nuclear extract and identified 579 (26.05%) individuals with significant levels of IgG antibodies to self-antigens. Next, we analyzed 639 representative sera from ANA-, ANA+, ILE and SLEs on an autoantigen proteomic array, which identified 33 antigens strongly recognized by IgG autoantibodies in ANA+ group as compared to ANA- normals. Of these 33, 19 antigens were non-nuclear or cytoplasmic proteins and 14 were nuclear proteins, including known SLE associated antigens such as dsDNA and Histones. Antigen cluster analysis showed that DNA and RNA clusters are more frequent in SLE and ILE group, while matrix proteins and antiphospholipid antibodies clusters are more frequent in ANA+ normals. Next, all the individuals were genotyped with immunochip and subset by target sequencing. Quantitative genetic association test identified SNPs associated with ANA and auto-antigens. The strongest association signal was observed at HLA, where SNP rs3117103 near BTNL2, rs9268832 near HLA-DRA and rs2395252 in HLA-DQA2 gene showed strongest (10-7) association. Other signals were in/near BANK1, PTTG1 and CR1L genes. RNA sequencing analysis identified ANA risk alleles associated with up regulation of HLA-DRB1 in monocytes and BANK1 in B cells. Our data suggest that functional variations in or near HLA DRB1 and BANK1 gene may be associated with development of humoral autoimmunity in general population.
Ikaros mediated gene regulation in human systemic lupus erythematosus (BA4P.138)
Authors: Nagpal Kamalpreet , Tsokos George
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. Genetic susceptibility and molecular aberrations create an abnormal T cell phenotype. One of the key components contributing to signaling defects in SLE pathogenesis is the serine threonine phosphatase (PP2A). The factors affecting expression of PP2A in SLE T cells thus warrant an extensive study. We have previously shown that the transcription factor Ikaros binds to a site in the first intron of PP2A and acts as a repressor of PP2A expression. Here, we build upon this key finding of a novel regulatory mechanism involved in the pathogenesis of SLE. This intronic Ikaros binding site in PP2A harbors a single nucleotide polymorphism. We report that Ikaros exhibits differential binding to this site. Using oligonucleotide binding assays as well as EMSA, we demonstrate defective binding of Ikaros to the site containing the SLE risk SNP as compared to WT sequence. Luciferase reporter assays show that promoter activity of PP2A is increased in presence of this SNP. We are further analyzing the recruitment of Ikaros to this site in individuals with either WT or the variant allele, to test our hypothesis that increased levels of PP2A observed in SLE patients is at least in part due to reduced Ikaros binding at this site. To conclude, we demonstrate that the repressor Ikaros binds with less avidity to a PP2A intronic SLE risk variant and allows increased PP2A expression, a key molecule in SLE pathogenesis.
Galectin-1 plays an essential role in intravenous tolerance in experimental autoimmune encephalomyelitis (BA4P.139)
Authors: Mari Elisabeth , Rasouli Javad , Zhang Guang-Xian , Ciric Bogoljub , Rostami A
Published: January 2023

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the central nervous system (CNS). EAE can be induced by immunizing mice with myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55, in adjuvant. It has been previously demonstrated that intravenous (i.v.) injection of MOG35-55 post immunization induction suppresses disease development, a phenomenon called i.v. tolerance. The glycan-binding protein galectin-1, produced primarily by regulatory T cells plays an immunoregulatory role in EAE by inducing tolerogenic DCs and IL-10-producing regulatory type 1 T cells (Tr1). To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 peptide i.v. to wild-type (WT) and galectin-1-/- (Gal1-/-) mice with ongoing EAE. MOG35-55 rapidly suppressed disease in the WT, but not in the Gal1-/- mice. Expression of pro-inflammatory cytokines IFN-gamma and IL-17A were reduced in the CNS and periphery of tolerized WT mice, while the number of Tr1 cells increased in the periphery. In contrast, i.v. MOG35-55 significantly increased IFN-gamma and IL-17A in the CNS and periphery of Gal1-/-mice and these mice had markedly fewer Tr1 cells in the periphery. Our data suggest that galectin-1 mediated i.v. tolerance induction is dependent on the downregulation of Th1 and Th17 pro-inflammatory responses, and enhanced development of Tr1 cells.
STAT3 signaling is essential for the germinal center maintenance and pathogenesis of lupus (BA4P.122)
Authors: Ding Chuanlin , Dascani Paul , Yan Jun
Published: January 2023

Antibody maturation as well as memory B and plasma cell differentiation occurs primarily in the germinal center (GC). Lupus may develop as a result of enhanced GC activity. Many factors involved in GC reaction, including T follicular helper (Tfh) cells, IL-21, IL-6, play critical roles in lupus pathogenesis. These inflammatory cytokines predominantly activate the STAT3 signaling pathway. Here, we demonstrate that STAT3 signaling in B cells is essential for antibody response as well as GC formation and maintenance. Increased cell apoptosis and down-regulated Bcl-xL expression are found in the STAT3 deficient GC B cells. The Tfh cell response is significantly decreased in B cell STAT3-deficient immunized mice. Further, STAT3 deficiency in autoreactive B cells results in decreased autoantibody production upon immunization with apoptotic cells. Results obtained using B cell STAT3 deficient MRL.Fas(lpr) mice suggest that STAT3 signaling contributes to the pathogenesis of lupus by regulation of autoantibody production. Our findings thus provide new insights into the role of B cell STAT3 signaling in the GC maintenance and pathogenesis of lupus.
Function of CK2 kinase activity in Th17 cell polarization and EAE (BA4P.125)
Authors: Gibson Sara , Qin Hongwei , Liu Yudong , Buel Amber , Benveniste Etty
Published: January 2023

Auto-reactive CD4+ T cells drive many autoimmune diseases including multiple sclerosis and colitis. Th1 and Th17 cells are pathogenic in the context of autoimmunity while Treg cells promote disease resolution. CK2 is a constitutively expressed and highly conserved serine/threonine kinase that promotes a number of pro-survival and pro-inflammatory signaling pathways including PI3K/Akt/mTOR, NF-κB, and JAK/STAT. These pathways are essential for CD4+ T cell activation and polarization, but little is known about how CK2 functions in T cells. Here we demonstrate that protein levels of the major catalytic subunit of CK2, CK2α, and CK2 kinase activity are elevated in murine CD4+ T cells polarized to the Th1 and Th17 phenotypes in vitro compared to naïve CD4+ T cells. Furthermore, CK2α expression is elevated in CD4+ T cells that are activated and infiltrate the central nervous system during EAE, a murine model of MS. Using the small molecule inhibitor CX-4945, which targets CK2α, we demonstrate that CK2 kinase activity promotes Th17 cell polarization in vitro, but not the polarization of Th1 or Treg cells. Additionally, in vivo treatment with CX-4945 during EAE resulted in a dampened MOG-specific Th17 response and a decrease in IL-17A+ and IL-17A+IFN-γ+ CD4+ T cells present in the central nervous system. Our data implicate CK2 as a novel regulator of T cell differentiation, and suggest that CK2 could be targeted in Th17 cell driven autoimmune disorders.