bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressive Th17 responses in patients with active tuberculosis is associated with reduced IL-6R expression on CD4+ T cells (37.51)
Authors: Chen Xinchun , Zhang Mingxia , Liao Mingfeng , Zhou Boping
Published: January 2023

The frequencies of IFN-γ-, IL-4-, IL-17-, FoxP3- and IL-6R- expressing CD4+ T cells in blood and/or pleural effusion sample of healthy donors, subjects with latent tuberculosis infection and patients with active tuberculosis, were analyzed by flow cytometry. The frequency of Th17 cells in patients with active tuberculosis is significantly lower than those in healthy donors and individuals with latent tuberculosis infection. The reduction of Th17 response was significantly more profound in patients with severe tuberculosis disease compared to those with pulmonary tuberculosis only. The frequency of Th17 cells was significantly lower in pleural effusion samples than in cohort peripheral blood from patients with tuberculous pleurisy. Correlation analysis showed that suppressive Th17 response observed in patients with active tuberculosis was significantly correlated with the reduced expression of interleukin-6 receptor on CD4+ T cells, but not the concentration of cytokine TGF-βand IL-6. consistently, in vitro study showed that Mycobacterium tuberculosis products inhibit the expression of interleukin-6 receptor on CD4+ T cells. Our results demonstrated that decreased Th17 responses were associated with the clinical outcome of Mycobacterium tuberculosis infection. Suppression of Th17 response through down-regulation of interleukin-6 receptor expression may be an important mechanism in the development of active tuberculosis.
IL-18, TNF-α, and IFN-γ are involved in a network of cytokine interactions that promote control of Mycobacterium tuberculosis by human macrophages (37.52)
Authors: Robinson Cory , Nau Gerard
Published: January 2023

Mycobacterium tuberculosis (MTB) is one of the most successful human pathogens and has infected approximately one-third of the global population. Understanding fundamental aspects of positive immune responses is pivotal to developing new strategies to combat infections. We previously published that treatment of primary human macrophages with interleukin (IL)-12 and a soluble reagent to neutralize IL-27 restricts MTB growth during infection. To understand this at the mechanistic level, we have investigated the immunological parameters that are essential in the control of MTB growth. IL-18, tumor necrosis (TNF)-α, and interferon (IFN)-γ are among the cytokines produced by human macrophages during infection when IL-12 is supplied and IL-27 is neutralized. We have explored the contribution of each of these cytokines and found them all to play a significant role in controlling MTB growth in human macrophages. IL-18 is important for achieving optimal levels of IFN-γ production in response to IL-12 and MTB. Similarly, IFN-γ enhances TNF-α expression and secretion. Furthermore, quantitative assays have shown that IFN-γ increases expression of the TNF receptor. In turn, TNF-α augments expression of the receptor for IFN-γ resulting in more receptor chains at the cell surface that are available for cytokine-induced signaling. These results suggest that IL-18, TNF-α, and IFN-γ interact in cooperative fashion to restrict growth of MTB in primary human macrophages.
Enhancing innate immune defense against Staphylococcus aureus infection by therapeutic targeting of virulence regulation (37.55)
Authors: Sully Erin , Alexander Susan , Hall Pamela , Gresham Hattie
Published: January 2023

Staphylococcus aureus is a medically significant pathogen that uses both cell-associated and secreted virulence factors to evade innate immune host defense. Because antibiotic resistance is an emerging and increasing problem with this pathogen, an alternative strategy for ameliorating the infection is to enhance host defense by inhibiting production of these virulence factors. We screened libraries of small molecules for inhibition of a pheromone-dependent quorum sensing operon, agr, which regulates expression of over 80 different genes including toxins and capsule that directly antagonize phagocyte-dependent host defense. A uricosuric drug used to treat gout, benzbromarone, inhibited pheromone dependent virulence without affecting bacterial viability in multiple S. aureus strains. When complexed with cyclodextrin as a vehicle, it inhibited S. aureus infection in a mouse model of dermonecrosis. More detailed in vitro studies revealed that the drug/cyclodextrin complex affected expression of both cell-associated and secreted virulence factors making the bacteria more susceptible to killing by oxidants and acid. Even after 50 generations of bacterial growth in the presence of benzbromarone, pheromone dependent virulence was still inhibited in the presence of the drug. These data indicate that signaling in bacterial pathogens can be targeted to enable killing and clearance by the innate immune system while limiting the development of induced resistance.
Immunoglobulin Class Switching During RSV Infection Is PKR-Dependent (38.1)
Authors: Thakur Sheetal , Zallinger Zachary , Corn-Minor Radiah , Johnson Teresa , Graham Barney
Published: January 2023

Adaptive immune responses play a critical role in protection against many pathogenic viruses including respiratory synctial virus (RSV). Previous studies from our laboratory have established protein kinase R (PKR) as an important mediator of RSV-induced innate immunity and immunopathology. Thus far, very little is known about the role of PKR in adaptive immune mechanisms involving B cells following RSV infection. Therefore, in this report the role of PKR in regulation of RSV-induced immunoglobulin (Ig) class switching in a mouse model was investigated. Wild type (WT) and PKR-deficient (PKR-/-) mice were infected with RSV intratracheally (1x 107 pfu/mouse) and serum Ig levels were determined at 3, 7 and 14 days postinfection. Increased serum levels of IgA, IgG and IgE were observed in WT mice compared to the PKR-/- mice. Furthermore, western blot analysis on extracts from RSV infected cells, showed that serum from infected WT mice contained significantly greater levels of RSV-specific IgG and IgA than serum from PKR-/-mice. Several immunoregulatory cytokines including TGF-β, IL-12 and IFN-γ significantly lower in PKR-/- mice compared to the WT mice. These cytokines are known to be critical factors in determining the modulation of B cell activation, differentiation which includes class switch recombination. Taken together, these results demonstrate that PKR plays an important role in virus specific Ig responses.
Protective human MAbs to a highly conserved conformational determinant on influenza M2e (38.14)
Authors: Grandea Andres , Olsen Ole , Cox Tom , Renshaw Mark , Hammond Philip , Chan-Hui Po-Ying , Mitcham Jennifer , Cieplak Witold , Stewart Shaun , Grantham Michael , Pekosz Andrew , Hatta Masato , Kawaoka Yoshihiro , Moyle Matthew
Published: January 2023

Specific antibody responses to the annual influenza vaccine can protect against infection if the vaccine and the infective strain are antigenically well-matched. However, due to the variability of the influenza genome, emergent strains arise for which the vaccine proves to be ineffective, and moreover, necessitate its yearly reformulation. Efforts have been made into identifying epitopes within more conserved elements of the influenza virus as an alternative attack on a wider range of strains. Through our platform of examining monoclonal antibodies (MAbs) expressed from plasmacytes derived from memory B cells from normal healthy human donors, we have found a previously unknown conformational antigenic determinant within the relatively invariable ectodomain of M2 (M2e) of influenza A. The MAbs bind to virus and cell-surface expressed M2, but not to the corresponding M2e peptide. The M2e region recognized is in almost all strains isolated to date. Furthermore, in a mouse model of infection, the MAbs can protect against lethal challenge. These results suggest that naturally expressed M2 can elicit a broadly cross-reactive and protective antibody response. And, the human recombinant anti-M2e MAbs might serve as therapeutic agents against infection.
ATAK Cells Require Intact Cytochrome Oxidase Activity to Kill Fungi (38.18)
Authors: Lin Lin , Luo Guanpingsheng , Baquir Beverlie , Ibrahim Ashraf S. , Fu Yue , Spellberg Brad
Published: January 2023

Invasive fungal infections cause high mortality rates in neutropenic patients. We developing an immunotherapy based on activation of HL-60 cells into Activated Targeted Killer (ATAK) cells. We have shown that ATAK cells phagocytize and kill C. albicans in vitro, and protect neutropenic mice from invasive candidiasis and aspergillosis. However, the mechanism by which ATAK cells kill fungi were unclear. Because we have recently found that gp91phox deficient mice were hypersusceptible to invasive candidiasis, we used siRNA to suppress gp91-phox expression in ATAK cells to define impact of suppression of NADPH-superoxide formation on fungal killing. We use a lentivirus to stably transfect a gp91-phox-targeting siRNA construct into HL-60 cells. Stable transfectants had significantly suppressed mRNA level (by RT-PCR) and protein expression (by western blot) of gp91-phox. To confirm functional suppression, we compared the superoxide production in wild-type ATAK cells and gp91-phox-/- cells by DHR123 staining and flow cytometry. We found the gp91-phox -/-cells had markedly reduced superoxide generation. In in vitro killing assays, gp91-phox-/- ATAK cells had marked reductions in their killing of C. albicans relative to wild type cells. Finally, gp91-phox deficient ATAK cells were unable to protect neutropenic mice from invasive candidiasis or invasive aspergillosis. Thus, ATAK cell fungal killing and in vivo efficacy requires intact superoxide production by the NADPH-oxidase system.
Enhancement of virus-specific expansion of transgenic CD8 T cells in aged mice by dendritic cells (38.3)
Authors: Jiang Jiu , Fisher Erin , Bennett Andrew , Murasko Donna
Published: January 2023

Aging is associated with a decreased CD8 T cell response to virus infection. Although both intrinsic and extrinsic factors have been shown to contribute to the decrease, the mechanisms are still largely unknown. In this study, the role of dendritic cells (DCs) in the age-associated decrease was examined. Influenza-specific TCR transgenic CD8 T cells of young mice demonstrated limited expansion in response to influenza infection when adoptively transferred to aged compared to young mice. This decreased response in aged mice could be significantly enhanced when DCs of young mice were co-transferred. Co-transfer of DCs had no impact in young recipient mice. Adoptive transfer of the DCs also increased the endogenous CD8 T cell response of intact aged mice, although to a lesser degree. These results suggest that the diminished CD8 T cell response to virus infection in aged mice is partially attributable to age-associated changes in DCs.
Alterations in immunodominance of the Streptococcus mutans P1 adhesin: lessons learned from immunomodulatory monoclonal antibodies (38.4)
Authors: Robinette Rebekah , Oli Monika , Brady L. Jeannine , McArthur William
Published: January 2023

S. mutans is the bacterial agent of human dental caries. P1 (aka AgI/II, PAc) is a surface localized multifunctional protein adhesin that has been studied as an anti-caries vaccine antigen. Our laboratory has identified several anti-P1 MAbs that redirect the host immune response via an Fc-independent mechanism toward one of improved inhibition of bacterial adherence when administered as part of an immune complex (IC) with bacterial cells in BALB/c mice. A common feature of our immunomodulatory MAbs is a requirement for certain intramolecular interactions between discontinuous segments of P1 for their epitope formation. Binding of P1 by these MAbs appears to cause a structural alteration of the cell surface adhesin exposing normally cryptic and protective epitopes. We now utilize information gained by evaluating changes in serum Abs after immunization with IC to design and evaluate structural variants of the P1 protein itself as more effective immunogens. Also, an anti-AgI/II MAb previously reported as a passive immunotherapy in clinical trials was evaluated and shown to be immunomodulatory. Results varied with MAb concentration, thus providing an explanation of long-term protection observed in human subjects and the difficulty in reproducing these results in all studies. Our results indicate a delicate balance exists between P1 structure and the availability of putative T helper cell epitopes, but suggests development of a protein-based vaccine is achievable. (NIH DE13882)
Mice with the Xid defect have impaired control against Borrelia burgdorferi infection but are more resistant to carditis (38.5)
Authors: Malkiel Susan , Kuhlow Christopher , Mena Patricio , Monsalve Gloria , Benach Jorge
Published: January 2023

The antibody response is the main form of protection against Borrelia. Antibodies are functional in the elimination of the spirochetes and in resolution of disease. T cell-independent antibodies have been shown to have a protective role in defending the host against the Lyme disease spirochete B. burgdorferi. To determine the role of B1 cells in this antibody response, X-linked immunodeficient (Xid) CBA/N mice were used because they are severely deficient in B1 cells. Spirochete burdens were enhanced in the Xid mice compared to CBA/Ca controls at 3, 4 and 8 weeks postinfection, which may be explained by reduced levels of B. burgdorferi-specific IgM in the Xid mice. Meanwhile, the antibody repertoire of the IgM response to B.burgdorferi antigens appeared similar in both groups of mice by immunoblot. Despite impaired pathogen clearance, the Xids developed comparable arthritis but less carditis than controls. These results suggest that B1 cells play a dichotomous role in protecting the host against infection while promoting inflammation in the heart.
Computational analysis for prediction of HLA-DR-promiscuous regions and epitopes of Mycobacterium tuberculosis protein Rv1980c, and their recognition by human T cell lines (39.1)
Authors: Mustafa AbuSalim
Published: January 2023

Rv1980c, a major secreted antigen of Mycobacterium tuberculosis, is absent in many vaccine strains of BCG, and hence could be useful in the diagnosis or as a new vaccine against tuberculosis. However, to have world-wide application, Rv1980c should have HLA-promiscuous recognition by human Th1 cells. The aim of this study was to determine HLA-promiscuous regions/epitopes of Rv1980c using a computational method, and confirm the predictions experimentally. The sequence of Rv1980c protein was analyzed for binding to 51 HLA-DR alleles using PROPRED, a computational method. The prediction results were experimentally verified by testing 20-mer synthetic peptides corresponding to the predicted HLA-DR binding regions/epitopes with T-cell lines established from PPD-positive and HLA-heterogeneous healthy subjects in Th1-cell assays (antigen-induced proliferation and Interferon-gamma secretion). The PROPRED analysis showed that Rv1980c could bind to all 51 HLA-DR alleles with eight HLA-DR binding regions (9 to 35 aa in length), and five regions (aa 20-44, aa 68-102, aa 132-146, aa 164-186 and aa 194-202) being HLA-DR promiscuous. The results of testing synthetic peptides with T cell lines in Th1 cell assays showed that four peptides belonging to four HLA-promiscuous regions (aa 21-40, aa 81-100, aa 171-190 and aa 191-202) were immunodominant and HLA-DR promiscuous. These results show that PROPRED analysis was useful in identifying HLA- promiscuous regions/epitopes of Rv1980c.
Hepatitis C Virus-specific immune response among needle-stuck Egyptian healthcare workers at high risk of infection without viremia or seroconversion (39.15)
Authors: Abdelwahab Sayed , Sobhy Maha , Rewisha Eman , Galal Iman , Zakaria Zainab , Mahmoud Mohamed , Capone Stefania , Folgori Antonella , Hashem Mohamed , El-Kamary Samer , Strickland G. Thomas , Cortese Riccardo , Nicosia Alfredo
Published: January 2023

Hepatitis C Virus (HCV)-specific cell-mediated immunity (CMI) was reported in exposed individuals e.g. IV drug users without seroconversion or viremia. We investigated the HCV-specific CMI response in seronegative, aviremic healthcare workers (HCWs) at the National Liver Institute who are at high risk of becoming infected with HCV after occupational exposure as more than 70% of their patients are HCV-infected. We quantified the CMI responses in 15 Egyptian HCWs with a recent history of a needle stick injury and who remained seronegative and aviremic for at least four months. We used ex vivo enzyme-linked immunospot (ELISPOT) assay to quantify interferon gamma (IFNγ) production in response to 7 HCV genotype 4 overlapping 15mer peptide pools and phenotyped the responding cells by flow cytometry. A positive HCV-specific IFN-γ responses (>55 spot forming cells (SFCs)/million PBMC) was elicited for 2-6 HCV pools in 8 (53%) of the HCW while 7 (47%) subjects responded to one or none of the pools tested with a total mean of 1069 (SEM ±239) and 118 (±17) IFNγ SFC, respectively. In summary, the majority of HCW demonstrated HCV-specific T cell responses for multiple HCV peptides and remained uninfected despite their high-risk of exposure. These responses suggest that the percentage of subjects clearing the infection is much higher than reported and their potential role in protection from HCV infection is important for understanding HCV pathogenesis and vaccine development.
Not just neutralizing Abs: prevention and cure of a viral disease by antibodies that block the biological function of a virulence factor (39.21)
Authors: Xu Renhuan , Sigal Luis
Published: January 2023

It is currently though that the neutralizing antibodies (Abs) are the most effective way to prevent viral infection. However, our recent work has shown that mousepox (the mouse equivalent of human smallpox) can be prevented by immunization with the Type I IFN binding protein (T1-IFNbp) of ectromelia virus (the agent of mousepox), which is a secreted non structural protein that can also attach to the surface of cells. Here we demonstrate that Abs to the T1-IFNbp are capable of preventing lethal mousepox when administered as late as 5 days post infection. The mechanism of protection was not dependent on viral particle neutralization or ADCC, but on blocking the biological function of the T1-IFNbp, which restored IFN signaling transduction in the liver as demonstrated by a dramatic increase in the transcription of IFN inducible genes such as Mx1 and IRF7. The increase IFN signaling led to reduced viral load and liver pathology and survival. Our results are the first to demonstrate that Abs directed to non-structural virulence factors can be highly effective at preventing and treating a viral disease even though they do not neutralize the viral particle.
Discordant immunodominant patterns of HIV-specific CD8 T cells defined by cytokine production or proliferative capacity in HIV elite controllers (39.4)
Authors: Ndhlovu Zaza , Proudfoot Jacqueline , Kaufmann Daniel , Walker Bruce
Published: January 2023

Several studies suggest that CTL proliferation is one of the functions specifically associated with elite control of HIV, but IFN-γ is generally used to define HIV epitope-specific CTL immunodominance. We performed multi-dimensional intra-donor analysis of HIV CTL-specific cytokine production and proliferation against panels of HLA-matched optimal epitopes. 9 elite controllers (EC) and 8 subjects with progressive infection (CP) were studied. Our analysis revealed two main immunodominance patterns defined by either predominant IFN-γ production or proliferative capacity with very different hierarchies of responses. In the EC, 40% of epitope-specific CTL that produced IFN-γ after 6 h stimulation did not proliferate whereas 47% of CTL with strong proliferative capacity did not produce IFN-γ after 6h stimulation. The contrasting patterns of immunodominance were associated with different phenotypes of memory and exhaustion markers. HIV CP usually had little HIV-specific proliferative capacity as compared to EC (21% vs 7% of tested epitopes, p=0.009). Our data indicate that immunodominance patterns of HIV-specific CD8 T cell responses differ markedly based on an IFN-γ read out assay compared to functional proliferation. The intra-individual heterogeneity of HIV-specific CTL responses shows that not all epitope specific responses are functionally similar. Assessing CTL functions at the single epitope level can facilitate identification of features critical for antiviral function.
Maine ScienceCorps fellows bring immunological research experiences into rural high school classrooms(41.1)
Authors: Tait Heidi , Cochrane Kelly
Published: January 2023

Teams of graduate students from the Departments of Applied Medical Sciences and Biology at the University of Southern Maine (USM), funded by the National Science Foundation’s Graduate STEM Fellows in K-12 Education (GK-12) program, together with faculty mentors design projects related to their graduate research to provide authentic laboratory-based research experiences in rural Maine high schools. Over the course of the school year the high school students gain hands-on experience with research methods that are applicable to current scientific inquiry in the context of immunology and molecular biology. The projects culminate in the opportunity for the students to present their research in poster format at Thinking Matters, a spring research symposium at USM. Throughout this experience the students learn about the scientific process how it is applied to questions posed by scientists around the world today, and in what ways these discoveries contribute to their understanding of the natural world. The scientific questions we have brought to the classroom involve Stephen Pelsue’s laboratory at USM, where they work on elucidating the role of Ttc7 (tetratricopeptide repeat domain7) in immune function. In this particular project the students are learning about the Ttc7fsn/fsn mouse model of lupus, how antibodies are made, the mechanics of autoimmune disease, and about applications of this research and the laboratory techniques used.
Case Study That Investigates the Role Environmental Toxins Play in Flu Infection (41.7)
Authors: Begy Elizabeth
Published: January 2023

There is growing evidence that environmental pollutants can cause cells in the body to work differently than normal, changing the immune system's ability to respond to respiratory infections. During infection with Influenza A virus, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; a dioxin and environmental toxin) enhances neutrophil recruitment and accumulation in the lungs, leading to detrimental inflammation. While neutrophils are necessary in the resolution of infection, excessive neutrophil recruitment generally has adverse effects. Within the first activity of this unit, students will gain a better understanding of the difference between the innate versus the adaptive immune response by completing cell differential counts for patients in various stages of flu infection. After graphing and analyzing cell differential data, students will explore how computers process the same data using flow cytometry. Next, students will investigate how TCDD plays a role during flu infection in laboratory mice. Students will apply what they learn with the mouse model to research dioxin issues in the environment and the subsequent effects on the human population. In culmination, students will write and perform a Public Service Announcement (PSA) regarding the immune response to the flu and the role that environmental toxins, such as TCDD, play in these infections.
DC-SIGN targeting carbohydrate moieties identified on 67 kDa mfa-1 fimbriae of mucosal pathogen Porphyromonas gingivalis (42.7)
Authors: Zeituni Amir , Scisci Elizabeth , McCaig William , Thanassi David , Cutler Christopher
Published: January 2023

We recently reported that the oral mucosal pathogen Porphyromonas gingivalis targets the C- type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased CD4+ T cell response (Zeituni et al 2009); moreover, DC invasion and immunosuppression are dependent upon expression of the 67 kDa mfa-1 fimbriae by P. gingivalis . We have now purified mfa-1 by ion exchange chromatography and sequenced it by tandem mass spectrometry (MS/MS), confirming its identify and revealing two putative N-glycosylation motifs as well as numerous O-glycosylation motifs. We further show mfa-1 is glycosylated by ProQ staining. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that mfa-1 contains fucose (1.35 nmol/mg) mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg) and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy reveals mfa-1 form oligomeric strains approximately 200 nm in length, which could be involved in targeting/ cross-linking DC-SIGN. These findings shed further light on mechanisms of invasion and immunosuppression by this unique mucosal pathogen.
Transducing the affinity of antigen recognition into graded structural modifications of the antibody product (43.21)
Authors: Kaattari Stephen , Ye Jianmin , Bromage Erin
Published: January 2023

We have found that the affinity of B cell interaction with its cognate antigen governs the degree of post-translational modification of the IgM product in trout. Affinity-based, antigen-driven lymphocytic selection and induction by the antigen, TNP-LPS, not only induced high affinity antibodies in vitro, but these antibodies also possessed a greater degree of disulfide polymerization. Affinity purification of high affinity vs. low affinity serum antibodies also simultaneously resulted in the partitioning of highly polymerized antibodies into a high affinity fraction, while the lightly polymerized antibodies partitioned into the low affinity fraction. The in vivo functional relevance of this distinctive post-translational modification was then revealed upon the transfer of labeled antibodies to naïve animals. These latter studies demonstrated that highly polymerized antibodies possessed longer half-lives. Finally, the degree of intermonomeric disulfide polymerization occurring at the C-terminal Cys 578, appears proportional to the concentration of available mannosyl residues, ostensibly at the proximal N-linked glycosylation site to Cys 578. This latter finding suggests mechanism(s) by which polymerization and half-life can be regulated via the affinity of BCR recognition. This research was supported by NIFA (National Institute of Food and Agriculture) Grant Nos. 2002-35204-11685, 2005-01594-16271 and VIMS intramural support.
Rapid evolution of a primate-restricted inflammasome regulator (43.25)
Authors: Harton Jonathan , Fuchs Travis , Atianand Maninjay
Published: January 2023

Pyrin-only protein 2 (POP2) is a small protein consisting solely of a pyrin domain capable of inhibiting IL-1β processing by preventing inflammasome assembly. Comparative genomic analysis reveals that POP2 is absent from the genomes of not only mice and rats, but those of other domestic mammals and new world monkeys as well. However, POP2 is present in the genomes of those primate species most closely related to humans. Interestingly, chimpanzee POP2 is identical to human POP2 at both the DNA and protein level but macaque POP2, although highly conserved, is not. Our analysis demonstrates that POP2 arose rapidly and recently in the mammalian genome and is likely derived from duplication and contraction of an extant NLR gene. Our findings support the hypothesis that mammalian NLR loci involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures driving their evolution. As POP2 is capable of regulating processes linked to innate immunity and inflammation, its highly restricted and conserved presence in the genomes of Old World primates suggests that further regulation of these events was critical in primate evolution.
Podoplanin interactions with CLEC-2 regulate dendritic cell migration (44.4)
Authors: Pinner Sophie , Mourao-Sa Diego , Turley Shannon
Published: January 2023

The migration of dendritic cells (DCs) from peripheral tissues to lymph nodes (LNs) is critical for the generation of protective immune responses and maintenance of self-tolerance. In our studies of DC trafficking we observed that migratory DCs, arriving in LNs from inflamed skin, exhibited elevated surface levels of the C-type lectin, CLEC-2. CLEC-2 activation in DCs was found to induce actin polymerization and increased protrusive activity, directly linking CLEC-2 to altered cytoskeletal dynamics and DC motility. CLEC-2 binds to podoplanin, a 38kDa surface glycoprotein required for correct formation of lymphatic vessels. Interestingly, podoplanin is normally expressed by both lymphatic endothelium (LECs) and fibroblastic reticular cells (FRCs); two stromal cell subtypes that directly interact with migratory DCs en route to the LN paracortex. Using a 3D in vitro culture system combined with time-lapse confocal microscopy we found that FRCs promote migration of DCs in a CCR7-independent manner. Podoplanin localizes to sites of contact between FRCs and DCs, and is required for DC migration along FRC scaffolds. Furthermore, hydrodynamic intradermal injection of podoplanin siRNA, used to silence podoplanin expression by LECs and FRCs, significantly impaired migration of DCs from skin to draining LNs. Our data illuminate important new features of DC-stromal cell interactions and reveal a critical role for podoplanin and its receptor CLEC-2 in regulating DC trafficking to LNs.
Depletion of dendritic cells enhances susceptibility to cell-free infection of HTLV-1 in CD11c-DTR transgenic mice (45.11)
Authors: Rahman Saifur , Manuel Sharron , Khan Zafar , Wigdahl Brian , Acheampong Edward , Jain Pooja
Published: January 2023

Human T-cell leukemia virus type 1 (HTLV-1) is associated with HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia. The genesis of these diseases is believed to be associated with the route and mode (cell-free versus cell-associated) of primary infection. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Mo-MLV to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-DTR transgenic mouse that permits conditional transient depletion of CD11c+ DCs. We infected these mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free, but not cell-associated HTLV-1 in both the CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA. Moreover, depletion of DCs significantly dampened the cellular immune response against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of ATL and HAM/TSP after the initial infection.
Vaccine with cationic lipid DNA adjuvant (JVRS-100) provides durable protection against heterosubtypic influenza strain (45.15)
Authors: Vaughn Marla , Callejo Bernadette , Hill Tracy , Fairman Jeffery
Published: January 2023

Influenza A infection causes annual substantial morbidity and mortality worldwide. Current vaccines, are administered either unadjuvanted or adjuvanted with aluminum hydroxide (alum). Efficacy is highly dependent on close matching of the hemagglutinin and neuraminidase surface proteins of the vaccine with currently circulating virus. The optimal adjuvant for influenza would elicit a robust, durable antibody response and provide protection against strains of drifted or heterosubtypic strains of influenza. An adjuvant consisting of cationic lipid DNA complexes (JVRS-100) has been evaluated with whole inactivated influenza in mice. Heat-inactivated HKx31 (H3N2) or PR/8/34 (H1N1) influenza virus (5µg) with or without the JVRS-100 adjuvant (20µg) alone was administered intramuscularly at week 0 and 2. At three months post the final injection animals were either terminated for immunogenicity tests or challenged intranasally with live HKx31 (1xMLD50). Vaccination with JVRS-100 resulted in a significant increase in hemagglutination inhibiting (HAI) antibodies and an IgG2a isotype switch compared to virus alone. Furthermore, the animals vaccinated with the heterosuptypic strain plus adjuvant had decreased morbidity and mortality compared to the non-adjuvanted group. The results suggest that the JVRS-100 adjuvant increases the magnitude, durability and quality of the immune response, making it an attractive candidate for adjuvanting influenza vaccines.
IRF3 is an integral component in the host defense against Pseudomonas aeruginosa lung infection in mice. (45.6)
Authors: Lin Tong-Jun , Carrigan Svetlana
Published: January 2023

Pseudomonas aeruginosa is a major opportunistic pathogen. Host defense mechanisms involved in P. aeruginosa lung infection remains incompletely defined. Interferon regulatory factor 3 (IRF3) is a transcription factor and is primarily associated with host defense against viral infections. A role of IRF3 in P. aeruginosa infection has not been reported previously. Here we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lung in mice. P. aeruginosa infection induced IRF3 nucleus translocation, activation of ISRE and production of IFNβ, suggesting that P. aeruginosa induces the IRF3-ISRE-IFN pathway activation. In vitro, macrophages from IRF3 deficient mice showed complete inhibition on the production of CCL5 (RANTES) and CCL10 (IP-10), partial inhibition of CXCL1 (KC) and TNF and no effect on CXCL2 (MIP-2) in response to P. aeruginosa stimulation. In vivo, IRF3 deficient mice showed complete inhibition of CCL5 production and partial or no effects on other cytokine and chemokine production in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that recruitment of neutrophils and macrophages into the airspace was reduced, while B cell, T cell, NK cells and NKT cells infiltrations were unaffected in IRF3 deficient mice in response to P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulate neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.
Signals derived from intestinal microbial communities augment anti-viral immunity (46.11)
Authors: Abt Michael , Beiting Dan , Kobuley Dmytro , Wherry John , Artis David
Published: January 2023

Alterations in intestinal microbial communities in humans are associated with enhanced susceptibility to inflammatory diseases suggesting that signals derived from commensal bacteria may influence the development and/or function of the immune system. Supporting this, germ-free (GF) mice exhibit reduced numbers of lymphocytes in the periphery and intestinal intraepithelial compartment. However, whether alterations in the composition of commensal bacteria influence immunity to infection remains poorly defined. To test this, mice housed under conventional (CNV) or GF conditions were infected with acute LCMV and the development of antigen-specific CD8+ T cell responses were analyzed. At day 7 p.i., GF mice exhibited a significant reduction in the frequency and number of LCMV-specific CD8+ T cells in multiple tissues including the spleen and intestinal intraepithelial compartment. The diminished CD8+ T cell response was not the result of inherent developmental defects in GF mice as depletion of intestinal bacteria in CNV mice via antibiotics also resulted in diminished LCMV-specific CD8+ T cell responses and impaired viral clearance. Further, adoptive transfer of CFSE-labeled LCMV-specific CD8+ T cells revealed a delay in the activation and expansion of LCMV-specific CD8+ T cells in antibiotic-treated mice. Collectively, these studies indicate that signals derived from intestinal bacteria aid in the induction of a rapid immune response required for immunity to viral infection.
Evaluation of the botulinum neurotoxin neutralizing efficiency in immunoglobulin purified from clinical volunteers vaccinated with recombinant botulinum vaccine (46.5)
Authors: Shearer Jeffry , Vassar Michelle , Niemuth Nancy , Swiderski William , Holley H. , Metcalfe Karen , Hart Mary
Published: January 2023

Efficacy of the Recombinant Botulinum Vaccine A/B (rBV A/B) against lethal intoxication with botulinum neurotoxin complex (BoNT) cannot be evaluated directly in humans; therefore, efficacy will be based on meeting the requirements of the FDA Animal Rule. Serum BoNT neutralizing antibody concentration (NAC) is being proposed as a correlate of protection to evaluate efficacy in nonclinical models. Under this approach, NAC levels in animals vaccinated either with rBV A/B or passively immunized with BoNT neutralizing antibody will be used to estimate the potential level of protection in humans with similar NAC levels. Estimating the level of protection based on NAC requires establishing NAC neutralizing efficiency (the amount of BoNT neutralized per NAC unit). The neutralizing efficiency of human BoNT neutralizing antibody was evaluated using immunoglobulin purified from pooled sera of clinical volunteers vaccinated with rBV A/B. Solutions of immunoglobulin were prepared at three different NAC levels, mixed with serial dilutions of BoNT serotype A or serotype B and injected into mice. At each NAC level, 50% and 99% neutralization of BoNT was determined by probit analysis of mortality dose-response curves. A simple linearly proportional relationship does not exist between NAC level and the amount of BoNT neutralized. Extrapolations of level of protection must consider variability in BoNT neutralizing efficiency at different NAC levels.
A novel subset of lung cells responds to IL-33 and mediates innate Th2-type immune responses (48.4)
Authors: Bartemes Kathleen , Iijima Koji , Kobayashi Takao , Kephart Gail , Kita Hirohito
Published: January 2023

Interleukin (IL)-1 family member cytokines are likely pivotal as interfaces between innate and adaptive immune responses. IL-33, an IL-1 family member, is implicated in airway Th2-type responses, including eosinophilia, increased mucus production and hyperresponsiveness. Our goal was to elucidate the immunological mechanisms involved in IL-33-mediated Th2-type responses in the lungs. Airway exposure of naïve BALB/c mice to IL-33 resulted in bronchoalveolar lavage (BAL) eosinophilia and increased lung levels of IL-5 and IL-13, but not IL-4 or IFNγ. Naïve Rag1-/- mice showed similar airway eosinophilia and cytokine responses as wild-type mice, suggesting a role for innate immune cells. By FACS analysis, IL-5-producing lung cells lacked CD3, B220 and other lineage markers (i.e. Lin-), but they expressed CD44 and CD25; markers for NK cells, mast cells, basophils and eosinophils were undetectable. FACS-sorted Lin-CD25+CD44+ lung cells expressed GATA-3 and the IL-33 receptor, T1/ST2, and responded to IL-33 by producing IL-5 and IL-13; morphologically, these lung cells resembled lymphocytes. In vitro blockade of CD25+ cells with anti-CD25 mAb (PC61) abrogated IL-33-induced Th2 cytokine production. Thus, mouse lungs contain a novel subset of innate immune cells that respond to IL-33. These airway cells may initiate early Th2-type immune responses and induce asthma and other allergic diseases.
Expression of co-stimulatory TNFR2 enhances resistance of CD4+FoxP3- conventional T cells to suppression by CD4+FoxP3+ regulatory T cells (49.13)
Authors: Chen Xin , Hamano Ryoko , Subleski Jeffrey , Hurwitz Arthur , Howard O.M.Zack , Oppenheim Joost
Published: January 2023

Our previous study showed that TNFR2 is preferentially expressed by CD4+FoxP3+ regulatory T (Treg) cells and expression of this molecule identified the maximally suppressive Treg cells. TNFR2 is also expressed by a small fraction of CD4+FoxP3- conventional T (Tconv) cells in normal mice and its expression is up-regulated by T cell activation. Our data indicate that TNFR2 also co-stimulates Tconv cells. In this study, by using FoxP3/gfp KI mice, we showed that TNFR2 signaling did not induce FoxP3- CD4 cells to become suppressive. TNFR2 is associated with greater suppressive functions when expressed by Treg cells and is associated with greater resistance to suppression when expressed by Tconv cells, because TNFR2+ Tconv cells were potently suppressed by FoxP3+TNFR2+ Treg cells, while resisting suppression by FoxP3+TNFR2- Treg cells. Furthermore, in mice with 4T1 breast tumor and LLC, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist the suppression mediated by LN-derived Treg cells. However, they remained susceptible to inhibition by more suppressive tumor infiltrating Treg cells which expressed higher levels of TNFR2. Intratumoral Treg cells express more TNFR2 and they are able to overcome the inhibition-resistance of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment. (Funded by NCI Contract N01-CO-12400)
Re-programming of IL-10 signal transduction: an inflammation-induced mechanism to control T cell susceptibility to suppression (49.18)
Authors: Raimondi Giorgio , Kowalkowski Frank , Kane Lawrence , Thomson Angus
Published: January 2023

Modulation of regulatory T cell (Treg) suppressive function may play an important role in the development of autoimmune diseases, tumor growth and transplant outcome. Currently, little is known about the molecular mechanisms that render T cells refractory to suppression. Since IL-10 is an important mediator of Treg activity, we investigated the influence of pro-inflammatory mediators on the IL-10 signaling pathway in T cells. The mouse CD4 T cell line D10 was incubated for various periods with the supernatant of bone marrow-derived dendritic cells stimulated overnight with the TLR4 ligand LPS (MATSup). We then measured the integrity of the IL-10 signaling pathway by assessing phosphorylation of the transcription factor STAT3 in response to titrated amounts of IL-10. Even after 24h incubation with MATSup, phosphorylated STAT3 was readily detected in T cells in response to IL-10. Surprisingly, measurement of cytoplasmic levels of total STAT3 and of pro-inflammatory STAT1 revealed short- term (3 to 6 hours) reduction of both transcription factors, followed by upregulation with an altered ratio between STAT3 and STAT1 in favor of the latter. This altered ratio appeared within 12 hours and was preserved for at least 24 hours. These results suggest that pro-inflammatory mediators re-program the IL-10 (and probably other) signaling pathway in T cells, reducing their susceptibility to suppression. Interference with this process may represent an effective immunomodulatory strategy.
Strong CD28 co-stimulation Limits Generation of Regulatory T cells from Naïve T cells through Lck and PI3K signaling (49.4)
Authors: Semple Kenrick , Nguyen Antony , Yu Xue-Zhong
Published: January 2023

CD28 co-stimulation is required for the generation of regulatory T cells (Tregs) in the thymus as well as in the periphery through Lck-signaling. However, high level of CD28 limits the generation of Tregs from naïve CD4 T cells, although underneath mechanism(s) has not been defined. Here we investigated the role of CD28-mediated signaling pathways in the down-regulation of Treg generation. To address the question, we used a series of transgenic (Tg) mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk. Under the culture condition with TGFβ that favors Treg generation, the generation of Tregs (CD25+Foxp3+) from naïve CD4 T cells with WT CD28 Tg (CD25-Foxp3-) was inversely related with the level of CD28 co-stimulation mediated by anti-CD28 or B7. Regardless of exogenous IL-2, the suppression of Treg generation caused by strong CD28 signals was intact in the T cells with CD28 Tg incapable of binding Itk, compromised in those incapable of binding PI3K, and essentially abrogated in those incapable of binding Lck. The essential role of Lck and contributory role of PI3K in CD28-mediated down-regulation of Treg generation were confirmed by using Lck- or PI3K-specific inhibitors. These results reveal novel mechanistic insight how CD28 can negatively regulate Treg generation, which may provide rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling.
TORC1 and TORC2 selectively regulate CD4+ T helper cell lineage differentiation (50.35)
Authors: Delgoffe Greg , Waickman Adam , Pollizzi Kristen , Xiao Bo , Worley Paul , Powell Jonathan
Published: January 2023

The mammalian target of rapamycin (mTOR) is a highly conserved serine-threonine kinase that regulates cell division, survival, and translation. Downstream mTOR signals via two functionally distinct complexes, TORC1 and TORC2. We have previously shown T cells deficient in mTOR fail to differentiate into Th1, Th2, or Th17 cells and instead become Foxp3+ regulatory cells. We further dissected mTOR signaling in T cell differentiation by deleting Rheb (the activator of TORC1) and rictor (an essential TORC2 subunit). T cells lacking Rheb selectively lack TORC1 activity, and, like mTOR mutants, fail to differentiate into Th1 or Th17 cells when skewed. However, Rheb-deficient T cells retain their ability to become Th2 cells. This is due in part to diminished STAT4 and STAT3 activation, but elevated STAT6 activation. Alternatively, rictor-deficient cells specifically lack TORC2 activity, and fail to become Th2 cells when skewed. However, they retain the ability to become Th1 and Th17 cells, with a pattern of STAT activation that inversely correlates with TORC1 mutants. Interestingly, neither TORC1 nor TORC2 deficiency alone is sufficient to induce Treg differentiation. In vivo, TORC1 deficiency protects against EAE while TORC2 deficiency leads to accelerated disease. Our data support a novel paradigm in which TCR engagement in the absence of mTOR defaults to a regulatory fate, and TORC1 and TORC2 activation selectively regulate T cell effector lineage differentiation.