bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecules involved in the NFκB signaling pathway are down regulated in neonatal alveolar macrophages resulting in reduced response to Pneumocystis infection (37.7)
Authors: Kurkjian Cathryn , Hollifield Melissa , Garvy Beth
Published: January 2023

Neonates are unable to clear Pneumocystis (PC) infection with the same efficiency as adults. The alveolar macrophage (AM) phenotype of neonates during infection varies from that of adults, leading to a delayed inflammatory response and a slower clearance of infection. Upon infection with PC, neonatal AMs appear to maintain an alternatively activated phenotype, where as adults tend to adopt a classical AM phenotype. Previous data from our lab has shown that an elevated expression of TGFβ in neonatal lungs suppresses the ability of AMs to respond to PC. Use of a conditional smad4 KO model in which TGFβ signaling is defective in macrophages allowed us to show that blocking the TGFβ signaling pathway improves clearance of PC in neonatal mice. To further examine the response of neonatal AMs, in vitro immunoflourescence assays were performed to examine NFκB translocation to the nucleus. We found that, while neonatal AMs translocate NFκB in response to zymosan, they remain unresponsive to PC cysts. This data suggests that the unresponsiveness of neonatal AMs to PC infection may be due in part to reduced response of macrophages to PC cysts, in addition to the immunosuppressed lung environment. We hypothesize that the down regulation of molecules involved with NFκB translocation to the nucleus results in the reduced response of AMs to PC.
Cell-intrinsic Transforming Growth Factor-β signaling mediates virus-specific CD8+ T cell deletion and viral persistence in vivo (39.12)
Authors: Tinoco Roberto , Alcalde Victor , Yang Yating , Sauer Karsten , Zuniga Elina
Published: January 2023

Chronic viral infections represent a major biomedical problem. Although, in both humans and mice, viral persistence has been associated with insufficient numbers and/or function of CD8+ T cells, the underlying mechanisms are still unclear. By using Lymphocytic choriomeningitis virus in its natural murine host, we found that enhanced and sustained Transforming Growth Factor-β (TGF- β),expression and phosphorylation of its signaling mediator, Smad-2, were distinctive features of virus-specific CD8+ T cells during chronic versus acute viral infections in vivo. Selective attenuation of TGF-β pathway in T cells decreased the expression of the pro-apoptotic protein Bim and increased survival and numbers of virus-specific CD8+ T cells. Under these conditions, virus-specific CD8+ T cells had enhanced cytotoxicity, increased production of anti-viral cytokines, and down-regulation of inhibitory molecules. The end result was rapid virus eradication and generation of an effective memory T cell response that protected the host upon subsequent challenge. Notably, we found that cell-intrinsic TGF-β signaling was responsible for virus-specific-CD8+ T cell apoptosis but was not necessary for their functional exhaustion. Our findings demonstrate that sustained TGF-β-Smad signaling is a hallmark and master regulator of CD8+ T cell responses during chronic viral infections in vivo.
Enhanced CD8+ T-cell immunity following sequential flavivirus vaccines (39.19)
Authors: Singh Rekha , Green Sharone
Published: January 2023

Flaviviruses are arthropod-borne, RNA viruses of the Flaviviridae family. Flavivirus antigen-specific CD8+ T-cells are important in virus clearance. Primary exposure to a virus causes priming of CD8+ T-cells by virus-derived immunodominant peptides, leading to clonal differentiation and proliferation of effector cells followed by contraction and memory generation. ChimeriVax™ Flavivirus vaccines contain the nonstructural genes of the yellow fever (YF) vaccine and the prM and E genes of heterologous flaviviruses such as Japanese encephalitis (JE) and West Nile (WN) viruses. These chimeric vaccines induce both humoral and cell mediated immunity, however their potential to generate cross-reactive CD8+ T-cell responses to heterologous flaviviruses has not been investigated. In this study, mice were immunized with YF, YF/JE, or YF/WN vaccines followed by secondary homologous or heterologous immunization. We examined the hierarchy and function of CD8+ T-cell responses to a variable envelope epitope with those directed against a conserved immunodominant YF NS3 epitope. Using cell based immunological assays (ELISPOT and ICS), we found that the secondary immunization with heterologous chimeric flavivirus vaccines generate an enhanced cross-reactive CD8+ T-cell response that was dependent on the sequence of the primary immunization. Present results suggest that controlled exposure to multiple related flavivirus vaccines may lead to enhanced protection against related flaviviruses.
Severity of Epstein-Barr Virus (EBV)-induced infectious mononucleosis correlates with frequency of cross-reactive influenza M1-specific memory CD8 T cells (39.31)
Authors: Aslan Nuray , Watkin Levi , Sullivan John , Luzuriaga Katherine , Selin Liisa
Published: January 2023

Cross-reactive memory T cells specific to previously encountered influenza A contribute to the characterisitic lymphoproliferation of EBV-associated infectious mononucleosis (IM). In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BRLF-1190 (10/10) and BMLF-1280 (14/26). Disease severity of IM did not correlate with viral load. Interestingly, instead disease severity directly correlated with the percentage of Flu-M1-specific CD8 T cells but not with either EBV-BMLF-1, or -BRLF-1-specific responses. In addition, 8/19 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF-1 and -BMLF-1 responses. These cross-reactive T cell responses were observed in all IM patients but different patterns of cross-reactivity occurred in each individual concerning the specific epitopes involved and their time of appearance after infection. A number of unique features of the TCR of the highly cross-reactive BRLF-1 epitope were noted. BRLF-1 T cell responses were of very high avidity. There was tremendous variation in the Vbeta/Valpha usage between different individuals, even in the same individual over time and this could be further modulated by peptide concentration in vitro. Cross-reactive BRLF-1 responses only became apparent at high antigen doses such as might occur during different phases of IM.
Structural Determinants of IFNα Distinguish Antiviral and Potentially Toxic Functions. (39.7)
Authors: Vazquez Nancy , Schmeisser Hana , Bekisz Joseph , Zoon Kathryn , Wahl Sharon
Published: January 2023

Interferon alpha (IFNα), a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of human immunodeficiency virus (HIV), exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3(APOBEC3) family members. Although IFNα therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFNα receptor binding sites, we generated IFNα hybrids and mutants and determined that structural changes in the C-loop influencing receptor interactions alter IFN’s ability to limit retroviral replication. Selective IFNα constructs differentially block HIV replication and their directional magnitude of inhibition correlates with elevated APOBEC3 levels with less significant changes in transcription of other antiviral molecules such as TRIM22 and PKR. Besides participation of the JAK-STAT pathway, we uncover previously unappreciated roles for NFκB, CAMK and PI3K signaling pathways in induction of APOBEC3 by the IFN constructs. Importantly, certain mutants exhibited reduced toxicity as reflected by induced indoleamine 2,3-dioxygenase and TNFα compared with intact IFN, suggesting discreet and shared intracellular signaling pathways. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable the design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects.
The CD8+ T cell response to Brucella melitensis during acute vs. chronic infection (40.24)
Authors: Durward Marina , Harms Jerome , Magnani Diogo , Eskra Linda , Splitter Gary
Published: January 2023

In areas of endemic brucellosis, prevention of infection via vaccine would be far more cost-effective than the World Health Organization suggested regimen of antibiotics. As with other intracellular organisms, subunit or killed vaccines present very limited efficacy. The rationale for an improved Brucella melitensis vaccine requires a greater understanding of the host-pathogen relationship. Using bioinformatics coupled with practical experiments in the BALB/c model of brucellosis, we have identified two Brucella epitopes and show their capacity to elicit specific CD8+ T cells that kill effectively in vivo. Additionally, as seen by microarray, gene transcripts that contain these epitopes are significantly and differentially expressed following Brucella infection of macrophages. Specific CD8+ T cells are present and functional following infection; however, reactivation of disease still occurs in up to 30% of human patients after antibiotic treatment. Our current work using Brucella specific MHC Class I tetramers focuses on the presence and persistence of CD8+ T cell memory during chronic brucellosis. Investigation of the functional potential of this memory fraction of CD8+ T cells will fill a gap in our understanding of the immunological response to this often asymptomatic, persistent infection.
Researching Type 1 Diabetes (41.10)
Authors: Judson Peter , Piccirillo Ciriaco
Published: January 2023

Research into the immunoregulation of type I diabetes (T1D), (an autoimmune disease that results in the destruction of insulin producing B cells in the pancreas), is the focus of this curriculum unit. The unit entitled “Researching Type I Diabetes” has been developed for high school students. This unit is designed to be used in classes such as health science, biology, or honors biology. The unit begins with a lesson on the immunoregulation of T1D via the use of a roundhouse diagram activity. Students then learn about the NOD mouse as a model in T1D research, and the legal and ethical aspects of using animals in medical research, to complete a persuasive writing activity involving bioethics. The third lesson requires students to follow a mouse dissection protocol. Following this protocol, students dissect the major organs of the mouse’s immune system (thymus and spleen) to isolate peripheral lymphatic nodes. Then the students observe a teacher demonstration of flow cytometry and cell sorting and why they are essential tools in the understanding of the immunoregulation of the disease. The culminating component of this unit is a “Web Quest,” in which students role-play the various roles involved in clinical and experimental research of T1D. The roles include: scientists, T1D patients, graduate students, physicians, and institutional review board members. Consequently, the students produce and present the results of their “Web Quest” to their classmates in a PowerPoint format.
Effect of a compromised inflammatory response on influenza virus infection (42.17)
Authors: Campbell Gillian , Dransfield Ian , Nash Tony , Dutia Bernadette
Published: January 2023

The inflammatory response to influenza virus infection in vivo represents a major determinant of the damaging immunopathology associated with cytokine storms and fatal infections. We hypothesised that production of inflammatory cytokines such as inducible nitric oxide synthetase and TNF-α by classically activated macrophages plays a major role in development of severe disease. However, alternatively activated macrophages might ameliorate symptoms. We therefore generated polarized macrophages from wild type and IFN-γR-/- mice using IFNγ and IL-4. Following infection with influenza A/WSN/33, we monitored the levels of virus and assessed the impact on cytokine expression profiles in the different macrophage populations. Infection of macrophages resulted in a predominantly inflammatory phenotype in both classically and alternative activated populations. However, alternatively activated macrophages were more readily infected and exhibited reduced viability. In vivo infection showed that wild type mice produce increasing levels of inflammatory cytokines as viral lung titres decline. Pilot studies suggest that IFN-γR-/- mice, which lack the ability to produce a classical macrophage response, are less susceptible to infection, showing no clinical symptoms or elevated pro-inflammatory cytokines and are able to clear virus more readily. Experiments are ongoing to elucidate the role of different macrophage phenotypes in control of influenza virus infection and immunopathology.
NLRP3 directs inflammasome activation in response to live F. tularensis infection (42.20)
Authors: Atianand Maninjay , Shah Aaloki , Duffy Ellen , Kar Supriya , Malik Meenakshi , Harton Jonathan
Published: January 2023

Detection of intracellular pathogens is facilitated by members of the nucleotide binding-leucine rich receptor (NLR) family. The role of ASC/Caspase-1 axis for protective innate immunity against Francisella infection is well established, however the NLR(s) involved in initiation of innate immune responses during Francisella infection are yet unknown. Here we demonstrate that human NLRP3 mediates inflammasome dependent IL-1β production following Francisella infection. Notably, both human NLRP3 and mouse Nlrp3 are sufficient to assemble functional inflammasomes in HEK293T cells infected with Francisella. Furthermore, our results also support the existence of more than one Francisella-responsive cytoplasmic inflammasome complex and suggest that intrinsic differences in bacterial strains as well as monocyte-macrophage differentiation status influence inflammasome-dependent innate immune responses to Francisella.
Herpetic Stromal Keratitis (HSK) lesion severity correlates with the amount of pro-inflammatory neuropeptide Substance P in the cornea (42.21)
Authors: Twardy Brandon , Channappanavar Rudragouda , Suvas Susmit
Published: January 2023

The cornea is richly supplied by nerve fibers that express a variety of biologically active neuropeptides. In this report, we looked into the possibility that after ocular herpes simplex virus-1 (HSV-1) infection of C57BL/6, the level of expression of neuropeptide SP in the cornea correlates with the development of HSK lesions. Our results showed that after ocular HSV-1 infection of C57BL/6 mice, about 60-70% of the infected corneas developed severe HSK lesions (corneal opacity ≥3 and angiogenesis ≥8) whereas, 30-40% developed mild HSK lesions (corneal opacity ≤1 and angiogenesis ≤5). No significant difference in the viral load was determined when analyzed between the corneas with mild or severe HSK lesions at different time-points post-infection. However, a strong influx of CD4 T cells and neutrophils were evident in the corneas with severe HSK lesions when compared to those with mild lesions at day 15 post-infection. In addition, corneas with mild HSK lesions had lower levels of IL-6, IL-2, IFN-g and IL-1 Ra in comparison to the corneas with severe HSK lesions as determined on day 15 post-infection. Most importantly, corneas with severe HSK lesions had significantly higher levels of neuropeptide SP in comparison those with mild HSK lesions, when determined on day 15 post-infection by using Immunofluorescence and EIA technique. We are looking into the possibility that neutralization of SP in the cornea impedes the development of severe HSK lesions in a mouse model.
Toll-like receptor 2 (TLR2) is critical for controlling local herpes simplex virus replication and for the generation of potent T lymphocyte immune responses (42.23)
Authors: Kollias Christina , Jennings Stephen
Published: January 2023

Herpes simplex virus (HSV) is a ubiquitous human pathogen that can cause a devastating encephalitis in rare instances [Herpes simplex encephalitis (HSE)]. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors capable of early detection of invading pathogens. The importance of TLR signaling in resistance to HSV has recently been studied using genetic knock-out animals. Endosomally-expressed TLR9 recognizes HSV DNA and is important for early type I interferon production and prevention of central nervous system (CNS) infection. In contrast, the role of surface-expressed TLR2 in HSV immunity is unclear. We set out to study the role of TLR2 signaling in a cutaneous lip infection model of HSV in mice. Here we report the role of TLR2 signaling in response to HSV as measured by viral replication in the skin and in the CNS. Additionally, the role of TLR2 signaling on the generation of adaptive immune responses is reported. Future experiments aim to analyze the specific role of TLR2 signaling at the level of microglia. We plan to assess levels of inflammatory mediators in the brain as well as leukocyte recruitment to the CNS. These studies are designed to delineate the role of TLR2 signaling in different topological environments in a model of viral-induced encephalitis, with the hopes of developing novel therapies for patients with HSE.
Roles of MHC class Ia and class Ib in hsp70 mediated anti-tumor responses in the frog Xenopus (43.11)
Authors: Nedelkovska Hristina , Goyos Ana , Easterhoff David , Maggirwar Sanjay , Robert Jacques
Published: January 2023

The heat shock proteins (hsps) gp96 and hsp70 mediate potent antigen-dependent anti-tumor responses in both mammals and Xenopus. We have developed an adoptive cell transfer assay using peritoneal leukocytes as antigen presenting cells (APCs), and shown that hsp72, but not hsc73, is as potent as gp96 to prime T cell responses in vivo against the Xenopus thymic tumor 15/0 that is tumorigenic when transplanted in MHC-compatible Xenopus clones. The 15/0 tumor expresses several nonclassical MHC class Ib genes and β2-m, however it does not express classical MHC class Ia. Despite the lack of class Ia expression by 15/0, Xenopus adults generate potent cytotoxic CD8 T cell effectors (CCU-CTLs) that specifically recognize and kill the 15/0 tumor. We postulate that hsp72, but not hsc73, can prime class Ib-mediated anti-tumor CCU-CTL responses by interacting with APCs. To reveal the roles of class Ia and class Ib in this process we silenced surface expression of both molecules on APCs and assessed the effects in priming hsp72- or hsc73-mediated tumor immunity by adoptive transfer. To further elucidate the involvement of class Ia and class Ib in hsp70-mediated T cell function in vivo, we developed reliable transgenic techniques for our Xenopus clones and obtained F0 animals with silenced β2-m expression in vivo. This study allows us to gain better perspective into the intricate relationship between the two hsps, class Ia and class Ib molecules in T cell function as well as tumor immunity.
Neospora caninum high mobility group box-1 (HMGB1) stimulates tumor necrosis factor-alpha (TNF-alpha) production by the murine macrophage cell line RAW264.7 (43.26)
Authors: Cao Lili , Fetterer Raymond , Zhang Xichen , Tuo Wenbin
Published: January 2023

Neospora caninum is a protozoan parasite that causes abortion in cattle. One of the possible mechanisms by which N. caninum induces pregnancy failure is by eliciting host inflammatory responses. Two putative genes encoding the high mobility group box-1 proteins (HMGB1, 98AA) are present in the Neospora genome and have a 51% nucleic acid sequence homology. These proteins are of non-histone nuclear origin and belong to the “alarmin” family which are inducers of proinflammatory cytokines, and therefore may be involved in provoking host inflammatory responses during pregnancy. Recombinant proteins were expressed in E. coli, purified by Nickel column and treated with Polymixin B to remove endotoxin. Antibodies raised against the NcHMGB-1 detected endogenous NcHMGB1 of expected size, confirming the presence of the proteins in tachyzoites. Lysate from E. coli transfected by plasmid alone was similarly purified and used as control. The results showed that both rNcHMGB1 proteins stimulated TNF-alpha in RAW264.7 cells in a dose dependent fashion. A unrelated Neospora protein (89AA) was used as a control, but TNF-alpha was not stimulated in RAW264.7 cells by this protein. The data indicate that the NcHMGB1 is active in cross-regulating host immunity and may be involved in eliciting uncontrolled inflammatory responses which induce pregnancy failure in cattle.
Interaction of children's acetaminophen with antigen and antibody from bovine and swine albumin sources (43.8)
Authors: Vezzoli Gary , Sinclair Jennifer , Mason Charles , Brulotte Joe
Published: January 2023

We have conducted optical, structural, and charge transport studies of the interaction of children's strength tylenol with both antigen and antibody sources from bovine and swine albumin over the physiological temperature range. Our results show that acetaminophen is chemically and structurally stable over this temperature range and up to 108F, however, is adversely affected by low-to-moderate concentration of NaCl(aq)forming black crystallites of unestablished structure. Our charge-transport and real-time visual and optical microsocopy data indicate a strong interaction between acetaminophen and antibodies from both sources, especially swine albumin. Recovered product from these experiments show formation of various species, including sliver-shaped yellow crystals, and a multi criss-crossed channel structured species appearing near interface/boundry locations of the sample. The pronounced absence of antibody serum in the regions of acetaminophen diffusion support Czech Republic work reporting in the British Medical Journal, Lancet, October 16th 2009, that acetaminophen caused a weakening of a multi-purpose vaccine produced by GlaxoSmithKline. Our work shows that acetaminonphen interacts with bovine antigen in a non-Euclidean geometry, similar to a 3-cusp hypocycloid (deltoid). Because interaction with antigen shows an unusual time-dependent geometry, the interaction with antibodies shows long-term kinetic dependence.
α4β7 integrin is essential for contact hypersensitivity by regulating migration of T cells to inflammatory skin. (44.11)
Authors: Ohmatsu Hanako , Kadono Takafumi , Sugaya Makoto , Tomita Manabu , Kai Hiromichi , Miyagaki Tomomitsu , Saeki Hidehisa , Tamaki Kunihiko , Steeber Douglas , Tedder Thomas , Sato Shinichi
Published: January 2023

β7 integrin, a cell adhesion molecule, is present in the form of α4β7 integrin or αEβ7 integrin. α4β7 integrin is expressed on most leucocytes and is essential for their migration to gut-associated lymphoid tissues by interacting with its primary ligand, MAdCAM-1, which is preferentially expressed in gut-associated lymphoid tissues. Although the importance of α4β7 integrin in intestinal inflammation has been established, its role in cutaneous inflammation remains to be elucidated. Here we show that β7 integrin-deficient mice, not αE integrin-deficient mice, are defective in contact hypersensitivity (CHS) responses. β7 integrin deficiency does not affect irritant contact dermatitis. The distribution, migration, and function of antigen presenting cells from β7 integrin-deficient mice are comparable to those from wild-type mice. Moreover, sensitized β7 integrin-deficient T cells are able to respond to antigen stimuli in vitro, and elicit CHS responses when directly injected into the skin. However, they are defective in reaching the skin under inflammatory conditions, resulting in reduced CHS responses when intravenously injected. Furthermore, T cells treated with anti-α4β7 integrin neutralizing antibody elicit impaired CHS responses. These results indicate a novel role of α4β7 integrin in regulating T cell migration to inflammatory skin.
Development and Migration of Pre-Plasma Cells in the Mouse Lymph Node (44.5)
Authors: Fooksman David , Skokos Dimitris , Dustin Michael , Nussenzweig Michel
Published: January 2023

Plasma cells (PCs) play important roles in the acute response to infection and the long-term protection of the host by acting as antibody factories. In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PC) in lymph nodes of mice by intravital two-photon microscopy using a Blimp-1-YFP transgene. Pre-PCs differentiate in the germinal center and migrate to the medullary cords where they become sessile plasma cells. Their migration has been shown to be chemokine-dependent based on in vitro chemotaxis assays and histological sections using chemokine receptor-deficient cells. Here we identify a novel migration pattern for pre-PCs, which is unique among previously-described lymphocytes and uses an alternative pathway for motility.
Sex Differences in the Innate Immune Response to Heart-Passaged Coxsackievirus B3: Microarray Analysis (45.16)
Authors: Onyimba Jennifer , Coronado Michael , Frisancho J. , Shin Jaewook , Fairweather DeLisa
Published: January 2023

Recent studies have shown that innate immunity influences the progression from viral infection to autoimmunity. The incidence and severity of autoimmune disease differs between men and women, including the heart inflammatory autoimmune disease myocarditis which is increased in men. To better understand the reason for increased heart disease in men, we studied sex differences in the innate immune response to heart-passaged coxsackievirus B3 (CVB3) in BALB/c mice by microarray. Five male and female BALB/c mice were infected intraperitoneally (ip) with CVB3 and spleens harvested 12 hours (h) later during the innate immune response and compared to uninfected controls. Gene expression was determined using an Affymetrix Mouse Gene 1.0 ST Array. We found that 24 genes were significantly increased in CVB3 infected males vs. females (>1.5 fold change). Androgen receptor (AR) mRNA was significantly decreased in infected males vs. infected females indicating activation of the receptor. Heat shock protein 90 (HSP90) and haptoglobin (Hp) mRNA were significantly increased in infected males vs. infected females. AR, HSP90 and Hp have been associated with increased heart disease in men. Significant gene changes were verified using qRT-PCR. Thus, we found that several genes important for the development of clinical heart disease are upregulated in males during the innate immune response. Funded in part by the Johns Hopkins University Diversity Summer Internship Program and NIH R01 HL087033.
Increased levels and function of specific T regulatory cells among rheumatoid arthritis patients that developed active tuberculosis (45.22)
Authors: Junqueira-Kipnis Ana Paula , Silva Bruna , Tannus-Silva Daniela , Ribeiro Elizangela , Rabahi Marcelo
Published: January 2023

Rheumatoid arthritis (RA) patients present an autoimmune disease characterized by excessive production of TNF-α, and the treatment is based on immunosuppressant and/or anti-TNF-α therapy. In this sense, TB latent RA patients are susceptible to develop active disease. The aim of this work was to evaluate the specificity of Treg cells among TBRA patients compared to Th17 and Th1 cells. It was recruited: TB (21), RA (24) and TBRA (4) patients and 24 health controls matched by age and sex. PBMC were cultivated with M. tuberculosis antigens (ESAT-6, HSP-X, and GLcB). TBRA patients presented twice the percentage of specific Treg IL-10+ cells than TB patients (TBRA=11.1±5.1 and TB=6.1±1.5). Similar results were observed between TBRA (17.3±3.1) and TB (17.8±9.8) patients for specific Treg TGF-B+ cells. Treg cells from both TBRA and TB patients were able to reduce proliferation of autologous CD4 CFSE marked cells. M. tuberculosis specific TCD4+IFN-γ+ were similar among TB patients (6.9±1.6), and TBRA patients (5.3±1.1). TB patients presented specific Th17 cells (TCD4+ IL-17+) (3.9±1.6) as well as TBRA patients (6.2±2.4). However, RA patients showed a polyclonal activation of Th1 cells (specific=7.3±1.8, median=6.9±2.1) and, Th17 cells (specific=2.7±1.1, median= 3.3±1.5). Patients with TBRA unlike RA or TB patients presented a higher level of specific Treg and Th17 subsets. TregIL-10+ cells from TBRA individuals may be induced to control the higher levels of specific Th17 cells.
MviN mediates Francisella tularensis virulence through the inhibition of inflammasome activation. (45.5)
Authors: Ulland Tyler , Buchan Blake , Jones Bradley , Nauseef William , Sutterwala Fayyaz
Published: January 2023

The mechanisms by which the facultative intracellular pathogen Francisella tularensis can evade innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, that functions as a F. tularensis virulence factor. In vivo infection of mice with a F. tularensis LVS mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with F. tularensis LVS. In vitro intracellular replication in macrophages or the ability to induce the production of TNFα or IL-6 was not effected by the mviN mutation. In contrast, we found that the mviN mutant induced increased caspase-1-dependent IL-1β secretion and cytotoxicity in macrophages compared to F. tularensis LVS. The compromised in vivo virulence associated with the mutation of mviN was dependent upon the production of IL-1β, as caspase-1-deficient mice did not exhibit preferential survival following infection. This study identifies mviN as a novel F. tularensis virulence factor through its ability to inhibit the activation of the macrophage caspase-1 inflammasome.
A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing Abs but does not influence natural intestinal immunity (46.4)
Authors: Yuki Yoshikazu , Nochi Tomonori , Katakai Yuko , Shibata Hiroaki , Tokuhara Daisuke , Mejima Mio , Kurokawa Shio , Takahashi Yuko , Hatai Hirotsugu , Chubachi Aya , Nakanishi Ushio , Terao Kenji , Kiyono Hiroshi
Published: January 2023

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. Here, we examined whether the rice-based vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the 3 cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 months, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Interestingly, the macaques harbored naturally acquired intestinal secretory IgA (SIgA) Abs that were capable of reacting with CT and homologous heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli (ETEC) and that had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 month to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent exposure to the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing, naturally occurring CT- and LT-reactive SIgA Abs in nonhuman primates.
An Oral Bacterial-Based Diarrheal Vaccine Stimulates TGF-β-Producing Regulatory T (Treg) Cells and Confers Protection Against Experimental Autoimmune Encephalomyelitis (EAE) (47.4)
Authors: Jun SangMu , Ochoa-Rapáraz Javier , Mierzejewska Dagmara , Pascual David
Published: January 2023

Oral Salmonella vaccine expressing enterotoxigenic E. coli colonization factor antigen 1 (CFA/I) fimbriae induced production of Treg cells. Adoptive transfer of CD25+ Treg cells obtained from oral Salmonella-CFA/I-vaccinated mice conferred superior protection to proteolipid protein (PLP)139-151-dependent EAE to those from Salmonella vector-immunized mice; this was attributed to their differential cytokine profiles. Salmonella-CFA/I-vaccinated mice produced more TGF-β than Salmonella vector-immunized mice. To test the hypothesis Salmonella-CFA/I-induced Treg cells, independent of PLP-specificity, confers protection via TGF-β, SJL/J mice were immunized with either Salmonella-CFA/I or Salmonella vector. Two wks later, CD25+ and CD25- CD4+ T cells were sorted, adoptively transferred to naïve SJL/J mice, concomitantly treated with anti-TGF-β mAb or isotype IgG, and then induced with EAE. Mice adoptively transferred with Treg cells neutralized of their TGF-β showed earlier disease onset and greater disease severity than mice adoptively transferred with Treg cells treated with IgG control. Moreover, greater proinflammatory cytokine production was observed. This work shows Treg cells can be induced to high potency by simply vaccinating against irrelevant Ags using a live, bacterial vaccine, thus, offering a novel approach to treat autoimmune diseases independent of the auto-Ag. Supported by NIH AI-41123.
Requirement of microbiota adjuvanticity for T cell spontaneous proliferation and induction of experimental colitis (47.8)
Authors: Feng Ting , Wang Lanfang , Schoeb Trenton , Elson Charles , Cong Yingzi
Published: January 2023

Homeostatic T cell proliferation has been implicated in various immune-related diseases, including experimental colitis. However, little is known about how microbiota regulate T cell proliferation in lymphopenic hosts, and the role of T cell spontaneous proliferation (SP) in the pathogenesis of colitis. In this report, we show that both microbiota adjuvants and antigens are required for chronic intestinal inflammation. Microbiota ligands provide adjuvant activity to stimulate T cell SP by activating dendritic cells (DC) to produce IL-6 via a Myd88-dependent pathway. This was demonstrated by SP of adoptively transferred T cells in specific pathogen free (SPF) RAG-/- mice but not in germ-free RAG-/- mice. Reconstitution with cecal bacterial lysate-pulsed wild type DC but not with IL-6-/- or Myd88-/- DC restored T cell SP in germ-free RAG-/- mice. Adoptive transfer of CBir1 TCR transgenic (CBir1 Tg) T cells, which are specific for an immunodominant commensal bacterial flagellin antigen, induced colitis in SPF RAG-/- mice. Blockade of CBir1 Tg T cell SP by adoptive transfer of bulk OT II CD4+ T cells abrogated colitis development. Although adoptively transferred OT II T cells underwent SP in RAG-/- mice, the recipient mice failed to develop colitis due to the lack of cognate antigen in the intestinal lumen. Collectively, our data demonstrate that induction of colitis requires both microbiota adjuvant-driven T cell SP and microbiota antigen-specific T cell proliferation.
NFATc2 is an intrinsic negative regulator of proliferation in effector T cells and is necessary for extrinsic control by regulatory T cells (49.17)
Authors: Modiano Jaime , Highfill Sarah , Zhou Qing , Lewellen Mitzi , Highfill Steven , Bucher Christoph , Blazar Bruce
Published: January 2023

In addition to its effect to limit the number of cell division cycles and the accumulation of memory T cells, recent data suggest NFATc2 may be required to enable development and function of regulatory T cells (Tregs). Here, we verified loss of NFATc2 relieved intrinsic negative regulation: effector T cells (CD4+ /CD25-) from NFATc2 knockout (KO) mice showed more facile in vitro proliferation than effector T cells from WT or heterozygous mice and, in vivo had significantly more activated (CD69+ ) and memory phenotype (CD44hi ) T cells. In contrast, we found no differences in the number of Tregs (CD4+ /CD25+ /FoxP3+ ) between NFATc2 KO and WT mice. However, NFATc2 KO Tregs were less able to functionally suppress NFATc2 KO and WT effector cells in vitro. In contrast to NFATc2 KO Tregs, WT Tregs effectively suppressed proliferation of NFATc2 KO T cells, indicating that NFATc2 KO Tregs are hypofunctional. The reduced suppressive capacity of NFATc2 KO vs WT Tregs was not due to global T cell hyperproliferation since Tregs from Tob1 KO mice, that also have hyperproliferative T cells, were more potent suppressors than Tregs from WTs or NFATc2 KOs. The functional deficit of NFATc2 Tregs could not be replicated by treating WT Tregs with the NFAT inhibitor, cyclosporin A. Instead, Treg function was directly correlated with reduced expression of CTLA4 in NFATc2 vs WT Tregs. Our data support the premise that NFATc2 is required for Treg function, possibly by controlling CTLA4 expression.
Phosphoinositide 3-kinase p110delta isoform is essential for effective CTL responses during influenza virus infection (50.13)
Authors: Gracias Donald , Boesteanu Alina , Norton Jillian , Mueller Yvonne , Turner Martin , Katsikis Peter
Published: January 2023

Phosphoinositide 3-kinases (PI3K) have been known to play an important role in various functional aspects of immune cells. Class IA PI3K are heterodimers composed of a catalytic and a regulatory subunit. The catalytic isoform p110δ of class IA PI3K is preferentially expressed in hematopoietic cells and can regulate CD4+ T cells, B cells, Natural Killer and regulatory T cells. However, the role of p110δ in CD8+ T cell function has not yet been characterized. We have found that p110δ plays an important role in CD8+ T cell responses. Infection of p110δ deficient (p110δ -/-) mice with influenza virus resulted in decreased numbers of lung NP366-374- specific CD8+ T cells when compared to wild type mice (0.6±0.1x106 vs. 1.0±0.2x106, respectively, p=0.03) on day 10. To determine whether the impaired response was intrinsic to CD8+ T cells, OT-I and p110δ -/- OT-I cells were transferred into congenically mismatched recipients. These mice were infected with SIINFEKL peptide expressing influenza virus strain WSN-OVA257-264, and at day 10, lungs were harvested and analyzed for OVA257-264- specific CD8+ T cell responses. In contrast to mice transferred with control OT-I cells, those transferred with p110δ -/- OT-I cells showed a significant 5 - fold reduction in number of donor OVA257-264- specific CD8+ T cells (4.5±1.0x106 vs. 0.9±0.2x106, respectively, p=0.01). Our study shows that PI3K p110δ isoform is critical for in vivo CD8+ T cell responses.
BTLA regulates HVEM signaling in lymphocytes (50.22)
Authors: Calemine-Fenaux Jill , Hutchinson Leah , Vendel Andrew , Eaton Dan
Published: January 2023

The binding partner of the co-inhibitory receptor, BTLA (B and T Lymphocyte Attenuator) is the co-stimulatory receptor, HVEM (Herpes Virus Entry Mediator). We explored the biological relevance of these two opposing receptor binding partners on lymphocyte activity. Here, we report both receptors, which are endogenously expressed on primary lymphocytes, bind in a cis conformation. While BTLA is constitutively activated, HVEM does not induce its typical stimulatory response, i.e. up-regulation of NFκB signaling, in a stable co-expressing cell line. Even in the presence of the co-stimulatory HVEM ligand, LIGHT, NFκB levels remain unchanged while BTLA maintains its phosphorylation state. HVEM induced stimulatory activity was only observed in primary T cells when BTLA was neutralized with an antagonist antibody. Taken together, these data suggest BTLA is the predominant receptor of the two binding partners.
The Th17 immune response is controlled by a c-Rel−RORγ−RORγT transcriptional axis (50.36)
Authors: Ruan Qingguo , Kameswaran Vasumathi , Zheng Shijun , Wang Junmei , Liou Hsiou-Chi , Beg Amer , Chen Youhai
Published: January 2023

The Th17 cells utilize the retinoid-related orphan receptor-γ (Rorg) gene to specify their differentiation and lineage-specific function. However, how Rorg is switched on during Th17 differentiation is unknown. We report here that the transcription factor c-Rel drives Th17 differentiation by binding to, and activating, two distinct Rorg promoters that control RORγT and RORγ expression, respectively. Similar to RORγT, RORγ is selectively expressed in Th17 cells and is effective in specifying the Th17 phenotype. c-Rel-deficient T cells are significantly compromised in Th17 differentiation, and c-Rel-deficient mice are defective in Th17 responses, and resistant to autoimmune diseases. Thus, Th17 immunity is controlled by a c-Rel−RORγ−RORγT axis, and strategies targeting Rel/NF-κB can be effective for controlling Th17 cell-mediated diseases.
A distinct pattern of mRNA expression by monocytes is found in a subset of children with autism spectrum disorders (ASD) who present with recurrent infection, repeated loss of cognitive skills, and persistent gastrointestinal (GI) symptoms. (51.16)
Authors: Jyonouchi Harumi , Geng Lee , Streck Deanna , Toruner Gokce
Published: January 2023

A subset of ASD children (ASD Test Group) characterized by repeated loss of cognitive skills triggered by infection and frequent persistent GI symptoms were studied. We evaluated mRNA expression by peripheral blood (PB) monocytes in ASD test group (N=20, median 8 y) , ASD controls(N=22, median 7.6 y) , and normal control children (N=10, median 5.5 y) as well as the mothers of the ASD test (N=19) and ASD control (N=22) groups. We found that 568 genes were up-regulated and 72 genes were down-regulated (>2 fold) in the ASD test group versus 469 up-regulated and 57 down-regulated in the control groups. Only the ASD control group displayed up-regulation of CCL7 and CCL2 and down-regulation of IL-6 and CD86. The ASD test group mothers revealed up-regulation of 175 genes including CCL7 and down-regulation of 57 genes as compared to the ASD control mothers. The ASD test group also revealed decreased spontaneous production of IL-6 and IL-1β by PB mononuclear cells compared to controls. The 16/20 ASD test group children with persistent GI symptoms revealed up-regulation of CCL7 (>5 fold) and CCL2 and down-regulation of inflammatory mediators (IL-6, IL-23, IL-1α/IL-1β, CD86, and other chemokines) as compared to ASD and normal controls. Our results indicate a distinct pattern of mRNA expression in PB monocytes in the ASD subset with characteristic clinical features. Interestingly, CCL7 and CCL2 are up-regulated in the brain in various neurodegenerative disorders.
Epitope-specificity and effector function analysis of T and B cell response elicited by a novel single-cycle West Nile virus vaccine candidate (52.8)
Authors: Winkelmann Evandro , Xu Fangling , Nelson Michelle , Bourne Nigel , Mason Peter , Milligan Gregg
Published: January 2023

We have previously reported the development and characterization of RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine to prevent West Nile disease. RepliVAX inoculation elicits a strong, protective immune response in mice, hamsters, and non-human primates; however, the magnitude, epitope specificity, and functionality of the WNV-specific T and B cell responses elicited by immunization have not been characterized. Here we have begun to determine the ability of RepliVAX to elicit polyfunctional WNV-specific T cells. Vigorous WNV-specific CD8+ cytolytic T cell responses were detected in vivo in RepliVAX immunized mice at day 4 and 6 post inoculation. In addition to CTL activity, our initial results detected populations of CD8+ effector T cells secreting IFN-γ and/or TNF-α. Future studies will analyze the epitope-specificity and effector functions of both WNV epitope-specific CD4+ and CD8+ T cells from RepliVAX immunized mice by simultaneous measurement of IFN-γ, TNF-α, and IL-2 production. Importantly, RepliVAX vaccination resulted in development of vigorous CD8+ memory T cell responses. High numbers of NS4B epitope-specific CD8+ memory T cells were detected in spleens of immune C57BL/6J mice 30 days after inoculation with RepliVAX. We have also developed methods to quantify WNV E and NS1 protein-specific antibody secreting cell responses by ELISPOT. Together, these studies should provide an in depth analysis of immune effector function elicited by RepliVAX immunization.
The influence of prolactin on the inflammatory component of lupus nephritis. (83.11)
Authors: Dugan Amy , Morel Laurence
Published: January 2023

A positive correlation between serum prolactin (PRL) and lupus disease activity such as IgG anti-DNA antibody deposition in the kidney has previously been shown. While the presence of immune complex deposition is required for the initiation of lupus nephritis (LN), renal MCP-1 expression and the subsequent infiltration and activation of macrophages ultimately results in inflammation and full-blown LN. The influence of PRL on the inflammatory component of LN has not been reported. Mice were 6-8 month-old males and were either C57Bl/6 wild-type (WT), C57Bl/6 PRL KO, or BcN (lupus strain). Renal proximal tubule cells were isolated and the PRL receptor expression was determined by RT-PCR and immunofluorescence microscopy. Renal proximal tubule cells from C57Bl/6 WT mice were stimulated with PRL and the amount of MCP-1 in the media was determined by ELISA. Cell surface expression and the mRNA expression of all 4 isoforms of the PRL receptor were comparable in the 3 genotypes. Proximal tubule cells produced significantly more MCP-1 when stimulated with 1000 ng/ml of PRL compared to 0 ng/ml (p<0.01). Our results indicate that PRL has the potential to directly stimulate proximal tubule cell MCP-1 production/release. Whether lower doses of PRL would have a greater impact in the presence of immune complexes is unknown. Because the renal tubule cell isolation technique most likely removes immune complexes, an in vivo model of SLE to ascertain whether PRL .....
Delayed central tolerance of IGRP specific T cells is mediated by IGRP-expressing peripheral dendritic cells (83.28)
Authors: Krishnamurthy Balasubramanian , Fynch Stacey , Graham Kate , Thomas Helen , Santamaria Pere , Kay Thomas
Published: January 2023

Prevention of autoimmunity requires the elimination of self-reactive T cells during their development in the thymus and maturation in the periphery. Due to central and peripheral tolerance, transgenic NOD mice that overexpress islet specific glucose 6 phosphatase catalytic subunit related protein (IGRP) in antigen presenting cells, under the control of the class II MHC promoter, have no IGRP specific T cells. Peripheral tolerance of IGRP-specific T cells was mediated by both stromal cells and bone marrow derived APCs. However, central tolerance was mediated only by bone marrow-derived APCs. Moreover, central tolerance was age dependent, occurring only after 10 weeks and was not related to thymic involution, change in the absolute number of dendritic cells or the amount of IGRP expression. Age-dependent central tolerance was mediated by an increased proportion of thymus homing, signal regulatory protein alpha-positive CD8alpha negative dendritic cells. This provides in vivo data to indicate the potential feasibility of inducing central tolerance with DC therapy. They also raise the possibility that the efficiency of central tolerance might increase with age.